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The Hunt for Chromosomal
Determinants of Maleness—
A gene mapping story…….
The HY Antigen Factor (Eichwald & Clarence)
1955: tissue grafts using inbred mice
found: transplants from male to female always
rejected transplants from female to male always
accepted
-Conclusion: females produce antibodies against
male tissue.
Until 1986, people thought that the HY antigen
was the genetic determinant for maleness.
http://biology.queensu.ca/courses/bio210/L3.html
BUT early gene
mapping showed
individuals with only long
arm of the Y had HY
but were female,
whereas patients with
the short arm had no
HY but were male.
The SRY Gene
"positional cloning"
David Page et al at MIT; used
patients from infertility clinics
(no sperm)
These were males, genetically
XX with a tiny piece of short
arm of Y translocated onto one
of X chromosomes
The SRY gene was discovered
by 2 groups separately in 1990.
Berta et al., (1990) Nature 348
448-45
Jager et al., (1990) Nature
348 452- 53
Subtractive hybridization
Deletion mapping
Zinc finger gene probes
Deletion Hydridization
It was found by studying a
human XY female. It turned out
that she had a deletion in the Y
chromosome that did not allow
testis development.
Deletion
mapping of the
gene for the
testisdetermining
factor(s) in
humans
Evidence
1) XY patients who are
female have mutations in
the SRY region.
2) A mutant line of mice
with XY females has
deletion of corresponding
Sry gene.
There are men who
appear to be normal men,
but have an XX
chromosomal combination
(about 1 out of every
20,000 males), and women
who appear to be normal
women but have the XY
combination.
In every Olympics
since sex testing was
initiated, several women
have been disqualified
because they were
identified as XY.
In 1990, Sinclair and
colleagues narrowed the region to
a 35,000 base-pair domain of the
small arm of the Y chromosome.
http://zygote.swarthmore.edu/sex2.html
SRY includes a single exon
that acts as a transcription
factor…..a protein product
recognizes and binds
specifically to the DNA
sequence: AACAAT
Four siblings with testicular feminization
syndrome. All four subjects in this
photograph have 44 autosomes plus an
X and a Y, (so they are genotypically
MALES (!)) but they have inherited the
recessive sex-linked allele conferring
insensitivity to androgens (male hormones).
http://www.cbs.dtu.dk/dave/roanoke/bio101ch15.htm
3) Experiment
-take fertilized mouse embryos, mixture of XX
and XY, inject with many copies of cloned and
purified mouse Sry gene.
-implant embryos back in mothers.
-gene will be incorporated into genome and
expressed in some but not all baby mice,
-produced 2 XX male mice. These were Sry
transgenic but also sterile (because lacked
other missing Y genes.)
SRY, the
master switch
for male
development in
early
embryogenesis.
Sex determining region Y
Gene symbol : SRY Location : Yp11.3
SRY encodes a 223 amino acid zinc
finger transcription factor that is a
member of the high mobility group (HMG)box family of DNA binding proteins. The
protein is expressed during testis
development for only 2 days.
How SRY Works
1994 used cloned Sry gene
as a probe to detect
location in body tissue of
Sry messenger RNA
-found gene active only in
testis
-Used PCR to
determine when active
- Sry m RNA detected
10.5 - 11.5 days after
fertilization
The protein product of the SRY gene binds
to DNA and in doing so distorts it
dramatically out of shape.
This alters the properties of the DNA and
likely alters the expression of a number of
genes, leading to testis formation.
SRY mutations give rise to
XY females with gonadal
dysgenesis.
Translocations of part of the Y
chromosome containing SRY give
rise to XX males.
In addition to SRY, the
DMRT1 gene on chromosome 9 is
involved in determining testis
development. And unlike the SRY
gene, it has to be present in two
copies to work properly. XY
humans who have lost one copy of
the gene fail to develop testes
and show sex-reversal, despite
having a functional SRY gene.
http://bmbus3.leeds.ac.uk/~bmbndr/nicola/nicodoc.htm
In females one of the X-chromosomes is inactivated
in every cell. The process is called Lyonization after
Mary Lyon who discovered it.
Inactivation occurs during embryogenesis. The
process starts at the "X inactivation center" and
spreads along the chromosome.
X Inactivation appears to be random in somatic cells.
(mosaic pattern)
XIST is located within the "X inactivation center"
and only expressed by the inactive X chromosome.
XIST is transcribed but not translated. It appears
to act as RNA by binding to the x chromosome and
promoting its movement to the nuclear periphery.
Methylation of the inactive X genes maintains
inactivity.
Evolutionary
studies have
shown that the Y
was once the
homologous pair
for X.
The y chromosome contains
mainly degraded copies of X
linked genes and large regions of
repeated sequences.