MBLG 2771 lecture 9 - University of Sydney

MBLG 2771 lecture 9 - University of Sydney

PHAR2811 Dales lecture 3 Genome Structure COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice

Questions from the last lecture The role of methotrexate in the inhibition of thymidylate synthase Methotrexate is a competitive inhibitor of the enzyme that converts dihydrofolate (DHF) to tetrahydrofolate (THF) (dihydrofolate reductase, DHFR) It binds to the enzyme with ~1000 times the affinity of DHF...making the inhibition almost irreversible Folate conversions

DHF reductase Dihydrofolate (DHF) Tetrahydrofolate, (THF) Thymidylate synthase Serine hydroxymethyl transferase

N5, N10-methylene THF H H2N N N H H

N CH2 O N H R etc

NADPH + H+ Dihydrofolate, DHF DHF reductase TMP Thymidylate Synthase NADP+ H

H2N N N H H H dUMP Serine Hydroxmethyl

Transferase O N CH2 H N H

H H2N N N H H CH2 CH2 O

etc Tetrahydrofolate, THF H N R N R

Serine Glycine etc This methylene group is transferred to dUMP at C6 Prokaryotes

The genome of prokaryotes is extremely efficient. There are 4.6 million base pairs in your average E. coli If the average bacterial protein has a molecular weight of ~40,000 D how many different proteins does the average E. coli make? Prokaryotes To do this calculation you need to know: The average mol. Wt. of an amino acid ~100

This means the average protein has 400 amino acids Which means 1200 bases + promoter and terminator sequences ~1500 bp. 4.6 X 106/1500 = ~3000 different proteins. In humans if the whole genome was coding The genome has 3*109 bp and the average protein subunit is 50,000 So 500 aa = 1500 bp = 3000 bp with large promoter regions which makes the maths easy

3*10 ^9/3*10^3 = 1*10^6 or 1 million different proteins. We only make about 30,000 different proteins so there is a discrepancy Prokaryotes versus Eukaryotes Prokaryotes have no room for redundant sequences. Their survival depends on rapid proliferation when nutrients are available Complex multi-cellular eukaryotes depend for survival on quick responses, adjusting to changes in the environment.

Prokaryotes versus Eukaryotes E. coli can divide every 20 min if conditions are optimal The human cell takes 18 to 24 h to go through the cell cycle once. The human genome only has about 2% coding regions. The gene density is much lower!! Chromosome Characteristics Chromosomes vary in number between species. The chromosome number is a

combination of the haploid number (n) X the number of sets. Algae and fungi are haploid; most animals and plants are diploid. The number of pairs of chromosomes in different species genomes is bizarre. What do these life forms have in common? Chromosome Characteristics Species Genome size in # Haploid

Megabases (Mb) chromosomes E. coli 4.6 1 circular yeast 13 16

cow human 39 3 000 23 alligator 16

carp 52 salamander 90 000 14 Chromosome Characteristics Chromosomes vary in size within a species. Within the human genome there is a

four fold difference in the size of the chromosomes. Centromere: the region of the chromosome where the spindle fibres attach. Repetitive satellite DNA is often found around the centromere. Telomere: ends of the chromosome, containing a distinct repeating sequence, which enables the ends of the chromosome to replicate. Centromere Characteristics

The relative position of the centromere is constant, which means that the ratio of the lengths of the two arms is constant for each chromosome. This ratio is an important parameter for chromosome identification, and also, the ratio of lengths of the two arms allows classification of chromosomes into several basic morphologic types: Centromere Characteristics Chromosome Banding

Chromosomes can be stained with special dyes which give a consistent and unique pattern like a bar-code for each chromosome; so much so that the bands have been numbered. The most common stain used is a Giesma stain. This stain, when applied after mild proteolytic treatment (trypsin) gives light (G-light) and dark (G-dark) bands. The Human Genome The ps and qs of chromosomes

There are 2 arms on the chromosome denoted p and q For most chromosomes the short arm is the petit or p arm The longer arm is the q or queue arm Numbering is done from the centromere along one of the arms Chromosome Banding When viewed at the lowest resolution only a few bands appear. These are numbered p1, p2, p3 etc counting from the centromere. If the stained chromosomes are viewed at higher

resolution many sub-bands are revealed. So the labelling then goes p11, p12, p13. So if your DNA marker may be given a position on the chromosome with a set of numbers like 17p23. This means the locus is on chromosome 17 on the short p arm in sub-band 23. Some terms: The general material which makes up the chromosomes is called chromatin This is composed of DNA and protein. Heterochromatin contains DNA which

is more tightly packaged or condensed and probably is transcriptionally inert. Euchromatin contains most active genes; those actively transcribed. Chromosome packaging at the molecular level. Each chromosome contains a single molecule of DNA This DNA is wound around small proteins called histones These proteins have lots of lysine and

arginine residues, making them very positively charged at pH 7 (and high pIs ~12) Histones There are 5 major histone variants: H1, H2A, H2B, H3 and H4. Two molecules each of H2A + H2B + H3 + H4 make up an octamer which the DNA wraps around with 1.7 turns. This structure is known as a nucleosome. Each nucleosome has an H1 associated and a linker section of DNA, like beads on

a thread. Histone Octamer Histones The major force holding the association of histones to DNA is electrostatic. To separate the histones from the DNA, chromatin is treated with high ionic strength solutions. The high salt reduces the electrostatic interactions and the protein dissociates from the DNA.

Higher order Packaging Figure 11.28 A model for chromosome structure, human chromosome 4. The 2-nm DNA helix is wound twice around histone octamers to form 10-nm nucleosomes, each of which contains 160 bp (80 per turn). These nucleosomes are then wound in solenoid fashion with six nucleosomes per turn to form a 30nm filament. In this model, the 30-nm filament forms long DNA loops, each

containing about 60,000 bp, which are attached at their base to the nuclear matrix. Eighteen of these loops are then wound radially around the circumference of a single turn to form a miniband unit of a chromosome. Approximately 106 of these minibands occur in each chromatid of human chromosome 4 at mitosis. The cell cycle At interphase (G1, S and

G2) the chromosomes look like a plate of spaghetti, entangled and dispersed throughout the nucleus At M phase the newly replicated daughter chromatids condense and line up. Chromosomes at interphase

The role of histones Shield the negative charges of the phosphates Allow bending and DNA wrapping Restrict access to transcription The interaction between the histones and the DNA is dynamic and non-base sequence specific Histone Remodeling Influences the DNA accessibility for transcription Can be one of the first events when switching on a set of genes.

Nucleosome remodeling complexes Nucleosome positioning Histone modification Histone modifications Phosphorylation; serine residues Methylation, adding CH3 Acetylation, lysine residues Histone modifications Phosphorylation; serine residues Methylation, adding CH3

Acetylation, lysine residues Acetylation Transferring an acetyl group to the amino side chain of lysine residues Histone acetyl Transferases (HATs) Histone deacetylases (HDAs) Lysine NH O C

CH H2 C NH C O H2 C

H2 C H2 C +NH3 Acetylated Lysine NH O C

CH O H2 C NH C O H2 C

H2 C H2 C NH C CH3

What effect would acetylation have on DNA accessibility? It neutralises the positive charge of the lysine side chain The histone will not have as much affinity for the DNA phosphates (negative) The nucleosome packing will be looser DNA more accessible for transcription Deacetylases will pack it up again!

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