The Structure of the Genome

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

The Structure of the Genome

Denaturation, Renaturation and Complexity


DNA denaturation (melting)

• strands held together by weak, noncovalent bonds

• strands start to separate at specific temperature– Within a few degrees, process is complete– solution contains single stranded molecules – higher single strand absorbance @ 260 nm– hydrophobic base interactions reduced– bases absorb photons more efficiently

• Melting temperature (Tm) is at half denatured

• Tm increases with G-C content (%G + %C)

• A-T-rich sections melt before G-C-rich segments

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 10.15



DNA renaturation

• J. Marmur (Harvard, 1960) – first described– Slowly cool heat-denatured DNA

– or drop temperature quickly to ~25°C below Tm &

incubate awhile

– Complementary single-stranded DNAs can reassociate or reanneal

• Renaturation very useful – genome complexity: variety & copy number

– hybridization: molecular identification


DNA renaturation

• Renaturation governed by– Ionic strength of the solution

– Temperature

– Time

– DNA concentration

– Size of the interacting molecules


Complexity of viral & bacterial genomes

• SV40:5.4 x 103 bp T4:1.8 x 105 E. coli:4.5 x 106 – Force all DNAs through tiny orifice under high

– Random shear ~1000 bp

– Reanneal at same DNA concentration (mg/ml)

• smaller genome - faster renaturation – more copies of small genomes

– Increases chance of collision between complementary fragments

– Viral/bacterial genomes - symmetrical curve



Complexity of eukaryotic genome

• Various nucleotide sequences in eukaryotic DNA fragments are present at very different concentrations

• first indication that eukaryotic DNA has much more complex organization– Curves show 3 broad DNA sequence classes

– differ in copy number



Highly repetitive DNA

• present in at least 105 copies per genome

• ~10% of total vertebrate DNA • this fraction reanneals very fast• Usually short (a few 100 bp at

most)• Usually in Tandem: over & over

uninterrupted


Highly repetitive DNA: 3 kinds

• Satellite DNAs – 5 to 100’s bp long

– repeated vast number of times in tandem;

– form very large clusters of up to several million bp

– usually unique base composition

– “satellite” band in gradient centrifugation



• Minisatellite DNAs – ~12 - 100 bp long

– form clusters of up to 3000 repeats

– shorter stretches than satellites

– unstable copy number over generations

– locus length is highly variable in population

– even among family members

– polymorphic: used for DNA fingerprinting

– polymorphism implicated in cancer & diabetes



• Microsatellite DNAs – 1 - 5 bp long; present in small clusters of ~50-100

bp long– Scattered evenly throughout DNA – at least 30,000 different loci in human genome– extremely high mutation rate– ethnic polymorphism in human populations– African origin?

• African’s should have greater sequence variation

• Appears to be true in 60 microsatellites

– Involved in inherited diseases (FRAX, Hunt.)

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E HP Figure 1


Where are satellite sequences located?

• Mary Lou Pardue & Joseph Gall (Yale) – develop in situ hybridization– used to locate satellites on chromosomes– spread chrmosomes on slide– treat with hot salt solution to separate the strands– treat with labeled satellite DNA probe– Satellite DNAs in centromeric region, telomeres

• Fluorescent in situ hybridization (FISH) – – better resolution than with radiolabel– biotin probe; fluorescent avidin (binds biotin)– map specific sequences along DNA

Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 10.22a


Moderately repeated DNA

• 20 to 80% of total DNA, depending on organism

• Repeated within genome a few times to tens of thousands of times– distinct families– some code for known RNAs or proteins

• tRNAs, rRNAs, histone mRNAs– typically identical to one another – located in tandem array– RNAs & histones are needed in large amounts– Histones needed in such large amounts in early

development


Moderately repeated DNA

• Repeated DNAs that lack coding functions – represents bulk of moderately repetitive DNAs

– scattered (interspersed) not tandem

– SINEs (short interspersed elements) - usually <500 bp long; ex. in humans: Alu

– LINEs (long interspersed elements) - usually >1000 bp long; ex. in humans: L1

– Sequences of both vary greatly between species

– Functions unknown


Nonrepeated (single copy) DNA

• 70% of human DNA fragments of 1000 bp length

• Very slow to hybridize– Represent Mendelian genes– Contain code for virtually all proteins but histones

• Genes coding for polypeptides – Globins, actins, myosins, collagens, tubulins,

integrins, probably most other eukaryote proteins – Each member of multigene family is encoded by

different but related nonrepeated sequence

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The Structure of the Genome