sex determination F2009 - esf.edu · the Lac operon’s promoter, therefore increases ... trp operon in textbook) 4 ... sex determination F2009.ppt Author:

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Gene expression

• Review sessions wt Jackie: 3-5PM Thursdays, room 8 Illick

• Previous: www.esf.edu/efb/powell/Gen_L8_3.pdf www.esf.edu/efb/powell/Gen_L8_6.pdf

www.esf.edu/efb/powell/Gen_L9_3.pdfwww.esf.edu/efb/powell/Gen_L9_6.pdf

www.esf.edu/efb/powell/Gen_L10_3.pdfwww.esf.edu/efb/powell/Gen_L10_6.pdf

Next Tuesday:www.esf.edu/efb/powell/Gen_L11_3.pdfwww.esf.edu/efb/powell/Gen_L11_6.pdf

Lac operon in E. coli

• What if glucose and lactose are present?– Note: takes less resources to use

glucose• Lac operon is not turned on• Catabolite Repression

– A product of glucose catabolism inhibitsthe formation cAMP (cyclic adenosinemonophosphate)

– Increase glucose = decrease cAMP– Decrease glucose = increase cAMP

Lac operon in E. coli

• How does cAMP effect the Lac operon?• cAMP binds to a protein called CAP (catabolite

activator protein)• cAMP/CAP increases binding of RNA polymerase to

the Lac operon’s promoter, therefore increasesexpression

• cAMP/CAP - example of positive control (required foractivation)

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Catabolite repression of Lac operon(Fig 17-8 in your textbook)

Glucose present, no lactose

No lac mRNA

(Low cAMP)

Glucose present, lactose present

little lac mRNA transcribed

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No glucose present, lactose present

Abundant lac mRNA

Comparison of negative & positivecontrol

• negative control– Repressor protein - prevents expression

• Positive control– Activator protein - enhances expression

Inducible vs. repressible systems

• Lac operon is an example of aninducible system (gene is normally off)

• Repressible systems work in a similarway, but their expression is normally on(example: trp operon in textbook)

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Gene regulation

• Simple to complex• Human genes average 5 cis-acting

elements which bind as many trans-acting regulatory proteins. Some geneshave up to 20.

Regulatory cascades

Gene Aon off

Regulatory cascades

Gene Aon off

Gene(s)

gene(s)

gene(s)

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Regulatory cascades

Gene Aon off

gene(s)

gene(s)

gene(s)

Developmental Gene Regulation

• Governed by regulatory cascades• Master switches

– Genes that commit development down onepath or the other

– Example: sex determination

Gene regulation in Eukaryotes

• So far looked at model system– Prokaryotic lac operon

• Eukaryotic systems similar, but much morecomplex

• Eukaryotes have 3 RNA polymerases– RNA polymerase II transcribes all mRNA

• RNA polymerase must bind to the promoterfor transcription to initiate– Many proteins that assist in binding

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Eukaryotic promoters

• Have additional cis-acting elements• Enhancers - enhance RNA pol. binding• Silencers - inhibit RNA pol. binding• Both act like a “volume switch” turning

expression up or down (not just on or off)• Often a given enhancer or silencer activity is

tissue specific• These elements can be located close or far,

upstream or downstream from core promoter

Core promoter & proximal elements

• Core promoter - transcription start site, 30bases upstream to a TATA box– Where the RNA polymerase binds to DNA

• Promoter proximal elements - 100-200 basesupstream from transcription start site– CCAAT box– GC-rich region

• These are the minimal promoter sequencesneeded for transcription initiation

Core promoter & proximal elements

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Complexity of a eukaryotic promoter

Enhancers help control physiology &development

• In eukaryotic organisms, cells in onepart of the body can control geneexpression in cells of other parts of thebody (global gene control)

• Example: the human endocrine system– Produce steroid hormones– Genes (cell#1) > enzymes > hormones >

travel through blood > receptor protein(cell#2) > binds to enhancer on gene

peptide hormones

Endocrine organs:

Sex determinationFight/flightMetabolic rate Sugar regulationGrowthEtc.

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Hormone responsive enhancers(inducible system, positive control)

Development & sex determination

• Opportunities forgene regulation

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Development of male vs. female

Sex determination

Fruit fly Human

XX

XY

female female

male maleAppear the same

aneuploids

XXY

XO

female male

femalemale

Shows sex isdetermined bydifferentmechanisms

Review of gene expression

pre-mRNA

transcription by RNA polymerase

RNA processingmessenger RNA (mRNA)

translation

polypeptide (protein) post-translationalprocessing

DNA

genetranscriptional unitPromoter

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Drosophila (fruit fly)

• Basis of sex determination: X:A ratio of a gene– X = X chromosome copy– A = autosome (non-sex chromosome) copy

• Female: X:A ratio of one• Male: X:A ratio of 0.5• How can the developing embryo count the

chromosome copies of genes?• Model: what is thought to happen from current

evidence

Drosophila• 2 genes encode for sex determining transcription

factors (TF)– Transcription factor helps to bind RNA polymerase to

DNA• Autosomal copy - has no functional DNA binding

domain• X chromosome copy - has a DNA binding domain• These TFs will form dimers in the cell

– Dimers - combination of two polypeptides– Must have two functional polypeptides to bind to DNA

transcription factors (TF)Female: 1:1 ratio Male: 1:2 ratio

X chromosomes X chromosomesautosomes autosomes

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active Inactive



active Inactive

Much more inactive than active

Sxl gene (female specific splicing)

Female: Male:

PEPLPEPL

intron

Two promotersLate & Early

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Female: Male:

PEPLPEPL

Many active Tfsturn on early promoter


Female: Male:

PEPLPEPL


Splicing ofmRNA


Female: Male:

PEPLPEPL


Splicing ofmRNA

Sxlprotein

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Female: Male:

PEPLPEPL


Splicing ofmRNA

Sxlprotein

Feedback to maintain splicingwhen late promoter is on.


Female: Male:

PEPLPEPL


Splicing ofmRNA

Sxlprotein


Not enough active Tfsto turn on earlypromoter


Female: Male:

PEPLPEPL


Splicing ofmRNA

Sxlprotein



Unspliced mRNA

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Female: Male:

PEPLPEPL


Splicing ofmRNA

Sxlprotein



Unspliced mRNA

UGA stop codon preventstranslation of Sxl protein

Drosophila

• Therefore XXY = female, X0 = male– XXY ratio is 1:1– X0 ratio is 1:2 (or 0.5)

Sxl master control switch

• Controls its own splicing and also starts acascade of gene expression

• tra gene - transformer– Female (active Sxl) - forms active tra RNA binding

protein– Male (no Sxl) - tra inactive

• Tra controls the RNA splicing of the dsx gene– Two forms of protein product

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dsx gene (negative control, repressible system)

• Female dsx protein– Represses male-specific structural genes

(many)• Male dsx protein

– Represses female-specific structural genes(many)

CascadeFemale: X:A = 1 Male: X:A = 0.5

Sxl (on) Sxl (off)


Sxl (on) Sxl (off)

tra (on) tra (off)

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Sxl (on) Sxl (off)

tra (on) tra (off)

dsx mRNAfemale splice

dsx mRNAmale splice


Sxl (on) Sxl (off)

tra (on) tra (off)


dsx mRNAmale splice

dsx-F protein dsx-M protein


Sxl (on) Sxl (off)

tra (on) tra (off)


dsx mRNAmale splice

dsx-F protein dsx-M proteinRepression ofmale-specific genes

Repression offemale-specific genes

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Drosophila• Null mutation of Sxl?• Male• Null mutation of Tra?• Male• Therefore, male is default pathway• Null mutation of dsx• Characteristics of both male and female

– No suppression of genes

Female: X:A = 1 Male: X:A = 0.5

Sxl (on) Sxl (off)

tra (on) tra (off)


dsx mRNAmale splice

dsx-F protein dsx-M protein

Repression ofmale-specific genes

Repression offemale-specific genes

Sex determination in mammals

Comparison: Drosophila MammalsX:A ratio


Comparison: Drosophila MammalsX:A ratio Y chromosome

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Comparison: Drosophila MammalsX:A ratio Y chromosome

autonomous non-autonomous every cell global


Comparison: Drosophila MammalsX:A ratio Y chromosomeautonomous non-autonomousevery cell global

mosaics: male and female male or female


Comparison: Drosophila MammalsX:A ratio Y chromosomeautonomous non-autonomousevery cell global

mosaics: male and female male or female

Why? - secondary sexual phenotypes are driven by the presence or absence of the testes

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Human development

• Gonads form within first two months fromconception

• If Y chromosome is present, then testes form• If no Y, then ovaries form• testes secrete testosterone (steroid hormone)• Testosterone binds to androgen-receptors in

all the cells of the body (global)• These act as transcription factors which

enhance male specific gene expression

Human development

• If no testosterone, androgen-receptors are notactivated, therefore female genes are expressed bydefault

• What would happen if a null mutation occurred in theandrogen-receptor gene?

• Testicular feminization (Tfm) syndrome– XY female– Female secondary characteristics, but has testes

instead of ovaries (demo. of global affect)• Several types of mutations that effect sex

determination

(Caster)

Human development

• Testosterone - developmentally specific for sometraits– developmental timing is important

• Female + testosterone ≠ male– Facial hair, increased muscle mass, more

aggressive• What drives the gonads to form testes?• SRY gene (old name TDF: testes determining factor)

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SRY gene

• Sex-determining region of Y chromosome• Identified by studying sex reversal

– XX males• Caused by translocation of portion of Y

chromosome onto an X chromosome• Proved SRY gene is the sex-determining

master switch by cloning into XX mice

SRY gene

PCR markers (Zfy-1 = Y chromosome)

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sex determination F2009 - esf.edu · the Lac operon’s promoter, therefore increases ... trp operon in textbook) 4 ... sex determination F2009.ppt Author: