Cell Biology Lecture Notes Introduction: A.

Definition of a cell: fundamental structural and functional unit of all living organisms B. Characteristics of cells: 1) Contain highly organized molecular and biochemical systems and are used to store information 2) Use energy 3) Ca able of movement !) "ense environmental changes #) Can du licate $transfer genetic information to offs ring) %) Ca able of self&regulation &'ost cells are microsco ic $invisible to the na(ed eye) and thus) a microsco e is needed to vie* most cells. C) +istory: &,iscovery of the cell follo*ed by the develo ment of the microsco e A. 1%%#&-obert +oo(e& observed cells from the fruiting bodies of fungi B. Anton van .ee*enhoe(& observed a variety of cells and called them /animalcules/ C. 10312s&3heodor "ch*ann and 'atthias "chleiden develo ed the cell theory &Cell Theory states: 1. 2. 3. All living organisms are com osed of cells Cells are the functional units of living organisms Cells arise from ree4isting cells via division

,) Louis Pasteur&develo ed the theory of s ontaneous generation that is that cells could develo from non&living matter &Also *or(ed on roblem associated *ith the fermentation of 5rench *ine &10#6&develo ed a artial sterilization rocess called asteurization& involves heating at a moderate tem eratures to reduce the number of living microorganisms 7) 10%#-Mendel&demonstrated that cellular traits $ henoty es) *ere inherited & "eed sha e and color in garden eas & 8amed /5ather of 9enetics/

5) 1061&Johan Freidrick Miescher&isolated nucleic acids from cells /nuclein/

9) 100:& !"lt#an& urified nucleic acids

+) 1:!!&$s%ald "&ery) Colin 'ac.eod) and 'ac.yn 'cCarty& &,emonstrated that ,8A *as the heredity molecule &,8A could transform bacterial cells ;) 1:#2&Alfred 'ershey and 'artha Chase&also demonstrated that ,8A *as the heredity molecule &-adioactive ,8A from a virus *as able to infect and transform bacterial cells <) 1:#3&<ames (atson and 5rancis Crick&develo ed the 3&, structure of ,8A

=) 1:#0&'atte* Meselson and 5ran( )tahl&demonstrated that ,8A re licated by a semi conservative method
.) 1:%1&Brenner* Jaco+* Meselson&discovered -8A ') 1:%%&Niren+erg and ,horana&elucidated the chemical nature of the genetic code 8) 1:62&1:63&Berg* Boyer* and Cohen& discovered gene cloning >) 1:6#&-il+ert and )anger&develo ed chemical techni?ues to ra idly se?uence ,8A Cell )tructure: ;. ;;. ;;;. 'ost cells are microsco ic and cannot be seen by the na(ed eye. 'icrosco es *ere develo ed to visualize cells. -esolution is the minimum distance *here 2 ob@ects can be visually se arated

&Unresolved &Aartially resolved &-esolved &,e ends on: a. b. c. Bavelength of light -efractive inde4 of the medium >f the light

&3he na(ed eye can resolve t*o se arate ob@ects se arated by 211 um 'etric system: &1 meter C 3.3 feet) 1 (m C 113 m) 1cm C 11&2 m) 1mm C 11&3 m) 1um C 11&% m) 1nm C 11&: m) 1 A C 11&11 m) 1 m C 11&12 m

;D. .ight microsco e: &Can resolve t*o ob@ects 111&211 nm a art $including cells and large sub cellular organelles) &Uses different light sources and atterns of image formation a. b. Bright field d) differential interference ,ar( field e) fluorescence c) hase contrast

D. 7lectron 'icrosco e: &Uses a beam of electrons $e&) rather than light as an illumination source A. 3ransmission 7lectron 'icrosco e .T/M0 &7lectrons forming the image focused through the s ecimen &"hort *avelength of e& beam im roves the resolution of 37' to # A $.#nm) &Can resolve small sub cellular organelles and large roteins B. "canning 7lectron 'icrosco e .)/M0 &Used to e4amine surfaces of cells or isolated cellular structures &e& beam /scans/ the s ecimen &-esolution # to 11 nm Prokaryotic Cells& small and rimitive bacteria and blue&green algae $cyanobacteria) 9ree(: AroCbefore (aryonCnucleus &.ac(s s ecialized internal membrane&bound com artments (no*n as organelles &Cell membrane& functions in trans ort) the movement of substances in and out of the cell) and in energy roduction $brea(do*n of large molecules) hotosynthesis) &Cell *all& gives structural strength $rigidity) to the cell &Ca sule& @elly&li(e substance *hich rotects the cell *all from environmental damage &8ucleiod& contains a single circular molecule of ,8A $stores genetic information) &Cyto lasm& region surrounding the nucleiod and *ithin the cell membrane &Contains ribosomes and -8A $site of rotein synthesis) &Dacuole $vesicles)$blue&green algae)&site of hotosynthesis $storage) &5lagellum& rotein fiber the functions in movement

/ukaryotic Cell& $euCtrue (aryonCnucleus) 1. 2. 3. Aossesses a com le4 membrane system +as a true nucleus ,istinct membrane&bound intracellular com artments called organelles &8ucleus& dar(&staining body *ithin the cell by enclosed an intracellular membrane called the nuclear envelo e &8uclear envelo e contains ores) *hich are filled *ith a ring of roteins called annulus &Contains ,8A in the form of chromatin fibers &,8A is linear $linear ,8A E roteins C chromosome) &8ucleolus& a cell organelle in the nucleus that disa ears during art of cell division. Contains r-8A genes &8ucleus also contains -8A $m-8A) r-8A) and t-8A) &3ranscri tion& conversion of genetic information from ,8A to -8A occurs in the nucleus &,8A re lication&du lication of genetic material &Cyto1las#: ma@or ortion of the roto lasmic substance *ithin the cell membrane a. -ibosomes&a cyto lasmic article that contains -8A and rotein and is involved in the rocess of rotein synthesis

&3ranslocation& rocess *hich ta(es lace in the cyto lasm and converts genetic information in -8A into roteins &-ibosomes can either be freely sus ended in the cyto lasm or attached to intracellular membranes a. 7ndo lasmic reticulum $7-)& a net*or( of intracellular membranes *here secreting roteins are synthesized &-ough 7-& the 7- E ribosom es &"mooth 7-& the 7*ithout ribosom es & 5unction

s in the brea(do *n of fats attached to the rough 7- in the 9olgi com le4 a. 9olgi a aratus&a membranous organelle that ac(ages and sorts ne*ly synthesized secretory roteins .ysosome& organelle *hich contains digestive enzymes e. 'itochondrion&semiautonomous eu(aryotic cell organelle &"ite of res iration &Consists of an outer membrane and a convoluted inner membrane &"ite of A3A roduction *ithin the cell a. b. 'icrobody&organelle *ithin a cell containing s ecialized enzymes *hose functions involve hydrogen ero4ide $ ero4isome) 'icrotubules&com osed of tubulin

a.

h. 'icrofilaments&com osed of actin &Both $g and h) are involved in cellular movement a. b. Plant cell organelles: &Chloro last& involved in hotosynthesis &Central vacuole& rovides su ort to the lant via osmotic ressure ;ntercellular&includes flagella and cilia ;ntracellular& cyto lasmic streaming

&Cell *all& com osed of cellulose) *hich rovides e4tra strength and rigidity i. " ecialized rotozoan cell organelle: &Contractile vacuole& used to maintain ro er osmotic ressure and secretes *aste and e4cess + 2> &3*o ty es of nuclei 1) 'acronucleus& involved in ase4ual re roduction

2) 'icronucleus& involved in se4ual re roduction Che#ical Bonds: ;. ! ty es of molecules ma(e u cells: 1) Carbohydrates 2) .i ids 3) Aroteins !) 8ucleic acids ;;. Biological macromolecules are held together by several different ty es of bonds: 1) ;onic bond&a transfer of electrons 2) Covalent bond&the sharing of electrons 3) +&bonds&*ea( attraction *hen +E serves as a bridge bet*een 2 electronegative atoms by a covalent bond and electrostatic attraction !) 8on olar associations&hydro hobic vs. +ydro hilic #) Dan der Baals&a momentary di ole that *ill affect the electron distribution of neighboring molecules "cids and Bases: .e*is definition: 1. 2. 3. !. #. %. 6. Acid&a substance that can ta(e u an electron air to form a covalent bond Base&a substance that can donate an electron air to form a covalent bond +2> dissociates into +E ions and >+& F+EG E F>+&G C 1411&1! molesHliter $') + C &log11 F+EG Acid + is from 1 to 6 Base + is from 6 to 1!

&Condensation reaction&*hen t*o molecules are combined into one molecule *ith the release of one *ater molecule & A E B CC C E +2> 74: 2 amino acids are @oined together to form a di e tide molecule &+ydrolysis reaction&*hen one molecule is bro(en into t*o molecules *ith the addition of *ater molecule & C E +2> CC A E B 74: disaccharide maltose E *ater CC 2 glucose molecules

eacti&e $rganic Molecules:

1. +ydro4yl grou & strongly olar and highly reactive

2. Carbonyl grou & *ea(ly olar and highly reactive 3. Aldehyde !. =etone #. Carbo4yl grou & strongly olar and acts as an acid %. Amino grou & olar and acts as a base 6. Ahos hate grou & acidic and olar 0. "ulfhydral grou & readily o4idized &3*o sulfhydral grou s can bond together to form a disulfide bond Car+ohydrates: A. 5unction: 1. 2. 3. "tore energy $starches in lants H glycogen in animals) Arovides rigidity to lant cells $cellulose) ;nvolved in cell&cell communication $glyco roteins)

B. "tructure &Carbohydrates have a characteristic content of C) +) > atoms in the ratio of 1C:2+:1> 1) 'onosaccharide is the subunit of a carbohydrate 2) ,isaccharide contains 2 monosaccharide subunits 3) >ligosaccharide contains 2&11 monosaccharide subunits !) Aolysaccharide contains I11 monosaccharide subunits C. 'ost carbohydrate subunits contains 3 carbons $triose)) # carbons $ entose)) or % carbons $he4ose) glucose & glucose & glucose ,. 2 common monosaccharides are: fructose and galactose

7. ,isaccharide C 2 monosaccharide subunits lin(ed together &74: maltose C 2 glucose molecules lin(ed together &9lycosidic bond C is the bond bet*een 2 carbohydrate subunits formed by the eliminated of *ater 5. 74am les of Aolysaccharides:

1) Cellulose& com rises lant cell *allsJ molecule com osed of re eating B& glucose units $monomers) held together by B 1C! lin(ages 2) "tarch& $ rimary storage com ound in lants) is a macromolecule com osed of re eating & glucose units held together by 1C! lin(ages 3) 9lycogen& $ rimary storage com ound in animals) is a branched macromolecule com osed of re eating 1C! and 1C% glycosidic lin(ages "ucrose:

.actose:

&'ost monosaccharides can e4ist in alternative forms *hen molecules) *hich are attached to the carbon chain) can be oriented in different ositions &"tereoisomers & t*o molecules) *hich have the same molecular formula and the same chemical formula and hysical ro erties) but are different in the s atial arrangement of atoms &'ost carbohydrates e4ist in , and . forms

Li1ids: A. ,efinition& fats or fat&li(e substances that are insoluble in *ater and soluble in non olar solvents li(e acetone) ether) chloroform and benzene. B. 5unction: 1) Arimary com onent of cell membranes 2) "tore energy C. 3here are 3 ty es of li ids: neutral li ids) hos holi ids) and steroids ,. 8eutral li ids $fats and oils) 1) Com osed of fatty acids and glycerol $alcohol) 2) 5atty acid is a long) unbranched chain of carbon atoms attached by hydrogen and other grou s and a terminal carbo4yl grou 3) C+3$C+2)nC>>+ saturated fatty acid since the carbons have the ma4imum ossible K of + atoms !) C+3$C+2)nC+CC+$C+2)nC>>+ unsaturated fatty acid because of the one double C&C bond. #) "tructure of neutral li ids

& 9lycerol has 3 >+ grou s each of *hich is attached to a fatty acid 7. Ahos holi ids& rimary li ids in cell membranes &'ost common hos holi id is a hos hoglyceride &2 fatty acids E glycerol E hos hate grou &>ne end is hydro hobicJ the other end is hydro hilic Ccalled am hi athic $am hi hilic) &3o satisfy these solubility ro erties) hos holi ids arrange themselves into a li id bilayer &3he li id bilayer is the basic arrangement of the cell membrane 5. "teroids &Based on a frame*or( of ! carbon ring

&"terols are the most abundant grou of steroids

&A non olar side chain is attached at one end of the ring structure and a olar side grou is attached to the o osite end of the ring structure &-2 is a olar unit and - is a non olar unit &3he combination of olar and non olar side grou s gives hos holi ids dual solubility ro erties &An e4am le of a sterol is cholesterol

&Cholesterol is an im ortant com onent of the cell membrane in all animal cells &Cholesterol also can be de osited inside arteries causing bloc(age) *hich contributes to the disease arteriosclerosis $hardening of the arteries) &+ormones are steroids and they lay ma@or roles in cell regulation) cell metabolism) and cell gro*th ;. Aroteins carry out many cellular functions: 1) Arovide cellular su ort $cytos(eleton: microtubules L tubulinJ microfilaments & actin)

2) As enzymes) they catalyze cellular reactions 3) "tabilize and control gene activity via interactions *ith other roteins and nucleic acids $,8A or -8A) !) Used in cell trans ort and cell recognition $cell membrane) #) ;nvolved in cell&cell trans ort via secretory roteins and hormones ;;. Aroteins are com osed of subunit structures called amino acids A. 21 ma@or biological amino acids B. 9eneral structure of amino acid

C. 21 amino acids & commit to memory:

1) "tructure 2) 8ame 3) 3 letter abbreviation !) 1 letter abbreviation ,. Amino acids can be lin(ed together in chains of 2 or more units

&Ae tide bond & is a bond in *hich the carbo4yl grou of one amino acid is @oined to the amino grou of a second amino acid via a condensation reaction &Ae tide & is a chain com osed of 2 or more amino acids and contains one or more e tide bonds &74: di e tide C chain com osed of 2 amino acids &3ri e tide C chain com osed of 3 amino acids &Aoly e tide is an amino acid chain com osed of 3 or more amino acids &3he amino acid se?uence of a oly e tide chain is called the rimary structure of a rotein 7. "econdary structure of roteins & is the conformation im osed on the oly e tide chain by hydrogen bonding bet*een amino acids & 3here e4ists hysical constraints on the rotation of the al ha carbon atoms that flan( the e tide bond & ;t has been determined that there are only 2 or 3 sta+le arrange#ents of a#ino acids *hich conform to these restraints 1. 2. 3. Al ha heli4 Beta strands $sheets) -andom coil & 3hese arrangements com ose the secondary structure of a oly e tide chain &"econdary structure & is the arrangement of al ha helices) beta sheets) and random coils in a oly e tide chain 1) Al ha heli4 & common structural motif of a oly e tide chain in *hich the linear se?uence of amino acids folds into a right&handed heli4 &+eli4 is stabilized by internal hydrogen bonding bet*een bac(bone atoms 2) Beta sheet & common structural motif of a oly e tide chain) *hich is com osed of beta) strands that are oriented in an anti arallel fashion &"tabilized by internal hydrogen bonds &Beta strand & is an e4tended zigzag arrangement of amino acids in a oly e tide chain &Beta barrel & is a cylindrical arrangement of beta sheets &74am le of a rotein that is com osed rimarily of beta sheets is the sil( rotein secreted by sil( *orms $contributes to the high strength of sil( fibers) 3) -andom coil & an irregular configuration of amino acids *ithin a oly e tide chain &Usually com osed of roline & cannot fit into an al ha heli4 or beta sheet &Allo*s the rotein to bend and fle4

&Allo*s the rotein to com act into its most stable energetic structure 5) 3ertiary structure of roteins &3he three&dimensional arrangement of a oly e tide chain *ithin a rotein $monomeric rotein) 9) Muaternary structure of rotein &3hree&dimensional relationshi bet*een 2 or more oly e tide chains *ithin a com le4 rotein &74: coiled coil N tri le heli4 &,imer C 2 subunits &+omodimer C identical subunits &+eterodimer C distinct subunits &'ultimeric rotein & com osed of 2 or more subunits $identical or distinct) Nucleic acids: ;. ,efinition & a large) chain&li(e macromolecule containing hos horic acid) sugar) and a nitrogenous base &2 e4am les are deo4yribonucleic acid $,8A) N ribonucleic acid $-8A) a. "ugar is #&carbon sugar called a entose ribose deo4yribose

b. Ahos horic acid is com osed of one or more hos hate grou s $A>!&) c. 8itrogenous base C 2 ty es urine yrimidine

1. 3*o common urine bases) adenine and guanine

%&amino urine $adenine) 2&amino&%&hydro4y urine $guanine)

2. 3hree common yrimidine bases) cytosine) thymine) and uracil !&amino&2&hydro4y yrimidine 2)!&dihydro4y yrimidine 2)!&dihydro4y&#&methyl yrimidine $cytosine) $uracil) $thymine)

;;. 8ucleoside & macromolecule com osed of a nitrogenous base @oined to a entose ;;;. 8ucleotide & is a macromolecule com osed of a nitrogenous base) a entose) and lin(ed $esterified) to one or more hos hate grou s &#2&ribo$deo4yribo) & nucleotide & Ahos hate is lin(ed to the 32 >+ of the entose C 32&ribo$deo4yribo) & nucleotide ;D. 8omenclature of nucleosides: A&deo4yriboside C deo4yadenosine A&riboside C adenosine 9&deo4yriboside C deo4yguanosine 9&riboside C guanosine C&deo4yriboside C deo4ycytidine C&riboside C cytidine 3&deo4yriboside C deo4ythymidine U&riboside C uridine D. 8omenclature of nucleotides in ,8A base & deo4yribose & hos hate #2&dA'A C deo4yadenosine&#2&A>! #2&d9'A C deo4yguanosine&#2&A>!

#2&d3'A C deo4ythymidine&#2&A>! #2&dC'A C deo4ycytidine&#2&A>! D;. 8omenclature of nucleotides in -8A base & ribose & hos hate #2&A'A C adenosine&#2& A>! #2&9'A C guanosine&#2& A>! #2&U'A C uridine&#2& A>! #2&C'A C cytidine&#2& A>! D;;. 8omenclature of nucleoside tri hos hates A3A C adenosine&#2&tri hos hate dA3A C deo4yadenosine&#2&tri hos hate D;;;. 8ucleic acids -8A and ,8A &8ucleotides held in chains by bridging a hos hate grou that e4tends bet*een the #2&carbon of one sugar *ith the 32&carbon of a second sugar $held together by a hos hodiester bond) &Aroduces a bac(bone chain of alternating sugar and A>! grou s &,8A e4ists in a double heli4 that contains 2 intert*ined chains of nucleotides &-8A is single&stranded /n4y#es: 1. -evie* thermodynamics &5irst and second la* of thermodynamics &-eversible reactions &Cou ling reactions &"tandard free energy change 2. ,efinition of an enzyme & is a rotein *hich increases the rate of a s ontaneous reaction $catalyzes the reaction) a) .o*ers the activation energy of the transition state b) -eaction *ould roceed *ithout the enzyme

c) 7nzyme cannot ma(e a reaction occur that *ould not roceed s ontaneously *ithout the enzyme d) 7nzymes do not alter the e?uilibrium of a reversible reaction e) 7nzymes increase the rate at *hich a reaction reaches e?uilibrium Classification of en4y#es: 1. >4idoreductases & catalyzes a reaction in *hich electrons are removed from the substrate are donated directly to molecular o4ygen &Catalyzes o4idation&reduction reactions &Act on alcohols) (etones) aldehydes) amines) etc. 2. 3ransferase & catalyze the transfer of functional grou s &"ulfhydral) glycosyl) aldehyde) acyl) etc.

3. +ydrolases & catalyze hydrolysis reactions &9lycosidic bonds &Ae tide bonds !. .yases & catalyze the addition of grou s to double bonds & CCC) CC8) CC> &74: AA lyases involved in re airing ,8A #. ;somerases & catalyze an intramolecular rearrangement &Catalyzed isomerization reaction &;somerization & rearrangement of atomic grou *ithin the same molecule *ithout any loss or gain of atoms %. .igases & a grou of enzymes that catalyze reactions in *hich a bond is formed bet*een 2 substrate molecules using energy $A3A) obtained from the cleavage of a yro hos hate bond &74: ,8A ligase N -8A ligase

Characteristics of en4y#atic 1roteins: &7nzymes combine briefly *ith reactants during an enzyme&catalyzed reaction $enzyme&substrate com le4) &7nzymes are released unchanged after catalyzing the conversion of reactants to roducts

&7nzymes are s ecific in their activityJ each enzyme catalyzes the reaction of a single ty e of molecule or a grou of closely related molecules &7nzymes are saturated by high substrate concentrations &'any enzymes contain non rotein grou s called cofactors &;norganic cofactors C metal ions &>rganic cofactors C coenzymes $e4: vitamins) ;. Activation energy: the energy in e4cess of the ground state that must be added to a molecular system to allo* a chemical reaction to start $e.g. roc( on a cliff must be ushed to roll do*n the hill) &>ne *ay to su ly energy is to heat the reactants

&"econd *ay is to add a catalyst &Catalyst forms a com le4 *ith the reactant) thus bringing the reactants closer together so they can react &;n biological systems a catalyst is called an enzyme) *hich lo*ers the activation energy of the system &9ra h:

;;. 3hree enzymatic mechanisms *hich can contribute to the formation of a transition state is a catalyzed biological reaction a) 7nzyme brings reacting molecules into close ro4imity b) 7nzyme orients reactants into ositions to induce favorable interactions c) 7nzymes alter the chemical environment of the reactants to romote interaction &7.g. is to create a non olar environment &5avorable condition for reactant non olar molecules ;;;. 5actors affecting enzyme activity

a) "ubstrate concentration b) 7nzyme inhibition & Com etitive & 8oncom etitive c) 3em erature & o timum tem . !1C &;f tem . increases *ill denature the roteins d) + & o timum + 6 for most enzymes ;D. "ubstrate concentration & reactants $substrates) roducts &As the reaction roceeds) the concentration of substrate decreases *ith time &;f *e increase the concentration of substrate) *e *ill increase the rate of an enzymatic reaction to a oint &5rom this oint) if add more substrate) the rate of the reaction *ill not increase &3he enzyme is said to be saturated *ith substrate &3his effect is seen *ith all enzymes &9ra h:

&Ahenomenon led 'ichaelis and 'enten $1:13) to the e4 lanation that the enzyme $7) and the substrate $") form a reversible com le4 7". 3he 7" then brea(s do*n to give the roduct A and the free enzyme &3hey derived an e4 lanation to describe enzyme reactions 6. ,erivation of the 'ichaelis 'enten 7?uation:

/n4y#e inhi+ition: &'odel for an enzyme&catalyzed reaction re?uires that the enzyme and substrate form a recognizable chemical com le4 7" $transition state) &;n general) enzymes are much larger in size than substrate molecules &Aortion of the enzyme that com le4es *ith the substrate are called the active center $site) of the enzyme &;t is not (no*n e4actly ho* enzymes lo*er the energy re?uired to form the transition state or 7" com le4 &Be do (no* that the com le4 e4ists by an e4 eriment com leted by .erner and "chultz *ho generated Ab2s against molecules *hich closely resemble a theoretical transition state $transition state analogs&act as enzymes by catalyzing reactions C abzymes) &A good amount of chemical information concerning the active sites of enzymes has been obtained by studying enzyme inhibitors &Chemical inhibitor & is a substance that re resses or sto s a chemical reaction &;nhibition & reduction in the rate of enzymatic activity 7 E " 7" 7 E A 7 E ; 7; no roducts a) Com etitive inhibition & b) 8oncom etitive inhibition &

1. Com etitive inhibition & the inhibition of enzyme activity caused by the com etition of an inhibitor *ith a substrate for the active $catalytic) site on the enzymeJ im airment of the function of an enzyme due to its reaction *ith a substance chemically related to its normal substrate &Dery common situation in the study of drug action &'any drugs *or( by com eting *ith the normal substrate molecules for the active site of enzymes &.ine*eaver&Bur(e lot:

2. 8oncom etitive inhibition & inhibition of enzyme activity by a substance that does not com ete *ith the normal substrate for the active site and thus cannot be reduced by increasing the substrate concentration &.ine*eaver&Bur(e lot:

egulation of /n4y#atic "cti&ity: &;n living cells) chemical e?uilibria for reactions are seldom) if ever) reached &,ue to the fact that cellular chemical reactions are cou led to form metabolic ath*ays ABC,7 &3hree ma@or (no*n mechanisms by *hich enzyme activities a ear to be regulated: a) Change in the rate of synthesis of the enzyme &;nduction of the enzyme N re ression of the enzyme contribute to gene regulation b) 5eedbac( inhibition of the enzyme & cellular control mechanism by *hich the end roduct of a series of metabolic reactions inhibits the activity of an earlier enzyme in the metabolic ath*ayJ thus) *hen the end roduct accumulates) its further roduction ceases c) Allosteric regulation $inhibition): &>ccurs by reversible combination of substances *ith sites on the enzyme other than the active site &7nzyme is called an allosteric enzyme & an enzyme *hose active site can be altered by the binding of a small molecule at a non&overla ing site &7nzyme can be activated by this binding C allosteric activation &7nzyme can be inhibited by this binding C allosteric inhibition N" +ased en4y#es .ri+o4y#es0: &5irst discovered in the rotozoan 3etrahymena &-8A molecule involved in s licing $or modifying) -8A intermediate structures &,iscovered by 3om Cech DN" structure: &"tructure of ,8A *as deduced by 4&ray diffraction and ro osed by Batson and Cric( 5 characteristics: 1) Contains 2 nucleotide chains *hich *ind into a right&handed double heli4 2) Bac(bone is com osed of alternating sugar and hos hate grou s se arated by 1.1nn 3) " ace is filled by a urine: yrimidine base air) *hich lies in a flat) lane er endicular to the bac(bone !) 7ach turn of the double heli4 is 11 b

#) 3he b are held together by +&bonds %) 3here are 2 surface grooves on the heli4) ma@or groove and a minor groove 6) 3he nucleotide chains of the heli4 run in o #2 32 32 #2 0)3his anti arallel arrangement re?uires that a ne* ,8A chain being co ied from an e4isting strand must run in the o osite direction &Aarent strand C tem late &8e* strand C com lementary strand Modifications to DN" )tructure: 1. Alternative ,8A forms $B&,8A) &.eft&handed heli4 $O&,8A)&involved in gene transcri tion and gene recombination 2. Chemical modification &# methyl cytosine&involved in gene regulation &3he methylation of ,8A leads to a shut do*n of transcri tion 3. "u ercoiling&com action of ,8A &"u ercoils induced by a grou of enzymes called to oisomerases&an enzyme that changes the e4tent of su ercoiling of the ,8A com le4 &3hese enzymes can also remove su ercoils or rela4 them &"u ercoiling lays a ma@or role in gene regulation) transcri tion) and ,8A re lication Proteins "ssociated %ith DN": A. +istones&basic roteins rich in arginine and lysine that occur in close association *ith the nuclear ,8A of most eu(aryotic organisms 1. 'a@or ty es of histones & +1) +2A) +2B) +3) and +! &3he last four are associated *ith each other and together *ith ,8A they form a structure called a nucleosome 2. 8ucleosome&the functional unit of chromatin in eu(aryotes &8ucleosomes ac( ,8A in a stable coiled form osite directions

&Contribute to gene regulation $changes structure during rocess of ,8A re lication) and transcri tion) 3. 8ucleosome structure !. Core article&of a nucleosome consists of 2 each of the four core histone roteins 2 +2A) 2+2B) 2 +3) and 2 +! lus 2 left hand turns of ,8A that is *ound around the roteins #. .in(er&is the ,8A that lin(s one nucleosome to the ne4t %. +1 associates *ith ,8A at the end entering and leaving the core article

B. 8onhistone Aroteins& roteins other than histones that are associated *ith ,8A in chromatin 1. 3y es of nonhistone roteins a) 3ranscri tion regulatory factors& roteins regulating in the rocess of transcri tion b) 7nzymes catalyzing reactions of transcri tion) re lication) recombination) ,8A re air) and modification of ,8A and chromosomal roteins c) Aroteins contributing to maintenance of chromatin structure

$rgani4ation of Nucleoso#es %ith Chro#atin Fi+ers: &8ucleosomes form solenoid structures to further com act chromatin &3his structure de ends on +1 &;n humans 1 meter of ,8A is com acted 11! fold Chro#atin in Inter1hase nucleus: &7uchromatin&loosely ac(ed regions of chromatin fibersC*here genes are e4 ressed &+eterochromatin&densely ac(ed regions of chromatin fibersCinactive genes DN" e1lication: &3he rocess *here the arental ,8A molecule is du licated into 2 e4act co ies &Arocess occurs by a semi&conservative mechanism &"emi&conservative re lication& is the roduction of a double&stranded ,8A molecule containing one ne* strand and one arental strand

&;llustrated by 'eselson and "tahl &3hey labeled ,8A in dividing bacterial cells *ith an isoto e of nitrogen $1#8) &-emove label and gre* cell in 1!8 &After one generation) isolated ,8A and found that the ,8A *as com osed of one strand of 1!8 and one strand of 1#8 &;f ,8A re licated by a conservative mechanism) ,8A *ould e4ist in t*o forms $ 1!8 and 1#8) $&er&ie% of Mechanis#: 1. ,8A re lication is catalyzed by an enzyme ,8A olymerase 2. ,8A olymerase re?uires a short stretch of nucleotides to initiate re lication called a rimer 3. -e lication of ne* strand occurs in a #2 32 direction !. ,8A olymerase binds to the 32 >+ grou of the sugar of the rimer and recognizes the first base to be co ied #. Binding of the base favors the lin(age bet*een the 32 >+ grou of the rimer and the innermost $al ha) hos hate grou bound to the #2 carbon of the incoming base &3he beta and gamma A>! are removed &3he al ha hos hate is bound to the o4ygen of the 32 >+ grou on the sugar of the rimer creating a #2 32 hos hodiester bond or lin(age bet*een the rimer and the added nucleotide &3his is a lin(age bet*een 2 alcohols and a A>! grou %. ,8A olymerase moves to the ne4t base on the tem late and re eats ste s ! and # &,uring this rocess) each nucleotide is added to an e4 osed 32&>+ grou &A 32&>+ grou is al*ays resent at the ne*est end of the gro*ing chain &"ynthesis roceeds in a #2 32 fashion Funda#ental Features of DN" e1lication: 1. 3*o nucleotide chains of tem late ,8A double heli4 must un*ind and se arate rior to re lication 2. Un*inding and re lication must roceed in the same direction 3. -e lication of the 32 #2 tem late occurs in a #2 32 direction) *hich roduces a continuous) or leading strand !. -e lication of the #2 32 tem late also occurs in a #2 32 fashion

&5orms short lengths or >(aza(i fragments &Aroduces a discontinuous or lagging strand &5ragments are then @oined together *ith covalent bonds catalyzed by the enzyme ,8A ligase )te1s in DN" e1lication: 1. Un*inding of ,8A double heli4 &Catalyzed by enzymes that re?uire energy $A3A) called ,8A helicase &ss,8A region is then stabilized by ,8A binding roteins &un*inding induces su ercoils into the ,8A molecule &su ercoils are removed ty e of enzyme ,8A to oisomerase that introduces single or double&stranded brea(s in the ,8A & rimer&is a short nucleotide se?uence $often -8A) that is aired *ith one strand of ,8A and rovides a free 32&>+ at *hich a ,8A olymerase starts synthesis of a ne* ,8A chain 2. after un*inding) -8A rimers are attached to ss,8A through the actions of a s ecialized -8A olymerase $ rimase) & rimase layers #&11 nucleotides in length in a #2 32 direction do*n a strand & rimer rovides a free 32&>+ grou that is necessary for ,8A olymerase to o erate 3. ,8A olymerase then directs the incor oration of the ro er base during the #2 32 re lication of the tem late strands $leading and lagging strands) &in addition to its olymerization function) ,8A olymerase also has a roofreading function &,8A olymerase searches for ,8A b mismatches) &accuracy of ,8A re lication is 1H116 bases Prokaryotic DN" 1oly#erases: &; and ;;Cfunction in ,8A re air &;;;C,8A re lication /ukaryotic DN" Poly#erases: & C,8A re lication & C,8A re air

& C,8A re lication in mitochondria and chloro lasts & C,8A re lication & C,8A re air & roofread function& carried out in a 32 #2 or a #2 32 &e4onuclease&enzyme that removes nucleotides one at a time from the end of a nucleotide chain &endonuclease&an enzyme that catalyzes the cleavage of ,8A at a s ecific internal site $restriction enzyme) &,8A can e4ist as a circular or a linear molecule &re lication of linear end of ,8A is carried out by an enzyme com osed of -8A called a telomerase &telomerase adds re eated nucleic acid se?uences to the end of ,8A &re eating ends are used for rimer synthesis to generate du licate ,8A) *hich corres onds to the se?uence end of the linear ,8A molecule N": 1. 3ranscri tion: &the synthesis of m-8A) r-8A) and t-8A from a ,8A tem late &messenger -8A $m-8A)&is the ribonucleic acid $-8A) that s ecifies the amino acid se?uence for a articular oly e tide chain &ss-8A &ribosomal -8A $r-8A)&is the ribonucleic acid of various sizes that ma(es u &constitutes u to :1P of the total -8A of a cell &ss-8A) *ith helical regions formed by b bet*een com lementary regions *ithin the strand &transfer -8A $t-8A)&is the ribonucleic acid involved in carrying amino acids to the ribosomes during translation &for each amino acid there are one or more corres onding t-8A2s that can bind it s ecifically 2. the enzyme that catalyzes transcri tion is called -8A olymerase 3. each -8A is transcribed from ,8A in a recursor form C $hn-8A) heterogeneous nuclear -8A) re& m-8A) or re&-8A !. the conversion of hn-8A m-8A) r-8A) and t-8A is called -8A rocessing &ste s: art of the ribosomes

1. addition of a #2 ca 2. addition of a 32 oly&A tail 3. s licing out of introns !. -8A structure &single stranded molecule com osed of ribonucleotides &molecule can form a variety of double&helical structures&*hich lay a ma@or role in gene e4 ression &structures: hair in stem loo

&double&helical hair in structures contain inverted re eats $;-) &*hich are symmetrically reversed se?uences $ alindrone se?uences) & rimary structure&se?uence of nitrogenous bases &secondary structure& double&helical structure derived from base& airing of inverted re eats &tertiary structure& 3&, structure such as r-8A and t-8A &2o and 3o structures are im ortant as recognition sites for enzymatic) structural) and regulatory roteins active at all levels of -8A function from transcri tion) rocessing) and -8A trans ort through rotein translation #. -8A transcri tion: &re?uires: 1) a ,8A tem late 2) 'g2E or 'n2E 3) 5our -8A nucleoside tri hos hates $A3A) C3A) 93A) U3A) a)-8A olymerase binds to the ,8A tem late and recognizes the first base $e4. 9uanine) b)-8A olymerase recognizes and binds to C3A $b *ith guanine)J undergoes a conformational change

c)-8A olymerase reads the ne4t base $adenine) d)-8A olymerase binds U3A $b *ith adenine) e)-8A olymerase catalyzes the lin(age bet*een C3A and U3A &terminal t*o hos hates of U3A $ and ) are removed &al ha hos hate binds to the >+ at carbon 3 of the ribose sugar &enzyme reaction creates a hos hodiester lin(age bet*een the 32 carbon of the first sugar and the #2 carbon of the second sugar f)re eat ste s b e for the ne4t base g)at one end of the ,8A tem late) ne*ly synthesized -8A molecule and the enzyme are released from the ,8A tem late) and transcri tion is terminated &the first nucleotide retains 3 hos hates $lin(ed to the #2carbon of sugar)CC#2 end of the molecule &last nucleotide has an >+ grou at the 3 carbon of the sugarCC32 end of the molecule &since the #2 carbon mar(s the beginning and a 32 >+ mar(s the end of a -8A molecule transcri tion is said to roceed in a #2 32 direction %. 3hree hases of transcri tion: 1. initiation&attachment of -8A olymerase to the ,8A tem late and binding by the enzyme of the first nucleotide in the ne* strand 2. elongation&reaction in *hich -8A nucleotides are added se?uentially according to the ,8A tem late 3. termination&end of -8A olymerization *here -8A olymerase and the ne*ly synthesized transcri t are released from the tem late Transcri1tion: I! Initiation: &35;;, is a com le4 multimeric rotein) *hich includes one oly e tide called the 3A3A&binding rotein$3BA) *hich binds to the 3A3A se?uence in the romoter &u stream ,8A se?uences: &transcri tion starts at E1 on the ,8A tem late a)3A3A bo4 functions in: &initiation of transcri tion and the binding of -8A olymerase

&located &21 to &31 bases from the start oint of transcri tion b)CAA3 bo4& se?uence &01 b u stream from the start of transcri tion &functions: se?uence binds regulatory roteins that modify the rate of transcri t initiation c)further u stream $or do*nstream) $11112s of b ) there are ,8A se?uences &enhancers&a ,8A se?uence that can stimulate transcri tion at an a reciable distance from the site *here it is located. &contains ,8A se?uences *hich bind regulatory roteins &coactivators&/ada ter/ molecules that integrate signals from activators or re ressors and binds or relays to basal transcri tion factors &re ressors&bind to selected sets of genes at sites (no*n as silencers) *hich slo* the rate of transcri tion 1. -8A olymerase binds to a s ecific region of ,8A called the romoter *ith the aid of transcri tion factors & romoter& region of the gene that signals -8A olymerase binding and the initiation of transcri tion &transcri tion factors&accessory roteins not art of -8A olymerase that are re?uired to initiate and regulate the rocess of transcri tion 2. only one of the 2 ,8A nucleotide chains contains the correct romoter se?uence and acts as the tem late & romoter length is determined by a techni?ue called ,8A foot rinting 3. -8A olymerase binds the first -8A nucleotide $creates the #2 end of the transcri t) II! /longation: &this is a olymerization reaction &during elongation) -8A olymerase: 1. binds a nucleoside tri hos hate and matches it to the tem late 2. s lits t*o hos hate grou s from the nucleoside tri hos hate and forms the hos hodiester lin(age 3. moves to the ne4t tem late base &during elongation) some of the initiation factors are re laced by elongation factors &-8A olymerase li(e ,8A olymerase has a roofread function and uses a 32 #2 e4onuclease activity to remove incorrect or base mis airs during transcri tion III! Ter#ination:

1. there are s ecific termination ,8A se?uences) *hich signal the end of transcri tion 2. termination factors&function in releasing -8A olymerase and elongation factors from the ,8A tem late and also release the transcri t from the tem late 3 Ty1es of N" 1oly#erases: 1. -8A olymerase ; & transcribes r-8A 2. -8A olymerase ;; & transcribes m-8A 3. -8A olymerase ;;; & transcribes #" r-8A) t-8A) and sn-8A2s )tructure and Processing of # N": 1. m-8A transcri tion is the first ste in the se?uence of events leading to rotein synthesis 2. m-8A is transcribed from the ,8A tem late as a recursor molecule called re&m-8A or hn-8A 3. "tructure of an m-8A molecule: a. #2 ca & consists of 3 nucleotides *here the first is an inverted or reversed guanisine nucleotide &ca is added immediately after transcri tion &3 functions of the ca : 1. aids in the initiation of translation 2. acts as a signal) *hich is recognized by the nuclear envelo e 3. rotects the -8A from degrading enzymes called nucleases b. #2 untranslated region $#2 U3-)&,8A se?uence that is transcribed into -8A but the -8A se?uence is not translated into an amino acid se?uence &functions of the #2U3-: 1. can increase or inhibit translation of -8A molecule 2. contains se?uences *hich are recognized by ribosomes and accessory roteins for initiation of translation&called consensus se?uences c. coding se?uences&an -8A se?uence that begins *ith AU9 initiator codon 'et and terminates *ith terminator codons UA9) U9A) or UAA. &also called transcribed se?uences they are com osed of e4ons and introns &e4on&is a segment *ithin a gene that carries art of the coding information for a rotein $,8A se?uences that are eventually translated into amino acid residues)

&intron&is a region of a gene) *hich is transcribed into a -8A transcri t that is removed but not translated) by s licing $,8A se?uences *hich are not eventually translated into amino acid residues) &introns are also called intervening se?uences &s licing&is the e4cision of a segment of -8A follo*ed by a re@oining of the remaining fragments $e4ons) *hich is catalyzed by ,8A or -8A ligase d. 32 untranslated region $32U3-)& -8A se?uence *hich follo*s the coding se?uence and consists of -8A molecules that are not translated into amino acids 1. 32U3- contains a short AAUAAA that signals the end of transcri tion & rocessing signal&fi4es length of 32U3- and indicates the site for attachment of the oly$A) tail & oly$A) tail&unbro(en string of adenine nucleotides$31&211) added to the 32 end of the -8A molecule by an enzyme oly$A) olymerase $may function in stability and aid in translationQQ) 2. 32U3- se?uences rovide stability for the -8A molecule Processing Pre-# N"6s: &occurs in the nucleus 1. addition of #2ca $*hile transcri tion is occurring) and 32 olymerase $A) tail $after transcri tion) 2. removal of introns by s licing a)occurs by interactions of the hn-8A *ith a s liceosome&a nuclear com le4 com osed of -8A $in the form of sn-8A) and roteins &ste s: 1)s liceosome binds to the intron 2)induces a conformational change in the intron *here the intervening se?uence forms a lariat or branched -8A structure 3)intron is removed and the t*o neighboring e4ons are @oined $by -8A ligase) &there are consensus&s licing se?uences at the boundaries of the intron & #2 9U se?uence and a 32 A9 se?uence called a 9UHA9 boundary se?uence &-ibosomal -8A $r-8A)&most abundant -8A found in the cell &identified in "uedberg units *hich is e4 ressed in the sedimentation constant $") 1" C 11 &13 sec

&the sedimentation constant is ro ortional to the rate of sedimentation $migration) of a molecule in a given centrifugal field and is related to the ring and sha e of the molecule &the faster the molecule descends the larger the " number and in general) the higher the molecular *eight &eu(aryotic ribosomes& com osed of 2 subunits each subunit consists of one or more -8A s ecies and 31&!1 different roteins 7 ty1es of r N": a)10" r-8A is associated *ith the smaller subunit b)20") #.0") and #" r-8A is associated *ith the larger subunit &20") 10") and #.0" r-8A are transcribed as a recursor -8A molecule by -8A olymerase ; &.oo( at re r-8A gene schematic &intragenic s acer vs. ;ntrons &#" r-8A is transcribed se arately as a hn r-8A molecule by -8A olymerase ;;;R re #"r-8A Large 1re-r N" genes .)tructure of r N"6s0: 1. gene encoding large re&r-8A2s occurs in multi le co ies in all eu(aryotes &concentrated in clusters on eu(aryotic chromosomes &in contrast to m-8A genes) *hich occur largely in single co ies &each re eat in the large re&r-8A gene cluster consists of a coding segment and a long intergenic s acer $region that is not transcribed) &intergenic s acers contain se?uences *hich signal initiation and termination of transcri tion 2. each gene *ithin the cluster contains one co y of 10") #.0") and 20" r-8A coding se?uences &coding se?uences se arated by intragenic s acers $se?uences *hich are transcribed but are later removed during re&r-8A rocessing) &2 rimary romoter se?uences: 1. core romoter element &31 to E1# 2. u stream romoter element &1#1 to &#1 &no 3A3A or CAA3 bo4es &order of se?uences #2 32 are: enhancer romoter transcribed intragenic s acer 10" coding se?uence transcribed intragenic s acer #.0" coding se?uence transcribed intragenic

s acer 20" coding se?uence $e4ons and introns) intergenic s acer $not transcribed) contains terminator se?uences Transcri1tion and Processing of Pre-r N": 1. binding of -8A olymerase ; to #2 flan(ing romoter $needs transcri tion factors) & ".1 transcri tion factor&binds *ith olymerase ; to the romoter&contains 3A3A binding rotein &u stream binding factor&UB5&binds to core and u stream romoter elements &stimulates binding of both ".1 and -8A olymerase ; to the romoter 2. transcri tion continues through the large re&r-8A gene &co ies both coding se?uences and s acers *ithout interru tion 3. transcri tion sto s at intergenic s acer $terminator signals) !. -8A rocessing follo*s transcri tion & re&r-8A is cut to 10") #.0") and 20" r-8A molecules &first ste liberates 10" E #.0" @oined to 20" CIthen #.0" and 20" are se arated &reactions thought to be catalyzed by -8A e4onucleases and endonucleases #. some re&r-8A contain introns *hich must be removed by s licing &not *ell understood but it is thought to involve secondary structures of -8A molecules &self&s licing of -8A $ribozymes) &grou ; intron removal $3etrahymena)&de ends on the attern of secondary structures set u by inverted se?uences in the 3etrahymena intron &grou ;; intron removal $mitochondria and chloro lasts)&forms an intermediate lariat structure similar to removal of m-8A introns 8) r N": &gene encoding #" re&r-8A also occurs in clustered) multi le co ies $BhyQ 3he cell needs a lot of ribosomes) &each gene contains a 121 b coding se?uence se arated by a nontranscribed intergenic s acer & romoter *ithin the transcribed gene contains 2 consensus se?uences &gene transcribed by -8A olymerase ;;; Transfer N" .t N"0:

&small molecules 6#&:# nucleotides *ith a distinct /cloverleaf/ structure &acce tor stem& functions in the binding of the amino acid to the t-8A

&modified bases: 1. influence the fidelity of codon&anticodon airing 2. ensures bases read three at a time &actual 3&, structure by 4&ray diffraction sho*s t-8A molecule is l&sha ed t N" genes: &occur in clustered) multi le co ies &each gene in a cluster consists of : 1. a short transcribed s acer $3&11 bases) 2. coding se?uence 3. 32transcribed s acer &co ies of genes se arated by nontranscribed intergenic s acer S& romoter is *ithin the transcribed gene and is s lit into 2 bloc(s: A and B &transcribed by -8A olymerase ;;; &some t-8A genes contain introns &immediately follo*ing the 32 side of the se?uence corres onding to the anticodon Transcri1tion and Processing of t N"6s: &cleavage of #2 s acer catalyzed by -8ase A $-8A endonuclease) &-8ase A contains some sn-8A E roteins both of *hich are re?uired &addition of the CCA se?uence *hich is added by t-8A nucleotidyl transferase&uses C3A and A3A as an energy source &introns removed by an -8A endonuclease &recognizes e4on boundaries &e4on ends are @oined by -8A ligase

Thus there are 3 distinct s1licing 1ath%ays: 1. intron removal from re&m-8A de ends on recognition of consensus se?uences at intron boundaries $ artici ation of sn-8A2s) 2. removal of introns from re&r-8A de ends on recognition of 2o structure in the intron 3. intron removal from re&t-8A2s recognition of the intron and location of the s licing reactions de end rimarily on regions forming the e4ons )#all nuclear N"6s.sn N"6s0: &U sn-8A & artici ates in re&m-8A and r-8A rocessing and s licing $uridine is a fre?uent base) &'1 sn-8A&forms art of the -8ase A com le4 *hich rocesses re&t-8A )#all cyto1las#ic N"6s .sc N"6s0: &form art of the signal recognition article &this ribonucleo rotein com le4 artici ates in the reaction attaching ribosomes to the endo lasmic reticulum during the synthesis of secretory roteins sn N"6s and sc N"6s: &transcribed by -8A olymerase ;; $sn) and ;;; $sc) &U sn-8A2s @oined to several oly e tides to form ribonucleo roteins C sn-8A2s or snur s &sc-8A2s @oined to oly e tides called scyr s &codon&a tri let of ad@acent bases in a olynucleotide chain of a m-8A molecule that codes for a s ecific amino acidsJ the basic unit of genetic code s ecifying an amino acid for incor oration into a oly e tide chain &anticodon&a se?uence of 3 nucleotides in a t-8A molecule that is com lementary to the codon tri let in m-8A Translation: &the assembly chains *ith m-8A serving as the tem late) a rocess that occurs at the ribosomes Four Phases of Translation: 1. Charging $Activation) reaction &amino acid is @oined to a t-8A molecule through a high energy bond 2. ;nitiation

& rocess *here the large and small ribosomal subunits assemble *ith a m-8A molecule and a s ecialized charged aminoacyl&t-8A called an initiator t-8A & rocess catalyzed by a series of roteins called initiation factors 3. 7longation & rocess *here the aminoacyl&t-8A com le4 binds to the ribosome in se?uence according to the m-8A code. As the se?uential airing roceeds) amino acids are transferred fro t-8A2s into a gradually lengthening oly e tide chain &catalyzed by a series of roteins called elongation factors !. 3ermination & rocess *here m-8A and the com leted oly e tide are released and the ribosomal subunits se arate & rocess catalyzed by a series of roteins called termination factors SAll four hases re?uire energy in the for of A3A or 93A Charging eaction ."" "cti&ation0: &activation ta(es lace in 2 ste s $both ste s catalyzed by the same enzyme an aminoacyl&t-8A synthetase) a. AA E A3A AA&A'A$al ha) E AiAi $beta and gamma) &called aminoacyl&adenylate com le4 b. AA&A'A E t-8A AA&t-8A E A'A &high energy molecule $#111&6111 calHmol) & rovides the energy to form e tide lin(age during oly e tide lengthening &aa is lin(ed to t-8A by a covalent bond to the terminal adenine of the CCA at the 32 end of the acce tor stem of t-8A molecule &there are 21 different aminoacyl&t-8A synthetases one for each of the 21 amino acids &8>37: the amino acid itself does not recognize the m-8A code) only the anticodon of the t-8A )tudy +y Li1#an .9:;30: &cysteine @oined to cysteine&t-8A by the ro er synthetase &cysteine&t-8A *as treated *ith +2 and 8i &"+ grou changed to + $Cys Ala)

&thus Cys&t-8A *as changed *ith Ala &this com le4 added to a test tube $in vitro) system that *ould synthesize +b) resulting +b contained Ala in lace of Cys &code had been fooled into inserting the *rong amino acid ;. ;nitiation $# ste s) a) ribosome se arates into large and small subunits b) 'et&t-8Ai E 93A 'et&t-8Ai&93A com le4 &'et&t-8Ai is uni?ue since it *ill associate *ith the A binding site on the ribosome c) 'et&t-8Ai&93A associates *ith the small subunit of the ribosome d) m-8A is added $re?uires energy in the form of A3A) &also attaches using the #2 ca *hich also involves an initiation factor &small subunit *ill scan do*nstream from the ca for the first AU9 codon e) addition of the large ribosomal subunit $re?uires energy in the form of 93A) &com leted ribosome *ith m-8A and 'et&t-8Ai attached is no* ready elongation ;;. 7longation $# ste s) &Arocess *here amino acids add one at a time to a gro*ing oly e tide chain -)tudy +y Dint4is .9:;30: &7longation roceeds from 8+2 C>>+ direction &.abeled -BC *ith 3+&.eu &Amino acid se?uences of +b in the -BC2s *as already (no*n &"am les *ere ta(en at different times bet*een ! and %1 min &;solated +b se arated oly e tide chain $al ha and beta) &Cleaved end chain into fragments $used try sin) &"e arated fragment by a er chromatogra hy &At ! min only C fragment contained 3+ &At 31 min C half fragment *as labeled

&At %1 min all fragment *as labeled 1. Aminoacyl&t-8A lin(s to 93A $catalyzed by an elongation factor) 2. Aminoacyl&t-8A binds to the ribosome at the A site &A site&binds aminoacyl&t-8A $t-8A2s *ith a single lin(ed amino acid) &A site& binds t-8A2s lin(ed to a oly e tide chain $ e tidyl&t-8A2s) &,uring initiation) the initiator t-8A attaches to the Asite) even though it carries only a single amino acid a) 7longation factor and 93A bind to ribosome *ith the aminoacyl&t-8A b) Aminoacyl&t-8A binding de ends on a correct codon: anticodon air *ith m-8A ositioned at A site c) 93A 9,A E Ai d) 7longation factor and 9,A released e) 7nergy released by 93A hydrolysis contributes to the binding of aminoacyl&t-8A at the A site 3. 5ormation of e tide bond bet*een amino acids at A and A site a. Catalyzed by e tidyl transferase $large ribosomal subunit) a. b. c. d. e. 'et at A site lin(s to amino acid at A site 7nergy re?uired by 'et&t-8A com le4 derived from the charging reaction Both amino acids of di e tide are attached to t-8A at the A site t-8A at the A site is converted from an aminoacyl&t-8A into a e tidyl&t-8A /7m ty/ t-8A remains at the A site

!. a) em ty t-8A in A site is released b) Ae tidyl&t-8A moves from A to Asite &-e?uires 93A hydrolysis and is catalyzed by elongation factor #. -ibosome moves along the m-8A through a distance e?uivalent to one codon &3his movement is called translocation &Carries the codon formerly at the A site) *ith it attached e tidyl&t-8A) to the A site and e4 oses a ne* codon at the A site &3ranslocation re?uires 93A hydrolysis &3 elongation factors are involved in elongation

&3hey act as enzymes do) binding to reacting molecules s eeding their interaction) and releasing unchanged at the end of the reaction &"everal ribosomes may be engaged in elongation for a given m-8A molecule m-8A E attached ribosomes C ;;;. 3ermination: 1. Arotein synthesis ste *hen A site of ribosome encounters one of three terminator codons &Aoly e tide is released from the ribosome &7m ty t-8A at the A site is e@ected &-ibosomal subunits se arate from the m-8A &3hese reactions are catalyzed by a single termination factor -5 or release factor 2. 3erminator codon causes -5 and 93A to bind at A site instead of aminoacyl&t-8A 3. -5 binding stimulates hydrolysis of the bond holding the oly e tide chain to the t-8A at the A site $93A 9,A E A) !. "ince no ne* amino acid is located at the A site) hydrolysis frees the oly e tide chain

,iffusion: is the movement of molecules across a concentration gradient from the area of higher concentration to the area of lo*er concentration. >smosis: the assage of a solvent $*ater) through a membrane from a dilute solution into a more concentrated one. >smotic ressure: is the force resulting from differences in solute concentrations on o semi ermeable membrane. "olute&is the substance dissolved in a solution. "olvent&is the substance used for dissolving another substance. "olute concentration&is the total number of dissolved articles for unit volume of solution. Descri1tions of relati&e solute concentrations: +y ertonic&is a solution *hose osmotic ressure is greater than that of a standard solution. osite sides of a

+y otonic&is a solution *hose osmotic ressure is less than that of a standard 1. ;sotonic& is a solution *hose osmotic ressure is e?ual to that of a standard one e4: salt solution has same osmotic ressure as blood Alasmolysis&is the shrin(ing of the roto lasm of a cell due to the loss of *ater by osmosis. Aassive trans ort $diffusion)&assisted movement of molecules from areas of high concentration to areas of lo* concentration. ate of diffusion de1ends on: 1. "olubility of the ermeant $in membranes) hydro hobic molecules diffuse faster) 2. 'olecular structure of the ermeant. 3. 'olecular size of the ermeant 5acilitated diffusion&is the movement of molecules from a region of high concentration to one of lo* concentration that occurs more ra idly than it *ould through the basic concentration gradientJ at an enhanced rate. Active trans ort&the movement of materials across cell membranes from regions of lo*er concentration to regions of higher concentration) re?uiring the e4 enditure of metabolic energy. Fluid #osaic #odel: &Aro osed in 1:62 by "inger and 8icholson &;s the currently acce ted model of the structure of the cyto lasmic membrane that describes this membrane as a li id bi&layer of roteins distributed in a mosaic&li(e attern on the surface and in the interior of the membrane) *ith lateral as *ell as transverse movement of roteins occurring through the structure. &.i id bi&layer is am hi athic&molecule having of hydro hobic and hydro hilic regions $dual solubility ro erties) &+ydro hobic regions) concentrated in the membrane interior) create a barrier that revents free movement of olar molecules across the membrane. &+ydro hilic regions face the *atery& olar surface of the membrane &;n addition to roteins and li ids) carbohydrates are also resent in cell membranes in the form of glyco roteins and glycoli ids. Li1ids .in #e#+rane0: 1. Ahos hoglycerides: &'ost abundant li id in membranes &Com osed of t*o fatty acids chains) a hos hate grou ) and an alcohol or glycerol grou

&Am hi athic $ olar and non olar grou s) 2. " hingoli ids $glycoli ids): &.i id *ith attached carbohydrates grou s &Bac(bone structure is called a s hingosine *hich is a serine attached to a fatty acid &8+2 grou of serine attaches to a second fatty acid chain&>+ grou of serine &;f >+ grou is unbound it is called a ceramide &>+ grou can attach to a hos hate&alcohol molecule is called a s hingomyelin $2 fatty acid chains E serine E hos hate grou E choline) &>+ grou can also attach to a carbohydrate grou ) *hich is called a glycos hingoli id 3. 9lycos hingoli ids have 2 functions: a) 5unction in membrane stabilization b) Also function in nerve cells $gangliosides) &3here are human diseases related to malfunction in the metabolism of glycerol as such as neurological disorders $3ay&"achTs disease) !. "terol: &Based on frame*or( of ! carbon ring &Aredominant sterol is cholesterol a) 'olecules disturb the close ac(ing of hydrocarbon chains of membrane hos holi ids b) 'embranes containing cholesterol remain fluid at lo* tem eratures rather than /freezing/ into nonfluid forms c) Cholesterol also increases the fle4ibility and mechanical stability of membranes Me#+rane 1roteins: 1. 11&#1 different ty es of membrane roteins: 11)111 to 2#1)111 molecular *eight 2. +ydro hobic $hydro hilic) amino acids are clustered this forms an al ha heli4 that can s an a membrane &3hese transmembrane segments act as anchors to hold the rotein in the li id bi&layer &A membrane rotein can be com osed of several transmembrane segments) *hich e4tend bac( and forth through the li id bi&layer

&7ach segment connected by a stretch of hydro hilic amino acids that are resent on either the outside or the inside of the membrane &"ome membrane roteins may have re eating amino acid se?uences) *hich can form secondary structure such as an al ha heli4 &Arrangement can lace hydro hobic amino acids on one side and hydro hilic amino acids on the other &Can create a olar channel $this is used to trans ort molecules through the membrane) these roteins are called 3rans ort roteins &'embrane roteins can also contain attached carbohydrate grou s that are straight or branched $2& %1 units) &Carbohydrates are usually on cell membrane surfaces &Cell membrane E surface glyco roteins C glycocaly4 A. 5unctions of the glycocaly4: 1. Add stability to membrane by hydrogen bonding 2. Act as recognition sites $bind signal molecules) of membrane rece tors 3. Cell&cell adhesion Cell #e#+rane function: 1. "inger ro osed an alternating confirmation model to e4 lain ho* trans ort roteins carry out facilitated diffusion A. 3rans ort rotein shifts bet*een 2 conformations *ithin the membrane B. ;nitially) roteins folded so the e4 osed binding site is facing the region of higher concentration &+igh affinity state binds strongly to trans orted molecule &Binding of trans orted molecule to binding site occurs via random collisions C. Binding of trans orted molecule to binding site causes a conformational change of the integral membrane rotein so the binding site containing the trans orted molecule is e4 osed to the site of the membrane *here there is a lo* concentration of the trans orted molecule Clo* affinity state ,) .o* affinity state allo*s the trans orted molecule to be released 7) -eturns the integral membrane rotein to the high affinity conformation

Cellular 1rotein trans1orters:

1. >rganic trans orters&facilitates the diffusion of small organic molecules $7. U. glucose) 2. Anion carriers&facilitates the diffusion of negatively charged inorganic grou s 3. ;on trans orters& $trans orts sodium) otassium) calcium) chlorine) &'ost ion channels are gated $com osed of roteins)&control and can e4ist in o en) close) or intermediate states &9ated&ion channels&conduct nerve im ulses &All trans orter roteins are 1. 2. ;ntegral membrane roteins containing several al ha&helical segments that zigzag bac( and forth across the membrane 9lyco roteins $ lasma membrane) *ith carbohydrate grou s directed to*ard the cell e4terior

A) 9lucose trans orter&si4 different ty es: &Consists of: 1. A single rotein *ith 12 al ha helical segments) *hich s an the cell membrane 2. "egment 6) 0) and 11 from trans orter ore &9lucose more concentrated outside of cell $seven U.) &9lucose immediately converted to glucose&%& hos hate *hen it is in the cyto lasm &9lucose&%& hos hate cannot ass bac( through membrane &"ome glucose trans orters regulated by hormone insulin) *hich stimulates muscle and fat cells to allo* glucose to enter B) Aassive ion rotein trans orters: &Conduct charged ions sodium) otassium or calcium and also chlorine ions &'ost on channels are o en or closed by gates &3here are three ty es of gates: 1) Doltage&gated channel & o ens or closes in res onse to changes in the voltage difference across the membrane housing the channel &"odium) otassium) calcium involved in conduction of nerve im ulses

2) .igand&gated channel&o ens or closes in res onse to binding s ecific control molecules $hormones) neurotransmitters) $a chlorine&ligand channel defect involved in cystic fibrosis) 3) 'echanosensitive $stretch&gated) channel&o ens or closes in res onse to mechanical stress on the membraneJ gates occur in multi le) closely related forms 7. U.: mammalian brain cells have three related voltage&gated sodium channels 7. U.: I 31 ty es of otassium channels $some voltage some ligand) "cti&e Trans1ort: &"ubstance trans orted actively sodium) roteins) calcium $other ions)J glucose $si4 carbon shutters)J amino acids 1) 9oes against chemical or electrical gradient 2) -e?uires energy and is sensitive to metabolic factors 3) ,e ends on the resence of functional membrane roteins !) Can be saturated #) ;s s ecific for certain substances or closely related grou s of substances &Active 3rans ort um s $directly de endent on A3A) $direct active 3rans ort um s): 1) A&ty e um & tem orarily binds a hos hate grou removed from A3A during the um ing cycle 2) D&ty e um $v C vesicle) & um brea(s do*n A3A as their energy source from active trans ort &Aum s do not bond a hos hate $from A3A) during their um ing action &74am les are 5151 A3Aase in mitochondria ;. A&ty e um s: 1) Aroton A3Aase um &moves rotons out of cell $A3Aase can *or( in both directions) 2) Calcium&A3Aase um & ushes calcium out of cell &Aushes calcium into cyto lasm 3) "odium H otassium&A3Aase&moves three sodiums out*ardJ moves otassium 2 in*ard &A charge is set u outside the membrane

;;. > eration of sodium H otassium A3Aase um : 1) Aum binds and hydrolyzes A3A &Ahos hate from A3A transferred to trans orter $A3A to A,A E Ai still bound) & Ahos horylated um is a high energy com le4 2) Ahos hate binding converts sodium&binding site to its high affinity state &Binds three sodiums on the cyto lasmic side of membrane 3) "odium binding triggers /a conformational change/ of the trans orter so the sodium&binding site is forcing the medium outside the cell !) "odium binding site changes from a high affinity "tate to a lo* affinity "tate for sodium &-eleases sodium to medium outside of the cell #) Aotassium binding site is altered to a high affinity state and otassium outside the cell binds to the trans orter %) Binding of otassium induces a conformational change of the trans orter so that the otassium&binding site faces the cyto lasmic of the cell &-emaining hos hate grou released into cyto lasmic 6) Aotassium released into the cyto lasm and a trans orter conformational change reverts the otassium&binding site to a lo* affinity state ;;;. ;ndirect active 3rans ort um s $sugars) amino acids) and ions): &Use an ion concentration gradient maintained by another) direct active trans ort) as an energy source &Cotrans ort& molecules carried in or out of cells by indirect active trans ort al*ays moves across membrane *ith ions *hich su ly the driving force for trans ort 1) 2 ty es of indirect active 3rans ort: a) "ym ort& cotrans orted substance moves in the same direction as the driving ion $7. U. of molecules trans orted in this fashion are sugars) amino acids) and ions) b) Anti ort& cotrans orted substance moves in the o sugars) amino acids) and ions) &8ote: there are size limitations ;D. Cell surface: &Cell surface contains glycoli ids and glyco roteins &"erves as site of recognition osite direction to the diving ions $also

&7. U. ma@or systems com atibility com le4 $'+C) the genetic region in human beings that controls tissue com atibility and the develo ment and activation of art of the immune res onse &Aresents intends to the immune system &7. U. blood ty e&an immunologically distinct) genetically determined set of antigens $glyco roteins) on the surface of -BCTs $A) B) AB) and >) 1. "urface rece tors and intracellular signaling: &"urface rece tors are membrane&s anning glyco roteins&segments e4tending on both sides of the cell membrane &-ecognize and bind signal molecules in e4tracellular medium &"egment on outside recognizes the signal $ e tide hormones) gro*th factors) neurotransmitters) &Binding by the rece tors triggers a com le4 internal res onse in cell receiving the signal &;ncrease or decrease in rate of trans ort &"ecretion &>4idative metabolism &;nitiation of cell division &Cell movement D. Characteristics of rece tors&res onse signaling ath*ays: 1) "ignal model does not enetrate into the cell to induce a res onse $binding to a surface rece tors is sufficient) 2) 8o res onse is roduced if the signal model is directly in@ected into the cyto lasm 3) -ece tors remain in the cell membraneJ it does not enter the cell to initiate the res onse &". 3. A. 3. ". &signal transduction and blan( of transduction !) 3here are a limited number of internal res onse ath*ays that share several common features a) 1 shared feature of all is the use of rotein (inases in internal res onse ath*ays 1)& rotein (inase is an enzyme) *hich attaches hos hate grou s derived from A3A to s ecific target roteins 2)&edition of the hos hate grou inhibits or stimulates the activity of the target rotein or roteins in carrying out a cellular reaction &74am les of target roteins:

A) 7nzymes carrying out critical ste s in metabolic ath*ays B) 3rans ort roteins $ion channels) C) -e resents all roteins ,) Aroteins regulating gene activity 7) >ther rece tors 3)& rotein (inases usually function in the final ste s of a reaction se?uence activated by a rece tors &Arotein (inases may be associated *ith membrane or sus ended in solution in the cyto lasm or nucleus b) Arotein hos hatases&enzymes that remove hos hate grou s from target roteins $counterbalance the activity of rotein (inases) D;. -ece tors *ith integral rotein (inase activity: &74am les: 1) ;nsulin&controls glucose u ta(e and metabolism by the cell 2) 7 idermal gro*th factor $795) 3) Alatelet&derived gro*th factor $A,95) &795 and A,95 regulate cell gro*th and division in animals 1. "tructure $transmembrane rece tors) &74tracellular&contains binding site for the signal &;ntracellular $cyto lasmic)&contains rotein (inase site &74tracellular and intracellular connected by al ha heli4 *hich s ans the membrane 2. Binding of signal induces conformational change of intracellular rece tors) *hich binds hos hate grou s to tyrosine $y) residues on the rece tors or to tyrosine residues on target roteins $tyrosine rotein (inases) 3. Addition of hos hate grou s to the rece tors is called self& hos horylation of rece tors C auto hos horylation can lead to activation of other rotein (inases in the cyto lasm !. Addition of hos hate to target roteins may either stimulate or inhibit activity of target roteins in the metabolic ath*ay D;;. 74am le of rece tors *ith integral rotein (inase activity: &795 rece tor&involves the -as rotein

1. Binding of hormone or signal or gro*th factor at the cell surfaces induces a conformational change that activate the rotein (inase site 2. Ahos hate grou s bind to rece tor com le4 in cyto lasm 3. Ahos hate grou s on rece tor are recognized by an ada ter rotein !. Ada ter E a hos horylated rece tor activate a second rotein $guanine nucleotide release factor&9-5) #. Activated 9-5 removes 9,A from -as rotein&-as in the /off state/ is bound to 9,A %. -as rotein binds 93A and becomes activated /on state/ 6. Activated -as can activate one or more rotein (inases) *hich leads to the hos horylation of target roteins &;f rece tor is bound to e4tracellular signal) -as stays on and the ath*ay stays on. &;f rece tor becomes unbound to e4tracellular signal) -as is turned off and ath*ay shuts off. &3here e4ists altered $mutant) forms of -as that have defective 93Aase activityJ rotein is continually on &Aresent in many ty es of cancer ;U. -ece tors *ith se arate rotein (inase activity 1. -ece tors are com le4 roteins *ith seven transmembrane segments 2. Binding and e4tracellular signal module) termed the 1st messenger in these ath*ays) activates an enzymatic site on the cyto lasmic end of the rece tors 3. Cyto lasmic site catalyzes the first ste in the series of se arate reactions leading to the activation of one or more rotein (inases reaction series: a) Arimary reaction series follo*ing activation of rece tor follo*s one of t*o ath*ays b) 5irst ste is activation of a 9 rotein &9 rotein&one of a large family of heterotrimeric 93A&binding roteins that are im ortant intermediaries in cell&signaling ath*ays. Usually activated by the binding of a hormone or other signaling ligand to a 6& ass transmembrane rece tor rotein c) 9 rotein activates an enzyme (no*n as an effector d) 7ffector generates one or more 2nd messengers&small molecules that activate rotein (inases &3hese rotein (inases are activated by the addition of A>! grou s to serine or threonine amino acids - 1roteins:

&Bind 93A and 9,A &Consists of three oly e tide chains associated *ith cyto lasmic side of the lasma membrane as a heterotrimer ;. Aeri heral roteins 1) Al ha chain contains 93A H 9,A binding site &9,A boundJ al ha chain bound to beta and gamma chainsJ al ha chain inactive &;f />ff/ state) it is a trimer 2) ;f 1st messenger bound to rece tor) 93A becomes bound to al ha chain $re laces 9,A)&activates al ha chain that releases from beta and gamma chains 3) Al ha chain activates effector !) 93A &I9,A and then al ha chain then shuts off $reactivated by another molecule of 93A) on and off states ;;. 3*o ma@or 2nd messenger ath*ays that utilize 9 roteins: &Both ma@or ath*ays rece tor&res onse ath*ays generate 2nd messengers to activate rotein (inases $effectors and second messengers are different) 1) c A ' A&cyclic A'A & nucleotide that is generated from A3A in res onse to hormonal stimulation of cell surface rece tors &cA'A acts as a signaling molecule by activating a (inase it is hydrolyzed to A'A by a hos hodiesterase &7ffector adenylate cyclase *ill activate c A'A as the second messenger &;n this ath*ay hormones and neurotransmitters act as the 1st messenger &Arotein (inases activated by c A'A are called rotein (inase A. &>ne e4am le of a cA'A rece tor&res onse ath*ay is the series of reactions use to brea(do*n glycogen&glucose 2) ;nsA3 H ,A9 ath*ay: &3he second ath*ay generates t*o 2nd messengers generated through the brea(do*n of a membrane hos holi id) hos hatidyl inositol $catalyzed by the effector hos holi ase c) &;nositol tri hos hate $;nsA3) &,iacylglycerol $,A9) ;;;. > eration $mechanism) of ;nsA3 H,A9 ath*ay

a) Binding of hormone or neurotransmitters activate rece tors b) Activation allo*s 93A to be bound to the rece tor c) ,issociation of al ha chain of 9 rotein and binding of al ha chain to hos holi ase c d) Activation of hos holi ase c causes the brea(do*n of hos hatidyl inositol to ;nsA3 and ,A9 e) ;nsA3 is released from the cell membrane diffuses through the cyto lasm to the endo lasmic reticulum &;nsA3 binds to the ;nsA3 rece tor $ligand&gated calcium channelJ ;nsA3 is the ligand) &;nsA3 binding o ens the channel and releases calcium to the cyto lasm f) Calcium acts as a su lemental second messenger in the ath*ay activating rotein (inases

g) 3he other 2nd messenger) ,A9) is in the lasma membrane &Arimary function is to activate rotein (inases &Activation ta(es lace on the inner side of the lasma membrane h) ,A9 activates rotein (inases $ rotein (inase C family) $C Ccalcium activated (inases) $serineHthreonine rotein (inases) ;D. 3argets of rotein (inase C: a) Chromosomal roteins b) 3rans ort roteins c) Contractile and cytos(eletal roteins d) Aroteins regulating secretion and endocytosis e) 7nzymes&metabolic &=inases may inhibit or stimulate a metabolic ath*ay &Calcium may interact *ith a control rotein called calmodulin &Ubi?uitous calcium&binding rotein *here binding to other roteins is governed by changes in intracellular calcium concentrations &;ts binding modifies the activity of many target enzymes and membrane trans ort roteins &Calmodulin&recognizes a 21 amino acid se?uence that contains ositive amino acids $-) =) +) that alternate *ith hydro hobic amino acids *hich forms and am hi athic al ha heli4 Cell adhesion:

&Cell attachments are accom lished using glyco roteins&CA'&cell adhesion molecule &;ntegral glyco roteins that s an the cell membrane &Can bind to similar CA'Ts from neighboring cells $homoty ic binding) &Can bind to different CA'Ts $heteroty ic binding) &CA'Ts are im ortant for: 1) Aro er antibody function in the immune system 2) Cell&cell connection in the nervous system &,uring embryonic develo ment) cell adhesion is ?uic(ly follo*ed by the formation of intercellular @unctions. ;. Cell @unctions &" ecialized regions of connection bet*een t*o cells or bet*een a cell in the e4tracellular matri4 1. Adhesive @unctions&cell&cell @unction that holds cell together in hel maintain cells in their fi4ed osition in tissues a) 2 ty es of adhesion @unctions: 1) ,esmosome&s ecialized cell &cell @unctions characterized by dense la?ues of rotein into *hich intermediate filaments in the t*o ad@oining cell insert under the cell membrane &Abundant in s(in) muscle) and e ithelial cells a) +emidesmosome $half desmosome)&is a s ecialized cell @unction bet*een a cell and the basal lamina $e4tracellular matri4) of neighboring cells. &74am le: collagen underlying e ithelial cells 2) Adheren @unctions&heart muscle) line body cavities &Cell @unction in *hich the cyto lasmic face of the lasma membrane is attached to actin filaments

2. 3ight @unction&cell&cell @unctions that seals ad@acent cells together) reventing the assage of most dissolved molecules from one side to the other 3. 9a $communicating @unction)&cell&cell @unction that allo*s ions and small molecules to ass the cyto lasm of one cell to the cyto lasm of a neighboring cell

/<tracellular #atri<: &Com le4 net*or( of olysaccharides and roteins su &5unctions: 1) "u ort orted by the cell

2) -egulation of cell division 3) Adhesion !) Cell motility #) Cell differentiation &Com osed of fibers and surrounding net*or( &5ibers&long) semi crystalline elements at rovide resistance to stretching &8et*or(&interloc(ed assembly of branched molecules that holds the rigid fibers in lace $elastic) "ni#al e<tracellular #atri<: &Com osed of collagens $fibers)J roteoglycans $net*or() C both glyco roteins &Collagen&fibers rotein rich in glycine and roline that is a ma@or com onent of the e4tracellular matri4 in connective tissues &74ists in many forms: &3y e ;& $most common) is found in s(in) tendon) and bone &3y e ;;&found in cartilage &3y e ;D& resent in the basal lamina &Collagen com osed of three al ha&helical oly e tide chains &Al ha chains regularly s aced glycine residues ;. Assembly of collagen &"ynthesized secretory rotein 1. Al ha oly e tide chains of collagen molecule are assembled on the ribosomes attached to membranes of the endo lasmic reticulum 2. Carbohydrate) oligosaccharide) grou s are added at the endo lasmic reticulum via s ecific enzymes 3. Collagen synthesized as a recursor molecule called rocollagen

!. Arocollagen trans orted to the 9olgi com le4 *here it is ac(aged into secretion vesicles #. 5usion of the vesicles *ith the lasma membrane releases the rocollagen to the cell e4terior %. ;n e4tracellular regions) rocessing enzymes remove the e4tra amino acid se?uences of rocollagen molecules &Arocollagen $soluble)&I collagen $insoluble) ;;. Aroteoglycans &9lyco roteins *ith high $01P&:1P) content of carbohydrates &'acromolecules consisting of one or more glycosaminoglycan chains $9A9) attached to a core rotein) *hich is modified by sulfhydral grou s &9A9&is an unbranched chain) *hich consists of hundreds of re eating 2&sugar units &5unction of roteoglycans: &3ra and im ede the flo* of *ater molecules &'ost abundant in cartilage& rovides fle4ibility and elasticity to this structure ;;;. 'olecules lin(ing collagen and roteoglycans to the cell surface &5ibronectin and laminin are glyco roteins &Collagen) roteoglycans) fibronectin) and laminin can bind to all surface rece tors called integrins&membrane&s anning glyco roteins &;ntegrin&is a member of a large family of transmembrane roteins involved in the adhesion of cells to the e4tracellular matri4 &"tructures that tie cells to the e4tracellular matri4 and also form connections *ith the cytos(eleton in the cyto lasm &'any integrins are used in signal transduction ath*ays to trigger internal cellular res onses from e4ternal stimuli &;ntegrins lay roles in cell gro*th) cell division) and cell differentiation &>4idative hos horylation& rocess in the mitochondria in *hich A3A formation is driven by the transfer of electrons from organic molecules to molecular o4ygen. Arocess involves the intermediate generation of a + gradient across a membrane and chemiosmotic cou ling

es1iration .+reathing0 ;. Com osed of: 1. 9lycolysis $cyto lasm) 2. Aentose cycle $cyto lasm) 3. Citric acid cycle $matri4 of mitochondria) ;;. ,ivided into three arts: 1. A series of reactions) *hich rovide a source of electrons at) elevated energy levels 2. An electron trans ort system) embedded in a membrane) that uses the energy of the electrons to build a roton gradient across the membrane 3. An A3A&synthesized enzyme embedded in a membrane that uses a roton gradient as an energy source to convert A,A to A3A ;;;. 'itochondria structure: &'embrane&bound cell organelle that carries out o4idative hos horylation and roduces A3A 1. Contains t*o se arate membrane systems) outer and inner boundary membranes $cristae) 2. ;nner most com artment is the matri4 ;D. >4idative reactions: 1. -lycolysis $7mbden&'eyerhoff ath*ay) &Ubi?uitous metabolic ath*ay in the cytosol $cyto lasmic) in *hich sugars are incom letely degraded *ith roduction of A3A /sugar s litting/ 1) ;n 11 ste s) glucose is s lit in half and converted to 2 3&carbon acids $ yruvic acids) A) 2 coenzymes are reduced B) 8et formation of 2 A3A molecules C) >riginal carbons 1 and %) 2 and #) and 3 and ! of glucose become e?uivalent 2) All enzymes are soluble and the *hole ath*ay is carried out in the cyto lasm 3) Ayruvic acid highly reactive and roceeds to form further roducts A) "ubstrate for the citric acid cycle $aerobic res iration)

&-educed coenzyme is eventually o4idized by o4ygen B) Anaerobic res iration: &2 ty es: 1) .actic acid formation& &Ayruvate is reduced to lactic acid &Ayruvate E 8A,+ &I lactate E 8A,E &>ccurs in muscle cells or heart cells in animals& tem orary storage for electrons 2) Alcohol fermentation $yeast): &Ayruvic acid is decarbo4ylated $releases carbons 3 and !) and reduced to ethyl alcohol &Ayruvate E 8A,+ &I ethyl alcohol E C>2 E 8A, E &"trict anaerobes& $bacteria) fungi) can roduce A3A only by fermentation &5acultative anaerobes&can s*itch bet*een fermentation and o4idation ath*ays &"trict aerobes&unable to carry out fermentation !) 3he net gain of 2 A3A molecules er glucose yields free energy &Aotential free energy still in yruvic acid &Vields a ro4imately 2P 2. Citric acid cycle $ yruvate o4idationJ 3CA cycleJ =rebTs cycle): &Central metabolic ath*ay found in all aerobic organisms &>4idizes acetyl grou s derived from sugars $glucose) to C>2 and *ater &>ccurs in mitochondria $matri4) in eu(aryotic cells 1) -eactions all occur in mitochondria $or the membranes) starting *ith the substrate $ yruvate) generated in cyto lasm

2) Aath*ay is a cycle of reactions involving the interconversion of 11 organic acids. C>2 and reducing o*er are formed from yruvic acid) *ith one for carbon intermediate being used u and one regenerated for each turn of the cycle &-emoves to electrons) 2 hydrogens) and moves one carbon $C>2)J acetyl grou s transferred to coenzyme A acetyl grou s&energy source for 3CA cycle&electrons from 8A,+ 1) Carbons released as C>2 in the cycle derives from carbons 3 and ! of glucose 2) Ayruvate o4idation catalyzed by a com le4 rotein called yruvate dehydrogenase com le4 3) 3he 2&carbon roduct of decarbo4ylation of yruvic acid condenses *ith a !&carbon acid $o4aloacetic acid) to form a %&carbon acid) citric acid !) "ubse?uently) the carbon of yruvic acid $originally carbons 2 or # of glucose) and then lastly the methyl carbon of yruvate $from carbons 1 or % of glucose) are metabolized to C>2 &3he sum of the reactions for one turn of the cycle is: 1) Ayruvate E 3 +2> E # coenzymesE E A,A E Ai 3 C>2 E # coenzymes. +2 E A3A 2) C>2 is released as a gas or solution) reducing o*er transferred to electron trans ort chain to o4ygen &7lectrons transfer is cou led to generation of A3A 3. Pento 1hos1hate 1ath%ay $ entose hos hate shunt): &Aath*ay *here glucose is o4idized and converted to a #&carbon sugar $ entose) $ribose&#& hos hate) &Aath*ay also reduces 8A,AE &I 8A,A+ &Aath*ay generates reducing o*er in the form of 8A,A+ D. 'itochondria electron trans ort system: 1. Contains ! large rotein &based com le4es that *or( in se?uence to deliver electrons from 8A,+ and 5A,+ 2 to o4ygen 2. 7ach com le4 contains:

A) A ma@or enzyme that catalyzes electron transfer B) 8on rotein organic grou s $ rosthetic grou s) that directly e4ce t and release electrons carried by the com le4 C) "tructural roteins that contribute to movement of rotons across the membrane &8A,AE C nicotinamide adenine dinucleotide hos hate &Coenzyme closely related to 8A,E that is used e4tensively in biosynthetic ath*ays &Ubi?uinone&electron carrier 3. "ystem also contains t*o carriers) *hich shuttle electrons bet*een the com le4es: cytochrome C) and ubi?uinone $coenzyme M) A) ! com le4es: &Com le4 ;&conducts electrons from 8A,+ to ubi?uinone &Com le4 ; contains: A) 7nzyme 8A,+ dehydrogenase&o4idizes 8A,+ B) 7lectrons from 8A,+ transferred to 5'8 $ rosthetic grou bound to com le4) C) 5'8+2 o4idized to ubi?uinone &Com le4 ; contains I 31 roteinsJ 6 encoded by mitochondrial ,8A) rest encoded by nucleus &5'8G&flavin mononucleotide &5A,&flavin adenine dinucleotide &$Ditamin B) riboflavin &I 2 hos hates &I ribose &I adenine &-educed form by adding hydrogens to central nitrogen of isoallo4azine ring &Com le4 ;;&carries electrons from succinate $3CA cycle) to ubi?uinone &Com le4 ;; contains: A) 7nzyme succinate dehydrogenase $catalyzes reaction % of 3CA cycle) &"uccinate &I fumerate B) 7lectrons transferred to 5A, &I ubi?uinone C) Contains three oly e tide subunits E $contain iron and sulfur) $iron Hsulfur centers) E succinate dehydrogenase

,) All ! subunits encoded in the cell nucleus &Com le4 ;;;&conducts electrons from ubi?uinone to cytochrome C &Com le4 ;;; contains: A) Cytochrome C reductase&catalyzes transfer of electrons from ubi?uinone to cytochrome C B) Contains 11 oly e tides: 1 $cytochrome B) encoded by mitochondrial ,8A) other iron H sulfur subunits &Com le4 ;D&conducts electrons from cytochrome C to o4ygen &Com le4 ;D contains: &! subunits E 13 oly e tides A) Cytochrome A B) Cytochrome A3 C) 2 co er center subunits CuA and CuB lin(ed by 3 oly e tides $;) ;;) ;;;) ;;; encoded by mitochondrial ,8A &11 additional $au4iliary subunits) encoded by nuclear ,8A D;. Aroton um ing by electron trans ort chain: 1. 'itochondrial electrons trans ort system um s rotons from the matri4 into the intermembrane com artment as electrons flo* from 8A,+ H 5A,+ to o4ygen A. ! rotons are ushed across the inner boundary membrane as 2 electrons move through com le4 ; B. 5rom matri4 to intermembrane com artment 2. 3*o rotons use t*o electrons move through com le4es ;; or ;D 3. Aroton gradient develo ed through electron trans ort is the energy source for the 5151&A3Aase $A,A E hos hate A3A)&catalyzes the conversion )tructure of F=F9 "TPase .adenosine tri1hos1hatase0: &Can be se arated into 2 sub arts 51 and 51 &51&cou ling factor 1 &51&oligomycin&sensitive factor&drug that inhibits A3Aase &5151 A3Aase is a lolli o &sha ed article &Contains a head iece connected to a basal unit or stal(

&51&site of A3A synthesis &51&su lies rotons $membrane channel) &5151 A3Aase of 7.coli contains 0 subunits &51 has five subunits &51 has three subunit& roton channels com osed of %&12 C subunits Photosynthesis: &Arocess in *hich light energy is absorbed by s ecialized igments of a cell and is converted to chemical energy 1. Com osed of light and dar( reactions &.ight reactions&re?uires light &Ahotoreduction& roducts are reduced carriers and o4ygen &8oncyclic hoto hos horylation&A3A &Cyclic hoto hos horylation&93A & A3A and reduced carriers are used in dar( reactions to convert C>2 to carbohydrates &Conversion of inorganic C>2 to organic com ounds 2. Arocess in eu(aryotes $ lants and algae) occurs in chloro lasts &'embrane&bound organelles *here the biochemical conversion of light energy to A3A occurs

;. "tructure of chloro lasts: 1. "urrounded by t*o continuous membranes) outer and inner boundary membranes &'embrane se arated by intermembrane com artment 2. Boundary membranes enclose an inner com artment called the stroma $analogous to mitochondria matri4) 3. Bithin stroma) there is a third membrane system consists of flattened sac(s called thyla(oids !. 3hyla(oids ranged in stac(s called grana #. "tac(s are connected by membrane called stromal lamellae ;;. 3he light reactions:

1. .ight energy $ hotons) is absorbed by igment molecules in the chloro lasts $chloro hyll) causing electrons in their ground state to move to a ne* orbital of higher energy $e4cited state) A) ;n hotosynthesis) light is converted to chemical energy through the transfer of electrons) e4cited by *hite absor tion in chloro hyll) to stable orbit in rimary rece tor molecules B) Also e4ist accessory igment molecules in addition to chloro hyll) *hich can absorb light energy called carotenoids 1) Aass energy to chloro hyll molecules &Chloro hyll and carotenoids are li ids bound to thyla(oids membranes and stromal lamellae &Chloro hylls are molecules based on a tetra yrole ring &2 ma@or chloro hylls a and b &7lectrons in ring can absorb light at different *avelengths 2) .ight reactions organized into 2 com le4es: hotosystem ; and hotosystem ;; &7ach system $in chloro lasts) contain: A) #1 to 111 chloro hyll a molecules lus a small number of carotenes B) 21 oly e tides carry out structural) enzymatic) and regulatory functions &-eaction center&one or 2 chloro hyll a molecules *ithin the hotosystems *here e4cited electrons are transferred to stable orbitals C) Also contain a se?uence of carrier molecules that are involved in the trans ort of electrons a*ay from rimary acce tors ,) Ahotosystem ;; contains the mechanism for s litting *ater into rotons) electrons and o4ygen) this is called hotolysis 7) .ight&harvesting com le4es $.+Cs)& internal membranes of chloro lasts) *hich contain igment& rotein assemblies that act as accessory light&gathering /antennas/ &,o not have reaction centers H rimary acce tors

&,o not convert light to chemical energy &Aass on energy of absorb light to the to hotosystems &.+C&1&associated *ith hotosystem ; &.+C&2&associated *ith hotosystem ;; 2. "tructure of hotosystem ;: A) Contains 111 chloro hyll a molecules E 1% beta&carotenes E 21 additional roteins in a light& absorbing structure called the core antenna B) .ight energy assed from core antenna to the reaction center&s ecialized chloro hyll a molecule called A611&absorbs light ma4imally at *avelength C 611 nm 3. "tructure of hotosystem ;;: A) Contains !1 chloro hyll a molecules E beta&carotenes E 22 roteins B) -eaction center contains a s ecialized form of chloro hyll a) A%01&absorbs light ma4imally at *avelength of %01 nm C) Ahotolysis associated *ith 3 eri heral oly e tides and ! manganese atoms com le4ed into a manganese center &Calcium and chlorine necessary cofactors for the center !. "tructure of the light&harvesting com le4es $.+Cs): A) .+C&1 com le4 $antenna) contains about 01 to 121 molecules of chloro hyll a and chloro hyll b E four oly e tides B) .+C&2 com le4 contains about #1 chloro hyll a and b molecules $1:1 ratio) E ! oly e tides lus a beta& carotene &7nergy of hotons absorbed by the .+Cs is assed through the 2 hotosystems C) 7lectron trans ort system lin(s the 2 hotosystems &2 hotosystems are lin(ed in chloro lasts by an electron trans ort system that conducts electrons from one system to the ne4t and delivers electrons to 8A,AE at the end of the ath*ay &Ahotosystems are arranged in a O& ath*ay #. O ath*ay: A) 7lectrons enter ath*ay $ hotosystem ;;) from the brea(do*n of *ater to rotons) electrons) and o4ygen $ hotolysis)

1) Carried to reaction center by electron carrier O &*ater &I electrons &I O &I A%01 &I A%01 $e4cited) &O E tyrosine residue on ,1 oly e tide 2) After e4citation) electrons travel from A%01 to a rimary rece tor of hotosystem ;; $Ma) $ lasto?uinone) &I 2nd lasto?uinone $Mb) $final electron carrier in hotosystem ;;) B) 7lectrons move from Mb to a ool of lasto?uinone molecules $AM ool) &Analogous to ?uinone carrier in mitochondria &7lectrons move from AM ool to 3 carriers &Cytochrome b% &;ron H sulfur rotein &Cytochrome f&related to cytochrome C &B% H f com le4&analogous to com le4 ;;; of mitochondrial system &7lectrons move from b% H f com le4 to final carrier *hich lin(s the hotosystems) lastocyanin $AC)& shuttles electrons from b% H f com le4 to hotosystem ;&analogous to cytochrome C of mitochondrial electron trans ort system C) 7lectrons flo* from AC to A611 chloro hyll at reaction center of hotosystem 1 1) After e4citation by light) electrons are transferred from A611 to rimary rece tor of hotosystems. ; $modified chloro hyll called A1) 2) 7lectrons flo* from A1 to A1) a ?uinone derived from vitamin = 3) 7lectrons then flo* through 3 iron H sulfur centers in hotosystem 1 !) 7lectrons ass from hotosystem 1 to roduction) an iron H sulfur rotein that access a se arate electron care #) 7lectron airs from ferredo4in to 5A, to 8A,A &5A, is a coenzyme) *hich is art of ferredo4in&8A,A o4idoreductase ;;;. 5eatures of hotosynthesis: 1. 7lectrons receive 2 in uts of energy &7nergy reduces 8A,A &Used to establish roton gradient $ roton um s from the stroma into the thyla(oids com artments)

&>ne in hotosystem ;; &>ne in hotosystem ; 2. 7lectron flo* in the O ath*ay from *ater to 8A,A is called noncyclic hotosynthesis $noncyclic hoto hos horylation) 3. Arocess of hotolysis utilizes *ater as an electron source &Also roduces o4ygen !. 7lectrons can also flo* in a cycle *ithin a closed circuit of hotosystem 1 $cyclic hoto hos horylation) &7lectrons flo* from ferredo4in bac( to the b% H f com le4 $instead of going to 8A,A E) &Cycle um s additional rotons each time electrons flo* through the b% H f com le4 &Aroton gradient drives the synthesis of one A3A molecule for each electron air) thus) A3A is formed *ithout reduction of 8A,A E #. Aroton gradient of O ath*ay: &Aroton roduced during: A) Ahotolysis B) As electrons ass from ferredo4in to 8A,A via ferredo4in&8A,A o4idoreductase carrier C) As electrons flo* through b% H f com le4 %. Chloro lasts use the roton gradient to roduce A3A &Contains a C51&C51 A3Aase $similar to 5151 A3Aase in mitochondria) 6. "ummary of hotosynthesis and the chemiosmotic hy othesis &Aro osed by ,r. 'itchell in 1:%2&connection bet*een roton gradient and A3A synthesis in chloro lasts A) "u orting features H evidence: 1) Ahoto hos horylation de ends on the resence of closed membranous vesicles $chloro lasts) 2) 7lectron trans ort through hotosystem ;; and hotosystem 1) or cyclic trans ort around hotosystem 1) results in an accumulation of rotons inside isolated thyla(oids or enzyme vesicles derived from thyla(oids 3) ,estruction of the roton gradient by agents that increase ermeability of thyla(oid membranes to rotons sto s A3A synthesis &;m osition of an artificial roton gradient across thyla(oid membranes stimulates A3A synthesis

;D. ,ar( reactions: &Chemical energy roduced in the light reactions is used in the dar( reactions $do not re?uire light) to convert C>2 into carbohydrate and other organic roducts &-e?uire roducts of light reactions $A3A H 8A,A+) &Calvin cycle $Calvin&Benson cycle)&ma@or metabolic ath*ay by *hich C>2 is fi4ed during hotosynthesis) C3 cycle&uses C>2) A3A) 8A,A+ as reactants &-eleases A,A) 8A,A E) and 3 A9A. as roducts &0 ste ath*ay

&3rans(etolase&transfers 2 carbon molecules from one sugar to another &3ransaldolase&transfers 3 carbon molecules from one sugar to another &Aldolase&transfers dihydro4yacetone hos hate to a variety of aldehydes

1. C! cycle: &Alternative dar( reaction ath*ay &,ue to the fact that the (ey regulatory enzyme -UB;"C> can also act as an o4ygenase &C! cycle can (ee C>2 bac( into the C3 cycle &C! cycle occurs *hen o4ygen levels are high in lants &>4ygen stimulates o4ygen as activity of -UB;"C> &>ne roduct of su ort ath*ay) malate) diffuses to cell *here o4ygen levels are lo*

&'alate &I yruvate *ith loss of C>2) *hich enters C3 cycle in cells *here o4ygen content is lo*

Microtu+ules: ;. 3*o structures involved in cell movement: 1) 'icrotubules& unbranched cylinders about 2# nm in diameter *ith an o en central channel and is com osed of tubulin $ rotein) 2) 'icrofilaments& are e4tremely fine unbranched solid fibers about #&6 nm in diameter and are com osed of the rotein actin

&Both act se arately and together to roduce cellular movement &7U: 1) 5lagellaHcilia beating&microtubules 2) 'uscle contraction&microfilaments 3) Cyto lasmic streaming&microfilaments !) 'ovement of mitochondriaHvesicles&microtubules #) Both involved in cell division &'icrotubules divide and distribute chromosomes &'icrofilaments divide the cyto lasm &'olecular mechanisms for movement occur through an active sliding ;;. 'icrotubule structure: &Consists of a circle of 13 s herical subunits &"ubunits line u in arallel) length*ise ro*s called rotofilaments &3hese rotofilaments form a *all &7ach *all article is a unit of tubulin &+eterodimer of 2 tubulin roteins $al ha N beta) &'icrotubules can occur as doublets or tri lets &Consist of one com lete microtubule $13 subunits) *ith one $doublet) or t*o $tri let) artial) C& sha ed microtubules fused to it at one side &,oublets occur in flagellaHcilia &3ri lets occur in centrioles $structures that give rise to flagella and anchor them in the cyto lasm)Hbasal bodies 1. Assembly of 3ubulin into 'icrotubules: &-eversible reaction that is in e?uilibrium A) ,e ends on concentration of: 1) 93A

2) Ca2E c) + 2. 'icrotubule&associated roteins $'AA2s): A) >ne end of a microtubule can assemble or disassemble 2 to ! times faster than the other C olarity &3he end *ith the faster rate of assembly or disassembly is the lus end of a microtubule &3he end *ith the slo*er rate is the minus end B) "table microtubules can achieve a balance oint at *hich tubulin heterodimers add to the lus end *hile the minus end is disassembling &+eterodimers can migrate through a microtubule or treadmill through a microtubule from one end to the other C) .arge scale roduction of microtubules occur in distinct structures called microtubule organizing centers $'3>C2s) &3here are ! ty es: a) Centrioles $basal bodies)&generate microtubules of flagella b) Cell centers $centrosomesHasters)&dense material located near the nucleus and organize cyto lasmic microtubules c) =inetochores& late&li(e structures on the surface of chromosomes $nuclear division) d) " indle ole bodies&give rise to structure $s indles) in fungal cells that are involved in nuclear division &'3>C2s rovide a nucleation center for heterodimer olymerization 3. 5unction of 'icrotubules&convert chemical energy to mechanical energy by slidingCinvolved in cell movement &,ynein&member of a family of large motor roteins that undergo A3A&de endent movement along microtubules via a crossbridge cycling event &Com osed of 2 arms e4tending !. Crossbridge mechanism $hy othesis): A) ,ynein arms undergo conformational changes that ad@ust their angle of attachment to the microtubule surface bet*een a ro4imately :12 and !#2 B) A3A binding) but not hydrolysis) is re?uired to release dynein arms attached in the :12 osition

C) A3A is hydrolyzed at some oint after release of dynein armsJ hydrolysis recedes or accom anies movement of the arms to the !#2 osition #) -elease of the roducts of A3A hydrolysis $A,A E Ai) does not occur until dynein arms reattach to an ad@acent microtubule after A3A hydrolysis #. "tructure of 'icrotubules in 5lagellaHCilia: &Consist of a circle of nine fused) double microtubules) the eri heral doublets) arranged around 2 central microtubules) the central singletsC:E2 system $flagellar a4oneme) A) 7ach of the 2 central singlets is a com lete microtubule B) Aeri heral doublets contain one com lete '3 $A subtubule) and one artial '3 $B subtubule) C) Connecting elements: 1) Central singlets connected by a bridge and a surrounding sheath 2) A subtubule of each doublet is connected to the central sheath by a s o(e 3) 8e4in lin( connects eri heral doublets&A subtubule of one doublet to B subtubule of the ne4t doublet ,) ,ynein arms connected to A subtubule doublet 7) 'icrotubules of cilia and flagella arise from structures or basal bodies called centrioles 1) Centrioles contain a related :E2 system 2) 8ine sets of tri let '32s ma(e u the outer circle of the centriole &A subtubule Ccom lete '3 &BNC subtubules C artial '32s Microfila#ents: &Actin filaments ;. 5unction in: 1) Cytos(eletal su 2) 'ovement a) Cyto lasmic streaming& segments of the cyto lasm flo* actively and directionally from one region of another b) Contraction& sliding microfilaments forcibly constrict or shorten cell segments or *hole cells ort

;;. "tructure of 'icrofilaments: 1) Actin& abundant rotein that forms '52s in eu(aryotic cells. &3he monomeric form is called globular or 9&actinJ the olymeric form is filamentous or 5&actin &Actin can olymerize to '52s &Aolymers have olarity $rates of assembly)$similar to '32s) &Alus end adds subunits 124 faster than minus end disassembles subunits &Aolarity can be determined by myosin decoration develo ed by ,r. +u4ley in 1:%3 &'yosin decoration&'5 reacted *ith myosin) *hich roduces an /arro*head/ attern that is characteristic of microfilaments &-eflect binding of myosin segment to actin &/Arro*s/ oint to*ard the minus end of a '5 or the / ointed/ end &> osite end / lus/ is called the barbed end 2) Actin binding roteins $ABA2s) &3here are I %1 ABA2s *hich function to shift olymerization reactions to unassembled subunits &Bloc(ing olymerization &Ca ing '5 subunits &"evering or brea(ing '5 subunits A) ABA2s regulated by: 1)Ca2EHcalmodulin com le4 2) Ahos horylation $(inases) &'yosin& ty e of motor rotein that uses A3A to drive movement along actin filaments $6 classes of myosin) using a crossbridge cycle &'yosin ;& is *idely distributed *ithin the cell) often membrane&bound) and is not assembled into filaments &'yosin;;& is a very large rotein that forms the thic( filaments of s(eletal muscle that slide over actin filaments during contraction 3. 'yosin: A) Com osed of 1 or 2 large oly e tide chains /myosin heavy chains/ and 1 or 2 smaller oly e tide chains /myosin light chains/

B) +eavy chains com osed of a head and tailJ the light chains associate *ith the head C) +ead contains: &Actin binding site &+ydrolyzes A3A&I A,A ,) 'yosin ;; can be s lit into 2 arts by rotein&digesting enzymes $ rotease) such as try sin &+ead fragment C heavy meromyosin $+'') fragment & contains A3Aase activity &3ail fragment C light meromyosin $.'') contains binding sites that assemble myosin ;; into su erstructures called thic( filaments !. 'yosin Crossbridge Cycle: &;s an attach& ull&release cycle) *hich generates '5&based motion &Cycle is the o*er source for microfilament sliding *hich is res onsible for muscle contraction &"imilar to crossbridge mechanism utilized by '32s A) -egulation of crossbridging: &2 ath*ays regulate myosin crossbridging cycle: 1) Actin&lin(ed regulation& involves roteins that bind to actin and form regulatory units located on the surfaces of microfilaments a) Actin&lin(ed regulation&de ends on 2 roteins) tro omyosin and tro onin &3hese t*o roteins control the crossbridging cycle by undergoing CaEE& de endent conformational changes &Changes alternately bloc( and e4 ose the sites on actin bound by myosin during the crossbridging cycle 2) 'yosin&lin(ed regulation& based on the regulatory myosin light chains a) Both are controlled by FCaEEG in cyto lasm 1)Ca2E ions bind to calmodulin 2) Calmodulin undergoes a conformational change and activates the myosin light chain (inase $'.C (inase)

3) Activated '.C (inase adds A>! grou s derived from A3A to the myosin light chains&crossbridging cycle is turned /on/ !) ;f Ca2E is released from calmodulin) there is an inhibition of '.C (inase activity $no hos horylation activity) #) A>! grou s are removed from the regulatory light chains by an enzyme '.C hos hatase %) Crossbridging sto s Cytoskeleton: &Com osed of '32s) '52s) ;52s and additional su ort roteins

&74tend from lasma membrane to the nuclear envelo e &1H3 of total K of roteins are cytos(eletal roteins ;. ;ntermediate 5ilaments: &,iameter of 11 nm $bet*een '5 and '3) 1. # classes in animal cells: A) =eratin&e ithelial cells) line body cavities B) Dimentin&blood) bone) cartilage C) ,esmin&muscle ,) 8eurofilament&neurons 7) 9lial filament &>bserved in vertebrates) invertebrates) slime molds) rotozoa) but not lants 2. 5ilaments assembled from a variety of roteins: A) Cyto(eratins B) Dimentin C) ,esmin ,) 8eurofilament 7) 9lial fibrillary acids

3. Assembly of ;ntermediate filaments: A) "ubunits assemble into a tetramer $consists of 2 coiled&coiled oly e tide chains) &3etramer associate to form a rotofilament) *hich is an octamer of intermediate filament oly e tide chains $contains 2 ro*s of tetramers) B) Assembly re?uires accessory roteins &;5&associated roteins $;5AA2s) &>ne e4am le is filaggrin&crosslin(ed rotein !. 'a@or Cytos(eletal 5unctions: A) "u B) "u C) "u orts lasma membrane orts outer cyto lasm $corte4) orts the inner cyto lasm bet*een the corte4 and the nucleus

$rgani4ation of Prokaryotic -eno#e: ;. % ma@or differences from the eu(aryotic: 1) Aro(aryotic genomes $com lete set of genetic information organized into a chromosome) are much smaller than eu(aryotic genomes &E.coli& circular chromosome !)611 (b &+umans& 23 linear chromosomes 3)111)111 (b 2) ;n bacteria circular chromosome) all genes are genetically lin(ed &3here are no telomeres 3) ;n ro(aryotes) almost all of the ,8A is used as genes or used as coding se?uences !) ;n ro(aryotes) there is a lasmid grou ing of functionally related genes into o erons #) ;ntrons are e4tremely rare in ro(aryotes %) Aro(aryotic cells contain e4trachromosomal ieces of ,8A called lasmids ;;. Arrangement of Coding "e?uences in Aro(aryotes $o erons):

&m-8A genes arranged in o erons&a grou or cluster of structural genes *hose coordinated e4 ression is controlled by a regulator gene 1. > erons: A) > erons are transcribed as a single m-8A B) Cistron& a segment of genetic nucleic acid that codes for a s ecific oly e tide chain C) > erons are controlled by a single romoter) *hich lies in front $#2) of the transcribed segment &Aromoter contains ,8A $consensus) se?uences that rovide recognition and binding sites for -8A olymerase &-egulate the rate of initiation of transcri tion &;ndicate the start oint for transcri tion 2. Aromoter se?uences: A) 2 consensus se?uences 1)&11 se?uence 3A3A33 $Aribno* bo4) is about 11 nucleotides u stream $#/) from the start oint of transcri tion 2)&3# se?uence 339ACA $3# nucleotides u stream from the start oint of transcri tion &3hese t*o se?uences set a basal rate for transcri tion &Basal level ad@usted by regulatory roteins $re ressorsHactivators) a) -e ressors are regulatory roteins *hich reduce the level of transcri tion b) Activators are regulatory roteins *hich elevate the level of transcri tion 3. 74am le of a bacterial o eron $lac o eron)&lactose metabolism A) Com osed of 3 genes: &z& codes for beta&galactosidase&brea(s do*n lactose to glucose N galactose &y& galactoside ermease &a& thiogalactoside transacetylase &y N a involved in rotein trans ort &>rganized into romoter and coding se?uences

B) > erator&a section of an o eron involved in the control of the synthesis of the gene roducts encoded *ithin the o eron $region of ,8A)J a regulatory gene that binds *ith a regulatory rotein to turn on and off transcri tion of a s ecified region of ,8A &-egulatory rotein) *hich binds to the o erator and inhibits the transcri tion of structural genes) is called a re ressor &7ncoded by a se arate gene &;n this e4am le of the lactose o eron) there e4ists a lactose re ressor) *hich binds to the o erator in the absence of lactose

;;. .actose -e ressor: &Also has a binding site for lactose &Bhen lactose is resent) *ill bind to re ressor induces a conformational change of the re ressor molecule to *hich *ill not bind to the o erator) thus) transcri tion *ill roceed &,rs. <acob and 'onod first discovered re ressor regulation in the 1:%12s &5ollo*ing transcri tion $ olycistronic message&m-8A that codes for more than one cistron)) translation occurs *here information of the m-8A is converted to a oly e tide chain. $Arocessed to mature functional roteins $rgani4ation of /ukaryotic -eno#e: &9enome&entire collection of genes and all other functional and nonfunctional ,8A se?uences in the nucleus ;. 5unctional se?uences: &5or e4am le) encode m-8A) r-8A) t-8A) snHsc -8A2s &-e lication origins &-egulatory element 3A3A CAA3 bo4 enhancers for gene e4 ression ;;. 8onfunctional se?uences: &Consist of re etitive se?uences&,8A se?uences) *hich are re eated thousands or millions of times &Arrangement of functionalJ and nonfunctional se?uences are not fi4ed in the genome &74isting se?uences have been observed to move from one location to another Cgenetic rearrangement 1. 74am les: &Aroduction of immunoglobulins

&74 ression of mating genes in yeast &74 ression of surface glyco roteins in rotozoa ;;;. >rganization ,efinitions: &Chromosome& linear ,8A molecule E histones E nonhistone roteins &3elomeres& end of a chromosome &Centromere& holds 2 chromosome arms together &+a loid genome& contains one co y of each chromosome &,i loid genome& contains 2 co ies of each chromosome &3ri loid& contains 3 co ies $common in lants) &3etra loid& contains ! co ies $common in lants) &Asuedogene& nonfunctional co ies of e4 ressed genes ;D. >rganization of Coding "e?uences: 1. m-8A genes A) 01P of coding ,8A codes for m-8A B) 'ost occur in single co y $one co y er genome) C) "ome m-8A genes have multi le co ies &'yosin) tubulin) histones) collagens) +b2s) immunoglobulins 2. r-8A) t-8A) snHsc-8A genes &74ist as gene families in multi le co ies D. -e etitive 8oncoding "e?uences: &,iscovered in the 1:%12s by ,rs. Britton and =ohne &,evelo ed a method for detecting re etitive ,8A se?uencesC reassociation (inetics 1. Under certain conditions) the ,8A double heli4 *ill brea( do*n to a random coil or single stranded molecule A) +eat B) +igh +

&Both of these brea( the + bonds holding the base airs) discharges the hydro hobic interactions bet*een the bases) ermitting the t*o strands to se arate from each other C) 74 osure to chemicals such as urea or dimethyl sulfo4ide &3his rocess is called ,8A denaturation &-everse rocess of denaturation is renaturation or reannealing &"ince renaturation is limited by the fre?uency of collision of com lementary se?uences) the rate at *hich it occurs *ill be slo*er as the size of the ,8A molecule increases &74: AC- reaction &'ost common method to measure ,8A denaturation is based on the hy erchromic effect of ,8A 2. Conce ts: A) 8ucleic acids have strong absor tion $o tical density) ma4ima at 2%1 nm

&Absorbance $turbidity of a solution)& the fraction of incident light absorbed by a solution at a given *avelength and is related to the thic(ness of the absorbing layer and the concentration of the absorbing molecule &Absorbance is measured ?uantitatively by the .amert&Beer .a*: log ;oH;Cecl ;o Cintensity of incident light ; Cintensity of transmitted light e Cmolar e4tinction coefficient $litersHmole&cm) c Cconcentration of absorbing molecule l Cthic(ness of the light absorbing sam le B) Absorbance of double stranded ,8A is not the sum of the absorbance for each of the bases &3he hydro hobic interactions and + bonding bet*een the stuc( bases reduces the >, $absorbance) reading C) Bhen the ,8A double heli4 is bro(en or denatured these forces are destroyed and the o tical density of the solution of ,8A is increased because each base no* contributes its normal o tical density 1) ;ncrease in >, is called the hy erchromic effect:

&,ifferent ,8A2s can have different melting rofiles &5or e4am le) a ,8A molecule that contains a high P of A3 b *ill have a lo*er 3m than a ,8A molecule *ith a high P of 9C b . 2) 74am le: a) +eat a ,8A solution to denature double heli4 b) Cool solution 11&1#2 belo* the 3m $renaturation) c) 3he se arated strands *ill collide *ith each other and once in a *hile the com lementary regions *ill find each other and renature CC double heli4 ,) -ate of renaturation is limited by the rate at *hich collisions occur bet*een com lementary se?uences 1) 3his is a 2nd order rate function based on: rate C (FAGFBG or rate C (FAG2 F G Cthe concentration of single com lementary strands $at time t) 2) ,8A of an organism is characterized by the value of Cot at W $#1P) reassociation &-eassociation of mammalian ,8A ?uite different from the bacterial ,8A $uniform size) &3here is a ra idly reassociating fraction C re resents re etitive ,8A se?uences &3here is a moderately reassociating fraction C re resent moderately re eated se?uences &3here is a slo*ly reassociating fraction C re resents single co y ,8A se?uences D;. 3y es of -e etitive ,8A "e?uences: 1. 'oderately -e eated "e?uences $111&1111): A) 5unctional se?uences: 1) 9ene coding for r-8A) t-8A and re eated m-8A genes 2) -e eated genes at the telomeres &5unction to stabilize ends of the chromosomes B) 8onfunctional se?uences 1) Asuedogene&mutated nonfunctional genes arising from gene du lication events

2) Celo genes &"ome co ies contain a -8A olymerase ;;; romoter &Active in transcri tion &5unction of most un(no*n 2. +ighly -e eated "e?uences $113 to 11%): &+ighly conserved *ithin a s ecies &Account for the large content of genomes in eu(aryotic cells &74: snails have more ,8A than mammals N lants &5unction of the se?uences still not understood A) 9enetic rearrangement & ,8A se?uences can move around *ithin the genome &Aroduction of antibodies &74 ression of mating genes in yeast &74 ression of surface glyco roteins in rotozoa &>ne source of genetic rearrangement occurs by trans osable elements& segments of ,8A that can move from one osition in a genome to another 3. 3*o classes of trans osable elements: A) 3rans osons&,8A se?uence that moves by a mechanism that cuts the se?uence from its original location in the genome and inserts it in a different site &Contains inserted re eat ,8A se?uences at each end B) -etrotrans oson $retro oson) & ,8A se?uence *hich du licates and moves to ne* locations via an -8A intermediate ,8A Ltranscri tion -8A &&reverse transcri tase c,8A &&/integrase/ inserted into genome &-everse transcri tase & enzyme first found in retroviruses that ma(es a double stranded ,8A co y from a single&stranded -8A tem late molecule &Arimary source of trans osable elements are retroviruses &Can be harmless or athogenic $A;,"Hcancer)

DN" re1air>#utations:

&,8A double heli4 is constantly being modified C ,8A damage &3his ultimately causes 01 & :1P of human cancers ;. 2 'a@or Classes of ,8A ,amage: 1. " ontaneous A) " ontaneous ,8A ,amage: 1) Can occur via: a) 7rrors in base& airing during ,8A re lication b) 7rrors in roofreading $32 & #2 e4onuclease function of ,8A ol.) c) 7rrors in accessory roteins $e4: ,8A binding roteins) d) Aost ,8A re lication mismatch errors &Cells eliminate ,8A damage $lesions that damage) by molecular ,8A re air &5irst described in 1:!# by ,r. =elner &5ound that visible light rotected microorganisms from the lethal effects of UD radiation 2. 7nvironmental &3hese factors may overla ;;. 5our different ty es of ,8A re air mechanisms: 1) ,irect re air of ,8A 2) Base e4cision re air 3) 8ucleotide e4cision re air !) 'ismatch re air 1. ,irect re air of ,8A: A) 3here are 3 ty es: 1) " ore hoto roduct re air &-estricted to 1 s ecies of bacteria Bacillus subtilus &-e airs cyclobutane dimers in s ores and re?uires light

2) Ahotoreactivation &;s the enzymatic reversal to monomers of yrimidine cyclobutane dimers &,imers are formed by the UD com onent of sunlight and are the ma@or cause of s(in cancer &,imers are removed from ,8A by an enzyme called hotoliase) *hich binds to the yrimidine dimer in ,8A) absorbs) a blue&light hoton) and s lits the cyclobutane ring by electron transfer from the hotoe4cited 5A,+2 at the active site $not in humans) 3) Al(yl transfer &,8A al(ylation adducts: &2 ma@or mutant al(ylation sights on ,8A bases are at the >% osition of guanine and the >! osition of thymine &'ethyl grou s can be attached via a variety of reactions during normal cell metabolism &,uring ,8A re lication) the ,8A olymerase *ill match $base& air) >% methyl guanine *ith thymine. ;f ,8A mismatch is not corrected) the 9C base air *ill be re laced *ith an A3 base air $mutation). &,uring ,8A re lication) the ,8A olymerase *ill match $base& air) >! methyl thymine *ith guanine. ;f ,8A mismatch is not corrected) the A3 base air *ill be re laced *ith a 9C base air $mutation) &5ortunately) all cells have enzymes that remove methyl grou s from ,8A. 3he ma@or enzyme is called >%& methylguanine&,8A&methyl transferase $'9'3) &3ransfers methyl grou s from 1%&methyl guanine and 1! methyl thymine to a cysteine amino acid in the active site of the enzyme &3his transfer is an irreversible reaction /suicide reaction/ thusJ '9'3 is not a true enzyme er se $not a catalyst) &'9'3 found in all s ecies &+umans have one enzyme &E. coli has 2 enzymes

&>% methyl guanine is cytoto4ic) mutagenic) and tumoragenic) tumor cells *hich lac( '9'3 activity form tumors. 3his '9'3 li(ely lays a significant role in cancer revention.

2. Base e4cision re air: A) A ,8A adduct $modified or damaged ,8A base) is removed by a s ecial enzyme called a ,8A glycosylase *hich leaves an em ty base site) in the ,8A double heli4 $AA site) B) AA&deo4yribose is removed by 2 enzymes called AA endonucleases &AA liase&enzyme that catalyzes the addition of functional grou s to double bonds &AA hydrolase&enzyme that catalyzes hydrolysis reactions C) 3he missing nucleotide is then re laced using a ,8A olymerase and the ,8A double heli4 is sealed together using a ,8A ligase ,) Base e4cision re air system or re airs the follo*ing ,8A base adduct: 1) Uracil $results from the deamination of cytosine) 2) +ydro4ymethyl uracil $o4idation of thymine) 3) 3hymine glycols $damaged thymine e4am le thymine dimers) !) 0 hydro4yguanine $o4idation roduct of guanine) 3. 8ucleotide e4cision re air: &Bor(s together *ith base e4cision re air &-emoves large bul(y ,8A adducts &Adduct formed *ith: cis latin) nitrosoureas) nitrogen mustard C anti&cancer drugs A) ;n nucleotide e4cision re air: 1) A multisubunit A3A&de endent nuclease $e4cision nuclease) e4cinuclease) ma(es 2 cuts or incisions) one on either side of the ,8A adduct 2) 74cises or removes an oligonucleotide *hich contains the adduct 3) 3he e4cised oligonucleotide is then re laced by co ying the com lementary ,8A strand using ,8A olymerase !) ,8A double heli4 is then sealed together using ,8A ligase !. 'ismatch re air:

A) Both ro(aryotic and eu(aryotic cells are ca able of re airing mismatched ,8A base airs after re lication B) 'olecular mechanisms of mismatch re air rocesses are related to the molecular mechanisms of base e4cision re air and nucleotide e4cision re air C) ;t is the function of the cellular mismatch re air system to recognize ,8A base mis airs and to eliminate these biosynthetic mista(es from ne*ly synthesized ,8A strands ,) 'ismatched re air systems rely on secondary signals *ithin the ,8A double heli4 to identify the ne*ly synthesized ,8A strand) *hich by definition contains the re lication errors 7) "ignal is in a 9A3C se?uenceJ the adenine base is methylated $in arental 9A3C se?uence)J ne*ly synthesized 9A3C is not methylated 1) ! roteins recognized these base mis airs on ne* strand $including an endonuclease) 2) >ligonucleotide se?uence $either 3 T or # T to the base mis air) is removed from the ne*ly synthesized strand $this involves 3 roteins&includes a helicase& introduces or removes ,8A su ercoils) and an e4onuclease 3) A correct strand is resynthesized using ,8A olymerase !) ,8A double heli4 is sealed together using ,8A ligase ;;;. 'utations: &'utation&is the original source of genetic variation caused by a change in a ,8A base or a chromosome &" ontaneous mutations& is those that a ear *ithout e4 lanation &;nduced mutations& is those attributed to a articular mutagenic $chemical and Hor hysical) agent &'utations can be introduced into an organismTs ,8A as a result of unre aired ,8A damage &'utations can have a variety of distinct cellular andHor molecular effects &Alter the structure of a rotein H-8A &Cause a change in gene e4 ression &7ffect can range from undetectable to lethality of an organism 1. 'utations and mutants: some definitions: &'utation&is a heritable change in the se?uence of an organismTs genome &9enome&full com lement of an organismTs genetic material &'utant&is an organism that carries one or more mutations in its genome

&9ene&basic unit of heredity &Allele&alternate form of a gene &,ifferent mutations that are located at the same gene $genetic locus) are said to be alleles of each other &.ocus&ma ortion of a gene

&9enoty e&is the genetic information that an organism encodes in its genome &Ahenoty e&observable characteristics of an organism &A mutation that as multi le effects on an organismTs henoty e is said to be leiotro hic &Bild&ty e genoty e&refers to the normal or arental observable characteristics of an organism $ henoty e) $o erational definition) &"cientists use a su erscri t lus sign *hen referring to the *ild&ty e gene $e4: lacO E) &A mutation that changes the henoty e from *ild&ty e to a mutant genoty e is said to be a for*ard mutation &A mutation that causes change of genoty e from mutant to *ild&ty e is said to be a reverse mutation &'utants can be obtained by selection or screening &;f a condition can be arranged in *hich all the members of the o ulation die or fail to gro* e4ce t for the desired class of mutants) the mutant can be identified or obtained by selection &74am le: selection for am icillin resistant bacterial colonies $or bacterial cells *hich contain UC 1:) &;f it is necessary to e4amine all the members of the o ulation to identify the mutant having the desired henoty e) the mutants are identified or obtained by screening &A mutagen is an agent $chemical H hysical) $biological viruses trans osable elements) that leads to an increase in the fre?uency of occurrence of mutations &3he rocess by *hich mutations are roduced is referred to as mutagenesis &'utagenesis that occurs *ithout treatment of the organism *ith an e4ogenous mutagen is referred to as s ontaneous mutagenesis &3hese mutations can occur because of re lication errors &>ccur as a conse?uence of ,8A lesions that are introduced into an organism2s genome during normal gro*th of the cell &'utagenesis that results from treatment of an organism *ith a chemical) hysical) or biological mutagen is referred to as chemical mutagenesisJ historically called UD mutagenesis

&9eneral term is induced mutagenesis &" ontaneous mutations and induced mutations can be the sameJ they refer to ho* they *ere identified or obtained &;f the resence of a articular adduct $modification or lesion) in ,8A can result in a mutation at the site *here the adduct is located) the ,8A adduct is termed a remutagenic lesion: e4: >%met9 N >!met3 &'utations occurring at sites in ,8A *here there *as not (no*n to be a ,8A adduct are referred to as untargeted mutations &'utation fre?uency&refers to the ro ortion of mutants in a o ulation &'utation rate&e4 resses mutations giving a articular observable henoty e er ,8A re lication) this can be e4 ressed as mutations er base air er cell division $re lication) &9enomic mutation rate&e4 resses mutations er genome er ,8A re lication $cell division) 2. 3*o classes of mutations&different ty es of mutations: A) "im le mutations $ oint mutations) & are mutations that result from the substitution of 1 base air for another or from the addition or deletion of one or a small number of base airs &'ost commonly results or arise from e4 osures to hysical or chemical mutagen B) Com le4 mutations&involve e4tensive changes in ,8A structure 1) ,eletion&is a mutation *hich involves a loss of one or more base airsJ *here a chromosomal segment or gene is missing 2) ;nsertion&a mutation *hich involves the addition of genetic materialJ *here a chromosomal segment or genes are added 3) ,u lication& situation in *hich a chromosomal segment or gene is re eated &.in(ed&tandem du lication &>r not lin(ed !) ;nversion&alteration in gene se?uence in a chromosome &Aericentric inversion includes the centromere &Aaracentric inversion does not &5urthermore) for organisms that have I1 chromosome) mutational events can occur that involve the @oining of the ,8A of one chromosome to the ,8A of another) thereby causing a chromosomal rearrangement $e4: a translocation& movement of a chromosome segment from one chromosome to another chromosome)

3. 3y es of oint mutations: A) Base substitution mutations&result from the substitution of one base air for another 1) 3ransition mutation& a mutation that involves a change from one urine for another or one yrimidine for another 9C A3 A3 9C 2) 3ransfusion mutation& a mutation that involves the interchange of a urine for a yrimidine or the interchange of a yrimidine for a urine 9C 3A 9C C9 A3 3A A3 C9 B) 'issense mutations& is base substitution mutations that change the codon for one amino acid to that for another amino acid 1) 7ffects of missense mutations: a) 8o detectable effect b) Aartial loss of function c) 9ain of function d) Alteration of function e) Change in rotein stability &3em erature effect & + effect f) Com lete loss of function C) "ilent or neutral mutations& is *hen a base substitution mutation changes a codon from one amino acid to a different codon for the same amino acid &,ue to the degeneracy of the genetic code ,) 8onsense mutations& is mutations that change the codon for an amino acid to one of the three sto codons &3A9) 3AA) 39A C usually lead to com lete or artial loss of function of the gene roduct &Ahenoty ic effects of nonsense mutations can be altered or su second mutation C nonsense su resser ressed by a

&8onsense su resser& is a mutation occurring *ithin a gene encoding a t-8A and alters the anticodon of that t-8A so that it can recognize a nonsense codon

&;n a cell that contains a nonsense mutation and a nonsense su resser) *hen the nonsense codon is read) at least a fraction of the time the amino acid carried by the nonsense su resser t-8A is inserted into the gro*ing oly e tide chain &Arevents chain termination &Arotein functional &Ahenoty e of the mutant organism is *ild&ty e even though its genoty e differs from that of its *ild&ty e arent by the resence of both the nonsense codon and the nonsense su resser mutations 7) AdditionH,eletion of base airs: &5rameshift mutations& mutations that alter or shift the translational reading frame $not in grou s of 3) Cancer: ;. ;ntroduction: &Cancer is the leading cause of death for *omen in the U.".& due to the consistent decline in heart disease mortality in the last !1 years &Cancer *ill be the leading cause of death in the U.". by the year 2111. &'ost common cancers are caused by tobacco and alcohol 1. Cancer& a malignant tumor resulting from a rogressive series of genetic and cellular events *hich occur in a single clone of cells due to alterations in a limited number of s ecific genes A) >ncogenes B) 3umor su resser genes

C) 3umor susce tibility genes &Cancer) or the rocess of carcinogenesis) can be divided into 2 or more stages de ending on the ty e of cancer &;nitiation&is a clonal e4 ansion of cells that gain a selective advantage by genetic changes in the cells $mutations) re&cancerous state &Aromotion $ rogression)& *hen there is an accumulation of genetic alterations in these re&cancerous cells &3hese alterations are caused by initial genetic changes and can also occur by e4 osure of the cells to tumor romoting com ounds such as horbol esters &3hese alterations cause the henoty e of these cells to change into more malignant ones

&'easurement of age&de endent cancers suggest that at least 2 or more than 6 inde endent events are necessary for the develo ment of malignant tumors ;;. 3here are t*o ty es of tumors: 1) Benign $ rimary or remalignant) tumors are gro*ths) *hich remain confined to its normal location &74: *arts 2) 'alignant $cancerous) tumors are gro*ths that can invade non&tumorous tissues via the circulatory or lym hatic systems &'ovement of cancerous tumors to non&tumor tissues is called metastasis &'ore than 111 different (inds of human cancers are recognized and historically classified according to their cellular origin. ;;;. 'ost cancers are divided into # ma@or grou s 1) Carcinomas 2) "arcomas 3) .eu(emiasHlym homas !) Carcinomas: &Com rise a ro4imately :1P of human cancers &Arise from e ithelial cells #) "arcomas) leu(emias) lym homas: &"arcomas are cancers of connective tissue or muscle tissueJ develo from mesodermal cells andHor circulatory cells of the blood and lym h system &.eu(emias are cancers derived from hemo oietic cells $blood bone marro* cells) &.ym homas are cancers derived from lym h cells &3umors are further classified to their tissue of origin & ;s a multiste ath*ay that leads to colon cancer

&3his ath*ay includes # genes: & #3) ,,C) AAC) ras ( &;n this ath*ay there must be at least 6 inde endent genetic events $2 each for #3) ,,C) and AAC and one for ras () for a normal cell to change to a metastatic tumor

&;t has been (no*n for several years that changes in ,8A and not changes in other subcellular com onents such as li ids and roteins are res onsible for the formation of cancerous tumors &3ransgenic mouse cells changed into tumor cells &;t *as further sho*n that the ,8A in the human cancer cells contained an activated oncogene) *hich *as not resent in the non&tumor mouse cells. +o*ever) the transgenic tumor mouse cells contained the activated human oncogene ;;;. Chromosomal rearrangements &'any genetic changes) *hich lead to cancer) are large chromosomal rearrangements that are visible under the light microsco e. +uman chromosomes can be re ared in culture latesHtubes and there are s ecific bonding atterns that are uni?ue to each chromosome and these atterns are genetically inherited &Chromosomal aberrations $genetic rearrangements)) *hich lead to cancer) can be translocated bet*een chromosomes) deletion of all or art of a chromosome or an inversion *ithin a chromosome or other more com le4 rearrangements &3hus) a molecular characterization of chromosomal rearrangement associated *ith cancer should lead to the identification of genes involved in cancer &3his molecular characterization has lead to the cloning of ,8A se?uences at s ecific arts of human chromosomes *hich are associated *ith cancer and &A molecular ma of the human genome is being constructed using overla ing ,8A fragments &;t is im ortant to determine *hich ,8A se?uences are res onsible for the transformation from normal cells to metastatic tumor cells &Bur(itt2s lym homa. ;n this case there is a reci rocal translocation bet*een chromosome 0 and 1! and laces a roto&oncogene ne4t to an Ab gene &3his event causes the activation of a roto&oncogene to an oncogene ;D. >ncogenes: 1. 7arly 1:012s ,r. Bisho discovered oncogenes: A) 9ene that causes a tumor B) 5irst discovered in retroviruses $ras gene) C) Aroduct of an oncogene is re?uired for the transforming activity of the virus $ability of the virus to change normal cells into tumor cells) ,) >ncogene act in a dominant manner &+eterozygote is tumoragenic

&"tudies on oncogenes in the 1:012s led to the discovery that the host cell has an inactive form of the oncogene $ roto&oncogene) 2. Aroto&oncogene: A) Cellular counter art of the oncogene B) 8ot e4 ressed in the cell or e4 ressed at lo* levels relative to the activated viral oncogene C) Cellular roto&oncogene can be activated to an oncogene by mutation ,) 'utational event can be a sim le base substitution or a more com le4 chromosomal rearrangement 7) Aroducts of oncogenes are called onco roteinsJ these roteins carry out a variety of cellular functions: 1) Arotein tyrosine (inases& enzyme that hos horylates tyrosine residues& involved in signal transduction ath*ays 2) 9ro*th factors& signal molecules that are involved in regulatory cell gro*th 3) 9 roteins& heterotrimeric 93A&binding roteins that are involved in signal transduction ath*ays !) 3ranscri tion factors& roteins that function in modulating gene e4 ression &'ore than three&dozen tumor viruses have been identified and this is a list of 2: retroviruses) the name of their transforming oncogene and their onco rotein gene roduct &>ncogenes such as ras+) raf) @un) and fos can function in signal transduction ath*ays *here a chemical or hysical signal is recognized by the cell &3he cell *ill eventually start roducing a s ecific set of roteins in res onse to the signal 3. "ignal transduction ath*ay using an oncogene: A) -elaying of a signal by conversion from one hysical or chemical to anotherJ the rocess by *hich a cell converts an e4tracellular signal into a res onse 1) 9ro*th factor binds to a rece tor 2) Binding activates a tyrosine rotein (inase 3) Activates -as !) -as activates) in se?uence) 3 cyto lasmic rotein (inases: -af) '7= $mitogen e4tracellular&signal&regulated (inase)) 'AA= $mitogen activating rotein (inases) &"erineHthreonine rotein (inases called intracellular transducers

&'AA= a rotein (inase that erforms a crucial ste in relaying signals from the lasma membrane to the nucleus. =inase activated by a *ide range of roliferation or differentiation inducing signals #) Activation of 'AA= $ hos horylation) allo*s the (inase to move from the cyto lasm through the nuclear envelo e into the nucleus %) 'AA= then activates <un and 7l( ; $transcri tion factors) by hos horylation 6) <un and 7l( ; *ill bind to the romoters of @un and fos genes res ectively and these 2 genes *ill be e4 ressed 0)And begin to roduce @un and fos m-8A *hich *ill then be trans orted out to the cyto lasm *here the :) 'essages $m-8A) *ill be translated to <un and 5os roteins 11)<un and 5os *ill bind together to form a $heterodimers) transcri tional activating com le4 $AA&1 com le4) 11) AA&1 com le4 *ill be trans orted from the cyto lasm through the nuclear envelo e to the nucleus 12) AA&1 com le4 *ill activate a number of target genes $QQ) *) *hich leads to cell transformation) or the conversion of normal cells to tumor cells &3he early ste s in this signal transduction ath*ay have been elucidated in great detail) but the actual mechanisms of cellular transformation *hich arises from the e4 ression of host cell target genes is still oorly understood. !. 3umor su resser genes: resser genes: 1) ;n contrast to inactive cellular roto&oncogenes) tumor su resser genes are highly active in normal cells and they function to re ress cell gro*th 2) ;nactivation of a tumor su resser gene by mutation leads to a deregulation of cellular gro*th resser genes)

A) # features of tumor su

3) Cancers) *hich are formed due to the inactivation of tumor su are inherited in a dominant fashion !) +o*ever) tumor su

resser genes act in a recessive manner $heterozygote is non tumoragenic)

#) 5ollo*ing inactivation of the first allele by mutation) the chromosome containing the second) and only other *ild ty e allele is usually lost &Arocess of chromosomal loss is called loss of heterozygosity $.>+) B) +uman cancer:

1) #3&is the most common target of mutations in human cancer $ #3 mutations are in I #1P of cancerous tumors) a) Cellular #3& a ears to regulate cell division and mutations in this gene lead to deregulated cell gro*th &+as a /hot s ot/ for 9 3 transversions $benzo yrene) b) 'utations in the -B 1 gene are associated *ith retinoblastoma& human childhood disease and involves the formation of a tumor on the retina c) 'utations in the ,,C gene are associated *ith colon cancer &3he recessive nature of tumor su resser genes can be visualized through the construction of cell hybrids. ;n this e4 eriment) normal cells are fused *ith tumor cells that contain a mutation in a tumor su resser gene. 3he resulting cell hybrid is non&tumoragenic. 3his result demonstrates that the *ild& ty e allele $form of the gene) from the normal cell is sufficient to restore a normal $non tumoragenic) henoty e &Cell hybrids are ty ically unstable &;f the *ild&ty e tumor su resser gene is lost) the hybrid cell is transformed bac( to a tumor cell resser gene) -B 1

&-etinoblastoma) *hich is caused by the inactivation of a tumor su

&3his disease is inherited in an autosomal $not se4&lin(ed) dominant manner #. 3he autosomal dominance A) After inactivation of one tumor su resser allele by mutation) the second allele is inactivated by a non&dis@unctional) event during mitosis *here the chromosome containing the *ild&ty e tumor su resser allele is lost and the chromosome that contains the mutant $inactivated) allele is du licated &8ondis@unction& defined as an abnormal se aration of chromosomes in mitosis $meiosis) B) 3his rocess is termed loss of heterozygosity $.>+) C) .>+ can be detected by a change of gene e4 ression of closely lin(ed loci and by the absence of chromosomal ,8A se?uences that are lin(ed to the tumor su resser genes &;ndividuals *ho have a family history of cancers) *hich are associated *ith tumor su resser genes) are more li(ely to develo cancers than individuals that have no history of family cancers &;n heredity cancer associated *ith tumor su already resent in the germ line resser genes) the first mutation is

&3his individual only needs to ac?uire a second somatic mutation that *ill lead to the formation of a cancerous tumor.

&+o*ever) in s oradic cancers) the develo ment of a tumor re?uires at least 2 somatic mutation events in the tumor su resser gene. &3hus) one can see that individuals *ho already carry a mutant form of a tumor su resser gene are much more li(ely to develo cancer since only one somatic mutation) rather than t*o) is needed for tumor formation. &=aryoty e& a hotogra hic or ictorial re resentation of the chromosomes in a given individual &-B1 is recessive in nature because if a *ild&ty e -B1 gene is introduced into a retinoblastoma tumor $transvection)Q &3ransfection& introduction of ,8A into a non&bacterial cell the resulting cell is non&tumoragenic %. 3umor susce tibility genes: A) 9enes do not actively cause cancer B) 'utations $inactivation) in these genes lead to an increased incident of cancer C) "everal of these genes are associated in ,8A re air &74am les defect in ,8A ligase) methyltransferase &Bith the identification of tumor susce tibility genes) studies have sho*n an interaction bet*een oncogenes) tumor su resser genes) ,8A re air genes) and genes involved in regulating the cell cycle $cell cycle genes) &Activation of a tumor su resser gene $ #3) and an oncogene $ 21) after the cell has been e4 osed to agents $e4: UD radiation) that can damage ,8A. &3he roducts of these genes can interact *ith cellular roteins to arrest the cell rior to mitotic division $in order to re air ,8A damage). &3he cell has sensory mechanisms $ #3H 21)) *hich can interru t normal cell division until genetic and subcellular damage can be re aired 6. 3he molecular characterization of cancer genes and develo ment of ne* ros ects to revent and treat cancer A) 'olecular techni?ues can be used to identify individuals *ith inherited susce tibilities to cancer &3hese techni?ues could reliably detect early re&malignant stages B) 3he develo ment of diagnostic a lications for the analysis of oncogenes has already been used in clinical ractice &Abnormalities of oncogenes have rovided useful assays $detection method) for monitoring the course of the disease during treatment $am lification by AC- of an oncogene fusion event $bcrHabl) to detect leu(emia cells C) 3he develo ment of drugs that target against oncogenes

&5or e4am le) the fusion of a retanoic acid rece tor to an oncogene leads to one form of leu(emia if you treat the atient *ith retanoic acid $-A) &-A *ill bind to the -A rece tor and this binding *ill revent activation of the oncogene &Bhen atients *ith this ty e of leu(emia are treated *ith -A treatment leads $I :1P) to remission of the tumor

Transcri1tion Factors: &"tructural motifs $structural design)$families) found in transcri tion factors& au4iliary roteins $beyond the 0 to 1! distinct oly e tides that ma(e u the olymerases) that assist in the o eration of eu(aryotic olymerases ;. .eucine zi &.eucine zi er& is a stretch of amino acids rich in leucine residues that rovide a dimerization motif er roteins can form dimers through a hydro hobic interface

&,imer formation re?uired for ,8A binding &c&@unHc&fos C leucine zi er roteins &+eterodimers& zi ered airs of nonidentical roteins $used to turn other genes /on/H/off/) &+eterodimers of c&<unHc&5os onco roteins bind ,8A much more strongly than homodimers of c& 5os or c&<un alone ;;. +eli4&loo &heli4 $+.+ rotein)& a family of roteins in *hich a ortion of the oly e tide forms 2 al ha& helices se arated by a loo $the +.+ domain) &3his domain acts as a se?uence&s ecific ,8A&binding domain &c&myc encodes a +.+ transcri tion factors ;;;. Oinc finger rotein& has a re eated motif of amino acids $rich in cytosineHhistidine) that bind to On & rotein can bind to ,8A andHor -8A forms zinc fingers ;D. heli4&turn&heli4& a highly conserved family of se?uences $%1 amino acidsH 101 b 2s)$homeobo4) in) length $homeodomain) &bind ,8A in a se?uence&s ecific manner &heli4 3 C is the recognition heli4 and ma(es im ortant contacts *ith ,8A &helices 1N2 C ma(es contact *ith other roteins eco#+inant DN" technology:

&enables individual fragments of ,8A from any genome to be inserted into vector ,8A molecules $e.g. lasmids) viruses) and these fragments can be introduced into bacteria $e.g. transformation) and then am lified &donor organism& organism under study that donates ,8A &e4: fruit fly Drosophila melanogaster &vector ,8A molecules& lasmidsHviruses that can acce t foreign ,8A se?uencesJ ca able of re licating &transformation& introduction of vector ,8A $ lasmid ,8A) into a bacterial cell &transfection& introduction of a virus into a host cell &e4: introducing lambda bacterio hage$virus that infects bacteria) into bacterial cells &recombinant ,8A$chimeric ,8AJ after 9ree( monster Chimera)& vector molecule E ,8A insert &recombinant $,8A) clone& a large o ulation of identical ,8A inserts

;. "te s in roducing -ecombinant ,8A molecules: 1) ;solating ,8A& both donor and vector ,8A &donor ,8A isolated by /genomic re / rocedures &vector ,8A: 1) lasmid also isolated using a variety of rocedures /mini/) /midi/) /ma4i/ re s 2 )viruses can also be urified using / hage re s/ 2) Cutting ,8A: &e4onucleases& cleave nucleotides one at a time from the end of a olynucleotide chainJ they may be s ecific for either #2 or 32 end of ,8A or -8A &endonucleases& cleave bonds *ithin a nucleic acid chain &restriction enzymes& endonucleases that recognize s ecific short se?uences of $usually) unmethylated ,8A and cleave the double&stranded molecule & roduced by bacteria as a defense mechanism against hages $ art of the restrictionHmodification system) &enzymes cut ,8A into fragments of a size suitable for cloning &enzymes can ma(e /staggered/ cuts) *hich generate single&stranded /stic(y/ ends conducive to the formation of recombinant ,8A

&most restriction enzymes either recognize a s ecific ! b /four cutters/ or a s ecific %b /si4 cutters/ ,8A se?uence &/! cutters/ *ill cut once) on average) every !! bases C 2#% bases &/% cutters/ *ill cut once every !% bases C !1:% bases &2 e4am les of restriction enzymes: &named after the organism from *hich it *as identified or urified from 1) 7co -1 & from bacterium Escherichia coli 2) +ind ;;; & from bacterium Haemophilus influenzae &both are % cutters &7co -1 C recognizes a % b alindrome se?uence

&both ,8A strands have the same nucleotide se?uence but in anti arallel orientation #2 & 9AA33C & 32 CI #2 & 9 AA33C & 32 32 & C33AA9 & #2 CI 32 & C33AA 9 & #2 &staggered cut& leaves a air of identical single&stranded /stic(y ends/ &ends can + bond to a com lementary se?uence 3) <oining ,8A: &donor ,8A $foreign ,8A) E vector ,8A C digested *ith restriction enzymeHmi4ed CI recombinant ,8A &@oin fragments $via + bonding of com lementary se?uences)$annealing)J catalyzed by enzyme ,8A ligase &seals nic(s that have available 32&>+ and #/&A>! termini &uses A3A as an energy source !) Am lifying recombinant ,8A: &recombinant lasmid ,8A is introduced into host cells by transformation &vector *ill re licate $donor ,8A *ill also re licate) &*ill have multi le co ies of vector in cell &cell *ill also re licate $cell division) &from 1 to 11: cellsHml

;;. Cloning a s ecific gene: 1. Choosing a cloning vector: &must be small $for mani ulationHconvenience) &must be able to re licate &must contain convenient restriction sites &must be able to identifyHrecover recombinant molecules 2. Cloning vectors: A) Alasmids: &autonomous) self&re licating) e4trachromosomal circular ,8A &distinct from bacterial chromosomes &1st lasmid that *as identified *as the 5 lasmid 1) e isome& can e4ist in 2 forms a)self&re licating b)integration into chromosome &se4 lasmid involved in bacterial con@ugation& the union of 2 bacterial cells) during *hich chromosomal material is transferred from the donor$+fr) to the reci ient$5&) cell

2) e4am les of lasmids: a) B- 322& contains 2 drug resistance genes 1) tct- C tetracycline resistance &tetracycline& binds to a rotein on the 31" subunit of the ribosome and inhibits ribosomal translocation 2) am - C am icillin resistance &am icillin& inhibits enzyme that are involved

in the synthesis of the cell *all &bla& codes for an enzyme that is secreted to cell *all and brea(s do*n am &donor ,8A can be inserted into tct- gene C insertional inactivation &select for tct"Ham - colonies b) UC lasmids: 1) lasmids have a mutation in a re lication gene $ro gene) *hich leads to increased co y number 2) has a olycloning site $ olylin(erJ multi le cloning site) &a ,8A se?uence that *as genetically constructed in vitro and contains many sites) *hich are recognized by restriction endonucleases 3) olylin(er inserted $insertion as in&frame *ith B&gal coding se?uence) in the lac O gene $B&galactosidase) &vector contains ,8A se?uence encoding the 1st 1!% amino acids of B& galactosidase) thus) UC vectors e4 ress the 8+2 terminal of B& galactosidase !) reci ient cells $e.g. ,+# al ha) e4 ress the C>>+ terminal fragment #) active B&galactosidase rotein is a homotetramer &multimeric enzyme *ith each oly e tide consisting of 1163 amino acids in length %) the 2 fragments *ill then @oin together in trans to form a functional gene roduct $ artial roteins coded by 2 fragments unite

to form a functional B&gal rotein) C called al ha&com lementation 6) thus) one can detect lacO e4 ression $B&gal activity) A) vector E cells $e.g. ,+# al ha)$lac&) CI B&gal activity $lacE) &B&gal activity in the resence of a colorless substrate called U&9A. $#&bromo&!&chlero&3& indoyl&B&,&galactoside) *ill roduce a colored $chromogenic& roduces a colored igment)$blue) roduct B) vector E insert E cells CI no B&gal activity C *hite colonies &colony& a visible clone of cells 0) insertion of a fragment of foreign ,8A into the olycloning site of UC results in roduction of an amino&terminal fragment that is not ca able of al ha& com lementation :) UC lasmids usually acce t foreign ,8A u to 11 (b 3. Bacterio hage: &viruses that infect bacteria A) lambda&convenient cloning vector 1)acce ts foreign ,8A usually 11&1# (b 2)central ortion of hage genome not re?uired for re lication ac(aging &can be removed using restriction enzyme 3)lambda can be am lified to get large amounts of foreign ,8A B) cloning in lambda: 1) nonessential region of genome discarded $leaving leftHright arms) 2) foreign ,8A digested into 11&1# (b fragments

3) foreign ,8A ligated into arms $ends of arms connected to form a long linear molecule consisting of multi le hage genomes $concatenated ,8A) &concatemer&series of unit genomes re eated in tandem !) ,8A then laced into a viral head $ca sid C com osed of viral roteins) in vitro ac(aging C) Cosmids: &hybrid vectors of lambda hages E lasmids &can re licate in a cell li(e a lasmid or be ac(aged li(e a hage &cosmids can acce t foreign ,8A u to !# (b $reason that most of the lambda genome has been deleted) &ho*ever) cosmids still contain the signal se?uences that romote hage headstuffing $cos sites) &shuttle vectors& is a lasmid constructed to have origins for ,8A re lication for 2 hosts $7. coliH". cerevisiae) so that it can be used to carry foreign se?uences in either ro(aryotes or eu(aryotes ,) "ingle&stranded hages $'13): &useful in ,8A se?uencing &e4 ression vectors& *here cloned genes can be transcribed and translated into a rotein &o en reading frame $>-5)& a ,8A se?uence that can be converted to an amino acid se?uence $via -8A) &can synthesize a foreign rotein in a bacterial cell $remember) bacteria cannot rocess introns C thus) clone cannot contain introns) ;;;. VAC2s &yeast artificial chromosomes: &foreign ,8A E yeast centromere se?uences E yeast telomere se?uences &can be assembled into artificial chromosomes $u to 1111 (b) &VAC2s use yeast cells as hosts ;D. BAC2s & bacterial artificial chromosomes: &based on 5 factor $se4 factor) &acce t u to 311 (b $usually 111 (b) &one advantage of BAC2s over VAC2s is that one uses bacterial cells rather than yeast cells

D. CloningHconstructing a ,8A library: &source of foreign ,8A &library& is a set of cloned fragments together re resenting the entire genome &/shotgun/ cloning& e4 erimenter clones $isolates) a large sam le of ,8A fragments ho ing that one of the clones *ill contain a /hit/ the desired gene 1. ,ifferent ty es of libraries: A) based on: 1) ty e of vector 2) source of ,8A 3) different cloning vectors carry different amounts of ,8A !) choice of vector de ends on size of genome a) small genomes C lasmidsH hages b) middle genomes C cosmids c) largest genomes C VAC2sHBAC2s B) genomic library& contains entire genome $introns) e4ons) etc.) C) c,8A library $com lementary ,8A)& is synthetic ,8A made from m-8A $catalyzed by an enzyme called reverse transcri tase& originally isolated from retrovirus $tumor virus) m-8A Lreverse transcri tase ss,8A L,8A ol ds,8A &thus) for e4am le) if a scientist (no*s that a articular gene is highly e4 ressed $i.e. there is lots of rotein) in a articular tissue of an organism) then one can construct a c,8A library using the m-8A from that tissue 2. Using Arobes to isolate s ecific clones: & robe& a nucleic acid molecule that can be detected by radioactive $32AH3+) methods or chemiluminescent methods & robes de end on the natural tendency of one single&strand of nucleic acid to find and base& air $hybridize) to a com lementary base se?uence via + bonding A) Bhere does robe come fromQ 1) you can synthesize it 2) you can get it from a friend /clone by hone/

3. Cloning "te s: A) transfer coloniesH la?ues & containing foreign ,8A of interest form the etri dishes to membrane filters com osed of nitrocellulose or nylon C colony liftsH la?ue lifts &,8A must be denatured on filters often by chemical treatment $e.g. 8a>+) B) bath membrane *ith solution containing robe & robe must be denatured first& heat it C) remove robeH develo filters to detect resence of com lementary ,8A se?uences &"outhern blotting $,8A&,8A hybridization)& describes the rocedure for transferring denatured ,8A from an agarose gel to a nitrocellulose filter *here it can be hybridized *ith a com lementary nucleic acid &restriction digests of genomic ,8A roduces a *ide array of ,8A fragments differing in size &*ill loo( li(e a smear on an agarose gel &a robe can detect a s ecific ,8A restriction fragment 1) se arate digested genomic ,8A using agarose gel electro horiesis $gel fractionation)$,8A fractionation) 2) lay absorbent membrane $nitrocelluloseHnylon)on to of the gel &,8A transferredH/blotted/ from gel to membranes by ca illary action $,8A stays in the same osition) 3) e4 ose membrane to a robe of interest $radioactive robe) !) remove robe and lay a hotogra hic film over membrane &e4 ose and develo film &*ill see a s ecific band on this film &Autoradiogra hy& rocess that detects radioactively labeled molecules by their effect in creating an image on hotogra hic film &"outhern blotting C ,8A&,8A hybridization &8orthern blotting C ,8A&-8A hybridization &Bestern blotting C rotein&AB interactions !. 5inding s ecific clones by functional com lementation:

&cloned genes can be detected through their ability to confer a missing function on a transformation reci ient &ecto ic e4 ression& describes the e4 ression of a gene in a tissue in *hich it is not usually e4 ressed &ecto ic insertion& insertion of recombinant ,8A $vector E insert) into a reci ient2s genome at a location that is different from its original site & ositional cloning& any method that uses information about a gene2s location or osition on a chromosome in order to isolate that gene &chromosome *al(ing& describes the se?uential isolation of clones carrying overla ing se?uences of ,8A allo*ing large regions of the chromosome to be s anned. Bal(ing is often erformed in order to reach a articular locus of interest A) Cloning a 9ene by 3agging& can isolate a s ecific gene by inducing a mutation in that gene$insertional mutagen C trans osable element) 1) A&element & clone genes in Drosophila 2) 3y1 element & clone genes in yeast

Cellular re1roduction: &according to cell theory) ne* cells originate from other living cells & rocess C cell division C cellular re roduction &mother$ arental) cell daughter$offs ring) cell ;. -evie* cell cycle: 1. Control of cell cycle$2 control events in cell cycle): A) initiation of ,8A re lication *hich occurs at the transition bet*een 91H" B) initiation of mitosis *hich occurs at the transition bet*een 92H' &research on factors controlling the cell cycle began in yeast *ith the discovery of a gene called cdc2 &budding yeast Saccharomyces cerevisiae &fission yeast Schizosaccharomyces pombe &revealed a large K of genes *hose roducts function to maintain the ro er cell cycle &*ere identified$ I 01 genes identified) as conditional mutations $ts C tem erature sensitive) called cdc mutations $cell cycle division)

&*ill gro* normally at lo* tem s $ ermissive tem & 232C) &*ill sto gro*ing at higher tem $restrictive tem 3%2C) &a articular cdc mutant *ill sto gro*ing at a s ecific time in the cell cycle $all cells *ill e4hibit a similar mor hological henoty e) &in contrast to a ty ical ts mutation) cell *ill sto at varying oints in the cell cycle $numerous henoty es) C) restriction $/start/) oint& during the 91 hase of the cell cycle) the oint at *hich the cell is committed to divide 1) factors influencing restriction oint: a) cell size& ratio of cyto lasmic volume to genomic size b) regulatory roteins& function as a molecular cloc( to carry out the cell cycle &ty es of regulatory roteins: A) rotein (inases &an enzyme that regulates the activity of another rotein) or target molecule) by adding a A>! derived from A3A B) cyclin&de endent (inases $Cd(2s)& a rotein that is active only *hen attached to a articular cyclin C) cyclins& roteins *hose concentration fluctuates over time $amount of rotein varies in a cycle C eriodic fluctuations) 2. entry into ' hase $mitotic hase) triggered by the activation of a rotein (inase called maturation& romoting factor $'A5) a) 'A5 contains 2 subunits: 1)catalytic subunit A3A A,A E A>! $serHthr) 2)regulatory subunit C cyclin &&first yeast cdc mutation C cdc2 $S. pombe)Hcdc20 $S. cerevisiae)

&*hen mutated) cell sto s gro*ing at : 1)either @ust rior to ,8A re lication or 2)@ust rior to mitosis & roduct of cdc2 is catalytic unit of 'A5 &cdc2 regulated at end of 91 and at end of 92$determined by tem erature&shift e4 eriment) &both cell cycle stages re resent oints in the cell cycle *hen a cell becomes committed to beginning crucial events a),8A re lication b)nuclear division & assage through these oints re?uires the transient activation of Cd(2s by s ecific cyclins & assage through "3A-3 $91H" restriction oint) re?uires the activation of cdc2 by a 91 cyclin $cig2) ,) cdc2 cig2 dimer & assage through the second oint of commitment @ust before the end of 92 re?uires activation of cdc2 by a different grou of cyclins $mitotic cyclins)$cdc13) 7) cdc2 cdc13 dimer & Arogression through the cell cycle re?uires the hos horylation and de ho s horylation of certain critical amino acids & rior to mitosis$92H' chec( oint)& Cd( subunit must be hos horylated at threonine&1%1 C catalyzed by CA= $Cd(&activating (inase) &there is also hos horylation at threonine 1!Htyrosine1# C catalyzed by *ee1 and mi(1 &this inhibits CA= activity &active *ee1 or mi(1 roduces an inactive cdc2 cdc13 enzyme com le4 &tyrosine1#Hthreonine 1! A>!2s can be removed by a hos hatase cdc2#

&thus) the cdc2# hos hatase and *ee1Hmi(1 (inases act as com etitors to one another) one stimulating cdc2 and the other inhibiting its activity ;;. rotein (inasesHcontrol of cell division: 1) the cyclin is synthesized throughout the cycle and accumulates during inter hase 2) Cyclin attaches to Cd( $cdc2) and the rotein com le4 is activated at the end of inter hase 3) 3he active com le4) 'A5 $maturation romoting factor)) coordinates mitosis by hos horylating various roteins that include rotein (inases hos hatases !) >ne of the roteins activated by 'A5 is a cyclin degrading enzyme that destroys 'A5 activity #) 3he Cd( com onent of 'A5 is recycled) its (inase activity restored by association *ith a ne* cyclin that accumulates during inter hase

Cyto1las#ic Me#+rane )yste#: &internal cyto lasmic structures first observed by biologists in the 1:!12s *ith the develo ment of the electron microsco es $scientists sa* membrane&bound vesicles of varying electron densities &electron microsco es ortray static) not dynamic rocesses &cisternae & flattened membrane&bound com artment &cyto lasm of eu(aryotic cells subdivided into a variety of distinct com artments $bounded by membranes) &endomembrane system & functionally and structurally interrelated grou of membranous cyto lasmic organelles including the endo lasmic reticulum) 9olgi com le4) endosomes) lysosomes) and vacuoles $nuclear envelo e can be included & not a cyto lasmic organelle) &trans ort vesicles & the shuttles) formed by budding from a membrane com artment) that carry materials bet*een organelles &budding fission cycle & vesicles move through the cyto lasm in a directed manner on trac(s $cytos(eleton then fuse to the ne4t com artment ;. ,istinct cyto lasmic ath*ays: 1. biosynthetic ath*ay $secretory ath*ay) & route through the cyto lasm by *hich materials are synthesized in the endo lasmic reticulum or 9olgi com le4) modified during assage through the 9olgi com le4) and trans orted *ithin the cyto lasm to various destinations such as the lasma membrane) lysosome) or a large vacuole $ lant cell) $alternate term secretory ath*ay has been used because many of the materials synthesized in the ath*ay are destined to be discharged outside the cell) &this includes the flo* of li ids) carbohydrates) and roteins

A) 2 ty es of secretory ath*ays: 1)constitutive secretion & discharge of materials synthesized in the cell into the e4tracellular in a continued) unregulated manner $contributes to formation of lasma membraneHe4tracellular matri4) 2)regulated secretion & discharge of materials synthesized in the cell that have been stored in membrane&bound secretory granules in the eri heral regions of the cyto lasm) occurring in res onse to an a ro riate stimulus & roductionHrelease of hormones) digestive enzymes) or neurotransmitters 2. 7ndocytic ath*ay & route for moving materials from outside the cell $and from the membrane surface of the cell) to com artments) such as endosomes $e4: lysosomes)) located *ithin the cell interior &trans ort re?uires traffic atterns: e4: salivary secretory vesicles digestive enzymes lysosomes & roteins are targeted through sorting signals & recognized by rece tors in the *alls of trans ort vesicles

;;. "tudy of Cytomembranes: 1) Autoradiogra hy $historical) & a techni?ue for visualizing biochemical rocesses by allo*ing an investigator to determine the location of radioactively labeled materials *ithin a cell. 3issue sections containing radioactive isoto es are covered *ith a thin layer of hotogra hic emulsion) *hich is e4 osed by radiation emanating from the tissue. "ites in the cell&containing radioactivity are revealed under the microsco e by silver grains after develo ment of the overlying emulsion 2) ulse&chase e4 eriments & are erformed by incubating cells very briefly $ ulse) *ith a radioactively labeled recursor $of some ath*ay or macromolecule)J then the fate of the label is follo*ed during a subse?uent incubation *ith a nonlabeled $chase) recursor 3) cell fractionation & se aration of vesicles derived from fusion of the ends of membrane fragments that form u on homogenization of cells. Desicles formed from each of the different membranous organelles $nuclei) mitochondria) lasma membrane) endo lasmic reticula) etc.) have different biochemicalH hysical ro erties that allo* this se aration &e.g. cyto lasm &$homogenization) fragment fuse into vesicles &microsomes & a heterogeneous collection of similar&sized vesicles formed from the endomembrane system $ rimarily the endo lasmic reticulum or 9olgi com le4) !) Use of mutants $yeast) & ,r. -andy "chelman &isolation of secretory defective mutants $cloned genesJ analyzed gene roducts such as roteins) &revealed that com onents of endomembrane system

&highly conserved $yeast) lants) human have similar secretory roteins) $can correct yeast defects *ith mammalian gene)

/ndo1las#ic eticulu# ./ 0: &rough 7- & attached ribosomes &smooth 7- & no ribosomes ;. fluid of cyto lasm & divided by 7- into 2 com artments: 1) luminal $cisternal) s ace & s ace bet*een 7- membranes 2) cytosolic s ace &region outside 7- membranes &ribosomes face cytosolic s ace ;;. "mooth 7- $"7-) & resent in s(eletal muscle) (idney tubules) steroid& roducing endocrine glands 1. 5unctions: A) synthesis of steroid hormones in the endocrine cells of the gonadHadrenal corte4 B) deto4ification $liver) &o4ygenases $A!#1 system) & converts hydro hobic molecules to hydro hilic molecules &benzo yrene BA,7 $carcinogen) C) converts glucose&%&A>! glucose $liver) &allo*s glucose to be released into the bloodstream ,) se?uesters CaEE & can be used in a variety of cellular reactions ;;;. -ough 7-: 1. 5unctions: A) site of synthesis of secretory roteins B) secretory roteins contain a signal se?uence at 8&terminus $%&21 non olar amino acids) C) triggers attachment of ribosome to 7- membraneHmovement of nascent oly e tide into cisternal s ace $signal hy othesis) 1) m-8A translated into oly e tide on free ribosome $*H signal se?uence)

2) signal se?uence recognized by signal recognition article $"-A) &% oly e tides &6#. -8A $sc-8A) &sto s translationH rotein folding 3) "-A E oly e tide C "-A com le4 binds to 7- rece tor on the cytosolic side of 7&translation then roceeds !) "-A released $re?uires energy 93A) $9 roteins) $second messenger ath*ays) & oly e tide enters cisternae $translocation) #) signal se?uence is removed using signal e tidase %) addition of oligosaccharides $oligosaccharyl transferase) 6) rotein ro erly folded $uses s ecialized enzymes e.g. disulfide isomerase) 2. 9lycosylators: 8 & lin(ages to as aragine > & lin(ages to serineHthreonine &addition of sugars to a gro*ing oligosaccharide chain catalyzed by glycosyltransferases & enzymes that transfer a monosaccharide to a sugar rece tor A) 9olgi com le4 & 10:0 Camillo 9olgi & 8obel Arize $1:1%) 1) 8et*or( of smooth membranes organized into a characteristic mor hology) consisting of flattened) disli(e cisternae *ith dilated rims and associated vesicles and tubules 2) 9olgi cisternae are olarized: &cisternae closest to 7- C cis face &o osite cisternae C trans face

3) 9olgi com le4 & rocessing lant) modification of roteins &divided into ! functionally distinct com artments: 1)cis cisternae 2)medial cisternae

3)trans cisternae !)trans 9olgi 8et*or( $398) !) Aassage of molecules from 7- through 9olgi: &coated vesicles & are vesicles *hose membrane has on its surface a layer of rotein coat &C>A2s & non&clathrin&coated vesicles $ art of non discriminatory ath*ay) &re?uires 93A as energy source &-etaining roteins in 7- & roteins contain $.ys) As &9lu&.eu) &e.g. disulfide isomerase &molecular cha erones & involved in rotein folding and remain inside com artment &3argeting vesicles during trans ort occurs by s ecific interactions bet*een roteins located on membranes of vesicles $D&"8A-7 roteins) and rece tors on target membrane $t&"8A-7 roteins) &e.g. D&"8A-7 from 7- binds to t&"8A-7 cis 9olgi not t&"8A-7 in lysosome) #) 3rans 9olgi 8et*or( $398) & a net*or( of interconnected tubular elements at the trans end of the 9olgi com le4 that sorts and targets roteins for delivery to their ultimate cellularHe4tracellular destination &2 ty es of vesicles: 1)nonselective non&clathrin 2)selective) clathrin&coated vesicles &74ocytosis & rocess of membrane fusion and content discharge during *hich the membrane of a secretory granule come into contact *ith the overlying lasma membrane *ith *hich it fuses) thereby forming an o ening through *hich the contents of the granule can be released ;D. Asymmetry of 'embrane: &membranes arise from re&e4isting membranes &asymmetric membranes & established in 7- & membrane asses by rocess of buddingHfusion 9olgi vesicles &;ntegral membrane roteins contain hydro hobic amino acids) *hich anchor rotein in membrane $sto &transfer se?uences)

&"ynthesis of membrane li ids &synthesized in 7&e4ce tions: s hingomyelin and glycoli ids & start in 7-) finishes in the 9olgi 1. Ahos holi ids: &enzymes are located on 7- as integral roteinsJ active site faces cytosol &inserted into W of bilayer facing cytosol &move to o osite W of bilayer under direction of fli ases

& hos holi id e4change roteins & trans ort hos holi ids from one com artment to another com artment

D. Cellular u ta(e: 1) hagocytosis & engulfment of articulate material 2) endocytosis & rocess by *hich roteins at the surface of the cell are internalized) being trans orted into the cell *ithin membranous vesicles a) bul(& hase & unregulated endocytosis b) rece tor&mediated $-'5) & s ecific endocytosis