1. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E The Structure of the Genome Denaturation, Renaturation and Complexity

2. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 (T m ) is at half denatured T m increases with G-C content (%G + %C) A-T-rich sections melt before G-C-rich segments

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

4. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 10.16

5. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E DNA renaturation J. Marmur (Harvard, 1960) – first described –Slowly cool heat-denatured DNA –or drop temperature quickly to ~25°C below T m & incubate awhile –Complementary single-stranded DNAs can reassociate or reanneal Renaturation very useful –genome complexity: variety & copy number –hybridization: molecular identification

6. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E DNA renaturation Renaturation governed by –Ionic strength of the solution –Temperature –Time –DNA concentration –Size of the interacting molecules

7. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Complexity of viral & bacterial genomes SV40:5.4 x 10 3 bp T4:1.8 x 10 5 E. coli:4.5 x 10 6 –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

8. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 10.19

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

10. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 10.20

11. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Highly repetitive DNA present in at least 10 5 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

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

13. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Highly repetitive DNA: 3 kinds 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

14. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Highly repetitive DNA: 3 kinds 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.)

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

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

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

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

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

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