Genomics, 1996 Sep 1, 36(2), 305 - 15 Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse; McKay MJ et al.; The rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks . The isolation of mouse and human putative homologs of rad21 is reported here . Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins showed that the similarity was distributed across the length of the proteins, with more highly conserved regions at both termini . The mHR21(sp) (mouse homolog of Rad21, S . pombe) and hHR21(sp) (human homolog of Rad21, S . pombe) predicted proteins were 96% identical, whereas the human and S . pombe proteins were 25% identical and 47% similar . RNA blot analysis showed that mHR21sp mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus . In addition to a 3.1-kb constitutive mRNA transcript, a 2.2-kb transcript was present at a high level in postmeiotic spermatids, while expression of the 3.1-kb mRNA in testis was confined to the meiotic compartment . hHR21sp mRNA was cell cycle regulated in human cells, increasing in late S phase to a peak in G2 phase . The level of hHR21sp transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation . In situ hybridization showed that mHR21sp resided on chromosome 15D3, whereas hHR21sp localized to the syntenic 8q24 region . Elevated expression of mHR21sp in testis and thymus supports a possible role for the rad21 mammalian homologs in V(D)J and meiotic recombination, respectively . Cell cycle regulation of rad21, retained from S . pombe to human, is consistent with a conservation of function between S . pombe and human rad21 genes. Nucleic Acids Res, 1996 Sep 1, 24(17), 3307 - 12 Molecular cloning and functional analysis of a Schizosaccharomyces pombe homologue of Escherichia coli endonuclease III; Roldan-Arjona T et al.; The Escherichia coli endonuclease III (Nth-Eco) protein is involved in the removal of damaged pyrimidine residues from DNA by base excision repair . It is an iron-sulphur enzyme possessing both DNA glycosylase and apurinic/apyrimidinic lyase activities . A database homology search identified an open reading frame in genomic sequences of Schizosaccharomyces pombe which encodes a protein highly similar to Nth-Eco . The gene has been subcloned in an expression vector and the protein purified to apparent homogeneity . The S.pombe Nth homologue (Nth-Spo) is a 40.2 kDa protein of 355 amino acids . Nth-Spo possesses glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polymers . The eukaryotic protein removes urea more efficiently than the prokaryotic enzyme, whereas its efficiency in excising thymine glycol is lower . A nicking assay was used to show that the enzyme also exhibits an AP lyase activity on UV- and gamma-irradiated DNA substrates . These findings show that Nth protein is structurally and functionally conserved from bacteria to fission yeast. RNA, 1996 Sep, 2(9), 909 - 18 Localization of modified nucleotides in Schizosaccharomyces pombe spliceosomal small nuclear RNAs: modified nucleotides are clustered in functionally important regions; Gu J et al.; The specific and dynamic RNA:RNA interactions between pre-mRNA and small nuclear RNAs (snRNAs), especially U2, U5, and U6 snRNAs, form the catalytic core and are at the heart of the spliceosome formation . The functionally important regions in the snRNAs correspond to the highly modified regions in snRNAs from human, rat, and plant cells . To better understand the importance of the modifications of snRNAs, we identified and localized the modified nucleotides in the five spliceosomal snRNAs of Schizosaccharomyces pombe cells . Twenty-two modified nucleotides, including base methylations, 2'-O-methylations, and pseudouridines, were found in the five spliceosomal snRNAs . The conservation of modified nucleotides between human and S . pombe snRNAs is striking . In addition, most of the modified nucleotides are in or around positions that form hydrogen bonds with the pre-mRNA or with other snRNAs . The results are consistent with the suggestion that modified nucleotides are clustered around functionally important regions of the spliceosomal snRNAs . These data provide the basis for further functional studies on posttranscriptional modifications in spliceosomal snRNAs. Nat Genet, 1996 Sep, 14(1), 42 - 9 Yeast SAS silencing genes and human genes associated with AML and HIV-1 Tat interactions are homologous with acetyltransferases; Reifsnyder C et al.; Silencing is an epigenetic form of transcriptional regulation whereby genes are heritably, but not necessarily permanently, inactivated . We have identified the Saccharomyces cerevisiae genes SAS2 and SAS3 through a screen for enhancers of sir1 epigenetic silencing defects . SAS2, SAS3 and a Schizosaccharomyces pombe homologue are closely related to several human genes, including one associated with acute myeloid leukaemia arising from the recurrent translocation t(8;16)(p11;p13) and one implicated in HIV-1 Tat interactions . All of these genes encode proteins with an atypical zinc finger and well-conserved similarities to acetyltransferases . Sequence similarities and yeast mutant phenotypes suggest that SAS-like genes function in transcriptional regulation and cell-cycle exit and reveal novel connections between transcriptional silencing and human disease. Curr Genet, 1996 Sep, 30(4), 284 - 93 Requirement of S . pombe exonuclease II, a homologue of S . cerevisiae Sep1, for normal mitotic growth and viability; Szankasi P et al.; Exonuclease II (ExoII) from Schizosaccharomyces pombe is a 5'-->3' single-stranded DNA exonuclease . We have cloned its gene, exo2, whose nucleotide sequence revealed that ExoII is a homologue of the multifunctional Saccharomyces cerevisiae Sep1 protein (also called Kem1, Xrn1, Rar5, Dst2) . S . pombe exo2 null mutants were cold-sensitive for growth, had increased cell size at the restrictive temperature, were hypersensitive to the mitotic inhibitor thiabendazol and to caffeine, and died rapidly in stationary phase . Many of these phenotypes are similar to those of sep1 (kem1 or xrn1) mutants of S . cerevisiae . In contrast, the exo2 mutation had only a moderate effect on progression through meiosis and no significant effect on meiotic recombination . We discuss possible functions of the multifunctional ExoII protein. Mol Cell Biol, 1996 Sep, 16(9), 4869 - 78 The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases; Brill S et al.; We previously described IQGAP1 as a human protein related to a putative Ras GTPase-activating protein (RasGAP) from the fission yeast Schizosaccharomyces pombe . Here we report the identification of a liver-specific human protein that is 62% identical to IQGAP1 . Like IQGAP1, the novel IQGAP2 protein harbors an N-terminal calponin homology motif which functions as an F-actin binding domain in members of the spectrin, filamin, and fimbrin families . Both IQGAPs also harbor several copies of a novel 50- to 55-amino-acid repeat, a single WW domain, and four IQ motifs and have 25% sequence identity with almost the entire S . pombe sar1 RasGAP homolog . As predicted by the presence of IQ motifs, IQGAP2 binds calmodulin . However, neither full-length nor truncated IQGAP2 stimulated the GTPase activity of Ras or its close relatives . Instead, IQGAP2 binds Cdc42 and Racl but not RhoA . This interaction involves the C-terminal half of IQGAP2 and appears to be independent of the nucleotide binding status of the GTPases . Although IQGAP2 shows no GAP activity towards Cdc42 and Rac1, the protein did inhibit both the intrinsic and RhoGAP-stimulated GTP hydrolysis rates of Cdc42 and Rac1, suggesting an alternative mechanism via which IQGAPs might modulate signaling by these GTPases . Since IQGAPs harbor a potential actin binding domain, they could play roles in the Cdc42 and Rac1 controlled generation of specific actin structures. J Virol, 1996 Sep, 70(9), 5821 - 6 Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe; Zhao Y et al.; The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest . In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression . The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S . pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression . Induction of HIV-1 vpr gene expression affected S . pombe at the colonial, cellular, and molecular levels . Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest . Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes . The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr . Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes . This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr . Together, these data showed that the HIV-1 Vpr-induced cellular changes in S . pombe are similar to those observed in human cells . Therefore, the S . pombe system is suited for further investigation of the HIV-1 vpr gene functions. Mol Gen Genet, 1996 Aug 27, 252(1-2), 20 - 32 The G protein beta subunit Gpb1 of Schizosaccharomyces pombe is a negative regulator of sexual development; Kim DU et al.; A Schizosaccharomyces pombe homolog of mammalian genes encoding G protein beta subunits, gpb1+, was cloned by the polymerase chain reaction using primer pairs that correspond to sequences conserved in several G beta genes of other species followed by screening of genomic and cDNA libraries . The gpb1 gene encodes 317 amino acids that show 47% homology with human G beta 1 and G beta 2 and 40% homology with Saccharomyces cerevisiae G beta protein . Disruption of the gpb1 gene indicated that this gene is not required for vegetative cell growth . However, gpb1-disrupted haploid cells mated and sporulated faster than wild-type cells, both in sporulation (MEA) and in complex medium (YE): when examined 23 h after transfer to sporulation medium, 35% of gpb1-disrupted haploid pairs had undergone conjugation and sporulation, whereas only 3-5% of wild-type haploid pairs had done so . Overexpression of the gpb1 gene suppressed this facilitated conjugation and sporulation phenotype of gpb1-disrupted cells but did not cause any obvious effect in wild-type cells . Co-disruption of one of the two S . pombe G alpha-subunit genes, gpa2, in the gpb1-disrupted cells did not change the accelerated conjugation and sporulation phenotype of the gpb1- cells . However, co-disruption of the ras1 gene abolished the gpb1- phenotype . These results suggest that Gpb1 is a negative regulator of conjugation and sporulation that apparently works upstream of Ras1 function in S . pombe . The possible relationship of Gpb1 to two previously identified, putative G alpha proteins of S . pombe is discussed. Dev Biol, 1996 Aug 25, 178(1), 90 - 100 Tosca: a Drosophila gene encoding a nuclease specifically expressed in the female germline; Digilio FA et al.; We describe here a Drosophila gene, tosca (tos), that is specifically expressed in the female germline . tos mRNA accumulates selectively within the pro-oocyte in germarial region 2 and persists throughout oogenesis . In the early embryo, the maternally supplied tos mRNA is evenly distributed at the syncytial blastoderm stage, but is excluded from the forming cells when cellularization begins . tos product is the first Drosophila member of the RAD2 protein family, a group of related DNA repair nucleases conserved from yeast to humans . Within the family, Tos is more closely related to ExoI, a Schizosaccharomyces pombe 5'-->3' double-stranded DNA exonuclease specifically induced in meiotic prophase I . The definite oocyte localization of tos transcript during meiosis and its ubiquitous distribution in early embryos suggest that tos may play a role in mismatch repair during genetic recombination and early cleavage divisions. J Biol Chem, 1996 Aug 23, 271(34), 20868 - 78 Purification, gene cloning, and reconstitution of the heterotrimeric single-stranded DNA-binding protein from Schizosaccharomyces pombe; Ishiai M et al.; We have purified a single-stranded DNA-binding protein (SSB) from Schizosaccharomyces pombe (Sp) and have shown that it is composed of three subunits of 68, 30, and 12 kDa . The SpSSB supports T antigen-dependent unwinding of SV40 ori containing DNA, but is not functional in the SV40 in vitro replication reaction . All three genes that encode the SpSSB subunit have been isolated . The cloned cDNA of the ssb1(+), encoding the p68 subunit, contains 609 amino acids (68.3 kDa), while that of the ssb2(+), encoding the p30 subunit, contains a 279 amino acids (30.3 kDa) . The genomic DNA clone of the p12 subunit gene (ssb3(+)) has 2 introns and an open reading frame of 104 amino acids (11.8 kDa) . Significant homology is observed among the largest and middle subunits of eukaryotic SSBs, but there is poor homology among the smallest subunits . In addition, we have reconstituted the SpSSB complex by coexpression of all three subunits in Escherichia coli . The reconstituted complex is active in single-stranded DNA binding and the T antigen-dependent unwinding of SV40 ori DNA . Finally, we observed a cell cycle-dependent phosphorylation pattern of the p30 subunit of SpSSB, which is similar to that observed for the human and Saccharomyces cerevisiae SSB. Biochim Biophys Acta, 1996 Aug 15, 1296(1), 69 - 75 Phosphoglycerate mutase from Schizosaccharomyces pombe: development of an expression system and characterisation of three histidine mutants of the enzyme; Nairn J et al.; The small, monomeric, phosphoglycerate mutase (PGAM) from Schizosaccharomyces pombe has been overexpressed in a strain of Saccharomyces cerevisiae in which the gene encoding PGAM has been deleted, with a yield of purified enzyme of 10-15 mg per litre cell culture . Three mutants in which histidine residues in S . pombe PGAM have been substituted by glutamine have been purified and characterised . Two mutants (H151Q and H196Q) have kinetic and structural properties very similar to wild-type enzyme, consistent with the proposed location of these (non-conserved) histidines on the surface of the enzyme . The third mutant (H163Q) involving a histidine thought to be part of the active site has greatly reduced mutase and phosphatase activities . Mass spectrometry shows that the phosphorylated form of the H163Q is several 100-times more stable towards hydrolysis than the phosphorylated form of wild-type enzyme . The H163Q mutant appears to be structurally quite distinct from wild-type enzyme . 600 MHz 1D proton NMR spectra of good quality have been obtained for wild-type enzyme and the H151Q and H196Q mutants. Proc Natl Acad Sci U S A, 1996 Aug 6, 93(16), 8278 - 83 p34cdc2 kinase activity is maintained upon activation of the replication checkpoint in Schizosaccharomyces pombe; Knudsen KE et al.; All eukaryotes use feedback controls to order and coordinate cell cycle events . In Schizosaccharomyces pombe, several classes of checkpoint genes serve to ensure that DNA replication is complete and free of error before the onset of mitosis . Wild-type cells normally arrest upon inhibition of DNA synthesis or in response to DNA damage, although the exact mechanisms controlling this arrest are unclear . Genetic evidence in fission yeast suggests that the dependence of mitosis upon completion of DNA replication is linked to the regulation of the p34cdc2 cyclin-dependent kinase . It has been hypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has never been shown biochemically . We analyzed the activity of p34cdc2 in wild-type and checkpoint-defective cells treated with a DNA synthesis inhibitor . Using standard in vitro assays we demonstrate that p34cdc2 kinase activity is maintained in wild-type cells arrested at the replication checkpoint . We also used a novel in vivo assay for p34cdc2 kinase activity, in which we expressed a fragment of the human retinoblastoma tumor suppressor protein in fission yeast . Phosphorylation of this fragment of the human retinoblastoma tumor suppressor protein is dependent on p34cdc2 kinase activity, and this activity is also maintained in cells arrested at the replication checkpoint . These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is not dependent on the attenuation of p34cdc2 activity. Yeast, 1996 Aug, 12(10), 983 - 90 Characterization of cwl1+, a gene from Schizosaccharomyces pombe whose overexpression causes cell lysis; Godoy C et al.; From a Schizosaccharomyces pombe genomic library we have isolated the gene cwl1+ that causes cell lysis when it is overexpressed in the absence of an osmotic stabilizer . Southern hybridization showed that cwl1+ exists as a single copy in the S . pombe genome . The cwl1+ gene nucleotide sequence revealed a putative open reading frame of 924 bp encoding a polypeptide of 308 amino acids with a calculated Mt of 27000 . The cwl1+ DNA hybridizes to a major RNA transcript of 1.5 kb whose 5' end maps at a position 452 bp upstream from the predicted translation start . Comparison of the amino acid sequence with those included in the current databases, showed no significant similarity to any known sequences . Cells overexpressing the cwl1+ gene under the control of the S . pombe nmt inducible promoter displayed a reduced cell wall content, were unable to separate after division and lysed drastically in the absence of osmotic stabilizer . Disruption of the cwl1+ gene caused no noticeable phenotype. Yeast, 1996 Aug, 12(10), 977 - 81 The translation initiation factor eIF4A from Schizosaccharomyces pombe is closely related to its mammalian counterpart; Fischli A et al.; We have isolated a cDNA clone encoding eIF4A from Schizosaccharomyces pombe . The deduced protein sequence is similar in length and sequence to other eIF4A proteins and exhibits highest similarity with the mammalian eIF4A protein . Hybridization with genomic DNA reveals two eIF4A genes located on two different chromosomes. Yeast, 1996 Aug, 12(10), 925 - 37 Cloning, mapping and characterization of a genomic copy of the Lipomyces kononenkoae alpha-amylase-encoding gene (LKA1); Steyn AJ et al.; The expression in Saccharomyces cerevisiae and Schizosaccharomyces pombe of a cDNA copy of the Lipomyces kononenkoae IGC4052B alpha-amylase gene (LKA1), linked to the phosphoglycerate kinase gene (PGK1) promoter, resulted in the extracellular production of biologically active alpha-amylase (LKA1) . However, transformation of S . cerevisiae and Schiz . pombe with a cosmid clone containing the complete genomic copy of LKA1, expressed from its native promoter, did not result in secretion of active alpha-amylase by any of the transformants . When the cDNA copy of LKA1 was expressed in S . cerevisiae under control of the wild-type L, kononenkoae promoter, biologically active alpha-amylase was secreted into the culture medium, indicating the recognition of the LKA1 promoter in S . cerevisiae . Sequence analysis of the GC-rich LKA1 promoter revealed canonical sequences that are homologous to the TATAAA, CAAT and CCAAT boxes and GCN4-binding sites that are present in several promoter sequences of S . cerevisiae . Primer extension analysis of LKA1 transcripts in L . kononenkoae indicated major initiation sites at nucleotides -64 and -65 . S . cerevisiae and Schiz . pombe cells transformed with a plasmid containing the open reading frame of the genomic copy of LKA1, linked to the PGK1 promoter, did not produce alpha-amylase . Polymerase chain reaction mapping and sequence analysis revealed the presence of a 61-bp intron in the genomic copy of LKA1 that impaired synthesis of biologically active alpha-amylase in S . cerevisiae and Schiz . pombe . This intron contains donor, acceptor and branch sequences that correlate with the consensus sequences identified in the introns of split genes from Schiz . pombe and mammals . Pulsed-field gradient gel electrophoresis resolved at least eight chromosomal DNAs for L . kononenkoae IGC4052B and chromoblot analysis indicated that LKA1 is located on the second smallest chromosome, designated chromosome II. Plant J, 1996 Aug, 10(2), 269 - 79 Arabidopsis profilins are functionally similar to yeast profilins: identification of a vascular bundle-specific profilin and a pollen-specific profilin; Christensen HE et al.; Four members of the Arabidopsis profilin (pfn) multigene family have been cloned, sequenced and analyzed . By RNA gel blot analysis it has been shown that these four genes fall into two groups: one group (pfn1 and pfn2) is expressed in all organs of the plant and the other group (pfn3 and pfn4) in floral tissues only . Based on amino acid sequence alignment Arabidopsis profilins can be divided into the same two groups: PFN1 and PFN2 are 89% identical and PFN3 and PFN4 are 91% identical . Between these two groups they are 71-75% identical . The Arabidopsis profilins bind poly-L-proline and can complement both the Saccharomyces cerevisiae profilin deletion mutant and the Schizosaccharomyces pombe cdc3-124/profilin mutation, showing that the plant profilins are functionally similar to yeast profilins despite the low amino acid sequence homology . Analysis of pfn promoter-GUS fusion genes in transgenic Arabidopsis shows that pfn2 is specifically expressed in the vascular bundles of roots, hypocotyls, cotyledons, leaves, sepals, petals, stamen filaments and stalks of developing seeds, whereas expression of pfn4 is restricted to mature and germinating pollen grains. Mol Cell Biol, 1996 Aug, 16(8), 4573 - 83 Dephosphorylation of threonine 169 of Cdc28 is not required for exit from mitosis but may be necessary for start in Saccharomyces cerevisiae; Lim HH et al.; Entry into mitosis requires activation of cdc2 kinase brought on by its association with cyclin B, phosphorylation of the conserved threonine (Thr-167 in Schizosaccharomyces pombe) in the T loop, and dephosphorylation of the tyrosine residue at position 15 . Exit from mitosis, on the other hand, is induced by inactivation of cdc2 activity via cyclin destruction . It has been suggested that in addition to cyclin degradation, dephosphorylation of Thr-167 may also be required for exit from the M phase . Here we show that Saccharomyces cerevisiae cells expressing cdc28-E169 (a CDC28 allele in which the equivalent threonine, Thr-169, has been replaced by glutamic acid) are able to degrade mitotic cyclin Clb2, inactivate the Cdc28/Clb2 kinase, and disassemble the anaphase spindles, suggesting that they exit mitosis normally . The cdc28-E169 allele is active with respect to its mitotic functions, since it complements the mitosis-defective cdc28-1N allele . Whereas replacement of Thr-169 with serine affects neither Start nor the mitotic activity of Cdc28, replacement with glutamic acid or alanine renders Cdc28 inactive for Start-related functions . Coimmunoprecipitation experiments show that although Cdc28-E169 associates with mitotic cyclin Clb2, it fails to associate with the G1 cyclin Cln2 . Thus, an unmodified threonine at position 169 in Cdc28 is important for interaction with G1 cyclins . We propose that in S . cerevisiae, dephosphorylation of Thr-169 is not required for exit from mitosis but may be necessary for commitment to the subsequent division cycle. Curr Genet, 1996 Aug, 30(3), 224 - 31 The mus-8 gene of Neurospora crassa encodes a structural and functional homolog of the Rad6 protein of Saccharomyces cerevisiae; Soshi T et al.; We cloned a DNA repair gene, mus-8, of Neurospora crassa and sequenced the genomic DNA and cDNA . Nucleotide-sequence analysis indicated that the mus-8 gene contains an open reading frame (ORF) of 456 bp, interrupted by three small introns . The deduced amino-acid sequence showed that the mus-8 gene encodes a 17 kDa protein which has 77.5% and 83.3% identity to the Rad6 protein of Saccharomyces cerevisiae and the rhp6(+) protein of Schizosaccharomyces pombe, respectively . The Rad6 protein is a ubiquitin-conjugating enzyme (E2) and is required for DNA repair, mutagenesis, and sporulation in yeast . Introduction of the mus-8 gene into a S . cerevisiae rad6 mutant resulted in significant recovery of DNA repair functions, especially UV-mutagenesis, and also sporulation, both of which are defective in the rad6 mutant . It is therefore postulated that mus-8 of Neurospora has a function very similar to that demonstrated for RAD6 of S . cerevisiae. J Biotechnol, 1996 Jul 31, 48(3), 259 - 67 Production of cadmium sulphide microcrystallites in batch cultivation by Schizosaccharomyces pombe; Williams P et al.; Cadmium sulphate was added to separate batch cultures of Schizosaccharomyces pombe during different growth phases to determine the effect on cadmium sulphide microcrystallite production . Exit gas analysis was used to determine the impact on metabolism . Addition during the early-exponential growth phase resulted in an immediate intracellular uptake of cadmium, followed by rapid efflux from the cells, permanent reduction in cell metabolism and a lower intracellular inorganic sulphide content . This response was not suitable for cadmium sulphide microcrystallite production . Stationary phase cultures did not induce cadmium sulphide microcrystallite production . However, the addition of cadmium sulphate to a culture during the mid-exponential growth phase increased the intracellular cadmium and inorganic sulphide concentrations for approximately 8 h before reaching a saturation level for the cell . This resulted in a significant level of cadmium sulphide microcrystallite production. Curr Genet, 1996 Jul 31, 30(2), 151 - 8 Comparison of three 3' non-coding regions in Schizosaccharomyces pombe expression vectors: efficiencies of transcription termination and mRNA 3'-end formation; Patrikakis M et al.; Analysis of an established Schizosaccharomyces pombe episomal shuttle vector suggested that inefficient transcription termination was deleterious to plasmid function . We undertook a study to determine if transcription in the presence and absence of 3'-processing within a vector could affect the ability of the plasmid to transform, transcribe and translate the RNA produced . This report provides an analysis of the effects that three S . pombe 3' non-coding regions have on the transformation and expression efficiencies of a fission yeast plasmid vector . The 3' regions from adh1, act1 and ura4 were tested for their ability to terminate and process adh1 promoter-driven transcription of a lacZ reporter gene . Differences between the 3'-processing sequences were observed, with transcription termination mediated by the ura4 3' region being more efficient than termination by the 3' regions of adh1 and act1 . We show that plasmids containing inefficient transcription termination signals result in readthrough transcription and reduced transformation efficiencies . In addition, the readthrough transcripts containing 3' non-coding regions show impaired translation efficiencies . We describe an S . pombe vector (pURAS) with a high transformation efficiency that directs the production of highly translatable, discrete-sized transcripts. Mol Gen Genet, 1996 Jul 26, 251(6), 635 - 46 Novel alleles of cdc13 and cdc2 isolated as suppressors of mitotic catastrophe in Schizosaccharomyces pombe; Berry LD et al.; Cell cycle control in the fission yeast Schizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including the cdc2, cdc13, cdc25, wee1, and mik1 gene products . Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Miky1 play a negative regulatory role . Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1 . Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2 . Although we did not identify such molecules, we isolated a number of alleles of both cdc2 and cdc13, including a novel wee allele of cdc2, cdc2-5w . Here, we characterize cdc2-5w and two alleles of cdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1. J Biol Chem, 1996 Jul 19, 271(29), 17062 - 6 Targeting the plant alternative oxidase protein to Schizosaccharomyces pombe mitochondria confers cyanide-insensitive respiration; Albury MS et al.; The Sauromatum guttatum alternative oxidase has been expressed in Schizosaccharomyces pombe under the control of the thiamine-repressible nmt1 promoter . Alternative oxidase protein and activity were detected both in spheroplasts and isolated mitochondria, indicating that the enzyme is expressed in a functional form and confers cyanide-resistant respiration to S . pombe, which is sensitive to inhibition by octyl-gallate . Protein import studies revealed that the precursor form of the alternative oxidase protein is efficiently imported into isolated mitochondria and processed to its mature form comparable to that observed with potato mitochondria . Western blot analysis and respiratory studies revealed that the alternative oxidase protein is expressed in the inner mitochondrial membrane in its reduced (active) form . Treatment of mitochondria with diamide and dithiothreitol resulted in interconversion of the reduced and oxidized species and modulation of respiratory activity . The addition of pyruvate did not effect either the respiratory rate or expression of the reduced species of the protein . To our knowledge this is the first time that the alternative oxidase has been effectively targeted to and integrated into the inner mitochondrial membrane of S . pombe, and we conclude that the expression of a single polypeptide is sufficient for alternative oxidase activity. J Biol Chem, 1996 Jul 5, 271(27), 15971 - 80 Schizosaccharomyces pombe proliferating cell nuclear antigen mutations affect DNA polymerase delta processivity; Arroyo MP et al.; We introduced nine site-directed mutations into seven conserved fission yeast proliferative cell nuclear antigen (PCNA) residues, Leu2, Asp63, Arg64, Gly69, Gln201, Glu259, and Glu260, either as single or as double mutants . Both the recombinant wild type and mutant PCNAs were able to form homotrimers in solution and to sustain growth of a null pcna strain (Deltapcna) . Wild type Schizosaccharomyces pombe PCNA and PCNA proteins with mutations in Asp63, Gln201, Glu259, or Glu260 to Ala were able to stimulate DNA synthetic activity and to enhance the processivity of calf thymus DNA polymerase delta holoenzyme similar to calf thymus PCNA . Mutations of Leu2 to Val or Arg64 to Ala, either singly or as a double mutant, yielded PCNA mutant proteins that had reduced capacity in enhancing the processivity of DNA polymerase delta but showed no deficiency in stimulation of the ATPase activity of replication factor C . S . pombe Deltapcna strains sustained by these two mutant-pcna alleles had moderate defects in growth and displayed elongated phenotypes . These cells, however, were not sensitive to UV irradiation . Together, these in vitro and in vivo studies suggest that the side chains of Leu2 and Arg64 in one face of the PCNA trimer ring structure are two of the several sites involved in tethering DNA polymerase delta for processive DNA synthesis during DNA replication. J Membr Biol, 1996 Jul, 152(2), 169 - 81 The SpTRK gene encodes a potassium-specific transport protein TKHp in Schizosaccharomyces pombe; Lichtenberg-Frate H et al.; Complementary DNAs involved in potassium transport in Schizosaccharomyces pombe were selected by complementation of defective K+ uptake in a trk1 trk2 mutant of Saccharomyces cerevisiae . Here we describe the SpTRK gene that encodes a protein of 833 amino acids . The predicted structure contains 12 putative membrane-spanning domains and resembles various high- and low-affinity systems for K+ transport in yeasts and plants . TKHp, the product of SpTRK exhibits high homology to TRK1 and TRK2 of Saccharomyces cerevisiae as well as to HKT1 of Triticum aestivum, but is not related to HAK1 of another ascomycete, Schwanniomyces occidentalis, suggesting that different routes for potassium uptake evolved independently . This protein is a potassium-specific transporter since functional analysis of the SpTRK complemented mutant strain of Sacch . cerevisiae revealed potassium transport affinities and uptake characteristics similar to those obtained in wild-type Sch . pombe . Patch-clamp analysis in the whole-cell mode confirmed the TKHp-mediated inward current in the complemented strain . The inward current increased by acidification of the extracellular medium thereby suggesting a mechanism of K+H+ cotransport . The inward current is not detectable when external K+ is substituted by Na+, documenting a distinct cation specificity of the protein. Yeast, 1996 Jul, 12(9), 869 - 75 Analysis of a 62 kb DNA sequence of chromosome X reveals 36 open reading frames and a gene cluster with a counterpart on chromosome XI; Huang ME et al.; We have sequenced a 61.989 bp stretch located between genes RAD7 and FIP1 of Saccharomyces cerevisiae chromosome X . This stretch contains 36 open reading frames (ORFs) of at least 100 codons . Fourteen of these correspond to sequences previously published as HIT1, CDC8, YAP17, CBF1, NAT1, RPA12, CCT5, TOR1, RFC2, PEM2, CDC11, MIR1, STE18 and GRR1 . The proteins deduced from four ORFs (YJR059w, YJR065c, YJR075w, YJR078w) have significant similarity to proteins of known function from yeast or other organisms, including S . cerevisiae serine/threonine-specific protein kinase . Schizosaccharomyces pombe Act2 protein, S . cerevisiae mannosyltransferase OCH1 protein and mouse indoleamine 2,3-dioxygenase, respectively . Four of the remaining 18 ORFs have similarity to proteins with unknown function, six are weakly similar to other known sequences, while another eight exhibit no similarity to any known sequence . In addition, three tRNA genes have been recognized . Three genes clustered within 22 kb (YJR059w, YJR061w and TOR1) have counterparts arranged within 15 kb on the left arm of chromosome XI. Yeast, 1996 Jul, 12(9), 839 - 48 Identification of ACT4, a novel essential actin-related gene in the yeast Saccharomyces cerevisiae; Huang ME et al.; Actin molecules are major cytoskeleton components of all eukaryotic cells . All conventional actins that have been identified so far are 374-376 amino acids in size and exhibit at least 70% amino acid sequence identity when compared with one another . In the yeast Saccharomyces cerevisiae, one conventional actin gene ACT1 and three so-called actin-related genes, ACT2, ACT3 and ACT5, have been identified . We report here the discovery of a new actin-related gene in this organism, which we have named ACT4 . The deduced protein, Act4, of 449 amino acids, exhibits only 33.4%, 26.7%, 23.4% and 29.2% identity to Act1, Act2, Act3 and Act5, respectively . In contrast, it is 68.4% identical to the product of the Schizosaccharomyces pombe Act2 gene and has a similar level of identity to other Sch . pombe Act2 homologues . This places Act4 in the Arp3 family of actin-related proteins . ACT4 gene disruption and tetrad analysis demonstrate that this gene is essential for the vegetative growth of yeast cells . The act4 mutants exhibit heterogenous morphological phenotypes . We hypothesize that Act4 may have multiple roles in the cell cycle. J Cell Sci, 1996 Jul, 109 ( Pt 7), 1927 - 35 The wis1 signal transduction pathway is required for expression of cAMP-repressed genes in fission yeast; Stettler S et al.; The wis1 protein kinase of Schizosaccharomyces pombe is a member of the MAP kinase kinase family . Loss of wis1 function has previously been reported to lead to a delay in the G2-mitosis transition, loss of viability in stationary phase, and hypersensitivity to osmotic shock . It acts at least in part by activating the MAP kinase homologue sty1; loss-of-function sty1 mutants share many phenotypes with wis1 deletion mutants . We show here that, in addition, loss of wis1 function leads to defective conjugation, and to suppression of the hyperconjugation phenotype of the pat1-114 mutation . Consistent with this, the induction of the mei2 gene, which is normally induced by nitrogen starvation, is defective in wis1 mutants . In wild-type cells, nitrogen starvation leads to mei2 induction through a fall in intracellular cyclic AMP (cAMP) level and activity of the cAMP-dependent protein kinase . We show here that wis1 function is required for mei2 induction following nitrogen starvation . Expression of the fbp1 gene is negatively regulated by cAMP in response to glucose limitation: induction of fbp1 also requires wis1 and sty1 function . Loss of wis1 is epistatic over increased fbp1 expression brought about by loss of adenylate cyclase (git2/cyr1) or cAMP-dependent protein kinase (pka1) function . These observations can be explained by a model in which the pka1 pathway negatively regulates the wis1 pathway, or the two pathways might act independently on downstream targets . The latter explanation is supported, at least as regards regulation of cell division, by the observation that loss of function of the regulatory subunit of the cAMP-dependent protein kinase (cgs1) brings about a modest increase in cell length at division in both wis1+ and wis1 delta genetic backgrounds. J Cell Sci, 1996 Jul, 109 ( Pt 7), 1919 - 25 The Schizosaccharomyces pombe pyp1 protein tyrosine phosphatase negatively regulates nutrient monitoring pathways; Dal Santo P et al.; The Schizosaccharomyces pombe pyp1+ gene, encoding a protein tyrosine phosphatase (pyp1), was isolated as a high copy number suppressor of a mutation that results in reduced cAMP-dependent protein kinase (PKA) activity . Overexpression of pyp1+ inhibits both transcription of the fbp1 gene, which is negatively regulated by a glucose-induced activation of PKA, and sexual development, which is negatively regulated by PKA through a nitrogen- and glucose-monitoring mechanism . Overexpression of a catalytically inactive form of pyp1 has little effect on either process . Previous studies suggest that overexpression of pyp1+ results in a mitotic delay by positively regulating wee1 activity . We show that pyp1 repression of fbp1 transcription is independent of wee1 . The direct role of the pyp1 protein is to dephosphorylate and inactivate the sty1/spc1 mitogen-activated protein kinase (MAPK) that is activated by the wis1 MAPK kinase . As overexpression of pyp1+ has no further effect upon the mitotic delay observed in a wis1 deletion strain, the role of pyp1 appears to be restricted to negative regulation of the sty1/spc1 MAPK . This study indicates that pyp1 negatively regulates fbp1 transcription, sexual development and mitosis by inactivation of the sty1/spc1 MAPK, but that bifurcations downstream of the MAPK separate these processes as seen by the differential role for the wee1 gene. Biosci Biotechnol Biochem, 1996 Jul, 60(7), 1156 - 9 Isolation and characterization of a glycosylation mutant from Schizosaccharomyces pombe; Takegawa K et al.; N-Linked oligosaccharides were elongated by glycosylation with mannose and galactose residues in the secretory pathway of Schizosaccharomyces pombe . The wild-type S . pombe cells were agglutinated by the additions of not only concanavalin A lectin, which is specific for mannose residues, but also PNA (from Arachis hypogaea) and RCA (Ricinus communis) lectins, which are specific for terminal galactose residues . By PNA-binding selection, we isolated an S . pombe mutant defective in protein glycosylation . The mutant cells, named gms1, were not agglutinated by PNA or RCA . In contrast, agglutination of the gms1 cells by the addition of concanavalin A was markedly increased . Structural studies on N-linked oligosaccharides from gms1 mutant cells showed that the number of alpha-1,2-linked galactose residues wes markedly reduced, and unsubstituted alpha-1,6-linked polymannose outer chains were attached to the core oligosaccharides. Biosci Biotechnol Biochem, 1996 Jul, 60(7), 1087 - 92 Purification and some properties of phospholipase B from Schizosaccharomyces pombe; Oishi H et al.; Phospholipase B from Schizosaccharomyces pombe was purified by ammonium sulfate fractionation and chromatographed on phenyl-Sepharose CL-4B, DEAE-Toyopearl 650M, and TSK gel G4000SW columns . The purified enzyme was a glycoprotein with molecular weight of approximately 300,000 and 100,000-150,000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively . The isoelectric point was pH 4.7 . The optimum pH of the enzyme was 2.5 and no activity was detected at neutral and alkaline pHs . The enzyme was not heat-stable . Enzyme activity was slightly stimulated by divalent ions except Fe2+ and 0.1% sodium deoxycholate, and inhibited by Fe2+, Fe3+, 0.1% sodium dodecyl sulfate, and 0.01% cetyltrimethylammonium bromide . The enzyme hydrolyzed mono- and diacylphospholipids, and phosphatidylinositol was hydrolyzed most preferentially . Triglyceride was not hydrolyzed . The enzyme also had acyltransferase activity on lysophosphatidylcholine, forming the corresponding diacylphosphatidylcholine. Nucleic Acids Res, 1996 Jul 1, 24(13), 2585 - 91 The Schizosaccharomyces pombe pla1 gene encodes a poly(A) polymerase and can functionally replace its Saccharomyces cerevisiae homologue; Ohnacker M et al.; We have isolated the poly(A) polymerase (PAP) encoding gene pla1 {for poly(A) polymerase} from the fission yeast Schizosaccharomyces pombe . Protein sequence alignments with other poly(A) polymerases reveal that pla1 is more closely related to Saccharomyces cerevisiae PAP than to bovine PAP . The two yeast poly(A) polymerases share significant sequence homology not only in the generally conserved N-terminal part but also in the C-terminus . Furthermore, pla1 rescues a S . cerevisiae PAP1 disruption mutant . An extract from the complemented strain is active in the specific in vitro polyadenylation assay . In contrast, recombinant PLA1 protein can not replace bovine PAP in the mammalian in vitro polyadenylation assay . These results indicate a high degree of conservation of the polyadenylation machinery among the evolutionary diverged budding and fission yeasts. Mol Cell Biol, 1996 Jul, 16(7), 3420 - 8 Schizosaccharomyces pombe map1+ encodes a MADS-box-family protein required for cell-type-specific gene expression; Yabana N et al.; We cloned the Schizosaccharomyces pombe map1 gene by virtue of its ability to stimulate transcription of the sxa2 gene, which encodes a carboxypeptidase expressed specifically in h- cells in response to mating-pheromone signaling . The cloned gene had a coding capacity of 398 amino acids split by two introns, and the deduced product was a protein of the MADS box family . This gene was most similar to Saccharomyces cerevisiae MCM1, which regulates cell-type-specific gene expression in budding yeast cells . Disruption of the S . pombe gene did not affect vegetative cell growth but conferred sterility . It blocked the mating ability of h+ cells completely and that of h- cells partially . Genetic and sequencing analysis indicated that the cloned gene is map1}, which was originally defined by a mutation that caused h+-speciftic sterility . Northern (RNA) blot analysis showed that the function of map1 is absolutely essential for the expression of h+-specific genes and is required for the full activation of h--specific gene expression . Overexpression of map1 resulted in enhanced transcription of cell-type-specilic genes, but the range of genes affected by Map1 was restricted by the mating type of the cell . Results of yeast two-hybrid analysis suggested that Map1 may physically interact with Mat1-Pc, the product of the h(+)-specific mating-type gene mat1-Pc . On the basis of these observations, we speculate that Map1 may be a transcriptional regulator of cell-type-specific genes similar to S . cerevisiae MCM1, whose activity is modulated by the oil and alpha2 mating-type gene products. J Biol Chem, 1996 Jun 28, 271(26), 15341 - 5 Heterologous complementation of a Rieske iron-sulfur protein-deficient Saccharomyces cerevisiae by the Rip1 gene of Schizosaccharomyces pombe; di Rago JP et al.; A cDNA carrying the Rip1 gene, which encodes the Rieske iron-sulfur protein of Schizosaccharomyces pombe, has been cloned by complementing the respiratory deficiency of a Saccharomyces cerevisiae strain in which the endogenous copy of the RIP1 gene has been deleted . The deduced amino acid sequences of the S . pombe and S . cerevisiae iron-sulfur proteins are 50% identical, with the highest region of identity being in the C termini of the proteins, where the 2Fe:2S cluster is bound . When expressed in the S . cerevisiae deletion strain, the S . pombe iron-sulfur protein restores 25-30% of the ubiquinol-cytochrome c reductase activity . The kinetics of cytochrome c reduction, the effects of inhibitors which act at defined sites in the cytochrome bc1 complex, and the optical properties of cytochrome b in membranes from the S . cerevisiae deletion strain complemented with S . pombe iron-sulfur protein indicate that the S . pombe protein interacts with cytochrome b to restore an apparently normal ubiquinol oxidase site, but that interaction between the iron-sulfur protein and cytochrome c1 is partially impaired . This is the first heterologous replacement of an electron transfer protein in a respiratory enzyme complex in S . cerevisiae. Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6308 - 13 Structural basis for selectivity of the isoquinoline sulfonamide family of protein kinase inhibitors; Xu RM et al.; A large family of isoquinoline sulfonamide compounds inhibits protein kinases by competing with adenosine triphosphates(ATP), yet interferes little with the activity of other ATP-using enzymes such as ATPases and adenylate cyclases . One such compound, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CK17), is selective for casein kinase-1 isolated from a variety of sources . Here we report the crystal structure of the catalytic domain of Schizosaccharomyces pombe casein kinase-1 complexed with CK17, refined to a crystallographic R-factor of 17.8% at 2.5 angstrom resolution . The structure provides new insights into the mechanism of the ATP-competing inhibition and the origin of their selectivity toward different protein kinases . Selectivity for protein kinases versus other enzymes is achieved by hydrophobic contacts and the hydrogen bond with isoquinoline ring . We propose that the hydrogen bond involving the ring nitrogen-2 atom of the isoquinoline must be preserved, but that the ring can flip depending on the chemical substituents at ring positions 5 and 8 . Selectivity for individual members of the protein kinase family is achieved primarily by interactions with these substituents. Mol Gen Genet, 1996 Jun 24, 251(4), 483 - 92 The genetics of the repair of 5-azacytidine-mediated DNA damage in the fission yeast Schizosaccharomyces pombe; Hegde V et al.; We have recently demonstrated that Schizosaccharomyces pombe cells treated with the nucleoside analogue 5-azacytidine (5-azaC) require previously characterised G2 checkpoint mechanisms for survival . Here we present a survey of known DNA repair mutations which defines those genes required for survival in the presence of 5-azaC . Using a combination of single-mutant and epistasis analyses we find that the excision, mismatch and recombinational repair pathways are all required in some degree for the repair of 5-azaC-mediated DNA damage . There are distinct differences in the epistatic interactions of several of the repair mutations with respect to 5-azaC-mediated DNA damage relative to UV-mediated DNA damage. Nucleic Acids Res, 1996 Jun 15, 24(12), 2404 - 10 A novel family of TRF (DNA topoisomerase I-related function) genes required for proper nuclear segregation; Castano IB et al.; We recently reported the identification of a gene, TRF4 (for DNA topoisomerase related function), in a screen for mutations that are synthetically lethal with mutations in DNA topoisomerase I (top1) . Here we describe the isolation of a second member of the TRF4 gene family, TRF5 . Overexpression of TRF5 complements the inviability of top1 trf4 double mutants . The predicted Trf5 protein is 55% identical and 72% similar to Trf4p . As with Trf4p, a region of Trf5p is homologous to the catalytically dispensable N-terminus of Top1p . The TRF4/5 function is essential as trf4 trf5 double mutants are inviable . A trf4 (ts) trf5 double mutant is hypersensitive to the anti-microtubule agent thiabendazole at a semi-permissive temperature, suggesting that TRF4/5 function is required at the time of mitosis . Examination of nuclear morphology in a trf4 (ts) trf5 mutant at a restrictive temperature reveals the presence of many cells undergoing aberrant nuclear division, as well as many anucleate cells, demonstrating that the TRF4/5 function is required for proper mitosis . Database searches reveal the existence of probable Schizosaccharomyces pombe and human homologs of Trf4p, indicating that TRF4 is the canonical member of a gene family that is highly conserved evolutionarily. Nucleic Acids Res, 1996 Jun 15, 24(12), 2377 - 86 Purification and characterization of the Pac1 ribonuclease of Schizosaccharomyces pombe; Rotondo G et al.; The pac1+ gene of the fission yeast Schizosaccharomyces pombe is essential for viability and its overexpression induces sterility and suppresses mutations in the pat1+ and snm1+ genes . The pac1+ gene encodes a protein that is structurally similar to RNase III from Escherichia coli, but its normal function is unknown . We report here the purification and characterization of the Pac1 protein after overexpression in E . coli . The purified protein is a highly active, double-strand-specific endoribonuclease that converts long double-stranded RNAs into short oligonucleotides and also cleaves a small hairpin RNA substrate . The Pac1 RNase is inhibited by a variety of double- and single-stranded polynucleotides, but polycytidylic acid greatly enhances activity and also promotes cleavage specificity . The Pac1 RNase produces 5'-phosphate termini and requires Mg2+; Mn2+ supports activity but causes a loss of cleavage specificity . Optimal activity was obtained at pH 8.5, at low ionic strength, in the presence of a reducing agent . The enzyme is relatively insensitive to N-ethylmaleimide but is strongly inhibited by ethidium bromide and vanadyl ribonucleoside complexes . The properties of the Pac1 RNase support the hypothesis that it is a eukaryotic homolog of RNase III. Gene, 1996 Jun 12, 172(1), 137 - 41 Candida albicans CDK1 and CYB1: cDNA homologues of the cdc2/CDC28 and cdc13/CLB1/CLB2 cell cycle control genes; Damagnez V et al.; Major transitions in the eukaryotic cell cycle are regulated by the cyclin-dependent protein kinases (CDK) . In particular, the G2/M transition is initiated by the activity of a complex formed by a CDK of the Cdc2/Cdc28 family and B-type cyclins of the Cdc13/C1b family in the yeasts, Schizosaccharomyces pombe (Sp) and Saccharomyces cerevisiae (Sc) . To study the molecular mechanisms that control the G2/M transition in the dimorphic pathogenic yeast, Candida albicans, we have cloned and characterized cDNAs corresponding to CDK1 and CYB1 . The CDK1 cDNA encodes a 317-amino-acid (aa) protein that shares 76.8 and 62.3% identity with the Sc CDC28 and Sp cdc2 gene products, respectively . The CYB1 cDNA encodes a 493-aa protein that is 34.8, 34.4 and 35.5% identical to Sc C1b1 and C1b2, and to Sp Cdc13, respectively . Cyb1 contains characteristic mitotic destruction and cyclin boxes . The CDK1 and CYB1 cDNAs are functional homologues, as they are able to complement Sp cdc2 and cdc13 temperature-sensitive (ts) mutations, respectively, and their gene products interact in vivo in Sc to form an active histone H1 kinase. Gene, 1996 Jun 12, 172(1), 125 - 9 Isolation of an HSP12-homologous gene of Schizosaccharomyces pombe suppressing a temperature-sensitive mutant allele of cdc4; Jang YJ et al.; Defects in the Schizosaccharomyces pombe (Sp) cell cycle-controlling genes prevent the cell cycle progression . Mutations in one of the late septation genes, cdc4, cause Sp cells to arrest at cytokinesis and result in an elongated cellular morphology . By functional complementation of one of the temperature-sensitive (ts) mutant alleles of cdc4, cdc4-31, with a Sp cDNA library, a novel gene, scf1, which suppresses the elongated ts phenotype of cdc4-31, was isolated . DNA sequence analysis of the cDNA revealed homology with a small heat-shock protein family, HSP12, of Saccharomyces cerevisiae (Sc) . Expression of this gene is highly induced, both at 37 degrees C and in the stationary phase of cell growth . It is likely that scf1 is expressed in stress conditions such as heat-shock or nutritional limitation . The phenotypic suppression of cdc4-31 by a small HSP12 homolog, Scf1, suggests that the functional loss of cdc4, which is involved in formation of the F-actin contractile ring, can be prevented or repaired by one of the small HSP . This implies that an HSP might be involved in late cell plate formation or in stabilization of the Sp F-actin contractile ring structure. Biochim Biophys Acta, 1996 Jun 7, 1307(2), 129 - 31 Cloning, sequencing and regulation of a cDNA encoding a small heat-shock protein from Schizosaccharomyces pombe; Orlandi I et al.; We have isolated a Schizosaccharomyces pombe cDNA encoding a small heat-shock protein, designated Hsp9 . The deduced amino acid sequence shares significant homology with the Saccharomyces cerevisiae Hsp12 gene product . Northern blot analysis identified a 600-base transcript which is expressed at a low level in S . pombe exponentially growing cells, but is strongly induced by heat-shock and upon entry into the stationary phase . An increase in the transcript level is also observed in response to glucose deprivation. Cell Struct Funct, 1996 Jun, 21(3), 167 - 74 Schizosaccharomyces pombe is more sensitive to pressure stress than Saccharomyces cerevisiae; Sato M et al.; The effects of hydrostatic pressure on ultrastructure, microtubules and microfilaments of Schizosaccharomyces pombe were investigated by fluorescence microscopy, conventional electron microscopy and immunoelectron microscopy . Cells were treated with hydrostatic pressure from 0.1 to 400 MPa for 10 min at room temperature . The nuclear membrane was disrupted at above 100 MPa . At 150 MPa the matrixes of mitochondria had an electron dense area . At 250 MPa the cytoplasmic substances changed dramatically, the cellular organelles could hardly be detected and the fragmented nuclear membrane was barely visible . The fluorescence in alpha-tubulin was lost in most of the cells at 100 MPa . The gold particles for anti alpha-tubulin were not visible in the cells at the same level . Cell cycle specific actin distribution was lost even at 50 MPa, although actin dots localized at the central region remained unchanged . Thick actin cables appeared at 100 MPa . Complete depolymerization of F-actin was observed at 150 MPa . These results suggest that S . pombe cells were more sensitive than Saccharomyces cerevisiae cells . The damage to microtubules and nuclear membrane caused by hydrostatic pressure was though to be followed by breakdown of nuclear division apparatus and the inhibition of nuclear division . This damage might contribute to the frequent formation of polyploidy in S . pombe. Glycobiology, 1996 Jun, 6(4), 453 - 61 Trehalose-P synthase of mycobacteria: its substrate specificity is affected by polyanions; Pan YT et al.; The trehalose-P synthase was purified to near homogeneity from the cytoplasmic fraction of Mycobacterium smegmatis . At the final stage of purification, the enzyme preparation showed one major band of 59 kDa on SDS gels . The 59 kDa band became labeled with N3-UDP{32P}-glucose, and this labeling was inhibited in a concentration-dependent manner by either unlabeled UDP-glucose or GDP-glucose . The native enzyme also had a molecular weight of about 60 kDa by gel filtration, indicating that the active enzyme is a monomer . The 59 kDa protein was subjected to endoproteinase Lys-C digestion, and three peptides isolated by HPLC were sequenced . The sequences of 56 amino acids in these three peptides showed 60% identity to the trehalose-P synthases of Saccharomyces cerevesiae and Schizosaccharomyces pombe . The purified mycobacterial enzyme catalyzed the synthesis of trehalose-P from glucose-6-P and a variety of nucleoside diphosphate glucose derivatives, depending on whether a polyanion was absent or present . Thus, UDP-glucose and GDP-glucose were the best glucosyl donors, but maximum activity with UDP-glucose required the presence of a polyanion such as heparin, whereas activity with GDP-glucose was relatively independent of polyanion . The presence of heparin in the incubation mixture increased the affinity of the enzyme for UDP-glucose by a factor of 100, or more . However, the affinity for GDP-glucose was only twofold better in the presence of heparin . The purified synthase also utilized ADP-glucose and CDP-glucose, but the K(m) for these glucosyl donors was quite high even in the presence of polyanion . The effect of heparin on UDP-glucose activity was dose-dependent and maximum at about 1-2 micrograms of heparin/incubation . However, the size of the heparin molecule (i.e., the number of monosaccharide residues) was critical for activation, and only those heparins with 18 or more monosaccharide units were effective in stimulating activity. J Cell Sci, 1996 Jun, 109 ( Pt 6), 1647 - 53 The kinetics of the B cyclin p56cdc13 and the phosphatase p80cdc25 during the cell cycle of the fission yeast Schizosaccharomyces pombe; Creanor J et al.; The levels of the B cyclin p56cdc13 and the phosphatase p80cdc25 have been followed in selection-synchronised cultures of Schizosaccharomyces pombe wild-type and wee1 mutant cells . p56cdc13 has also been followed in induction-synchronised cells of the mutant cdc2-33 . The main conclusions are: (1) cdc13 levels in wild-type cells start to rise from base line at about mid-G2, reach a peak before mitosis and then fall slowly through G1 . Cells exit mitosis with appreciable levels of cdc13 . (2) cdc13 levels in wee1 cells fall to zero in interphase . They also start to rise at the beginning of G2, which may be related to the absence of a mitotic size control . (3) cdc25 starts to rise later and reaches a peak after mitosis . This is not what would be expected from a simple mitotic inducer and suggests that cdc25 has an important function at the end of mitosis . (4) An upper (heavier) band of cdc25 peaks at the same time as the main band but rises and falls more rapidly . If this is a hyperphosphorylated form, its timing shows that it is most unlikely to function in the ways shown for such a form in eggs and mammalian cells . (5) Experiments with the mutant cdc10-129 and with hydroxyurea show that the initial signal to begin synthesis of cdc13 originates at Start . (6) In induction synchrony, where G2 spans across cell division, there is evidence that some events in one cycle cannot start in the previous one . (7) Revised timings are given for the times of mitosis in these cultures. Plant Mol Biol, 1996 Jun, 31(3), 585 - 93 Thi1, a thiamine biosynthetic gene in Arabidopsis thaliana, complements bacterial defects in DNA repair; Machado CR et al.; An Arabidopsis thaliana cDNA was isolated by complementation of the Escherichia coli mutant strain BW535 (xth, nfo, nth), which is defective in DNA base excision repair pathways . This cDNA partially complements the methyl methane sulfonate (MMS) sensitive phenotype of BW535 . It also partially corrects the UV-sensitive phenotype of E . coli AB1886 (uvrA) and restores its ability to reactivate UV-irradiated lambda phage . It has an insert of ca . 1.3 kb with an open reading frame of 1047 bp (predicting a protein with a molecular mass of 36 kDa) . This cDNA presents a high homology to a stress related gene from two species of Fusarium (sti35) and to genes whose products participate in the thiamine biosynthesis pathway, THI4, from Saccharomyces cerevisiae and nmt2 from Schizosaccharomyces pombe . The Arabidopsis predicted polypeptide has homology to several protein motifs: amino-terminal chloroplast transit peptide, dinucleotide binding site, DNA binding and bacterial DNA polymerases . The auxotrophy for thiamine in the yeast thi4::URA3 disruption strain is complemented by the Arabidopsis gene . Thus, the cloned gene, named thi1, is likely to function in the biosynthesis of thiamine in plants . The data presented in this work indicate that thi1 may also be involved in DNA damage tolerance in plant cells. Microbiology, 1996 Jun, 142 ( Pt 6), 1399 - 407 Metabolic fluxes in chemostat cultures of Schizosaccharomyces pombe grown on mixtures of glucose and ethanol; de Jong-Gubbels P et al.; Simultaneous utilization of glucose and ethanol by the yeast Schizosaccharomyces pombe CBS 356 was studied in aerobic chemostat cultures . In glucose-limited cultures, respirofermentative metabolism occurred at growth rates above 0.16 h-1 . Although Sch . pombe lacks a functional glyoxylate cycle and therefore cannot utilize ethanol as a sole carbon source, ethanol was co-consumed by glucose-limited chemostat cultures . As a result, biomass yields increased, but not up to the theoretical value {0.92 g biomass (g glucose)-1} expected if all of the acetyl-CoA produced from glucose was instead synthesized from ethanol . When ethanol accounted for more than 30% of the substrate carbon in the mixed feed, it was incompletely utilized . In mixed-substrate cultures with a saturating ethanol fraction in the feed, the increase of the biomass yield as a result of ethanol consumption was highest at low dilution rates . This was not due to an increased specific rate of ethanol consumption at low growth rates; rather, the longer residence times at low dilution rates allowed Sch . pombe to utilize a larger fraction of the available ethanol, part of which was oxidized to acetate . Activities of gluconeogenic and glyoxylate-cycle enzymes were not detected in cell-free extracts of any of the cultures . Activities of acetaldehyde dehydrogenase and acetyl-CoA synthetase were low and of the same order of magnitude as the in vivo rates of acetate activation to acetyl-CoA . The results show that ethanol is a poor substrate for Sch . pombe, even as an auxiliary energy source. Biosci Biotechnol Biochem, 1996 Jun, 60(6), 994 - 9 Genetic analysis of the sam mutations, which induce sexual development with no requirement for nutritional starvation in fission yeast; Katayama S et al.; The cAMP pathway and the Ras pathway are the two major pathways to sexual development in the fission yeast Schizosaccharomyces pombe . To understand the cAMP pathway or the related pathway, we analyzed mutants that display a phenotype similar to cyr1-, that is, hyper-sporulation . Nine mutants termed sam (sporulation abnormal mutant), which are highly inclined to sexual development despite the presence of nitrogen sources, were partially characterized . Cyclic AMP was detected in all nine sam mutant cells, and over-expression of the adenylyl cyclase gene (cyr1) failed to suppress the hyper-sporulation phenotype of these sam mutants, suggesting that none of the sam mutants were likely to be allelic to cyr1 . Epistatic tests of sam mutants showed that they were divided into two dominant and seven recessive mutants . Dominants were able to make spores in sam/sam+ heterodiploid cells upon abundant nutrients . Both two dominant mutants bypassed the inability to make spores in ras1 deficient diploid cells, suppressed the deficiency to execute sporulation in byr2 deficient diploid cells, but failed to suppress the byr1 deficiency . Two dominant mutations seem not to occur within the byr2 gene. J Cell Biol, 1996 Jun, 133(6), 1307 - 19 A novel suppressor of ras1 in fission yeast, byr4, is a dosage-dependent inhibitor of cytokinesis; Song K et al.; A novel gene, designated byr4, was identified in Schizosaccharomyces pombe that affects the mitotic cell cycle and shows genetic interactions with the ras1 signaling pathways . Null alleles of byr4 cause cell cycle arrest in late mitosis and permit multiple rounds of septation . The multiple septa typically divide two nuclei, but the nuclei frequently do not stain equally with 4',6-diamidino-2-phenylindole (DAPI), suggesting that byr4 is required for proper karyokinesis . Overexpression of byr4 inhibits cytokinesis, but cell cycle progression continues leading to multinucleate cells . When byr4 is overexpressed, the early steps in the cytokinesis pathway, including formation of the medial F-actin ring, occur normally; however, the later steps in the pathway, including contraction of the F-actin ring, septation, and rearrangement of the medial F-actin following mitosis, rarely occur, byr4 shows two genetic interactions with ras1 . The inhibition of cytokinesis by byr4 overexpression was exacerbated by null alleles of ras1 and scd1, suggesting a link between pathways needed for cell polarity and cytokinesis . Overexpression of byr4 also partially bypasses the need for ras1 for sporulation . The electrophoretic mobility of the byr4 protein varied in response to mutants that perturb cytokinesis and karyokinesis, suggesting interactions between byr4 and these gene products . A more rapidly migrating byr4 protein was found in cells with mutations in cdc16, which undergo repeated septation, and in cdc15, which fail to form a medial F-actin ring in mitosis . A slower migrating byr4 protein was found in cells with a mutation in the beta-tubulin gene, which arrests cells at the metaphase-anaphase transition. Nucleic Acids Res, 1996 Jun 1, 24(11), 2059 - 66 An essential domain in Saccharomyces cerevisiae U14 snoRNA is absent in vertebrates, but conserved in other yeasts; Samarsky DA et al.; U14 is a small nucleolar RNA (snoRNA) required for early cleavages of eukaryotic precursor rRNA . The U14 RNA from Saccharomyces cerevisiae is distinguished from its vertebrate homologues by the presence of a stem-loop domain that is essential for function . This element, known as the Y-domain, is located in the U14 sequence between two universal sequences that base pair with 18S rRNA . Sequence data obtained for the U14 homologues from four additional phylogenetically distinct yeasts showed the Y-domain is not unique to S.cerevisiae . Comparison of the five Y-domain sequences revealed a common stem-loop structure with a conserved loop sequence that includes eight invariant nucleotides . Conservation of these features suggests that the Y-domain is a recognition signal for an essential interaction . Several plant U14 RNAs were found to contain similar structures, though with an unrelated consensus sequence in the loop portion . The U14 gene from the most distantly related yeast, Schizosaccharomyces pombe, was found to be active in S.cerevisiae, showing that Y-domain function is conserved and that U14 function can be provided by variants in which the essential elements are embedded in dissimilar flanking sequences . This last result suggests that U14 function may be determined solely by the essential elements. Nucleic Acids Res, 1996 Jun 1, 24(11), 2005 - 10 Identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme UBC9; Yasugi T et al.; Ubiquitin conjugating enzymes (UBCs) are a family of proteins directly involved in ubiquitination of proteins . Ubiquitination is known to be involved in control of a variety of cellular processes, including cell proliferation, through the targeting of key regulatory proteins for degradation . The ubc9 gene of the yeast Saccharomyces cerevisiae (Scubc9) is an essential gene which is required for cell cycle progression and is involved in the degradation of S phase and M phase cyclins . We have identified a human homolog of Scubc9 (termed hubc9) using the two hybrid screen for proteins that interact with the human papillomavirus type 16 E1 replication protein . The hubc9 encoded protein shares a very high degree of amino acid sequence similarity with ScUBC9 and with the homologous hus5+ gene product of Schizosaccharomyces pombe . Genetic complementation experiments in a S.cerevisiae ubc9ts mutant reveal that hUBC9 can substitute for the function of ScUBC9 required for cell cycle progression. Curr Genet, 1996 Jun, 30(1), 83 - 8 Electrophoretic karyotype of the amylolytic yeast Lipomyces starkeyi and cloning, sequencing and chromosomal localization of its TRP1 gene; Bignell GR et al.; The genome of the amylolytic yeast strain Lipomyces starkeyi NCYC 1436 was analysed using contour-clamped homogeneous electric field gel electrophoresis (CHEF) . The banding pattern under a variety of running conditions indicating the presence of 11 different chromosome-sized DNA molecules . The sizes of these chromosome bands were determined by comparison with chromosomes from standard strains of Schizosaccharomyces pombe and Saccharomyces cerevisiae . The chromosomal bands were estimated to be within the range 0.7-2.8 Mb, with the genome (excluding mitochondrial DNA) estimated at 15 Mb . The molecular cloning of the TRP1 gene, isolated from a genomic library of this strain, is also reported: the gene was present on a 6.5-kb Sau3A DNA fragment, and complemented the trpC gene of E . coli . The DNA sequence was determined (EMBL accession No . Z68292) and compared to other tryptophan biosynthetic genes encoding N-(5'-phosphoribosyl) anthranilate isomerase (PRAI) activity . The gene was also used as a probe in hybridization studies, and by this means, its chromosomal location was identified. Mol Cell Biol, 1996 Jun, 16(6), 2870 - 7 Activation and regulation of the Spc1 stress-activated protein kinase in Schizosaccharomyces pombe; Degols G et al.; Spc1, an osmotic-stress-stimulated mitogen-activated protein kinase (MAPK) homolog in the fission yeast Schizosaccharomyces pombe, is required for the induction of mitosis and survival in high-osmolarity conditions . Spc1, also known as Sty1, is activated by Wis1 MAPK kinase and inhibited by Pyp1 tyrosine phosphatase . Spc1 is most closely related to Saccharomyces cerevisiae Hog1 and mammalian p38 kinases . Whereas Hog1 is specifically responsive to osmotic stress, we report here that Spc1 is activated by multiple forms of stress, including high temperature and oxidative stress . In this regard Spc1 is more similar to mammalian p38 . Activation of Spc1 is crucial for survival of various forms of stress . Spc1 regulates expression of genes encoding stress-related proteins such as glycerol-3-phosphate dehydrogenase (gpd1+) and trehalose-6-phosphate synthase (tps1+) . Spc1 also promotes expression of pyp2+, which encodes a tyrosine phosphatase postulated as a negative regulator of Spc1 . This proposal is supported by the finding that Spc1 associates with Pyp2 in vivo and that the amount of Spc1 tyrosine phosphorylation is lower in a Pyp2-overproducing strain than in the wild type . Moreover, the level of stress-stimulated gpd1+ expression is higher in delta pyp2 mutants than in the wild type . These findings demonstrate that Spc1 promotes expression of genes involved in stress survival and that of regulation may be commonly employed to modulate MAPK signal transduction pathways in eukaryotic species. Nature, 1996 May 30, 381(6581), 438 - 41 Cut2 proteolysis required for sister-chromatid seperation in fission yeast; Funabiki H et al.; Although mitotic cyclins are well-known substrates for ubiquitin-mediated proteolysis at the metaphase-anaphase transition, their degradation is not essential for separation of sister chromatids; several lines of evidence suggest that proteolysis of other protein(s) is required, however . Here we report the anaphase-specific proteolysis of the Schizosaccharomyces pombe Cut2 protein, which is essential for sister-chromatid separation . Cut2 is located in the nucleus, where it is concentrated along the short metaphase spindle . The rapid degradation of Cut2 at anaphase requires its amino-terminal region and the activity of Cut9 (ref . 14), a component of the 20S cyclosome/anaphase-promoting complex (APC), which is necessary for cyclin destruction . Expression of non-degradable Cut2 blocks sister-chromatid separation but not cell-cycle progression . This defect can be overcome by grafting the N terminus of cyclin B onto the truncated Cut2, demonstrating that the regulated proteolysis of Cut2 is essential for sister-chromatid separation. FEBS Lett, 1996 May 27, 387(1), 89 - 93 Characterization of the NHA1 gene encoding a Na+/H+-antiporter of the yeast Saccharomyces cerevisiae; Prior C et al.; The NHA1 gene (2958 nt) encoding a putative Na(+)/H(+) antiporter (986 aa) in Saccharomyces cerevisiae was cloned by selection based on increased NaCl tolerance . The putative protein is highly similar to sodium/proton antiporters from Schizosaccharomyces pombe (gene sod2), and Zygosaccharomyces rouxii (gene Z-SOD2) . Overexpression of the NHA1 gene results in higher and partially pH-dependent tolerance to sodium and lithium; its disruption leads to an increased sensitivity towards these ions. Gene, 1996 May 24, 171(1), 119 - 22 Amiloride toxicity in the fission yeast Schizosaccharomyces pombe is released by thiamine and mutations in the thiamine-repressible gene car1; Niederberger C et al.; Amiloride (Am) inhibits growth in the fission yeast Schizosaccharomyces pombe . We show that the toxic effect of this drug is relieved by low concentrations of thiamine (Th) and that the pyrimidine moiety of the Th molecule is responsible for growth inhibition release . A putative membrane protein encoded by the car1 gene is the target for Am action . It is responsible for Am sensitivity and is involved in the utilization of Th and its biosynthetic precursor, 4-amino-5-hydroxymethyl-2-methylpyrimidine . Its expression is repressed by Th and is under the genetic control of the genes, thi1, tnr1, tnr2 and tnr3, which have previously been shown to be responsible for the transcriptional control of genes involved in the biosynthesis and dephosphorylation of Th. Mol Gen Genet, 1996 May 23, 251(2), 204 - 10 Cloning by functional complementation, and inactivation, of the Schizosaccharomyces pombe homologue of the Saccharomyces cerevisiae gene ABC1; Bonnefoy N et al.; The Saccharomyces cerevisiae gene ABC1 is required for the correct functioning of the bc1 complex of the mitochondrial respiratory chain . By functional complementation of a S . cerevisiae abc1(-) mutant, we have cloned a Schizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein . Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution . The cloned cDNA corresponds to a single S . pombe gene abc1Sp, located on chromosome II, expression of which is not regulated by the carbon source . Inactivation of the abc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of the S . cerevisiae ABC1 gene . The inactivated strain shows a drastic decrease in the bc1 complex activity . a decrease in cytochrome aa3 and a slow growth phenotype . To our knowledge, this is the first example of the inactivation of a respiratory gene in S . pombe . Our results highlight the fact that S . pombe growth is highly dependent upon respiration, and that S . pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes. Mol Gen Genet, 1996 May 23, 251(2), 167 - 75 Identification of the DNA damage-responsive elements of the rhp51+ gene, a recA and RAD51 homolog from the fission yeast Schizosaccharomyces pombe; Jang YK et al.; The Schizosaccharomyces pombe rhp51+ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and the Saccharomyces cerevisiae RAD51 protein . Levels of rhp51+ mRNA increase following several types of DNA damage or inhibition of DNA synthesis . An rhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulating rhp51+ expression in response to DNA damage . Two elements, designated DRE1 and DRE2 (for damage-responsive element), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes from S . cerevisiae . However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene . A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function . The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa . DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins . These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility of rhp51+ expression. Mol Gen Genet, 1996 May 23, 251(2), 153 - 60 Two-hybrid interaction of a human UBC9 homolog with centromere proteins of Saccharomyces cerevisiae; Jiang W et al.; Using a two-hybrid system, we cloned a human cDNA encoding a ubiquitin-conjugating enzyme (UBC), hUBC9, which interacts specifically with all three subunits of the Saccharomyces cerevisiae centromere DNA-binding core complex, CBF3 . The hUBC9 protein shows highest homology to a new member of the UBC family: 54% identity to S . cerevisiae Ubc9p and 64% identity to Schizosaccharomyces pombe (Sp) hus5 . Overexpression of hUBC9 partially suppresses a S . cerevisiae ubc9 temperature-sensitive mutation, indicating that the UBC9 gene family is also functionally conserved . Like hUBC9, Sphus5 also interacts specifically with all three subunits of the CBF3 complex . However, S . cerevisiae Ubc9p interacts only with the Cbf3p subunit (64 kDa) of the CBF3 complex, indicating the specificity of the interaction between S . cerevisiae Ubc9 and Cbf3p proteins . The function of Ubc9p in the G2/M phase of S . cerevisiae could be related to regulation of centromere proteins in chromosome segregation in mitosis . Therefore, the ubiquitination process and centromere function may be linked to chromosome segregation . We also provide further in vivo evidence that Mck1p, a protein kinase, is specifically associated with the centromere proteins Cbf2p and Cbf5p, which were previously shown to interact in vitro. Nucleic Acids Res, 1996 May 15, 24(10), 1849 - 54 The small subunit of the splicing factor U2AF is conserved in fission yeast; Wentz-Hunter K et al.; The human splicing factor U2 auxiliary factor (hsU2AF) is comprised of two interacting subunits of 65 and 35 kDa . Previously we identified the Schizosaccharomyces pombe homolog, spU2AF59, of the human large subunit . We have screened a fission yeast cDNA library in search of proteins that interact with spU2AF59 using the yeast two-hybrid system and have identified a homolog of the hsU2AF35 subunit . The S . pombe U2AF small subunit is a single copy gene that encodes a protein which shares 55% amino acid identity and 17% similarity with the human small subunit . Unlike the human protein, the yeast protein lacks an arginine/serine-rich region . The predicted molecular mass of the spU2AF small subunit is 23 kDa . The region of spU2AF59 that interacts with spU2AF23 is similar to the region in which the human small and large subunits interact. Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 5031 - 6 Functional expression of the Schizosaccharomyces pombe Na+/H+ antiporter gene, sod2, in Saccharomyces cerevisiae; Hahnenberger KM et al.; In the fission yeast, Schizosaccharomyces pombe, tolerance to high sodium and lithium concentrations requires the functioning of the sod2, Na+/H+ antiporter . We have directly measured the activity of this antiporter and demonstrated reconstitution of the activity in gene deletion strains . In addition, we have shown that it can be transferred to, and its antiporter activity detected in, the budding yeast, Saccharomyces cerevisiae, where it also confers sodium and lithium tolerance . Proton flux through the S . pombe Na+/H+ antiporter was directly measured using microphysiometry . The direction of transmembrane proton flux mediated by this antiporter was reversible, with protons being imported or exported in response to the external concentration of sodium . This bidirectional activity was also detected in S . cerevisiae strains expressing sod2 and expression of this gene complemented the sodium and lithium sensitivity resulting from inactivation of the ENA1/PMR2 encoded Na+-exporting ATPases . This suggests that antiporters or sodium pumps can be utilized interchangeably by S . cerevisiae to regulate internal sodium concentration . Potent inhibitors of mammalian Na+/H+ exchangers were found to have no effect on sod2 activity . The proton flux mediated by sod2 was also found to be unaffected by perturbation of membrane potential or the plasma membrane proton gradient. Biochem Mol Biol Int, 1996 May, 39(1), 127 - 35 Molecular cloning of GAF2, a Schizosaccharomyces pombe GATA factor, which has two zinc-finger sequences; Hoe KL et al.; By low stringency screening of a lambda-Shizosaccharomyces pombe genomic library, we have cloned a GATA factor homologous gene, gaf2+, within a 3.1-kb EcoRI fragment . The gaf2 ORF predicts a protein of M(r) 61 kDa consisting of intronless 564 amino acids corresponding to 1,692 bp . Gaf2 has two zinc-fingers as Urbs1 of Ustilago maydis, whereas most of fungal GATA factors have only one zinc-finger . The separation between two zinc-fingers of Gaf2 is rather long . In addition to gaf2, the sequence analysis revealed a Val-tRNA gene in the 3'-flanking region of gaf2 . Northern blot analysis indicated that the gaf2 gene is transcribed constitutively irrespective of the nitrogen source in a medium. Yeast, 1996 May, 12(6), 555 - 64 Isolation and characterization of Schizosaccharomyces pombe fragile mutants; Belda F et al.; Three Schizosaccharomyces pombe fragile mutants requiring the presence of an osmotic stabilizer to grow, that lyse when transferred into hypotonic solutions and that secrete to the extracellular medium more protein than the parental strain were isolated . In the three mutants, the fragile phenotype segregated in a Mendelian fashion, indicating a single chromosomal gene mutation, and behaved as a recessive character . By complementation analysis, the three fragile mutants fell in a single complementation group, defining the same gene (SRB1) . Mutations of this gene are responsible for alterations in the cells such as fragile character, increase in the cell wall porosity, changes in the cell morphology and floc-forming ability . The study of the three srb1 alleles indicated that the degree of these alterations is proportional to a significant decrease in the galactomannan fraction of the mutants cell wall . The data presented in this report suggest that the product of the SRB1 gene is critical for the maintenance of the integrity and structure of Sz . pombe cell wall. Yeast, 1996 May, 12(6), 523 - 9 Review: the Cct eukaryotic chaperonin subunits of Saccharomyces cerevisiae and other yeasts; Stoldt V et al.; All eight of the CCT1-CCT8 genes encoding the subunits of the Cct chaperonin complex in Saccharomyces cerevisiae have been identified, including three that were uncovered by the systematic sequencing of the yeast genome . Although most of the properties of the eukaryotic Cct chaperonin have been elucidated with mammalian systems in vitro, studies with S . cerevisiae conditional mutants revealed that Cct is required for assembly of microtubules and actin in vivo . Cct subunits from the other yeasts, Candida albicans and Schizosaccharomyces pombe, also have been identified from partial and complete DNA sequencing of genes . Cct8p from C . albicans, the only other completely sequenced Cct protein from a fungal species other than S . cerevisiae, is 72% and 61% similar to the S . cerevisiae and mouse Cct8 proteins, respectively. Biosci Biotechnol Biochem, 1996 May, 60(5), 918 - 20 Construction and characterization of a deletion mutant of gpd2 that encodes an isozyme of NADH-dependent glycerol-3-phosphate dehydrogenase in fission yeast; Yamada H et al.; Schizosaccharomyces pombe has two genes each encoding an isozyme of NADH-dependent glycerol-3-phosphate dehydrogenases (gpd1+ and gpd2+) . To gain an insight into the function of these genes, here we constructed a gpd2 deletion mutant, in addition to the previously constructed gpd1 deletion mutant . We showed that the gpd1+ and gpd2+ gene-products are both functional in terms of the de novo glycerol synthesis . Furthermore, the gpd1(+)-mediated glycerol production is primarily responsible for the osmoregulation, but the gpd2+ gene is not . Interestingly, however, the gpd2 deletion mutant had histidine- or lysine-auxotrophy for growth on a minimal medium. RNA, 1996 May, 2(5), 404 - 18 Mutational analysis of U1 function in Schizosaccharomyces pombe: pre-mRNAs differ in the extent and nature of their requirements for this snRNA in vivo; Alvarez CJ et al.; The U1 snRNP is known to play a critical role in spliceosome assembly, at least in part through base pairing of its RNA moiety to the substrate, but many details remain to be elucidated . To further dissect U1 snRNA function, we have analyzed 14 single point mutations in the six nucleotides complementary to the 5' splice site for their effects on growth and splicing in the fission yeast Schizosaccharomyces pombe . Three of the four alleles previously found to support growth of Saccharomyces cerevisiae are lethal in S . pombe, implying a more critical role for the 5' end of U1 in fission yeast . Furthermore, a comparison of phenotypes for individual nucleotide substitutions suggests that the two yeasts use different strategies to modulate the extent of pairing between U1 and the 5' splice site . The importance of U1 function in S . pombe is further underscored by the lethality of several single point mutants not examined previously in S . cerevisiae . In total, only three alleles complement the U1 gene disruption, and these strains are temperature-sensitive for growth . Each viable mutant was tested for impaired splicing of three different S . pombe introns . Among these, only the second intron of the cdc2 gene (cdc2-I2) showed dramatic accumulation of linear precursor . Notably, cdc2-I2 is spliced inefficiently even in cells containing wild-type U1, at least in part due to the presence of a stable hairpin encompassing its 5' splice site . Although point mutations at the 5' end of U1 have no discernible effect on splicing of pre-U6, significant accumulation of unspliced RNA is observed in a metabolic depletion experiment . Taken together, these observations indicate that the repertoire of U1 activities is used to varying extents for splicing of different pre-mRNAs in fission yeast. Curr Genet, 1996 May, 29(6), 530 - 6 The distance-dependence of the fission yeast ade6-M26 marker effect in two-factor crosses; Zahn-Zabal M et al.; Random spore analysis of crosses between a strain bearing the ade6-M26 hotspot mutation and strains bearing other ade6 mutations was performed . Recombinant prototroph frequencies increase with increasing distance from M26 for mutations both 5' and 3' of M26 . Maximum prototroph frequencies are obtained for mutations lying more than 700 nucleotides downstream from M26 . Similar results are obtained for crosses with the ade6-M375 control mutation, but the prototroph frequencies are lower . The factor of stimulation of recombination by M26 as compared to the M375 control (M26 marker effect) also displays distance-dependence . These results are discussed in the context of the mechanism of M26 recombination, as well as in relation to recombination initiation, hybrid DNA formation, and mismatch repair at ade6.Keywords Conversion middle dot M26 hotspot middle dot Recombination middle dot Schizosaccharomyces pombe Nucleic Acids Res, 1996 May 1, 24(9), 1669 - 75 Cloning and characterization of RAD17, a gene controlling cell cycle responses to DNA damage in Saccharomyces cerevisiae; Siede W et al.; Mutants of the yeast Saccharomyces cerevisiae defective in the RAD17 gene are sensitive to ultraviolet (UV) and gamma radiation and manifest a defect in G2 arrest following radiation treatment . We have cloned the RAD17 gene by complementation of the UV sensitivity of a rad17-1 mutant and identified an ORF of 1.2 kb encoding a predicted gene product of 45.4 kDa with homology to the Schizosaccharomyces pombe rad1+ gene product and to Ustilago maydis Rec1, a known 3'->5'exonuclease . The RAD17 transcript is cell cycle regulated, with maximum steady-state levels during late G1 . The rad17-1 mutation represents a missense mutation that maps to a conserved region of the gene . A rad17 disruption mutant grows normally and manifests levels of UV sensitivity similar that of the rad17-1 strain . As previously observed with other genes involved in G2 arrest (such as RAD9 and RAD24), RAD17 regulates radiation-induced G1 checkpoints at at least two possible arrest stages . One is equivalent to or upstream of START, the other at or downstream of the Cdc4 execution point . However, the temperature sensitivity of the cell cycle mutant dna1-1 (a G1 arrest mutant) is not influenced by inactivation of RAD17. Int J Radiat Biol, 1996 May, 69(5), 565 - 73 Effect of B-type cyclin over-expression on radiation-induced mitotic delay in the fission yeast; Rowley R et al.; Exposure to ionizing radiation temporarily blocks eukaryotic cell cycle progression at the G2/M boundary (G2 delay) . The delay probably provides time for repair of DNA damage before chromosome segregation and is thus an active response, indicative of a checkpoint control function . Transition from G2 into mitosis is normally controlled by the activity of a cyclin-dependent kinase, cdc2 in the fission yeast (Schizosaccharomyces pombe) . Genetic and cell kinetic evidence suggest that irradiation may impose mitotic delay by inactivation of the cdc2 product, p34cdc2 . The activity of p34cdc2 in G2 is regulated by phosphorylation and association with a B-type cyclin, the product of the cdc13 gene, p56cdc13 . Previous work does not support a major role for changes in phosphorylation of p34cdc2 in the induction of mitotic delay . Alternatively the kinase may be regulated by changes in the activity/availability of p56cdc13 . We have therefore tested the effect of high level, episomal expression of the cdc13 gene on the induction of mitotic delay . No influence of this procedure on the duration of delay was detected, either in a wild-type cell cycle background, or the mutants wee1-50 and cdc2-3w, which show abnormal phosphorylation of p34cdc2. Biochemistry, 1996 Apr 30, 35(17), 5577 - 85 Is the function of the cdc2 kinase subunit proteins tuned by their propensities to oligomerize? Conformational states in solution of the cdc2 kinase partners p13suc1 and p9cksphy; Birck C et al.; The cdc2 kinase subunit (cks) proteins play an essential function in the control of mitosis through their molecular complexes with the cdc2 kinase . In this work, we characterize the conformational state(s) in solution of the cks proteins p13suc1 from Schizosaccharomyces pombe and p9cksphy from Physarum polycephalum . Monomers of p13suc1 and p9cksphy were found to be markedly nonglobular, presumably with a long, nonfolded C-terminal moiety . This was in contrast to the previously published structure of p13suc1, derived from crystallographic studies on a zinc-promoted p13suc1 dimer, in which the individual p13suc1 subunits had a globular conformation . This disparity was resolved when we found that the globular p13suc1 fold undergoes a conformational transition into nonglobular monomers upon dissociation of the dimers following chelation of the zinc ions by ethylenediaminetetraacetic acid (EDTA) . We also found that p13suc1, but not p9cksphy, forms stable dimers in the absence of metal ions . The topology of these EDTA-insensitive dimers likely resembles that of the human p9ckshs2 protein, characterized by beta 4 strand exchange from each nonglobular monomer. Biochemistry, 1996 Apr 30, 35(17), 5426 - 34 Probing the function of Asp128 in the lower molecular weight protein-tyrosine phosphatase-catalyzed reaction . A pre-steady-state and steady-state kinetic investigation; Wu L et al.; The role of Asp128 in the catalytic mechanism of the low Mr protein-tyrosine phosphatase (PTPase) from the fission yeast Schizosaccharomyces pombe has been investigated by a combination of site-directed mutagenesis and pre-steady-state and steady-state kinetic analysis . The corresponding aspartic acid in the bovine enzyme is located on a loop adjacent to the phosphate-binding loop and forms a hydrogen bond with the oxygen atom of the bound sulfate or phosphate that is structurally homologous to the ester oxygen in substrates {Su et al . (1994) Nature 370, 575-578; Zhang, M., et al . (1994) Biochemistry 33, 11097-11105} . Asp128 has been replaced by a Glu, an Asn, and an Ala . The kcat for the hydrolysis of p-nitrophenyl phosphate (pNPP) decreases by factors of 6.7, 400, and 650 for the mutants D128E, D128N, and D128A, respectively . Compared to the wild type, the binding affinity for phosphate is decreased 2 and 4.3-fold, respectively, for the D128A and D128N mutants, whereas no change in affinity is observed for the D128E mutant . An evaluation of the burst kinetics demonstrates that Asp128 plays a role in both the phosphoenzyme intermediate formation (k2) and breakdown (k3) . Thus, substitution at Asp128 by a Glu, an Asn, or an Ala reduces k2 by 17, 7480, and 11900 and reduces k3 by 6.2, 380, and 40 . The greater effect on k2 and k3 is consistent with a dissociative transition-state for the low M(r)PTPase-catalyzed reaction . Results from the rapid kinetics, partition experiments, and leaving group dependence experiments suggest that for the wild type and D128E mutant, the rate-limiting step is k3, whereas k2 has become rate-limiting for the D128N mutant . With the exception of pNPP, k2 may also be rate-limiting for D128A . Taken together, these results are consistent with Asp128 or Glu128 acting as a general acid to donate a proton to the phenolate leaving group in the phosphorylation step, and the carboxylate side chain plays a role as a general base to activate a nucleophilic water molecule in the dephosphorylation step . The presence of the general acid ensures productive partitioning toward phosphoenzyme formation . In the absence of the general acid, the nature of the transition-state for the phosphorylation step is sensitive to the pKa of the attacking active site thiol group and changes with the structure of the leaving group. J Biol Chem, 1996 Apr 26, 271(17), 10017 - 22 Reduction of BiP levels decreases heterologous protein secretion in Saccharomyces cerevisiae; Robinson AS et al.; Increased levels of the endoplasmic reticulum-resident protein folding chaperone BiP would be expected to either increase protein secretory capacity by improved solubilization of folding precursors or decrease secretory capacity by binding and retaining misfolded proteins . To address this question, the relationship between BiP levels and heterologous secretion in yeast was determined . A yeast strain was constructed in which BiP expression is tunable from 5 to 250% of wild-type levels, and this strain was used to explore the effect of varying BiP level on overall secretion of three heterologous proteins: human granulocyte colony-stimulating factor, Schizosaccharomyces pombe acid phosphatase, and bovine pancreatic trypsin inhibitor . For all three proteins examined, reduction in BiP expression below wild-type level diminished overall secretion, whereas 5-fold BiP overexpression from a constitutive glycolytic promoter did not substantially increase or decrease secretion titers . These results are consistent with a positive role for BiP in promoting membrane translocation and solubilization of folding precursors but are inconsistent with a negative role in proofreading and improper retention of heterologous secreted proteins. Gene, 1996 Apr 17, 170(1), 153 - 4 The Schizosaccharomyces pombe spqM gene is a new member of the Qm transcription factor family; Masson JY et al.; The Qm family of proteins, which are found in a wide variety of species such as budding yeast, plants and humans, are believed to play a role in gene expression . Here, we report the isolation ofaa gene, spqM, from the fission yeast Schizosaccharomyces pombe, whose deduced amino-acid sequence shared 71.6 to 61.36% identity with members of the Qm family . The high degree of conservation of the Qm members suggest that they were selectively conserved, because of an important biological role. Gene, 1996 Apr 17, 170(1), 113 - 7 Schizosaccharomyces pombe rad23 is allelic with swi10, a mating-type switching/radioresistance gene that shares sequence homology with human and mouse ERCC1; Hang H et al.; Schizosaccharomyces pombe (Sp) rad23-1 mutant cells are extremely sensitive to UV light and ionizing radiation . A genomic DNA fragment that contains wild-type (wt) rad23 has been cloned . The DNA sequence of this cloned gene has been determined and was found to be identical to the previously characterized mating-type switching/radioresistance gene, swi10 . Complementation tests between rad23-1 and swi10-154 mutant cells exclusively produce UV-sensitive progeny and confirm that these two genes are allelic . The DNA sequences of rad23-1 and swi10-154 reveal that each contains a single, unique point mutation . In rad23-1, Glu231 changes to a stop codon, resulting in the production of a truncated protein . In swi10-154, a G to A transition mutation is within a splice consensus sequence for intron 1 . Therefore, the corresponding mRNA is incapable of being processed appropriately. Nucleic Acids Res, 1996 Apr 15, 24(8), 1481 - 8 The gamma subfamily of DNA polymerases: cloning of a developmentally regulated cDNA encoding Xenopus laevis mitochondrial DNA polymerase gamma; Ye F et al.; We used the known sequence of the Saccharomyces cerevisiae DNA polymerase gamma to clone the genes or cDNAs encoding this enzyme in two other yeasts, Pychia pastoris and Schizosaccharomyces pombe, and one higher eukaryote, Xenopus laevis . To confirm the identity of the final X.laevis clone, two antisera raised against peptide sequences were shown to react with DNA polymerase gamma purified from X.laevis oocyte mitochondria . A developmentally regulated 4.6 kb mRNA is recognized on Northern blots of oocyte RNA using the X.laevis cDNA . Comparison of the four DNA polymerase gamma gene sequences revealed several highly conserved sequence blocks, comprising an N-terminal 3'-->5'exonuclease domain and a C-terminal polymerase active center interspersed with gamma-specific gene sequences . The consensus sequences for the DNA polymerase gamma exonuclease and polymerase domains show extensive sequence similarity to DNA polymerase I from Escherichia coli . Sequence conservation is greatest for residues located near the active centers of the exo and pol domains of the E.coli DNA polymerase I structure . The domain separating the exonuclease and polymerase active sites is larger in DNA polymerase gamma than in other members of family A (DNA polymerase I-like) polymerases . The S.cerevisiae DNA polymerase gamma is atypical in that it includes a 240 residue C-terminal extension that is not found in the other members of the DNA polymerase gamma family, or in other family A DNA polymerases. Nucleic Acids Res, 1996 Apr 15, 24(8), 1412 - 9 DNA-protein interactions at the telomeric repeats of Schizosaccharomyces pombe; Duffy M et al.; Gel retardation assays using a probe containing the repeat region of a Schizosaccharomyces pombe chromosomal telomere identified four specific DNA- protein complexes in S . pombe total protein extracts (I, I', IIa and IIb) . The proteins responsible for these complexes bound to the telomeric repeat region irrespective of whether or not the repeats were in close proximity to the end of a DNA molecule, and none of them bound strongly to single-stranded DNA . The protein responsible for complex I (TeRF I) was separated from the activity responsible for complexes IIa and IIb (TeRF II) using heparin-Sepharose chromatography . Both factors were efficiently cross-competed by an oligonucleotide containing the 18 bp sequence 5'-GGTTACAGGTTACAGGTT-3', which corresponds to two complete telomeric repeat units . Mutation of the T residues at positions 4 and 11 in the oligonucleotide dramatically reduced binding by TeRF II, but had no affect on binding by TeRF I . The protein responsible for complex I' did not bind strongly to either the wild-type or mutant oligonucleotide. J Mol Biol, 1996 Apr 12, 257(4), 804 - 13 Activation of a yeast pseudo DNA methyltransferase by deletion of a single amino acid; Pinarbasi E et al.; The biological methylation cytosine bases in DNA is central to such diverse phenomena as restriction and modification in bacteria, repeat induced point-mutation (RIPing) in fungi and for programming gene expression patterns in vertebrates . Structural studies on HhaI DNA methyltransferase, together with the sequence comparisons of around 40 cytosine-specific DNA methyltransferases, have recently provided a molecular framework for understanding the mechanism of action of the related group of enzymes that catalyse this base modification . There are, however, a number of organisms, including Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster, which have no detectable DNA methylation . Here we report that the product of the pmt1 gene recently identified in S . pombe, which contains most of the primary structure elements of a typical cytosine-specific DNA methyltransferase, is catalytically inert owing to the insertion of a Ser residue between the Pro-Cys motif found at the active site of all such DNA methyltransferases . Following deletion of this Ser residue, catalytic activity is restored and, using a range of DNA binding experiments, it is shown that the enzyme recognises and methylates the sequence CC(A/T)GG, the same sequence that is modified by the product of the Escherichia coli dcm gene . The pmt gene of S . pombe therefore encodes a pseudo DNA methyltranferase, which we have called psiM.SpoI. J Biol Chem, 1996 Apr 12, 271(15), 9166 - 71 Characterization of a nuclear protein conferring brefeldin A resistance in Schizosaccharomyces pombe; Turi TG et al.; The fungal metabolite brefeldin A disrupts protein secretion and causes the redistribution of the Golgi complex to the endoplasmic reticulum . Previously we isolated six genes that, when present in multiple copies, confer brefeldin A resistance to wild type Schizosaccharomyces pombe . Here we describe the characterization of one of these genes, hba1 . This gene encodes an essential protein that shares homology with the mammalian protein RanBP1 and the protein encoded by the Saccharomyces cerevisiae gene YRB1 and contains a peptide motif present in several proteins found within the nuclear pore complex . The protein encoded by hba1 is localized to the nucleus, and it was determined that this protein is phosphorylated in vivo . The characterization of hba1 thus demonstrates a novel mechanism of drug resistance in S . pombe. J Mol Biol, 1996 Apr 5, 257(3), 618 - 31 Three-dimensional structure of mammalian casein kinase I: molecular basis for phosphate recognition; Longenecker KL et al.; The three-dimensional structure for the catalytic region of the mammalian protein kinase, casein kinase I delta (CKI delta), has been solved by X-ray crystallography to a resolution of 2.3 A . A truncation mutant of CKI delta lacking the C-terminal autoinhibitory region was expressed in Escherichia coli, purified, and crystallized . The structure was solved by molecular replacement using the crystal structure of the catalytic domain of a CKI homolog from Schizosaccharomyces pombe, Cki1 . A tungstate derivative confirmed the initial molecular replacement solution and identified an anion binding site which may contribute to the unique substrate specificity of CKI . Like other protein kinases, the catalytic domain of CKI is composed of two lobes with a cleft between them for binding ATP . Comparison of the mammalian and yeast CKI structures suggests that a rotation of the N-terminal domain occurs upon ATP binding . This domain motion is similar, but not identical, to that observed in cAMP-dependent protein kinase upon binding ATP . Although Cki1 has many similarities to CKI delta over the catalytic domain, these two forms of CKI likely perform different functions in vivo . Relating the primary sequences of other CKI enzymes to the three-dimensional architecture of CKI delta reveals a catalytic face that is especially conserved among the subset of CKI family members associated with the regulation of DNA repair. Oncogene, 1996 Apr 4, 12(7), 1513 - 20 Analysis of human c-Abl tyrosine kinase activity and regulation in S . pombe; Walkenhorst J et al.; c-Abl protein tyrosine kinase activity is tightly regulated in vertebrate cells . Several mutations, including deletions of the SH3 domain, can activate abl and convert it into an oncogene . To study c-Abl activity in a cellular environment likely to lack specific regulators, we have expressed human c-Abl in Schizosaccharomyces pombe in an inducible fashion . c-Abl, but not a kinase inactive form of the molecule, causes growth arrest followed by death of the cells . Concomitant to Abl expression we observed extensive phosphorylation of endogenous proteins on tyrosine . Mutations in the SH2 domain or in the autophosphorylation site dramatically reduce the ability of Abl to confer the growth arrest phenotype and to phosphorylate endogenous proteins, suggesting a fundamental role of these structures in the activity of the enzyme . An SH3 domain deletion mutant of Abl is equally active as wild type c-Abl in yeast, even under conditions allowing detection of subtle differences . These results demonstrate that there is no intrinsic regulation of c-Abl kinase activity via the SH3 domain and suggest that the inhibitory effect of the SH3 domain observed in mammalian cells is medicated by a factor that is absent in fission yeast . Expression of Ab1 S.pombe provides a novel quantitative assay for ab1 activity and regulation. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2958 - 63 Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes; Kovalenko OV et al.; Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned . The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae . The Hsubc9 gene complements a ubc9 mutation of S . cerevisiae . It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus . According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51 . A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein . In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis. Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 2850 - 5 cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein; Cimprich KA et al.; A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified . One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA . Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S . cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression . We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family . Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa . Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24 . FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins. Genes Cells, 1996 Apr, 1(4), 391 - 408 Schizosaccharomyces pombe gad7+ encodes a phosphoprotein with a bZIP domain, which is required for proper G1 arrest and gene expression under nitrogen starvation; Kanoh J et al.; BACKGROUND: Fission yeast cells arrest at G1 phase when starved of nitrogen . The molecular mechanism that ensures this arrest is poorly understood . We took a genetic approach to this problem . RESULTS: The fission yeast gad7-1 mutant failed to arrest at G1 when starved of nitrogen, and was poor in mating and sporulation . The gad7 gene was cloned by complementation . The deduced gad7 gene product was a bZIP protein of 566 amino acids, which could bind to the CRE (cAMP response element) sequence in vitro . Disruption of gad7 resulted in the same phenotypes as gad7-1 . Expression of ste11, which encodes a key transcription factor for sexual development, was not inducible in the disruptant . Gad7 was co-immunoprecipitated with another bZIP protein Pcr1, suggesting that the two proteins form a heterodimer in vivo . Gad7 was phosphorylated, and the state of its phosphorylation appeared to be modified in pka1delta or wis1delta cells . CONCLUSIONS: Gad7, a CRE-binding protein that cooperates with Pcr1, is required for proper G1 arrest and gene expression under nitrogen starvation . Gad7 is a phosphoprotein, whose activity may be regulated by protein kinases including the cAMP-dependent protein kinase (Pka1) and Wis1 osmosensory MAP kinase kinase. Arch Microbiol, 1996 Apr,