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.     

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.     

Ribosomal DNA of Caenorhabditis elegans

We have characterized the organization of the genes coding for 18 S, 5·8 S and 26 S ribosomal RNAs in the nematode Caenorhabditis elegans. These ribosomal genes, present in about 55 copies per haploid genome, alternate in a repeating tandem array. The repeating unit is only 7000 base-pairs, containing a non-transcribed spacer of no more than 1000 base-pairs. Most of the repeating units have identical restriction maps, but one repeat contains a deletion of 2900 base-pairs, which eliminates all or part of the 18 S coding region. We have found no difference in the major ribosomal DNA restriction endonuclease cleavage patterns between two interbreeding strains of C. elegans, but found differences between C. elegans and the closely related Caenorhabditis briggsae.     

Cloning and sequence determination of the gene encoding the largest subunit of the fission yeast Schizosaccharomyces pombe RNA polymerase I

The gene encoding the largest subunit of RNA polymerase I (SPRPA190) was cloned from the fission yeast Schizosaccharomyces pombe by cross-hybridization with a probe containing part of the corresponding Saccharomyces cerevisiae gene RPA190. The SPRPA190 gene is present in a single copy per haploid genome and is essential for cell growth. The polypeptide encoded by this gene, as deduced from the nucleotide sequence of the uninterrupted coding frame, consists of 1689 amino acids and its calculated Mr is 189,300. The amino acid identity between the subunits of the two yeast species is 50%. Amino acid sequence conservation covers the regions previously suggested to be functionally important for the S. cerevisiae enzyme. In addition, two markedly hydrophilic regions recognized in the S. cerevisiae polypeptide can also be recognized in the S. pombe polypeptide in approximately the same positions, even though the amino acid sequences in these regions are diverged from each other. In the 5'-flanking region of the gene, several nucleotide sequence elements are detected which are also found in the two S. pombe ribosomal protein genes so far sequenced.     

Dictyostelium discoideum acidic ribosomal phosphoproteins: identification and in vitro phosphorylation

Four acidic phosphoproteins from the ribosomes of the slime mold Dictyostelium discoideum have been identified and partially characterized. These proteins are selectively released from ribosomal particles by salt/ethanol washes, have low molecular weight and acidic pI, and tend to aggregate in solution to form homodimers. These features correspond to proteins of different origins that have been included in the conserved family of eukaryotic A-ribosomal proteins, and, therefore, we have named them Dictyostelium ribosomal proteins A1, A2, A3 and A4. We also demonstrate that Dictyostelium ribosomal A-proteins are specifically phosphorylated in vitro by a type II casein kinase previously identified in Dictyostelium. Isoelectric focusing separation has permitted us to identify four proteins (or P-proteins) that may consist of the phosphorylated forms of A-proteins. A-proteins from Dictyostelium and yeast do not present immunological cross-reactivity. Dictyostelium A-proteins contain, therefore, some specific features in their amino acid sequence that distinguish them from other members of the conserved eukaryotic A-protein family; this conclusion is coherent with data deduced from the nucleotide sequence of cDNA clones encoding two Dictyostelium A-proteins (P1 and P2) which we have recently reported.     

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.     

The 5S RNA genes of Schizosaccharomyces pombe.

The genomic arrangement and sequences of S. pombe 5S RNA genes are reported here. The 5S gene sequences appear to be dispersed within the genome, and are found independently of other rRNA genes. The sequences of two 5S genes examined show identical coding regions of 119 base pairs but have widely varying flanking sequences. A tRNAAsp gene is found in the 3' flanking region of one of the 5S genes. The tRNAAsp gene is faithfully transcribed in an X. laevis in vitro system, while the 5S genes are not transcribed in this system. The phylogenetic position of S. pombe is examined through comparison of 5S RNA sequences.     

Organization, primary structure, and evolution of histone H2A and H2B genes of the fission yeast Schizosaccharomyces pombe.

The histone H2A and H2B genes of the fission yeast Schizosaccharomyces pombe were cloned and sequenced. Southern blot and sequence analyses showed that, unlike other eucaryotes, Saccharomyces cerevisiae included, S. pombe has unequal numbers of these genes, containing two histone H2A genes (H2A-alpha and -beta) and only one H2B gene (H2B-alpha) per haploid genome. H2A- and H2B-alpha are adjacent to each other and are divergently transcribed. H2A-beta has no other histone gene in close proximity. Preceding both H2A-alpha and -beta is a highly conserved 19-base-pair sequence (5'-CATCAC/AAACCCTAACCCTG-3'). The H2A DNA sequences encode two histone H2A subtypes differing in amino acid sequence (three residues) and size (H2A-alpha, 131 residues; H2A-beta, 130 residues). H2B-alpha codes for a 125-amino-acid protein. Sequence evolution is extensive between S. pombe and S. cerevisiae and displays unique patterns of divergence. Certain N-terminal sequences normally divergent between eucaryotes are conserved between the two yeasts. In contrast, the normally conserved hydrophobic core of H2A is as divergent between the yeasts as between S. pombe and calf.     

Interspersion of histone and 5S RNA genes in Artemia

Four recombinant lambda phage containing histone genes were selected from a library of Artemia genomic DNA fragments. The histone gene organization of Artemia resembles that of other invertebrates in that all five genes are clustered and repeated in tandem with approximate repeat lengths of 8.5 kb and 9.3 kb. Each recombinant lambda phage isolate hybridizes with five histone mRNAs and unexpectedly also with 5S ribosomal RNA. Hybridization kinetics have shown the number of histone genes to be about 95-100 copies per haploid genome. An identical number of copies was determined for a hybridization probe containing the 5S gene but no histone genes. We have not found any evidence for a separate set of repeated 5S genes outside this histone + 5S block.     

Numbers of 5S and tRNA genes in macro- and micronuclei of Tetrahymena pyriformis

Macronuclei of Tetrahymena pyriformis contain approximately 200 copies of the genes for 25S and 17S ribosomal RNA (rRNA) per haploid genome. Micronuclei, however, contain only a few copies of the rRNA genes per haploid complement. Since macronuclei develop from, products of meiosis, fertilization and division of micronuclei, we suggested that the multiple copies of the rRNA genes in macronuclei are generated by amplification of the small number of genes in micronuclei (Yao et al., 1974). This process provides a simple mechanism for maintaining the homogeneity of the repeated rRNA genes. To test if amplification is a general mechanism operating on all repeated genes in Tetrahymena, we have examined the numbers of 5S RNA and tRNA genes in macro- and micronuclei. 5S RNA was purified by polyacrylamide gel electrophoresis and hybridized to saturation against macro- and micronuclear DNA. Approximately 0.013–0.014% of macronuclear DNA and about 0.009% of micronuclear DNA is complementary to 5S RNA. After correcting for the differences in the DNA sequence complexities between the two nuclei, we calculate that there are 300–350 5S genes per haploid macro- or micronuclear genome. From these data we conclude that there is little or no detectable amplification of the 5S genes in macronuclei relative to micronuclei. Similar studies using tRNA indicate that these genes are also highly repeated in both nuclei; about 800 genes are present per haploid genome. Thus, amplification from a small number of genes can be excluded as the mechanism for generating the repeated copies of the 5S and tRNA genes in Tetrahymena and it is likely that another, as yet unidentified, mechanism operates to maintain the homogeneity of these genes.     

Isolation and structural analysis of ribosomal protein genes in Xenopus laevis. Homology between sequences present in the gene and in several different messenger RNAs

The ribosomal protein genes are present in two to four copies per haploid genome of Xenopus laevis. Using cloned complementary DNA probes, we have isolated, from a genomic library of X. laevis, several clones containing genes for two different ribosomal proteins (L1 and L14). These genes contain intervening sequences. In the case of the L1 gene, the exons are 100 to 200 base-pairs long and the introns, on average, 400 base-pairs. Along the genomic fragments, two different classes of repetitive DNA are present: highly and middle repetitive DNA. Both are evolutionarily unstable as shown by hybridization to Xenopus tropicalis DNA. Several introns of the gene coding for protein L1 contain middle repetitive sequences. Hybridization and hybrid-released translation experiments have shown that sequences inside the two genes hybridize to several poly(A) messenger RNAs. Some of the products encoded by these mRNA have electrophoretic properties of ribosomal proteins.     

Vesicle-mediated protein transport pathways to the vacuole in Schizosaccharomyces pombe

The vacuole of Saccharomyces cerevisiae plays essential roles not only for osmoregulation and ion homeostasis but also down-regulation (degradation) of cell surface proteins and protein and organellar turnover. Genetic selections and genome-wide screens in S. cerevisiae have resulted in the identification of a large number of genes required for delivery of proteins to the vacuole. Although the complete genome sequence of the fission yeast Schizosaccharomyces pombe has been reported, there have been few reports on the proteins required for vacuolar protein transport and vacuolar biogenesis in S. pombe. Recent progress in the S. pombe genome project of has revealed that most of the genes required for vacuolar biogenesis and protein transport are conserved between S. pombe and S. cerevisiae. This suggests that the basic machinery of vesicle-mediated protein delivery to the vacuole is conserved between the two yeasts. Identification and characterization of the fission yeast counterparts of the budding yeast Vps and Vps-related proteins have facilitated our understanding of protein transport pathways to the vacuole in S. pombe. This review focuses on the recent advances in vesicle-mediated protein transport to the vacuole in S. pombe.     

Inter- and intra-strain variation in the 5.8S ribosomal RNA and internal transcribed spacer sequences of Entamoeba histolytica and comparison with Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens

The ribosomal RNA genes in Entamoeba histolytica are located on circular DNA molecules in about 200 copies per genome equivalent. Nucleotide sequence analysis of the 5.8S rRNA gene and the flanking internal transcribed spacers was carried out to determine the degree of sequence divergence in the multiple rRNA gene copies of a given strain; amongst three different E. histolytica strains (HM-1:IMSS, Rahman and HK-9); and amongst four species of Entamoeba (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens). The results show that all rRNA gene copies of a given strain are identical. Few nucleotide positions varied between strains of a species but the differences were very pronounced amongst species. In general, the internal transcribed spacer 2 sequence was more variable and may be useful for strain- and species-identification. The 5.8S rRNA gene and the internal transcribed spacer 2 of E. invadens were unusually small in size.     

Nucleotide sequences of the 5S ribosomal RNA genes and their adjacent regions in Schizosaccharomyces pombe.

The organization of 5S ribosomal RNA (rRNA) genes in the genome of Schizosaccharomyces pombe has been investigated by restriction and hybridization analyses. The 5S rRNA genes were not linked to the other three species of rRNA genes which formed a repeating unit of 6.9 megadaltons, but located in other regions surrounded by heterogeneous sequences. The 5S rRNA gene organization in S. pombe is therefore different from those in other yeasts; Saccharomyces cerevisiae and Torulopsis utilis. Four restriction segments of different sizes each containing a single 5S rRNA gene were cloned on a bacterial plasmid, and the sequences in and around the RNA coding regions were determined. In the RNA coding regions, the sequences in four clones were identical with an exception that one residue has been substituted in one clone. In the flanking regions, the sequences were extremely rich in the AT-content and highly heterogeneous. The sequences were also markedly different from those in the corresponding regions of the other two yeasts. THe presence of T-clusters in the regions immediately after the RNA coding sequences was only notable homology among the four clones and the other two yeasts.     

A new estimate of human ribosomal gene number.

Radioactively labelled DNAs (5 X 10(6) cpm/mug) complementary to human 18 S and 28 S ribosomal RNA were synthesized using RNA-directed DNA polymerase (EC 2.7.7.7). These complementary DNAs were used to measure human ribosomal gene numbers by two independent methods, both of which indicated numbers at least four-fold lower than those previously reported. First, the kinetics of the annealing of the complementary DNAs with total human placental DNA indicated that the number of both 18-S and 28-S ribosomal genes per haploid genome is approximately 50. Second, saturation experiments in which a constant amount of DNA was annealed with increasing amounts of complementary DNA also indicated that the number of 28 S ribosomal RNA genes in human placental and spleen DNA is is about 50 per haploid genome.