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Biomedical subjects

J R Hawkins

Publications and source records attributed to J R Hawkins.

At least 37 records · Page 2Linked to original sources
PubMed1996

Establishment and characterization of conditionally immortalized cells from the mouse urogenital ridge.

Cell cultures from the urogenital ridge have been established to facilitate the study of the regulation and downstream interactions of Sry in mammalian sex determination. Cells have been explanted from transgenic mice carrying a temperature sensitive SV40 large T-antigen, and established in ongoing cultures. Analysis of the cells in these cultures at the electron microscope level reveals multiple cell types that compare to the cell types found in vivo during this period of development. Primordial germ cells, that are simultaneously explanted in the course of these experiments, also survive in culture. The explants undergo a morphogenetic organization into branching cord-like structures when cells are trypsinized and plated in extracellular matrix (Matrigel). We analyzed the expression of a number of molecular markers of the fetal gonad during monolayer culture, during in vitro morphogenesis in Matrigel, and in clonal lines derived from the complex explants. This analysis included Sry which is found to be expressed in some cultures from XY urogenital ridges that have been maintained for as long as 8 months.

Alleles↗
PubMed1994

Sex determination.

The cloning of the 'testis determining gene', SRY, promised a revolution in the understanding of sex determination in humans. The failure to isolate further genes involved in sex determination has been a disappointment. The biology of SRY, however, has kept the field exciting. The discoveries of sex reversing SRY mutations with variable penetrance, but with full expressivity, rapid SRY sequence evolution and circular Sry transcripts could not have been predicted.

Amino Acid Sequence↗
PubMed1993

Mutational analysis of SRY in XY females.

The Y chromosome located gene SRY (sex determining region Y gene) was identified in the search for the mammalian testis determining factor (TDF). Approaches for evaluating SRY as a candidate for TDF included the finding of mutations in SRY in the genomes of patients with failed testis development (XY females or 46,XY gonadal dysgenesis) and the production of female to male sex reversed mice transgenic for the mouse homologue of SRY, [Sry]. Since the initial use of XY females in the proof of SRY/TDF identity, many more patients have been analysed using different techniques and more mutations identified. A total of 11 mutations in SRY have now been described, all in the DNA-binding HMG-box region of the gene, and all in patients with apparently complete gonadal dysgenesis. Surprisingly, three familial SRY mutations have been identified, where the phenotype is either fertile male or sterile sex-reversed female. Estimates of the proportion of XY females mutant for SRY average at approximately 15%. Reasons for the low frequency of SRY mutations in XY sex reversal could be the presence of mutations in regions of SRY not yet discovered, the occurrence of mutations that give the same phenotype, perhaps in genes close to SRY in the testis determining pathway, or incorrect diagnosis of complete gonadal dysgenesis.

Amino Acid Sequence↗
PubMed1993

Deletion 9p and sex reversal.

We report a case of a female infant with a de novo deletion of the short arm of chromosome 9, sex reversal, and an apparently intact SRY gene. Sex reversal has been reported in a number of subjects with a normal Y chromosome and a deletion of the terminal segment of the short arm of chromosome 9. The factors controlling early development of the male testes are unknown. There are likely to be many genes involved and we present additional evidence that one of these is situated on the end of the short arm of chromosome 9.

Base Sequence↗
PubMed1992

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↗
PubMed1992

Molecular cytogenetic analysis of XX males using Y-specific DNA sequences, including SRY.

XX maleness is the most common condition in which testes develop in the absence of a cytogenetically detectable Y chromosome. Using molecular techniques, it is possible to detect Yp sequences in the majority of XX males. In this study, we could detect Y-specific sequences, including the sex-determining region of the Y chromosome (SRY), using fluorescence in situ hybridization. In 5 out of 6 previously unpublished XX males, SRY was translocated onto the terminal part of an X chromosome. This is the first report in which translocation of an SRY-bearing fragment to an X chromosome in XX males could be directly demonstrated.

Adolescent↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

Cloning and mutational analysis of SRY.

A candidate for the male sex-determining gene has recently been isolated. This sex-determining gene (SRY) has been found to be mutated in some individuals with failed testis development, and, in mouse transgenesis, the SRY murine homologue (Sry) causes female-to-male sex reversal. The cloning of SRY should facilitate the characterisation of other genes in the testis-determining pathway and provide information on the mechanism of mammalian developmental decisions.

Animals↗
PubMed1992

Genetic determinants of testis development in normal and abnormal individuals.

A candidate for the male sex determining gene 2 has recently been isolated. This gene, named SRY, has an expression profile consistent with a role in testis determination, is mutated in some XY females, and causes female to male sex reversal in transgenic mice. The cloning of this gene should facilitate the characterization of other genes in the testis determining pathway.

Animals↗
PubMed1992

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↗
PubMed1991

A 9-base pair deletion in COL1A1 in a lethal variant of osteogenesis imperfecta.

A proband with lethal osteogenesis imperfecta has been investigated for the causative defect at the levels of collagen protein, mRNA, and DNA. Analysis of type I collagen synthesized by the proband's fibroblasts showed excessive post-translational modification of alpha 1(I) chains along the entire length of the helix. Oververmodification of alpha chains could be prevented by incubation of the cells at 30 rather than 37 degrees C, and the thermal stability of the triple helix, as determined by protease digestion, was normal. RNase A cleavage of RNA:RNA hybrids formed between the proband's mRNA and antisense RNA derived from normal pro-alpha 1(I) chain cDNA clones was used to locate an abnormality to exon 43 of the proband's pro-alpha 1(I) collagen gene (COL1A1). The nucleotide sequence of the corresponding gene region showed, in one allele, the deletion of 9 base pairs, not present in either parent, within a repeating sequence of exon 43. The mutation causes the loss of one of three consecutive Gly-Ala-Pro triplets at positions 868-876, but does not otherwise disrupt the Gly-X-Y sequence. Procollagen processing in fibroblast cultures and susceptibility of the mutant collagen I to cleavage with vertebrate collagenase were normal, indicating that the slippage of collagen chains by one Gly-X-Y triplet does not abolish amino-propeptidase and collagenase cleavage sites. How the mutation produces the lethal osteogenesis imperfecta phenotype is not entirely clear; the data suggest that the interaction of alpha chains immediately prior to helix formation may be affected.

Amino Acid Sequence↗
PubMed1991

Testis-determining factor and Y-linked sex reversal.

A gene named SRY, isolated last year from the sex-determining region of the human Y chromosome, satisfies many of the criteria expected of the testis-determining factor gene. Mutations in SRY have been found in XY females, strongly implicating SRY as the testis-determining gene.

Animals↗
PubMed1990

Genetic evidence equating SRY and the testis-determining factor.

The testis-determining factor gene (TDF) lies on the Y chromosome and is responsible for initiating male sex determination. SRY is a gene located in the sex-determining region of the human and mouse Y chromosomes and has many of the properties expected for TDF. Sex reversal in XY females results from the failure of the testis determination or differentiation pathways. Some XY females, with gonadal dysgenesis, have lost the sex-determining region from the Y chromosome by terminal exchange between the sex chromosomes or by other deletions. If SRY is TDF, it would be predicted that some sex-reversed XY females, without Y chromosome deletions, will have suffered mutations in SRY. We have tested human XY females and normal XY males for alterations in SRY using the single-strand conformation polymorphism assay and subsequent DNA sequencing. A de novo mutation was found in the SRY gene of one XY female: this mutation was not present in the patient's normal father and brother. A second variant was found in the SRY gene of another XY female, but in this case the normal father shared the same alteration. The variant in the second case may be fortuitously associated with, or predisposing towards sex reversal; the de novo mutation associated with sex reversal provides compelling evidence that SRY is required for male sex determination.

Amino Acid Sequence↗
PubMed1990

A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif.

A search of a 35-kilobase region of the human Y chromosome necessary for male sex determination has resulted in the identification of a new gene. This gene is conserved and Y-specific among a wide range of mammals, and encodes a testis-specific transcript. It shares homology with the mating-type protein, Mc, from the fission yeast Schizosaccharomyces pombe and a conserved DNA-binding motif present in the nuclear high-mobility-group proteins HMG1 and HMG2. This gene has been termed SRY (for sex-determining region Y) and proposed to be a candidate for the elusive testis-determining gene, TDF.

Amino Acid Sequence↗