Professional Documents
Culture Documents
Genome Organization 1
Genome Organization 1
Chapter 12
Virus/Phage Models
fX174
Single-stranded DNA circle
T-even phage
Linear double-stranded DNA
Lambda (l)
Linear ds DNA that circularizes upon entry into cell
Polyoma virus
Double-stranded DNA circle
Bacterial Models
Mycoplasma species
500,000 bp
Bacterial Chromosomes
Commonly circular ds DNA
E. coli about 1.2 mm in circumference
DNA Supercoiling
1963 observation that polyoma virus DNA preparations had 3 different types of DNA which had uniquely different sedimentation velocities 1965, two are circular, one linear
One circular molecule is underwound
Negatively supercoiled (more than 10.4 bp/turn of helix)
Topoisomers
Created by topoisomerases
Types I and II
Polytene Chromosomes
Balbiani, 1881 Certain dipteran cells (e.g. Drosophila salivary glands) Replication without cell division
1000 to 5000 DNA duplexes per chromosome Differences in chromosome structure/packaging creates a banded appearance after staining
Bands once thought to represent genes Bands undergo localized uncoiling during gene expression (puff)
Polytene Chromosome
Chromosome Puff
Lampbrush Chromosomes
Vertebrate oocytes, some insect spermatocytes
Diplotene of meiosis
Look like a brush used to clean kerosene lamp chimneys Useful for studies of crossing over and gene expression
Lampbrush Chromosomes
Chromatin Structure
Chromatin proteins subdivided into histones and nonhistones Histones
Very high contents of lysine + arginine (20-30%) Amino acid sequences very conserved between species
Histone IV differs by one amino acid between pea and cow
Chromatin Structure
Olins and Olins used electron microscopy to observe beads on a string in the mid 1970s Nuclease studies revealed most sensitive sites on DNA in chromatin to be spaced at multiples of about 200 bp Studies showed that histones H2A, H2B, H3 and H4 could interact to form tetramers/octamers
EM Studies
Beads on a string Originally called Nu bodies, now nucleosomes
Nucleosomes
Basic building block of eukaryotic chromatin structure
Octamer of 2 each of H2A, H2B, H3 and H4 About 147 bp of DNA wrapped around histone core particle Linker DNA between core particles gives total of about 200 bp per nucleosome Histone H1 is on the outside at the point of DNA entry/exit to the core particle
Nucleosome Structure
Very regular Highly conserved Can be crystallized for X-ray analysis
DNA Condensation
DNA duplex 2 nm in diameter Nucleosome string about 11 nm in diameter Nucleosomes form coiled solenoid-shaped fiber with 6 nucleosomes per spiral
30 nm fiber
30 nm fiber then folded/looped to form 300 nm fiber 300 nm fibers coiled/folded to form 700 nm fiber that forms chromatid in meiosis/mitosis
Chromatin Remodeling
Chromatin structure is dynamic Induced change in chromatin structure
Replication, gene expression
Histone modification
Acetylation by histone acetyltransferase (HAT) Methylation by methyl transferases Phosphorylation by kinases
DNA modifications
Methylation of cytosine (5-methyl C) in CpG islands
Heterochromatin
1928, staining differences in nuclei lead to terms euchromatin and heterochromatin Heterochromatin
Dark staining Genetically inactive
Few genes, those present repressed Replicates late in S phase Centromeres and telomeres are heterochromatic Portion of Y and inactivated X chromosomes Position effect when genes are translocated to location adjacent to heterochromatin
G-banding
Trypsin treatment before Giemsa staining Provides a unique series of bands along each chromosome Provided uniform nomenclature for human chromosomes/locations in 1971
C-Banding
G-Banding
Satellite DNAs
Some DNAs, when subjected to buoyant density analysis, give a density profile that is not a single peak as expected for random variations in the AT/CG content from one region of DNA to another
Some densities are over represented
Satellite DNAs
Short sequence tandem repeats Heterochromatic regions, mostly associated with centromeres
CEN sequences
First isolated/studied in yeast
About 125 bp, highly conserved between chromosomes, mutations often not a good idea
Mammals
Not very conserved between chromosomes Perhaps not essential for centromere function
More on CEN
Drosophila have 10 bp sequence (AATAACATAG) tandemly repeated on all chromosomes Humans have 171 alphoid family sequence that can be repeated to give up to 3 million bp region
But neither the sequence nor repeat number is very well conserved among primates
Telomeric DNA
Telomeres found on ends of eukaryotic chromosomes
Render natural chromosome ends inert with regard to interactions by other chromosome ends such as those resulting from chromosome breakage
Also telomere-associated sequences (also repetitive) of unknown functions Short repeats synthesized by telomerase
Pseudogenes
Evolutionary vestiges of duplicated genes that failed to become useful
Mutations have generally rendered them inexpressible