DNA Organization in Chromosomes

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

Haemophilus influenzae Escherichia coli

l Phage and Chromosome

Bacterial Chromosomes
Commonly circular ds DNA
E. coli about 1.2 mm in circumference

Found in nucleoid region

Associated with DNA-binding proteins
Small positively charged, similar to histones HU and H

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

DNA Organization In Eukaryotes

DNA protein complex called chromatin
Human chromosomes about 19,000 to 73,000 microns in length, total about 2 meters/cell Nucleus about 5-10 microns in diameter Condensation about 10,000X

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

Humans have about 25 million nucleosomes/cell

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

Levels of DNA Condensation

DNA 11 nm fiber 30 nm fiber 300 nm fiber 700 nm fiber

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

Chromosome Banding of Mitotic Chromosomes

C-banding
Giemsa stain only stains centromeric regions

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

Banding Map of Human X Chromosome

Repetitive DNA Sequences

Not all DNA sequences occur at 1 copy her haploid genome equivalent
Can be highly or middle repetitive Mostly nongenic but middle repetitive does include some multiple copy genes such as rDNA Highly repetitive sequences can be 10% or more of higher eukaryotic genomes
Functions, if any, often unknown

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

Buoyant Density Analysis

In Situ Hybridization With Mouse Satellite DNA Probe

Centromeric DNA Sequences

Centromeres
Responsible for sister chromatid adhesion Site of kinetochore assembly
Mitotic separation failure rate seems to be less than one per 100,000 cell divisions

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

Yeast CEN Sequences

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

Sequence composed of short repeat segments

50 repeats of GGGGTT in Tetrahymena
GGGATT in humans Transposable elements in Drosophila

Also telomere-associated sequences (also repetitive) of unknown functions Short repeats synthesized by telomerase

Middle Repetitive Sequences

Middle or moderately repetitive sequences
Recognized by C0t analysis

Variable number tandem repeats (or minisatellites)

Repeats of 15 to 100 bp to give total tandem repeat length of 1,000 to 20,000 bp Scattered throughout the genome of higher eukaryotes Original basis for DNA fingerprinting

Short Tandem Repeats (STRs) or microsatellites

Commonly di-, tri-, or tetranucleotide repeats Basis for present DNA profiling technology and gene mapping studies

Repetitive Transposed Sequences

Dispersed repetitive sequences Short interspersed elements (SINES)
Perhaps 500,000 in human genome (5% total) Includes Alu elements 200-300 bp in length

Long interspersed elements (LINES)

L1 family in humans about 6400 bp in length
100,000 copies (lines also about 5% of genome)

Retroposons (transpose by first being transcribed into RNA)

Pseudogenes
Evolutionary vestiges of duplicated genes that failed to become useful
Mutations have generally rendered them inexpressible

Including other noncoding DNA sequences

Less than 10% or a sea urchins DNA encodes genes, 5-10% in Drosophila and less than 5% in humans