A novel double nucleotide substitution in the HMG box of the SRY gene associated with Swyer syndrome

We describe a novel double nucleotide substitution in the SRY gene of a 46,XY female with gonadal dysgenesis or Swyer syndrome. The SRY sequence was analysed by both the single-strand conformational polymorphism assay and direct DNA sequencing of products from the polymerase chain reaction. A double nucleotide substitution was identified at codon 18 of the conserved HMG box motif, causing an arginine to asparagine amino-acid substitution. The altered residue is situated in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. Since the mutation abolishes one HhaI recognition site, the results were confirmed by HhaI restriction mapping. No other mutations were found in the remaining regions of the gene. The corresponding DNA region from the patient’s brother was analysed and found to be normal. We conclude that the SRY mutation in the reported XY female occurred de novo and is associated with sex reversal. Received: 16 December 1996 / Accepted: 5 May 1997     

The biochemical role of SRY in sex determination

The human sex-determining gene on the Y chromosome, termed SRY, has recently been isolated by positional cloning; compelling evidence now exists equating SRY with the testis-determing factor, TDF. The SRY gene product is an HMG box protein whose DNA-binding activity is vital for testis formation as sex-reversed patients with SRY mutations lack this activity in vitro. The in vivo DNA target for SRY, however, remains elusive. Here, we show, by gel retardation analysis, that SRY recognises specific DNA sequences and that such sequences exist upstream of the AMH promoter, a potential downstream target for SRY. We also describe the DNA bending and cruciform DNA-binding functions of SRY and propose a model for the potential action of SRY in the “HMG-1-rich” mammalian nucleus. © 1994 Wiley-Liss, Inc.     

The mechanism of sequence non-specific DNA binding of HMG1/2-box B in HMG1 with DNA

The DNA binding mechanism of box B in HMG1, a member of the sequence non-specific DNA binding HMG1/2-box family of proteins, has been examined by both mutation analyses and molecular modeling techniques. Substitution of the residue 102F, which is characteristically exposed to solvent, with a small hydrophobic amino acid affected its DNA binding activity. However, no additional effect was observed by the further mutation of flanking 101F. Molecular dynamics simulation and modeling studies revealed that 102F intercalates into DNA base-pairs, being supported by the flanking 101F. The mutants with a small hydrophobic residue at position 102 tolerated the substitution for 101F because the side chain at position 102 is too short to intercalate. Thus the intercalation of 102F and the positive effect of the flanking 101F residue are important for the sequence non-specific DNA binding of the HMG1/2-box. The conserved basic residues of 95K, 96R and 109R were also examined for their roles in DNA binding. These residues interacted with DNA mainly by electrostatic interaction and maintained the location of the box on the DNA, which prescribed the intercalation of 102F. The DNA intercalation by HMG1 consists of an ingenious mechanism which brings DNA conformational changes necessary for biological functions.     

A novel missense mutation (S18N) in the 5′ non-HMG box region of the SRY gene in a patient with partial gonadal dysgenesis and his normal male relatives

Mutations in the sex-determining region of the Y chromosome (the SRY gene) have been reported in low frequency in patients with 46,XY gonadal dysgenesis. We investigated 21 Brazilian 46,XY sex-reversed patients, who presented either complete or partial gonadal dysgenesis or embryonic testicular regression syndrome. Using Southern blotting, polymerase chain reaction, denaturing gradient gel electrophoresis and direct sequencing, we analyzed deletions and point mutations in the SRY gene. We found a missense mutation at codon 18 upstream of the 5′ border of the HMG box of the SRY gene in one patient with partial gonadal dysgenesis. This variant sequence was also found in DNA obtained from blood and sperm cells of his father and in blood cells of his normal brother. The S18N mutation was not found in 50 normal males, ruling out the possibility of a common polymorphism. We identified a novel familial missense mutation (S18N) in the 5’ non-HMG box of the SRY gene in 1 of 21 patients with 46,XY sex reversal. Received: 6 May 1997 / Accepted: 2 October 1997     

XY sex reversal associated with a nonsense mutation in SRY.

Sex determination in humans is mediated through the expression of a testis-determining gene on the Y chromosome. In humans, a candidate gene for the testis-determining factor (TDF) that encodes a protein with a putative DNA-binding motif and has been isolated is termed SRY. Here we describe an XY sex-reversed female with pure gonadal dysgenesis who harbors a de novo nonsense mutation in the SRY open reading frame (SRY-orf). This single-basepair substitution results directly in the formation of a termination codon in the putative SRY DNA-binding motif, presumably leading to a nonfunctional gene product. This brings the number of reported XY sex-reversed females with de novo mutations in the known SRY-orf to three, each occurring in the putative DNA-binding domain. This provides further evidence to support SRY being TDF in humans and also indicates the functional importance of the putative DNA-binding domain of the SRY protein.     

Related function of mouse SOX3, SOX9, and SRY HMG domains assayed by male sex determination

Sox genes encode proteins related to each other, and to the sex determining gene Sry, by the presence of a DNA binding motif known as the HMG domain. Although HMG domains can bind to related DNA sequences, Sox gene products may achieve target gene specificity by binding to preferred target sequences or by interacting with specific partner proteins. To assess their functional similarities, we replaced the HMG box of Sry with the HMG box of Sox3 or Sox9 and tested whether these constructs caused sex reversal in XX mice. Our results indicate that such chimeric transgenes can functionally replace Sry and elicit development of testis cords, male patterns of gene expression, and elaboration of male secondary sexual characteristics. This implies that chimeric SRY proteins with SOX HMG domains can bind to and regulate SRY target genes and that potential SRY partner factor interactions are not disrupted by HMG domain substitutions. genesis 28:111-124, 2000.     

Mutational analysis of SRY: nonsense and missense mutations in XY sex reversal

Summary 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.     

A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.

The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA. We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing. The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations. The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins. Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay. The difference suggests that the two activities of association-promotion and DNA bending are distinct. These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin.     

A testis-specific gene encoding a nuclear high-mobility-group box protein located in elongating spermatids.

A cDNA encoding a DNA-binding protein has been isolated by screening a mouse testicular expression cDNA library with a concatemer of a 12-bp putative protein-binding element present in the promoter of the testis-specific gene PGK-2. Sequence analysis of the isolated cDNA indicated the presence of an open reading frame that encodes a protein with two conserved DNA-binding motifs known as the high-mobility-group (HMG) boxes. Northern (RNA) blot analysis demonstrated that expression of the gene is restricted to the postpuberal testis. The DNA-binding activity and sequence specificity of the recombinant HMG protein were confirmed by DNA mobility shift assay using the initial concatemer of the PGK-2 promoter element as a probe as well as the wild-type or mutated versions of the 12-bp element within its natural sequence context. Immunocytochemical staining of adult testis sections with polyclonal antisera recognizing this recombinant HMG protein demonstrated that it is located predominantly in the nuclei of elongated spermatids at steps 9 and 10. These results suggest that this novel HMG box protein gene may be involved in the regulation of gene expression of the haploid male genome. The gene from which the cDNA was derived has been termed testis-specific HMG (tsHMG).     

Amplification and application of the HMG box of bovine SRY gene for sex determination

A fast and reliable method for bovine sexing has been developed through amplification of the bovine high motility group (HMG) box of the sex-determining region of the Y chromosome gene (SRY). Oligonucleotide primers were designed according to the conserved bovine SRY HMG box sequence motif. In agarose gel electrophoresis, a normal bull showed 1 SRY band, and a normal cow showed no SRY band. After optimization, the PCR procedure for sex determination was applied to 14 embryo biopsies. The biopsied embryos were transferred into 14 recipient cows on the same day (day 7 of the estrus cycle) that the embryos were collected and sex of the calf was confirmed after parturition. Nine calves were born and anatomical sex corresponded to those sex determined by PCR in all cases (100% accuracy). Thus, this study showed for the first time that the present method can be applied in bovine breeding programs to facilitate manipulation of the sex ratio of offspring and also allows a quick diagnosis for the XY-bovine offspring by amplification of the HMG box of the bovine SRY gene.     

水牛SRY基因的克隆及序列分析

根据荷斯坦牛SRY基因设计一对引物,采用聚合酶链式反应（PCR）技术,以中国沼泽型水牛（Swamp Buffalo）基因组DNA为模板,扩增得到SRY（Sex Deterimation region of Y chromosome）全序列约2005bp,其中1-504bp为5’启动子区,1196-2005bp为3’侧翼序列,在505-1195bp为SRY的外显子,编码229个氨基酸。在SRY HMG box区域设计探针,用地高辛标记后分别与雄性、雌性水牛基因组DNA进行Southern 杂交,结果显示该段序列只在雄性DNA样本中有杂交信号,证明SRY基因为雄性特异。BLAST比对结果显示与牛属动物SRY基因的同源性为96％,其中SRY基因HMG box区域同源性高达99%,说明SRY基因具有高度的进化保守性     

SOX14 is a candidate gene for limb defects associated with BPES and Möbius syndrome

Members of the SOX gene family encode proteins with homology to the HMG box DNA-binding domain of SRY, the Y-linked testis-determining gene. SOX genes are expressed during embryogenesis and are involved in the development of a wide range of different tissues. Mutations in SRY, SOX9 and SOX10 have been shown to be responsible for XY sex reversal, campomelic dysplasia and Waardenburg-Hirschsprung disease, respectively. It is likely that mutations in other SOX genes are responsible for a variety of human genetic diseases. SOX14 has been identified from a human genomic library and the mouse and chicken sequences obtained by polymerase chain reaction amplification. The SOX14 amino acid sequence is highly conserved across these species, suggesting an important role for this protein in vertebrate development. SOX14 is expressed in the neural tube and apical ectodermal ridge of the developing chicken limb. This is the only SOX gene known to be expressed in the apical ectodermal ridge, a structure that directs outgrowth of the embryonic limb bud. Human SOX14 is localised to a 1.15-Mb yeast artificial chromosome on chromosome 3q23, close to loci for BPES (blepharophimosis, ptosis, epicanthus inversus syndrome) and Mobius syndrome. Although SOX14 maps outside these loci, its expression pattern and chromosomal localisation suggest that it is a candidate gene for the limb defects frequently associated with these syndromes.