Human genome project. Variation is now the theme.
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Chromosome Mapping↗
Biomedical subjects
P N Goodfellow
Publications and source records attributed to P N Goodfellow.
Evolution of sex determination and the Y chromosome: SRY-related sequences in marsupials.
In mammals, testis determination is under the control of the testis-determining factor borne by the Y chromosome. SRY, a gene cloned from the sex-determining region of the human Y chromosome, has been equated with the testis-determining factor in man and mouse. We have used a human SRY probe to identify and clone related genes from the Y chromosome of two marsupial species. Comparisons of eutherian and metatherian Y-located SRY sequences suggest rapid evolution of these genes, especially outside the region encoding the DNA-binding HMG box. The SRY homologues, together with the mouse Ube1y homologues, are the first genes to be identified on the marsupial Y chromosome.
Amino Acid Sequence↗
Hidden messages in genetic maps.
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Animals↗
DNA binding activity of recombinant SRY from normal males and XY females.
The protein encoded by the human testis determining gene, SRY, contains a high mobility group (HMG) box related to that present in the T cell-specific, DNA-binding protein TCF-1. Recombinant SRY protein was able to bind to the same core sequence AACAAAG recognized by TCF-1 in a sequence dependent manner. In five XY females point mutations were found in the region encoding the HMG box. In four cases DNA binding activity of mutant SRY protein was negligible; in the fifth case DNA binding was reduced. These results imply that the DNA binding activity of SRY is required for sex determination.
Amino Acid Sequence↗
SRY, like HMG1, recognizes sharp angles in DNA.
HMG boxes are DNA binding domains present in chromatin proteins, general transcription factors for nucleolar and mitochondrial RNA polymerases, and gene- and tissue-specific transcriptional regulators. The HMG boxes of HMG1, an abundant component of chromatin, interact specifically with four-way junctions, DNA structures that are cross-shaped and contain angles of approximately 60 and 120 degrees between their arms. We show here also that the HMG box of SRY, the protein that determines the expression of male-specific genes in humans, recognizes four-way junction DNAs irrespective of their sequence. In addition, when SRY binds to linear duplex DNA containing its specific target AACAAAG, it produces a sharp bend. Therefore, the interaction between HMG boxes and DNA appears to be predominantly structure-specific. The production of the recognition of a kink in DNA can serve several distinct functions, such as the repair of DNA lesions, the folding of DNA segments with bound transcriptional factors into productive complexes or the wrapping of DNA in chromatin.
Amino Acid Sequence↗
The role of the sex-determining region of the Y chromosome (SRY) in the etiology of 46,XX true hermaphroditism.
The syndrome of 46,XX true hermaphroditism is a clinical condition in which both ovarian and testicular tissue are found in one individual. Both Mullerian and Wolffian structures are usually present, and external genitalia are often ambiguous. Two alternative mechanisms have been proposed to explain the development of testicular tissue in these subjects: (1) translocation of chromosomal material encoding the testicular determination factor (TDF) from the Y to the X chromosome or to an autosome, or (2) an autosomal dominant mutation that permits testicular determination in the absence of TDF. We have investigated five subjects with 46,XX true hermaphroditism. Four individuals had a normal 46,XX karyotype; one subject (307) had an apparent terminal deletion of the short arm of one X chromosome. Genomic DNA was isolated from these individuals and subjected to Southern blot analysis. Only subject 307 had Y chromosomal sequences that included the pseudoautosomal boundary, SRY (sex-determining region of Y), ZFY (Y gene encoding a zinc finger protein), and DXYS5 (an anonymous locus on the distal short arm of Y) but lacked sequences for DYZ5 (proximal short arm of Y) and for the long arm probes DYZ1 and DYZ2. The genomic DNA of the other four subjects lacked detectable Y chromosomal sequences when assayed either by Southern blotting or after polymerase chain reaction amplification. Our data demonstrate that 46,XX true hermaphroditism is a genetically heterogeneous condition, some subjects having TDF sequences but most not. The 46,XX subjects without SRY may have a mutation of an autosomal gene that permits testicular determination in the absence of TDF.
Base Sequence↗
Mutational analysis of SRY: nonsense and missense mutations in XY sex reversal.
XY females (n = 17) were analysed for mutations in SRY (sex-determining region Y gene), a gene that has recently been equated with the testis determining factor (TDF). SRY sequences were amplified by the polymerase chain reaction (PCR) and analysed by both the single strand conformational polymorphism assay (SSCP) and DNA sequencing. The DNA from two individuals gave altered SSCP patterns; only these two individuals showed any DNA sequence variation. In both cases, a single base change was found, one altering a tryptophan codon to a stop codon, the other causing a glycine to arginine amino acid substitution. These substitutions lie in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. The corresponding regions of DNA from the father of one individual and the paternal uncle of the other, were sequenced and found to be normal. Thus, in both cases, sex reversal is associated with de novo mutations in SRY. Combining this data with two previously published reports, a total of 40 XY females have now been analysed for mutations in SRY. The number of de novo mutations in SRY is now doubled to four, adding further strength to the argument that SRY is TDF.
Base Sequence↗
A familial mutation in the testis-determining gene SRY shared by both sexes.
A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRYF109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic background or environmental factors.
Amino Acid Sequence↗
Characterization of the central region containing the X-inactivation center and terminal region of the mouse X chromosome using irradiation and fusion gene transfer hybrids.
The irradiation and fusion gene transfer (IFGT) procedure provides a means of isolating subchromosomal fragments for use in the mapping of loci and for cloning probes from a particular area of a chromosome. Using this procedure, two large panels of somatic cell hybrids that contain mouse X Chromosome (Chr) fragments have been generated. These hybrid panels were generated by irradiating the monochromosomal mouse-hamster hybrid HYBX, which retains the mouse X Chr, with either 10 K or 50 K rads of X-irradiation followed by fusion with a recipient Chinese hamster cell line. IFGT hybrids retaining mouse material were generated at high frequency. These hybrids were used to orient loci in the X-inactivation center region that had not been resolvable in our interspecies backcross panel and also to map, within the terminal region of the X Chr, repeat elements detected by the probe p15-4. These hybrids not only complement existing interspecies meiotic mapping panels for the detailed analysis of specific regions of particular chromosomes, but also provide a potential source of material for chromosome-specific probe isolation.
Animals↗
The human genetic map.
The introduction of new technology and increased effort from around the world is driving the completion of the human gene map. In parallel with the creation of the map, we are beginning to see the bio-medical benefits that are a direct consequence of learning more about our own genome.
Animals↗
Isolation of DNA markers from a region between incontinentia pigmenti 1 (IP1) X-chromosomal translocation breakpoints by a comparative PCR analysis of a radiation hybrid subclone mapping panel.
A strategy based on the use of human-specific interspersed repetitive sequence (IRS)-PCR amplification was used to isolate regional DNA markers in the vicinity of the incontinentia pigmenti 1 (IP1) locus. A radiation hybrid (RH) resulting from a fusion of an irradiated X-only somatic cell hybrid (C12D) and a thymidine kinase deficient (TK-) hamster cell line (a23) was identified as containing multiple X chromosome fragments, including DNA markers spanning IP1 X-chromosomal translocation breakpoints within region Xp11.21. From this RH, a panel of subclones was constructed and analyzed by IRS-PCR amplification to (a) identify subclones containing a reduced number of X chromosome fragments spanning the IP1 breakpoints and (b) construct a mapping panel to assist in identifying regional DNA markers in the vicinity of the IP1 locus. By using this strategy, we have isolated three different IRS-PCR amplification products that map to a region between IP1 X chromosome translocation breakpoints. A total of nine DNA sequences have now been mapped to this region; using these DNA markers for PFGE analyses, we obtained a probe order DXS14-DXS422-MTHFDL1-DXS705. These DNA markers provide a starting point for identifying overlapping genomic sequences spanning the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should assist the molecular analysis of this locus.
Animals↗
The human genetic map.
The introduction of new technology and increased effort from around the world is driving the completion of the human gene map. In parallel with the creation of the map, we are beginning to see the biomedical benefits that are a direct consequence of learning more about our own genome.
Base Sequence↗
Telomere-associated chromosome fragmentation: applications in genome manipulation and analysis.
Telomere-associated chromosome fragmentation (TACF) is a new approach for chromosome mapping based on the non-targeted introduction of cloned telomeres into mammalian cells. TACF has been used to generate a panel of somatic cell hybrids with nested terminal deletions of the long arm of the human X chromosome, extending from Xq26 to the centromere. This panel has been characterized using a series of X chromosome loci. Recovery of the end clones by plasmid rescue produces a telomeric marker for each cell line and partial sequencing will allow the generation of sequence tagged sites (STSs). TACF provides a powerful and widely applicable method for genome analysis, a general way of manipulating mammalian chromosomes and a first step towards constructing artificial mammalian chromosomes.
Animals↗
Sex reversal in a child with a 46,X,Yp+ karyotype: support for the existence of a gene(s), located in distal Xp, involved in testis formation.
We report on a sex reversed Japanese child with a 46,X,Yp+ karyotype, minor dysmorphic features, and no testicular development. The Yp+ chromosome was derived by translocation of an Xp fragment (Xp21-Xp22.3) to Yp11.3. This has resulted in deletion of distal part of the Y chromosome pseudoautosomal region (DXYS15-telomere) and duplication of the X specific region (DXS84-PABX) and proximal part of the pseudoautosomal region (MIC2-DXYS17). No deletion of the Y specific region was detected nor was any mutation found in SRY. Cytogenetic analysis suggests that the proximal part of the Xp fragment is the most distal part of the short arm of the Yp+ chromosome (Xp21----Xp 22.3::Yp11.3----Yqter). No chromosomal mosaicism was detected. These results are similar to previous reports of sex reversal in four subjects with a 46,Y,Xp+ karyotype. We conclude that the sex reversal is a direct, or indirect, consequence of having two active copies of the distal part of Xp and may indicate the presence of a gene(s) which acts in the testis determination or differentiation pathway.
Base Sequence↗
Isolation of new probes from Xq12-->q13: an example of the screening of reference libraries with Alu-PCR products from radiation hybrids.
In order to isolate new probes from the juxtacentromeric region of the long arm of the human X chromosome, we used Alu-mediated polymerase chain reaction (Alu-PCR) products as probes to directly screen a chromosome X-specific gridded cosmid library. These Alu-PCR products were synthesized from radiation hybrids containing the loci DXS159, PGK1, and PGK1P1. This approach allowed us to select 18 cosmids capable of hybridizing with at least two Alu-PCR products. Four cosmids hybridized to more than three Alu-PCR products. Three of these four cosmids were contiguous, and the fourth was independent. Two cosmids that hybridized with two Alu-PCR products were further characterized. Physical mapping indicated that all of these clones are located in the expected region on Xq, confirming the validity of our approach.
Base Sequence↗
Evidence for increased prevalence of SRY mutations in XY females with complete rather than partial gonadal dysgenesis.
The Y chromosome gene SRY (sex-determining region, Y gene) has been equated with the mammalian testis-determining factor. The SRY gene of five subjects with 46,XY complete gonadal dysgenesis (46,XY karyotype, completely female external genitalia, normal Müllerian ducts, and streak gonads) was evaluated for possible mutations in the coding region by using both single-strand conformation polymorphism (SSCP) assay and DNA sequencing. Mutations were identified in three subjects, of which two gave altered SSCP patterns. Two of them were point mutations causing amino acid substitutions, and the third was a single-base deletion causing a frameshift. All three mutations caused alterations in the putative DNA-binding region of the SRY protein. Genomic DNA was obtained from the fathers of two of the three mutant patients: one mutation was demonstrated to be de novo, and the other was inherited. The presence of SRY mutations in three of five patients suggests that the frequency of SRY mutations in XY females is higher than current estimates.
Base Sequence↗
DNA fingerprinting. New variations on the theme.
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Alleles↗