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.     

A cloned bovine cDNA encodes an alternate form of the catalytic subunit of cAMP-dependent protein kinase

While attempting to isolate a cDNA clone for the catalytic subunit of the bovine cAMP-dependent protein kinase, we have isolated cDNAs which code for a protein slightly different than the known amino acid sequence. The alternate cDNA was identified by screening a bovine pituitary cDNA library using synthetic oligonucleotides predicted from the known amino acid sequence of the catalytic subunit. The cDNA which we identified, encodes a protein which is 93% identical to the known amino acid sequence of the bovine catalytic subunit. It seems likely that this cDNA represents a previously undiscovered catalytic subunit of the cAMP-dependent protein kinase. The mRNA for the alternate catalytic subunit is different in size from the mRNA coding for the previously known catalytic subunit and also has a different tissue distribution. These findings suggest that there are at least two different genes for the catalytic subunit. The differences in amino acid sequence and tissue distribution suggest the possibility of important functional differences in the two enzymes.     

Isolation and sequence analysis of a cDNA clone encoding a type-1 protein phosphatase catalytic subunit: homology with protein phosphatase 2A

A 1.5 kb clone containing the full-length coding sequence of a type-1 protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The protein sequence deduced from the cDNA contains 311 residues and has a molecular mass of 35.4 kDa. A single mRNA species at 1.6 kb was visualized by Northern blotting. The type-1 protein phosphatase was strikingly homologous to protein phosphatase 2A, 49% of the amino acids between residues 11 and 280 being identical. The first 10 and last 31 residues were dissimilar. Residues 1-101 of the type-1 protein phosphatase also showed 21% sequence identity with a region of mammalian alkaline phosphatases.     

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.     

SSL2, a suppressor of a stem-loop mutation in the HIS4 leader encodes the yeast homolog of human ERCC-3.

Reversion of haploid, His4- yeast containing a stem-loop mutation in the 5' UTR that blocks HIS4 translation initiation identified four unlinked suppressor genes, SSL1-SSL4, which restore His4+ expression. The SSL2 gene encodes an essential 95 kd protein with ATP-dependent helicase motifs. SSL2 protein is 54% identical to the protein encoded by the human gene, ERCC-3, for which a defective form causes xeroderma pigmentosum and Cockayne's syndrome. An SSL2 allele made to resemble the defective ERCC-3 gene confers UV light hypersensitivity to yeast cells. Hence, SSL2 is the functional homolog of ERCC-3. However, the SSL2 suppressor gene does not restore HIS4 expression by removal of the stem-loop from DNA or the mRNA. We propose that SSL2 and ERCC-3 may have two functions, one defined by a UV repair defect, and a second essential function that is related to gene expression.     

A multicopy suppressor of a cell cycle defect in S. pombe encodes a heat shock-inducible 40 kDa cyclophilin-like protein.

Cyclophilins are peptidyl-prolyl cis-trans isomerases (PPIases) which have been implicated in intracellular protein folding, transport and assembly. Cyclophilins are also known as the intracellular receptors for the immunosuppressive drug cyclosporin A (CsA). The most common type of cyclophilins are the 18 kDa cytosolic proteins containing only the highly conserved core domain for PPIase and CsA binding activities. The wis2+ gene of the fission yeast Schizosaccharomyces pombe was isolated as a multicopy suppressor of wee1-50 cdc25-22 win1-1, a triple mutant strain which exhibits a cell cycle defect phenotype. Sequence analysis of wis2+ reveals that it encodes a 40 kDa cyclophilin-like protein, homologous to the mammalian cyclophilin 40. The 18 kDa cyclophilin domain (CyP-18) of wis2 is followed by a C-terminal region of 188 amino acids. The C-terminal region of wis2 is essential for suppression of the triple mutant defect. Furthermore this region of the protein is able to confer suppression activity on the 18 kDa S.pombe cyclophilin, cyp1, since a hybrid protein consisting of an 18 kDa S.pombe cyclophilin (cyp1) fused to the C-terminus of wis2 shows suppression activity. We also demonstrate that the level of wis2+ mRNA increases 10- to 20-fold upon heat shock of S.pombe cells suggesting a role for wis2+ in the heat-shock response.     

A nuclear yeast gene (GCY) encodes a polypeptide with high homology to a vertebrate eye lens protein

We describe here the nuclear gene for a yeast protein showing unexpectedly high homology with mammalian aldo/keto reductases as well as with p-crystallin, one of the prominent proteins of the frog eye lens. Although it could be proven that the gene occurs as a single copy in the haploid yeast genome, replacement of the intact by a disrupted, nonfunctional allele led to no obvious phenotype, indicating that the gene is dispensable. The gene was assigned to chromosome XV. It is transcribed in vivo into an mRNA of about 1300 bases with a coding capacity for a protein of 312 amino acids (estimated Mr 35,000).     

Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker

We have isolated a gene (NOP2) encoding a nucleolar protein during a search for previously unidentified nuclear proteins in the yeast Saccharomyces cerevisiae. The protein encoded by NOP2 (Nop2p) has a predicted molecular mass of 70 kD, migrates at 90 kD by SDS-PAGE, and is essential for cell viability. Nop2p shows significant amino acid sequence homology to a human proliferation-associated nucleolar protein, p120. Approximately half of Nop2p exhibits 67% amino acid sequence identity to p120. Analysis of subcellular fractions indicates that Nop2p is located primarily in the nucleus, and nuclear fractionation studies suggest that Nop2p is associated with the nucleolus. Indirect immunofluorescence localization of Nop2p shows a nucleolar-staining pattern, which is heterogeneous in appearance, and a faint staining of the cytoplasm. The expression of NOP2 during the transition from stationary phase growth arrest to rapid growth was measured, and compared to the expression of TCM1, which encodes the ribosomal protein L3. Nop2p protein levels are markedly upregulated during the onset of growth, compared to the levels of ribosomal protein L3, which remain relatively constant. NOP2 mRNA levels also increase during the onset of growth, accompanied by a similar increase in the levels of TCM1 mRNA. The consequences of overexpressing NOP2 from the GAL10 promoter on a multicopy plasmid were investigated. Although NOP2 overexpression produced no discernible growth phenotype and had no effect on ribosome subunit synthesis, overexpression was found to influence the morphology of the nucleolus, as judged by electron microscopy. Overexpression caused the nucleolus to become detached from the nuclear envelope and to become more rounded and/or fragmented in appearance. These findings suggest roles for NOP2 in nucleolar function during the onset of growth, and in the maintenance of nucleolar structure.     

A nuclear protein in Schizosaccharomyces pombe with homology to the human tumour suppressor Fhit has decapping activity

A number of eukaryotic proteins are already known to orchestrate key steps of mRNA metabolism and translation via interactions with the 5' m7GpppN cap. We have characterized a new type of histidine triad (HIT) motif protein (Nhm1) that co-purifies with the cap-binding complex eIF4F of Schizosaccharomyces pombe. Nhm1 is an RNA-binding protein that binds to m7GTP-Sepharose, albeit with lower specificity and affinity for methylated GTP than is typical for the cap-binding protein known as eukaryotic initiation factor 4E. Sequence searches have revealed that proteins with strong sequence similarity over all regions of the new protein exist in a wide range of eukaryotes, yet none has been characterized up to now. However, other proteins that share specific motifs with Nhm1 include the human Fhit tumour suppressor protein and the diadenosine 5', 5"'-P1, P4-tetraphosphate asymmetrical hydrolase of S. pombe. Our experimental work also reveals that Nhm1 inhibits translation in a cell-free extract prepared from S. pombe, and that it is therefore a putative translational modulator. On the other hand, purified Nhm1 manifests mRNA decapping activity, yet is physically distinct from the Saccharomyces cerevisiae decapping enzyme Dcp1. Moreover, fluorescence and immunofluorescence microscopy show that Nhm1 is predominantly, although not exclusively, nuclear. We conclude that Nhm1 has evolved as a special branch of the HIT motif superfamily that has the potential to influence both the metabolism and the translation of mRNA, and that its presence in S. pombe suggests the utilization of a novel decapping pathway.