Molecular Genetics Prof. Haim Cohen, Haim.Cohen@biu.ac.il 8383, Nano Bldg Room 804A Today we will discuss about DNA and the concept of mutations, complementation and complementation groups

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

Mutation First, conceptual basis of the connection between mutation (Gene, or DNA change), and phenotype

Neurospora crassa

Neurospora crassa

גיבורים

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

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

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

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

x x x Gene1 Gene2 Gene3 Precursor X Y Z One gene one enzyme hypothesis. The first exciting insight into the function of genes Mutant Ornithine Citruline Arginine arg-1 arg-2 arg-3 + + + - + + - - + Supplement x x x Gene1 Gene2 Gene3 Precursor X Y Z Precursor --------ornithine----------citruline----------arginine ARG1 ARG2 ARG3

In bacteria - Introduce an isolated (cloned) gene by transformation In vitro 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.)

Complementation assays r 1 X r2 ?

Complementation assays r 1 X r2 ? Wild-type (WT)

gtg cat ctg act cct gag gag One gene – one gene product (here a protein) From gene to gene product function, from mutation to phenotype gtg cat ctg act cct gag gag Peptide bond

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

gtg cat ctg act cct gag gag From gene to gene product function, from mutation to phenotype gtg cat ctg act cct g t g gag MUTANT –Sickle cell anemia gtg cat ctg act cct g t g gag

wild type Hemoglobin-S sickle cell

From gene to gene product function, from mutation to phenotype

gtg cat ctg act cct gag gag gene ->gene product function, mutation to phenotype, In bacterial enzyme, . . . or in gene for complex Human syndrome gtg cat ctg act cct gug gag MUTANT –Sickle cell anemia אנמיה חרמשית gtg cat ctg act cct gug gag

Xeroderma pigmentosum

המופיליה B חוסר ב- Factor IX חוסר ב- Factor IX Simple (even spontaneous) changes, huge consequences – how to find them?

Gene for Factor IX (HemoB) Factor IX (HemoB)

Phenotypic Rescue: Introduce an isolated (cloned) gene by viral (engineered) Infection with Hemo.B gene (encodes clotting Factor IX).

Evolution from “1 gene, 1 protein” to… 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

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

Regulatory Coding

Coding Promoter

Transcriptional controls on initiation, elongation, termination of RNA 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.

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.)

Ribosome Binding site

Animation ch-9 transl…nSteps TRANSLATION/Shine-Dalgarno

Shine-Delgarno

Example: Eukaryotic RNA's are spliced - one of several post Post transcriptional modifications of the RNAs Example: Eukaryotic RNA's are spliced - one of several post transcriptional alterations

Splicing site

Polyaden. site

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

Mutation are the driving force of nature