The nucleotide sequence of 5S ribosomal RNA from Schizosaccharomyces pombe

The nucleotide sequence of 5S rRNA from the fission yeast, S. pombe, has been established by post labeling procedures combined with cataloging RNase T1- and A-oligonucleotides derived from unlabeled 5S rRNA. The sequence consists of 119 nucleotides without a modified base and shows more dissimilarities (at 38 positions) from that of S. cerevisiae than from that of humans (at 33 positions).     

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.     

The 5.8S RNA gene sequence and the ribosomal repeat of Schizosaccharomyces pombe

We have characterized the rRNA gene repeat in Schizosaccharomyces pombe. This repeat, which does not contain the 5S RNA gene, is found in a 10.4 kb HindIII DNA fragment. We have determined the nucleotide sequences of the S. pombe 5.8S RNA gene and intergenic spacers from two different 10.4 kb DNA fragments. Analysis of isolated total cellular 5.8S RNA revealed the presence of eight species of 5.8S RNA, differing in the number of nucleotides at the 5'-end. The eight 4.8S RNA species vary in length from 158 to 165 nucleotides. Apart from the heterogeneity observed at the 5'-end, the sequence of the eight 5.8S RNA species appears to be identical and is the same sequence as coded for by the 5.8S genes. The gene sequence shows great homology to the 5.8S RNA genes or S. cerevisiae and N. crassa. Most of the base differences are confined to the highly variable stem though to be involved in co-axial helix stacking with the 25S RNA, where base pairing is nearly identical despite the sequence differences. Secondary structure models are examined in light of 5.8S RNA oligonucleotide conservation across species from yeasts to higher eukaryotes.     

Characterization of Schizosaccharomyces pombe RNA triphosphatase

RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the β–γ phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast Schizosaccharomyces pombe. Pct1p hydrolyzes the γ phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and P_i in the presence of manganese or cobalt (K_m = 19 µM ATP; k_cat = 67 s^–1). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I_0.5 = 1 mM), pyrophosphate (I_0.5 = 0.4 mM) and tripolyphosphate (I_0.5 = 30 µM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.     

The nucleotide sequence of the major glutamate transfer RNA from Schizosaccharomyces pombe.

The nucleotide sequence of glutamate tRNA1 from Schizosaccharomyces pombe was determined to be pU-C-C-G-U-U-G-U-m1G-G-U-C-C-A-A-C-G-G-C-D-A-G-G-A-U-U-C-G-U-C-G-C-U-U-U*-C-A-C-C-G-A-C-G-G-G-A-G-m5C-G-G-G-G-T-psi-C-G-A-C-U-C-C-C-C-G-C-A-A-C-G-G-A-G-C-C-AOH. The sequence differs markedly from that of S. cerevisiae tRNAGlu. S. pombe glutamate tRNA1 can be aminoacylated by the homologous glutaminyl-tRNA synthetase as well as by the corresponding enzyme from S. cerevisiae.     

Identification of ras-related, YPT family genes in Schizosaccharomyces pombe.

Screening for genes homologous to ras in Schizosaccharomyces pombe resulted in the isolation of a homolog of Saccharomyces cerevisiae YPT1. This S. pombe gene, named ypt3, has a coding capacity of 214 amino acids interrupted by two introns, and is essential for cell growth. Two more YPT1 homologs were isolated from S. pombe using a part of the ypt3 gene as the probe. One of them, named ypt1, is highly homologous to S. cerevisiae YPT1 and mouse ypt1 and is essential for cell growth. This gene has four introns and encodes 203 amino acids. Its cDNA placed downstream of the S. cerevisiae GAL7 promoter could complement S. cerevisiae ypt1-, indicating that Sp ypt1 and Sc YPT1 are functionally homologous. The other isolate, named ryh1, and a fourth homolog, ypt2, have been characterized by Gallwitz and co-workers. The ypt1, ypt2 and ypt3 genes, but not ryh1, constitute a family, their products having double cysteine as their C terminus and serine in place of a glycine residue highly conserved in ras proteins (mammalian Gly-12 or S. pombe Gly-17). The physiological roles of these genes appear to be distinct because each of them is indispensable for cell growth.     

The 26S proteasome of the fission yeast Schizosaccharomyces pombe

The 26S proteasome is the multiprotein complex that degrades proteins that have been marked for destruction by the ubiquitin pathway. It is made up of two multisubunit complexes, the 20S catalytic core and the 19S regulatory complex. We describe the isolation and characterization of conditional mutants in the regulatory complex and their use to investigate interactions between different subunits. In addition we have investigated the localization of the 26S proteasome in fission yeast, by immunofluorescence in fixed cells and live cells with the use of a GFP-tagged subunit. Surprisingly, we find that in mitotic cells the 26S proteasome occupies a discrete intracellular compartment, the nuclear periphery. Electron microscopic analysis demonstrates that the complex resides inside the nuclear envelope. During meiosis the localization showed a more dynamic distribution. In meiosis I the proteasome remained around the nuclear periphery. However, during meiosis II there was a dramatic relocalization: initially, the signal occupied the area between the dividing nuclei, but at the end of mitosis the signal dispersed, returning to the nuclear periphery on ascospore formation. This observation implies that the nuclear periphery is a major site of proteolysis in yeast during mitotic growth and raises important questions about the function of the 26S proteasome in protein degradation.     

Inactivation of nonsense suppressor transfer RNA genes in Schizosaccharomyces pombe. Intergenic conversion and hot spots of mutation

Intergenic conversion is a mechanism for the concerted evolution of repeated DNA sequences. A new approach for the isolation of intergenic convertants of serine tRNA genes in the yeast Schizosaccharomyces pombe is described. Contrary to a previous scheme, the intergenic conversion events studied in this case need not result in functional tRNA genes. The procedure utilizes crosses of strains that are homozygous for an active UGA suppressor tRNA gene, and the resulting progeny spores are screened for loss of suppressor activity. In this way, intergenic convertants of a tRNA gene are identified that inherit varying stretches of DNA sequence from either of two other tRNA genes. The information transferred between genes includes anticodon and intron sequences. Two of the three tRNA genes involved in these information transfers are located on different chromosomes. The results indicate that intergenic conversion is a conservative process. No infidelity is observed in the nucleotide sequence transfers. This provides further evidence for the hypothesis that intergenic conversion and allelic conversion are the result of the same molecular mechanism. The screening procedure for intergenic revertants also yields spontaneous mutations that inactivate the suppressor tRNA gene. Point mutations and insertions of A occur at various sites at low frequency. In contrast, A insertions at one specific site occur with high frequency in each of the three tRNA genes. This new type of mutation hot spot is found also in vegetative cells.     

The genes for 5 S ribosomal RNA and transfer RNA in Tetrahymena pyriformis.

In the present communication a characterization of the 5 S rRNA genes and the tRNA genes of Tetrahymena pyriformis has been performed. The number of 5 S rRNA and tRNA genes in the macromolecular DNA has been established. Furthermore no sequence homology is observed for these genes. The number of both types of genes does not change significantly under starvation conditions. The genomic organization of the 5 S rRNA and tRNA genes has been investigated. From in vivo replication studies it is concluded, that replication of both 5 S rRNA and tRNA genes takes place throughout the whole S-period.     

The site of 5S RNA genes in primates

A major site of genes for 5S RNA has been localized in representative members of the family Pongidae by means of hybridization in situ. These genes are shown to be concentrated in the most distal bands of the primate chromosome arm homologous to human chromosome 1q.