DNA Organization in Chromosomes

Chapter 12

Virus/Phage Models

X174– Single-stranded DNA circle

• T-even phage– Linear double-stranded DNA

• Lambda ()– 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

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

Preview

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 urchin’s DNA encodes

genes, 5-10% in Drosophila and less than 5% in humans