Genetics PCB 3063

1
Genetics - PCB 3063

• Problem set
• Today we will finish some of our introduction to
  sex determination, discuss some of the
  complexities caused by chromosomal sex
  determination, and introduce mitosis and the cell
  cycle.
• Today Sex Chromosomes and Mitosis
• 1. What is the impact of chromosomal sex
  determination upon genetics?
• 2. What is the phenomenon of dosage compensation
  and how does it relate to sex chromosomes?
• 3. How do eukaryotic cells regulate their
  proliferation?

2
Testis-determining factorA mammalian Y
chromosome gene

• SRY encodes a protein that binds to DNA and
  regulates gene expression.
• Leads to sex reversal when present in females
• Related genes, called Sox genes, are present on
  autosomes.
• One of the few genes on the mammalian Y
  chromosome.

3
With the exception of SRY, mammalian Y
chromosomes have few genes.

• Despite these differences, there are Y chromosome
  pseudoautosomal regions that allow it to pair
  with the X during meiosis.
• Without these regions, there would be no
  mechanism to pair the sex chromosomes.
• Males are not hemizygous for genes in the
  pseudoautosomal region.
• These genes escape the X chromosome inactivation
  that results from the mammalian dosage
  compensation mechanism.
• http//www.hhmi.org/news/page5a.html

4
Why doesnt X recombine with Y?

• Other than pseudoautosomal regions (and the rare
  recombinants used to localize SRY) the X and Y do
  not undergo recombination. Why not?
• Remember, mating type can be determined by a
  single gene in the fungi (MAT a and a in
  Saccharomyces cerevisiae)
• This gene is on chromosome 3, and this chromosome
  recombines freely.
• However, imagine a gene necessary for
  spermatogenesis linked to a sex determining gene.
• If such a gene is defective, it will have no
  impact on females but it will have an impact upon
  males. Recombination would allow defective
  alleles to segregate with the male phenotype -
  resulting in sterile males!
• However, this problem would not be that strong
  unless the gene was sexually anatagonistic - it
  was selected against in females but advantageous
  in males.

5
Sexually Antagonistic Genes

• Sexually antagonistic genes need not result in
  male sterility.
• Imagine a male trait (such as bright color in
  guppies) that makes them more attractive to
  females.
• This trait might be selected for in males,
  because females would preferentially mate with
  males having the phenotype.
• However, the trait might make female guppies more
  subject to predation. Thus, it would be selected
  against in females.
• If the trait were on the Y chromosome and there
  was no recombination between X and Y, there is no
  conflict, because it would show holandric
  inheritance.
• Indeed, research from the late 1920s showed that
  such traits in guppies were Y-linked.

6
Sexually Antagonistic Genes

• Sexually antagonistic genes need not result in
  male sterility.
• Such a model could also select for X-linked
  sexually antagonistic genes.
• When hemizygous, recessive alleles could be
  expressed, but females (if the system is XY)
  could be sheltered by dominant alleles on the
  other X chromosome.
• So, is there evidence for the clustering of genes
  involved in male functions on sex chromosomes?

7
Sexually Antagonistic Genes

• There are a number of genes involved in male
  function on the sex chromosomes.
• This supports the sexually antagonistic gene
  model.
• Wang, P. J., McCarrey, J. R., Yang, F. Page, D.
  C. (2001) An abundance of X-linked genes
  expressed in spermatogonia. Nature Genet.
  27422-426

The mouse genome showing genes expressed in germ
cells - BLUE for spermatogonia (male germ cells)
only RED for male and female germ cells.
8
Y chromosome recombination blocks accumulated in
mammals

• Four distinct evolutionary strata are evident
  in human sex chromosomes.
• Recombination between genes will cause them to
  remain similar.
• Thus, when recombination ceases, the genes will
  accumulate differences due to mutation.
• Comparing genes on the X and Y allows the timing
  of divergence - and hence cessation of
  recombination - to be established.

9
W Chromosome Recombination Blocks Accumulated in
Birds

• A similar stepwise cessation of recombination can
  be seen in the ZW chromosomes of birds.
• For a sex-linked CHD gene, only one form could be
  found in ratites, but there were two forms in
  other birds, and the W-linked gene in chickens
  (for example) was more like the W-linked form in
  a falcon than either were like the Z chromosome
  form.
• Distinct results were found for a W-linked gene
  called ATP5A1. Here W and Z forms within the same
  group of birds resembled each other more.
• So recombination of ZW near CHD stopped in the
  common ancestor of chickens and gulls, while
  recombination near the ATP5A1 locus stopped later
  (and independently) in these birds.

10
Excellent Evidence for this Model can be Found in
Recently Evolved Y Chromosomes
E.g., the Carica papaya L. Y chromosome
Silene latifolia (White Campion)
Papaya
11
Sex Determination in Drosophila

• In contrast to mammals, X0 Drosophila develop as
  (sterile) males.
• This indicates that there is no gene on the
  Drosophila Y chromosome that determines male
  development, like the SRY gene.
• Instead, elegant experiments by Calvin Bridges
  showed that the ratio of XA ratio (the ratio of
  X chromosomes to autosomes) determines sex in
  Drosophila.
• This was done using crosses of flies that had
  undergone nondisjunction. Bridges results were

12
XA ratio in Drosophila

• Neither the number of X chromosomes nor the
  presence of a Y determine sex in Drosophila.
• Low XA ratios cause flies to develop as males or
  metamales (weak and sterile)
• Intermediate XA ratios cause an intersex
  phenotype (male-specific sex-combs formed
  improperly, genitalia malformed, and low level
  expression of yolk protein)
• High XA ratios cause flies to develop as females
  or metafemales (metafemales are weak and sterile,
  often not emerging from pupal cases)
• Numerator and denominator elements on the X
  chromosomes and autosomes respectively allow
  flies to determine the XA ratio.
• These are gene that encode products allowing the
  chromosomes to be counted.
• X chromosome numerator elements include three
  sisterless genes (a, b, and c).

13
Drosophila sex determination

• The XA ratio acts to change the form of the
  sex-lethal gene product
• The sex-lethal (sxl) gene was originally called
  female-lethal because of its phenotype.
• The sxl gene has introns that can be spliced in
  two alternative ways. If it is spliced in the
  female specific manner, a protein (Sxl) is
  synthesized.
• This regulates a cascade of alternative intron
  splicing that ultimately generates a male or
  female form of the doublesex gene product.
• The product of the doublesex (dsx) gene, Dsx, has
  two forms (DsxM and DsxF) that have both positive
  and negative regulatory roles in sex
  determination.

14
Invertebrate Sex Determination

• The product of dsx appears to have an ancient
  role in sex determination while the sxl gene
  product has a role limited to Drosophila and
  relatives.
• The C. elegans dsx (called mab-3) has a role in
  male sex determination.
• The housefly (Musca domestica) sxl gene does not
  play a role in sex determination.

Richard Roberts
1993 Nobel Laureates for the discovery of split
genes
Paul Sharp
15
Sex Chromosome Genes - Summary

• Given the existence of systems based upon the XA
  ratio, the basis for X0 sex determination is
  clear.
• The presence of male fertility genes on the
  Drosophila Y chromosome could still be explained
  by the model of selection on sexually
  antagonistic genes, because only males will carry
  the non-recombining Y.
• The mechanism of sex determination in birds is
  presently unclear.
• The Z-linked DMRT1 gene is conserved across phyla
  as a gene involved in sexual differentiation and
  is expressed early in male development,
  suggesting that the number of Z chromosomes might
  regulate avian sex.
• However, at least one gene (the PKCIW gene) that
  is present on the W chromosome and expressed only
  in female birds has been found - could it or some
  other similar gene be analogous to SRY in
  activity?

16
Dosage Compensation Revisited

• As we discussed both today and last Thurs., the
  the heterogametic sex is hemizygous for genes on
  the sex chromosome that is present in both sexes.
• X in mammals or Z in birds.
• To leads to a need to compensate for different
  numbers of gene copies that are present on sex
  chromosomes.
• This is a phenomenon called DOSAGE COMPENSATION.
• Multiple mechanisms for dosage compensation
  exist.
• For now, lets restrict our discussion to
  mammalian dosage compensation, which involves X
  chromosome inactivation.
• A condensed body was observed in the nuclei of
  female mammals - this condensed chromosome was
  called the BARR BODY to honor Murray Barr.

17
X Chromosome Inactivation

• The Barr body was hypothesized to be an inactive
  X chromosome (Mary Lyon).
• Individuals with unusual X chromosome karyotypes
  have one fewer Barr bodies than X chromosomes.
• Electrophoretic analysis of the product of an
  X-linked gene (G6Pd) provided additional for the
  Lyon hypothesis.
• This gene has two alleles that can be
  distinguished by electrophoresis (A and B)

• AB heterodimers could not be found in females,
  suggesting only one allele is expressed in each
  cell.
• Thus, female mammals are mosaics, with different
  X chromosomes active in each cell.

18
How are X Chromosomes Inactivated?

• The X inactivation center (XIC) contains the XIST
  gene.
• The XIST gene is active on the inactive X
  chromosome.
• Unlike many genes, the XIST gene does not encode
  a protein. Instead, it is an RNA that associates
  with the Barr body. The exact mechanism is
  unclear.
• Some genes on the X chromosome are not
  inactivated.
• Genes in the pseudoautosomal region of the Y
  chromosome, as well as those in the
  non-recombining region of the Y chromosome that
  have counterparts on the X chromosome, escape X
  chromosome inactivation.
• Can you predict which genes might escape X
  inactivation and which ones might not escape X
  inactivation?

19
Do All Organisms Inactivate X (or W) Chromosomes?

• Drosophila dosage compensation involves 2-fold
  chromosome-wide up-regulation of essentially all
  genes on the single male X chromosome.
• This hypertranscription involves the association
  of a complex containing at least 5 proteins
  male-specific lethal-1, -2, -3 (msl1, msl2 and
  msl3), maleless (mle) and males absent on the
  first (mof) and at least two non-coding RNAs,
  roX1 and roX2
• C. elegans and related nematodes also use a
  chromosome counting mechanism for sex
  determination.
• But it involves a gene called xol-1, which halves
  the level of transcription in XX hermaphrodites
  (C. elegans has males X0 and hermaphrodites
  XX).

20
C. elegans Dosage Compensation

• In C. elegans, and related nematodes also use a
  chromosome counting mechanism for sex
  determination.
• It involves a gene called xol-1, which halves the
  level of transcription in XX hermaphrodites (C.
  elegans has males X0 and hermaphrodites XX).
• Overexpression of xol-1 causes a hermaphrodite
  specific lethality.
• The absence of sufficient xol-1 expression causes
  a male specific lethality.
• Why do you think this is?

21
C. elegans Dosage Compensation

• In C. elegans, and related nematodes also use a
  chromosome counting mechanism for sex
  determination.
• It involves a gene called xol-1, which halves the
  level of transcription in XX hermaphrodites (C.
  elegans has males X0 and hermaphrodites XX).
• Overexpression of xol-1 causes a hermaphrodite
  specific lethality.
• The repression is blocked, so X chromosome genes
  are themselves overexpressed.
• The absence of sufficient xol-1 expression causes
  a male specific lethality.
• This causes the expression of X chromosome genes
  in males.
• In both cases a lethal phenotype results.

22
The Cell Cycle

• The regulation of cell division has been the
  subject of intensive study.
• In fact, the 1991 Nobel prize in
  medicine/physiology was recently awarded for
  basic work to understand the regulation of
  cellular division.
• The importance of this work reflects the
  importance of cell division regulation in the
  development of cancer.

Leland Hartwell
Tim Hunt
Sir Paul Nurse
23
The Cell Cycle

• The regulation of cell division has been the
  subject of intensive study.
• In fact, the 1991 Nobel prize in
  medicine/physiology was recently awarded for
  basic work to understand the regulation of
  cellular division.
• The importance of this work reflects the
  importance of cell division regulation in the
  development of cancer.
• Cell division is divided into 4 phases.
• Interphase
• Two Gap phases G1 and G2
• S phase DNA synthesis
• Mitosis M phase

24
Interphase and the Cell Cycle

• A number of important events (e.g., DNA
  synthesis) occur during interphase.
• Now, the cell cycle is divided into four phases
• G1 phase - the first gap phase.
• S phase - DNA synthesis phase.
• G2 phase - the second gap phase.
• M phase - mitosis, itself subdivided.
• Non-dividing vertebrate cells are thought to exit
  the cell cycle and enter G0 arrest.
• G0 cells appear to have specific requirements to
  resume proliferation.

25
Control of the Cell Cycle

• Within the cell cycle, the product of the CDC2
  gene regulates progression.
• CDC2 encodes a cyclin-dependent kinase.
• This enzyme (cyclin-dependent kinase) transfers a
  phosphate from ATP to proteins.
• This reaction is called phosphorylation.
• Ser, Thr, and Tyr residues can be phosphorylated.
• The CDC2 gene product is a Ser/Thr kinase.
• The CDC2 gene product is a cyclin-dependent
  kinase because it requires a protein called
  cyclin for full activity.
• These proteins associate with cyclin-dependent
  kinase to activate the phosphorylation activity.

26
Control of the Cell Cycle

• Within the cell cycle, the product of the CDC2
  gene regulates progression.
• CDC2 encodes a cyclin-dependent kinase.
• This enzyme (cyclin-dependent kinase) transfers a
  phosphate from ATP to proteins.
• The CDC2 gene product is a cyclin-dependent
  kinase because it requires a protein called
  cyclin (discovered by Hunt) for full activity.
• The CDC genes were originally identified in yeast
  (both S. cerevisiae and S. pombe) in genetic
  screens (discovered by Hartwell and Nurse).
• Mutant yeast that stop at specific points in the
  cell cycle were identified.

27
Temperature Sensitive Alleles

• Mutations that block progress through the cell
  cycle would be lethal mutations.
• Therefore, mutations in genes such as CDC2 would
  not be viable.
• To allow researchers to examine genes with lethal
  phenotypes temperature-sensitive alleles are
  often examined.
• CDC genes were identified by exposing yeast to
  mutagens and screening those yeast that die at a
  high temperature (usually 37C) to find those
  that arrest at a specific point in the cell
  cycle.
• The CDC2 gene was identified because it causes
  such a block.
• The mutants grow at the permissive temperature
  (30C).

28
Temperature Sensitive Alleles

• These temperature-sensitive alleles allowed
  analyses of the cell cycle.

• Part A of this figure shows wild type S. pombe
  cells. The nucleus is stained with a fluorescent
  dye that binds to DNA.
• Part B shows S. pombe cells with a
  temperature-sensitive allele of a cdc gene.
• The cells in part B are at the restrictive
  temperature - note the act that the cells are
  elongated because their division is inhibited.

29
Yeast cdc Mutants

• Yeast cdc mutants (in S. pombe and S. cerevisiae)
  ultimately revealed the genes involved in
  progress through the cell cycle.

• This figure shows the S. pombe cell cycle, with
  the nucleus highlighted using a fluorescent
  stain.
• Similar results were obtained using cdc mutants
  to analyze the S. cerevisiae cell cycle, though
  many specifics differ.

30
Control of the Cell Cycle

• Within the cell cycle, the product of the CDC2
  gene regulates progression.
• The CDC2 gene product is a cyclin-dependent
  kinase that cyclin for full activity.
• The CDC genes were originally identified in yeast
  in genetic screens.
• There are multiple genes encoding cyclins in most
  eukaryotes.
• Different cyclins are required at different
  points in the cell cycle.
• Cyclins accumulate until they trigger the kinase
  and then they are degraded.
• This allows the cell cycle to move in a single
  direction.

31
Checkpoints and the Cell Cycle

• During the cell cycle, some events are delayed
  until all necessary conditions are in place.
• Since cells do not normally proceed in the cell
  cycle until the necessary conditions have been
  checked off these points where the cell will
  stop are called CHECKPOINTS.
• Examples of CHECKPOINTS include the checks on DNA
  replication and checks on the alignment of
  chromosomes along the metaphase plate.
• In addition to checkpoints, a variety of signals
  can regulate cell division.
• These include growth factors in multicellular
  organisms and nutrients in unicellular organisms.
• These signals ultimately regulate the
  accumulation of cyclins during the cell cycle.

32
Checkpoints and the Cell Cycle

• The cell must assure itself that DNA replication
  is complete before it enters mitosis. If there is
  DNA damage, the cell cycle will arrest until it
  has been repaired.
• This arrest is mediated by the inhibition of the
  CDC2 gene product.
• CDC2 gene product is itself regulated by
  phosphorylation - a specific tyrosine residue is
  phosphorylated under some conditions this
  phosphorylation inhibits cyclin-dependent kinase
  activity.
• Mutants with a phenylalanine at this position
  enter mitosis even when DNA is damaged.
• Other DNA damage checkpoint mechanisms? An area
  of active research.

33
Chromosome Structure

• Before we discuss the cell cycle and mitosis
  lets discuss the structures of chromosomes.
• This is a condensed chromosome.
• Chromosomes condense during mitosis in most
  organisms.
• This is chromosome has duplicated.

34
Mitosis

• Mitosis (M phase) is the phase of the cell cycle
  in which chromosomes are partitioned to daughter
  cells.

• Progress through M phase involves four major
  phases.
• Prophase
• Metaphase
• Anaphase
• Telophase
• These terms come from the Greek (pro- before
  meta- mid ana- back telo- end).

35
The Process of Mitosis

• Division of the nucleus occurs during mitosis.
• Mitosis can be subdivided into 4 different
  stages
• Prophase chromosomes condense, centrioles
  separate and migrate to the poles of the cell.
  The nuclear envelope disintegrates and the
  mitotic spindle forms.
• The mitotic spindle is made up of microtubles,
  composed of tubulin and accessory proteins.
• Spindle fibers attach to the kinetochores of
  chromosomes. Kinetochores are found at the
  centromere of chromosomes.
• Specific differences in different organisms, but
  the overall processes are similar.
• Metaphase chromosomes line up along the
  metaphase plate.

36
The Process of Mitosis

• Mitosis can be subdivided into 4 different
  stages
• Anaphase the centromeres of the chromosomes
  divide, this will convert each chromosome from
  two sister chromatids and one centromere into two
  progeny chromosomes.
• The separation of chromosomes during anaphase
  produced two sets of progeny chromosomes
  reflecting the complete complement of the
  original cell.
• Telophase The chromosomes near the poles and the
  nuclear envelope reforms. The chromosomes and
  microtubules of the spindle return to their
  interphase forms.
• In many cases, telophase is correlated with cell
  division but this is not always the case.
• If cells do not divide a syncitium will form.

37
For Tuesday...

• Complete your problem set.
• We will discuss meiosis and the segregation of
  alleles during sexual crosses.
• We will discuss different types of interactions
  among alleles.
• This material is covered in the first part of
  chapter 4.