molecular genetics first step: dna is the genetic material – dna, not protein of the...
Post on 15-Jan-2016
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Molecular Genetics
• First step: DNA is the genetic material
– DNA, NOT protein of the chromosomes/chromatin
– PROVEN WITH TRANSFORMATION AND TRANSDUCTION, THAT WE JUST MASTERED
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First, conceptual basis of the connection between mutation
(Gene, or DNA change),and phenotype
Mutation
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One gene one enzyme hypothesis. The first exciting insight into the function of genes
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Mutant Ornithine Citruline Arginine
arg-1
arg-2
arg-3
+ + +
- + +
- - +
Supplement
One gene one enzyme hypothesis. The first exciting insight into the function of genes
Precursor X Y ZGene1 Gene2 Gene3x x x
NOT like above, where the DNA must recombine and replace the endogenous copy, BUT where the DNA is extrachromosomal and persists as an episome (plasmid, F’, etc.)
In vitro
In bacteria - Introduce an isolated (cloned) gene by transformation
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Because many drug-resistant mutations are recessive, cloning by complementation is often feasible
xGenomic DNA library
Each mutant is transformed with the library
x
x
x
x
x
Mutation in this gene is responsible for the drug resistance phenotype
in mutant #1
Mutant #1
Complementation of the drug resistance phenotype
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Mutagenesis
Plate to select for phenotype of interest
Complementation groups
Complementation groups
First, we need to catalogue our mutants to complementation groups (Total of 138 mutants were isolated in the original CTF screen).
x xMate
xx
Diploid still shows CTF phenotype
Mutant#1 Mutant#2
Mutant#1 and Mutant#2are mutated in the same gene
Same complementation group
Diploid
x xMate
Mutant#3 Mutant#4 Diploid
xx
Diploid dont show CTF phenotype
Mutant#3 and Mutant#4are mutated in different genes
Different complementation groups
Complementation groups
Chromosome Transmission Fidelity (Chromosome Transmission Fidelity (ctfctf) Mutants) Mutants
Total # of mutant isolates: 138
19 Complementation Groups 10137 Undesignated (single member) 37
Estimated total # of genes represented ~ 50 ctf genes
Complementation groups
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One gene – one gene product (here a protein)
gtg cat ctg act cct gag gag
From gene to gene product function, from mutation to phenotype
Pe
ptid
e b
on
d
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A single b.p. change in the hemoglobin gene leads to all the phenotypes of Sickle Cell Anemia
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gtg cat ctg act cct gag gag
gtg cat ctg act cct g t g gag
MUTANT –Sickle cell anemia
gtg cat ctg act cct g t g gagFrom gene to gene product function, from mutation to phenotype
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wild type Hemoglobin-S sickle cell
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From gene to gene product function, from mutation to phenotype
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gtg cat ctg act cct gag gag
gtg cat ctg act cct gug gag
MUTANT –Sickle cell anemia אנמיה חרמשית
gtg cat ctg act cct gug gag
gene ->gene product function, mutation to phenotype, In bacterial enzyme, . . . or in gene for complex Human syndrome
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Xeroderma pigmentosum
Bהמופיליה חוסר ב-
Factor IX
Simple (even spontaneous) changes, huge consequences – how to find them?
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Factor IX(HemoB)
Gene for Factor IX(HemoB)
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PhenotypicRescue:Introduce an isolated (cloned) gene by viral(engineered)Infection withHemo.B gene(encodes clotting Factor IX).
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Evolution from “1 gene, 1 protein” to…
• 1 gene, 1 protein– Haemoglobin
• 1 gene, 1 polypeptide
• 1 gene, 1 gene product– tRNAs, rRNAs, snRNAs, miRNAs, …
Additionally – next: (e.g. for proteins)- many sites are susceptible to mutations outside of coding region 28
Genes contain much more information than the structural region
(or "coding region for proteins") of the gene product.
They include information directing the proper timing and placement of the
expression of the gene product
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Regulatory Coding
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CodingPromoter31
Example: Prokaryotes and Eukaryotes have highly conserved necessary promoter elements specific distances from transcription start, enhancers which act from varying distances. Specifically: many eukaryotic genes have a "TATA box" 30 bases from the start site and a CCAAT box 70-80 base pairs from the start site in their promoter.
Transcriptional controls on initiation, elongation, termination of RNA
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Translational controls on initiation, elongation, termination, localization
Example: Here too, a large degree of control is exercised at initiation, and the "non-translated leader" portion of RNA includes control information.
Starts are always at Met (AUG), but specifically, in Prokaryotes: true start sites are preceded by a consensus sequence know as a ribosome binding site, or Shine - Dalgarno sequence - AGGAGGU (prok.)
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RibosomeBinding site35
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Animation ch-9 transl…nSteps
TRANSLATION/Shine-Dalgarno37
Shine-Delgarno
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Example: Eukaryotic RNA's are spliced - one of several post transcriptional alterations
Post transcriptional modifications of the RNAs
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Splicing site40
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Polyaden.site 42
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Post-translational modifications, processing, packaging and trafficking of proteins
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Darwin’s Finches