The essential DNA-binding protein sap1 of Schizosaccharomyces pombe contains two independent oligomerization interfaces that dictate the relative orientation of the DNA-binding domain.

The sap1 gene from Schizosaccharomyces pombe, which is essential for mating-type switching and for growth, encodes a sequence-specific DNA-binding protein with no homology to other known proteins. We have used a reiterative selection procedure to isolate binding sites for sap1, using a bacterially expressed protein and randomized double-strand oligonucleotides. The sap1 homodimer preferentially selects a pentameric motif, TA(A/G)CG, organized as a direct repeat and spaced by 5 nucleotides. Removal of a C-terminal dimerization domain abolishes recognition of the direct repeat and creates a new specificity for a DNA sequence containing the same pentameric motif but organized as an inverted repeat. We present evidence that the orientation of the DNA-binding domain is controlled by two independent oligomerization interfaces. The C-terminal dimerization domain allows a head-to-tail organization of the DNA-binding domains in solution, while an N-terminal domain is involved in a cooperative interaction on the DNA target between pairs of dimers.

Identification of the DNA-binding domains of the switch-activating-protein Sap1 from S.pombe by random point mutations screening in E.coli.

Mating type switching in fission yeast, Schizosaccharomyces pombe, is initiated by a site-specific double-strand break (DSB) at the mat1 locus. The DSB is controlled from a distance by cis- and trans-acting elements. The switch-activating protein, Sap1 binds to the SAS1 cis-acting element which controls the frequency of the DSB at the mat1 locus and, consequently the efficiency of mating type switching. We developed a general method for screening randomly mutagenized expression libraries of DNA-binding protein in E.coli. Sap1 gene was mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA was expressed in E.coli and screened for SAS1-recognition. This method was used to isolated 16 point mutations that abolished SAS1 interaction together with 18 mutations that did not affect binding. The position of these point mutations allowed the identification of three protein domains located in the N-terminal part of Sap1 that are essential for DNA-binding. Deletions and biochemical analysis showed that Sap1 is a dimer both in solution and when bound to SAS1 sequence. The dimerization domain was localized C-terminally to the three domains described above and when used in exess it inhibited DNA binding.

E. coli 4.5S RNA is part of a ribonucleoprotein particle that has properties related to signal recognition particle.

E. coli 4.5S RNA and P48 have been shown to be homologous to SRP7S RNA and SRP54, respectively. Here we report that expression of human SRP7S in E. coli can suppress the lethality caused by depletion of 4.5S RNA. In E. coli, both RNAs are associated with P48. In vitro, both E. coli P48 and SRP54 specifically bind to 4.5S RNA. Strains depleted of 4.5S RNA strongly accumulate pre-beta-lactamase and fail to accumulate maltose binding protein. These effects commence well before any growth defect is observed and are suppressed by expression of human SRP7S. Strains overproducing P48 also accumulate pre-beta-lactamase. 4.5S RNA and P48 are components of a ribonucleoprotein particle that we propose to be required for the secretion of some proteins.

Two genes encode 7SL RNAs in the yeast Yarrowia lipolytica.

We have identified an abundant cytoplasmic 7S RNA in crude extracts of the yeast Yarrowia lipolytica. A cDNA probe was prepared from this RNA and used to screen a genomic library. The DNA sequence of a positive clone was determined and the end positions of the 7S RNA gene established by comparison with the sequence of the extremities of 7S RNA. This gene, designated SCR2, encodes a 270-nucleotide RNA that can be folded into a secondary structure similar to that of 7SL RNAs. This RNA is 94.4% homologous to a previously identified 7S RNA from this yeast, but is encoded by a separate gene with highly divergent flanking sequences.

Schizosaccharomyces pombe U4 small nuclear RNA closely resembles vertebrate U4 and is required for growth.

The single-copy gene snu4, which encodes the small nuclear RNA (snRNA) U4, has been cloned and sequenced. Schizosaccharomyces pombe U4 is 128 nucleotides in length, similar in size to vertebrate U4 and shows substantial primary and secondary structure homology. The gene lacks sequences closely resembling vertebrate snRNA transcription signals, but has a TATA box at -33 to -30; TATA sequences flanked by several additional conserved nucleotides are found in the same position in the 5' regions of other snRNA genes from Schiz. pombe. The cloned snu4 gene was disrupted by transposon mutagenesis and used to replace one chromosomal copy of snu4 in a diploid strain. On sporulation snu4- haploid strains could not be recovered, demonstrating that U4 is required, at least for spore germination. Haploid snu4- strains are viable if they also carry snu4+ on a replicating plasmid but are unable to loose the plasmid under non-selective growth, demonstrating a continuous requirement for U4 for viability.

7SL RNA from Schizosaccharomyces pombe is encoded by a single copy essential gene.

We have identified an abundant ribonucleoprotein particle from Schizosaccharomyces pombe with properties related to those of the vertebrate signal recognition particle (SRP), including cytoplasmic localization, association with microsomes and ribosomes at low, but not high, salt concentrations and high resistance to micrococcal nuclease. The 256-nucleotide RNA component carries a 5'-triphosphate group and shows close secondary structure, and limited primary sequence homology to vertebrate 7SL RNA. 7SL-like RNAs were also detected in a number of other fungi. The single copy gene (SRP7) encoding S.pombe 7SL was disrupted by insertion of a transposon carrying the selective marker LEU2, and the disrupted gene was used to replace one chromosomal SRP7 gene in a diploid strain. Haploid srp7[unk] strains fail to germinate.

Expression of the hepatitis B virus large envelope protein in Saccharomyces cerevisiae.

The yeast vector pPV2 has been constructed for inducible expression of non-fused proteins from the PHO5 promoter. Signals of the URA3 gene are used for transcription termination. The 226-amino-acid 'major' and the 389-amino-acid 'large' envelope protein of hepatitis B virus (HBV) have been produced in Saccharomyces cerevisiae following insertion of the S gene or of the entire pre-S region and the S gene, respectively, of HBV into pPV2. Although normally only a minor constituent of the viral envelope, the 'large' protein forms particles with cellular lipids similar to those composed of the 'major' envelope protein. Such particles carry pre-S1, pre-S2, and S-encoded epitopes and, in addition, a receptor for polymerized human serum albumin.

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase.

We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1-5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1-5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.

[Deafness due to renal failure. Clinicopathological study (author's transl)]. / Les surdités par insuffisance rénale: étude anatomo-clinique.

In this series of 54 cases of chronic renal failure, the largest number of curves showed a curve of a presbyacousia type (3/4 of the total). This hypoacousia may be related to the length of time for which the renal pathology has existed and, parat from group 0 (patients treated without haemodialysis nor grafting), may be correlated with the curve of the speed of nervous conduction. This latter correlation is particularly close in patients with a strictly isolated renal disease. The aetiology of this deafness would appear to be related to the premature aging provoked by chronic renal failure. The method of surface preparation applied to 5 petrous temporal bones from 3 patients was felt to be of particular value in the study of this pathology. Whilst one case of unilateral sudden deafness of vascular aetiology is reported, no vascular changes were seen in the other petrous temporal bones. Case 7-11 was particularly demonstrative since bilateral deafness with a rapidly progressive descending occurred in the presence of changes in the pre-ganglionic cochlear fibres charaterised by demyelinisation. This was associated with a cell loss of approximately 25% in the spiral ganglion and less marked demyelinisation of the fibres of the vestibular nerve.