A 10-cM YAC contig spanning GLC1A, the primary open-angle glaucoma locus at 1q23-q25.

Primary open-angle glaucoma is a complex of ocular disorders characterized by irreversible lesions of the optic nerve, open angle of the anterior chamber of the eye and elevated intraocular pressures. GLC1A, a locus involved in one form of this disease, has been mapped to an approximately 9-cM interval within 1q23-q25, between markers D1S445 and D1S416/D1S480. A 10-cM yeast artificial chromosome (YAC) contig spanning the whole region is described. This contig is based on 67 YACs, and 41 sequence tagged sites comprising 23 genetic markers, 16 YAC ends and 2 expressed sequence tags. The reagents reported in this study should be useful tools for the identification of the GLC1A gene by positional cloning.

Lamellar ichthyosis: further narrowing, physical and expression mapping of the chromosome 2 candidate locus.

Lamellar ichthyosis (LI) is an autosomal recessive genodermatosis which has been shown to be both clinically and genetically heterogeneous. Keratinocyte transglutaminase (or transglutaminase 1: TGM1) has been demonstrated to be the disease-causing gene in some families, whilst in others, a second unidentified LI gene was mapped to chromosome 2q33-35 (ICR2B locus). In this study, we present a physical map that encompasses the ICR2B locus, including the mapping of new microsatellite markers. Based on this new map, genotyping additional families highly suggests a reduction in size of the candidate interval. The final interval is covered by a single yeast artificial chromosome (937-H-3) which is 2.2Mb in length. Fine mapping of potential candidate transcripts was also focused on this region.

Evolutionary comparisons of three enzymes of the threonine biosynthetic pathway among several microbial species.

As an approach in the study of the evolution of threonine biosynthetic pathways throughout various organisms, the sequences of three enzymes, namely homoserine dehydrogenase, homoserine kinase and threonine synthase, originating from six organisms, namely Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Brevibacterium lactofermentum, Pseudomonas aeruginosa and Saccharomyces cerevisiae, were compared. As a general trend all three enzymatic activities were carried out by proteins sharing sequence relatedness (except for the homoserine kinase of P aeruginosa). Unexpectedly however, for each step one or two enzymes stood out of the main stream: i) for homoserine dehydrogenase, the yeast protein is atypically similar to the E coli enzyme; ii) for homoserine kinase, the P aeruginosa protein shares no similarity with any other species; and iii) for threonine synthase, the B subtilis protein is far distant from the enzymes of other species. Hence in contrast to other biosynthetic pathways such as the tryptophan one, the threonine pathway seems not to have evolved as a whole throughout different organisms but rather each step seems to have been subjected to multiple constraints including substrate-mediated ones and host-specific ones.

Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes.

Three genes from Pseudomonas aeruginosa involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrc genes lie at the thr locus of the P. aeruginosa chromosome map (31 min) and are likely to be organized in a bicistronic operon. The encoded proteins are quite similar to the Hom and TS proteins from other bacterial species. The thrB gene was located by pulsed-field gel electrophoresis experiments at 10 min on the chromosome map. The product of this gene does not share any similarity with other known ThrB proteins. No phenotype could be detected when the chromosomal thrB gene was inactivated by an insertion. Therefore the existence of isozymes for this activity is postulated. HDH activity was feedback inhibited by threonine; the expression of all three genes was constitutive. The overall organization of these three genes appears to differ from that in other bacterial species.

The human SRY transcript.

SRY encodes the Y-linked testis-determining factor in humans. A predominant 900 bp transcript originates from a single exon and encompasses the putative SRY coding sequence. We show that in human adult testis SRY transcription involves multiple start sites. In addition to a previously defined major initiation site, transcripts originating at least 410 bp upstream of this site were detected. Using a cDNA specific RT-PCR assay, embryonic and adult human tissues were screened for SRY expression. In humans, SRY transcription is not restricted to the presumptive and the mature gonadal tissues in the embryo and the adult respectively but can be detected in a range of other locations. Two human cell lines, NTERA-2 cl.D1 (NT2/D1) and Hep G2, have been identified which express SRY at similar levels to adult testis. The NT2/D1 SRY transcripts appear to have the same structure as those in adult testis. HMBA-induced differentiation of NT2/D1 cells results in a diminution of SRY mRNA, while transcription of SRY in retinoic acid differentiated NT2/D1 is unaffected.

Genotypic diagnosis of familial Mediterranean fever (FMF) using new microsatellite markers: example of two extensive non-Ashkenazi Jewish pedigrees.

Familial Mediterranean fever is an autosomal recessive disease characterised by multiple attacks of serosal inflammation in the absence of treatment. In the absence of timely diagnosis, renal amyloidosis is a life threatening complication. The diagnosis is often missed because no specific test is available. Early colchicine treatment prevents attacks and renal complications. The FMF gene (MEF) has been mapped to chromosome 16p 13.3 but has not yet been identified. We compared the suitability of a series of microsatellite markers (four of them were new) and propose the routine use of seven of these markers, exhibiting alleles in strong linkage disequilibrium with the disease and informative in 100% of diagnosed patients. Moreover, the discovery of a homozygous status for the 3-3-9 (or 3-3-18) haplotype at the core loci (D16S3070, D16S3082, and D16S3275), which was found in 73% non-Ashkenazi Jewish patients, points to a diagnosis of FMF, even in sporadic cases, with a risk of error of only 2.10(-5). Two extensive pedigrees covering most indications for genetic counselling are presented, showing that it is now possible both prospectively and retrospectively to identify members likely to have MEF mutations. With the help of this accurate test, colchicine treatment can be better targeted, especially where the symptomatology is mild or atypical.

A transcriptional Map of the FMF region.

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by attacks of fever and serositis, which affects primarily non-Ashkenazi Jews, Armenians, Turks, and Arabs. We present here a transcriptional map covering the FMF locus that we constructed in the course of the positional cloning of the gene responsible for this disease. This map was established from a contig constructed with YAC, BAC, and cosmid clones and covers about 500 kb of 16p13.3. It contains nine transcriptional units corresponding to known genes or to genes belonging to known gene families, 23 gene fragments characterized by partial sequences, and an endogenous retrovirus sequence. It thus considerably increases the number of genes in this interval and improves our knowledge concerning some of the genes or gene families present in this region. Data accumulated in this region were also used in a comparative study of different methods of exon detection.