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Developmental Biology Test I Review

Review Notes Focus on experiments discussed, describe o What are the questions addressed? Draw cadherins Draw formation of neural tube Look for Rohde essay questions (5+) Special questions o Make dilutions using V1C1=V2C2 o Generate a certain volume of solution with a specific molarity using the m.w. o Make percent solution using: @ RT @ 1atm, 1ml = 1g = 1 cc (of water) How does 1 mm3 fit in this relationship? How can this be used to make a 10% soln of SDS? o Generate complex solutions from stocks: Make a 250 ml solution of 100 mM Tris-HCl, 50 mM NaCl, 0.1% SDS and 1 mM EDTA using the following: 1 M Tris-HCl 5 M NaCl 10% SDS 0.5 M EDTA and then bringing it up to 250 ml with water o convert units (nm m) Root word questions (multiple choice)

Every organism has to function as it builds itself. The embryo (developing org b/t fert & birth) is the mediator between the genotype and the phenotype. o Mediates between inherited genes and the adult organism. Dev Bio studies initiation/construction rather than maint. o Questions of becoming *-Pg. 2, bottom: possible essay? 2 objectives development accomplishes: o Generates cellular diversity and order o Ensures continuity of life from one generation next *[Possible Essay p. 4] Six General Questions: o Differentiation: o Morphogenesis: o Growth: o Reproduction: o Evolution: o Environmental integration: Developmental history of the leopard frog:

Gametogenesis and fertilization are seasonal o Plants/insects in pond o Air and water temp Hours of daylight and temperature tells the female pituitary gland that it is spring o Secretes hormones stim ovary make estrogen liver, make/secrete yolk o Progesterone (ov) signals egg to resume meiosis (froz @ metaphase I); egg released for fertilization o *Egg has jelly coat: protection, attract sperm Jelly coat is species dependant Notes on fig 1.1: o (GAMETOGENESIS) Production of gametes o (FERTILIZATION) Gametes fuse, become zygote. Germ Plasm egg, already set up. o (CLEAVAGE) Rapid mitotic divisions; DNA synthesis and mitosis ONLY. Morula ball of cells, compaction Blastula Hollow ball; germ cells there. o (GASTRULATION) Formation of tube gut Invagination outer cells roll inward, forms the germ layers. Formation of the 3 germ layers Ectoderm: Skin and CNS o Secretes fluid, swelling at anterior end becomes brain in organogen. stage Endoderm: digestive tube and assoc. organs, includes lungs Mesoderm: Between the others, forms blood, kidney, gonads, bones, and CT o (ORGANOGENESIS) Interaction of the 3 germ layers All the different tissues/organs associated with each layer begin to develop. Formation of the neural tube; becomes CNS. Secretions from the ectoderm form brain. o (LARVAL STAGE) For dispersal of the young and feeding Germ cells move to their proper location from yolk sac, multiplying themselves along the way. Metamorphosis o (MATURITY) sexually mature. In humans, first 2 divisions in cleavage are controlled by maternal DNA. Sea urchin: blastula is just a hollow ball, after fertilization, divides and immediately becomes a larvae to swim away. o in frog, mass at bottom of ball, hast to live off yolk initially.

Be able to draw this:

From larval stage to adult: o initiated by hormones in tadpole thyroid gland. Most important things about meiosis: o Chromosomes replicate before cell division (each gene X 4) o Replicated chrom. (chromatid) held together by their kinetochores (centromeres) and the 4 homologues pair together. o Meiotic division separates chromatid pairs o 2nd meiotic division splits the kinetochore; each chromatid chromosome o The result is 4 GERM CELLS, each with a haploid nucleus. Triploblastic: 3 germ layers Diploblastic: lack true mesoderm (jellies)

Inductions: tissue interactions All 3 layers influence each other until reaching a certain level Notochord: rod of dorsal mesoderm that separates the embryo into R and L halves o Instructs ectoderm above it to form CNS [Cellular basis of Morphogenesis: p. 21] Fate Maps: trace cell lineages; follows an individual cell to maturity o Observe live embryos that are transparent o Apply vital dyes to region of interest o Fluorescent dyes can be detected in progeny o Genetic marking: Permanent way to mark cells; create mosaic embryos Or use antibodies to 1uail cell antigens Exogenous agents: chemicals, viruses, radiation, and hyperthermia: Teratogens

o Abnormalities caused by teratogens: Disruptions o Thalidomide Be able to ID these:

3 Postulates of Differential Gene Expression: o Every cell nucleus contains complete genome established in the fertilized egg. DNA of different cells is identical o The unused genes in differentiated cells are neither destroyed nor mutilated; retain the potential for being expressed o Only a small percentage of the genome is expressed in each cell type Totipotent total potential cell has the capacity to form an entire organism Pleuripotent - Cells that may still differentiate into various types of specialized tissue in the body. Evidence of Genomic Equivalence: o Metaplasia transformation of 1 cell type into another o Eye regeneration remove newt lens, regenerated o Cloning o Rule is that the genome is the same in every cell in the body EXCEPTION: Immunoglobin genes of B lymphocyteGene rearrangement occurs Different cell types make different proteins, even though = DNA o Different promoters, o Human genes: ~ 25,000 MECHANISM OF DIFF GENE EXPRESSION (How genes are activated in diff cell types at diff times) 1. Differential gene Xscptn regulates which of the nuclear genes are Xscribed into nucRNA 2. Selective nucRNA processing regulates which RNAs are Xscribed, which enter cytoplasm

3. Selective messenger RNA Xlation regulates which of the mRNAs in cytoplasm are Xlated 4. Differential protein mod regulates which prots are allowed to remain and/or fxn. DIFFERENCES IN PROK AND EUK GENES 1) Euk genes are contained in complex of DNA & protein (chromatin) o methylation can activate/repress Xscriptions; depends on AA on histone that was methylated o ex: various methylated residues on H3 lead to different regulations of Xscptn 2) Euk genes are not co-linear with peptide products: Have exons and introns o Gene consists of the following elements: Promoter region: binds RNApol and the subsequent initiation of Xscptn. Xscptn initiation site: cap sequence varies among genes; receives the 5 cap Xlation initiation site, ATG: after Xscptn initiation site and 5 UTR, which can determine the rate at which Xlation is initiated. Exon: Intron Exon Intron Exon Xlation termination codon (TAA): ribosome dissociates, protein is released. 3 UTR: includes AATAAA for Polyadenation, polyA tail: o confers stability on the mRNA o allows mRNA to exit nucleus o permits the mRNA to be Xlated Xscptn termination sequence PROMOTER STRUCTURE & FXN: o Lac Operon: The operator DNA is cis-regulatory element The repressor protein = trans-regulator; binds to cis-regulatory operator on another chromosome. o Cis-regulators: represent specific DNA seqs on a given chrom (act only on adjacent genes) Promoters: Located immediately upstream from Xscptn start site 100s of bp long Needed for binding of RNA pol II & accurate installation of Xscpnt RNA pol require at least 6 additional prots for efficient initiation of Xscptn Enhancers: o Trans-regulators: soluble molecules (incl prots & RNAs) that are made by one gene and interact with genes on the same or different chromosomes BASASL XSCPTN FACTORS: o -6 nuclear prots needed for RNA pol II proper Xscptn o Order of binding: 1. TFIID: foundation of Xscptn cplx o Prevents stabilization of nucleosomes o Antagonistic to HI histone o Binds to TATA via TATA binding protein (TBP) 30 bp upstream of mRNA initiation o No Xscptn if TFIID added later 2. TFIIA stabilizes TFIID binding

3. TFIIB and TFIIH join the complex on the TATA box; o Rate-limiting step for many genes o TFIIB: needed for addition of RNA pol II; 4. TFIIE and TFIIF bind RNA pol II before or during binding TFIID o TFIIE: unwinds DNA helix (helicase) o TFIIF: DNA-dependent ATPase (generates energy for Xscptn 5. RNA pol II is bound to TATA; ready to be released for Xscptn 6. TFIIH: provides releasing power o Phosphorylates each of 52 repeats in the CTD 7. CTD (carboxyterminal domain) of RNA pol II is phosphorylated by TFIIH and is released by TFIID; RNA pols II can now Xscribe mRNA.

TAFs (TATA binding protein-associated factors) 2 fxns: o Determine whether or not TFIID remains on promoter (No TFIID Xscptn) o Fxn as co-activator Bridge the enhancer-bound proteins to [basal] Xscptn cplx via prot-prot interaction o And TAFs are tissue-specific o 250 + 150 kDa TAFS critical for TBFs to remain bound to TATA (stabilize) Estrogen receptor activates by binding to 30-kDa TAF. Other *cell specific TFs* can activate Xscptn by stabilizing initiation complex. TATA-less promoters have SP1. o SP1 binds to GC-rich promoter element, then TFIID (directly or via TFIID) o TFIID can not start Xscptn

ENHANCERS- DNA activate utilization of the promoter. (turn it on) o Control efficiency and rate of Xscptn 1. Can activate only cis-linked promoters (on same chrom) at a great distance (some 50 kbp from promoter) Can be most anywhere on gene, not just on 5 side (3, intron, complementary strand) Bind to SPECIFIC trans-regulator proteins called TFs (NOT basal TFs) SILENCER- negative enhancer o TFs binding silencers repress Xscptn from cis-linked promoter. Can be positive or negative in different cell types; depends on the TFs that are present. Enhancer FXN: o Temporal or spatial o Reporter Gene: chloramphenical acetyltransferase (CAT) Bacterial gene Transfect hybrid gene into 5 promoter region of mammalian cells: 1. ovary cell (doesnt secrete insulin or chymotrypsin) 2. Insulin secreting cell 3. Pancreatic exocrine cell Now, measure activity of CAT marker. Conclusion: expression of genes in exocrine & endocrine cells of pancreas appear to be controlled by different enhancers. Another way to do this experiment: Fuse GFP reporter gene to enhancer element or -gal Shows where enhancers are used and important to the expression of that gene (in same region; completely different region) Combinatorial Association: o Enhancers are modular (an enhancer DNA seq can be expressed in other genes) o But co-dependent units within each cis-regulatory module, TFs work in a combinatorial fashion. (series of TFs are ALL needed for the transcription of a particular protein) Enhancer Region Modularity o Enhancer seqs on DNA are the same in every cell type; what differs is the combination of TF proteins that they bind to. Once bound TFs can enhance or suppress RNA pol activity Bind to several TFs; its the specific combination of TFs necessary to generate a particular cell type Same TF with different TFs will activate different promoters in different cells Same gene can have several enhancers with each enhancer binding to TFs that enable that same gene to be expressed in different cell types.

Gene for Pax6 critical in development of different tissues; has several enhancers (green) that direct Pax6 expression differentially in the pancreas, lens/cornea, retina, and neural tube. o When lacZ is fused to Pax6 enhancers for expression, -gal can be seen in those tissues. o Enhancer modularity, having multiple, separate enhancers, allows a protein to be expressed in several different tissues while not being expressed at all in others.

o Product of Pax6 gene is a TF that works in a combination with other TFs. o In absence of Pax6, pancreas endocrine cells dont develop properlyreduces hormone output o Pax6 enhancer binding site in 3rd intron (with Sox2 and L-Maf TFs) activate the crystallin gene (the last green area at +2218) only in head cells that will become the lens. o Pdx1 Specific for pancreatic region of endoderm TFs can be grouped based on similarity in structure. o TFs in a family share a common framework in their DNA-binding sites (ON THE PROTEIN OF THE XSCPTN FACTOR), and slight differences in the AA at the binding site, causing the binding site to recognize different DNA seqs. KNOW THE 4 FAMILIES, SUB-FAMILIES AND AT LEAST 1 REPRESENTATIVE TF FOR EACH! Family Representative TFs Some functions 1) Homeodomain: HoxHoxa-1, Hoxb-2, etc. Axis formation POUPit-1, Unc-86, Oct-2 Pituitary development; neural fate LIMLim-1, Forkhead Head development PaxPax1, 2, 3, etc. Neural specification; eye development 2) bHLH: MyoD, achaete, daughterless Muscle and nerve specification 3) leucine (bZip): C/EBP, AP1 Liver differentiation; fat cell specification 4) Zinc finger: StandardWT1, Krppel, Engrailed Kidney, gonad, and macrophage devel NucHrmn Rec-Glucocortid Receptor 2 sex det; craniofacl devel; limb devel Sry-SoxSry, SoxD, Sox2 Bend DNA; mamm. 1 sex determ Enhancers bind TFs and each enhancer can have binding sites for several TFs o First: TFs bind an enhancer w/its DNA binding domain; other TF domains bind other TFs and recruit histone-modifying enzymes Ex: Pax6, Sox2, L-Maf TFs combine with each other in lens cells to recruit a histone acetyltransferase that Xfers acetyl groups histones dissociate the nucleosomes in that area, activating gene Xscptn. Pax7 (muscle specific genes) -- Transactivating Thus, the displacement of nucleosomes along DNA makes it possible for the TFs to find their binding sites on enhancers. o Second: TFs stabilize Xscptn initiation complex enables RNA pol to bind to promoter Understand this figure: Not the names; just that the TFs stabilize Xscptn preinitiation complex

o Third: (MOST IMPT!!!) combinatorial fxn called Coordinated Gene expression the simultaneous expression of many cell-specific genes in a cell Ex: specific genes giving rise to the lens are activated @ same time b/c they have enhancers that bind Pax6 All other TFs might be gathered @ enhancer, but until Pax6 binds, THE LENS DOES NOT DEVELOP. Important fxn in development TRANSCRIPTION FACTOR DOMAINS o TFs have 3 major binding domains: DNA-binding domain recognizes DNA seq in enhancer Trans-activating domain activates/suppresses Xscptn of the gene with the bound promoter/enhancer. Usually enables TF to interact w/proteins that bind RNA pol (TFIIB, TFIIE) Protein-protein interaction domain allows TF activity to be modulated by TAFs or other TFs. TFs convert exocrine cells into insulin-secreting cells with 3 TFs (Pdx1, Ngn3, Mafa) o These TFs activate other TFs act in concert to turn pancreatic endodermal cells into insulinsecreting cells. Marfa -cells (insulin) Modification of histones can signal the recruitment of *proteins that can retain the memory of Xscptnal state from generation generation via mitosis o *Trithorax and Polycomb families o Polycomb proteins: bind to condensed nucleosomes KEEP GENE INACTIVE Act as repressor and stabilizer of repression by using MeXferase, which methylates H3. o Trithorax proteins: keeps genes active when bound to the nucleosomes of active genes. Helps retain memory of activation and works to counter Polycomb activity by modifying nucleosomes or alter nucleosome position on chromatin allows TFs to bind to previously condensed DNA. PIONEER TFs: starts unwinding DNA to find a promoter o Penetrate repressed chromatin and bind to their enhancer CRITICAL IN ESTABLISHING CERTAIN CELL LINEAGES. o Pioneer TF in muscle: Pbx acts as molecular beacon for MyoD. Silencers: regulatory elem, repress Xscptn o NRSE: Neural Restrictive Silencer Element Found only in genes expressed in Nervous system Binds a zinc-finger TF: NRSF (Neural Restrictive Silencer Factor) 1. NRSF found in all cells EXCEPT neural cells Seems NRSE seq is necessary for normal repression of neural genes in non-neural cells.

Thus: NRSF in non-neural cells suppress neural genes. Because there is no NRSF in a neural cell, neural genes can be expressed. DNA Methylation & Control of Xscptn o The fifth base is made enzymatically after DNA replication o Conversion can only occur when cytosine is followed by a quanosine [at a CpG sequence] Methylation during development: methylates different that encode different embryonic hemoglobins (-globin; -globin) o Methylation acts in 2 ways to repress gene expression: Block the binding of TFs to enhancers Some TFs can only bind to unmethylated enhancers for gene activation A methylated cytosine can recruit the binding of proteins that facilitate the methylation or deacetylation of histones => stabilizing the nucleosome Genomic Imprinting: recognition of methyl cytosines on one DNA strand & methylates the newly synthesized strand of DNA o Each pattern of methylation can be maintained after cell division => stably inherited in daughter cell. o Why its necessary for the C to be next to a G Dosage Compensation: o Promoter regions of numerous genes are methylated on the inactive X chrom and unmethylated on the active X chrom. DIFFERENTIAL RNA PROCESSING: to become active protein, the nRNA must: o Be processed into mRNA by removing introns o Be translocated from nucleus to cytoplasm o Be translated by ribosome o Some need posttranslational modifications to become active o ALTERNATIVE SPLICING produces a lot of different proteins with relatively few genes Differential RNA Processing o Censorship (RNA selection) selecting which nRNAs are processed into mRNAs that can transferred out of nucleus. o Differential (alternative) Splicing of mRNA precursor into messages that specify different proteins by using different combos of exons THUS 1 gene => entire family of proteins o A splice site has a consensus seq recognized by a spliceosome (consists of snRNAs and proteins called splicing factors) o Dscam gene can produce 38,016 proteins from 115 exons; multiple splice sites in each exon. Alt splicing in diff exons; when 2 dendrites from same axon touch, they are repelled. o Mechanisms of differential RNA processing involve: cis-acting seqs and trans-acting prot factors: Splicing enhancers Splicing silencers Trans-acting prots recognizes these sites and can activate or silence splicing Xlation Regulation o Stability depends on length of polyA tail o Some mRNAs are selectively stabilized @ specific times in specific cells ***MOST mRNAs FORM CIRCLES with 3 end brought to 5end.

Morphogenesis: organizing cells into multicellular arrangements such as tissues and organs. CELL ADHESION: o Differential cell affinity

Reaggregated cells become spatially segregated. Final position of reaggregation reflects the cells embryonic positions MESODERM migrates CENTRALLY TO EPIDERMIS NEURAL PLATE cells migrate CENTRALLY TO EPIDERMIS Aggregates of different cells will sort out Cells aggregate with smallest interfacial free energy; arrange in most thermodynamically stable pattern STRENGTH OF ADHESION differences in cells is all that is required for sorting to occur. CADHERIN-MEDIATED CELL ADHESION: DRAW!!!

E-cadherin (epithelial cadherin, also called uvomorulin and L-CAM) is expressed on all early mammalian embryonic cells, even at the 1-cell stage. Later, this molecule is restricted to epithelial tissues of embryos and adults. P-cadherin (placental cadherin) appears to be expressed primarily on the trophoblast cells (those placental cells of the mammalian embryo that contact the uterine wall) and on the uterine wall. It is possible that Pcadherin facilitates the connection of the embryo to the uterus, since P-cadherin on the uterine cells is seen to contact P-cadherin on the trophoblast cells of mouse embryos N-cadherin (neural cadherin) is first seen on mesodermal cells in the gastrulating embryo as they lose their E-cadherin expression. It is also highly expressed on the cells of the developing central nervous.

EP-cadherin (C-cadherin) has been found to be critical for maintaining adhesion between the blastomeres of the Xenopus blastula and is required for the normal movements of gastrulation Protocadherins are calcium-dependent adhesion proteins that differ from the classic cadherins in that they lack connections to the cytoskeleton through catenins. Protocadherins have been found to be very important in separating the notochord from the other mesodermal tissues during Xenopus gastrulation

TAFs Xscptn-associated factors; proteins required to facilitate efficient binding of Euk RNA pol to TATA seq in promoter.