Chapter One Cell Biology Basics

1.1 INTRODUCTION
Cell biology (also called cellular biology or formerly cytology) is an discipline that studies cell's physiological properties, structure, the organelles they contain, their interactions with their environment, their life cycle, division and death.

1.2 CELL
The cell is the basic unit of organisation or structure of all living matter. The organism with only one cell in their body are called unicellular organisms. The organism with many cells in their body are called multicellular organisms. Any cellular organism may contain only any one type of cell from the following type of cells :

Prokaryotic cells (pro primitive or before ; karyon nucleus) Eukaryotic cells (eu well; karyon nucleus)

COMPARISION BETWEEN PROKARYOTIC AND EUKARYOTIC CELLS

Characteristic Size of cell Prokaryotes Typically 0.2-2.0 m m in diameter Nucleus do not have a well-defined nucleus Membrane-enclosed organelles Absent Eukaryotes Typically 10-100 m m in diameter True nucleus, consisting of nuclear membrane & nucleoli Present; examples include lysosomes, Golgi complex, endoplasmic reticulum, mitochondria & chloroplasts Flagella Glycocalyx Cell wall Plasma membrane Cytoplasm Consist of two protein building Complex; consist of multiple blocks Present as a capsule or slime layer Usually present; chemically complex lacks sterols microtubules Present in some cells that lack a cell wall When present, chemically simple serve as receptors present

No carbohydrates and generally Sterols and carbohydrates that No cytosketeton or cytoplasmic Cytoskeleton; cytoplasmic

streaming Ribosomes Chromosome (DNA) arrangement Cell division Sexual reproduction Single circular chromosome; lacks histones Single circular chromosome; lacks histones Binary fission No meiosis; transfer of DNA fragments only (conjugation)

streaming Multiple linear chromosomes with histones Multiple linear chromosomes with histones Mitosis Involves meiosis

figure:1.3(diagram showing structure of prokaryotic and eukaryotic cell

1.3 ORGANELLES AT A GLANCE

Structure and function of the components of this basic organizer in living organisms.

Name of Organelle Major Function

Nucleus Nucleolus Chromosomes Controls all cell activities Synthesizes rRNA and assembles ribosomes Controls heredity

Other information

Etymology
central part of a thing little nut

Composed of DNA and proteins Chromatin- a collection of chromosomes

khroma "color" + soma "body." So called because the structures contain a substance that stains readily with basic dyes.

Nuclear Envelope and Encloses the Nuclear Pore Complex nucleoplasm and rest of the cell Mitochondria Produces energy (ATP) for the cell by cellular respiration

Double membrane Made of lipids and proteins

separates nucleus from Contains pores Double membrane structure mitos "thread" + Inner folded membranes are called cristae Area surrounded by cristae is called the matrix khondrion "little granule."

Ribosomes

Protein synthesis

Composed of rRNA Free ribosomes- those suspended in cytosol Bound ribosomes- those attached to ER

ribo(nucleic acid) + -some "body."

Endoplasmic Reticulem

Smooth ER1. important in the synthesis of lipids, 2. metabolism of carbohydrates, 3. detoxification of drugs and poisons; Rough ER- assists in the production of secretory proteins, most of which are glycoproteins (proteins attached to carbohydrates)

Membranous system of flattened sacs which are continuous with the nuclear envelope; Smooth ER- no ribosomes are present Rough ER- ribosomes are attached

reticulema netlike network

interconnected tubules and pattern or structure;

Vacuole

Stores and excretes water

Membrane bound sac plant cells, whereas there are many small vacuoles in animal cells-- called vesicles. Contractile vacuoles pump out excess water

vaccusempty

substances, especially Usually 1 large vacuole in

Golgi Appratus

Processes and packages substances for export from the cell and to other parts of the cell

Flattened membranous sacs

Lysosomes

Digests (hydrolyzes) substances

Membrane-bound sacs that lysopertaining to contain digestive enzymes dissolving+

Work best at pH 5 Cell Membrane Also known as a outer layer of a cell assists in the movement of molecules in and out the cell plays both a structural and protective role Cell Wall A structure that characteristically is found in plants and prokaryotes and not animals that plays a structural and protective role Centrioles Important in cell division Cilia and Flagella 1. Cell movement 2. Moves fluids over surface of tissues Cytoskeleton Structural support and Composed of microtubules, cell movement intermediate filaments, and microfilaments figure 1.1:diagram showin plant or animal cell Composed of 2 and proteins plasma membrane, this phospholipid layers

somebody cella "small room, hut," +membrane"parch ment,"

Composed of cellulose and cell"small lignin in eukaryotic cells room,"+wall interior partition,

Located in pairs Composed from center of microtubules.Found only in animal cells Composed of microtubules

1.4 BIOLOGICAL ENERGY

ATP stands for Adenosine Tri-Phosphate, and is the energy used by an organism in its daily operations. It consists of an adenosine molecule and three inorganic phosphates. After a simple reaction breaking down ATP to ADP, the energy released from the breaking of a molecular bond is the energy we use to keep ourselves alive. Respiration is the usual manner in which cells convert food (glucose) into a ATP.

The form of respiration usually employed by cells is aerobic respiration. Aerobic respiration requires oxygen. There are three steps to aerobic respiration; 1. Glycolysis (where glucose is converted into a molecule known as pyruvate). This produces a small amount of energy which is converted into ATP. 2. The Krebs cycle converts pyruvate into citrate, producing more ATP. 3. Oxidative phosphorylation is a chain reaction where energy stored in chemical intermediates produced in the Krebs cycle are converted into heat and yet more ATP. When cells lack oxygen, they produce ATP by anaerobic respiration. This is much less efficient than aerobic respiration at producing ATP, and has the added disadvantage that it produces toxic byproducts such as lactic acid.

1.5 CHROMOSOME
Chromosomes are genetic element of any cell playing a vital role in inheritance of a character and evolutionary development of species.

1.5.1 CHROMOSOME NUMBER

All animals have a characterisic number of chromosomes in their body cells called diploid or 2n number

These occur as homologous pairs. One member of each pair is acquired from gamete of each of the two parents.

The gametes contain the haploid number (n) of chromosomes.

Diploid numbers of some commonly studied organisms (as well as a few extreme examples)
Organism Homo sapiens (human) Mus musculus (house mouse) Drosophila melanogaster (fruit fly) Caenorhabditis elegans (microscopic roundworm) Saccharomyces cerevisiae (budding yeast) Arabidopsis thaliana (plant in the mustard family) Xenopus laevis (South African clawed frog) Canis familiaris (domestic dog) Gallus gallus (chicken) Zea mays (corn or maize) Number 46 40 8 12 32 10 36 78 78 20

Muntiacus reevesi (the Chinese muntjac, a deer) Muntiacus muntjac (its Indian cousin) Myrmecia pilosula (an ant) Parascaris equorum var. univalens (parasitic roundworm) Cambarus clarkii (a crayfish) Equisetum arvense (field horsetail, a plant)

23 6 2 2 200 16

1.5.2 LIGHT MICROSCOPY OF CHROMOSOME

Chromosomes are visible in the light microscope during mitosis and meiosis and can be conveniently studied at 1000 times magnification under microscope. Usually a chromosome consist of two limbs and an intervening nonstaining gap called centromere. Before cell gets ready to divide each chromosome gets duplicated (ie.during S phase of cell cycle). As mitosis begins, these duplicated chromosome condense into short structures which can be stained and easily visible under light microscope. These duplicated chromosomes are called dyads. While they are still attached, it is common to call the duplicated chromosomes sister chromatids. The Kinetochore is a complex of proteins formed at each centromere and serves as point of attachment for the spindle fibres.It helps in seperation of sister chromatids as mitosis proceeds into anaphase. The upper shorter limb is called P arm and the lower longer one q arm. Staining with the trypsin-giemsa method reveals a series of alternating light and dark bands called G bands. G bands are numbered and provide "addresses" for the assignment of gene loci.

1.5.3 STRUCTURE OF CHROMOSOME

A chromosome contains a single molecule of DNA along with several kinds of proteins. Octamers containing two each of histones H2A, H2B, H3 and H4 form a core around which 146 base pairs of helical DNA are wrapped nearly twice to form nucleosomes. Composed of DNA and histones, nucleosomes are primary structural units of chromatin. Almost all the DNA in the nucleus is packed in this form. Together with linker histone H1 (it links two core histones), chromatin is packed into a helical assembly of six nucleosomes per turn called solenoid. The unit of one nucleosome plus one bound H1 histone is referred to as a chromatosome. The condensed chromatin is further folded into giant supercoiled loops which finally form a

chromosome. figure:1.2(free hand diagram showing structure of chromosome

1.6 KARYOTYPING Karyotype is a collective term for arranging chromosomes on the basis of their size, centromeric position and banding pattern and it is species specific. The karyotype of the human female contains:

22 pairs of autosomes 1 pair of X chromosomes

The karyotype of the human male contains:

the same 22 pairs of autosomes one X chromosome one Y chromosome

Analysis of normal human karyotype will reveal following allosomic characteristics: Y chromosome The Y chromosome is the sex-determining chromosome in most mammals, including humans. In mammals, it contains the gene SRY, which triggers testis development, thus determining sex. The human Y chromosome is composed of about 60 million base pairs. X chromosome The sex chromosome associated with female characteristics in mammals, occurring paired in the female and single in the male. Lampbrush chromosome A large chromosome found especially in the immature eggs of amphibians, consisting of two long strands that form many brushlike loops along the main axis of the chromosome. Homologous chromosome A pair of chromosomes containing the same linear gene sequences, each derived from one parent. Humans normally have 22 pairs of homologous chromosomes and 2 X chromosomes (female) or 1 X and 1 Y chromosome (male).

Chromosome puffs A swelling at a site along the length of a polytene chromosome, where transcription is actively taking place. Chromosome rosette Right before metaphase, human chromosomes briefly form this wheel-shaped single ring structure. Chromosome satellite A small chromosomal segment separated from the main body of the chromosome by a secondary constriction; in humans it is usually associated with the short arm of an acrocentric chromosome. 1.7 DNA STRUCTURE AND REPLICATION DNA determines all the characteristics of an organism. It consist of genetic material that is passed on from one generation to another generation in a species so that the information within them can be passed on for the offspring to harness in their lifetime. 1.7.1 Structure Of DNA

Watson and Crick prosposed the double helcial structure of DNA molecule. A Deoxribonucleic acid (DNA) molecule consist of chemically linked sequence of nucleotides in helical form.

Each nucleotide is made up of a nitrogen base, a deoxyribose sugar and a phosphate group. There are four different types of nucleotide possible in a DNA sequence adenine (A), cytosine (C), guanine(G) and thymine (T).

Nucleotides are situated in adjacent pairs in the double helix. The following rules apply in regards to what nucleotides pair with one another.

There are four possible types of nucleotide, adenine, cytosine, guanine and thymine. Thymine and adenine can only make up a base pair. Guanine and cytosine can only make up a base pair. Therefore, thymine and cytosine would NOT make up a base pair, as is the case with adenine and guanine.

Coiling of DNA strands is helical like a circular staircase that always retains the same cylidrical diameter and width of steps.

DNA undergoes plectonemic coiling i.e., one which forms a wider groove (22 Amstrong) and a narrow groove (12 Amsstrong across).

The double helix is right-handed (B-type), the turns run clockwise looking along the helical axis.

Each step represents H-bonded nitrogenous bases. Although they are weaker than the usual chemical bonds, but many of them occuring together give high degree of stability and rigidity to the molecule.

Unlike the regular staircase, the DNA staircase is both polarized as well as grooved. It is further tightly linked with basic protein molecule called histones.

figure:1.4(diagram showing structure of dna nucleotides)

Figure 1.5(diagram showing binding between complementary base pairs

1.7.2 DNA REPLICATION Replication is the process of doubling chromosomal DNA or viral DNA using parental strand as a template. Replication to occur, the following must be available

The actual DNA to act as an exact template A pool of relevant and freely available nucleotides A supply of the relevant enzymes to stimulate reaction ATP to provide energy for these reactions

In the first step of replication, a special protein called helicase unwinds two parental strands of DNA double helix.

Next, a molecule of DNA polymerase binds to one strand of the DNA and start synthesizing new DNA strand from 5' to 3' direction of the new molecule (i.e., 3' to 5' direction of the original molecule).

This newly synthesized strand is called the leading strand and is necessary for forming new nucleotides and reforming a double helix.

Because DNA synthesis can only occur in the 5' to 3' direction, a second DNA polymerase molecule is used to bind to the other template strand as the double helix opens.

This molecule synthesizes discontinuous segments of polynucleotides, called Okazaki fragments.

Another enzyme, called DNA ligase, is responsible for stitching these fragments together into what is called the lagging strand.

There are many replication origin sites on a eukaryotic chromosome. Therefore, replication can begin at some origins earlier than at others. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, forming two new molecules.

Figure:1.6(diagram illustrating replication fork)

1.8 RNA SYNTHESIS AND PROCESSING

The process of synthesis of mRNA from DNA template is called transcription. This process is very similar to DNA replication. RNA polymerase is the most important enzyme directing synthesis of mRNA from DNA template. Synthesis of mRNA begins from a specific DNA sequence found at the beginning of the gene called promoter sequence. Promoter sequence instructs RNA polymerase from where to initiate synthesis of mRNA and in which direction it should continue. RNA polymerase then unwinds the DNA double helix at that point and begins synthesis of RNA strand complementary to one of the strand of DNA. This strand is called antisense or template strand where as the other strand is called sense or coding strand. Synthesis can then proceed in unidirectional manner. Processed eukaryotic meassages contain poly(A) addition signal (AAUAAA) at their 3' end followed by a string of adenine. This poly-A-site contributes not only to addition of poly (A) tail but also to transcription termination and release of RNA polymerase from DNA template. The upstream portion of newly formed mRNA then undergoes further modification called the posttranscriptional modification, and becomes mRNA. The downstream RNA becomes unstable and is rapidly degraded.

1.11 PROTEIN SYNTHESIS AND PROCESSING

The cellular machinery that is responsible for synthesis of proteins is ribosome. Ribosome consist of 50S smaller subunit and 80S larger subunt. The process of translating mRNA to protein begins when smaller subunit encounters the larger subunit. There are two sites in larger subunit for amino acid binding and thus be close enough to each other to form a bond. The "A site" accepts a new transfer RNA, or tRNA molecule or adaptor molecule that acts as a translator between mRNA and protein bearing an aminoacid. The "P site" binds the tRNA that becomes attached to the growing chain. Each tRNA has a specific acceptor site that binds a particular triplet of nucleotides, called a codon, and an anti-codon site that binds a sequence of three unpaired nucleotides. Each tRNA also has a specific charger protein, called an aminoacyl tRNA synthetase. This protein can only bind to that particular tRNA and attach the correct amino acid to the acceptor site The start signal for translation is the codon ATG, which codes for methionine. A tRNA charged with methionine binds to the translation start signal. The large subunit binds to the mRNA and the small subunit, and so begins elongation, the formation of the polypeptide chain. After the first charged tRNA appears in the A site, the ribosome shifts so that the tRNA is now in the P site. New charged tRNAs, corresponding the codons of the mRNA, enter the A site, and a bond is formed between the two amino acids. The first tRNA is now released, and the ribosome shifts again so that a tRNA carrying two amino acids is now in the P site. A new charged tRNA then binds to the A site. This process of elongation continues until the ribosome reaches what is called a stop codon, a triplet of nucleotides that signals the termination of translation. A protein will often undergo further modification, called post-translational modification. For example, it might be cleaved by a protein-cutting enzyme, called a protease, at a specific place or have a few of its amino acids altered.

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INPUTS FROM DR MATIN

1. RULES OF METABOLISM There are several rules for metabolism.

Metabolism occurs in small steps to release energy in small, usable packets. For example, consider sugar being converted to CO2. This releases a lot of energy, about 50 times more than is needed for any single anabolic conversion in the cell. So if it happened in one step, most of the energy released would be lost as heat. Instead, it happens in over 40 steps, and energy is released in about a dozen of them.

Because each metabolic conversion is determined by a gene, the pathways present in an organism are genetically determined. For example, bacteria have genes that code for enzymes that can take the carbon, hydrogen, and oxygen atoms in simple sugar and rearrange them to make ascorbic acid, vitamin C. We cant do this because we lack the enzymes. Other bacteria have the ability to take cellulose from wood or paper and convert it to sugar so they can use that for energy. Again, we lack the gene (and enzyme) to do this. Of course, there are many things we can do that bacteria cant. The point is, metabolism means phenotype, and this is determined by genes. 2. WHY IN BIOCHEMISTRY WE SAY THAT REACTIONS ARE OCURRING IN CYTOSOL and NOT CYTOPLASM Because Cytoplasm consists of Organelles and the intracellular fluid (ICF) or cytoplasmic matrix called Cytosol. We know that certain biochemical reaction occur exclusively in Organelles like Mitochondria ( Krebs cycle) ' and few occur in Cytosol (Glycolysis ) , while few occur in both ( Urea cycle ) .Suppose we say that certain reaction is occurring in Cytoplasm then we might get confused as to that particular reaction is occurring in Cytosol or Mitochondria AS CYTOPLASM = CYTOSOL+ ORGANELLES 3..Here is an INTERESTING OBSERVATION : ( Krebs cycle or Krebs Henseleit Cycle ?) The urea cycle is a cycle of biochemical reactions occurring in many animals that produces urea from ammonia (NH3). This cycle was the first metabolic cycle discovered (Hans Krebs and Kurt Henseleit, 1932), five years before the discovery of the TCA cycle,so Urea cycle is also called Krebs Henseleit Cycle which is not to be confused with the Krebs cycle also known as the citric acid cycle or the tricarboxylic acid cycle (TCA cycle), , or the Szent-GyrgyiKrebs cycle The name of this metabolic pathway is derived from citric acid (a type of tricarboxylic acid) that is first consumed and then regenerated by this sequence of reactions to complete the cycle. The components and reactions of the citric acid cycle were established in the 1930s by seminal work from the Nobel laureates Albert Szent-Gyrgyi and Hans Adolf Krebs.

4. REVERSE TRANSCRIPTION ( The process of Reverse Transcription is a complex one which is very well illustrated in site at www.med.sc.edu:85/flash/hiv-ltr-fn.html as animation ) The process by which DNA is synthesized from an RNA template by means of the enzyme reverse transcriptase. Reverse transcription begins when the viral particle enters the cytoplasm of a target cell. The viral RNA genome enters the cytoplasm as part of a nucleoprotein complex that has not been well characterized. The process of reverse transcription generates, in the cytoplasm, a linear DNA duplex via an intricate series of steps. This DNA is colinear with its RNA template, but it contains terminal duplications known as the long terminal repeats (LTRs) that are not present in viral RNA Extant models for reverse transcription propose that two specialized template switches known as strand-transfer reactions or jumps are required to generate the LTRs. Retroviral DNA synthesis is absolutely dependent on the two distinct enzymatic activities of RT: a DNA polymerase that can use either RNA or DNA as a template, and a nuclease, termed ribonuclease H (RNase H), that is specific for the RNA strand of RNA:DNA duplexes. Although a role for other proteins cannot be ruled out, and it is likely that certain viral proteins (e.g., nucleocapsid, NC) increase the efficiency of reverse transcription, all of the enzymatic functions required to complete the series of steps involved in the generation of a retroviral DNA can be attributed to either the DNA polymerase or the RNase H of RT Reverse Transcription can be broken down into the following steps : A tRNA primer binds to the primer binding site on the HIV RNA. Reverse Transcriptase (RT) starts at this binding site and copies RNA into a single strand of complementary DNA. At this point it only copies from the primer binding site back into the Long Term Repeat (LTR) , so all that has been copied so far is the LTR plus a little extra. RNase H degrades the section of the RNA which has been copied. This allows the tRNA / RT / ssDNA to come dissociate from the HIV RNA , and then reattach at the other end of the stretch of RNA - the fresh DNA copy of one LTR associates with the other LTR. RT then picks up where it left off, copying HIV's RNA genome into DNA. RNase H again joins in, degrading the RNA once it's copied. It leaves one section of RNA intact, a tiny stretch called the polypurine tract. The polypurine tract lies about two-thirds of the way through the HIV Genome . RT starts to create the second strand of DNA - it starts at the polypurine tract and makes a second strand of DNA to complement the code in the first strand.

RNase H now removes all the remaining RNA - the polypurine tract and the tRNA primer (which was until now still attached to one end of the fresh DNA). The DNA circularises - the two ends of the DNA are complementary and easily stick together to make the DNA a loop. RT finishes off its job, completing the second strand of the DNA and also completing the Long Term Repeats at each end. In the process of this the DNA loop breaks again, leaving a double-stranded DNA fragment with a Long Term Repeats at each end.

Fig : Steps of Reverse Transcription

5. HIV GENOME ( What is the difference between HIV-1 & 2 Genome wise ?)
The full HIV genome is encoded on one long strand of RNA. (In a free virus particle, there are actually two separate strands of RNA , but they're exactly the same!) This is the form it has when it is a free virus particle. When the virus is integrated into the host's DNA genome (as pro virus ) then its information too is encoded in DNA. The following image shows roughly how the genes are laid out in HIV (remember that HIV-1 and HIV -2 are quite different).

This diagram is based on a fantastic map of the HIV-1, HIV-2, and SIV genomes, available at hiv-web.lanl.gov/content/immunology/pdf/2000/intro/GenomeMaps.pdf

The genes in HIV's genome are as follows:

gag (coding for the viral capsid proteins) pol (notably, coding for reverse transcriptase) (NB. gag and pol together can be expressed in one long strand called "gag-pol") env (coding for HIV's envelope-associated proteins) And the regulatory genes: tat rev nef vif vpr vpu (N.B. not present in HIV-2) --Difference between HIV-1& 2 vpx (N.B. not present in HIV-1)

The HIV genome also has a " Long Term Repeat ( "LTR) at each end of its genome - not quite a gene, but a sequence ofRNA/DNA which is the same at either end and which serves some structural and regulatory purposes. LTR : The Long Terminal Repeat is something which is often found in strands of RNA or DNA is the Long Terminal Repeat. At each end of the string is the same sequence of code at each end of the string. Almost like the repeat at the start and finish of these sentences, almost like! There are two important functions for the LTR:

Firstly they are "sticky ends" (that's a biochemistry term) which the integrase protein uses to insert the HIV Genome into host DNA .

Secondly, they act as promoter/enhancers - when integrated into the host genome, they influence the cell machinery which transcribes DNA, to alter the amount of transcription which occurs. Protein binding sites in the LTR are involved with RNA initiation.

6. PLUS & NEGATIVE RNA VIRUSES Plus(sense) RNA the virus genome is the virus m-RNA Minus (sense) RNA the virus m-RNA is transcribed from the parental genome