The spontaneous mouse grey-lethal (gl) mutation is responsible for a coat color defect and for the development of the most severe autosomal recessive form of osteopetrosis. Using a positional cloning approach, we have mapped and isolated the gl locus from a approximately 1.5 cM genetic interval. The gl locus was identified in a bacterial artificial chromosome (BAC) contig by functional genetic complementation in transgenic mice. Genomic sequence analysis revealed that the gl mutation is a deletion resulting in complete loss of function. The unique approximately 3 kb wild-type transcript is expressed primarily in osteoclasts and melanocytes as well as in brain, kidney, thymus and spleen. The gl gene is predicted to encode a 338-amino acid type I transmembrane protein that localizes to the intracellular compartment. Mutation in the human GL gene leads to severe recessive osteopetrosis. Our studies show that mouse Gl protein function is absolutely required for osteoclast and melanocyte maturation and function.

Transfer RNAs of the extreme halophile Haloferax volcanii contain several modified nucleosides, among them 1-methylpseudouridine (m1 psi), pseudouridine (psi), 2'-0-methylcytosine (Cm) and 1-methylinosine (m1l), present in positions 54, 55, 56 and 57 of the psi-loop, respectively. At the same positions in tRNAs from eubacteria and eukaryotes, ribothymidine (T-54), pseudouridine (psi-55), non-modified cytosine (C-56) and non-modified adenosine or guanosine (A-57 or G-57) are found in the so-called T psi-loop. Using as substrate a T7 transcript of Haloferax volcanii tRNA(Ile) devoid of modified nucleosides, the enzymatic activities of several tRNA modification enzymes, including those for m1 psi-54, psi-55, Cm-56 and m1l-57, were detected in cell extracts of H.volcanii. Here, we demonstrate that modification of A-57 into m1l-57 in H.volcanii tRNA(Ile) occurs via a two-step enzymatic process. The first step corresponds to the formation of m1A-57 catalyzed by a S-adenosylmethionine-dependent tRNA methyltransferase, followed by the deamination of the 6-amino group of the adenine moiety by a 1-methyladenosine-57 deaminase. This enzymatic pathway differs from that leading to the formation of m1l-37 in the anticodon loop of eukaryotic tRNA(Ala). In the latter case, inosine-37 formation preceeds the S-adenosylmethionine-dependent methylation of l-37 into m1l-37. Thus, enzymatic strategies for catalyzing the formation of 1-methylinosine in tRNAs differ in organisms from distinct evolutionary kingdoms.

No abstract

To study the involvement of DNA mismatch repair genes in non-small cell lung cancer, matched normal and tumoral DNA samples from 31 patients were analyzed for both LOH and microsatellite instability with 34 markers at or linked to hMLH1 (3p21), hMSH2 (2p16), hMSH3 (5q11-q13), hMSH6 (2p16), hPMS1 (2q32), and hPMS2 (7p22) loci. Chromosomal regions 3p21 and 5q11-q13 were found to be hemizygously deleted in 55% and 42% of the patients, respectively. Sixty five percent of the patients deleted at hMLH1 were also deleted at hMSH3. The shortest regions of overlap for 3p21 and 5q11-q13 deletions delimited by D3S1561/D3S1612 and D5S2107/ D5S624, respectively, were restricted to genetic distances of only 1 cM. Currently, the hMLH1 (3p21) and hMSH3 (5q11-q13) genes are the only known candidates located within these regions. The mutational analysis of hMLH1 and hMSH3 in hemizygously deleted patients led to the detection of 2 new polymorphisms in hMSH3. The consequence of these allelic losses remains unclear, but the lack of inactivating mutation might explain that replication error, the hallmark of mismatch repair genes inactivation in cancer cells, was quasiabsent in tumors. We suggest that hMLH1 and hMSH3 genes could be involved in lung tumorigenesis through dosage effect in cellular functions other than replication error correction. Int. J. Cancer 77:173-180, 1998. Wiley-Liss, Inc.Lung cancer is the leading cause of death from cancer worldwide with a survival rate as low as 10% despite intensive treatment. About 75% of all cases fall into the broad category of non-small cell lung cancer (NSCLC) which includes squamous cell carcinoma, adenocarcinoma and large cell carcinoma (Carney and de Leij, 1988). Lung carcinomas are characterized by multiple genetic alterations, which include the activation of oncogenes and the loss or inactivation of tumor suppressor genes (reviewed by Gazdar et al., 1994). Mutations in the p53 gene , as well as deletions at chromosome 3p (Chiba et al., 1990) appear to constitute the most common alterations involved in lung carcinogenesis. Frequent allelic losses affecting several chromosomal arms also strongly suggest the involvement of other yet unknown tumor suppressor genes (Petersen et al., 1997).Another type of genetic alteration, revealed as microsatellite instability in tumors (reviewed by Speicher, 1995), was reported in up to 45% of small cell lung cancers (Merlo et al., 1994) and in up to 66% of NSCLC (Pifarre et al., 1997). In hereditary nonpolyposis colon cancer (HNPCC) this type of genetic instability underlies defects in replication errors correction due to inactivation of mismatch repair genes (reviewed by Peltomäki and de la Chapelle, 1997). Does the deficiency in DNA repair contribute to lung carcinogenesis? Like suppressor genes, inactivation of mismatch repair genes appears to occur in two steps. The first mutational event is either somatic (sporadic cancer) or germinal (hereditary cancer) followed by somatic mutations of the second allele. This second step may be revealed as LOH,...

We have isolated and sequenced the gene for a putative NADP-dependent glutamate dehydrogenase from the extremely halophilic archaebacterium Halobacterium salinarium. This gene is transcribed as a unique RNA molecule of about 1700 nucleotides. The 5' end of the transcript contains characteristic consensus transcription initiation and promoter sequences observed in halophilic archaebacteria. The encoded polypeptide, with a predicted length of 435 amino acids, shows significant overall homology and conservation of functional domains when compared with different eubacterial and eukaryotic glutamate dehydrogenases. Surprisingly, the archaebacterial protein shares a larger number of identical amino acid residues with homologous polypeptides from higher eukaryotes than with those from unicellular eukaryotes and eubacteria.

SummaryTo study the involvement of DNA mismatch-repair genes in sporadic breast cancer, matched normal and tumoral DNA samples of 22 patients were analysed for genetic instability and loss of heterozygosity (LOH) with 42 microsatellites at or linked to hMLH1 (3p21), hMSH2 (2p16), hMSH3 (5q11-q13), hMSH6 (2p16), hPMS1 (2q32) and hPMS2 (7p22) loci. Chromosomal regions 3p21 and 5q11-q13 were found hemizygously deleted in 46% and 23% of patients respectively. Half of the patients deleted at hMLH1 were also deleted at hMSH3. The shortest regions of overlapping (SRO) deletions were delimited by markers D3S1298 and D3S1266 at 3p21 and by D5S647 and D5S418 at 5q11-q13. Currently, the genes hMLH1 (3p21) and hMSH3 (5q11-q13) are the only known candidates located within these regions. The consequence of these allelic losses is still unclear because none of the breast cancers examined displayed microsatellite instability, a hallmark of mismatch-repair defect during replication error correction. We suggest that hMLH1 and hMSH3 could be involved in breast tumorigenesis through cellular functions other than replication error correction.

ted with the development of the disease. We examined 56

The structural gene pfTRM1 (GenBank accession no. AF051912), encoding tRNA(guanine-26, N 2- N 2) methyltransferase (EC 2.1.1.32) of the strictly anaerobic hyperthermophilic archaeon Pyrococcus furiosus, has been identified by sequence similarity to the TRM1 gene of Saccharomyces cerevisiae (YDR120c). The pfTRM1 gene in a 3.0 kb restriction DNA fragment of P.furiosus genomic DNA has been cloned by library screening using a PCR probe to the 5'-part of the corresponding ORF. Sequence analysis revealed an entire ORF of 1143 bp encoding a polypeptide of 381 residues (calculated molecular mass 43.3 kDa). The deduced amino acid sequence of this newly identified gene shares significant similarity with the TRM1- like genes of three other archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum and Archaeoglobus fulgidus), one eukaryon (Caenorhabditis elegans) and one hyperthermophilic eubacterium (Aquifex aeolicus). Two short consensus motifs for S-adenosyl-l-methionine binding are detected in the sequence of pfTrm1p. Cloning of the P.furiosus TRM1 gene in an Escherichia coli expression vector allowed expression of the recombinant protein (pfTrm1p) with an apparent molecular mass of 42 kDa. A protein extract from the transformed E.coli cells shows enzymatic activity for the quantitative formation of N 2, N 2-dimethylguanosine at position 26 in a transcript of yeast tRNAPhe used as substrate. The recombinant enzyme was also shown to modify bulk E.coli tRNAs in vivo.