Molecular cloning and analysis of the CRY1 gene: a yeast ribosomal protein gene.

Using cloned DNA from the vicinity of the yeast mating type locus (MAT) as a probe, the wild type allele of the cryptopleurine resistance gene CRY1 has been isolated by the technique of chromosome walking and has been shown to be identical to the gene for ribosomal protein 59. A recessive cryR1 allele has also been cloned, using the integration excision method. The genetic distance from MAT to CRY1 is 2.2 cM, while the physical distance is 21 kb, giving a ratio of about 10 kb/cM for this interval. The phenotypic expression of both plasmid borne alleles of the gene can be detected in vivo. The use of this gene as a hybridization probe to examine RNA processing defects in the rna 2, rna 3, rna 4, rna 8, and rna 11 mutants is also discussed.     

A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif.

The RNA1 gene from Saccharomyces cerevisiae is defined by the temperature-sensitive rna1-1 mutation that interferes with the maturation and/or nucleocytoplasmic transport of RNA. We describe the purification of a 44-kDa protein from the evolutionary distant fission yeast Schizosaccharomyces pombe and the cloning and sequence analysis of the corresponding gene. Although this protein shares only 42% sequence identity with the RNA1 gene product, it represents a functional homologue because the expression of the S. pombe gene in S. cerevisiae complements the rna1-1 defect. Disruption in S. pombe of the gene encoding the 44-kDa protein, for which we propose the name S. pombe rna1p, reveals that it is essential for growth. Our analysis of purified S. pombe rna1p represents the first biochemical characterization of an RNA1 gene product and reveals that it is a monomeric protein of globular shape. Cell fractionation and immunofluorescence microscopy indicate that rna1p is a cytoplasmic protein possibly enriched in the nuclear periphery. We identify a sequence motif of 29 residues, which is rich in leucine and repeated eight times both in S. pombe and in S. cerevisiae rna1p. Similar leucine-rich repeats present in a series of other proteins, e.g., the mammalian ribonuclease/angiogenin inhibitor, adenylyl cyclase from S. cerevisiae, the toll protein from Drosophila melanogaster, and the sds22 protein phosphatase regulatory subunit from S. pombe, are thought to be involved in protein-protein interactions. Thus rna1p may act as a scaffold protein possibly interacting in the nuclear periphery with a protein ligand that could be associated with exported RNA.     

Mutations inSTS1 suppress the defect in 3′ mRNA processing caused by therna15-2 mutation inSaccharomyces cerevisiae

In a search for proteins associated with Rna15p in processing the 3′ ends of messenger RNAs, we have looked for suppressors that correct, even partially, the thermosensitive growth defect of therna15-2 mutant. Mutations in a single locus that we namedSSM5, were able to suppress both the thermosensitivity of cell growth and the mRNA 3′ processing defect associated with therna15-2 mutation, but only slightly alleviated the thermosensitive growth defect of anrna14-1 mutant. Thessm5-1 mutant is sensitive to hydroxyurea at 37° C, a drug that inhibits DNA synthesis. By screening for complementation of the hydroxyurea-sensitive phenotype we cloned the corresponding wild-type gene and found that it corresponds to the essential geneSTS1 (also namedDBF8). Sts1p has an apparent molecular weight of 30 kDa and was confirmed to be a cytosolic protein by immunofluorescence analysis. Western blot analysis indicates that the thermosensitive mutant strainsrna15-2, rna14-1 andpap1-1 present a very low level of the Rna15p at 37° C. Thessm5-1 mutation restores the level of Rna15p in therna15-2 ssm5-1 double mutant. Use of the two-hybrid system suggests that Sts1p does not interact directly with Rna15p, but may be active as a homodimer. The present data suggest that Sts1p may play a role in the transport of Rna15p from the cytoplasm to the nucleus.     

Primary and secondary structure of black beetle virus RNA2, the genomic messenger for BBV coat protein precursor.

The nucleotide sequence of black beetle virion (BBV) RNA2 has been determined. RNA2 is 1399 b long. Its 5' terminus is capped. Its 3' terminus has an unidentified moiety that renders the RNA resistent to polyadenylation and ligation. The first AUG codon at base 23 is followed by an open reading frame for a protein 407 amino acids long, the predicted size of coat protein precursor. A second open reading frame for a putative protein 72 amino acid residues long begins at base 1110. No other large open reading frames exist. The 5' half of the RNA can be folded into a long, imperfect hairpin of high predicted stability. The 3' half of the RNA can fold into a complex set of multiply bifurcated stem and loop regions.     

The MAK11 protein is essential for cell growth and replication of M double-stranded RNA and is apparently a membrane-associated protein

MAK11 is a gene necessary for the maintenance of killer M1 double-stranded RNA, but not for other cellular double-stranded RNAs (L-A, L-BC, T, W). The DNA sequence of this gene revealed a 1407-base pair open reading frame, which corresponds to a 54-kDa protein. The C-terminal region is lysine-rich and is necessary for mak11-complementing activity. The N-terminal 24 amino acids of the open reading frame include 16 hydrophobic amino acids, 4 basic residues, and 4 neutral amino acids; this sequence could span a membrane. We constructed a MAK11-lacZ fusion that includes the entire MAK11 protein and complements the mak11-1 mutation. The fusion protein was localized in a membrane fraction as shown by centrifugation in Percoll gradients. The fusion protein could be released from the membrane fraction by salt washing. Western blotting of protein, isolated from the membrane fraction and purified by p-aminophenyl-beta-D-thiogalactoside-agarose column chromatography, revealed a fusion protein monomer of 170 kDa which agrees with the predicted molecular weight. While the mak11-1 mutation results in specific loss of M1 double-stranded RNA without any apparent growth defect, replacing a 792-base pair internal EcoRV fragment of MAK11 with the URA3 gene (gene disruption) resulted in a lethal mutation.     

Cloning,sequencing, and expression of the gene from bacillus circulans that codes for a heparinase that degrades both heparin and heparan sulfate

The gene, designated hep, coding for a heparinase that degrades both heparin and heparan sulfate, was cloned from Bacillus circulans HpT298. Nucleotide sequence analysis showed that the open reading frame of the hep gene consists of 3,150 bp, encoding a precursor protein of 1,050 amino acids with a molecular mass of 116.5 kDa. A homology search found that the deduced amino acid sequence has partial similarity with enzymes belonging to the family of acidic polysaccharide lyases that degrade chondroitin sulfate and hyaluronic acid. Recombinant mature heparinase (111.2 kDa) was produced by the addition of IPTG from Escherichia coli harboring pETHEP with an open reading frame of the mature hep gene and was purified to homogeneity by SDS-polyacrylamide gel electrophoresis. Analyses of substrate specificity and degraded disaccharides indicated that the recombinant enzyme acts on both heparin and HS, as does heparinase purified from the wild-type strain.     

Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle

vps3 mutants of the yeast Saccharomyces cerevisiae are impaired in the sorting of newly synthesized soluble vacuolar proteins and in the acidification of the vacuole (Rothman, J. H., and T. H. Stevens. Cell. 47:1041-1051; Rothman, J. H., C. T. Yamashiro, C. K. Raymond, P. M. Kane, and T. H. Stevens. 1989. J. Cell Biol. 109:93-100). The VPS3 gene, which was cloned using a novel selection procedure, encodes a low abundance, hydrophilic protein of 117 kD that most likely resides in the cytoplasm. Yeast strains bearing a deletion of the VPS3 gene (vps3-delta 1) are viable, yet their growth rate is significantly reduced relative to wild-type cells. Temperature shift experiments with strains carrying a temperature conditional vps3 allele demonstrate that cells rapidly lose the capacity to sort the vacuolar protein carboxypeptidase Y upon loss of VPS3 function. Vacuolar morphology was examined in wild-type and vps3-delta 1 yeast strains by fluorescence microscopy. The vacuoles in wild-type yeast cells are morphologically complex, and they appear to be actively partitioned between mother cells and buds during an early phase of bud growth. Vacuolar morphology in vps3-delta 1 mutants is significantly altered from the wild-type pattern, and the vacuolar segregation process seen in wild-type strains is defective in these mutants. With the exception of a vacuolar acidification defect, the phenotypes of vps3-delta 1 strains are significantly different from those of mutants lacking the vacuolar proton-translocating ATPase. These data demonstrate that the acidification defect in vps3-delta 1 cells is not the primary cause of the pleiotropic defects in vacuolar function observed in these mutants.     

Rna14p, a component of the yeast nuclear cleavage/polyadenylation factor I, is also localised in mitochondria

RNA14 was identified as a gene involved in premessenger RNA cleavage and polyadenylation. These processing steps take place in the nucleus, but the Rna14p protein is distributed in both the nucleus and the cytoplasm. By subcellular fractionation, we show here that the cytoplasmic fraction is localised in the mitochondria. In order to understand the role played by Rna14p in mitochondria, we have searched for new thermosensitive alleles of RNA14. We isolated thirteen new mutants. Some of them are deficient in mRNA cleavage and polyadenylation at the restrictive temperature - like the first mutant identified (rna14-1). However, others do not appear to be impaired in any of the steps in RNA metabolism investigated, nor do they appear to be involved in the replication or expression of mitochondrial DNA or in respiration. The localisation data strongly suggest that, besides an essential function in mRNA polyadenylation, the Rna14p protein has a non essential function in mitochondrial metabolism.     

Primary structure of non-histone protein HMG1 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.     

The DNA sequence of the nuclear gene coding for the 17-kd subunit VI of the yeast ubiquinol-cytochrome c reductase: a protein with an extremely high content of acidic amino acids

We have determined the DNA sequence of the nuclear gene coding for the 17-kd subunit VI of the ubiquinol-cytochrome c reductase. The reading frame found encodes a putative polypeptide of 17 394 daltons. This protein is highly unusual: 38% of its residues are acidic and 14% are basic amino acids. The most notable feature in the protein sequence is a stretch of 25 consecutive acidic amino acids. The polypeptide has homology with the 9-kd so-called 'hinge' protein of beef-heart complex III, which also has a cluster of acidic residues. Acidic amino acids are likely to be essential for the function of these proteins, since their degree of conservation is higher than that of other residues.     

The nucleotide sequence of the HEM1 gene and evidence for a precursor form of the mitochondrial 5-aminolevulinate synthase in Saccharomyces cerevisiae

The biosynthesis of yeast 5-aminolevulinate (ALA) synthase, a mitochondrial protein encoded by the nuclear HEM1 gene, has been studied in vitro in a cell-free translation system and in vivo in whole cells. In vitro translation of mRNA hybrid-selected by the cloned HEM1 gene, or of total RNA followed by immunoprecipitation with anti-(ALA synthase) antibody yielded a single polypeptide of higher molecular mass than the purified ALA synthase. This larger form, also seen in pulse-labeled cells, can be post-translationally processed by isolated mitochondria. These results show that the cytoplasmically made ALA synthase is synthesized with a cleavable extension which was estimated to be about 3.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The complete nucleotide sequence of the HEM1 gene and its flanking regions was determined. The 5' ends of the HEM1 mRNAs map from -76 to -63 nucleotides upstream of the translation initiation codon. The open reading frame of 1644 base pairs encodes a protein of 548 amino acids with a calculated Mr of 59,275. The predicted amino-terminal sequence of the protein is strongly basic (five basic and no acidic amino acids within the first 35 residues), rich in serine and threonine and must represent the transient presequence that targets this protein to the mitochondria. Comparison of deduced amino acid sequences indicates a clear homology between the mature yeast and chick embryo liver ALA synthases.     

Myb-binding protein 1a augments AhR-dependent gene expression

We have studied the mechanism by which an acidic domain (amino acids 515-583) of the aromatic hydrocarbon receptor (AhR) transactivates a target gene. Studies with glutathione S-transferase fusion proteins demonstrate that the wild-type acidic domain associates in vitro with Myb-binding protein 1a, whereas a mutant domain (F542A, I569A) does not. AhR-defective cells reconstituted with an AhR containing the wild-type acidic domain exhibit normal AhR function; however, cells reconstituted with an AhR containing the mutant acidic domain do not function normally. Transient transfection of Myb-binding protein 1a into mouse hepatoma cells is associated with augmentation of AhR-dependent gene expression. Such augmentation does not occur when Myb-binding protein 1a is transfected into AhR-defective cells that have been reconstituted with an AhR that lacks the acidic domain. We infer that 1) Myb-binding protein 1a associates with AhR, thereby enhancing transactivation, and 2) the presence of AhR's acidic domain is both necessary and sufficient for Myb-binding protein 1a to increase AhR-dependent gene expression.     

Primary structure of non-histone chromosomal protein HMG2 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone for the entire sequence of pig thymus non-histone protein HMG2 are described. cDNA the size of 1153 nucleotides contains an open reading frame of 627 nucleotides. The 5'-untranslated region of 146 nucleotides is extremely rich in GC residues whereas the 3'-untranslated region of 380 nucleotides is rich in AT residues. The open reading frame encodes 209 amino acids, which contain a unique continuous run of 23 acidic amino acids at the C-terminal. The deduced amino acid sequence is 79% homologous to that of HMG1 protein from the same source which we reported [Tsuda, K., Kikuchi, M., Mori, K., Waga, S., & Yoshida, M. (1988) Biochemistry 27, 6159-6163]. In addition, the hydropathy index profiles of both proteins are very similar, supporting that they have similar structural features. Northern analysis of poly(A+) RNA reveals that a single-sized mRNA codes for HMG2 protein. Southern analysis suggests that the HMG2 coding gene is homogeneous within the pig thymus genome.