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The DNA complementary to RNA (cDNA) of the ribosomal stalk protein BmP0 of the silkworrn Bombyx mori was isolated from a cDNA library and was subsequently expressed in the conditional PO-null mutant Saccharomyces cerevisiae D67dGP0, whose ribosomes also lack the other stalk components, proteins P1/P2. The transformed strain was able to grow under restrictive conditions, indicating that in the absence of the P1/P2 proteins BmP0 can bind to the yeast ribosomes and complement the lack of the endogenous YP0 protein. In addition, the binding capacity of the B. mori ribosomal stalk components to the ribosomal particle was studied by means of high salt treatment of purified ribosomes. The BmP0 protein retained its binding to the ribosome, suggesting a stable association with the rRNA, in contrast to the acidic proteins BmP1 and BmP2, which were easily released. The results clearly indicate that, as opposed to recent in vitro results, BmP0 does not require the presence of P1/P2 proteins in order to bind to the ribosome. (C) 2003 Elsevier B.V. All rights reserved.

The genes encoding the ribosomal P-proteins CcP0, CcP1 and CcP2 of Ceratitis capitata were expressed in the conditional P0-null strains W303dGP0 and D67dGP0 of Saccharomyces cerevisiae, the ribosomes of which contain either standard amounts or are totally deprived of the P1/P2 proteins, respectively. The presence of the CcP0 protein restored cell viability but reduced the growth rate. In the W303CcP0 strain, all four acidic yeast proteins were found on the ribosomes, but in notably less quantity, while a preferable binding of the YP1 alpha/YP2 beta pair was established. In the absence of the endogenous P1/P2 proteins in the D67CcP0 strain, the complementation capacity of the CcP0 protein was considerably reduced. The simultaneous expression of the three medfly genes resulted in alterations of the stalk composition: both the CcP1 and CcP2 proteins were found on the particles substituting the YP1 alpha and YP2 alpha proteins, respectively, but their presence did not alter the growth rate, except in the case of the YP1 alpha/beta defective strain, where a helping effect on the binding of the YP2 alpha and YP2 beta proteins on the ribosomes was confirmed. Therefore, the medfly ribosomal P-proteins complement the yeast P-protein deficient strains forming an heterogeneous ribosomal stalk, which, however, is not functionally equivalent to the endogenous one.

The genes of the acidic ribosomal proteins P1 and P2 (CcP1 and CcP2) of the medfly Ceratitis capitata were isolated from a genomic library using homologue DNA probes prepared by PCR. Sequencing and characterization of the two genes revealed strong similarities of the encoded amino acid sequence to the homologous proteins of Drosophila melanogaster and other eukaryotic species. The predicted amino acid sequences of the CcP1 and CcP2 proteins shared an almost identical carboxyl terminal sequence of 10 amino acids common to most known acidic ribosomal proteins. The CcP2 gene lacked intervening sequences in contrast to the CcP1 gene, which was interrupted by an intron of 188 nucleotides. Both genes were cloned in expression pT7 vectors and were expressed in Escherichia coli. The 17- and 15-kDa recombinant proteins reacted with a monoclonal antibody specific to the highly conserved carboxyl terminus of eukaryotic acidic ribosomal proteins, confirming their equivalence to these ribosomal components. Both recombinant proteins were electrophoretically identical to acidic proteins extracted from purified ribosomes of C. capitata. (C) 1999 Elsevier Science B.V. All rights reserved.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome IV has been determined. Apart from chromosome XII, which contains the 1-2 Mb rDNA cluster, chromosome IV is the longest S. cerevisiae chromosome. It was split into three parts, which were sequenced by a consortium from the European Community, the Sanger Centre, and groups from St Louis and Stanford in the United States. The sequence of 1,531,974 base pairs contains 796 predicted or known genes, 318 (39.9%) of which have been previously identified. Of the 478 new genes, 225 (28.3%) are homologous to previously identified genes and 253 (32%) have unknown functions or correspond to spurious open reading frames (ORFs). On average there is one gene approximately every two kilobases. Superimposed on alternating regional variations in G+C composition, there is a large central domain with a lower G+C content that contains all the yeast transposon (Ty) elements and most of the tRNA genes. Chromosome IV shares with chromosomes II, V, XII, XIII and XV some long clustered duplications which partly explain its origin.

A new ribonuclease from Saccharomyces cerevisiae, specific for poly(U) and poly(C) substrate, was purified near to homogeneity by successive fractionation with DEAE-Sepharose, Heparin-Sepharose and CM-Sepharose chromatography. The purified molecule detected by SDS/polyacrylimide gel electrophoresis has a molecular mass of 29 kDa. The optimum PH for the enzyme activity is 5.5-7 and its isoelectric point is 7.5. The purified enzyme was able to degrade 26S, 18S and 5S rRNAs as well as mRNA obtained from in vitro transcription. No catalytic activity was observed when the RNase was incubated with tRNA and double stranded substrate. Our findings suggest that this novel RNase may play an important role in the processing of RNA in Saccharomyces cerevisiae. (C) 1997 Elsevier Science B.V.