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We report here the complete sequence of the mitochondrial (mt) genome of the pathogenic yeast Candida glabrata. This 20 kb mt genome is the smallest among sequenced hemiascomycetous yeasts. Despite its compaction, the mt genome contains the genes encoding the apocytochrome b (COB), three subunits of ATP synthetase (ATP6, 8 and 9), three subunits of cytochrome oxidase (COX1, 2 and 3), the ribosomal protein VAR1, 23 tRNAs, small and large ribosomal RNAs and the RNA subunit of RNase P. Three group I introns each with an intronic open reading frame are present in the COX1 gene. This sequence is available under accession number AJ511533.

Aims: Although dilated cardiomyopathy is the most frequent form of cardiomyopathy, its aetiology is still poorly understood. In about 20-30% of cases the disease is familial with a large predominance of autosomal dominant transmission. Ten different chromosomal loci have been described for autosomal dominant forms of dilated cardiomyopathy. Only two genes have been associated with pure forms (without myopathy and/or conduction disorders) of the disease, the cardiac actin and the desmin genes. Our aim was to determine the proportion of dilated cardiomyopathy affected individuals carrying a mutation in one of these two genes., Methods and Results: We performed (1) a systematic polymerase chain reaction-SSCP-sequencing screening of the coding sequences of cardiac actin on DNA samples from 43 probands of dilated cardiomyopathy families and 43 sporadic cases; (2) a systematic polymerase chain reaction-SSCP-sequencing screening of the coding sequences of desmin combined with a search for the described missense mutation (Ile451Met) by restriction fragment length polymorphism analysis on DNA samples from 41 probands of dilated cardiomyopathy families and 22 sporadic cases., Conclusion: None of the patients presents a mutation in any of these two genes. Consequently, the proportion of European dilated cardiomyopathy affected individuals bearing a mutation in (1) the cardiac actin gene is less than 1.2%, (2) the desmin gene is less than 1.6%., (Copyright 2000 The European Society of Cardiology.)

Mutations of the ribosomal protein S19 (RPS19) gene were recently identified in 10 patients with Diamond Blackfan anemia (DBA). To determine the prevalence of mutations in this gene in DBA and to begin to define the molecular basis for the observed variable clinical phenotype of this disorder, the genomic sequence of the 6 exons and the 5' untranslated region of the RPS19 gene was directly assessed in DBA index cases from 172 new families. Mutations affecting the coding sequence of RPS19 or splice sites were found in 34 cases (19.7%), whereas mutations in noncoding regions were found in 8 patients (4.6%). Mutations included nonsense, missense, splice sites, and frameshift mutations. A hot spot for missense mutations was identified between codons 52 and 62 of the RPS19 gene in a new sequence consensus motif W-[YFW]-[YF]-x-R-[AT]-A-[SA]-x-[AL]-R-[HRK]-[ILV]-Y. No correlation between the nature of mutations and the different patterns of clinical expression, including age at presentation, presence of malformations, and therapeutic outcome, could be documented. Moreover, RPS19 mutations were also found in some first-degree relatives presenting only with isolated high erythrocyte adenosine deaminase activity and/or macrocytosis. The lack of a consistent relationship between the nature of the mutations and the clinical phenotype implies that yet unidentified factors modulate the phenotypic expression of the primary genetic defect in families with RPS19 mutations.

The PIP gene, localized in the 7q34 region that contains a number of fragile sites such as FRA 7H and FRA TI, codes for gp17/PIP, a protein secreted by breast apocrine tumors. We analyzed the integrity of this gene in 20 tumors of the urogenital tract. We found rearranged EcoRI fragments in 5 of 15 primary prostate carcinomas. No rearrangement was found in normal prostates derived from five patients undergoing prostatocystectomy during treatment of bladder cancers. By Southern blot hybridization with PIP gene exon-specific probes, the rearrangements were mapped at or near the 3' end of the gene. These abnormalities were found, not only in the neoplastic cells invading the prostatic tissues, but also in seminal vesicles without histologic tumoral features. These data suggest a critical role of the PIP gene or neighboring genes in prostate cancer.

Rheumatoid arthritis (RA), the most common autoimmune disease, is associated in families with other autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM). Its genetic component has been suggested by familial aggregation (lambdas = 5), twin studies, and segregation analysis. HLA, which is the only susceptibility locus known, has been estimated to account for one-third of this component. The aim of this paper was to identify new RA loci. A genome scan was performed with 114 European Caucasian RA sib pairs from 97 nuclear families. Linkage was significant only for HLA (P < 2.5.10(-5)) and nominal for 19 markers in 14 other regions (P < 0.05). Four of the loci implicated in IDDM potentially overlap with these regions: the putative IDDM6, IDDM9, IDDM13, and DXS998 loci. The first two of these candidate regions, defined in the RA genome scan by the markers D18S68-D18S61-D18S469 (18q22-23) and D3S1267 (3q13), respectively, were studied in 194 additional RA sib pairs from 164 nuclear families. Support for linkage to chromosome 3 only was extended significantly (P = 0.002). The analysis of all 261 families provided a linkage evidence of P = 0. 001 and suggested an interaction between this putative RA locus and HLA. This locus could account for 16% of the genetic component of RA. Candidate genes include those coding for CD80 and CD86, molecules involved in antigen-specific T cell recognition. In conclusion, this first genome scan in RA Caucasian families revealed 14 candidate regions, one of which was supported further by the study of a second set of families.

We have developed an integrated approach for the analysis of human cDNA libraries from neuromuscular tissues, based on the acquisition of primary structural, expression and mapping data. 26,938 sequence signatures (over 7 million bases) have been derived from both ends of skeletal muscle and brain cDNA clones. Primary redundancy analysis and classification of database similarities made it possible to characterize by structural data about 8,000 human gene transcripts, the majority of which is catalogued for the first time. Collecting hybridization signatures of complex cDNA probes derived from the tissues of origin to cDNA clones arrayed on high density filters provided a global and quantifiable view of the complexity and level of expression of the different transcripts. The development of 2,792 eSTS markers amplifiable by PCR defined the chromosomal localization of some 2,500 genes corresponding to the transcripts sequenced. The data collected are part of the corpus of the human gene transcript catalog and the genic map of the human genome.

Crotoxin, the main toxin from the venom of the South American rattlesnake Crotalus durissus terrificus, is a beta-neurotoxin which consists of the non-covalent association of two subunits: a phospholipase A2 subunit B (CB), and a non-enzymic subunit A (CA). We have previously purified and characterized several isoforms of each subunit of crotoxin in the venom collected from numerous snakes. Furthermore, three cDNAs encoding two CB isoforms and the precursor, pro-CA, of subunit A have been isolated from a cDNA library prepared from a single venom gland of Crotalus durissus terrificus. The aim of this study is to analyse an individual snake venom from an animal that has been used to construct a cDNA library. Several isoforms of subunit A and two isoforms of subunit B were isolated and compared to purified and characterized subunit isoforms from pooled venom. The result of this study showed that the multiplicity and the diversity of crotoxin isoforms result from post-translational modifications occurring on a precursor and from the expression of different messenger RNAs present in an individual snake. It allowed for the identification of the two CB isoforms encoding cDNAs expressed in the individual venom with two isoforms from pooled venom, CBc and probably CBa2, that belong to two classes of crotoxin complexes which can be distinguished biochemically and pharmacologically.

Previously, we deduced the amino acid sequence of a novel phospholipase-A2-like protein (PLA2) from the nucleotide sequence of a cDNA isolated from a library prepared from the venom gland of the Australian elapid Notechis scutatus scutatus. The corresponding protein has now been identified, purified from the venom and named Notechis 11'2. Its complete amino acid sequence has been determined by automated Edman degradation of both the whole protein and peptides generated by Staphylococcus aureus protease digestion and chemical cleavage at a tryptophan residue. As predicted from its sequence which contains all the residues putatively required for PLA2 activity, Notechis 11'2 exhibits an esterase activity, preferentially against neutral phospholipids. However, despite its sequence homology with other highly toxic PLA2 present in the venom of Notechis scutatus scutatus, notechis 11'2 has no lethal activity. This observation further supports the view that the lethal activity of PLA2 from Notechis scutatus scutatus is not due to the esterasic activity only.

We report the sequences of three cDNAs encoding the two subunits (CA and CB) of crotoxin, a neurotoxic phospholipase A2 from the venom of the South-American rattlesnake Crotalus durissus terrificus. CB is a basic and toxic phospholipase A2 and CA is an acidic, non toxic and non enzymatic three chain containing protein which enhances the lethal potency of CB. Two cDNAs encoding precursors of CB isoforms have been isolated from a cDNA library prepared from one venom gland. Both precursors are made of the same 16 residues signal peptide followed by a polypeptide of 122 amino acid residues. The two mature sequences differ from each other at eight positions and are in good agreement with the previous polypeptide sequence reported for CB. In the case of CA, the cDNA encodes a signal peptide identical to those found in CB precursors, followed by a polypeptide of 122 amino acids clearly homologous to phospholipases A2 and including three regions which correspond to the three chains of mature CA. This demonstrates that CA is generated from a phospholipase A2-like precursor, called pro-CA, by the removal of three peptides, leaving unchanged the molecule core cross-linked by disulfide bridges. The 5'-untranslated tracts of cDNAs encoding CA and CB are nearly identical and the 3'-untranslated tracts are very similar, suggesting that the mRNAs encoding the two crotoxin subunits may result from the alternative splicing of a single gene or from the existence of a recent gene conversion. Data have been analysed in light of recent results on other phospholipases A2 from different origins.

Crotoxin, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus, is a neurotoxic phospholipase A2 which exerts its pathophysiological action by blocking the neuromuscular transmission. Crotoxin acts primarily by altering the acetylcholine release from the nerves terminals through a mechanism which has not yet been elucidated. It also acts on postsynaptic membranes by stabilizing the acetylcholine receptor in an inactive conformation very similar to the desensitized state. Crotoxin is made of two dissimilar subunits: a basic and weakly toxic phospholipase A2 component-B, and an acidic and non toxic component-A which does not possess any enzymatic activity. Binding experiments showed that crotoxin subunits dissociate when crotoxin interacts with biological membranes: Component-B binds, whereas component-A appears free in solution. The phospholipase A2 subunit binds in a non saturable, non specific manner, on any kind of biological membranes, whereas in the presence of component-A it interacts only with a limited number of high affinity binding sites present on synaptic membranes but not on erythrocyte membranes. Although the target site (acceptor) of crotoxin has not yet been formally identified, binding experiments carried out with small unilamellar phospholipid vesicles of different compositions indicate that some negatively charged phospholipids like mono and diphosphoinositide phosphates might be an important component of crotoxin acceptor site. Crotoxin is in fact a mixture of several isoforms which have very similar but not identical polypeptide sequences. An individual Crotalus durissus terrificus snake is able to synthesize several crotoxin isoforms which may result of the expression of several isogenes and/or of post-translational events. When compared in quantitative manner, the crotoxin isoforms slightly but significantly differ in their enzymatic and pharmacological properties. Finally, immunochemical investigations carried out with polyclonal antibodies prepared against both crotoxin subunits, showed that non precipitating anti-component-B- antibodies (Fab) inhibit the phospholipase A2 activity of crotoxin and neutralize its lethal potency, suggesting that the catalytic and toxic sites of crotoxin are closely related.

The expression of tubulin, of glial fibrillary acidic protein of their encoding mRNAs were studied in primary cultures of mouse astrocytes. The effects of dibutyryl cyclic AMP on these parameters were also analyzed. (1) The concentration of both ?- and ?-tubulin chains markedly increased (3-fold) between 7 and 12 days of culture and then remained at the same high value at later stages. In contrast ?-tubulin mRNA concentration remained constant throughout the same culture period. (2) Glial fibrillary acidic protein concentration increased steadily reaching a 4-fold higher value at the end of the same culture period. The glial fibrillary acidic protein mRNA increased 3-fold during the first 2 weeks of culture, leveled off at day 18 and then decreased steadily returning to low values (3-fold decrease) at later stages. (3) Addition for 2 days, at day 5 or 19 of the culture, of either dibutyryl cyclic AMP or forskolin remained without effect on tubulin concentration whereas a 50% decrease in ?-tubulin mRNA was observed. In the same conditions the effects of these drugs on glial fibrillary acidic protein and its mRNA were not significant. These results suggest that the increase in tubulin concentration seen between days 7 and 12, as well as that of glial fibrillary acidic protein observed during the last weeks of the culture, are not secondary to an increase either of the rate of transcription or of the stability of their encoding mRNAs.