Vaccinia virus encodes an essential gene with strong homology to protein kinases

The B1 gene of vaccinia virus encodes a 34-kDa protein which is essential for viral replication. Temperature-sensitive mutants bearing lesions in this gene arrest at the stage of DNA replication during nonpermissive infections. In this report, the sequence of the 34-kDa open reading frame is presented, and the mutations in two ts alleles are identified. Analysis of the deduced protein sequence reveals strong homology with catalytic domains of numerous protein kinases. The lesion in one of the mutants alters an invariant glycine residue within one such domain.     

The Nop60B gene of Drosophila encodes an essential nucleolar protein that functions in yeast

The Cbf5 protein of Saccharomyces cerevisiae was originally identified as a low-affinity centromeric DNA-binding protein, and cbf5 mutants have a defect in rRNA synthesis. A closely related protein from mammals, NAP57, is a nucleolar protein that coimmunoprecipitates with the nucleolar phosphoprotein Nopp140. To study the function of this protein family in a higher eukaryote that is amenable to genetic approaches, the gene encoding a Drosophilamelanogaster homolog, Nop60B, was identified. The predicted Drosophila protein shares a high degree of sequence identity over a 380-residue region with both the mammalian and yeast proteins, and shares several conserved motifs with the prokaryotic tRNA pseudouridine 55 synthases. Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development. Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis. Nop60B mutants were generated and shown to be homozygous lethal. The Drosophila gene can rescue the lethal phenotype of yeast cbf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila.     

Isolation and sequencing of NOP1. A yeast gene encoding a nucleolar protein homologous to a human autoimmune antigen

We have identified the gene for the yeast nucleolar protein p38 and deduced the primary structure of p38 from its sequence. We propose the name NOP1 (nucleolar protein 1) for this gene. NOP1 encodes a 327 amino acid protein of 34,470 daltons and is flanked by potential promoter and polyadenylation sequences. Blot analyses indicate that the mRNA transcribed from NOP1 is approximately 1.3 kilobases in size and that there is one NOP1 gene per haploid genome. The amino-terminal sequence of p38 is homologous with the 31 known amino-terminal residues of the autoimmune antigen fibrillarin, confirming the previously observed similarity between p38 and this mammalian nucleolar protein. Consistent with this, p38 cross-reacts with serum from a patient with the autoimmune disease scleroderma. A putative nuclear localization signal can be identified in p38. Interestingly, a repetitive amino acid sequence motif begins near the amino terminus of p38. This motif is approximately 80 residues long, is rich in glycine and arginine, and shows striking sequence homology to mammalian nucleolins and certain nucleic acid binding proteins.     

Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase.

The MPS1 gene has been previously identified by a mutant allele that shows defects in spindle pole body (SPB) duplication and cell cycle control. The SPB is the centrosome-equivalent organelle in the yeast Saccharomyces cerevisiae, and it nucleates all the microtubules in the cell. We report the isolation of the MPS1 gene, which encodes an essential protein kinase homolog. The MPS1 open reading frame has been fused to those that encode the LexA protein or the GST protein and both of these constructs function in yeast. The fusion proteins have been affinity-purified from yeast extracts and the GST chimeric protein has been found to be a phosphoprotein. Both proteins have been used to demonstrate intrinsic in vitro protein kinase activity of Mps1p against exogenous substrates and itself (autophosphorylation). A mutation predicted to abolish kinase function not only eliminates in vitro protein kinase activity, but also behaves like a null mutation in vivo, suggesting that kinase activity contributes to the essential function of the protein. Phosphoamino acid analysis of substrates phosphorylated by Mps1p indicates that this kinase can phosphorylate serine, threonine and tyrosine residues, identifying Mps1p as a dual specificity protein kinase.     

A pathogen-responsive cDNA from potato encodes a protein with homology to a phosphate starvation-induced phosphatase

Infiltration of potato leaves with the phytopathogenic bacteria Pseudomonas syringae pv. maculicola induces local and systemic defense gene expression as well as increased resistance against subsequent pathogen attacks. By cDNA-AFLP a gene was identified that is activated locally in potato leaves in response to bacterial infiltration and after infection with Phytophthora infestans, the causal agent of late blight disease. The encoded protein has high homology to a phosphate starvation-induced acid phosphatase from tomato. Possibly, decreased phosphate availability after pathogen infection acts as a signal for the activation of the potato phosphatase gene.     

An essential yeast gene with homology to the exonuclease-encoding XRN1/KEM1 gene also encodes a protein with exoribonuclease activity.

An essential gene, designated HKE1/RAT1, has been isolated from the yeast Saccharomyces cerevisiae and characterized. The gene encodes a protein of 116 kDa (p116) and has significant homology to another yeast gene (XRN1/KEM1) encoding a related protein (p175) with 5'-->3' exonuclease activity as well as activities involving chromosomal DNA pairing and mechanics. Preliminary analysis of an hke1ts mutant reveals a precipitous decline in the translation of mRNA at the nonpermissive temperature. Sporulation of heterozygous HKE1/hke1::URA3 diploids reveals that this gene, unlike the highly related XRN1/KEM1 gene, is essential for cell viability. Overexpression of the homologous gene product, p175, failed to rescue cells lacking a functional p116. In vitro studies demonstrate that p116 is a protein with 5'-->3' exoribonuclease activity, a major activity of the related p175. An immunoreactive RNase activity of 116 kDa is abolished with antiserum against p116. Both the level of this protein and the RNase activity correlate with HKE1 gene dosage. The RNase activity purifies coincidentally with a previously described 116-kDa RNase having 5'-->3' exoribonuclease activity.     

Dictyostelium gnt15 encodes a protein with similarity to LARGE and plays an essential role in development

LARGE is a putative glycosyltransferase found to be mutated in mice with myodystrophy or patients with congenital muscular dystrophy. By homology searches, we identified in the Dictyostelium discoideum genome four open reading frames, i.e. gnt12-15, encoding proteins with sequence similarity to LARGE. Semi-quantitative RT-PCR analysis revealed distinct temporal expression patterns of the four gnt genes throughout Dictyostelium development. To explore the gene function, we performed targeted disruptions of gnt14 and gnt15. The gnt14(-) strains showed no obvious phenotypes. However, gnt15(-) cells grew slowly, changed in morphology, and displayed a developmental phenotype arresting at early stages. Compared with the wild type, gnt15(-) cells were more adhesive and exhibited altered levels of some surface adhesion molecules. Moreover, lectin-binding analysis demonstrated that gnt15 disruption affected profiles of membrane glycoproteins. Taken together, our data suggest that Gnt15 is essential for Dictyostelium development and may have a role in modulating cell adhesion and glycosylation.     

The vaccinia virus gene I2L encodes a membrane protein with an essential role in virion entry

The previously unstudied vaccinia virus gene I2L is conserved in all orthopoxviruses. We show here that the 8-kDa I2 protein is expressed at late times of infection, is tightly associated with membranes, and is encapsidated in mature virions. We have generated a recombinant virus in which I2 expression is dependent upon the inclusion of tetracycline in the culture medium. In the absence of I2, the biochemical events of the viral life cycle progress normally, and virion morphogenesis culminates in the production of mature virions. However, these virions show an ~400-fold reduction in specific infectivity due to an inability to enter target cells. Several proteins that have been previously identified as components of an essential entry/fusion complex are present at reduced levels in I2-deficient virions, although other membrane proteins, core proteins, and DNA are encapsidated at normal levels. A preliminary structure/function analysis of I2 has been performed using a transient complementation assay: the C-terminal hydrophobic domain is essential for protein stability, and several regions within the N-terminal hydrophilic domain are essential for biological competency. I2 is thus yet another component of the poxvirus virion that is essential for the complex process of entry into target cells.     

NOP3 is an essential yeast protein which is required for pre-rRNA processing

The four nucleolar proteins NOP1, SSB1, GAR1, and NSR1 of Saccharomyces cerevisiae share a repetitive domain composed of repeat units rich in glycine and arginine (GAR domain). We have cloned and sequenced a fifth member of this family, NOP3, and shown it to be essential for cell viability. The NOP3 open reading frame encodes a 415 amino acid protein with a predicted molecular mass of 45 kD, containing a GAR domain and an RNA recognition motif. NOP3-specific antibodies recognize a 60-kD protein by SDS-PAGE and decorate the nucleolus and the surrounding nucleoplasm. A conditional lethal mutation, GAL::nop3, was constructed; growth of the mutant strain in glucose medium represses NOP3 expression. In cells depleted of NOP3, production of cytoplasmic ribosomes is impaired. Northern analysis and pulse-chase labeling indicate that pre-rRNA processing is inhibited at the late steps, in which 27SB pre-rRNA is cleaved to 25S rRNA and 20S pre-rRNA to 18S rRNA.     

The Nop60B gene of Drosophila encodes an essential nucleolar protein that functions in yeast

The Cbf5 protein of Saccharomyces cerevisiae was originally identified as a low-affinity centromeric DNA-binding protein, and cbf5 mutants have a defect in rRNA synthesis. A closely related protein from mammals, NAP57, is a nucleolar protein that coimmunoprecipitates with the nucleolar phosphoprotein Nopp140. To study the function of this protein family in a higher eukaryote that is amenable to genetic approaches, the gene encoding a Drosophilamelanogaster homolog, Nop60B, was identified. The predicted Drosophila protein shares a high degree of sequence identity over a 380-residue region with both the mammalian and yeast proteins, and shares several conserved motifs with the prokaryotic tRNA pseudouridine 55 synthases. Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development. Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis. Nop60B mutants were generated and shown to be homozygous lethal. The Drosophila gene can rescue the lethal phenotype of yeast cbf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila. Received: 23 February 1998 / Accepted: 17 June 1998     

Yeast KEX2 genes encodes an endopeptidase homologous to subtilisin-like serine proteases

Yeast Saccharomyces cerevisiae KEX2 gene previously isolated, was characterized as the gene encoding a calcium-dependent endopeptidase required for processing of precursors of alpha-factor and killer toxin. In this study, we report the amino acid sequence of the KEX2 gene product deduced from nucleotide sequencing. Our results indicate that the KEX2 gene contains a 2,442-bp open reading frame encoding a polypeptide of 814 amino acids. The deduced amino acid sequence contains a region extensively homologous to the members of subtilisin-like serine protease family near the N-terminus. A putative membrane-spanning domain near the C-terminus was also detected. These facts indicate that the KEX2-encoded protein may function as a membrane-bound, subtilisin-like serine protease.     

DOMINO1, a member of a small plant-specific gene family, encodes a protein essential for nuclear and nucleolar functions

Arabidopsis embryos carrying the domino1 mutation grow slowly in comparison with wild type embryos and as a consequence reach only the globular stage at desiccation. The primary defect of the mutation at the cellular level is the large size of the nucleolus that can be observed soon after fertilization in the nuclei of both the embryo and the endosperm. The ultrastructure of mutant nucleoli is drastically different from wild type and points to a fault in ribosome biogenesis. DOMINO1 encodes a protein, which belongs to a plant-specific gene family sharing a common motif of unknown function, present in the tomato DEFECTIVE CHLOROPLASTS AND LEAVES (LeDCL) protein. Using a GFP protein fusion, we show that DOMINO1 is targeted to the nucleus. We propose that inactivation of DOMINO1 has a negative effect on ribosome biogenesis and on the rate of cell division.     

Yeast PPA2 gene encodes a mitochondrial inorganic pyrophosphatase that is essential for mitochondrial function.

We have cloned a gene encoding a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae by low stringency hybridization to PPA1, the yeast gene for cytoplasmic PPase. The new gene, PPA2, is located on chromosome 13 and encodes a protein whose sequence is 49% identical to the cytoplasmic enzyme. The protein differs from cytoplasmic PPase in that it has a leader sequence enriched in basic and hydroxylated residues, which is typically found in mitochondrial proteins. Yeast cells overproducing PPA2 had a 47-fold increase in mitochondrial PPase activity. This activity was further stimulated 3-fold by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which suggests that PPA2 is part of an energy-linked enzyme. Using gene disruptions, we found that PPA1 is required for cell growth. In contrast, cells disrupted for PPA2 are viable, but unable to grow on respiratory carbon sources. Fluorescence microscopy revealed that these cells have lost their mitochondrial DNA. We conclude that the mitochondrial PPase encoded by PPA2 is essential for mitochondrial function and maintenance of the mitochondrial genome.     

The yopM gene of Yersinia pestis encodes a released protein having homology with the human platelet surface protein GPIb alpha.

In Yersinia pestis KIM, there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2+ response virulence plasmid pCD1. Only Yops M and N are found in easily detectable amounts in the culture medium. In this study, we located and characterized the yopM gene to obtain clues about its role in the virulence of Y. pestis. Rabbit antibody was raised against Yops M and H, copurified from the supernatant of Y. pseudotuberculosis 43(pGW600, pCD1 yopE::Mu dI1[Apr lac]). This antiserum was adsorbed with an Escherichia coli clone that strongly expressed YopH. The resulting YopM-specific antibody was used to screen a HindIII library of pCD1. HindIII-F and several subclones from it expressed YopM in E. coli minicells. A DNA fragment of 1.39 kilobases from HindIII-F was sequenced and found to contain a 367-amino-acid open reading frame capable of encoding a protein with molecular mass (41,566 daltons) and isoelectric point (4.06) similar to those of YopM. The +1 site of the yopM gene was determined by primer extension. The DNA sequence contained repeating structures: 11 pairs of exact direct repeats, two exact inverted repeats, and three palindromes, ranging from 10 to 42 bases in size. One consensus 14-amino-acid sequence was repeated six times in the predicted protein sequence. The YopM sequence shares some significant homology with the von Willebrand factor- and thrombin-binding domain of the alpha chain of human platelet membrane glycoprotein Ib. These findings suggested a testable hypothesis for the function of YopM.