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Biomedical subjects

R Lovell-Badge

Publications and source records attributed to R Lovell-Badge.

At least 19 recordsLinked to original sources
PubMed1996

The HMG box of SRY is a calmodulin binding domain.

The HMG box domain of the testis determining factor, SRY, includes a basic amphiphilic sequence common to calmodulin (CaM) binding proteins. By affinity chromatography, native gel electrophoresis and fluorescence spectroscopy, we show the calcium-dependent binding of SRY to CaM. Binding occurs via the HMG box and an SRY peptide of residues 57-80 binds CaM like the intact domain. SRY/CaM complex formation is specifically inhibited by the SRY DNA binding site sequence, AACAAT, but not a mutated sequence. Fluorescence spectra of the SRY/CaM complex indicate 1:1 stoichiometry and that binding is accompanied by a conformational change in SRY. The A domain of HMG1 also binds CaM and we propose that CaM binding is a property of the wider HMG box family, including SOX and TCF/LEF proteins. These results suggest that CaM may regulate the DNA binding activity of HMG box transcription factors.

Amino Acid Sequence↗
PubMed1996

Mouse Dax1 expression is consistent with a role in sex determination as well as in adrenal and hypothalamus function.

Duplications of a chromosome Xp21 locus DSS (Dosage Sensitive Sex reversal) are associated with male to female sex reversal. An unusual member of the nuclear hormone receptor superfamily, DAX1, maps to the DSS critical region and is responsible for X-linked adrenal hypoplasia congenita. Here we describe the isolation of the mouse Dax1 gene and its pattern of expression during development. Expression was detected in the first stages of gonadal and adrenal differentiation and in the developing hypothalamus. Moreover, Dax1 expression is down-regulated coincident with overt differentiation in the testis, but persists in the developing ovary. Comparison of the predicted protein products of the human and mouse genes show that specific domains are evolving rapidly. Our results suggest a basis for adrenal insufficiency and hypogonadotropic hypogonadism in males affected by adrenal hypoplasia congenita and are consistent with a role for DAX1 in gonadal sex determination.

Adrenal Glands↗
PubMed1996

Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds.

Heterozygous mutations in SOX9 lead to a human dwarfism syndrome, Campomelic dysplasia. Consistent with a role in sex determination, we find that Sox9 expression closely follows differentiation of Sertoli cells in the mouse testis, in experimental sex reversal when fetal ovaries are grafted to adult kidneys and in the chick where there is no evidence for a Sry gene. Our results imply that Sox9 plays an essential role in sex determination, possibly immediately downstream of Sry in mammals, and that it functions as a critical Sertoli cell differentiation factor, perhaps in all vertebrates.

Amino Acid Sequence↗
PubMed1996

A comparison of the properties of Sox-3 with Sry and two related genes, Sox-1 and Sox-2.

The Sox gene family consists of a large number of embryonically expressed genes related via the possession of a 79-amino-acid DNA-binding domain known as the HMG box. Partial clones for the first three Sox genes (al-a3) were isolated by homology to the HMG box of the testis-determining gene Sry and are now termed Sox-1, Sox-2 and Sox-3, Sox-3 is highly conserved amongst mammalian species and is located on the X chromosome. This has led to the proposal that Sry evolved from Sox-3. We present the cloning and sequencing of Sox-1, Sox-2 and Sox-3 from the mouse and show that Sox-3 is most closely relate to Sry. We also confirm that mouse Sox-3 is located on the X chromosome between Hprt and Dmd. Analysis of the distribution of Sox-3 RNA shows that its main site of expression is in the developing central nervous system, suggesting a role for Sox-3 in neural development. Moreover, we demonstrate that Sox-3, as well as Sox-1 and Sox-2, are expressed in the urogenital ridge and that their protein products are able to bind the same DNA sequence motif as Sry in vitro, but with different affinities. These observations prompt discussion of an evolutionary link between the genes and support the model that Sry has evolved from Sox-3. However our findings imply that if this is true, then Sry has undergone concomitant changes resulting in loss of CNS expression and altered DNA-binding properties.

Amino Acid Sequence↗
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↗
PubMed1995

The molecular genetics of Sry and its role in mammalian sex determination.

The process of sex determination, by which is meant the decision as to whether an embryo develops as a male or a female, is considered as a paradigm of how gene action can influence developmental fate. In mammals the decision is dependent on the action of the testis determining gene present on the Y chromosome, now known to be the gene Sry. Sry is expressed for only a brief period in the mouse embryo and must act to initiate rather than maintain the pathway of gene activity required for testis differentiation. It probably acts within cells of the supporting cell lineage to direct their differentiation into Sertoli cells, rather than the granulosa cells characteristic of the ovary. Other lineages in the gonad then follow the male pathway. The nature of the Sry transcript in the genital ridge of mice has been determined and compared with that from the human gene which is dramatically different. The expression of Sry has been carefully examined during the critical stages of genital ridge development and compared to the expression of a number of other genes involved in gonadal development and male development such as that for anti-Mullerian hormone. This has defined the period in which Sry must act to between 11 and 11.5 days post coitum. The expression of Sry has also been examined in cases of sex reversal in the mouse. There is a dependence on level of expression and extent of testicular differentiation that suggests thresholds for both the amount of SRY per cell and the number of cells expressing the gene. The SRY protein interacts with DNA through an HMG box type of DNA binding domain, however at present no definite target genes have been found. Progress on strategies to find such genes is discussed.

Animals↗
PubMed1995

Involvement of SOX proteins in lens-specific activation of crystallin genes.

We have studied the mechanism of delta 1-crystallin gene activation, which occurs early in lens cell differentiation, and have previously shown that an essential element of the delta 1-crystallin enhancer is bound by a group of nuclear factors, delta EF2, among which delta EF2a is highly enriched in lens cells. In this report we show that the cDNA of delta EF2a codes for the chicken SOX-2 protein (cSOX-2), which is structurally related to the sex-determining factor SRY. Sox-2 is expressed at high levels in the early developing lens in both chicken and mouse embryos. Overexpression of delta EF2a/cSOX-2 increased delta 1-crystallin enhancer activity to a plateau in lens cells, but not in fibroblasts, consistent with the previously drawn conclusion that delta EF2a activates transcription only in concert with another factor present in the lens. This result supports the model that SOX proteins act as architectural components in the activating complex formed on an enhancer, as indicated for another HMG domain protein, lymphoid enhancer binding factor 1 (LEF-1). We also show that SOX protein binding is essential for lens-specific promoter activity of the mouse gamma F-crystallin gene. This work is the first to show delta- and gamma-crystallin genes as examples of direct regulatory targets of SOX proteins and provides evidence that diversified crystallin genes are regulated, at least partly, by a common mechanism.

Amino Acid Sequence↗
PubMed1995

Evolutionary conservation in the DNA-binding and -bending properties of HMG-boxes from SRY proteins of primates.

Mammalian sex determination is caused by the Y-chromosome gene SRY, which encodes a protein containing a DNA-binding domain (HMG-box) of about 70 amino acids (aa). The HMG-box is very conserved in a wide variety of mammals; conversely, the flanking non-box regions show a high degree of aa sequence divergence, even between closely related species. The HMG-box of human SRY binds sequence-specifically to linear DNA and produces a sharp bend; it also interacts with high affinity to kinked DNA structures irrespective of their sequences. Point mutations associated with sex reversal in XY human females fall within the HMG-box and either affect the affinity for DNA or modify the geometry of the DNA-protein complex. Here, we show that the DNA-binding and -bending properties of the HMG-boxes of SRY from human and seven different primates are extremely similar to each other. Together with other data, this suggests that the inability of mouse and human SRY to substitute for each other is due to differences in the conserved HMG-box, rather than the non-conserved flanking sequences.

Amino Acid Sequence↗
PubMed1995

The zebrafish Zf-Sox 19 protein: a novel member of the Sox family which reveals highly conserved motifs outside of the DNA-binding domain.

A cDNA encoding a zebrafish Sox protein (Sry-type high-mobility-group box) was isolated and sequenced. The sequence within the HMG box is close to those of the B subfamily comprising mouse Sox-1, Sox-2, Sox-3 and Sox-14. While much of the rest of the zebrafish protein is unique, there are blocks of amino acids showing considerable identity with regions of Sox-1, -2 and -3. These domains may represent conserved parts of the protein, required for interaction with other proteins, and strengthen the assignment of the zebrafish gene, termed Zf-sox 19, to the B subfamily.

Amino Acid Sequence↗
PubMed1995

The genes for a spliceosome protein (SAP62) and the anti-Müllerian hormone (AMH) are contiguous.

During an investigation of the regulatory potential of a region 5' of the mouse anti-müllerian hormone (Amh) gene, we identified a region of homology with the known cDNA sequence of a human spliceosome gene (SAP62). In mouse, the Sap62 termination codon (TGA) is just 434 bp 5' of the Amh start of translation (ATG); in the human the equivalent distance is 789 bp. RNase protection analysis shows the majority of Sap62 transcripts use an uncommon polyadenylation signal (ATTAAA) lying in the intragenic region, 87 bp 3' of the TGA. This analysis also shows that Sap62 is transcribed in all tissues examined, whereas specific Amh transcription initiating 10 bp 5' of the ATG is limited to the developing testis of the fetus from 11.5 days post coitum and in the ovary from 3 days post partum. However, in all tissues a significant number of Sap62 transcripts fail to polyadenylate in the intragenic region and continue through the Amh locus. This implies that the Amh locus is in an open chromatin state in all tissues despite a requirement for precise regulation. Human SAP62 can now be mapped to HSA19p and mouse Sap62 to MMU10.

Amino Acid Sequence↗
PubMed1995

Expression of Sry, the mouse sex determining gene.

In the mouse, Sry is expressed by germ cells in the adult testis and by somatic cells in the genital ridge. Transcripts in the former exist as circular RNA molecules of 1.23 kb, which are unlikely to be efficiently translated. We have used RNase protection to map the extent of the less abundant Sry transcript in the developing gonad. We demonstrate that it is a linear mRNA derived from a single exon. This begins in the unique region 5' of the protein coding region and extends several kilobases into the 3' arm of the large inverted repeat which bounds the Sry genomic locus. Knowledge of this transcript, which is very different from that of the human SRY gene, allows us to predict its protein product and reveals several features which may be involved in translational control. Our data is also consistent with there being two promoters for the Sry gene, a proximal one that gives functional transcripts in the genital ridge and a distal promoter used in germ cells in the adult testis. As RNase protection is a quantitative technique, a detailed timecourse of Sry expression was carried out using accurately staged samples. Sry transcripts are first detectable just after 10.5 days post coitum, they reach a peak at 11.5 days and then decline sharply so that none are detected 24 hours later. This was compared with anti-Müllerian hormone gene expression, an early marker of Sertoli cells and the first known downstream gene of Sry. Amh expression begins 20 hours after the onset of Sry expression at a time when Sry transcripts are at their peak. While this result does not prove a direct interaction between the two genes, it defines the critical period during which Sry must act to initiate Sertoli cell differentiation.

Amino Acid Sequence↗
PubMed1995

The SRY protein, like HMG 1, recognizes (CA)n sequences, an abundant repeat sequence in vertebrates.

The sex-determining region of the Y chromosome gene, sry is expressed in the foetal mouse for a brief period, just before testis differentiation, which could be consistent with negative autoregulation. SRY is a DNA binding protein which can bind to cruciform DNA and to linear DNA with a sequence specificity. We have examined if the Sry gene contain DNA binding sites for the SRY protein itself. We have found that in an in vitro assay, the SRY protein binds to several sites of the Sry gene and especially to a (CA)25 sequence and to a (CAG)30 repeat. These binding suggest that the function of SRY and in a general way HMG-box proteins may be mediated by an interaction with repeat sequences.

Animals↗
PubMed1994

Sex-reversing mutations affect the architecture of SRY-DNA complexes.

The testis determining factor, SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. We have analysed the biochemical properties of the DNA binding domains (HMG-boxes) of mutant SRY proteins from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination.

Base Sequence↗