Walter Just

Transcription of the Y chromosomal gene,Sry, in adult mouse brain

The Y chromosomal gene Sry encodes a putative transcription factor which appears to serve as a master switch initiating testicular development. Here we show that this gene is transcribed in hypothalamus, midbrain, and testis of adult male but not adult female mice. In contrast to its circular transcripts in adult testis, those in brain are linear and may be translated. We propose that Sry exerts a role in the regulation of sex differentiation of the mammalian nervous system.

Developmental profile of Sry transcripts in mouse brain

ABSTRACTTransient activation of the gene Sry in the gonadal ridge during a brief period of embryonic development is believed to function as a key signal for sex determination. However, a number of reports suggest that Sry expression is not as restricted in space and time as one would expect if its role was confined to directing male-specific differentiation in the early gonadal anlage. We have previously reported the occurrence of Sry/SRY transcripts in adult murine and human brain. The present communication is concerned with the study of the ontogenetic time course of Sry transcripts in mouse brain as detected by reverse transcription-polymerase chain reaction (RT-PCR). Particular emphasis was placed on the identification of two different forms of Sry mRNA, which can be linear or circular. To this aim, we used specific RT-PCR strategies to distinguish between both. Sry transcripts were found in male brain tissue of all ontogenetic stages investigated. Circular, presumably untranslatable, transcripts were found in embryonic brains of day 11 through 19. In contrast, postnatal Sry transcripts were linear, and thus translatable, and were found in diencephalon, midbrain, and cortex. The change from one transcript form to the other suggests that expression of the Sry gene in mouse brain is developmentally regulated, presumably by a switch in promoter selection. This supports the notion that Sry expression in brain is biologically significant.

A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories

Background The GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied on repeat-primed PCR (RP-PCR) methods for detection of the expansions. To investigate the inherent limitations of this technique, we compared methods and results of 14 laboratories. Methods The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or FTD) in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone; Southern blotting techniques were used as a reference. Results Using PCR-based techniques, 5 of the 14 laboratories got results in full accordance with the Southern blotting results. Only 50 of the 78 DNA samples got the same genotype result in all 14 laboratories. There was a high degree of false positive and false negative results, and at least one sample could not be genotyped at all in 9 of the 14 laboratories. The mean sensitivity of a combination of amplicon-length analysis and RP-PCR was 95.0% (73.9–100%), and the mean specificity was 98.0% (87.5–100%). Overall, a sensitivity and specificity of more than 95% was observed in only seven laboratories. Conclusions Because of the wide range seen in genotyping results, we recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. We propose that Southern blotting techniques should be the gold standard, and be made obligatory in a clinical diagnostic setting.

Sex determination in Ellobius lutescens: The story of an enigma

The unusual karyotype of Ellobius lutescens (2n = 17,X in males and females) has attracted permanent interest and prompted a series of hypotheses on sex determination in this species since its first description by Matthey (1953). The developing knowledge about the sex chromosomes and sex determination as well as the availability of new cytogenetic and molecular genetic techniques prompted studies to test the compatibility between current hypotheses and new findings and rendered modifications of the hypotheses necessary. After a long period dominated by the question what the sex chromosome constitution of this species might be and where the testis determining factor could be located, the presence of Sry had been eventually excluded and sex determination attributed to a hypothetical mutated gene acting downstream of Sry. An X-chromosomal or autosomal location of this gene can be assessed by cosegregation of sex with X-chromosome markers. Some preliminary results concerning X-chromosome dinucleotide repeat markers are reported. However, these markers were homomorphic in Ellobius lutescens. We now report evidence that Zfy is also missing in Ellobius lutescens and E. tancrei (males and females XX), a finding from which we conclude that the entire Y chromosome has been lost from these species. Perspectives concerning future studies are discussed.

Characterization of FMR1 Promoter Elements by In Vivo-Footprinting Analysis

Fragile X syndrome is associated with silencing of the FMR1 gene. We studied the transcriptional regulation, by analysis of the FMR1 promoter region for the presence of in vivo protein/DNA interactions and for cytosine methylation at the single-nucleotide level. Four protein-binding sites were present in the unmethylated promoter of the active FMR1 gene. In the methylated promoter of inactive genes no footprints were detected, and no evidence of active repression was found in the region investigated. We propose that the silencing of FMR1 gene transcription results from a lack of transcription-factor binding.

A comparison of the expression pattern of five genes of the family of small leucine-rich proteoglycans during mouse development.

For five members of the family of the small leucine‐rich proteoglycans (SLRPs), the expression pattern during fetal development was analyzed. RNA in situ hybridization on whole body sections of mouse embryos was performed for biglycan (Bgn), decorin (Dcn), fibromodulin (Fmod), chondroadherin (Chad), and lumican (Lum). Special attention was given to the question of whether these patterns coincide only with sites of collagen secretion in connective tissue during tissue modeling or if expression can be observed at specific sites of organ differentiation also. In general, Fmod, Lum, and Bgn are expressed at sites of cartilage and bone formation and interstitial tissue deposition; Chad is expressed only at sites of cartilage; and Dcn is expressed only at sites of interstitial tissue deposition. However, there are some distinct developmental stages where no collagen secretion is known to occur. For example, this applies for the expression of Fmod in the forming somites of stage 9.5 postconception (p.c.), for Dcn and Lum in later stage embryos in the pituitary gland and dorsal root ganglia, and for Bgn and Dcn during differentiation in the kidney. These studies provide further evidence for a role of these molecules during connective tissue organization but also for an involvement at specific sites of organ differentiation.

ZOO-FISH analysis in a species of the order Chiroptera: Glossophaga soricina (Phyllostomidae).

Glossophaga soricina is a flower-visiting bat which lives in the neotropics. The diploid chromosome number is 2n = 32 with a fundamental number of autosomal arms, FN, of 60. G. soricina belongs to the Microchiroptera which have a lower diploid DNA content and a higher AT composition in their DNA compared with other mammals. By ZOO-FISH analysis with human chromosome-specific DNA probes, the human autosomes were found conserved in 41 segments. This is an arrangement similar to other mammals which have been analyzed. Several chromosomal associations already known from ZOO-FISH studies in other species were also present in G. soricina.

Copy number variation of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development

Background SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia in combination with XY sex reversal. Studies in mice indicate that SOX9 acts as a testis-inducing transcription factor downstream of SRY, triggering Sertoli cell and testis differentiation. An SRY-dependent testis-specific enhancer for Sox9 has been identified only in mice. A previous study has implicated copy number variations (CNVs) of a 78 kb region 517–595 kb upstream of SOX9 in the aetiology of both 46,XY and 46,XX disorders of sex development (DSD). We wanted to better define this region for both disorders. Results By CNV analysis, we identified SOX9 upstream duplications in three cases of SRY-negative 46,XX DSD, which together with previously reported duplications define a 68 kb region, 516–584 kb upstream of SOX9, designated XXSR (XX sex reversal region). More importantly, we identified heterozygous deletions in four families with SRY-positive 46,XY DSD without skeletal phenotype, which define a 32.5 kb interval 607.1–639.6 kb upstream of SOX9, designated XY sex reversal region (XYSR). To localise the suspected testis-specific enhancer, XYSR subfragments were tested in cell transfection and transgenic experiments. While transgenic experiments remained inconclusive, a 1.9 kb SRY-responsive subfragment drove expression specifically in Sertoli-like cells. Conclusions Our results indicate that isolated 46,XY and 46,XX DSD can be assigned to two separate regulatory regions, XYSR and XXSR, far upstream of SOX9. The 1.9 kb SRY-responsive subfragment from the XYSR might constitute the core of the Sertoli-cell enhancer of human SOX9, representing the so far missing link in the genetic cascade of male sex determination.

The X-chromosomal human biglycan gene BGN is subject to X inactivation but is transcribed like an X-Y homologous gene

We report the mRNA and protein expression levels of human biglycan (BGN) in patients with different numbers of sex chromosomes. BGN maps to the distal long arm of the X chromosome, band Xq28, near the second pseudoautosomal region. BGN expression levels are reduced in 45,X Turner patients and increased in patients with additional sex chromosomes. This is suggestive of a pseudoautosomal gene or a gene that escapes X inactivation and that has an active Y chromosomal copy. However, we also provide evidence from hybrid cell lines that BGN is subject to X inactivation and that there is no homolog on the Y chromosome. This evidence excludes an escape from X inactivation. Moreover, additional Y chromosomes increase BGN expression levels, despite the absence of a Y chromosomal BGN gene. Therefore, another explanation has to be invoked. The “pseudoautosomal expression” of BGN may be attributed to a gene or genes that escape X inactivation and that regulate the transcriptional activity of BGN. This is the first report concerning an X chromosomal gene that does not show the conventional correlation between gene dosage and expression rate known from other X chromosomal genes.

Chromosomal evolution of Arvicolinae (Cricetidae, Rodentia). II. The genome homology of two mole voles (genus Ellobius), the field vole and golden hamster revealed by comparative chromosome painting

Using cross-species chromosome painting, we have carried out a comprehensive comparison of the karyotypes of two Ellobius species with unusual sex determination systems: the Transcaucasian mole vole, Ellobius lutescens (2n = 17, X in both sexes), and the northern mole vole, Ellobius talpinus (2n = 54, XX in both sexes). Both Ellobius species have highly rearranged karyotypes. The chromosomal paints from the field vole (Microtus agrestis) detected, in total, 34 and 32 homologous autosomal regions in E. lutescens and E. talpinus karyotypes, respectively. No difference in hybridization pattern of the X paint (as well as Y paint) probes on male and female chromosomes was discovered. The set of golden hamster (Mesocricetus auratus) chromosomal painting probes revealed 44 and 43 homologous autosomal regions in E. lutescens and E. talpinus karyotypes, respectively. A comparative chromosome map was established based on the results of cross-species chromosome painting and a hypothetical ancestral Ellobius karyotype was reconstructed. A considerable number of rearrangements were detected; 31 and 7 fusion/fission rearrangements differentiated the karyotypes of E. lutescens and E. talpinus from the ancestral Ellobius karyotype. It seems that inversions have played a minor role in the genome evolution of these Ellobius species.