MOLECULAR GENETICS LASS SESSIONS: 1. DNA, Genes, Chromatin 2. DNA Replication, Mutation, Repair 3. RNA Structure and Transcription 4. Eukaryotic Transcriptional Regulation 5. CLASS DISCUSSION – GENETIC DISEASES 6. RNA Processing 7. Protein Synthesis and the Genetic Code 8. Protein Synthesis and Protein Processin 9. CLASS DISCUSSION – GENETIC DISEASES 10. DNA Cloning and Isolating Genes
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MOLECULAR GENETICS CLASS SESSIONS: 1. DNA, Genes, Chromatin 2. DNA Replication, Mutation, Repair 3. RNA Structure and Transcription 4. Eukaryotic Transcriptional.
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MOLECULAR GENETICS
CLASS SESSIONS: 1. DNA, Genes, Chromatin 2. DNA Replication, Mutation, Repair 3. RNA Structure and Transcription 4. Eukaryotic Transcriptional Regulation 5. CLASS DISCUSSION – GENETIC DISEASES 6. RNA Processing 7. Protein Synthesis and the Genetic Code 8. Protein Synthesis and Protein Processing 9. CLASS DISCUSSION – GENETIC DISEASES10. DNA Cloning and Isolating Genes
a). Evidence that DNA is the genetic informationi). DNA transformation – know this termii). Transgenic experiments – know this processiii). Mutation alters phenotype – be able to define
genotype and phenotypeb). Structure of DNA
i). Structure of the bases, nucleosides, and nucleotidesii). Structure of the DNA double helixiii). Complementarity of the DNA strands
c). Chemistry of DNAi). Forces contributing to the stability of the double helixii). Denaturation of DNA
Chargaff’s rule: The content of A equals the content of T, and the content of G equals the content of C in double-stranded DNA from any species
Hydrogen bonding of the bases
Double-stranded DNA
Major groove
Minor groove
5’ 3’
5’ 3’3’ 5’
“B” DNA
Chemistry of DNA
Forces affecting the stability of the DNA double helix
• hydrophobic interactions - stabilize - hydrophobic inside and hydrophilic outside
• stacking interactions - stabilize - relatively weak but additive van der Waals forces
• hydrogen bonding - stabilize - relatively weak but additive and facilitates stacking
• electrostatic interactions - destabilize - contributed primarily by the (negative) phosphates - affect intrastrand and interstrand interactions - repulsion can be neutralized with positive charges
(e.g., positively charged Na+ ions or proteins)
Stacking interactions
Charge repulsion
Ch
arg
e re
pu
lsio
n
Model of double-stranded DNA showing three base pairs
Cot1/2 = 1 / k2 k2 = second-order rate constant Co = DNA concentration (initial) t1/2 = time for half reaction of each
component or fraction
50
100
0
% D
NA
re
ass
oc
iate
d
I I I I I I I I I
log Cot
fast (repeated)
intermediate (repeated)
slow (single-copy)
Kinetic fractions: fast intermediate slow
Cot1/2
Cot1/2
high k2
106 copies per genome ofa “low complexity” sequence
of e.g. 300 base pairs
1 copy per genome ofa “high complexity” sequence
of e.g. 300 x 106 base pairs
low k2
Type of DNA % of Genome Features
Single-copy (unique) ~75% Includes most genes 1
Repetitive Interspersed ~15% Interspersed throughout genome between
and within genes; includes Alu sequences 2
and VNTRs or mini (micro) satellites Satellite (tandem) ~10% Highly repeated, low complexity sequences
usually located in centromeres and telomeres
2 Alu sequences are about 300 bp in length and are repeated about 300,000 times in the genome. They can be found adjacent to or within genes in introns or nontranslated regions.
1 Some genes are repeated a few times to thousands-fold and thus would be in the repetitive DNA fraction
50
100
0
I I I I I I I I I
fast ~10%
intermediate ~15%
slow (single-copy) ~75%
Classes of repetitive DNA
Interspersed (dispersed) repeats (e.g., Alu sequences)
TTAGGGTTAGGGTTAGGGTTAGGG
Tandem repeats (e.g., microsatellites)
GCTGAGG GCTGAGGGCTGAGG
viruses
plasmids
bacteria
fungi
plants
algae
insects
mollusks
reptiles
birds
mammals
Genome sizes in nucleotide pairs (base-pairs)
104 108105 106 107 10111010109
The size of the humangenome is ~ 3 X 109 bp;almost all of its complexityis in single-copy DNA.
The human genome is thoughtto contain ~30,000 to 40,000 genes.
bony fish
amphibians
5’ 3’
promoter region
exons (filled and unfilled boxed regions)
introns (between exons)
transcribed region
translated region
mRNA structure
+1
Gene structure
The (exon-intron-exon)n structure of various genes
-globin
HGPRT(HPRT)
total = 1,660 bp; exons = 990 bp
histone
factor VIII
total = 400 bp; exon = 400 bp
total = 42,830 bp; exons = 1263 bp
total = ~186,000 bp; exons = ~9,000 bp
Properties of the human genome
Nuclear genome
• the haploid human genome has ~3 X 109 bp of DNA• single-copy DNA comprises ~75% of the human genome• the human genome contains ~30,000 to 40,000 genes• most genes are single-copy in the haploid genome• genes are composed of from 1 to >75 exons• genes vary in length from <100 to >2,300,000 bp• Alu sequences are present throughout the genome
Mitochondrial genome
• circular genome of ~17,000 bp• contains <40 genes
From Nussbaum, R.L. et al. "Thompson & Thompson Genetics in Medicine," 6th edition (Revised Reprint), Saunders, 2004.
LDL receptor gene
Alu repeats present within introns
Alu repeats in exons
4
4
4
5
5
5 6
6
6
Alu Alu
AluAlu
X
4 6Alu
unequalcrossing over
one product has a deleted exon 5(the other product is not shown)
Chromatin structure
EM of chromatin shows presence ofnucleosomes as “beads on a string”
Nucleosome structure
Nucleosome core (left)• 146 bp DNA; 1 3/4 turns of DNA• DNA is negatively supercoiled• two each: H2A, H2B, H3, H4 (histone octomer)
Nucleosome (right)• ~200 bp DNA; 2 turns of DNA plus spacer• also includes H1 histone
Histones (H1, H2A, H2B, H3, H4)• small proteins• arginine or lysine rich: positively charged• interact with negatively charged DNA• can be extensively modified - modifications in
general make them less positively chargedPhosphorylationPoly(ADP) ribosylationMethylationAcetylation
Hypoacetylation by histone deacetylase (facilitated by Rb)
“tight” nucleosomes assoc with transcriptional repression
Hyperacetylation by histone acetylase (facilitated by TFs)“loose” nucleosomes assoc with transcriptional activation
Nucleofilament structure
Condensation and decondensation of a chromosome in the cell cycle
Telomeres and aging
Metaphase chromosome
centromere
telomere telomere
telomere structure
young
senescent
Telomeres are protective“caps” on chromosomeends consisting of short5-8 bp tandemly repeatedGC-rich DNA sequences,that prevent chromosomesfrom fusing and causingkaryotypic rearrangements.
(TTAGGG)many
(TTAGGG)few
• telomerase (an enzyme) is required to maintain telomere length in germline cells
• most differentiated somatic cells have decreased levels of telomerase and therefore their chromosomes shorten with each cell division
<1 to >12 kb
Class Assignment (for discussion on Sept 9th)
Botchkina GI, et al.“Noninvasive detection of prostate cancer by quantitative analysis of telomerase activity.”Clin Cancer Res. May 1;11(9):3243-3249, 2005