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

1

First, conceptual basis of the connection between mutation

(Gene, or DNA change),and phenotype

Mutation

2

One gene one enzyme hypothesis. The first exciting insight into the function of genes

4

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

5

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

6

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

11

12

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

13

14

15

16

A single b.p. change in the hemoglobin gene leads to all the phenotypes of Sickle Cell Anemia

17

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

18

wild type Hemoglobin-S sickle cell

19

20

21

From gene to gene product function, from mutation to phenotype

22

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

23

24

Xeroderma pigmentosum

Bהמופיליה חוסר ב-

Factor IX

Simple (even spontaneous) changes, huge consequences – how to find them?

25

Factor IX(HemoB)

Gene for Factor IX(HemoB)

26

PhenotypicRescue:Introduce an isolated (cloned) gene by viral(engineered)Infection withHemo.B gene(encodes clotting Factor IX).

27

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

29

Regulatory Coding

30

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

32

33

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.)

34

RibosomeBinding site35

36

Animation ch-9 transl…nSteps

TRANSLATION/Shine-Dalgarno37

Shine-Delgarno

38

Example: Eukaryotic RNA's are spliced - one of several post transcriptional alterations

Post transcriptional modifications of the RNAs

39

Splicing site40

41

Polyaden.site 42

43

44

Post-translational modifications, processing, packaging and trafficking of proteins

45

46

47

Darwin’s Finches