Genome organization 1.ppt
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Transcript of Genome organization 1.ppt
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