FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 151 - 5 An archaebacterial homolog of pelota, a meiotic cell division protein in eukaryotes; Ragan MA et al.; An open reading frame (pelA) specifying a homolog of pelota and DOM34, proteins required for meiotic cell division in Drosophila melanogaster and Saccharomyces cerevisiae, respectively, has been cloned, sequenced and identified from the archaebacterium Sulfolobus solfataricus . The S . solfataricus PelA protein is about 20% identical with pelota, DOM34 and the hypothetical protein R74.6 of Caenorhabditis elegans . The presence of a pelota homolog in archaebacteria implies that the meiotic functions of the eukaryotic protein were co-opted from, or added to, other functions existing before the emergence of eukaryotes . The nuclear localization signal and negatively charged carboxy-terminus characteristic of eukaryotic pelota-like proteins are absent from the S . solfataricus homolog, and hence may be indicative of the acquired eukaryotic function(s). Cell, 1996 Nov 1, 87(3), 405 - 13 tRNA ligase is required for regulated mRNA splicing in the unfolded protein response; Sidrauski C et al.; The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers an intracellular signaling pathway, the unfolded protein response (UPR), that leads to increased transcription of genes encoding ER-resident proteins . Transcriptional activation is mediated by a dedicated transcription factor, Hac1p, whose activity is controlled by regulated splicing of its mRNA . We have identified a mutation in tRNA ligase that disrupts the UPR in the yeast Saccharomyces cerevisiae . In this mutant, splicing of HAC1 mRNA, but not tRNA, is blocked . In contrast, HAC1 mRNA splicing is not impaired in cells that are blocked in spliceosome-mediated mRNA splicing . Furthermore, the splice junctions of HAC1 mRNA do not conform to the consensus sequences of other yeast pre-mRNAs . Our results suggest that the regulated splicing of HAC1 mRNA occurs by a novel pathway, involving tRNA ligase and bypassing the spliceosome. Cell, 1996 Nov 1, 87(3), 391 - 404 A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response; Cox JS et al.; Cells respond to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing transcription of genes encoding ER-resident proteins . The information is transmitted from the ER lumen to the nucleus by an intracellular signaling pathway, the unfolded protein response (UPR) . We have identified a basic-leucine zipper transcription factor, Hac1p, that is required for the UPR and binds to the UPR element in the promoter of UPR-regulated genes . Surprisingly, Hac1p is found in UPR-activated cells only, and its level is controlled by regulated splicing of its mRNA . Splicing replaces the C-terminal tail of Hac1p with a different peptide that renders Hac1p more resistant to an otherwise extremely rapid ubiquitin-dependent degradation . We propose that the complex regulation of Hac1p expression serves to provide multiple levels at which the UPR can be controlled. Endocrinology, 1996 Nov, 137(11), 5126 - 34 Developmental expression of proprotein-processing endoprotease furin in rat pancreatic islets; Kayo T et al.; The expression of furin in pancreatic beta-cells induces faster cell growth and a decrease in differentiated beta-cell-specific characteristics . During the development of rat pancreatic islets, the prohormone convertases, PC2 and PC3, appear during late gestation and are expressed long after birth . We investigated the developmental change in another yeast Kex2 family endoprotease, furin, in rat islets in relation to islet cell growth . Furin had appeared by gestational day 18 and was distributed in islets as well as exocrine tissues . The expression of furin in islets increased toward the neonatal stage . Bromodeoxyuridine (BrdU) incorporation was also elevated in the perinatal period . On postnatal days 10 and 20, staining characteristics were attenuated . On day 25, immediately after weaning, furin staining began to localize in the beta-cell region, and staining in the alpha-cell region became fainter . On day 270, the staining in the alpha-cell region disappeared, and staining in the beta-cell region remained positive, but was weaker . We conclude that furin expression was greatest during the perinatal period, when BrdU incorporation into islets was maximal . Furin expression continued, however, even after the BrdU incorporation decreased . Thus, furin appears to control the proliferation as well as differentiation of islet cells. Mol Cell Biol, 1996 Nov, 16(11), 6603 - 16 Identification of a regulatory subcomplex in the guanine nucleotide exchange factor eIF2B that mediates inhibition by phosphorylated eIF2; Yang W et al.; Eukaryotic translation initiation factor 2B (eIF2B) is a five-subunit complex that catalyzes guanine nucleotide exchange on eIF2 . Phosphorylation of the alpha subunit of eIF2 {creating eIF2(alphaP}) converts eIF2 x GDP from a substrate to an inhibitor of eIF2B . We showed previously that the inhibitory effect of eIF2(alphaP) can be decreased by deletion of the eIF2B alpha subunit (encoded by GCN3) and by point mutations in the beta and delta subunits of eIF2B (encoded by GCD7 and GCD2, respectively) . These findings, plus sequence similarities among GCD2, GCD7, and GCN3, led us to propose that these proteins comprise a regulatory domain that interacts with eIF2(alphaP) and mediates the inhibition of eIF2B activity . Supporting this hypothesis, we report here that overexpression of GCD2, GCD7, and GCN3 specifically reduced the inhibitory effect of eIF2(alphaP) on translation initiation in vivo . The excess GCD2, GCD7, and GCN3 were coimmunoprecipitated from cell extracts, providing physical evidence that these three proteins can form a stable subcomplex . Formation of this subcomplex did not compensate for a loss of eIF2B function by mutation and in fact lowered eIF2B activity in strains lacking eIF2(alphaP) . These findings indicate that the trimeric subcomplex does not possess guanine nucleotide exchange activity; we propose, instead, that it interacts with eIF2(alphaP) and prevents the latter from inhibiting native eIF2B . Overexpressing only GCD2 and GCD7 also reduced eIF2(alphaP) toxicity, presumably by titrating GCN3 from eIF2B and producing the four-subunit form of eIF2B that is less sensitive to eIF2(alphaP) . This interpretation is supported by the fact that overexpressing GCD2 and GCD7 did not reduce eIF2(alphaP) toxicity in a strain lacking GCN3; however, it did suppress the impairment of eIF2B caused by the gcn3c-R104K mutation . An N-terminally truncated GCD2 protein interacted with other eIF2B subunits only when GCD7 and GCN3 were overexpressed, in accordance with the idea that the portion of GCD2 homologous to GCD7 and GCN3 is sufficient for complex formation by these three proteins . Together, our results provide strong evidence that GCN3, GCD7, and the C-terminal half of GCD2 comprise the regulatory domain in eIF2B. Mol Cell Biol, 1996 Nov, 16(11), 6593 - 602 p53-dependent association between cyclin G and the B' subunit of protein phosphatase 2A; Okamoto K et al.; We and others previously showed that cyclin G is a transcriptional target of the p53 tumor suppressor protein . However, cellular proteins which might form a complex with cyclin G have not yet been identified . To gain insight into the biological role of cyclin G, we used the yeast two-hybrid screen and isolated two mouse cDNAs encoding cyclin G-interacting proteins . Interestingly, both positive cDNAs encoded B' regulatory subunits of protein phosphatase 2A (PP2A) . One clone encodes B'alpha, while the other clone codes for a new member of the B' family, B'beta . B'beta is 70% identical to other members of the B' family . B'alpha associated both in vitro and in vivo with cyclin G but not with the other mammalian cyclins . Furthermore, cyclin G formed a complex with B'alpha only after induction of p53 in p53 temperature-sensitive cell lines . These results indicate that cyclin G forms a specific complex with the B' subunit of PP2A and that complex formation is regulated by p53 . Potential roles for the cyclin G-B' complex in p53-mediated pathways are discussed. Mol Cell Biol, 1996 Nov, 16(11), 6468 - 76 In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior; Shaaban SA et al.; We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S . A . Shaaban, B . M . Krupp, and B . D . Hall, Mol . Cell . Biol . 15:1467-1478, 1995) . The pattern of completed-transcript release was also characterized for three of the mutant enzymes . The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P . James, S . Whelen, and B . D . Hall, J . Biol . Chem . 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III . In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants . One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme . In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA(Tyr) gene . Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly . The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved . Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end . The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination. Mol Cell Biol, 1996 Nov, 16(11), 6457 - 67 Interaction of D-type cyclins with a novel myb-like transcription factor, DMP1; Hirai H et al.; The cyclin D-dependent kinases CDK4 and CDK6 trigger phosphorylation of the retinoblastoma protein (RB) late in G1 phase, helping to cancel its growth-suppressive function and thereby facilitating S-phase entry . Although specific inhibition of cyclin D-dependent kinase activity in vivo can prevent cells from entering S phase, it does not affect S-phase entry in cells lacking functional RB, implying that RB may be the only substrate of CDK4 and CDK6 whose phosphorylation is necessary for G1 exit . Using a yeast two-hybrid interactive screen, we have now isolated a novel cyclin D-interacting myb-like protein (designated DMP1), which binds specifically to the nonamer DNA consensus sequences CCCG(G/T)ATGT to activate transcription . A subset of these DMP1 recognition sequences containing a GGA trinucleotide core can also function as Ets-responsive elements . DMP1 mRNA and protein are ubiquitously expressed throughout the cell cycle in mouse tissues and in representative cell lines . DMP1 binds to D-type cyclins directly in vitro and when coexpressed in insect Sf9 cells . In both settings, it can be phosphorylated by cyclin D-dependent kinases, suggesting that its transcriptional activity may normally be regulated through such mechanisms . These results raise the possibility that cyclin D-dependent kinases regulate gene expression in an RB independent manner, thereby serving to link other genetic programs to the cell cycle clock. Mol Cell Biol, 1996 Nov, 16(11), 6295 - 302 Autophosphorylation sites participate in the activation of the double-stranded-RNA-activated protein kinase PKR; Taylor DR et al.; The interferon-induced RNA-dependent protein kinase PKR is found in cells in a latent state . In response to the binding of double-stranded RNA, the enzyme becomes activated and autophosphorylated on several serine and threonine residues . Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase . We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the activation of PKR . Mutation of one site, threonine 258, results in a kinase that is less efficient in autophosphorylation and in phosphorylating its substrate, the initiation factor eIF2, in vitro . The mutant kinase is also impaired in vivo, displaying reduced ability to inhibit protein synthesis in yeast and mammalian cells and to induce a slow-growth phenotype in Saccharomyces cerevisiae . Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect . Taken together with earlier results (S . B . Lee, S . R . Green, M . B . Mathews, and M . Esteban, Proc . Natl . Acad . Sci . USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via autophosphorylation. Mol Cell Biol, 1996 Nov, 16(11), 6208 - 17 Allele-specific suppression of a defective trans-Golgi network (TGN) localization signal in Kex2p identifies three genes involved in localization of TGN transmembrane proteins; Redding K et al.; Kex2 protease (Kex2p) and Ste13 dipeptidyl aminopeptidase (Ste13p) are required in Saccharomyces cerevisiae for maturation of the alpha-mating factor in a late Golgi compartment, most likely the yeast trans-Golgi network (TGN) . Previous studies identified a TGN localization signal (TLS) in the C-terminal cytosolic tail of Kex2p consisting of Tyr-713 and contextual sequences . Further analysis of the Kex2p TLS revealed similarity to the Ste13p TLS . Mutation of the Kex2p TLS results in transport of Kex2p to the vacuole by default . When expression of a GAL1 promoter-driven KEX2 gene is shut off in MAT(alpha) cells, the TGN becomes depleted of Kex2p, resulting in a gradual decline in mating competence which is greatly accelerated by TLS mutations . To identify the genes involved in localization of Kex2p, we isolated second-site suppressors of the rapid loss of mating competence observed upon shutting off expression of a TLS mutant form of Kex2p (Y713A) . Seven of 58 suppressors were allele specific, suppressing point mutations at Tyr-713 but not deletions of the TLS or entire C-terminal cytosolic tail . By linkage analysis, the allele-specific suppressors defined three genetic loci, SOI1, S0I2, and S0I3 . Pulse-chase analysis demonstrated that these suppressors increased net TGN retention of both Y713A Kex2p and a Ste13p-Pho8p fusion protein containing a point mutation in the Ste13p TLS . SOI1 suppressor alleles reduced the efficiency of localization of wild-type Kex2p to the TGN, implying an impaired ability to discriminate between the normal TLS and a mutant TLS . soi1 mutants also exhibited a recessive defect in vacuolar protein sorting . Suppressor alleles of S0I2 were dominant . These results suggest that the SOI1 and S0I2 genes encode regulators or components of the TLS recognition machinery. Mol Cell Biol, 1996 Nov, 16(11), 6200 - 7 Mutational analysis of the hsp70-interacting protein Hip; Prapapanich V et al.; The hsp70-interacting protein Hip participates in the assembly pathway for progesterone receptor complexes . During assembly, Hip appears at early assembly stages in a transient manner that parallels hsp70 interactions . In this study, a cDNA for human Hip was used to develop various mutant Hip forms in the initial mapping of functions to particular Hip structural elements . Hip regions targeted for deletion and/or truncation included the C-terminal region (which has some limited homology with Saccharomyces cerevisiae Sti1 and its vertebrate homolog p60), a glycine-glycine-methionine-proline (GGMP) tandem repeat, and a tetratricopeptide repeat (TPR) . Binding of Hip to hsp70's ATPase domain was lost with deletions from the TPR and from an adjoining highly charged region; correspondingly, these Hip mutant forms were not recovered in receptor complexes . Truncation of Hip's Sti1-related C terminus resulted in Hip binding to hsp70 in a manner suggestive of a misfolded peptide substrate; this hsp70 binding was localized to the GGMP tandem repeat . Mutants lacking either the C terminus or the GGMP tandem repeat were still recovered in receptor complexes . Truncations from Hip's N terminus resulted in an apparent loss of Hip homo-oligomerization, but these mutants retained association with hsp70 and were recovered in receptor complexes . This mutational analysis indicates that Hip's TPR is required for binding of Hip with hsp70's ATPase domain . In addition, some data suggest that hsp70's peptide-binding domain may alternately or concomitantly bind to Hip's GGMP repeat in a manner regulated by Sti1-related sequences. Mol Cell Biol, 1996 Nov, 16(11), 6110 - 20 The mismatch repair system reduces meiotic homeologous recombination and stimulates recombination-dependent chromosome loss; Chambers SR et al.; Efficient genetic recombination requires near-perfect homology between participating molecules . Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system . The effects of chromosomal divergence in diploids of Saccharomyces cerevisiae in which one copy of chromosome III is derived from a closely related species, Saccharomyces paradoxus, have been examined . Meiotic recombination between the diverged chromosomes is decreased by 25-fold . Spore viability is reduced with an observable increase in the number of tetrads with only two or three viable spores . Asci with only two viable spores are disomic for chromosome III, consistent with meiosis I nondisjunction of the homeologs . Asci with three viable spores are highly enriched for recombinants relative to tetrads with four viable spores . In 96% of the class with three viable spores, only one spore possesses a recombinant chromosome III, suggesting that the recombination process itself contributes to meiotic death . This phenomenon is dependent on the activities of the mismatch repair genes PMS1 and MSH2 . A model of mismatch-stimulated chromosome loss is proposed to account for this observation . As expected, crossing over is increased in pms1 and msh2 mutants . Furthermore, genetic exchange in pms1 msh2 double mutants is affected to a greater extent than in either mutant alone, suggesting that the two proteins act independently to inhibit homeologous recombination . All mismatch repair-deficient strains exhibited reductions in the rate of chromosome III nondisjunction. Mol Cell Biol, 1996 Nov, 16(11), 6083 - 95 Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site; Oyake T et al.; Members of the small Maf family (MafK, MafF, and MafG) are basic region leucine zipper (bZip) proteins that can function as transcriptional activators or repressors . The dimer compositions of their DNA binding forms determine whether the small Maf family proteins activate or repress transcription . Using a yeast two-hybrid screen with a GAL4-MafK fusion protein, we have identified two novel bZip transcription factors, Bach1 and Bach2, as heterodimerization partners of MafK . In addition to a Cap'n'collar-type bZip domain, these Bach proteins possess a BTB domain which is a protein interaction motif; Bach1 and Bach2 show significant similarity to each other in these regions but are otherwise divergent . Whereas expression of Bach1 appears ubiquitous, that of Bach2 is restricted to monocytes and neuronal cells . Bach proteins bind in vitro to NF-E2 binding sites, recognition elements for the hematopoietic transcription factor NF-E2, by forming heterodimers with MafK . Furthermore, a DNA binding complex that contained MafK as well as Bach2 or a protein related closely to Bach2 was found to be present in mouse brain cells . Bach1 and Bach2 function as transcription repressors in transfection assays using fibroblast cells, but they function as a transcriptional activator and repressor, respectively, in cultured erythroid cells . The results suggest that members of the Bach family play important roles in coordinating transcription activation and repression by MafK. Mol Cell Biol, 1996 Nov, 16(11), 6029 - 36 RIP-140 interacts with multiple nuclear receptors by means of two distinct sites; L'Horset F et al.; We have characterized two distinct binding sites, called site 1 and site 2, in the nuclear protein RIP-140 which interact with the ligand binding domain of the estrogen receptor both in solution and when the receptor is bound to DNA . Both sites are capable of independently interacting with other nuclear receptors, including the thyroid hormone and retinoic acid receptors, but they are not identical since the interaction with retinoid X receptor is mediated primarily by site 1 . The interaction is enhanced by agonists but not by antagonists, and the in vitro binding activities to a number of mutant receptors correlate with their abilities to stimulate transcription in vivo . When RIP-140 is fused to heterologous DNA binding domains, it is able to stimulate the transcription of reporter genes in both yeast and mammalian cells . Thus, RIP-140 is likely to function as a bridging protein between receptors and the basal transcription machinery and thereby stimulate the transcription of target genes. Diabetes, 1996 Nov, 45(11), 1556 - 62 Troglitazone inhibits fatty acid oxidation and esterification, and gluconeogenesis in isolated hepatocytes from starved rats; Fulgencio JP et al.; The effects of troglitazone and pioglitazone on glucose and fatty acid metabolism were studied in hepatocytes isolated from 24-h-starved rats . These thiazolidinediones inhibited long-chain fatty acid (oleate) oxidation and produced a very oxidized mitochondrial redox state . By contrast, thiazolidinediones did not affect the rate of medium-chain fatty acid (octanoate) oxidation or the activity of mitochondrial carnitine palmitoyltransferase (CPT) I . Thiazolidinediones inhibited selectively triglyceride synthesis but not phospholipid synthesis . The combined inhibition of oleate oxidation and esterification by troglitazone was due to a noncompetitive inhibition of mitochondrial and microsomal long-chain acyl-CoA synthetase (ACS) activities . It was suggested that troglitazone must be metabolized into its sulfo-conjugate derivative in liver cells to inhibit mitochondrial and microsomal ACS activities . Thiazolidinediones inhibited glucose production from lactate/pyruvate or from alanine . Analysis of gluconeogenic metabolite concentrations suggested that troglitazone would inhibit gluconeogenesis at the level of pyruvate carboxylase and glyceraldehyde-3-phosphate dehydrogenase reactions . It was concluded that 1) at a similar concentration, troglitazone was more efficient than pioglitazone to inhibit fatty acid metabolism and gluconeogenesis and 2) the inhibition of gluconeogenesis by troglitazone could be the result of the inhibition of long-chain fatty acid oxidation (decrease in acetyl-CoA, NADH-to-NAD+, and ATP-to-ADP ratios). Science, 1996 Nov 1, 274(5288), 774 - 7 PIN: an associated protein inhibitor of neuronal nitric oxide synthase; Jaffrey SR et al.; The neurotransmitter functions of nitric oxide are dependent on dynamic regulation of its biosynthetic enzyme, neuronal nitric oxide synthase (nNOS) . By means of a yeast two-hybrid screen, a 10-kilodalton protein was identified that physically interacts with and inhibits the activity of nNOS . This inhibitor, designated PIN, appears to be one of the most conserved proteins in nature, showing 92 percent amino acid identity with the nematode and rat homologs . Binding of PIN destabilizes the nNOS dimer, a conformation necessary for activity . These results suggest that PIN may regulate numerous biological processes through its effects on nitric oxide synthase activity. Med Pediatr Oncol, 1996 Nov, 27(5), 476 - 83 Genomic imprinting and Wilms' tumor; Moulton T et al.; The selective loss of maternal and reduplication of paternal chromosome 11p15.5 alleles in Wilms' tumors (WTs) points to the existence of a paternally imprinted tumor suppressor gene(s) and/or a maternally imprinted dose-dependent growth-promoting gene(s) in this chromosomal region . Two reciprocally imprinted chromosome 11p15.5 genes, H19, a candidate tumor suppressor gene, and IGF2, a candidate dominant oncogene, have been well-characterized in terms of their imprinting and expression status in WTs . Here we review and extend data indicating that a majority of WTs show a bipaternal epigenotype at these loci, with H19 inactive and IGF2 biallelically active . This can arise either through loss of heterozygosity (LOH) or by a non-LOH pathway involving localized biallelic hypermethylation of H19 DNA . Conversion to this bipaternal endpoint has recently been found to affect not only these two genes, but also at least one other imprinted 11p15.5 gene, KIP2 . Since 11p15.5 LOH and biallelic H19 hypermethylation can occur both early and late in tumor progression and since early loss is not associated with bilaterality or multifocality of WTs, these types of lesions appear to be permissive rather than rate-limiting in Wilms' tumorigenesis. Nature, 1996 Oct 31, 383(6603), 840 - 3 A meiotic recombination checkpoint controlled by mitotic checkpoint genes; Lydall D et al.; In budding yeast, meiotic recombination occurs at about 200 sites per cell and involves DNA double-strand break (DSB) intermediates . Here we provide evidence that a checkpoint control requiring the mitotic DNA-damage checkpoint genes RAD17, RAD24 and MEC1 ensures that meiotic recombination is complete before the first meiotic division (MI) . First, RAD17, RAD24 and MEC1 are required for the meiotic arrest caused by blocking the repair of DSBs with a mutation in the recA homologue DMC1 . Second, mec1 and rad24 single mutants (DMC1+) appear to undergo MI before all recombination events are complete . Curiously, the mitosis-specific checkpoint gene RAD9 is not required for meiotic arrest of dmc1 mutants . This shows that although mitotic and meiotic control mechanisms are related, they differ significantly . Rad17 and Rad24 proteins may contribute directly to formation of an arrest signal by association with single-strand DNA in mitosis and meiosis. Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12309 - 14 Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication; Hopwood B et al.; We report the isolation and characterization of CDC45, which encodes a polypeptide of 650 amino acids that is essential for the initiation of chromosomal DNA replication in the budding yeast, Saccharomyces cerevisiae . CDC45 genetically interacts with at least two members of the MCM (minichromosome maintenance) family of replication genes, CDC46 and CDC47, which are proposed to perform a role in restricting initiation of DNA replication to once per cell cycle . Like mutants in several MCM genes, alleles of CDC45 also show a severe minichromosome maintenance defect . Together, these observations imply that Cdc45p performs a role in the control of initiation events at chromosomal replication origins . We investigated this possibility further and present evidence demonstrating that Cdc45p is assembled into complexes with one MCM family member, Cdc46p/Mcm5p . These observations point to a role for Cdc45p in controlling the early steps of chromosomal DNA replication in conjunction with MCM polypeptide complexes . Unlike the MCMs, however, the subcellular localization of Cdc45p does not vary with the cell cycle, making it likely that Cdc45p interacts with MCMs only during the nuclear phase of MCM localization in G1. Mol Gen Genet, 1996 Oct 28, 252(6), 709 - 16 NUC-2, a component of the phosphate-regulated signal transduction pathway in Neurospora crassa, is an ankyrin repeat protein; Poleg Y et al.; In response to phosphorus limitation, the fungus Neurospora crassa synthesizes a number of enzymes that function to bring more phosphate into the cell . The NUC-2 protein appears to sense the availability of phosphate and transmits the signal downstream to the regulatory pathway . The nuc-2+ gene has been cloned by its ability to restore growth of a nuc-2 mutant under restrictive conditions of high pH and low phosphate concentration . We mapped the cloned gene to the right arm of linkage group II, consistent with the chromosomal position of the nuc-2 mutation as determined by classical genetic mapping . The nuc-2' open reading frame is interrupted by five introns and codes for a protein of 1066 amino acid residues . Its predicted amino acid sequence has high similarity to that of its homolog in Saccharomyces cerevisiae, PHO81 . Both proteins contain six ankyrin repeats, which have been implicated in the cyclin-dependent kinase inhibitory activity of PHO81 . The phenotypes of a nuc-2 mutant generated by repeat-induced point mutation and of a strain harboring a UV-induced nuc-2 allele are indistinguishable . Both are unable to grow under the restrictive conditions, a phenotype which is to some degree temperature dependent . The nuc-2+ gene is transcriptionally regulated . A 15-fold increase in the level of the nuc-2+ transcript occurs in response to a decrease in exogenous phosphate concentration. FEBS Lett, 1996 Oct 28, 396(1), 1 - 6 Yeasties and beasties: 7 years of genome sequencing; Thomas K; The Saccharomyces cerevisiae genome sequencing project was the first of many projects aimed at sequencing the entire genomes of model organisms . Since its initiation in 1989, there have been numerous debates about the validity of genome sequencing, especially with reference to the model organisms . Seven years on, I hope to satisfy some of the critics by demonstrating that, as a consequence of the mass of data now becoming available from such projects, and the beginning of the major collaborative effort to sequence the human genome, we are now entering an exciting and dynamic time for those involved not only in genome sequencing, but also in all areas of the biological sciences. J Mol Biol, 1996 Oct 25, 263(2), 163 - 80 Substrate recognition and induced DNA distortion by the PI-SceI endonuclease, an enzyme generated by protein splicing; Gimble FS et al.; PI-SceI, a double-stranded DNA endonuclease from Saccharomyces cerevisiae, is generated by protein splicing of an intein, which is an internal polypeptide within a larger precursor protein . The enzyme initiates the mobility of the intein by cleaving at inteinless alleles of the VMA1 gene . Genetic and biochemical studies reveal that the enzyme makes numerous base-specific and phosphate backbone contacts with its 31 bp asymmetrical recognition site . This site can be divided into two regions, both of which contain nucleotides that are essential for cleavage by PI-SceI . Region I contains the PI-SceI cleavage site while Region II includes an adjacent sequence that covers two helical turns . Mutational, interference and DNA mobility shift analyses demonstrate that Region II is sufficient for high-affinity PI-SceI binding . Within this region, PI-SceI uses primarily phosphate backbone and some major groove interactions to contact the DNA, while within Region I, protein binding involves predominantly major groove interactions that overlap and lie proximal to the cleavage site . Interestingly, DNA binding by PI-SceI induces DNA conformational changes within Region II that are entirely exclusive of Region I sequences . Furthermore, additional distortion occurs when PI-SceI binds to Region I in conjunction with Region II . The importance of this latter distortion in the cleavage pathway is underscored by substrate mutations at or near the cleavage site that reduce or eliminate both Region I DNA bending and substrate cleavage . Based on these findings, we propose a model in which sequence-specific contacts made by PI-SceI contribute to its localization to the cleavage site and to its stabilization of a DNA conformation that is required for catalysis . Finally, we discuss how the recognition characteristics of PI-SceI may have allowed the evolution of other endonucleases with altered, but similar, specificities. J Biol Chem, 1996 Oct 25, 271(43), 27044 - 8 Cloning and characterization of a Dictyostelium myosin I heavy chain kinase activated by Cdc42 and Rac; Lee SF et al.; The motile activities of the small, single-headed class I myosins (myosin I) from the lower eukaryotes Acanthamoeba and Dictyostelium are activated by phosphorylation of a single serine or threonine residue in the head domain of the heavy chain . Recently, we purified a myosin I heavy chain kinase (MIHCK) from Dictyostelium based on its ability to activate the Dictyostelium myosin ID isozyme (Lee, S . -F., and Cote, G . P . (1995) J . Biol . Chem . 270, 11776-11782) . The complete sequence of the Dictyostelium MIHCK has now been determined, revealing a protein of 98 kDa that is composed of an amino-terminal domain rich in proline, glutamine, and serine, a putative Cdc42/Rac binding motif, and a carboxyl-terminal kinase catalytic domain . MIHCK shares significant sequence identity with the Saccharomyces cerevisiae Ste20p kinase and the mammalian p21-activated kinase . Gel overlay assays and affinity chromatography experiments showed that MIHCK interacted with GTPgammaS (guanosine 5'-3-O-(thiotriphosphate))-labeled Cdc42 and Rac1 but not RhoA . In the presence of GTPgammaS-Rac1 MIHCK autophosphorylation increased from 1 to 9 mol of phosphate/mol, and the rate of Dictyostelium myosin ID phosphorylation was stimulated 10-fold . MIHCK may therefore provide a direct link between Cdc42/Rac signaling pathways and motile processes driven by myosin I molecules. J Biol Chem, 1996 Oct 25, 271(43), 27031 - 8 Acylation of glucosaminyl phosphatidylinositol revisited . Palmitoyl-CoA dependent palmitoylation of the inositol residue of a synthetic dioctanoyl glucosaminyl phosphatidylinositol by hamster membranes permits efficient mannosylation of the glucosamine residue; Doerrler WT et al.; Two critical steps in the assembly of yeast and mammalian glycosylphosphatidylinositol (GPI) anchor precursors are palmitoylation of the inositol residue and mannosylation of the glucosamine residue of the glucosaminyl phosphatidylinositol (GlcNalpha-PI) intermediate . Palmitoylation has been reported to be acyl-CoA dependent in yeast membranes (Costello, L . C., and Orlean, P . (1992) J . Biol . Chem . 267, 8599-8603) but strictly acyl-CoA independent in rodent membranes (Stevens, V . L., and Zhang, H . (1994) J . Biol . Chem . 269, 31397-31403), and thus poorly conserved . In addition, it was suggested that acylation must precede mannosylation in both yeast (Costello, L . C., and Orlean, P . (1992) J . Biol . Chem . 276, 8599-8603) and rodent (Urakaze, M., Kamitani, T., DeGasperi, R., Sugiyama, E., Chang, H.-M., Warren, C . D., and Yeh, E . T . H . (1992) J . Biol . Chem . 267, 6459-6462) cells because GlcNalpha-acyl-PI accumulates in vivo when mannosylation is blocked . However, GlcNalpha-acyl-PI accumulation would also be expected if mannosylation and acylation were independent of each other . These issues were addressed by the use of a synthetic dioctanoyl GlcNalpha-PI analogue (GlcNalpha-PI(C8)) as an in vitro substrate for GPI-synthesizing enzymes in Chinese hamster ovary cell membranes . GlcNalpha-PI(C8) was acylated in an manner requiring acyl-CoA . Thus, the process involving acyl-CoA reported for yeast has been conserved in mammals . Furthermore, both GlcNalpha-PI(C8) and GlcNalpha-acyl-PI(C8) could be mannosylated in vitro, but mannosylation of the latter was significantly more efficient . This provides direct support for the earlier suggestion that acylation precedes mannosylation in rodents cells . A similar result was also observed with the Saccharomyces cerevisiae mannosyltransferase . In contrast, it has been reported that mannosylation of endogenous GlcNalpha-PI by Trypansoma brucei membranes occurs without prior acylation . The same result was obtained with GlcNalpha-PI(C8), confirming that the mannosyltransferase of trypanosomes is divergent from those in yeasts and rodents. J Biol Chem, 1996 Oct 25, 271(43), 26939 - 46 Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport; Belden WJ et al.; COPII-coated endoplasmic reticulum (ER)-derived transport vesicles contain a distinct set of membrane-bound polypeptides . We have obtained the NH2-terminal amino acid sequence of polypeptide constituents found on purified vesicles and in this report investigate the 24- and 25-kDa species . The 24-kDa protein is identical to Emp24p, a type I transmembrane protein that is required for transport of a subset of secretory proteins from the ER to the Golgi complex (Schimmoller, F., Singer-Kruger, B., Schroder, S., Kruger, U., Barlowe, C., and Riezman, H . (1995) EMBO J . 14, 1329-1339) . The 25-kDa protein, termed Erv25p (ER vesicle protein of 25 kDa), corresponds to an open reading frame found on chromosome XIII of Saccharomyces cerevisiae . Erv25p shares overall sequence identity with Emp24p, but the two proteins are not functionally interchangeable . Antibodies directed against Erv25p reveal that Emp24p and Erv25p depend on each other for stability and form a protein complex that can be isolated after chemical cross-linking . Yeast strains lacking Erv25p (erv25Delta) are viable and display the same selective defect in transport of secretory proteins from the ER to Golgi complex as an emp24Delta strain . A cell-free assay that measures vesicle formation from ER membranes demonstrates that Erv25p and Emp24p are incorporated equally into ER-derived vesicles when COPII-coated budding is reconstituted . Vesicle formation from an erv25Delta strain, an emp24Delta strain and a double erv25Delta emp24Delta strain proceed at wild-type levels; however, incorporation of the Erv25p or the Emp24p protein into COPII-coated vesicles requires expression of both subunits . A potential model for transport of the Erv25p-Emp24p complex between the ER and Golgi compartments is discussed. Science, 1996 Oct 25, 274(5287), 624 - 7 Kap104p: a karyopherin involved in the nuclear transport of messenger RNA binding proteins; Aitchison JD et al.; A cytosolic yeast karyopherin, Kap104p, was isolated and shown to function in the nuclear import of a specific class of proteins . The protein bound directly to repeat-containing nucleoporins and to a cytosolic pool of two nuclear messenger RNA (mRNA) binding proteins, Nab2p and Nab4p . Depletion of Kap104p resulted in a rapid shift of Nab2p from the nucleus to the cytoplasm without affecting the localization of other nuclear proteins tested . This finding suggests that the major function of Kap104p lies in returning mRNA binding proteins to the nucleus after mRNA export. Science, 1996 Oct 25, 274(5287), 546, 563 - 7 Life with 6000 genes; Goffeau A et al.; The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration . The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes . In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history . The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes. FEBS Lett, 1996 Oct 21, 395(2-3), 183 - 7 Involvement of histone hyperacetylation in triggering DNA fragmentation of rat thymocytes undergoing apoptosis; Lee E et al.; The treatment of rat thymocytes with trichostatin A and sodium butyrate, which are inhibitors of histone deacetylase, resulted in an increase in DNA fragmentation in a concentration-dependent manner . A significant increase in DNA fragmentation induced by these compounds was observed after a lag time of 2 h . Analysis of the fragmented DNA revealed the production of approximately 50 kb DNA fragments and DNA ladders, the biochemical hallmarks of apoptotic cell death . Judging from a laser scanning microscopic analysis, the inhibitors of histone deacetylase induced nuclear condensation, the morphological feature of apoptosis . Biochemical and morphological analyses demonstrated that trichostatin A and sodium butyrate induced thymocyte apoptosis . Furthermore, hyperacetylation of nuclear histones was observed in thymocytes treated with the inhibitors of histone deacetylase . These effects of sodium butyrate and trichostatin A were seen 0.5 and 1 h, respectively, after incubation of the cells . These results thus indicate that hyperacetylation of nucleosomal histones precedes DNA fragmentation in thymocytes undergoing apoptosis induced by trichostatin A and sodium butyrate. FEBS Lett, 1996 Oct 21, 395(2-3), 109 - 12 Identification of the antigenic determinants of factors 8, 9, and 34 of genus Candida; Kobayashi H et al.; We investigated the antigenic determinants of factors 8, 9, and 34 of the genus Candida among pathogenic yeasts by enzyme-linked immunosorbent assay (ELISA) using mannans of Saccharomyces cerevisiae wild type and mutant types, mnn 1-mnn 4 and mnn 2 . Results of ELISA including antisera against the antigenic factors of genus Candida (Candida Check, latron; FAbs) indicated that these three types of mannan distinctly react with FAbs 34, 8 and 9, respectively . To identify the recognition sites of these FAbs, we compared the ability of various oligosaccharides to inhibit the binding of the mannans to FAbs . The results indicated that FAb 34 preferentially recognizes linear side chains containing a non-reducing terminal alpha-1,3-linked mannose residue, Man(alpha)1 --> 3Man(alpha)1 --> (2Man(alpha)1 --> )n(2Man) (n > or = 0), and that one of the recognition sites of FAb 9 is linear alpha-1,6-linked oligomannosyl series, Man(alpha)1 --> (6Man(alpha)1 --> )n(6Man) (n > or = 2) . On the other hand, the recognition site of FAb 8 apparently consisted of two alpha-1,2-linked oligomannosyl side chains and an alpha-1,6-linked mannose residue that originated from the mannan backbone, Man(alpha)1 --> 2Man(alpha)1 --> 2(Man(alpha)1 -->2Man(alpha)1 --> 6)Man. J Biol Chem, 1996 Oct 18, 271(42), 25731 - 4 The small GTP-binding protein rho activates c-Jun N-terminal kinases/stress-activated protein kinases in human kidney 293T cells . Evidence for a Pak-independent signaling pathway; Teramoto H et al.; Work from a number of laboratories has established a role for certain small GTP-binding proteins in controlling the enzymatic activity of a family of serine-threonine kinases known as mitogen-activated protein kinases (MAPKs) . MAPKs have been classified into three subfamilies: extracellular signal-regulated kinases (ERKs), also known as MAPKs; c-Jun N-terminal kinases (JNKs); and p38 kinase . Whereas Ras controls the activation of MAPKs, we and others have recently observed that in certain cells, the small GTP-binding proteins Rac1 and Cdc42 but not Rho regulate the activity of JNKs . Furthermore, because Rac1 and Cdc42 but not Rho bind and activate a kinase known as Pak1, it has been suggested that Pak1 is the most upstream component of the pathway linking these GTPases to JNK . However, in both yeast and mammalian cells, Rho1p, a Rho homologue, and RhoA, respectively, directly interact with a number of proteins, including kinases related to protein kinase C . In addition, in yeast, Rho1p controls the activity of a MAPK cascade involved in bud formation . Considering this diversity of target molecules for small GTP-binding proteins, their likely tissue specific distribution, and the potential role for Rho in signaling to a kinase cascade, we decided to extend our initial analysis, exploring the ability of Ras and Rho-related GTP-binding proteins to activate MAPK or JNK in a variety of cell lines . We found that in the human kidney epithelial cell line, 293T, Cdc42 and all Rho proteins, RhoA, RhoB, and RhoC, but not Rac or Ras can induce activation of JNK . Furthermore, we provide evidence that signaling from Rho proteins to JNK in 293T cells does not involve Pak1 . Taken together these findings demonstrate that Rho signals to JNK in a cell type-specific manner and suggest the existence of a novel, Pak1-independent signaling route communicating the Rho family of small GTP-binding proteins to the JNK pathway. Oncogene, 1996 Oct 17, 13(8), 1781 - 7 Three distinct domains in the HOX-11 homeobox oncoprotein are required for optimal transactivation; Zhang N et al.; HOX-11 (tcl-3) is a homeobox oncogene isolated from the breakpoint region of the t(10;14) chromosomal translocation recurring in T-cell acute lymphoblastic leukemia . Here we demonstrate that the HOX-11 homeoprotein mediates transactivation of reporter genes through various promoters in both mammalian and yeast cells . By deletion analysis, the transactivation domains of HOX-11 have been mapped to three amino acid stretches in the homeoprotein, the glycine-proline-rich region at the amino terminus, the homeodomain and the glutamine-rich region at the carboxyl terminus . The three distinct functional domains of HOX-11 act in concert for optimal transactivation . In addition, the homeodomain of HOX-11 appears to be differentially utilized in a promoter-dependent manner . Our data support the notion that the HOX-11 homeoprotein functions as an oncogenic transcription activator in leukemogenesis. Nature, 1996 Oct 17, 383(6601), 644 - 6 Capture of retrotransposon DNA at the sites of chromosomal double-strand breaks; Moore JK et al.; Non-homologous repair of broken chromosomes in Saccharomyces cerevisiae can be studied at a defined location by expressing the site-specific HO endonuclease that cuts the mating-type (MAT) locus . When homologous recombination is prevented, most double-strand breaks are repaired by non-homologous end-joinings similar to those observed in mammalian cells . About 1% of non-homologous repair events were exceptional, having 'captured' approximately 100 base pairs of DNA within the HO cleavage site . In each case, the insertion came from yeast's retrotransposon Tyl element . Four of the five contained the R-U5 region, which is the first part of Tyl messenger RNA to be converted to complementary DNA . The capture of cDNA fragments at the sites of double-strand breaks may account for the way that pseudogenes and long and short interspersed sequences (LINES and SINES) have been inserted at many locations in the mammalian genome. Nature, 1996 Oct 17, 383(6601), 641 - 4 Retrotransposon reverse-transcriptase-mediated repair of chromosomal breaks; Teng SC et al.; The abundance of short and long interspersed nuclear sequences (SINEs and LINEs) and pseudogenes in eukaryotic genomes indicates that reverse transcriptase (RT)-mediated phenomena are important in genome evolution . However, the mechanisms involved in their spread are largely unknown . We have developed a selection system in the yeast Saccharomyces cerevisiae to test whether RT-mediated events could be linked to the repair of double-strand breaks (DSBs) . Here we show that DSBs can be fixed by the insertion of complementary DNAs at the break site . In the presence of functional RT (from human L1, yeast Tyl or Crithidia CRE1), and in the absence of homologous recombination, an HO endonuclease-induced DSB at the mating type (MAT) locus is the primary site at which a marked cDNA is observed among surviving cells . The structure and junctional sequences of these insertions suggest that repair occurs primarily by non-homologous recombination . Our data support a role for endogenous retroelements in the repair of chromosomal breaks. Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11729 - 34 Molecular clock or erratic evolution? A tale of two genes; Ayala FJ et al.; We have investigated the evolution of glycerol-3-phosphate dehydrogenase (Gpdh) . The rate of amino acid replacements is 1 x 10(-10)/site/year when Drosophila species are compared . The rate is 2.7 times greater when Drosophila and Chymomyza species are compared; and about 5 times greater when any of those species are compared with the medfly Ceratitis capitata . This rate of 5 x 10(-10)/site/year is also the rate observed in comparisons between mammals, or between different animal phyla, or between the three multicellular kingdoms . We have also studied the evolution of Cu,Zn superoxide dismutase (Sod) . The rate of amino acid replacements is about 17 x 10(-10)/site/year when comparisons are made between dipterans or between mammals, but only 5 x 10(-10) when animal phyla are compared, and only 3 x 10(-10) when the multicellular kingdoms are compared . The apparent decrease by about a factor of 5 in the rate of SOD evolution as the divergence between species increases can be consistent with the molecular clock hypothesis by assuming the covarion hypothesis (namely, that the number of amino acids that can change is constant, but the set of such amino acids changes from time to time and from lineage to lineage) . However, we know of no model consistent with the molecular clock hypothesis that would account for the increase in the rate of GPDH evolution as the divergence between species increases. Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11534 - 9 A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases; Yang SW et al.; The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes . However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death . We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA . The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate . The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I. Biochemistry, 1996 Oct 15, 35(41), 13494 - 500 Involvement of the alpha subunit of farnesyl-protein transferase in substrate recognition; Pellicena P et al.; Using photoaffinity labeling, we have identified a region in mammalian farnesyl-protein transferase (FPTase) involved in substrate recognition . The photolabel used (Compound 1) is a peptide containing the photoactive amino acid p-benzoylphenylalanine (Bpa) . Upon exposure to UV light . Compound 1 inhibits FPTase activity in a time- and concentration-dependent manner . Photoinhibition of FPTase activity by Compound 1 is prevented by adding H-Ras to the reaction mixture, indicating that labeling is targeted to the enzyme active site . We used peptide mapping by HPLC, Edman sequencing, and matrix-assisted time-of-flight (MALDI-TOF) mass spectrometry to identify the site of interaction with radiolabeled Compound 1 . These experiments indicate that a specific region of the alpha subunit of the enzyme, Asp110-Arg112, is involved in substrate binding and suggest that Glu111 is likely to be the residue covalently modified by the photoaffinity label . Sequence alignments between yeast and mammalian FPTases reveal that Glu111 is conserved . The implications of this finding are discussed in light of previous mutagenesis studies on FPTase. FEBS Lett, 1996 Oct 14, 395(1), 6 - 10 Determination of the disulfide array of the first inducible antifungal peptide from insects: drosomycin from Drosophila melanogaster; Michaut L et al.; Drosomycin is a 44-residue antifungal peptide with four intramolecular disulfide bridges which have been isolated from immune-challenged Drosophila . To produce adequate amounts of this peptide for 3D-structure analysis, studies on the mode of action and activity spectrum, we expressed a synthetic cDNA in Saccharomyces cerevisiae . For this purpose, we used the mating factor alpha gene and concomitantly overexpressed the KEX2 gene to increase the yield of fully processed drosomycin . Using a combination of Edman degradation and mass spectrometry, we show that drosomycin shares the same array of intramolecular disulfide bridges than plant defensins, in addition to their sequence similarities. Biochem Pharmacol, 1996 Oct 11, 52(7), 1007 - 13 Isolation and characterization of rat liver microsomal R-ibuprofenoyl-CoA synthetase; Brugger R et al.; Microsomal long-chain acyl-CoA synthetase (EC 6.1.2.3.) has been suggested to be involved in the stereoselective formation of the CoA thioester of ibuprofen . In this study, we demonstrated that the microsomal enzyme from rat liver responsible for palmitoyl-CoA synthesis also catalyzes the formation of R-ibuprofenoyl-CoA in a Mg(2+)- and ATP-dependent process . Long-chain acyl-CoA synthetase from rat liver microsomes was purified to homogeneity as evidenced by SDS-gel electrophoresis . Simultaneous measurements of palmitoyl-CoA and R-ibuprofenoyl-CoA formation with HPLC in various fractions and purification steps during protein isolation revealed a high correlation between both activities . The purification procedure included solubilization of the microsomes obtained from rat livers with Triton X-100 and subsequent chromatography of the 100,000 x g supernatant on blue-sepharose, hydroxyapatite, and phosphocellulose . The purified enzyme exhibited an apparent molecular weight of 72 kDa as estimated by SDS gel electrophoresis, with specific activities of 71 nmol.min-1.mg-1 protein and 901 nmol.min-1.mg-1 protein for formation of R-ibuprofenoyl-CoA and palmitoyl-CoA, respectively . Palmitoyl-CoA formation catalyzed by the purified enzyme exhibited biphasic kinetics indicative of two isoforms, a high-affinity (KM 0.13 +/- 0.11 microM), low-capacity form and a low-affinity (KM 81 +/- 11.5 microM), high-capacity form . In contrast, measurement of R-ibuprofenoyl-CoA synthesis over a concentration range from 5 to 3000 microM showed the participation of a single CoA ligase with a KM of 184 +/- 19 microM, corresponding to the low-affinity isoform of palmitoyl-CoA synthesis with a marked enantioselectivity towards the R-form of ibuprofen . R-ibuprofenoyl-CoA formation of the enzyme preparation was inhibited by palmitic acid (KI 13.5 +/- 0.5 microM) and S-ibuprofen (KI 405 +/- 10 microM) . In summary, these data give strong evidence for the identity of R-ibuprofenoyl-CoA and long-chain acyl-CoA synthetase. Science, 1996 Oct 11, 274(5285), 252 - 5 Visualization of gene expression in living adult Drosophila; Calleja M et al.; To identify genes involved in the patterning of adult structures, Gal4-UAS (upstream activating site) technology was used to visualize patterns of gene expression directly in living flies . A large number of Gal4 insertion lines were generated and their expression patterns were studied . In addition to identifying several characterized developmental genes, the approach revealed previously unsuspected genetic subdivisions of the thorax, which may control the disposition of pattern elements . The boundary between two of these domains coincides with localized expression of the signaling molecule wingless. Science, 1996 Oct 11, 274(5285), 246 - 8 Identification of a human mitotic checkpoint gene: hsMAD2; Li Y et al.; In Saccharomyces cerevisiae, MAD2 is required for mitotic arrest if the spindle assembly is perturbed . The human homolog of MAD2 was isolated and shown to be a necessary component of the mitotic checkpoint in HeLa cells by antibody electroporation experiments . Human, or Homo sapiens, MAD2 (hsMAD2) was localized at the kinetochore after chromosome condensation but was no longer observed at the kinetochore in metaphase, suggesting that MAD2 might monitor the completeness of the spindle-kinetochore attachment . Finally, T47D, a human breast tumor cell line that is sensitive to taxol and nocodazole, had reduced MAD2 expression and failed to arrest in mitosis after nocodazole treatment . Thus, defects in the mitotic checkpoint may contribute to the sensitivity of certain tumors to mitotic spindle inhibitors. J Biol Chem, 1996 Oct 11, 271(41), 25152 - 6 A recombinant sickle hemoglobin triple mutant with independent inhibitory effects on polymerization; Himanen JP et al.; As part of a comprehensive effort to map the most important regions of sickle hemoglobin that are involved in polymerization, we have determined whether two sites previously shown to be involved, Leu-88(beta) and Lys-95(beta), had additive effects when substituted . The former site is part of the hydrophobic pocket that binds Val-6(beta), the natural mutation of HbS, and the latter site is a prominent part of the hemoglobin exterior . A sickle hemoglobin triple mutant with three amino acid substitutions on the beta-chain, E6V/L88A/K95I, has been expressed in yeast and characterized extensively . Its oxygen binding curve, cooperativity, response to allosteric effectors, and the alkaline Bohr effect showed that it was completely functional . The polymer solubility of the deoxy triple mutant, measured by a new micromethod requiring reduced amounts of hemoglobin, was identical to that of the E6V(beta)/K95I(beta) mutant, i.e . when the K95I(beta) substitution was present on the same tetramer together with the naturally occurring E6V(beta) substitution, the L88A(beta) replacement had no additive effect on polymer inhibition . The results suggest that Lys-95(beta) on the surface of the tetramer and its complementary binding region on the adjoining tetramer are potential targets for the design of an effective antisickling agent. Gene, 1996 Oct 10, 175(1-2), 105 - 8 Isolation of a Candida glabrata centromere and its use in construction of plasmid vectors; Kitada K et al.; A centromere has been isolated from Candida glabrata by functional selection based on the lethality of the SUP11 gene at high copy number . Nucleotide sequence analysis revealed a centromeric structure similar to that of Saccharomyces cerevisiae: the two highly conserved elements CDEI (8 bp) and CDEIII (26 bp) are separated by a 79-bp A+T-rich element, CDEII . Three centromere-bearing plasmid vectors with different selection markers have been constructed . These plasmids were highly stable in mitosis (< 1% loss rate per generation) and exist in one or two copies per cell. Cell, 1996 Oct 4, 87(1), 95 - 104 Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4; Verreault A et al.; Chromatin assembly factor 1 (CAF-1) assembles nucleosomes in a replication-dependent manner . The small subunit of CAF-1 (p48) is a member of a highly conserved subfamily of WD-repeat proteins . There are at least two members of this subfamily in both human (p46 and p48) and yeast cells (Hat2p, a subunit of the B-type H4 acetyltransferase, and Msi1p) . Human p48 can bind to histone H4 in the absence of CAF-1 p150 and p60 . p48, also a known subunit of a histone deacetylase, copurifies with a chromatin assembly complex (CAC), which contains the three subunits of CAF-1 (p150, p60, p48) and H3 and H4, and promotes DNA replication-dependent chromatin assembly . CAC histone H4 exhibits a novel pattern of lysine acetylation that overlaps with, but is distinct from, that reported for newly synthesized H4 isolated from nascent chromatin . Our data suggest that CAC is a key intermediate of the de novo nucleosome assembly pathway and that the p48 subunit participates in other aspects of histone metabolism. Cell, 1996 Oct 4, 87(1), 53 - 63 The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts; Coleman TR et al.; We have cloned a Xenopus Cdc6 homolog (Xcdc6) and characterized its role in DNA replication with Xenopus egg extracts . Immunodepletion of Xcdc6 abolishes chromosomal replication but not elongation on single-stranded DNA templates . Xcdc6 binds to chromatin at the beginning of interphase but disappears from chromatin upon initiation of replication . Immunodepletion studies indicate that binding of Xcdc6 to chromatin requires Xorc2, a component of the origin recognition complex . Moreover, Xmcm3 cannot bind to chromatin lacking Xcdc6, suggesting that Xorc2, Xcdc6, and Xmcm3 associate with the DNA sequentially . In postreplicative nuclei, Xcdc6 is associated with the nuclear envelope . These studies indicate that Xcdc6, is essential for initiation of replication in vertebrates and that interaction with the nuclear envelope may regulate its function. Cell, 1996 Oct 4, 87(1), 33 - 41 Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria; Bauer MF et al.; Tim23, an essential component of the protein import machinery of the inner membrane of mitochondria (TIM complex), forms dimers that display a dynamic behavior . Dimer formation is promoted by the membrane potential delta psi . Binding of a matrix targeting sequence to Tim23 triggers dimer dissociation . Monomeric Tim23 is present when a preprotein chain is in transit across the TIM complex . Dimerization of Tim23 is dependent on the second half of its N-terminal hydrophilic domain, which is exposed to the intermembrane space . This segment contains a heptad leucine repeat motif with a predicted capacity for dimer formation . We propose that Tim23 exerts a key function in protein import: Tim23 dimers formed in response to delta psi act as receptors for matrix targeting sequences on the surface of the inner membrane . The ensuring dissociation of Tim23 dimer triggers opening of the TIM channel and insertion of the preprotein. Cell, 1996 Oct 4, 87(1), 21 - 32 Importin provides a link between nuclear protein import and U snRNA export; Gorlich D et al.; Importin-alpha mediates nuclear protein import by binding nuclear localization signals and importin-beta . We find approximately 30% of SRP1p, the yeast importin-alpha, in a nuclear complex with the Saccharomyces cerevisiae nuclear cap-binding protein complex (CBC) . Similarly, a large fraction of Xenopus CBC is associated with importin-alpha in the nucleus . CBC promotes nuclear export of capped U snRNAs and shuttles between nucleus and cytoplasm . The CBC-importin-alpha complex binds specifically to capped RNA, suggesting that CBC might shuttle while bound to importin-alpha . Strikingly, importin-beta binding displaces the RNA from the CBC-importin-alpha complex . Thus, the commitment of CBC for nuclear reentry triggers the release of the export substrate into the cytoplasm . We provide evidence for a mechanism that ensures that importin-mediated RNA release is a specifically cytoplasmic event. J Biol Chem, 1996 Oct 4, 271(40), 24989 - 94 Histidyl-tRNA synthetase-related sequences in GCN2 protein kinase regulate in vitro phosphorylation of eIF-2; Zhu S et al.; In yeast, starvation for amino acids stimulates GCN2 phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2) . Phosphorylation of eIF-2alpha induces the translational expression of GCN4, a transcriptional activator of the general amino acid control pathway . It has been proposed that GCN2 sequences containing homology to histidyl-tRNA synthetases (HisRS) bind uncharged tRNA that accumulate during amino acid limitation and stimulate the activity of GCN2 kinase . In this report we address whether the HisRS-related sequences are required for GCN2 phosphorylation of eIF-2alpha in an in vitro assay . To measure the activity of GCN2 kinase in cellular extracts, we expressed and purified a truncated form of yeast eIF-2alpha . Phosphorylation of the recombinant eIF-2alpha substrate was dependent on both GCN2 kinase activity and the eIF-2alpha phosphorylation site, serine 51 . Mutations in the HisRS-related domain of GCN2, which have been shown to block phosphorylation of eIF-2alpha in vivo and the subsequent stimulation of the general control pathway, also greatly reduced eIF-2alpha phosphorylation in the in vitro assay . These results indicate that the HisRS-related sequences are required for activation of GCN2 kinase function. Nature, 1996 Oct 3, 383(6599), 434 - 7 Superoxide dismutase protects calcineurin from inactivation; Wang X et al.; Calcineurin is the only protein phosphatase known to be under the control of Ca2+ and calmodulin . It is targeted by immunosuppressive drugs and has a critical role in T-cell activation . It is specifically inhibited by immunosuppressant immunophilin complexes, which enabled its function in regulating a wide range of cellular responses to Ca2+-mobilizing signals to be identified . Calcineurin in situ is 10-20 times more active than in the purified form and is subject to a time- and Ca2+/calmodulin-dependent reversible inactivation that is facilitated by small, heat-stable molecules . Here we identify a factor that prevents the inactivation of calcineurin in vitro and in vivo as the enzyme superoxide dismutase, which indicates that inactivation may be the result of oxidative damage to the Fe-Zn active centre of calcineurin . The redox state of iron provides a mechanism to regulate calcineurin activity by desensitizing the enzyme and coupling Ca2+-dependent protein dephosphorylation to the redox state of the cell . The protection of calcineurin against inactivation by superoxide dismutase constitutes a new physiological role for this enzyme which enables the Ca2+-dependent regulation of cellular processes to be modulated by the redox potential. Genes Cells, 1996 Oct, 1(10), 895 - 904 Genetic interactions between CDC7 and CDC28: growth inhibition of cdc28-1N by Cdc7 point mutants; Ohtoshi A et al.; BACKGROUND: Cdc7 kinase of Saccharomyces cerevisiae, a nuclear phosphoprotein, regulates initiation of chromosomal DNA replication . Overexpression of kinase-negative Cdc7 point mutants (T281E, D182N and D163N) arrests the cell cycle of the wild-type Saccharomyces cerevisiae cells at the G1/S boundary . This is caused by titration of a regulatory protein, Dbf4, from the wild-type Cdc7, which leads to inactivation of its kinase activity . RESULTS: We report here that kinase-negative Cdc7 mutants, when overexpressed in cdc28-1N(ts) at a permissive temperature, not only inhibit DNA replication by inactivating the wild-type Cdc7 but may also disturb coordination between DNA replication and cell division . Suppression of growth inhibition under this condition requires co-expression of both Dbf4 and Cdc28, whereas Dbf4 alone can counteract the growth inhibition in the wild-type cells . In cdc28-1N(ts), co-expression of the wild-type Dbf4 rescues only the G1/S defect and results in accumulation of those cells with less than 1C DNA as well as 2C DNA . On the other hand, co-expression of Cdc28 alone leads to increase of those cells arrested at the G1/S boundary, as found typically in the wild-type . We also report that overexpression of T281A, a 'weak' allele of Cdc7, causes growth arrest in cdc28-1N(ts) cells, but not in the CDC28 wild-type cells . This suggests that T281A is inactive in cdc28-1N(ts) and is consistent with the idea that Cdc28 activates Cdc7 by phosphorylation . CONCLUSION: We conclude that two essential serine-threonine kinases, Cdc28 and Cdc7, genetically interact for initiation of the S phase and possibly for G2/M progression and/or S phase checkpoint control. Comput Appl Biosci, 1996 Oct, 12(5), 405 - 13 GenomeInspector: a new approach to detect correlation patterns of elements on genomic sequences; Quandt K et al.; MOTIVATION: Most of the sequences determined in current genome sequencing projects remain at least partially unannotated . The available software for DNA sequence analysis is usually limited to the prediction of individual elements (level 1 methods), but does not assess the context of different motifs . However, the functionality of biological units like promoters depends on the correct spatial organization of multiple individual elements . RESULTS: Here, we present a second-level software package called GenomeInspector {{}}, for further analysis of results obtained with level 1 methods (e.g . MatInspector {{}} or ConsInspector {{+}}) . One of the main features of this modular program is its ability to assess distance correlations between large sets of sequence elements which can be used for the identification and definition of basic patterns of functional units . The program provides an easy-to-use graphical user interface with direct comprehensive display of all results for megabase sequences . Sequence elements showing spatial correlations can be easily extracted and traced back to the nucleotide sequence with the program . GenomeInspector identified promoters of glycolytic enzymes in yeast {{as members of a subgroup with unusual location of an ABF1 site . Solely on the basis of distance correlation analysis, the program correctly selected those transcription factors within these promoters already known to be involved in the regulation of glycolytic enzymes, demonstrating the power of this method. Curr Biol, 1996 Oct 1, 6(10), 1256 - 64 Rho: theme and variations; Ridley AJ; In addition to their roles in organizing the actin cytoskeleton, members of the Rho family of GTP-binding proteins have recently been implicated in a plethora of other functions, including the activation of kinase cascades and transcription factors, and the control of endocytosis and secretion . Alongside this expansion is proposed functions has been the identification of multiple target proteins that interact directly with Rho, Rac or Cdc42 . Molecular connections are now being made along the signalling pathways activated by members of the Rho family. Curr Biol, 1996 Oct 1, 6(10), 1209 - 12 Cell cycle: cull and destroy; Jackson PK; A newly discovered family of proteins homologous to yeast Cdc53, called cullins, may play a key role in the targeting of cell-cycle regulators, such as cyclins, for destruction by ubiquitin-dependent proteolysis. Curr Biol, 1996 Oct 1, 6(10), 1222 - 5 Chromatin: a sticky silence; Marcand S et al.; Recent findings indicate that heterochromatin serves as a molecular sink for factors involved in chromatin-mediated repression of gene expression; long-range interactions that position a euchromatic gene near a heterochromatin domain influence its susceptibility to transcriptional silencing. J Cell Sci, 1996 Oct, 109 ( Pt 10), 2493 - 8 Neurofilament triplet protein interactions: evidence for the preferred formation of NF-L-containing dimers and a putative function for the end domains; Carpenter DA et al.; In this report we examine the molecular interactions that lead to formation of neurofilaments, the intermediate filaments in neurons . Using the yeast two-hybrid system, we found that the rod domains of all three NF triplet proteins interacted strongly with one another and with rod domains of the Type III IF proteins, vimentin and desmin . A slight preference toward NF-L-containing dimers was observed over ones not containing NF-L . Interactions among the full length NF triplet proteins exhibited more specificity . Full length NF-L had only a relatively weak interaction with another full length NF-L molecule, but reacted more robustly with full length NF-M or NF-H lacking only part of the head domain . No homologous or heterologous dimerization of NF-M and NF-H was detectable . These results support the hypothesis that neurofilaments are obligate heteropolymers and that heterodimeric subunits are the preferred building blocks . They further suggest that the mechanism that specifies heterodimeric interaction among the NF triplet proteins resides in the end domains. Mutat Res, 1996 Oct, 366(1), 45 - 63 The roles of telomeres and telomerase in cell life span; Counter CM; Telomeres cap and protect the ends of chromosomes from degradation and illegitimate recombination . The termini of a linear template cannot, however, be completely replicated by conventional DNA-dependent DNA polymerases, and thus in the absence of a mechanisms to counter this effect, telomeres of eukaryotic cells shorten every round of DNA replication . In humans and possibly other higher eukaryotes, telomere shortening may have been adopted to limit the life span of somatic cells . Human somatic cells have a finite proliferative capacity and enter a viable growth arrested state called senescence . Life span appears to be governed by cell division, not time . The regular loss of telomeric DNA could therefore serve as a mitotic clock in the senescence programme, counting cell divisions . In most eukaryotic organisms, however, telomere shortening can be countered by the de novo addition of telomeric repeats by the enzyme telomerase . Cells which are "immortal' such as the human germ line or tumour cell lines, established mouse cells, yeast and ciliates, all maintain a stable telomere length through the action of telomerase . Abolition of telomerase activity in such cells nevertheless results in telomere shortening, a process that eventually destabilizes the ends of chromosomes, leading to genomic instability and cell growth arrest or death . Therefore, loss of terminal DNA sequences may limit cell life span by two mechanisms: by acting as a mitotic clock and by denuding chromosomes of protective telomeric DNA necessary for cell viability. Genomics, 1996 Oct 1, 37(1), 135 - 9 Molecular cloning of RPA2, the gene encoding the second largest subunit of mouse RNA polymerase I; Seither P et al.; We have cloned the cDNA encoding the second largest subunit of RNA polymerase I, termed RPA2, from mouse cells . The cDNA has a 3978-nucleotide open reading frame encoding a polypeptide of 1136 amino acids with a calculated molecular mass of 128 kDa . A sequence alignment of mouse RPA2 with the corresponding gene from Drosophila melanogaster and yeast reveals a much lower sequence similarity of this subunit of RNA polymerase I (Pol I) compared to the second largest subunit of other eukaryotic RNA polymerases . Four Pol I-specific regions, termed I alpha-I delta, are conserved in the N-terminal part of RPA2 . The structural features of the different domains as well as the homology to essential functional domains found in other RNA polymerases are discussed. Int J Pept Protein Res, 1996 Oct, 48(4), 397 - 400 Construction of a dimeric DNA-binding peptide model by peptide-anthraquinone conjugation; Takenaka S et al.; A peptide-anthraquinone conjugate was designed and synthesized containing linked peptide chains composed of Asp-Pro-Ala-Ala-Leu-Lys-Arg-Ala-Arg-Asn-Thr-Glu-Ala- Ala-Arg-Arg-Ser-Arg-Ala-Arg-Lys-Leu-Gln-Arg-Met, representing the basic region of GCN4 . The two peptides were joined with anthraquinone at its 1- and 8-positions in a two-fold symmetric fashion, mimicking dimeric DNA-binding proteins . Experimental data indicated both an interaction of the anthraquinone moiety with the DNA double strand and an increase in the alpha-helicity of the peptide moieties of the ligand when it binds to DNA. Trends Biochem Sci, 1996 Oct, 21(10), 392 - 6 RNA turnover and the control of mitochondrial gene expression; Margossian SP et al.; Recent evidence suggests that RNA turnover in yeast mitochondria is important, not only to regulate RNA abundance, but also to facilitate group I intron splicing and suppress the potentially toxic effect of high levels of excised group I intron RNAs . Protein-assisted splicing of group I introns requires that splicing factors are 'actively' recycled, because of their tight binding to the intron RNA . The putative NTP-dependent RNA helicase Suv3p might promote this recycling and, at the same time, suppress intron overaccumulation because of the functional association of this protein with mtEXO, a novel 3'-5' exoribonuclease that can degrade excised group I intron RNAs. Trends Biochem Sci, 1996 Oct, 21(10), 383 - 7 Signaling phosphate starvation; Lenburg ME et al.; Phosphate starvation induces the transcription of several genes involved in phosphate metabolism in the budding yeast Saccharomyces cerevisiae . The signal transduction pathway that mediates this response consists of components that resemble those used to regulate the eukaryotic cell cycle; these include a cyclin-dependent kinase or CDK (Pho85), a cyclin (Pho80) and a CDK inhibitor (Pho81) . The possibility that this pathway mediates cell-cycle responses to phosphate starvation is discussed. Mol Microbiol, 1996 Oct, 22(1), 109 - 17 Functional in vivo studies of the Neurospora crassa cys-14 gene upstream region: importance of CYS3-binding sites for regulated expression; Li Q et al.; Sulphate transport in Neurospora crassa is achieved by two distinct sulphate permeases, I and II, encoded by the cys-13 and cys-14 genes, respectively . The synthesis of both sulphate permeases is subject to sulphur repression and requires the global positive-acting regulatory protein CYS3, CYS3, a bZIP DNA binding protein, regulates cys-14 expression at the transcriptional level and binds in vitro specifically to three DNA-recognition sites, A, B, and C, in the cys-14 upstream region . In vivo functional analysis of the cys-14 promoter was carried out with 5' deletions and by deletions or mutations of CYS3 DNA-binding sites . The most distal CYS3-binding site, C, located 1.4kb upstream of the transcriptional start site, is necessary and sufficient to mediate strong transcriptional activation by CYS3; moreover, site C was able to function equally well when it was located at variable distances upstream of the cys-14 gene . Site B, located 1 kb upstream, alone is able to support a moderate degree of cys-14 expression . Site A is not required and does not appear to play any functional role in cys-14 expression, even though it is in close proximity to the transcriptional start site . The presence of multiple copies of CYS3-binding elements A or B in the cys-14 promoter results in a parallel increase of regulated gene expression . When a transforming cys-14 gene becomes integrated at ectopic locations in the host genome, it can be expressed in an unregulated fashion, presumably by coming under the control of other promoter elements . Our results also suggested that at least one enzyme in the sulphate catabolic pathway requires a functional CYS3 protein for expression. Mol Biol Cell, 1996 Oct, 7(10), 1535 - 46 Cloning and functional characterization of mammalian homologues of the COPII component Sec23; Paccaud JP et al.; We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa . hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p . Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p . Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex . By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles . hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C . RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio . Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER . The exact function of hSec23Bp remains to be determined. Development, 1996 Oct, 122(10), 3075 - 84 PAR-2 is asymmetrically distributed and promotes association of P granules and PAR-1 with the cortex in C . elegans embryos; Boyd L et al.; The par genes participate in the process of establishing cellular asymmetries during the first cell cycle of Caenorhabditis elegans development . The par-2 gene is required for the unequal first cleavage and for asymmetries in cell cycle length and spindle orientation in the two resulting daughter cells . We have found that the PAR-2 protein is present in adult gonads and early embryos . In gonads, the protein is uniformly distributed at the cell cortex, and this subcellular localization depends on microfilaments . In the one-cell embryo, PAR-2 is localized to the posterior cortex and is partitioned into the posterior daughter, P1, at the first cleavage . PAR-2 exhibits a similar asymmetric cortical localization in P1, P2, and P3, the asymmetrically dividing blastomeres of germ line lineage . This distribution in embryos is very similar to that of PAR-1 protein . By analyzing the distribution of the PAR-2 protein in various par mutant backgrounds we found that proper asymmetric distribution of PAR-2 depends upon par-3 activity but not upon par-1 or par-4 . par-2 activity is required for proper cortical localization of PAR-1 and this effect requires wild-type par-3 gene activity . We also find that, although par-2 activity is not required for posterior localization of P granules at the one-cell stage, it is required for proper cortical association of P granules in P1. J Bone Miner Res, 1996 Oct, 11(10), 1498 - 507 The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H(+)-ATPase; David P et al.; Although bisphosphonates have been shown to be potent inhibitors of osteoclast-mediated bone resorption in vivo and in vitro and are used as therapeutic agents in hyper-resorptive bone diseases such as Paget disease or hypercalcemia of malignancy, their exact biochemical target(s) and mode(s) of action are for the most part still unknown . The resorption of bone requires solubilization of the mineral component of the matrix, achieved by acidification of the resorbing compartment by a vacuolar-type proton ATPase (V-ATPase) present in the ruffled border membrane of osteoclasts . Since we have shown that the V-ATPase is inhibited by both ADP and phosphate, which share structural characteristics with bisphosphonates, we hypothesized that inhibition of the osteoclast V-ATPase could be one of the mechanism(s) by which bisphosphonates inhibit bone resorption . Pyrophosphate and the bisphosphonates etidronate, alendronate, and YM-175 inhibited proton transport in membrane vesicles derived from chicken kidney and osteoclasts but with very low potency (IC50 > or = 5 mM) . In contrast, the ability of tiludronate to inhibit proton transport was 5-fold higher in kidney-derived vesicles (IC50 = 1.1 mM) and 10,000-fold higher in vesicles derived from osteoclasts (IC50 = 466 nM) . Tiludronate also potently inhibited proton transport in yeast microsomal preparations (IC50 = 3.5 microM) and inhibited the activity of purified yeast V-ATPase . The inhibition of the osteoclast V-ATPase-mediated proton transport by tiludronate was rapid, pH-dependent, and reversible . No change in membrane vesicle permeability to protons was detected . The inhibition was noncompetitive with respect to ATP, and tiludronate did not protect the pump from inactivation by N-ethylmaleimide, strongly suggesting that tiludronate does not bind to the catalytic site of the enzyme . It is concluded that tiludronate is a significantly more potent inhibitor of V-ATPase than other bisphosphonates and that it has a significant degree of selectivity for the avian osteoclast V-ATPase relative to the avian kidney V-ATPase. Genetics, 1996 Oct, 144(2), 495 - 510 SEC3 mutations are synthetically lethal with profilin mutations and cause defects in diploid-specific bud-site selection; Haarer BK et al.; Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those that result from complete deletion of the profilin gene . We have used a colony color-sectoring assay to identify mutations that cause pfy1-111, but not wild-type, cells to be inviable . These profilin synthetic lethal (psl) mutations result in various degrees of abnormal growth, morphology, and temperature sensitivity in PFY1 cells . We have examined psl1 strains in the most detail . Interestingly, these strains display a diploid-specific defect in bud-site selection; haploid strains bud normally, while homozygous diploid strains show a dramatic increase in random budding . We discovered that PSL1 is the late secretory gene, SEC3, and have found that mutations in several other late secretory genes are also synthetically lethal with pfy1-111 . Our results are likely to reflect an interdependence between the actin cytoskeleton and secretory processes in directing cell polarity and growth . Moreover, they indicate that the secretory pathway is especially crucial for maintaining budding polarity in diploids. Nucleic Acids Res, 1996 Oct 1, 24(19), 3821 - 8 The mouse poly(C)-binding protein exists in multiple isoforms and interacts with several RNA-binding proteins; Funke B et al.; The murine poly(C)-binding protein (mCBP) was previously shown to belong to the group of K-homology (KH) proteins by virtue of its homology to hnRNP-K . We have isolated cDNA-splice variants of mCBP which differ by two variable regions of 93 bp and/or 39 +/- 3 bp respectively . Both variable regions are located between the second and third KH-domain of mCBP . The characterization of a partial genomic clone enabled us to propose a model for the generation of the second variable region by the use of a putative alternative splice signal . The mCBP mRNA is expressed ubiquitously and the protein is found predominantly in the nucleus with the exception of the nucleoli . We have identified five proteins which interact with mCBP in the yeast two hybrid system: mouse y-box protein 1 (msy-1), y-box-binding protein, hnRNP-L, filamin and splicing factor 9G8 . The interaction between mCBP and splicing factor 9G8 was confirmed in vivo . These results suggest a function of mCBP in RNA metabolism. Mol Biol Evol, 1996 Oct, 13(8), 1128 - 32 Origin of the Mesozoa inferred from 18S rRNA gene sequences; Pawlowski J et al.; The phylum Mesozoa comprises small, simply organized wormlike parasites of marine invertebrates and is composed of two classes, the Rhombozoa and the Orthonectida . The origin of Mesozoa is uncertain; they are classically considered either as degenerate turbellarians or as primitive multicellular animals related to ciliated protists . In order to precisely determine the phylogenetic position of this group we sequenced the complete 18S rRNA gene of one rhombozoid, Dicyema sp., and one orthonectid, Rhopalura ophiocomae . The sequence analysis shows that the Mesozoa branch early in the animal evolution, closely to nematodes and myxozoans . Our data indicate probably separate origins of rhombozoids and orthonectids, suggesting that their placement in the same phylum needs to be revised. Mol Biol Evol, 1996 Oct, 13(8), 1059 - 66 Satellite DNA of the red flour beetle Tribolium castaneum--comparative study of satellites from the genus Tribolium; Ugarkovic D et al.; A highly abundant satellite DNA comprising 17% of the Tribolium castaneum (Insecta, Coleoptera) genome was cloned and sequenced . The satellite monomer is 360 bp long, has a high A+T content of 73%, and lacks significant internal substructures . The sequence variability is 3.6%, essentially due to random distribution of single-point mutations . The satellite is evenly distributed in the regions of centromeric heterochromatin of all 20 chromosomes, as shown by fluorescent in situ hybridization . Comparison of T . castaneum satellite with those from three different but congeneric species reveals the highest sequence similarity of 47.1% with the satellite from the sibling species Tribolium freemani . The phylogenetic relationships among Tribolium species deduced from satellite sequence agree with those based on karyological, chemotaxonomic, and hybridization data . This indicates a parallel in the divergence of satellites and some genetic and cytogenetic characters . Despite low mutual sequence similarity, which makes them species-specific, Tribolium satellites have a common structural characteristic: a block of about 95% A+T content, 20 to 42 bp long, flanked at one side by an inverted repeat which can potentially form a thermodynamically stable dyad structure . Since similar structural features are found in centromeric DNA of Saccharomyces cerevisiae and Chironomus pallidivittatus, their possible importance in centromere function may be inferred. Mol Pharmacol, 1996 Oct, 50(4), 709 - 15 Identification and characterization of novel somatostatin antagonists; Bass RT et al.; The study of the five somatostatin receptor subtypes (SSTx, where x is the subtype number) has been hampered by the lack of high affinity antagonists . Potent and selective antagonists would increase our understanding of SST structure, function, and regulation . In this study, the identification of novel disulfide-linked cyclic octapeptide antagonists of somatostatin is described . The antagonists contain a core structure of a DL-cysteine pair at positions 2 and 7 of the peptides . Substitution of a D-cysteine at position 2 with an L-cysteine converts the full antagonist into a full agonist . All somatostatin receptor subtypes are coupled to inhibition of adenylate cyclase . The functional properties of these peptides have been determined in radioligand binding assays, in functional coupling of the SST2 subtype to yeast pheromone response pathway, and in cAMP accumulations . One peptide antagonist {Ac-4-NO2-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)-D-Tyr-NH2} displays a binding affinity to SST2 comparable with that observed for the native hormone (Ki = 0.2 nM) and reverses somatostatin-mediated inhibition of cAMP accumulation in rat somatomammotroph GH4C1 cells, cells transfected with the SST2 and SST5 subtypes, as well as somatostatin-stimulated growth of yeast cells expressing the SST2 subtype . This class of somatostatin antagonists, which are the first to be described, should be useful for determination of somatostatin's diverse functions in vivo and in vitro. J Cell Biol, 1996 Oct, 135(1), 123 - 37 Redox-sensitive homodimerization of Pex11p: a proposed mechanism to regulate peroxisomal division; Marshall PA et al.; Pex11p (formerly Pmp27) has been implicated in peroxisomal proliferation (Erdmann, R., and G . Blobel . 1995 . J . Cell Biol . 128; 509-523; Marshall, P.A., Y.I . Krimkevich, R.H . Lark, J.M . Dyer, M . Veenhuis, and J.M . Goodman, 1995 . J . Cell Biol . 129; 345-355) . In its absence, peroxisomes in Saccharomyces cerevisiae fail to proliferate in response to oleic acid; instead, one or two large peroxisomes are formed . Conversely, overproduction of Pex11p causes an increase in peroxisomal number . In this report, we confirm the function of Pex11p in organelle proliferation by demonstrating that this protein can cause fragmentation in vivo of large peroxisomes into smaller organelles . Pex11p is on the inner surface of the peroxisomal membrane . It can form homodimers, and this species is more abundant in mature peroxisomes than in proliferating organelles . Removing one of the three cysteines in the protein inhibits homodimerization . This cysteine 3-->alanine mutation leads to an increase in number and a decrease in peroxisomal density, compared with the wild-type protein, in response to oleic acid . We propose that the active species is the "monomeric" form, and that the increasing oxidative metabolism within maturing peroxisomes causes dimer formation and inhibition of further organelle division. J Cell Biol, 1996 Oct, 135(1), 19 - 35 The organization of endoplasmic reticulum export complexes; Bannykh SI et al.; Export of cargo from the ER occurs through the formation of 60-70nm COPII-coated vesicular carriers . We have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of ER export sites in vivo and in vitro . We find that ER buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes . The basic organization of an export complex can be divided into an active COPII-containing budding zone on a single ER cisterna, which is adjacent to budding zones found on distantly connected ER cisternae . These budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain COPI coats involved in retrograde transport . Vesicles within these export complexes contain concentrated cargo molecules . The structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition . We conclude that budding from the ER through recruitment of COPII is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by COPI. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10620 - 5 High-resolution mapping of nucleoprotein complexes by site-specific protein-DNA photocrosslinking: organization of the human TBP-TFIIA-TFIIB-DNA quaternary complex; Lagrange T et al.; We have used a novel site-specific protein-DNA photocrosslinking procedure to define the positions of polypeptide chains relative to promoter DNA in binary, ternary, and quaternary complexes containing human TATA-binding protein, human or yeast transcription factor IIA (TFIIA), human transcription factor IIB (TFIIB), and promoter DNA . The results indicate that TFIIA and TFIIB make more extensive interactions with promoter DNA than previously anticipated . TATA-binding protein, TFIIA, and TFIIB surround promoter DNA for two turns of DNA helix and thus may form a "cylindrical clamp" effectively topologically linked to promoter DNA . Our results have implications for the energetics, DNA-sequence-specificity, and pathway of assembly of eukaryotic transcription complexes. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10604 - 8 Elongation factor TFIIS contains three structural domains: solution structure of domain II; Morin PE et al.; Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS . This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes . Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III . The two C-terminal domains (II and III) are required for transcription activity . The structure of domain III has been solved previously by using NMR spectroscopy . Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix . The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10578 - 83 Hepatitis B virus transactivator protein, HBx, associates with the components of TFIIH and stimulates the DNA helicase activity of TFIIH; Qadri I et al.; Human hepatitis B virus genome encodes a protein, termed HBx, that is widely recognized as a transcriptional transactivator . While HBx does not directly bind cis-acting transcriptional control elements, it has been shown to associate with cellular proteins that bind DNA . Because HBx transactivated a large number of viral/cellular transcriptional control elements, we looked for its targets within the components of the basal transcriptional machinery . This search led to the identification of its interactions with TFIIH . Here, we show that HBx interacts with yeast and mammalian TFIIH complexes both in vitro and in vivo . These interactions between HBx and the components of TFIIH are supported by several lines of evidence including results from immunoprocedures and direct methods of measuring interactions . We have identified ERCC3 and ERCC2 DNA helicase subunits of holoenzyme TFIIH as targets of HBx interactions . Furthermore, the DNA helicase activity of purified TFIIH from rat liver and, individually, the ERCC2 component of TFIIH is stimulated in the presence of HBx . These observations suggest a role for HBx in transcription and DNA repair. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10548 - 55 What determines the folding of the chromatin fiber? van Holde K, Zlatanova J. In this review, we attempt to summarize, in a critical manner, what is currently known about the processes of condensation and decondensation of chromatin fibers . We begin with a critical analysis of the possible mechanisms for condensation, considering both old and new evidence as to whether the linker DNA between nucleosomes bends or remains straight in the condensed structure . Concluding that the preponderance of evidence is for straight linkers, we ask what other fundamental process might allow condensation, and argue that there is evidence for linker histone-induced contraction of the internucleosome angle, as salt concentration is raised toward physiological levels . We also ask how certain specific regions of chromatin can become decondensed, even at physiological salt concentration, to allow transcription . We consider linker histone depletion and acetylation of the core histone tails, as possible mechanisms . On the basis of recent evidence, we suggest a unified model linking targeted acetylation of specific genomic regions to linker histone depletion, with unfolding of the condensed fiber as a consequence. Chromosoma, 1996 Oct, 105(4), 211 - 22 Identification of Porto-1, a new repeated sequence that localises close to the centromere of chromosome 2 of Drosophila melanogaster; Coelho PA et al.; We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA . Using primers based on the human CENP-B box present in alpha-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of specific DNA fragments were amplified from total genomic DNA . In situ hybridization to polytene and mitotic chromosomes showed these fragments to localise to centromeric and pericentromeric regions . Direct cloning of the amplified fragments into conventional plasmids proved unsuccessful . However, a recombinant P1 clone containing D . melanogaster genomic DNA that supports PCR amplification by the primers was identified . Molecular characterisation of this clone revealed a DNA fragment that localises primarily to the centromere of chromosome 2 . Sequence analysis indicated that this fragment contains at least four different repeats, including Rsp, transposable elements, Bari-1 and a new AT-rich repeated sequence that we have designated Porto-1 . Detailed fluorescence in situ hybridization analysis shows that Porto-1 is localised very close to the primary constriction of chromosome 2 . Sequence analysis suggests that this repeat was specifically amplified by our primers, although limited homology to the CENP-B box or CDEIII elements was found . In situ hybridization to a number of Drosophila species shows Porto-1 to be present only in D . melanogaster. Genes Dev, 1996 Oct 1, 10(19), 2491 - 504 Radical mutations reveal TATA-box binding protein surfaces required for activated transcription in vivo; Bryant GO et al.; Regions on the surface of human TATA-box binding protein (TBP) required for activated transcription in vivo were defined by construction of a library of 89 surface residue mutants with radical substitutions that were assayed for their ability to support activated transcription in vivo, basal transcription in vitro, and TFIIA and TFIIB binding in vitro . Four epitopes were identified in which substitutions in two to four neighboring surface residues greatly inhibited activated transcription in vivo . One epitope in which substitutions inhibited both basal and activated transcription (E284, L287) is the interface between TBP and TFIIB . Another (A184, N189, E191, R205) is the recently determined interface between TBP and TFIIA . Mutations in residues in this TFIIA interface greatly inhibit activated, but not basal transcription, demonstrating a requirement for the TFIIA-TBP interaction for activated transcription in vivo in mammalian cells . The remaining two activation epitopes (TBP helix 2 residues R231, R235, R239, plus F250; and G175, C176, P247) are probably interfaces with other proteins required for activated transcription . The library of mutants responded virtually identically to two different types of activators, GL4-E1A and GAL4-VP16, indicating that transcriptional activation by different classes of activators requires common interactions with TBP. Mol Cell Biol, 1996 Oct, 16(10), 5896 - 904 Length changes in the joining segment between domains 5 and 6 of a group II intron inhibit self-splicing and alter 3' splice site selection; Boulanger SC et al.; Domain 5 (D5) and domain 6 (D6) are adjacent folded hairpin substructures of self-splicing group II introns that appear to interact within the active ribozyme . Here we describe the effects of changing the length of the 3-nucleotide segment joining D5 to D6 {called J(56)3} on the splicing reactions of intron 5 gamma of the COXI gene of yeast mitochondrial DNA . Shortened variants J(56)0 and J(56)1 were defective in vitro for branching, and the second splicing step was performed inefficiently and inaccurately . The lengthened variant J(56)5 had a milder defect-splicing occurred at a reduced rate but with correct branching and a mostly accurate 3' splice junction choice . Yeast mitochondria were transformed with the J(56)5 allele, and the resulting yeast strain was respiration deficient because of ineffective aI5 gamma splicing . Respiration-competent revertants were recovered, and in one type a single joiner nucleotide was deleted while in the other type a nucleotide of D6 was deleted . Although these revertants still showed partial splicing blocks in vivo and in vitro, including a substantial defect in the second step of splicing, both spliced accurately in vivo . These results establish that a 3-nucleotide J(56) is optimal for this intron, especially for the accuracy of 3' splice junction selection, and indicate that D5 and D6 are probably not coaxially stacked. Mol Cell Biol, 1996 Oct, 16(10), 5857 - 64 Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K; Han J et al.; The E1B 19-kilodalton protein (19K protein) is a potent apoptosis inhibitor and the adenovirus homolog of Bcl-2 (E . White, Genes Dev . 10:1-15, 1996) . To obtain a better understanding of the biochemical mechanism by which the E1B 19K protein regulates apoptosis, proteins that interact with 19K have been identified; one of these is Bax (J . Han, P . Sabbatini, D . Perez, L . Rao, D . Mohda, and E . White, Genes Dev . 10:461-477, 1996), and another is Bak (S . N . Farrow, J . H . M . White, I . Martinou, T . Raven, K.-T . Pun, C . J . Grinham, J.-C . Martinou, and R . Brown, Nature (London) 374:731-733, 1995) . Bax and Bak are Bcl-2 family members which contain Bcl-2 homology regions 1, 2, and 3 (BH1, BH2, and BH3), which interact with E1B 19K and Bcl-2 and promote apoptosis . Like Bax and Bak, Nbk was cloned from a yeast two-hybrid screen for proteins that interact with E1B 19K . Nbk contained BH3 but not BH1 or BH2 . It also interacted with Bcl-2 but not with Bax . Both Bcl-2 and E1B 19K interacted with Nbk in vitro, and this interaction was highly specific . In vivo, the Nbk and E1B 19K proteins may colocalize with cytoplasmic and nuclear membranes . Nbk expression functionally antagonized 19K-mediated inhibition of apoptotic cell death and completely prevented transformation by E1A and E1B 19K . Nbk was sufficient for induction of apoptosis in the presence of mutant p53 and thus low levels of Bax, suggesting that Nbk functions independently of Bax to induce apoptosis . Nbk may therefore represent a novel death regulator which contains only a BH3 that interacts with and antagonizes apoptosis inhibitors such as the E1B 19K protein. Mol Cell Biol, 1996 Oct, 16(10), 5801 - 10 Purines are required at the 5' ends of newly initiated RNAs for optimal RNA polymerase III gene expression; Zecherle GN et al.; We have made specific alterations in the CAACAA element at the transcription start site of a Saccharomyces cerevisiae suppressor tRNA gene . The mutant genes were tested for their ability to suppress the ochre nonsense alleles ade2-1, lys4-1, and met4-1 . Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o . A 2-bp change, CTCCAA, which alters bases encoding the +1 and +2 nucleotides of pre-tRNA Tyr, had a strong deleterious effect in vivo, as did the more extensive change CTCCTC . In contrast, mutant genes bearing each of the possible single changes at nucleotide +1 retained normal suppression levels . The transcription start point could be shifted in a limited fashion in response to the specific sequences encountered by RNA polymerase III at the start site . ATP was preferentially utilized as the 5' nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the +1 nucleotide . Short oligopyrimidine RNAs formed on the CTCCTC allele could be repositioned in the active center of the newly formed ternary complex . Early postinitiation complexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive transcripts than similar complexes formed on SUP4-o . Our results suggest that the purine-rich sequences at the 5' ends of the nascent transcripts of many genes act to stabilize the early ternary complex. Mol Cell Biol, 1996 Oct, 16(10), 5772 - 81 Mad proteins contain a dominant transcription repression domain; Ayer DE et al.; Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Max and mSin3A or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3 . The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A . Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity . Here we show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region . A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites . In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains . When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities . Fusions between the GAL DNA binding domain and full-length mSin3 were also capable of repression . We show that the association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices . Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators. Mol Cell Biol, 1996 Oct, 16(10), 5708 - 16 Nuclear respiratory factors 1 and 2 utilize similar glutamine-containing clusters of hydrophobic residues to activate transcription; Gugneja S et al.; Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) are ubiquitous transcription factors that have been implicated in the control of nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication . Recently, both factors have been found to be major transcriptional determinants for a subset of these genes that define a class of simple promoters involved in respiratory chain expression . Here, functional domains required for transactivation by NRF-1 have been defined . An atypical nuclear localization signal resides in a conserved amino-terminal region adjacent to the DNA binding domain and consists of functionally redundant clusters of basic residues . A second domain in the carboxy-terminal half of the molecule is necessary for transcriptional activation . The activation domains of both NRF-1 and NRF-2 were extensively characterized by both deletion and alanine substitution mutagenesis . The results show that these domains do not fall into known classes defined by a preponderance of amino acid residues, including glutamines, prolines, or isoleucines, as found in other eukaryotic activators . Rather, in both factors, a series of tandemly arranged clusters of hydrophobic amino acids were required for activation . Although all of the functional clusters contain glutamines, the glutamines differ from the hydrophobic residues in that they are inconsequential for activation . Unlike the NRF-2 domain, which contains its essential hydrophobic motifs within 40 residues, the NRF-1 domain spans about 40% of the molecule and appears to have a bipartite structure . The findings indicate that NRF-1 and NRF-2 utilize similar hydrophobic structural motifs for activating transcription. Mol Cell Biol, 1996 Oct, 16(10), 5701 - 7 The transcription factor Swi5 regulates expression of the cyclin kinase inhibitor p40SIC1; Knapp D et al.; DNA replication in budding yeast cells depends on the activation of the Cdc28 kinase (Cdk1 of Saccharomyces cerevisiae) associated with B-type cyclins Clb1 to Clb6 . Activation of the kinase depends on proteolysis of the Cdk inhibitor p40SIC1 in late G1, which is mediated by the ubiquitin-conjugating enzyme Cdc34 and two other proteins, Cdc4 and Cdc53 . Inactivation of any one of these three proteins prevents p40SIC1 degradation and causes cells to arrest in G1 with active Cln kinases but no Clb-associated Cdc28 kinase activity . Deletion of SIC1 allows these mutants to replicate . p40SIC1 disappears at the G1/S transition and reappears only after nuclear division . Cell cycle-regulated proteolysis seems largely responsible for this pattern, but transcriptional control could also contribute; SIC1 RNA accumulates to high levels as cells exit M phase . To identify additional factors necessary for the inhibition of the Cdk1/Cdc28 kinase in G1, we isolated mutants that can replicate DNA in the absence of Cdc4 function . Mutations in three loci (SIC1, SWI5, and RIC3) were identified . We have shown that high SIC1 transcript levels at late M phase depend on Swi5 . Swi5 accumulates in the cytoplasm during S, G2, and M phases of the cell cycle but enters the nuclei at late anaphase . Our data suggest that cell cycle-regulated nuclear accumulation of Swi5 is responsible for the burst of SIC1 transcription at the end of anaphase . This transcriptional control may be important for inactivation of the Clb/Cdk1 kinase in G2/M transition and during the subsequent G1 period. Mol Cell Biol, 1996 Oct, 16(10), 5691 - 700 Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype; Moriggl R et al.; The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses . Seven members of the Stat gene family are known . MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells . Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes . We examined the functional properties of the carboxyl termini of these molecules . Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; Stat5a delta 750 did not . Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants . Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a delta 750 mutant . The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4 . Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells . We propose that Stat5a delta 750 and Stat5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters. Mol Cell Biol, 1996 Oct, 16(10), 5591 - 6 Surfeit locus gene homologs are widely distributed in invertebrate genomes; Armes N et al.; The mouse Sur