Manipulatingproteins 111109090227-phpapp01
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  • A NUCLEOSOME(11 nm fiber) is composed of histoneoctamer (2 each of H2A, H2B, H3, H4) around which is wound 176 DNA base pairs; nucleosomes are linked to each other by H1Larger fibers made of nucleosome groups are called SOLENOIDS (30 nm fiber)

Manipulatingproteins 111109090227-phpapp01 Manipulatingproteins 111109090227-phpapp01 Presentation Transcript

  • DNA is the stuff our genes are made of… The organization of total sum of genetic information (or genome) of an organism is in the form of double- stranded DNA, except that viruses may have single- stranded DNA, single-stranded RNA or double- stranded RNA genomes. In many viruses and prokaryotes, the genome is a single linear or circular molecule. In eukaryotes, the nuclear genome consists of linear chromosomes (usually as a diploid set) and the mitochondrial and chloroplast (in plants) genomes are small circular DNA molecules. In 1952, Watson and Crick proposed that DNA is a double helix which is known to have alternative forms.
  • (a) The B form of DNAhas ≈10.5 base pairs per helical turn. Adjacent stacked base pairs are 0.36 nm apart. (b) The more compact A form of DNA has 11 base pairs per turn and exhibits a large tilt of the base pairs with respect to the helix axis. (c) Z DNA is a left-handed double helix.
  • DNA supercoils can be removed bycleavage of one strand. (a) EM of SV40 viral DNA. When the SV40 circular DNA is isolated, the DNA duplex is underwound and assumes the supercoiled configuration. (b) If asupercoiled DNA is nicked, the strands can rewind, leading to loss of a supercoil. Topoisomerase I catalyzes this reaction and also reseals the broken ends. All the supercoils inisolated SV40 DNA can be removed by the sequential action of this enzyme, producing the relaxed-circle conformation.
  • In general, genome size increases with thecomplexity of organism.
  • 1. Base stacking interactions - hydrophobic interactions resulting from the individual base pairs’ stacking on top of each other in the nonpolar interior of the double helix, electrostatic forces between nearest- neighbor base pairs, and from van der Waals forces between the bases.2. Deoxyribose sugar- this is less reactive because of C-H bonds. Consequently, DNA has smaller grooves which hinder attachment of damaging enzymes that attack DNA.3. Hydrogen bonds- the sum of all the H-bonds between the paired bases leads to a stabilizing "zipper effect.“4. Protective "twisting" of the DNA helix and flexibility of the two strands - although the bases cannot rotate freely about the axis of their bonds with each other, they are able to rotate around their bonds with the sugars. This area of rotation is like a joint on a human arm. In DNA, there are several flexible bonds: a. bonds between oxygen and phosphorus in phosphate groups b. bonds linking the phosphate groups to the sugar rings c. bonds which link the sugar rings to the aromatic bases5. Interaction with histones- chains of DNA become even more stable, as they entwine with histones. DNA ribbons coil around histones for protection, like a string on a spool.
  •  The bacterial chromosome is localized in the nucleoid region of the cell (no nucleus) and is looped into negative coils. The loops are 50,000 to 100,000 bps in length (similar to eukaryotic chromosomes) which are held in place by RNA and small basic (histone- like) proteins. Plasmids, small negatively supercoiled circular DNA molecules, carry usually non- essential genes (often drug resistance).
  •  A chromosome is formed from a single, enormously long DNA molecule that contains a linear array of many genes. The human genome contains 3.2 × 109 DNA nucleotide pairs, divided between 22 different autosomes and 2 sex chromosomes.
  • Chromosomal banding patterns and multicolor FISH are used to analyze human anomalies Characteristic chromosomal translocations are associated withcertain genetic disorders and specific types of cancers. In nearly all patients with chronic myelogenous leukemia, the leukemic cells contain the Philadelphia chromosome, a shortenedchromosome 22 [der (22)], and an abnormally long chromosome 9 [der (9)]. These result from a translocation between normal chromosomes 9 and 22.
  • Comparative studies reveal that human genomes contain genes in the same order as another mammal, a feature called conserved synteny. Using chromosome banding/ painting, the phylogenetic history of our own chromosomes maybe reconstructed by comparing them with those from other mammals.
  • DNA in a eukaryotic chromosome contains genes, manyreplication origins, one centromere, and two telomeres. These sequences ensure that the chromosome can be replicated efficiently and passed on to daughter cells.
  • If each nucleotide pair is drawn as 1 mm as in (A), then the human genome would extend 3200 km (approximately 2000 miles), far enough to stretch across the center of Africa, the site of our human origins (red line in B). At this scale, there would be, on average, a protein-coding gene every 300 m. An average gene would extend for 30 m, but the codingHuman DNA, if fully extended, would have a total length of 1.7 m. If you unwrap all the sequences in this gene DNA you have in all your cells, you could would add up to only reach the moon ...6000 times! just over a meter.
  • Chromosome Organization
  •  Interphase chromosomes contain both condensed and more extended forms of chromatin  Constitutive heterochromatin - found in the centromere, nucleolar organizers (found in human chromosomes 13,14,15,21, and 22), repetitive or satellite DNA  Facultative heterochromatin- one of the homologues become heterochromatic, e.g. X chromosome becomes Barr body  Euchromatin – loosely packed, actively transcribed regions of the chromosome. Chromatin structure is dynamic: by temporarily altering its structure by using chromatin remodeling complexes and enzymes that modify histone tail, the cell can ensure that proteins involved in gene expression, replication, and repair have rapid, localized access to the necessary DNA
  • Different chromatin remodelingcomplexes disrupt and reformnucleosomes. The same complexmight catalyze both reactions. The DNA-binding proteins could be involved in gene expression, DNAreplication, or DNA repair.
  • Each histone can bemodified by the covalent attachment of different molecules.Histone H3, for example, can receive an acetyl group (Ac), amethyl group (Me), or a phosphate (P). Note that some positions(e.g., lysine 9 and 27) can be modified in more than one way.
  • Different combinations of histone tail modifications may constitute a type of “histone code.” Each marking conveys aspecific meaning to the stretch of chromatin on which it occurs. Only a few of the meanings of the modifications are known.
  •  Contains alpha satellite sequences (5,000- 15,000 copies of 171 base pair sequences). Position of centromere P and q arms
  • Within the centromere region, the actual location where the attachment of chromosomes to spindle fibers occurs is called the kinetochore and iscomposed of both DNA anda protein called CEN DNA. It can be moved from one chromosome to another and still provide the chromosome with the ability to segregate. CEN DNA consists of severalsub-domains, CDE-I, CDE-II and CDE-III. Additional analyses of the DNA andprotein components of thecentromere are necessary to fully understand themechanics of chromosome segregation.
  •  Telomeres are non-sticky regions that prevent fusion of chromosomes and DNAse from degrading their ends. They facilitate replication without loss of material. Most species have telomeric 3’G overhangs that form G- quartets (Hoogstein base- pairing) Contain tandem repeats which are highly conserved (TTAGGGG in man) These 500-3,000 repeats in normal cells shorten with age (biological fortune-tellers?)
  • The stability of the T- loop is largely dependent on theintegrity of associated telomere-specific proteins called the shelterin complex. TRF (telomeric repeat-binding factor) 1 and TRF2 bind to the double-stranded segment of telomeric DNA. POT1 (protein protection of telomeres 1) binds directly to the single-stranded telomeric DNA and interacts directly with TPP1 (tripeptidylpeptidase 1). Rap1 (repressor activator protein 1) binds TRF2, andTIN2 (TRF1-interacting nuclear factor 2) is a central component of the complex interacting with TRF1, TRF2 and TPP1.
  • TERRA (Telomeric repeat-containing RNA)Biogenesis, telomere association and displacement from telomeres. TERRA forms telomeric heterochromatin which may have roles (?) in telomerase regulation and in orchestrating chromatin remodelling throughout development and cellular differentiation. TERRA dysfunctionleads to RF collapse.
  • Most human cells lack telomerase. In normal cells that still produce functional p53 and have their cell-cycle checkpoints intact, thistriggers cell death. But a cell that has acquired ap53 mutation may ignore this signal and cause massive chromosomal damage. Some cells reactivate telomerase, which restores enoughchromosomal stability for cell survival. These damaged cells can then go on to accumulate the additional mutations needed to produce a cancer.
  •  The mitochondria and chloroplasts also have a DNA genome (or chromosome). These resemble procaryotic genomes (likely due to the endosymbiotic origin of these organelles) but are much smaller. The mitochondrial genome varies in size among eukaryotes (mammals =16.5 kb & 37 genes, yeast and plants are greater than 5X this). Chloroplasts are ~120 kb and have ~120 genes. DNA in ORGANELLES
  • DNA Can Undergo Reversible Strand SeparationDenaturation or “melting,”(unwinding and separationof DNA strands), can be induced by increasing thetemperature of a solution of DNA. Denaturation and renaturation of DNA are the basis of nucleic acid hybridization. Loss of the multiple weak interactions holding the strands together along the entire length of the DNA molecules lead to an abrupt change in the absorption of ultraviolet (UV) light. The melting temperature (Tm ) at which DNA strands will separate depends on several factors: a. When the ion concentration is low, shielding of negatively charged phosphate groups in the two strands by positively charged ions is decreased, thus increasing the repulsive forces between the strands and reducing the Tm.
  • b. A greater proportion of G-C pairs require higher temperatures to denature.c. pH extremes denature DNA at low temperature. At low pH, the bases become positively charged, repelling each other. At high pH, the bases become negatively charged, again repelling each other because of the similar charge.d. Agents that destabilize hydrogen bonds, such as formamide or urea, also lower the Tm.
  • Through the analysis of DNA renaturation studies, the large sizes of eukaryotic genomes reveal large amounts of repeated DNA.These undergo a complex pattern of re-annealing which revealsa large amount of repeated DNA sequences (fast annealing) and unique, non-repeated DNA (slow annealing).