Yeast ribosomal proteins: XIV. Complete nucleotide sequences of the two genes encoding Saccharomyces cerevisiae YL16.

We isolated and sequenced YL16A and YL16B encoding ribosomal protein YL16 of Saccharomyces cerevisiae. The two nucleotide sequences within coding regions retain 91.1% identity, and their predicted sequences of 176 amino acids show 93.8% identity. Out of the ribosomal protein sequences from various organisms currently available, no counterpart to YL16 could be found.     

Yeast ribosomal proteins. VIII. Isolation of two proteins and sequence characterization of twenty-four proteins from cytoplasmic ribosomes

Summary Two proteins, YL41 and YL43, were isolated from 80S ribosomes of Saccharomyces cerevisiae by filtration through a Sephacryl S-200 column and by chromatography on a column of carboxymethylcellulose. Their amino acid compositions are presented. Twenty-four proteins including these two proteins were subjected to sequence analyses by automated Edman degradation. Amino-terminal amino acid sequences were determined for 17 proteins, YS3, YS9, YS23, YS24, YS29, YL6, YL8, YL11, YL15, YL17, YL23, YL28, YL33, YL37, YL39, YL41, and YL43. YL41, which has a 72.7% lysine and arginine content, was found to be particular to eukaryotic ribosomes. The aminotermini of another seven proteins, YS2, YS5, YS8, YS12, YS13, YS20, and YS27, were suggested to be blocked.Comparison of the amino-terminal sequences with all other ribosomal protein sequences so far available indicates that YS9 shows sequence homology to rat liver ribosomal protein S8 (Wittmann-Liebold et al. 1979).     

Yeast ribosomal proteins: VII. Cytoplasmic ribosomal proteins from Schizosaccharomyces pombe

The cytoplasmic ribosomal proteins from a fission yeast Schizosaccharomyces pombe were analysed by two-dimensional polyacrylamide gel electrophoresis. Seventy-three protein species were identified in the 80S ribosome, and named SP-S1 to SP-S33 and SP-L1 to SP-L40 in the small and large subunits, respectively. Many of these proteins could be correlated to those of Saccharomyces cerevisiae on the basis of their electrophoretic mobilities. Eleven proteins were isolated from the 80S ribosome, and their amino acid compositions were determined. Of these, SP-S6, SP-L1, SP-L12, SP-L15, SP-L17, SP-L27, SP-L36 and SP-L40c and d were sequenced from their amino-termini. SP-S28 and SP-L2 appear to have their amino-termini blocked. These results were compared with the data available for the S. cerevisiae and rat liver ribosomal proteins. The S. cerevisiae counterparts of the eight proteins mentioned above were found to be YS4, YL1, YL10, YL14, YL35, YL40 and YL44c and d, respectively. The rat liver counterparts of SP-S6, SP-L1, SP-L27 and SP-L40c and d were the rat S6, L4, L37 and P2, respectively. Comparison of the partial sequences of these ribosomal proteins suggests that these two yeasts are relatively far apart, phylogenetically.     

Yeast ribosomal proteins: XII. YS11 of Saccharomyces cerevisiae is a homologue to E. coli S4 according to the gene analysis.

We isolated and sequenced a gene, YS11A, encoding ribosomal protein YS11 of Saccharomyces cerevisiae. YS11A is one of two functional copies of the YS11 gene, located on chromosome XVI and transcribed in a lower amount than the other copy which is located on chromosome II. The disruption of YS11A has no effect on the growth of yeast. The 5'-flanking region contains a similar sequence to consensus UASrpg and the T-rich region. The open reading frame is interrupted with an intron located near the 5'-end. The predicted amino acid sequence reveals that yeast YS11 is a homologue to E. coli S4, one of the ram proteins, three chloroplast S4s and others out of the ribosomal protein sequences currently available.     

Unbalanced regulation of the ribosomal 5 S RNA-binding protein in Saccharomyces cerevisiae expressing mutant 5 S rRNAs.

A gene encoding the 5 S rRNA-binding protein (YL3) in yeast (Saccharomyces cerevisiae) was further characterized with respect to its chromosomal localization, the controlling sequence regions, and the influence of 5 S rRNA gene expression. Sequence and chromosome blot analyses localized the gene on chromosome XVI immediately downstream of a cytochrome oxidase assembly gene, COXII. S1 nuclease protection studies identified two major initiation sites, 20 and 65 nucleotides upstream of the coding sequence, and a single polyadenylation site, 98 nucleotides downstream of the stop codon. Northern blot analyses and S1 nuclease protection indicated a normal pattern of gene regulation in media supporting alternate rates of growth, but significantly unbalanced regulation was observed in the presence of mutant 5 S rRNA genes which under-produce RNA and result in reduced growth rates. The results suggest a co-ordinating regulatory mechanism which maintains appropriate levels of 5 S rRNA-protein complex; an internal control region-like sequence in the upstream region of the YL3 gene is consistent with this feedback mechanism.     

The contribution of a zinc finger motif to the function of yeast ribosomal protein YL37a

Eukaryotic ribosomes have a large number of proteins but the exact nature of their contribution to the structure and to the function of the particle is not known. Of the 78 proteins in yeast ribosomes, six have zinc finger motifs of the C2-C2 variety. Both genes encoding the essential yeast ribosomal protein YL37a, which has such a zinc finger motif, were disrupteXXPd. The double deletion, which is lethal, can be rescued with a plasmid-encoded copy of a YL37a gene. Mutations were constructed in a plasmid-encoded copy of YL37a; the mutations caused the cysteine residues in the motif (at positions 39, 42, 57 and 60) to be replaced, one at a time, with serine. The cysteine residue at position 39, the first of the four in the motif, is essential for the function of YL37a, since a C39S mutation did not complement the null phenotype. However, plasmids encoding variants with C42S, C57S, or C60S mutations in the zinc finger motif were able to rescue the null mutant. YL37a binds zinc, but none of the mutant proteins, C39S, C42S, C57S, or C60S, was able to bind the metal. Thus, all four cysteine residues are essential for the binding of zinc; only one, C39, is essential for the function of the ribosomal protein.     

The Drosophila homologue of ribosomal protein L8.

We have cloned the gene encoding the Drosophila melanogaster homologue of ribosomal protein L8. It contains two introns: one in the 5' untranslated region and the second in the beginning of the ORF, and encodes a 256-residue protein which is highly conserved when compared with RpL8 proteins of other organisms. The gene is present as a single copy in the Drosophila genome and maps at position 62E6-7 on polytene chromosomes. It is expressed ubiquitously at all stages of development. It is located close to the gene encoding RpL12 and both are candidate targets of the Minute mutation, M(3)LS2, mapped in the region 62E-63A.     

Identification of the yeast nuclear gene for the mitochondrial homologue of bacterial ribosomal protein L16.

An open reading frame encoding a member of the L16 family of ribosomal proteins is adjacent to the URA7 gene on the left arm of chromosome II in Saccharomyces cerevisiae. The predicted L16-like polypeptide is basic (pl 11.12), contains 232 amino acids (26.52 kDa) and has 36% amino acid sequence identity to E. coli L16. Immunoblot analysis with polyclonal antibodies to the L16-like polypeptide showed specific cross-reaction with a 22,000 Mr mitochondrial polypeptide that co-sediments with the large subunit of the mitochondrial ribosome in sucrose density gradients. The levels of the L16 mRNA and protein varied in response to carbon source. In [rho degree] cells lacking mitochondrial rRNA, the L16 mRNA accumulated at normal levels, but the protein was barely detectable, indicating RNA-dependent accumulation of the L16 protein. Gene disruption experiments demonstrated that the yeast mitochondrial L16 is an essential ribosomal protein in vivo.     

海鲈核糖体蛋白L8cDNA的克隆与进化分析

本文构建了海鲈(Lateolabrax japonicus)头肾全长eDNA文库.PCR方法扩增得到海鲈的核糖体蛋白L8基因,全长848bp,编码257个氨基酸,含有L2及L2-C两个保守区.进化分析结果表明,以L8为参照的进化鉴定结果同经典的分子生物学标准18s鉴定结果十分相似,因此核糖体蛋白L8基因L8可以作为鉴定物种进化程度的新标准.     

Sequence encoding ribosomal protein L33 of Lactococcus lactis.

A cloned fragment from Lactococcus lactis chromosome encoding the L33 ribosomal protein was sequenced. Two incomplete open reading frames (ORFs) were also found: the upstream ORF shows similarity to the tetracycline-resistance protein (Tet) of Bacillus stearothermophilus, and the downstream ORF shows homology to a protein of Bacillus subtilis participating in sporulation (SpoVE), and to proteins of Escherichia coli involved in cell division (FtsW) and the maintenance of cell shape (RodA).     

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.     

Krb1, a Suppressor of Mak7-1 (a Mutant Rpl4a), Is Rpl4b, a Second Ribosomal Protein L4 Gene, on a Fragment of Saccharomyces Chromosome Xii

The mak7-1 mutant loses the killer toxin-encoding M(1) dsRNA. MAK7 is RPL4A, one of two genes encoding ribosomal protein L4. KRB1 is a dominant suppressor of mak7-1 that is tightly centromere-linked, but not linked to centromere markers of chromosomes I-XVI. Our orthogonal field agarose gel electrophoresis analysis of chromosomal DNA from strains with KRB1 shows a novel band of ~250 kb. This band hybridizes with an RPL4B-specific probe, but not an RPL4A (MAK7)-specific probe. The RPL4B-specific probe also hybridizes to chromosome XII where the original RPL4B is located. KRB1 is meiotically linked to this extra chromosome. Disruption of either the RPL4B gene on chromosome XII or that on the extra chromosome results in loss of the killer phenotype and a decreased concentration of free 60S subunits. Thus, the RPL4B on the extra chromosome is KRB1 and is active. The extra chromosome contains chromosome XII sequence between Lambda 5345 clone (ATCC70558) and Lambda 6639 clone (ATCC71085) of Olson's Lambda library, indicating that KRB1 represents a chromosomal rearrangement involving chromosome XII and explaining the earlier genetic data.     

Nuclear-encoded tobacco chloroplast ribosomal protein L24. Protein identification, sequence analysis of cDNAs encoding its cytoplasmic precursor, and mRNA and genomic DNA analysis.

Using a Nicotiana tabacum leaf cDNA library in the expression vector lambda gt11, two cDNAs encoding the full-length precursor polypeptide (M(r) 20,696) of tobacco chloroplast ribosomal protein L24 were identified and sequenced. These cDNAs encode a mature protein of 146 amino acids (M(r) 16,418) with a transit peptide of 41 amino acids (M(r) 4,278). The mature tobacco L24 protein has 78, 65, 45, and 35% sequence identity with ribosomal proteins L24 of pea, spinach, Bacillus subtilis, and Escherichia coli, respectively. The transit peptide of tobacco L24 is 54 and 57% identical with that of L24 chloroplast ribosomal proteins of pea and spinach, respectively. An expressed beta-galactosidase:L24 fusion protein, bound to nitrocellulose filters, was used as affinity matrix to purify monospecific antibody to L24 protein. Using this monospecific antibody protein L24 was identified among high performance liquid chromatography (HPLC)-purified tobacco chloroplast ribosome 50 S subunit proteins. The predicted amino terminus of the mature L24 protein was confirmed by partial sequencing of the HPLC-purified L24 protein. Northern blot analysis revealed a single mRNA band (0.85-0.90 kilobase) corresponding in size to full-length L24 cDNA. The presence of multiple genes for L24 is suggested by Southern blot hybridization and characterization of two cDNAs for L24 which only differ in their 3'-noncoding sequences.     

Molecular cloning of some components of the translation apparatus of fission yeast Schizosaccharomyces pombe and a list of its cytoplasm ic proteins genes]

Full-length cDNAs of four new genes encoding cytoplasmic ribosomal proteins L14 and L20 (large ribosomal subunit) and S1 and S27 (small ribosomal subunit) were isolated and sequenced during the analysis of the fission yeast Schizosaccharomyces pombe genome. One of the Sz. pombe genes encoding translation elongation factor EF-2 was also cloned and its precise position on chromosome I established. A unified nomenclature was proposed, and the list of all known genetic determinants encoding cytoplasmic ribosomal proteins of Sz. pombe was compiled. By now, 76 genes/cDNAs encoding different ribosomal proteins have been identified in the fission yeast genome. Among them, 35 genes are duplicated and three homologous genes are identified for each of the ribosomal proteins L2, L16, P1, and P2.     

Characterisation ofSaccharomyces cerevisiae genes encoding ribosomal protein YL6

We have characterised aSaccharomyces cerevisiae cDNA (cDNA13), originally isolated on the basis of the short half-life of the corresponding mRNA. We show here that its sequence is closely related to that of the genes encoding ribosomal proteins K37, KD4 and K5 ofSchizosaccharomyces pombe. ‘mRNA13’ also behaves like other mRNAs encoding ribosomal proteins, in that its abundance increases sharply when glucose is added to cells grown on ethanol (nutrient-up shift), and declines when cells are subjected to a mild heat-shock. Unspliced mRNA13 accumulates when cells bearing a temperature-sensitive splicing mutation are grown at the restrictive temperature. The gene(s) corresponding to cDNA13, like other ribosomal protein genes ofS. cerevisiae, thus contain an intron. Southern blot analysis indicates the presence of two separate loci related to cDNA13 in theS. cerevisiae genome. From the sequence of one of these, a complete polypeptide sequence was deduced. The first 40 amino acids are identical to those of YL6, aS. cerevisiae ribosomal protein characterised only by N-terminal protein sequence analysis. There is clear evidence within the genomic sequence for the predicted intron, and for elements similar to those that regulate expression of otherS. cerevisiae ribosomal protein genes.     

Expression of a micro-protein.

The smallest known open reading frame encodes the ribosomal protein L41, which in yeast is composed of only 24 amino acids, 17 of which are arginine or lysine. Because of the unique problems that might attend the translation of such a short open reading frame, we have investigated the properties and the translation of the mRNAs encoding L41. In Saccharomyces cerevisiae L41 is encoded by two linked genes, RPL41A and RPL41B. These genes give rise to mRNAs that have short 5' leaders of 18 and 22 nucleotides and rather long 3' leaders of 203 and 210 nucleotides not including their poly(A) tails. The mRNAs are translated exclusively on monosomes, suggesting that ribosomes do not remain attached to the mRNA after termination of translation. Calculations based on the abundance of ribosomes and of L41 mRNA indicate that the entire translation event, from initiation through termination, must occur in approximately 2 s. Termination of translation after only 25 codons does not subject the mRNAs encoding L41 to nonsense-mediated decay. Surprisingly, despite the L41 ribosomal protein being conserved from the archaea through the mammalia, S. cerevisiae can grow relatively normally after deletion of both RPL41A and RPL41B.