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Lecture 1

Sex Determination.


What is the two-fold cost of sex?

Only half of the offspring are able to bear children. Two individuals are needed to reproduce.


Describe the origin of asexual species.

Usually of recent evolutionary origin.


Give 2 examples of asexual organisms.

Aphids. Desert Grassland Whiptail.


What are the benefits of sex?

Increase of fitness through sexual selection and biparental care. Increased rate of evolution via mixing of alleles and sexual selection. Increased genetic diversity. Reduced sibling competition. Having homo/heterozygotes reduces the effect of cyclical environment changes.


Describe the sex chromosome configurations of Drosophila.

3 autosomes in one haploid set. Males XY; Females XX.


What is the function of Y in sex determination in Drosophila?

Not involved.


What is the initial step in Drosophila sex determination?

Evaluation of the X:A ratio: 1:2 in males, 2:2 (1:1) in females.


Describe the female determining pathway of Drosophila.

X:A = 1; Sex-lethal gene on (Sxl); Activate tra gene; dsx RNA spliced to female; Repression of male structural genes.


Describe the male determining pathway of Drosophila.

X:A =0.5; Sxl off; tra off; dsx default (male); DSX-M protein formed; Repression of female genes.


How are chromosomes counted to determine the X:A ratio?

X subunits (numerator) combine with other X and autosome subunits (denominator) to form dimers. XX homodimers are active, XA are inactive heterodimers and AA homodimers are also inactive. Sex pathway depends on concentration of active homodimers.


What is the role of Numerator-Denominator heterodimers?

Helix-loop-helix transcription factors.


Describe the Sxl switch.

In females, high conc of XX activates transcription at P(E) (early promoter) of Sxl; Early type SXL-protein forms; Early SXL blocks male Sxl exon when the P(L) (late promoter) transcribes the gene; Late female SXL-protein forms. The Sxl prtotein loop acts as memory to maintain female splicing pattern. In males, P(E) is not activated; Exon not blocked; Sxl transcribed by P(L) with all exons.


What effect would a null mutation in Sxl/tra/sis-b (numerator gene) have on the final phenotype?

Male produced.


How do gymnadomorphs arise?

Null mutation in dsx gives male + female features due to lack of repression of either. Every cell determines own sex, mosaicism possible; Feminisation of brain structures can change sexual orientation - expression of female specific tra in olfactory bulbs may cause recognition of opposite pheromones.


Describe the sex chromosome configurations in C. elegans.

No Y. Hermaphroditic XX; Male XO. Primary switch involves X:A ratio.


Describe the male determining pathway of C. elegans.

1:2 ratio; XOL-1 gene activated; Represses sdc (sex determination and dosage compensation) gene; No SDC-1/-2/-3 protein; Her-1 activity not repressed; Cascade activates; Repression tra-1; Male phenotype.


Describe the hermaphroditic determining pathway of C. elegans.

2:2 ratio; XOL-1 off; sdc on; SDC-1/-2/-3 made; Her-1 repressed; Cascade; Tra-1 activated; Female phenotype.


How is sex determined in mammals?

Pathway triggered by a primary genetic switch. Secondary sex determined by hormones and not cell-autonomous responses.


Describe the differentiation of gonads in mammals.

Female and male gonads stem from an indifferent gonad at ~7 weeks. If Y present - Mullerian duct degenerates. Wolffian duct forms Vas deferens. If Y absent - Wolffian duct degenerates. Mullerian duct forms fellopian tube.


What is the evidence for the Y chromosome being dominantly sex-determining in mammals?

Polyploid genotypes with multiple Y are all male. This may explain the skewed male:female ratio in populations.


Describe the structure of the Y chromosome.

60Mb compared to 160 of X. 26Mb of euchromatin.


Describe the genetic content involved in sex determination, of the Y chromosome.

SRY - Sex-determining region 1. Consists of TDF - Testis Determining Factor - DNA binding protein.


What is the Function of SRY?

Together with SF1, acts on an enhancer of Sox9 to promote differentiation of Sertoli cells from their precursors.


Describe the hypothesis proposed for X/Y differentiation.

Y accumulated TDF. Y accumulated male-advantage genes. Recombination between X and Y suppressed. Y mutation/deletionshortened it.


Describe the recombinationpattern of the Y chromosome.

Only 1 recombination event. At the Pseudoautosomal Region 1. Recombination may have lead to TDF going to Y.]


How can the presence of TDF on Y be tested?

Deletion mapping. Recombination doesn't occur everywhere so linkage mapping isn't possible.


How do XX males arise?

80% of XX males carry SRY. Female mice injected with 14Kb DNA (SRY) => Normal male development. Suggests SRY is a switch for male determining genes which are on X-chromosomes. 20% XX males arise from a GOF mutation downstream of the pathway.


How can XY females have a normally functioning SRY?

75% of XY females carry a LOF mutation downstream of the pathway.


How does SD complexity differ from invertebrates to vertebrates?

Vertebrate SD is a web of interregulating factors and not a straightforward cascade like in Drosophila.


Describe the sex chromosome configurations of 3 different examples of Rodents.

Japanese Spinous Country Rat: Female - XO; Male - XO (No SRY). Mole-vole: Female - XX; Male - XX. Creeping vole: Female - XO; Male - XY.


Describe the sex chromosome configuration of birds.

Female - ZW; Male - ZZ. W is mostly heterochromatic, like mammalian Y.


What is the basis of sex determination in birds?

Same downstream proteins as mammals. Dosage based. DMRT1 - a Z-linked gene is required for male development. Feminisation is caused by RNAi being activated by dicer enzyme cleaving mRNA.


Describe the basis of sex determination in reptiles.

Regulated by environmental conditions: Temp (1-4 degree margin)/Day length/Crowding. Post Conception Determination. Thermosensitive transcriptional regulation of aromatase gene. Aromatase cleaves androgens into oestrogens (feminisation).


Describe the function of DMRT1 homologues in humans, chickens and aligators.

Chr9 in humans - deleted in XY femalesl; Z-linked in chickens - Male development; Upregulated at male temperatures in aligators.


How is sex determination of Medaka fish different from related species?

XX/XY system observed. X and Y morphologically undistinguishable. Y contains unique 250Kb sequence inherited from an autosome - DMRT1 gene. Evolutionarily novel mechanism.


Describe the basis of sex determination in the platypus and it's evolutionary origin.

5X and 5Y chromosomes line up at meiosis. Homologous to mammalian X and bird ZW. Suggests mammalian XY may have evolved from ancestral ZW system.


Lecture 2

Dosage Compensation.


What are the main regulatory issues in sex determination?

Embryo must distinguish between karyotypes to initiate proper differentiation. Non-sex-specific genes must be dosed equally in each sex.


Give examples of whole chromosome abnormalities leading to incorrect dosage.

Aneuploidies - Trisomy: Most lethal except 13, 18 and 21.


Give examples of single gene abnormalities leading to incorrect dosage.

Hereditary Haemorrhagic Teleangiectasia - Bleeding spots due to blood vessel malformation. Caused by haploinsufficiency of endothelial cell surface receptors which disrupts TGF-Beta signalling used for proliferation.


Give 3 examples of dosage compensation.

Mammals - X inactivated in females; Drosophila - X double transcribed in males; C. elegans - XX transcription halved in hermaphrodites.


Describe the dosage compensation pathway in male Drosophila.

Male-specific lethal genes form proteins (MSL1/MSL2/MSL3/MLE/MOF (Histone acetyl transferase) and mRNAs (roX1/roX2). These components form multisubunit compensasomes which attach uniformly to the X chromosome causing increased gene expression via the action of histone-acetyl transferase.


Describe the dosage compensation pathway in female Drosophila.

Sxl suppresses msl-2 translation by binding to the 3'UTR which means that females do not express MSL2 protein. No compensasome forms causing normal transcription of both Xs.


Describe the dosage compensation pathway in male C. elegans.

1X:2A causes activation of xol-1 (XO-lethal gene 1). Xol-1 negatively regulates 3 sdc genes (sdc-2/sdc-3) which negatively regulate her-1 and dumpy genes (DPY-26/DPY-27/DPY-28/DPY-30). Without sdc genes the X chromosomes cannot be hypotranscribed.


Describe the dosage compensation pathway in female C.elegans.

2X:2A represses xol-1 which means that sdc can be transcribed. Her-1 and dumpy genes are repressed causing formation of the hypotranscribing complex which reduces gene expression in X chromosomes/


Describe the dosage compensation pathway in female humans.

One X of somatic cells is inactivated/heterochromatinised forming a Barr body via a process called lyonization. The rule of lyonization is that all but one X chromosome is inactivated (multiple Barr bodies per cell in XXXX females). Choice of chromosome is random but maintained in all daughter cells of the initial cell.


What are the problems faced by lyonisation?

Process must be initiated, which requires a counting mechanism; X must be chosen; Inactivation must be spread; Inactivation must be maintained.


Describe how X inactivation is regulated during reproduction.

Barr body reactivated before oogenesis; X chromosomes distributed into gametes; Egg fuses with sperm; If sperm also contains X - either paternal or maternal X is inactivated; All descentant cells carry the same inactivated X.


How may mosaicism occur during X inactivation? Give an example of Xi mosaicism.

If the zygote divides before inactivation and each cell has a different X inactivated. Calico cat.


What 3 DNA-modifying processes contribute to X inactivation?

Promoter methylation; Concentration of Histone macroH2A1 instead of H2A histones; Histone H4 hypoacetylation.


What is XIC?

X inactivation centre.


How was XIC mapped?

By translocation of autosomal DNA onto X until X could no longer inactivate (translocation in XIC).


Describe the function of XIC.

Part of the counting mechanism of X inactivation. 2 must be present, each on a different copy of X. Number of XICs is counted and only one allowed.


What are XISTs?

Xi-Specific Transcripts. Genes of XIC.


What is the function of XIST?

Causes X inactivation.


Describe the process by which XISTs inactivate X.

Transcript blocked from association with XIST gene to prevent auto-inhibition. XIST transcript stabilises and associates with the gene. Transcript RNA coats entire surface of X. Inactive state established. Condensation of chromatin (H3/H4 acetylation and macroH2A1 recruitment).


What evidence is there to support the role of XIST in inactivation of X?

XIST expression high before inactivation - causes inactivation and isn't a consequence. Skewed X inactivation in humans stems from a mutation in XIST promoter. XIST KO in mice causes inability to inactivate X. Introduction into mice as a transgene on chr.12 causes inactivation of chr.12.


Which topics of X inactivation are still unclear?

Counting; Heterochromatinisation; Silencing; Maintenance and escape of some Xi genes.


Describe the conservation patterns of X chromosomes.

High conservation within therian mammals (all but monotremes) - Synteny. Translocations onto autosomes cause imbalance of product hence are strongly selected against (maintain conservation).


What is Turner Syndrome?

XO female (45X). 99% mortality in utero.


What is the phenotype of Turner Syndrome?

Short; Glucose intolerant; Webbed neck; Ovarian failure before puberty.


What does Turner Syndrome suggest about X inactivation?

Not Xi genes are inactivated since having 1X in Turner Syndrome shows clear haploinsufficiency. Some genes ar X-Y homologous since no turner syndrome in males (1X) (SHOX gene of PAR1 - lack causes short stature.


Mice with XO show no abnormalities. Give 2 explanations.

Doses in males, females and XO females are equal; Toleration of dosage imbalance exists.


Give a human example of toleration of dosage imbalance.

STS (Steroid Sulphatase) gene is X linked but has no Y homology.


Why are some bird/butterfly genes of ZZ/ZW not compensated? Give an example.

May be sex-specific genes. DMRT1 causes feminisation when a single copy is present, hence females are hemizygous (ZW).


What are the common features of dosage compensation genes in all animals?

All modulate transctiption by changing chromatin conformation via the 3 processes. Drosophila and mammals both invoke non-translated RNAs (rox-1/rox-2 and XIST respectively).


How does gene expression of X in mammals compare to autosomes?

1X = 2A.


Why is gene expression upregulated in mammalian X?

Y degenerated therefore X needed to upregulate to compensate.


How are X upregulation and X activation associated?

X upregulation in males caused evolution of X inactivation in females to compensate for the increased gene expression.


Lecture 3

Mitochondrial DNA and the Y Chromosome.


What is the fate of mitochondria in fertilisation?

50-75 mitochondria of the sperm cell are eliminated with the entire midpiece. Maternal 10^5-10^6


What are the functions of mitochondria?

Oxidative phosphorylation. Fatty acid oxidation. Citric acid cycle.


Describe the mitochondrial genome.

Circular. Heavy H-strand on the outside. Light L-strand on the inside. 13 protein coding genes. 2 rRNA and 22 tRNA genes. 16,569bp. Some coding regions overlap. Polycistronic


What is the D-loop?

Only non-coding part of the mt genome. Contains 2 promoters. 7% of the genome.


What's interesting about synthesis and transcription of H and L strands?

Synthesis and transcription act in opposite directions.


Why can't mitochondria live outside of prokaryotic cells?

Mt genome isn't enough to sustain a mitochondrion. 85% of components are from nuclear genes including RNA polymerase and mtDNA polymerase.


What is the evidence for endosymbiosis of mitochondria?

Genome is essentially a reduced bacterial genome; Structural similarities between mt and bacterial proteins (ribosomes); Ribosomes of both are sensitive to chloramphenicol but not cycloheximide.


Describe the differences between mitochondrial and nuclear transcription.

Different codons cipher different amino acids: AGA/AGG: Arg in nuclear; Stop in mammal mt; Ser in drosophila mt.


What is the cause of mitochondrial diseases?

Mutations in mitochondrial genes.


How can a mother with a mild phenotype have daughters with various severities of phenotype?

Due to cytoplasmic segregation: Primordial germ cells of the daughter have low variance but bottleneck segregation produces primary oocytes with highly variant concentrations of affected mitochondria. Concentration of affected mt proportional to severity of phenotype.


Give 4 examples of mt diseases caused by point mutations/deletions.

MERRF - Myoclonous Epilepsy and Ragged Red Fibres; NARP - Neurogenic muscle weakness, Ataxia Retinitis Pigmentosa; MELAS - Mt Encephomyopathy, Lactic Acidosis, Strokelike symptoms; LHON - Lebers Hereditary Optic Neuropathy.


Why is mt mutation rate 10x greater than that of mammalian nuclei?

Replication close to Electron Transport Chain - plenty of free radicals; Not protected by histones; More rounds of replication; Single stranded for most of replication; Lack of endo-/exonuclease repair system.


How do mitochondria contribute to aging?

Amount of deleted/mutated DNA increases over time. This may affect oxphos causing death of cells including neurons hence causing neurodegenerative diseases (Alz/Park).


How have mt be used for phylogenetics?

Passed on from mother to daughter. Phylogenetic trees shiow recent african ancestor. High copy number means high preservation.


Describe the genetic structure of the Y chromosome.

Only 30Mb is euchromatin. Most exempt from recombination (Except for PAR1 (2.6Mb) and PAR2 (0.32Mb). SRY gene just below PAR1 involved in sex determination.


What is PAR1?

Pseudo-Autosomal Region 1.


Give examples of genes on PAR1?

Hox genes; Interleukin receptors; Acetyl Serotonin Methyl transferase; Adenine nucleotide translocator; Turner syndrome candidates.


What is the importance of PAR1 in segregation?

Recombination with X to allow inactivation excape of 12 X genes. Failure to recombine results in incorrect segregation. (Most 47 XXY failed to recombine).


What is hollandric inheritance?

Father-to-son inheritance of Y chromosome.


How can Y chromosome be used to see population divergence?

Haplogroup distribution of Y chromosomes can show the geographical map of the Y chromosome.


Give an example of Y-linked disorder.

Hairy ears - though may not be Y-linked since females don't grow facial hair anyway.


What causes Y-linked infertility?

Deletion of long arm (q) of Y (Azoospermia factor (Azf) gene).


How can infertility be heritable?

IVF of infertile sperm?


Give examples of Y-linked phenotypes.

Hypertension. CHD. Testicular Prostate Cancer.


What are the two gene categories on the Y chromosome?

Single copy - X homologues; Multi-copy - No homologue - testis-specific and tissue specific genes.


Will Y degenerate completely? Provide evidence.

No. Y has 100 genes compared to X's 1000. Primates have almost all XY genes. Became more compact in humans - not degenerated.