Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases.

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

Molecular cloning and analysis of yeast gene for cycloheximide resistance and ribosomal protein L29.

A cosmid clone bank of yeast DNA has been used to isolate the cycloheximide resistance gene cyh2 of Saccharomyces cerevisiae. A cosmid carrying this gene was identified by cross hybridization to another cloned gene, tsm437. The two genes, which are tightly linked genetically are both present on a 31 kb segment of cloned DNA. The cyh2 gene encodes ribosomal protein L29, a component of the large subunit. Blot hybridization analysis reveals that this gene is present as a single copy in the yeast genome, unlike many other yeast ribosomal protein genes which appear to be duplicated. The cyh2 gene also appears to contain an intervening sequence, a characteristic common to most yeast ribosomal protein genes that have been cloned.     

Drastic alteration of cycloheximide sensitivity by substitution of one amino acid in the L41 ribosomal protein of yeasts.

Cycloheximide is one of the antibiotics that inhibit protein synthesis in most eukaryotic cells. We have found that a yeast, Candida maltosa, is resistant to the drug because it possesses a cycloheximide-resistant ribosome, and we have isolated the gene responsible for this. In this study, we sequenced this gene and found that the gene encodes a protein homologous to the L41 ribosomal protein of Saccharomyces cerevisiae, whose amino acid sequence has already been reported. Two genes for L41 protein, named L41a and L41b, independently present in the genome of S. cerevisiae, were isolated. L41-related genes were also isolated from a few other yeast species. Each of these genes has an intron at the same site of the open reading frame. Comparison of their deduced amino acid sequences and their ability to confer cycloheximide resistance to S. cerevisiae, when introduced in a high-copy-number plasmid, suggested that the 56th amino acid residue of the L41 protein determines the sensitivity of the ribosome to cycloheximide; the amino acid is glutamine in the resistant ribosome, whereas that in the sensitive ribosome is proline. This was confirmed by constructing a cycloheximide-resistant strain of S. cerevisiae having a disrupted L41a gene and an L41b gene with a substitution of the glutamine codon for the proline codon.     

Ribosomal protein L2 in Saccharomyces cerevisiae is homologous to ribosomal protein L1 in Xenopus laevis. Isolation and characterization of the genes

By cross-hybridization with a cDNA probe for the Xenopus laevis ribosomal protein L1 we have been able to isolate the homologous genes from a Saccharomyces cerevisiae genomic library. We have shown that these genes code for a ribosomal protein which was previously named L2. In yeast, like in X. laevis, these genes are present in two copies per haploid genome and, unlike the vertebrate counterpart, they do not contain introns. Amino acid comparison of the X. laevis L1 and S. cerevisiae L2 proteins has shown the presence of a highly conserved protein domain embedded in very divergent sequences. Although these sequences are very poorly homologous, they confer an overall secondary structure and folding highly conserved in the two species.     

Yeast ribosomal protein L24 affects the kinetics of protein synthesis and ribosomal protein L39 improves translational accuracy, while mutants lacking both remain viable

Four mutant strains from Saccharomyces cerevisiae were used to study ribosome structure and function. They included a strain carrying deletions of the two genes encoding ribosomal protein L24, a strain carrying a mutation spb2 in the gene for ribosomal protein L39, a strain carrying a deletion of the gene for L39, and a mutant lacking both L24 and L39. The mutant lacking only L24 showed just 25% of the normal polyphenylalanine-synthesizing activity followed by a decrease in P-site binding, suggesting the possibility that protein L24 is involved in the kinetics of translation. Each of the two L39 mutants displayed a 4-fold increase of their error frequencies over the wild type. This was accompanied by a substantial increase in A-site binding, typical of error-prone mutants. The absence of L39 also increased sensitivity to paromomycin, decreased the ribosomal subunit ratio, and caused a cold-sensitive phenotype. Mutant cells lacking both ribosomal proteins remained viable. Their ribosomes showed reduced initial rates caused by the absence of L24 but a normal extent of polyphenylalanine synthesis and a substantial in vivo reduction in the amount of 80S ribosomes compared to wild type. Moreover, this mutant displayed decreased translational accuracy, hypersensitivity to the antibiotic paromomycin, and a cold-sensitive phenotype, all caused mainly by the deletion of L39. Protein L39 is the first protein of the 60S ribosomal subunit implicated in translational accuracy.     

A pair of Bacillus subtilis ribosomal protein genes mapping outside the principal ribosomal protein cluster.

Before now, the only ribosomal protein gene loci to be identified in Bacillus subtilis map within the principal ribosomal protein gene cluster at about 10 degrees on the linkage map. Using mutants with alterations in large subunit ribosomal proteins L20 or L24, I mapped the corresponding genes near leuA at approximately 240 degrees. The data were fully consistent with the fact that the genes for the two proteins were close together but not near any other ribosomal protein genes, as is also the case with the genes for the corresponding proteins of Escherichia coli.     

Identification of A1 protein as the fourth member of 13 kDa-type acidic ribosomal protein family in yeast Saccharomyces cerevisiae

The identity of protein A1 predicted by a cDNA clone from yeast Saccharomyces cerevisiae which has common carboxyl-terminus to 13 kDa-type acidic ribosomal proteins has been examined. The unique gene for A1 was isolated using the cDNA clone and found to possess two boxes similar to upstream activation sequences for ribosomal protein genes (UASrpg) in the 5'-flanking region. The in vitro-translation product directed by hybrid-selected mRNA with A1 cDNA comigrated with a minor component of split proteins from ribosome by electrofocusing. In addition, the mRNA level for A1 was found to be lower than other two major acidic ribosomal proteins suggesting that A1 is the fourth member of the protein family so far identified which is expressed at relatively low level.     

The 26S rRNA binding ribosomal protein equivalent to bacterial protein L11 is encoded by unspliced duplicated genes in Saccharomyces cerevisiae.

Transformant phages expressing L15, a yeast ribosomal protein which binds to 26S rRNA and interacts with the acidic ribosomal proteins, were isolated by screening a yeast cDNA expression library in lambda gt11 with specific monoclonal antibodies. Using yeast DNA HindIII fragments that hybridize with the cDNA insert from the L15-expressing clones, minilibraries were prepared in pUC18, which were afterward screened with the same cDNA probe. In this way, plasmids carrying two different types of genomic DNA inserts were obtained. The inserts were subcloned and sequenced and we found a similar coding sequence in both cases flanked by 5' and 3' regions with very low homology. Sequences homologous to the consensus TUF-binding UAS boxes are present in the 5' flanking regions of both genes. Southern analysis revealed the presence of two copies of the L15 gene in the Saccharomyces cerevisiae genome, which are located in different chromosomes. The encoded amino acid sequence corresponds, as expected, to protein L15 and shows a high similarity to bacterial ribosomal protein L11.     

The primary structure of rat ribosomal protein L17

The amino acid sequence of the rat 60S ribosomal subunit protein L17 was deduced from the sequence of nucleotides in two recombinant cDNAs. Ribosomal protein L17 has 184 amino acids and has a molecular weight of 21,383. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 17-19 copies of the L17 gene. The mRNA for the protein is about 720 nucleotides in length. Rat L17 is homologous to human L17 and related to Saccharomyces cerevisiae YL17, Halobacterium marismortui L23, Halobacterium halobium L22e, Escherichia coli L22 and other members of the prokaryotic L22 family.     

The primary structure of rat ribosomal protein L23.

The amino acid sequence of the rat 60S ribosomal subunit protein L23 was deduced from the sequence of nucleotides in two recombinant cDNAs. Ribosomal protein L23 has 140 amino acids and a molecular weight of 14,856. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7-9 copies of the L23 gene. The mRNA for the protein is about 600 nucleotides in length. Rat L23 is homologous to Saccharomyces cerevisiae L17a and related to Escherichia coli L14 and other members of the prokaryotic L14 family.