Genetics Set 1

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JACOB AND MONOD

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French researchers
Awarded Nobel Prize for their work on the lac operon

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LACTOSE METABOLISM IN E.COLI
Preferred Sugar for ATP is:
But can use____ if other is absent

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- Glucose
- Lactose (can be used but is less efficient)

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To utilize lactose as energy source E. coli needs:

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-Beta-galactosidase (cleaves lactose---> glucose and galactose)
-Permease (makes cell wall more permeable to lactose)
-Galactoside transacetylase (prevents toxic buildup of lactose products in cell??)

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E. Coli is most efficient to express enzymes when?

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- Lactose is Available
- And Glucose is unabailable

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SO..HOW DOES E. COLI Up-regulate gene expression for the 3 enzymes needed...?

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-When lactose is available
-When glucose is unavailable
-In a fast and coordinated way

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Lac Operon System in E. Coli

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Pic

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LAC OPERON IN E.COLI

Since Jacob and Monod’s initial discovery additional research has shown:

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-Three operator regions within promoter
-Repression x1,300 when all three functional (transcription at 1/1300 of level when lactose and cAMP/CAP present)
-Loss of individual operators reduces repression level

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HOW WAS LAC OPERON FUNCTION INVESTIGATED?

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-First gene regulatory system to be understood at molecular level
-Jacob and Monod inserted F’ plasmids into E. coli to create partial diploids (merozygotes)

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During the Lac Operon Function Investigation what did they find?

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-Inhibitor protein from I gene diffusible
-Functional I gene on one lac operon strand will repress transcription on both (provided both operators unmutated)
-Promoter/operator complex only controls genes downstream on same strand
-This is cis-dominance

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Eukaryote's Genome Structure and Organization Characteristics

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-Often multicellular, more complex
-May have multiple copies of genes
-Linear DNA complexed with histones
-Nuclear membrane separating DNA from cytoplasm
-Process primary RNA transcript
-mRNA has longer life in cytoplasm
-Always have monocistronic mRNA

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Gene Regulation in Eukaryotes

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Pic

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GENE REGULATION IN EUKARYOTES

Pre-transcriptional

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Gene methylation

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GENE REGULATION IN EUKARYOTES

Transcriptional

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-Hormones as activators
-Enhancers

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GENE REGULATION IN EUKARYOTES

Post-transcriptional

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Alternate RNA processing

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GENE REGULATION IN EUKARYOTES

Translational

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Masking mRNA

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GENE Methylation IN EUKARYOTES

Methyl group (CH3) added to

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cytosine in
5’-CG-3’ dinucleotides

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Eukaryotes:
Heavy methylation inactivates

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DNA

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Eukaryotes:
Inactivation reversible if

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methyl groups removed

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Eukaryotes:
Methylated DNA often accumulates

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Mutations

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During Gene Methlaytion of Eukaryotes you have high risks of?

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Permanent inactivation of rarely-transcribed genes

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DOSAGE COMPENSATION

One X chromosome in every somatic cell inactivated by ?

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methylation in female mammals

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DOSAGE COMPENSATION

Males and females have same number of

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active X-linked genes

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Inactivated genes found on the X chromosome in cats such as:

Tortoiseshell
Calico

Both are XbXo
But have different time frames when these genes are inactivated. When does this happen?

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-Calico: X chromosome inactivated early in embryonic development
-Tortoiseshell: X chromosome inactivated later in development

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TRANSCRIPTIONAL GENE REGULATION : HORMONES

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-Small molecules carried from endocrine cells to target cells
-Target cells contain receptor sites
-Most hormones effectors
-Possible modes of hormone action:
-Bind directly to enhancer or promoter DNA
-Bind to/activate effector
-Inactivate repressor
-Change chromatin structure

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GENE REGULATION BY STEROID HORMONES
Hormone non-polar

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can pass through cell membrane

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GENE REGULATION BY STEROID HORMONES

Can bind to where?

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Binds in cytoplasm to receptor protein (zinc finger motif)

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GENE REGULATION BY STEROID HORMONES
Receptor protein carries hormone into

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nucleus

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GENE REGULATION BY STEROID HORMONES

Zinc finger binds to

An initiates?

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HRE (hormone responsive element) DNA acceptor sequence in nucleus


Transcription

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GENE REGULATION BY STEROID HORMONES

Diagram

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Pic

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ZINC FINGER MOTIF

Picture

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-Proteins with zinc finger motif bind to DNA
-Polypeptide folded so Zn held between two cysteines and two histidines

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HRE for estrogen is an

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inverted repeat

5’ AGGTCANNNTGACCT 3’
3’ TCCAGTNNNACTGGA 5’

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TRANSCRIPTIONAL GENE REGULATION: ENHANCERS

Enhancer region can be found?

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upstream or downstream of promoter/structural gene

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TRANSCRIPTIONAL GENE REGULATION: ENHANCERS

Transcriptional activator binds to

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enhancer

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TRANSCRIPTIONAL GENE REGULATION: ENHANCERS

What does DNA do for initiation?

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DNA loops to form transcription initiation complex

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POST-TRANSCRIPTIONAL GENE REGULATION: ALTERNATE RNA SPLICING IN MOUSE ALPHA-AMYLASE

Picture

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pic

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TRANSLATIONAL GENE REGULATION : MASKED mRNA

Unfertilized sea urchin eggs store......

After fertilization proteins removed, and.....

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mRNA complexed with protein, inhibits translation



translation increases 50x