Circadian rhythm-related genes: implication in autoimmunity and type 1 diabetes.

Recent gene association and functional studies have proven the implication of several circadian rhythm-related genes in diabetes. Diabetes has been related to variation in central circadian regulation and peripheral oscillation. Different transcriptional regulators have been identified. Circadian genes are clearly implicated in metabolic pathways, pancreatic function and in type 2 diabetes. Much less evidence has been shown for the link between circadian regulation and type 1 diabetes. The hypothesis that circadian genes are involved in type 1 diabetes is reinforced by findings that the immune system undergoes circadian variation and that several autoimmune diseases are associated with circadian genes. Recent findings in the non-obese diabetic mouse model pinpoint to specific mechanisms controlling type 1 diabetes by the clock-related gene Arntl2 in the immune system.

Control of neurulation by the nucleosome assembly protein-1-like 2.

Neurulation is a complex process of histogenesis involving the precise temporal and spatial organization of gene expression. Genes influencing neurulation include proneural genes determining primary cell fate, neurogenic genes involved in lateral inhibition pathways and genes controlling the frequency of mitotic events. This is reflected in the aetiology and genetics of human and mouse neural tube defects, which are of both multifactorial and multigenic origin. The X-linked gene Nap1l2, specifically expressed in neurons, encodes a protein that is highly similar to the nucleosome assembly (NAP) and SET proteins. We inactivated Nap1l2 in mice by gene targeting, leading to embryonic lethality from mid-gestation onwards. Surviving mutant chimaeric embryos showed extensive surface ectoderm defects as well as the presence of open neural tubes and exposed brains similar to those observed in human spina bifida and anencephaly. These defects correlated with an overproduction of neuronal precursor cells. Protein expression studies showed that the Nap1l2 protein binds to condensing chromatin during S phase and in apoptotic cells, but remained cytoplasmic during G1 phase. Nap1l2 therefore likely represents a class of tissue-specific factors interacting with chromatin to regulate neuronal cell proliferation.

Detection of an elicitor on infection structures of Puccinia graminis using monoclonal antibodies.

The basidiomycetous fungus Puccinia graminis f. sp. tritici causes the stem rust disease of wheat. Resistance of wheat to the fungus is often associated with the hypersensitive reaction of infected host cells. A glycoprotein isolated from germ tube cell walls of the pathogen elicits a hypersensitive-like response when injected into wheat leaves. Infection structures morphologically identical to those grown on wheat were induced in the absence of the host plant, and indirect immunofluorescence together with specific monoclonal antibodies to the elicitor was employed to locate the antigen at fungal infection structures. No binding occurred to germ tubes or appressoria. The antibodies located the antigen only at that part of the fungal infection structure that develops endophytically in nature and, moreover, only at the youngest part of this structure. In rust-infected wheat leaves, the immunolabel appeared only at haustoria, the structures thought to be involved in specific recognition between host and parasite.

Construction of yeast artificial chromosomes containing barley DNA and the identification of clones carrying copies of the repeated element BIS-1.

Yeast artificial chromosome (YAC) cloning vectors allow the isolation and analysis of very large segments of DNA. Barley DNA was cleaved with the rare-cutting restriction enzyme MluI and fractionated according to size on a linear sucrose gradient or by pulsed field gel electrophoresis. DNA fragments of approximately 50-250 kb were ligated with the YAC vector pYAC-RC and transformed into yeast spheroplasts. The presence of recombinant YACs with barley DNA inserts was established, and a number of clones containing copies of the repeated element BIS-1 were isolated. These results show that YAC cloning techniques can be successfully applied to the analysis of the barley genome.

Three loci on mouse chromosome 6 influence onset and final incidence of type I diabetes in NOD.C3H congenic strains.

The development of insulin-dependent diabetes mellitus in both human and mouse is dependent on the interaction between genetic and environmental factors. The analysis of newly created NOD.C3H congenic strains for spontaneous and cyclophosphamide-induced diabetes has allowed the definition of three controlling genetic loci on mouse chromosome 6. A NOD-derived susceptibility allele at the Idd6 locus strongly influences the onset of diabetes in spontaneous diabetes. A NOD-derived resistance allele at the Idd19 locus affects the final diabetes incidence observed in both models, while a novel locus, provisionally termed Idd20, appears to control Idd19 in an epistatic manner. Decreased diabetes incidence is observed in CY-induced diabetes when Idd20 is homozygous for the C3H allele, while heterozygosity is associated with an increase in diabetes incidence. The Idd20, Idd19, and Idd6 candidate regions fall respectively within genetically defined intervals of 4, 7, and 4.5 cM on mouse chromosome 6. From our YAC contig, Idd6 would appear to localize within a ca. 1.5-Mb region on distal chromosome 6.

Transcription mapping in a 700-kb region around the DXS52 locus in Xq28: isolation of six novel transcripts and a novel ATPase isoform (hPMCA5).

The chromosomal band Xq28 has been a focus of interest in human genetics because > 20 hereditary diseases have been mapped to this region. However, about two-thirds of the disease genes remain uncloned. The region around the polymorphic DXS52 locus (ST14) within Xq28 lies in the candidate regions for several as-yet-uncloned disease genes. So far, only four melanoma antigen genes (MAGE) and the human biglycan (BGN) gene, have been mapped within the 700-kb stretch around DXS52, suggesting that more genes may reside in this region. By combining exon trapping and direct cDNA selection methods, we sought to identify novel transcripts around the DXS52 locus. In addition to recovering the MAGE and BGN genes, we isolated and mapped six putative novel genes (XAP103-XAP108), the caltractin gene, and a gene encoding a novel Ca(2+)-transporting ATPase isoform (hPMCA5). The newly isolated sequences were considered as representing parts of putative genes if they contained at least one unique exon-trap product and/or at least one expressed sequence tag (EST) from sequence data bases and if, in addition, they showed evidence of expressed RT-OCT and/or Northern blot analysis. Our data facilitated the integration of the transcription map with the physical map around the DXS52 locus. Future analysis of the novel genes as candidates for Barth syndrome (BTHS) and chondrodysplasia punctata (CDPX2) is in progress.

Transcriptional analysis of the candidate region for incontinentia pigmenti (IP2) in Xq28.

The hereditary form of incontinentia pigmenti (IP2) is a rare disorder characterized by abnormalities of the tissues and organs derived from the ectoderm and neuroectoderm and has been linked to Xq28 distal to the factor VIII gene (F8C). Four YAC clones covering the 1.1-Mb candidate region at the telomere of Xq28 were subjected to direct cDNA selection and Alu long-range PCR. The products of both methods were subsequently used to isolate 154 cosmid clones that were assembled into five cosmid contigs. This first-generation cosmid map covered the region almost entirely and was used as a basis for constructing a transcript map that was in turn integrated with the physical YAC and cosmid maps. To isolate specifically coding sequences, exon trapping and cDNA selection methods were combined. Exon trapping was carried out on YAC Alu-PCR products, YAC Alu long-range PCR products, and on pools of cosmids. The region-specific enriched cDNA library was then screened by using the exon trap products as complex probes. To ensure a more complete analysis, the products from cDNA selection experiments were also used to screen conventional oligo(dT) primed cDNA libraries. Twenty overlapping cDNA contigs were assembled and computer analyses were performed to identify EST hits, open reading frames, protein motifs, and protein sequence homologies. Five of the cDNA contigs corresponded to known sequences such as the factor VIII, c6.1A, and c6.1B. genes, and both distal copies of the factor VIII intron 22 repeat sequence. Expression patterns of the 15 new cDNA contigs were analyzed by Northern blot and RT-PCR studies and these data were integrated with expression data obtained from known EST sequences. Although a more detailed analysis of this 1.1-Mb region with respect to the structure and function of the genes will only ultimately be possible by a global sequencing approach, an analysis of all novel transcripts as candidate genes for incontinentia pigmenti is already in progress.

The melanoma antigen gene (MAGE) family is clustered in the chromosomal band Xq28.

The melanoma antigen gene (MAGE) family comprises 12 known genes, of which 6 are expressed in tumors. In the course of a systematic analysis of transcripts in Xq28, we have identified cDNAs related to different MAGE genes. Analysis of cell hybrids, ordered YACs, and cosmids showed that all MAGE genes are located in Xq28 and are clustered in three main intervals within 3.5 Mb. The six genes expressed in tumors are contained in the two intervals closest to the telomere and are highly homologous to each other. Analysis of different species suggests that human MAGE sequences are conserved in primates, but less well conserved in other vertebrate species.

The mouse Tsx gene is expressed in Sertoli cells of the adult testis and transiently in premeiotic germ cells during puberty.

Tsx is a gene of unknown function that was previously shown to be expressed specifically in the testis. In order to gain insight into the function of Tsx its pattern of expression was characterized with regard to both timing and cell type in the testis. Northern blot analysis of early postnatal testes showed not only that Tsx message was detectable shortly after birth, but that it increased substantially between 7 and 12 days postpartum (dpp), roughly coincident with the onset of meiosis in the mouse. Alternative Tsx transcripts, detected by RT-PCR, included a spliced form that first appeared at around 12 dpp. In situ hybridization revealed Tsx signal in the somatic Sertoli cells of the adult testis. Consistent with the data from Northern blots, in situ hybridization signal was first detectable in normal pubertal testes at 12 dpp. An anti-Tsx polyclonal antiserum specifically stained premeiotic germ cells in addition to Sertoli cells of pubertal testes at 16, 19, and 27 dpp. Tsx immunostaining in germ cells was nuclear, while Sertoli cells displayed signal throughout the cytoplasm and nucleus. In the adult, Tsx was detected exclusively in Sertoli cells. In contrast, in the adult testis of the oligotriche (olt) mutant, where spermatogenesis is blocked after meiosis, Tsx protein was still present in the spermatogonial nuclei of a subset of tubules. Taken together, these results demonstrate that Tsx expression is induced in both premeiotic germ cells and Sertoli cells during the first wave of spermatogenesis, but that expression is maintained at a detectable level only in Sertoli cells of the normal adult. The persistence of Tsx expression seen in spermatogonia of the adult olt mutant supports the hypothesis that during the first wave of normal spermatogenesis, the advent of a late-stage cell type, either elongating spermatid or spermatozoan, is responsible for extinguishing expression in spermatogonia in normal adult testis. To our knowledge, Tsx is the first gene to show a pattern of germ cell expression that is apparently specific to the pubertal testis.

A YAC clone map spanning 7.5 megabases of human chromosome band Xq28.

Xq28 has been of special interest in human genetics because a large number of diseases map to this region. As a step in the molecular analysis of the as yet uncloned disease genes, and as a test for the detailed analysis of larger regions of the genome, we have constructed YAC clone contigs covering the 7.5 Mb region between IDS to the telomere on the long arm of the human X chromosome. Contigs were assembled and verified by an integrated hybridization-based strategy. Data was combined from the physical map, from YAC and cosmid mapping experiments, and from the localization of specific transcripts in the region. Two gaps in the YAC map of 250 and 100 kb were covered in part by the aid of cosmid clones, but small gaps of 50 kb each remain. The cloned region is expected to contain yet unidentified genes for at least ten genetic diseases. The construction of ordered YAC clone contigs of Xq28 represents an important step in the molecular identification of these genes, and the further analysis of one of the genetically most interesting regions of the human genome.

A 900-kb cosmid contig and 10 new transcripts within the candidate region for myotubular myopathy (MTM1).

The X-linked myotubular myopathy locus (MTM1) has been assigned to the Xq28 region by linkage analysis. By observation of an interstitial deletion in a female patient, the candidate region could be further reduced to a region of 600 kb flanked by the markers DXS304 and DXS497. We describe here cosmid contigs covering a region of 900 kb, including the entire MTM1 candidate region. Cosmids from the region were used to construct an enriched cDNA library from this area. Filter grids carrying this library were then screened by hybridization with whole cosmid clones, with CpG island-containing fragments from linking clones located in the area, and with total exon trap products of cosmid clones from the candidate region. In this analysis, 10 new transcripts were identified and localized precisely within the map. Genes in this area are candidates for MTM1 and a number of other diseases localized by genetic linkage studies to the chromosomal band Xq28.