J Bacteriol, 1992 Nov, 174(22), 7398 - 406 Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes; Yanai K et al.; Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II . Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized . Southern blot analyses of the total genomic DNA from R . oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment . Two DNA fragments were isolated from the phage bank of R . oligosporus genomic DNA with the synthetic oligonucleotides as probes . The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively) . The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain . Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes . Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains . It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal. Mol Cell Biol, 1992 Nov, 12(11), 5197 - 205 Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing; Frank D et al.; To investigate the function of the U5 small nuclear ribonucleoprotein (snRNP) in pre-mRNA splicing, we have screened for factors that genetically interact with Saccharomyces cerevisiae U5 snRNA . We isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of the U5 snRNA and named these genes SLU, for synergistically lethal with U5 snRNA . SLU1 and SLU2 are essential for the first catalytic step of splicing, while SLU7 and SLU4 (an allele of PRP17 {U . Vijayraghavan, M . Company, and J . Abelson, Genes Dev . 3:1206-1216, 1989}) are required only for the second step of splicing . Furthermore, slu4-1 and slu7-1 are lethal in combination with mutations in PRP16 and PRP18, which also function in the second step, but not with mutations in factors required for the first catalytic step, such as PRP8 and PRP4 . We infer from these data that SLU4, SLU7, PRP18, PRP16, and the U5 snRNA interact functionally and that a major role of the U5 snRNP is to coordinate a set of factors that are required for the completion of the second catalytic step of splicing. Mol Cell Biol, 1992 Nov, 12(11), 5189 - 96 TFIIA induces conformational changes in TFIID via interactions with the basic repeat; Lee DK et al.; DNA-binding studies with Saccharomyces cerevisiae TFIID point mutants indicated that TFIIA interacts with the basic repeat region of TFIID and induces structural changes . The latter was shown by the ability of TFIIA to compensate for TFIID point mutants defective for DNA binding . Interaction with TFIIA also rendered TFIID binding temperature independent, thus mimicking the effect of removing the nonconserved N terminus of TFIID . In addition, N-terminal truncation of the TFIID point mutants defective for DNA binding mimicked the ability of TFIIA to restore DNA binding of those mutants . Taken together, these results suggest that TFIIA enhances TFIID binding to DNA by eliminating an otherwise inhibitory effect of the nonconserved N terminus of TFIID . Furthermore, analyses of TFIID contact points on DNA and binding studies with TATA-containing oligonucleotide probes showed that TFIIA decreases the effect of sequences flanking the adenovirus major late TATA element on TFIID binding to DNA, suggesting a possible role of TFIIA in allowing TFIID to recognize a wider variety of promoters. Mol Cell Biol, 1992 Nov, 12(11), 4981 - 7 A transcriptionally active form of GAL4 is phosphorylated and associated with GAL80; Parthun MR et al.; The GAL4 activator and GAL80 repressor proteins regulate the expression of yeast genes in response to galactose . A complex of the two proteins isolated from glucose-grown cells is inactive in an in vitro transcription reaction but binds DNA and blocks activation by the GAL4-VP16 chimeric activator . The complex purified from galactose-grown cells contains a mixture of phosphorylated and unphosphorylated forms of GAL4 . The galactose-induced form of GAL4 activates in vitro transcription to levels similar to those seen with GAL4-VP16 . The induced GAL4 complex is indistinguishable in size and apparent shape from the uninduced complex, consistent with a continued association with GAL80 . These results confirm in vivo analyses that correlate GAL4 phosphorylation with galactose induction and support a model of transcriptional activation that does not require GAL80 dissociation. J Cell Biol, 1992 Nov, 119(3), 513 - 21 SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex; Hardwick KG et al.; The ERD2 gene, which encodes the yeast HDEL (His-Asp-Glu-Leu) receptor, is essential for growth (Semenza, J . C., K . G . Hardwick, N . Dean, and H . R . B . Pelham . 1990 . Cell . 61:1349-1357; Lewis, M . J., D . J . Sweet, and H . R . B . Pelham . 1990 . Cell . 61:1359-1363) . SED5, when present in multiple copies, enables cells to grow in the absence of Erd2p . Sequence analysis of SED5 reveals no significant homology with ERD2 or other known genes . We have raised antibodies to Sed5p which specifically recognize a 39-kD integral membrane protein . A stretch of hydrophobic residues at the COOH terminus is predicted to hold Sed5p on the cytoplasmic face of intracellular membranes . Cells that are depleted of Sed5p are unable to transport carboxypeptidase Y to the Golgi complex, and stop growing after a dramatic accumulation of ER membranes and vesicles . We conclude that the SED5 gene is essential for growth and that Sed5p is required for ER to Golgi transport . When Sed5p is overexpressed the efficiency of ER to Golgi transport is reduced, vesicles accumulate, and cellular morphology is perturbed . Immunofluorescence studies reveal that the bulk of Sed5p is not found on ER membranes but on punctate structures throughout the cytoplasm, the number of which increases upon SED5 overexpression . We suggest that Sed5p has an essential role in vesicular transport between ER and Golgi compartments and that it may itself cycle between these organelles. EMBO J, 1992 Nov, 11(11), 3921 - 6 Crystal structure of human platelet-derived growth factor BB; Oefner C et al.; The crystal structure of the homodimeric BB isoform of human recombinant platelet-derived growth factor (PDGF-BB) has been determined by X-ray analysis to 3.0 A resolution . The polypeptide chain is folded into two highly twisted antiparallel pairs of beta-strands and contains an unusual knotted arrangement of three intramolecular disulfide bonds . Dimerization leads to the clustering of three surface loops at each end of the elongated dimer, which most probably form the receptor recognition sites. Arch Biochem Biophys, 1992 Nov 1, 298(2), 486 - 91 Lithocholate binding by Y and Y' proteins in bovine small intestine; Takikawa H et al.; Cytosolic proteins may play an important role in the intracellular transport of bile acids in enterocytes . The lithocholate binding properties of cytosolic protein from bovine small intestine were studied . Lithocholate binding was observed in the Y (45-50 kDa), Y' (30-35 kDa), and Z fractions (10-15 kDa) following gel filtration of cytosol . A Y protein with glutathione S-transferase activity (46 kDa) was purified by S-octyl-glutathione affinity chromatography and chromatofocusing (eluted at pH 7.5) of the Y fraction . Two Y' bile acid binding proteins with dihydrodiol dehydrogenase activity were partially purified from the Y' fraction by chromatofocusing and hydroxyapatite-HPLC . The lithocholate binding affinity of Y' protein (Kd < 0.35 microM) was higher than that of Y protein (Kd = 2 microM) and was comparable to that of Z protein (Kd = 0.2 microM) . The binding affinity of Y protein was higher for bilirubin (Kd = 2.5 microM) than that for BSP (Kd = 200 microM) . This was comparable to the binding affinity of bovine hepatic Y protein . These data indicate that Y' and Z proteins participate in the intracellular transport of bile acids from the brush border to the basolateral pole in enterocytes. EMBO J, 1992 Nov, 11(11), 4103 - 9 Single amino acid substitutions alter helix-loop-helix protein specificity for bases flanking the core CANNTG motif; Fisher F et al.; While all basic region/helix-loop-helix (bHLH) proteins bind the consensus CANNTG motif, other factors must be involved in determining regulatory specificity . In this report we show that bases outside this core 6 bp are involved in determining the specificity of binding . Thus, binding of the yeast bHLH protein PHO4, but not CPF-1, is inhibited by the presence of a T residue immediately 5' to their common CACGTG recognition sequence . PHO4 binding specificity is altered by mutation at any of three different positions in the basic region, including a single Glu to Asp substitution . The significance of these data for DNA-binding and transcription regulation by the bHLH family of transcription factors is discussed. FEBS Lett, 1992 Oct 26, 311(3), 295 - 8 cDNA encoding the chicken ortholog of the mouse dilute gene product . Sequence comparison reveals a myosin I subfamily with conserved C-terminal domains; Sanders G et al.; We report the cDNA-deduced primary structure of the chicken counterpart of the murine dilute gene product, a member of the myosin I family . Comparison of the chicken and mouse sequences reveals a distinct pattern of domains of high and low sequence conservation . An internal deletion of 25 amino acids probably reflects differential mRNA processing . Compared with other myosin heavy chain molecules, sequence similarity is highest with the MYO2 gene product of Saccharomyces cerevisiae . The MYO2 protein, implicated in vectorial vesicle transport, is homologous to the dilute protein over practically its entire length . In addition, the C-terminal domain of the dilute protein is highly similar to a putative glutamic acid decarboxylase sequence cloned from mouse brain . Alternatively, this closely related clone might represent an isoform of the dilute protein derived from a second gene, potentially involved in genetic conditions related to dilute. Gene, 1992 Oct 21, 120(2), 313 - 4 Sequences for two cDNAs encoding Arabidopsis thaliana eukaryotic protein synthesis initiation factor 4A; Metz AM et al.; Two distinct cDNAs encoding protein synthesis initiation factor 4A (eIF-4A) were isolated from an Arabidopsis thaliana cDNA library and sequenced . The deduced amino acid sequences from the two cDNAs were compared to eIF-4A from tobacco, mouse and Saccharomyces cerevisiae . The putative ATP-binding sites and RNA helicase motifs were identified. Biochemistry, 1992 Oct 20, 31(41), 9955 - 60 Isopentenyl-diphosphate isomerase: irreversible inhibition by 3-methyl-3,4-epoxybutyl diphosphate; Lu XJ et al.; Isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) catalyzes the 1,3-allylic rearrangement of the homoallylic substrate isopentenyl diphosphate (IPP) to its allylic isomer, dimethylallyl diphosphate (DMAPP) . Incubation of yeast IPP isomerase with 3-methyl-3,4-epoxybutyl diphosphate (EIPP) resulted in a time-dependent first-order loss of activity characteristic of an active-site-directed irreversible process, where k2 = 0.63 +/- 0.10 min-1 and KI = 0.37 +/- 0.11 microM . A 1:1 covalent E-I complex was formed upon incubation with {1-14C}EIPP . The inhibited enzyme was treated with trypsin to give two radioactive fragments, which were purified by reversed-phase HPLC on a C18 column . The modified amino acid in each fragment was identified as C139 by sequencing the radiolabeled peptides . Incubation of IPP isomerase with {2,4,5-13C3}EIPP gave a 13C-labeled E-I complex . A 1H-13C heteronuclear multiquantum correlation spectrum had strong cross-peaks at 1.2/28 and 2.9/48 ppm, which we assigned to the labeled methyl group and C(4) methylene, respectively, of the inhibitor . In addition, a weak signal at 2.17/42 ppm may be from the C(2) methylene . Comparison of these chemical shifts with those of a synthetic adduct isolated from treatment of EIPP with cysteine indicates C139 attacks C(4) of EIPP to generate a thioether linkage between the enzyme and the inhibitor. Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9910 - 4 Growth-regulated expression of D-type cyclin genes in human diploid fibroblasts; Won KA et al.; The human CCND1 cyclin D1/PRAD1 gene was previously identified by a genetic screen for G1 cyclin function in Saccharomyces cerevisiae and also was identified as the putative BCL1 oncogene . However, its role in human cell proliferation is not known . To determine if expression of human D-type cyclin genes correlates with the state of cell growth, we examined the level of mRNAs for CCND1 and a related gene, CCND3, in normal human diploid fibroblasts (HDF) . The levels of both mRNAs decrease upon serum depletion or at high cell densities . Following stimulation of quiescent fibroblasts with serum, the mRNA levels increase gradually to a peak at about 12 hr, prior to the onset of S phase . Induction of cyclin gene expression by serum is reduced concomitantly with the decline in FOS induction in aging HDFs, suggesting a possible relationship to the decrease in the proliferative response to mitogens during cellular senescence . Cycloheximide partially blocks the induction of CCND1 and CCND3 gene expression by serum, suggesting that both de novo protein synthesis-dependent and -independent pathways contribute to induction . Treatment of HDFs with defined growth factors suggests a correlation between CCND mRNA induction and DNA synthesis . However, induction of these genes is not sufficient for the transition from quiescence through G1 into S phase. Proc Natl Acad Sci U S A, 1992 Oct 15, 89(20), 9454 - 8 Cell cycle-dependent localization of casein kinase I to mitotic spindles; Brockman JL et al.; Casein kinase I (CKI) is a class of protein kinases ubiquitous to all eukaryotic cells . Recently, cDNA clones encoding several bovine CKI isoforms have been sequenced that show high sequence identity to the HRR25 gene product of the budding yeast Saccharomyces cerevisiae; HRR25 is required for normal cellular growth, nuclear segregation, DNA repair, and meiosis . We have raised polyclonal antibodies to a human erythroid 34-kDa CKI and have sequenced a portion of this kinase . The amino acid sequence identifies the CKI as the alpha-CKI isoform, which is 62% identical to the HRR25 protein kinase . By use of immunofluorescence, the alpha-CKI has been localized to vesicular cytosolic structures and to the centrosome in interphase cells . As cells progress into mitosis, centrospheric staining increases and, in mitosis, alpha-CKI associates with kinetochore fibers . This localization suggests that alpha-CKI, like HRR25, plays a role in the segregation of chromosomes during mitosis and may be cell cycle-regulated both in humans and in yeast. Nature, 1992 Oct 15, 359(6396), 650 - 2 Identification of an amino acid-base contact in the GCN4-DNA complex by bromouracil-mediated photocrosslinking; Blatter EE et al.; The bZIP DNA-binding proteins are characterized by a 50-amino-acid DNA binding and dimerization motif, consisting of a highly basic DNA-binding region ('b') followed by a leucine zipper dimerization region ('ZIP') . The best characterized bZIP DNA-binding protein is GCN4, a yeast transcriptional activator . GCN4 binds to a 9-base-pair two-fold-symmetric DNA site, 5'-A-4T-3G-2A-1C0T+1C+2A+3T+4-3' (refs 7-10) . A detailed model known as the 'induced helical fork' model has been proposed for the structure of the GCN4-DNA complex . Using a site-specific bromouracil-mediated photocrosslinking method, we show here that the alanine at position 238 of GCN4 contacts, or is close to, the thymine 5-methyl of A.T at position +3 of the DNA site in the GCN4-DNA complex . Our results strongly support the induced helical fork model . Our site-specific bromouracil-mediated photocrosslinking method requires no prior information regarding the structure of the protein or the structure of the protein-DNA complex and should be generalizable to DNA-binding proteins that interact with the DNA major groove. J Biol Chem, 1992 Oct 15, 267(29), 21072 - 9 Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum; Troll H et al.; Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was purified by hydroxyapatite and CM-cellulose chromatography . The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis . The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component . Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials . The channel displayed a characteristic voltage dependence for potentials higher than 20 mV . It switched to substates of smaller conductance and a selectivity different to that of the open state . The closed state was stabilized at low ionic strength . The cDNA sequence of mitochondrial porin from D . discoideum was determined . It showed little sequence similarities to other known mitochondrial porins . The functional similarity, however, was striking . Localization of the porin in the mitochondrial outer membrane was confirmed by immunogold labeling of cryosections of fixed cells. Gene, 1992 Oct 12, 120(1), 115 - 8 Characterization of the ilv-2 gene from Neurospora crassa encoding alpha-keto-beta-hydroxylacyl reductoisomerase; Sista H et al.; We have isolated the cDNA and corresponding genomic DNA for the ilv-2 locus of Neurospora crassa . This gene encodes alpha-keto-beta-hydroxylacyl reductoisomerase (Ilv-2), required for the synthesis of isoleucine and valine . The gene contains four introns, maps to the right arm of chromosome V, and encodes a protein of 400 amino acids (aa) . Alignment of the aa sequence of Ilv-2 with the two other known eukaryotic sequences encoding this enzyme reveals two conserved regions. Curr Opin Genet Dev, 1992 Oct, 2(5), 712 - 9 RNA splicing in lower eukaryotes; Woolford JL Jr et al.; Recently, cis-acting elements and trans-acting RNA and protein factors necessary for splicing nuclear pre-mRNAs, group II introns or group III introns, have been discovered, and new roles for the splicing factors have been elucidated . Parallels among the pathways for splicing these different classes of introns have been identified. Nucleic Acids Res, 1992 Oct 11, 20(19), 5173 - 9 The selective isolation of novel cDNAs encoded by the regions surrounding the human interleukin 4 and 5 genes; Morgan JG et al.; We have developed modifications to direct cDNA selection that allow the rapid and reproducible isolation of low abundance cDNAs encoded by large genomic clones . Biotinylated, cloned genomic DNAs are hybridized in solution with amplifiable cDNAs . The genomic clones and attached cDNAs are captured on streptavidin coated magnetic beads, the cDNAs are eluted and amplified . We have applied this protocol to a 425kb YAC that contains the human IL4 and IL5 genes . After two cycles of enrichment twenty-four cDNAs were evaluated, all of which were homologous to the YAC . DNA sequencing revealed that nine cDNAs were 100% homologous to the interferon regulatory factor 1 (IRF1) gene . Six clones were 70% homologous to the murine P600 gene, which is coexpressed with IL4 and IL5 in mouse Th2 cells . The nine remaining clones were unique within the sequence databases and were non redundant . All of the selected cDNAs were initially present at very low abundance and were enriched by as much as 100,000-fold in two cycles of enrichment . This modified selection technique should be readily applicable to the isolation of many candidate disease loci as well as the derivation of detailed transcription maps across large genomic regions. J Biol Chem, 1992 Oct 5, 267(28), 20217 - 24 Isolation and characterization of the rat liver actin N-acetylaminopeptidase; Sheff DR et al.; Actins from most eukaryotes undergo a unique post-translational modification of the amino terminus called "processing." Processing consists of the removal of an amino-terminal Ac-Met or Ac-Cys to leave an acidic amino-terminal residue . We have previously demonstrated that this reaction is not catalyzed by the ribosomally associated methionine aminopeptidase or by other previously described acetylaminopeptidases . Here we present the isolation and characterization of the actin N-acetylaminopeptidase (ANAP) from rat liver . A five-step purification protocol achieves a 4100-fold purification of the enzyme with an overall 8% recovery of activity . ANAP is a 77-kDa monomer with a pI of 4.6 . Using unprocessed yeast actin as a substrate, the Km of ANAP is 3.5 microM . Purified ANAP was used to generate a polyclonal antibody . The antibody has been used along with activity assays to demonstrate the presence of ANAP in a variety of rat tissues . Finally, evidence is presented that in mammals, ANAP may function with a second, as yet unpurified, component to process actin amino termini. J Biol Chem, 1992 Oct 5, 267(28), 20204 - 11 Tagetitoxin inhibition of RNA polymerase III transcription results from enhanced pausing at discrete sites and is template-dependent; Steinberg TH et al.; In yeast nuclear extracts, tagetitoxin inhibition of RNA polymerase III promoter-directed single- and multiple-round transcription is characterized by pausing or stalling of the elongation complex at several discrete points on the template . Paused ternary complexes isolated from tagetitoxin-inhibited reactions can be elongated to produce full-length RNA . The distribution of "tagetitoxin-enhanced" pause sites is distinct for each of the class III genes we have examined . These tagetitoxin-enhanced pause sites may also be intrinsic pause sites for the elongation complex . Tagetitoxin inhibition of in vitro transcription of the yeast SUP4 and SUP6 tRNA(Tyr) genes demonstrates template dependence and indicates that inhibition may occur after UMP incorporation . Factor-independent transcription by purified yeast RNA polymerase III can also be inhibited by tagetitoxin, and the degree of inhibition is template-dependent . Tagetitoxin may be most effective as an inhibitor under conditions where polymerase III tends to pause on the template . We propose that differences in tagetitoxin inhibition among class III genes may reflect differences in the number of stability of these pause sites. J Biol Chem, 1992 Oct 5, 267(28), 20168 - 74 Interactions between the catalytic centers and subunit interface of triosephosphate isomerase probed by refolding, active site modification, and subunit exchange; Sun AQ et al.; The effects of unfolding, refolding, and hybridization of triosephosphate isomerase (TPI) subunits from different species and subunits which have been specifically modified at the active site have been examined . These effects have been evaluated in terms of changes in catalytic parameters, CD spectra, and susceptibility to denaturation . Dissociation followed by reassociation yields an active dimer but with increased Km, reduced kcat, and increased susceptibility to inactivation and unfolding in denaturants . These data suggest that while the general structure of the refolded dimer is similar to the native enzyme, its complete original structure is not restored . Covalent reaction of the active site Glu165 with the substrate analogue 3-chloroacetol phosphate (CAP) results in dimers with increased susceptibility to unfolding and inactivation by denaturants (i.e . the rates of inactivation and unfolding are (TPICAP)2 greater than (TPI-TPICAP) greater than (TPI)2) . These data point to the interactions between the catalytic center and the subunit interface . Subunits of TPI from different species, in spite of structural differences at the subunit interface, hybridized to active heterodimers . Subunit hybridization was random among monomers from different mammals, preferential between yeast and mammalian or avian monomers . Hybridization did not occur between avian and mammalian monomers under these conditions . These data provide information on the elements in the interface of the dimer and the relationship of the catalytic center with the subunit interface. J Biol Chem, 1992 Oct 5, 267(28), 19806 - 12 Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules; Palmer DJ et al.; An investigation of the molecular properties of glycoprotein III has shown this to be a major component of molecular aggregates present in the membrane and soluble fractions of secretory vesicles from bovine adrenal medulla . These aggregates also contain components identified as glycoproteins H, J, and K which are molecular forms of Kex2-related proteases (glycoprotein H) and carboxypeptidase H (glycoprotein components J and K) and which have functions concerned with the processing of prohormones . A number of experiments indicated that these glycoproteins were associated . These components were coimmunoprecipitated from the soluble and membrane fractions of chromaffin granules . Purification of soluble glycoprotein III using wheat germ agglutinin-Sepharose resulted in the recovery of similar proportions of glycoproteins H, J, and K and gel filtration of the eluted material in combination with immunoprecipitation revealed the presence of heteroaggregates containing all of the glycoproteins . Similar results were obtained following octylglucoside solubilization of chromaffin granule membranes . Glycoprotein components III, H, J, and K were also found to have identical distributions following fractionation of chromaffin granule membranes with Triton X-114 . It was concluded that the aggregates seen in the soluble fraction reflect an association of these components in the chromaffin granule membrane . This raises the possibility that these interactions are important for the targetting of these glycoproteins to secretory granules. Cell, 1992 Oct 2, 71(1), 97 - 105 The translation machinery and 70 kd heat shock protein cooperate in protein synthesis; Nelson RJ et al.; The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches . The SSB hsp70s (Ssb1/2p) are associated with translating ribosomes . This association is disrupted by puromycin, suggesting that Ssb1/2p may bind directly to the nascent polypeptide . Mutant ssb1 ssb2 strains grow slowly, contain a low number of translating ribosomes, and are hypersensitive to several inhibitors of protein synthesis . The slow growth phenotype of ssb1 ssb2 mutants is suppressed by increased copy number of a gene encoding a novel translation elongation factor 1 alpha (EF-1 alpha)-like protein . We suggest that cytosolic hsp70 aids in the passage of the nascent polypeptide chain through the ribosome in a manner analogous to the role played by organelle-localized hsp70 in the transport of proteins across membranes. J Histochem Cytochem, 1992 Oct, 40(10), 1573 - 8 Measurements of mitochondrial deficiencies in living cells by microspectrofluorometry; Schneckenburger H et al.; Microspectrofluorometric methods were developed for detection of mitochondrial metabolites and marker molecules in living cells . After excitation in the near UV and blue spectral ranges, respiratory-deficient strains of Saccharomyces cerevisiae showed higher levels of intrinsic fluorescence than corresponding wild types . This may be attributed to an increased emission by NADH and flavin molecules of the mutants . After incubation with the mitochondrial marker rhodamine 123, there was a strong indication that an energy transfer from flavin to rhodamine molecules occurred, which was more pronounced for the respiratory-deficient yeast strains . Skin fibroblasts obtained from patients with mitochondrial diseases showed approximately the same levels of autofluorescence and energy transfer but higher variances than a control cell line . These higher variances may result from a coexistence of intact and defective mitochondria. Arch Biochem Biophys, 1992 Oct, 298(1), 198 - 203 Effect of mutations at Lys250, Arg251, and Lys253 of cytochrome P450 1A2 on the catalytic activities and the bindings of bifunctional axial ligands; Krainev AG et al.; Some eukaryotic cytochromes P450 (P450s) have a series of ionic amino acids, corresponding to Lys250, Arg251, and Lys253 residues in the P450 1A2 sequence . To understand the roles of those ionic amino acids in the catalytic function of P450, three single mutants, Lys250Leu, Arg251Leu, and Lys253Leu of P450 1A2 were obtained from yeast (Saccharomyces cerevisiae) expression system . Turnover numbers of the Arg251Leu mutant in dealkylation reactions of methoxy- and ethoxyresorufin catalyzed by the P450 reconstituted system were remarkably increased by sixfold compared to those of the wild type . The Lys250Leu and Lys253Leu mutants also showed turnover numbers higher than those of the wild type by three- to fourfold . Those catalytic activities were inhibited competitively by pyridine derivatives, nitrogenous axial ligands to the P450 heme . From those findings together with other spectral data, it was suggested that the ionic site of Lys250, Arg251, and Lys253 may be somehow located near the substrate recognition site and/or near the axial-ligand access channel of this enzyme. Mol Microbiol, 1992 Oct, 6(20), 2989 - 97 Determination of a specific region of the purine-cytosine permease involved in the recognition of its substrates; Bloch JC et al.; Three u.v.-induced mutants of the purine-cytosine permease gene of Saccharomyces cerevisiae, with altered apparent Michaelis constant of transport (Kmapp), were cloned and sequenced . One of the mutants had extensive nucleotide replacement, whereas the other two had a single mutation . To evaluate the contribution of the different amino acid replacements to the phenotype of the complex mutant, simpler mutants were created by site-directed mutagenesis . All the amino acid replacements found in the segment from amino acids 371 to 377 inclusive, contribute to the determination of the phenotype . According to the model postulated this segment lies on the cell surface . In particular, amino acids at position 374 and 377 modulate the affinity of the permease towards its substrates . In the wild-type, when asparagine is present at both of these positions, the lowest Kmapp values are found. Mol Endocrinol, 1992 Oct, 6(10), 1559 - 70 Testicular expression of PC4 in the rat: molecular diversity of a novel germ cell-specific Kex2/subtilisin-like proprotein convertase; Seidah NG et al.; The rat cDNA sequence of PC4 (rPC4), representing a new member of the Kex2/subtilisin-like proprotein convertases, demonstrated the presence of at least three rPC4 mRNAs resulting in the production of rPC4-A (654 amino acids), rPC4-B (619 amino acids), and rPC4-C (609 amino acids) with different C-terminal sequences . Analogous to rat PC4, three cDNAs were also found for the mouse PC4 . The observed molecular diversity of PC4 mRNA possibly results from the differential splicing and/or exon skipping of the parent gene . PC4 mRNA, with a major form at 2.8 kilobases, was highly abundant in the rat testis but could not be detected by Northern analysis in any other tissues including the central nervous system and peripheral tissues . Testicular cell separation studies combined with Northern analysis indicate the high expression levels of PC4 in germ cells but not in Leydig, Sertoli, or peritubular cells . In situ hybridization histochemistry confirms the site of PC4 gene expression as the pachytene spermatocytes and the round spermatids but not in the elongating spermatids . We also demonstrate the colocalization of PC4 with proenkephalin in testicular germ cells by in situ hybridization . A study of the ontogeny of PC4 indicated that PC4 mRNA was first expressed postnatally between days 19 and 22, coinciding with the first stages of spermiogenesis . The stage-specific expression of PC4 in testis indicates its potential role in the developmental maturation of germ cells and that this convertase may play a specific physiological function in reproduction. Mol Gen Genet, 1992 Oct, 235(1), 74 - 80 The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis; Rasmussen JB et al.; A spontaneous uridine-requiring auxotroph of Colletotrichum graminicola was recovered by selection for resistance to 5-fluoro-orotic acid . The auxotroph lacked orotate phosphoribosyl transferase (OPRTase) and was complemented with a clone from a cosmid library of C . graminicola DNA . A 3.1 kb HindIII-SalI fragment was subcloned from the cosmid and it could efficiently transform the auxotrophic strain to uridine prototrophy and integrate by site-specific recombination . This DNA fragment contains an open reading frame that is similar to OPRTase genes of the fungi Sordaria macrospora, Trichoderma reesei, Podospora anserina, and Saccharomyces cerevisiae . Based on the sequence similarities and the ability to restore uridine prototrophy, we conclude that the fragment contains the C . graminicola gene for OPRTase, which we have named PYR1 . Our results demonstrate that cloning by complementation is feasible in C . graminicola, that the gene for OPRTase from C . graminicola can be useful as a selectable marker in transformation of the fungus, and that the OPRTase gene product is similar to OPRTase from other fungi. Gene, 1992 Oct 1, 119(2), 163 - 73 Characterization of the rRNA-encoding genes and transcripts, and a group-I self-splicing intron in Pneumocystis carinii; Lin H et al.; Although Pneumocystis carinii is the most common opportunistic pathogen infecting individuals with AIDS, very little is known of the basic biology of the organism . We have examined the ribosomal RNA (rRNA) and the DNA encoding it (rDNA) in P . carinii in an attempt to clarify its taxonomic position and to begin to study its genetic processes . Electrophoretic analysis showed that the sizes of the P . carinii rRNAs are quite similar to the sizes of the corresponding rRNAs from Saccharomyces cerevisiae . Direct sequence analysis of approx . 60% of the 18S small subunit-rRNA (Ss-rRNA) confirmed that its sequence is similar to that of yeast-like fungi and that a putative group-I intron previously observed in the 18S rDNA is, in fact, excised from the mature rRNA . PCR analysis of the intron in P . carinii genomic DNA showed that each of the multiple rDNA genes bears the group-I intron and in vitro transcripts of the intron autocatalytically excise from the rRNA primary transcript in the presence of GTP . Finally, analogues of GTP inhibit the self-splicing reaction, indicating that the guanosine-binding site of the intron closely resembles that of other well-characterized group-I introns . Since no group-I introns have been found in higher eukaryotes, this self-splicing process represents a viable target for chemotherapy of P . carinii pneumonia (PCP). Exp Cell Res, 1992 Oct, 202(2), 501 - 6 Spermidine nuclear acetylation in rat hepatocytes and in logarithmically growing rat hepatoma cells: comparison with histone acetylation; Desiderio MA et al.; Spermidine acetylation has been studied in nuclear homogenates and in entire nuclei from rat hepatocytes and rat hepatoma tissue culture (HTC) cells, isolated at different stages of logarithmic growth, and compared to histone acetylation . Under all experimental conditions, N8-acetylspermidine was the predominant product of the reaction (90%) . Unlike histone, spermidine acetylation in HTC cell and hepatocyte entire nuclei was almost absent or strikingly reduced relative to acetylation using nuclear homogenates as the enzyme sources . This was due to the lack of a free minor pool of spermidine, most likely lost during the purification of entire nuclei . Thus, preincubation of intact nuclei in the presence of spermidine restored activities to values observed using nuclear sonicates . Spermidine acetylation in HTC cell nuclei fluctuated moderately during cell growth, being stimulated immediately after initiation of proliferation and decreasing progressively as cultures reached high cell density . This pattern corroborated that of N8-acetylspermidine intracellular accumulation induced by culturing cells in the presence of 1 mM 7-amino-2-heptanone, a competitive inhibitor of N8-acetylspermidine deacetylase . Histone acetylation during HTC cell growth was not markedly different qualitatively from that of spermidine . Moreover, spermidine and histone acetylations in hepatocyte nuclei were of the same order of magnitude as those seen in rat hepatoma cell nuclei . Finally, inhibition of deacetylation of N8-acetylspermidine had no apparent deleterious effects on cell and growth . It remains to be determined whether the acetylation step is of higher physiological importance, in particular, and as discussed in nuclear spermidine turnover. EMBO J, 1992 Oct, 11(10), 3721 - 9 Roles of PRP8 protein in the assembly of splicing complexes; Brown JD et al.; Three different approaches have been used to investigate the roles of the yeast U5 snRNP protein PRP8 in spliceosome assembly: genetic depletion of PRP8 protein in vivo, heat inactivation of temperature-sensitive prp8 protein in protoplasts and inhibition of PRP8 function with antibodies in vitro . In each case, U5 and U4/U6 snRNPs failed to assemble into the forming spliceosomes . In addition, extract prepared from PRP8-depleted cells and extract containing inactivated PRP8 protein had substantially reduced amounts of U4/U6.U5 triple snRNP complexes . Thus, functional PRP8 protein is required for the stable formation of U4/U6.U5 complexes without which spliceosomes fail to form . As spliceosome formation was also blocked by anti-PRP8 antibodies that apparently do not disrupt triple snRNPs, PRP8 protein may play a separate role in the assembly of triple snRNPs into spliceosomes . As a consequence of PRP8 depletion the levels of the U4, U5 and U6 snRNAs declined dramatically . We discuss this in the context of the known genetic interactions between PRP8 and putative RNA helicase (DEAD box protein) genes and propose that PRP8 protein plays a role in regulating dynamic RNA-RNA interactions in spliceosome assembly, possibly ensuring the correct directionality of these events. EMBO J, 1992 Oct, 11(10), 3533 - 40 VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation; Egerton M et al.; Activation of T cells through the T cell antigen receptor (TCR) results in the rapid tyrosine phosphorylation of a number of cellular proteins, one of the earliest being a 100 kDa protein . We have sought to identify this 100 kDa substrate by partially purifying the protein by antiphosphotyrosine (APT) affinity purification, in order to obtain amino acid sequence data and, using this information, to isolate the cDNA clone encoding the molecule . We report here that the amino acid sequence data showed pp100 to be the murine equivalent of porcine valosin containing protein (VCP), a finding confirmed from the cloning and sequencing of the murine pp100 cDNA . Sequence analysis has shown VCP to be a member of a family of ATP binding, homo-oligomeric proteins, and the mammalian homolog of Saccharomyces cerevisiae cdc48p, a protein essential to the completion of mitosis in yeast . We also provide proof that both endogenous and expressed murine VCP are tyrosine phosphorylated in response to T cell activation . Thus we have identified a novel component of the TCR mediated tyrosine kinase activation pathway that may provide a link between TCR ligation and cell cycle control. Curr Opin Biotechnol, 1992 Oct, 3(5), 560 - 5 Protein processing within the secretory pathway; Rehemtulla A et al.; Endoproteolytic cleavage of hormone and neuropeptide precursors, as well as many complex proteins, such as coagulation factors and viral glycoproteins, is a key process in the generation of bioactive polypeptides . These cleavages typically occur at the dibasic amino acid residues Lys-Arg or Arg-Arg . The enzymes responsible for the processing belong to a newly discovered family of serine proteases related to the bacterial subtilisins . These include PACE (furin), PC1/PC3, PC2 and PACE4, which have all been characterized functionally and structurally. Genes Dev, 1992 Oct, 6(10), 2001 - 9 HAP1 positive control mutants specific for one of two binding sites; Turcotte B et al.; The expression of the yeast CYC1 and CYC7 genes is controlled by the HAP1 activator . A GAL4-like zinc finger (residues 1-148) specifies binding to the dissimilar sites UAS1 (of CYC1) and CYC7, and an acidic domain (residues 1307-1483) is essential for activation of transcription . To analyze how HAP1 binds to UAS1 and CYC7, we performed saturation mutagenesis of the DNA-binding domain and recovered mutants with altered activity . Class 1 mutants had a reduced activity at both UAS1 and CYC7, and class 2 mutants selectively eliminated activity at CYC7 . Surprisingly, several mutants of both classes exhibited wild-type DNA binding, indicating that they were specifically defective in activation . These positive control (PC) mutants alter residues that bracket the zinc finger . We explain these mutants in a model involving cofactor proteins that bind UAS1 and CYC7 along with HAP1 . The existence of PC mutants that only affect activity at CYC7 raises the possibility that different cofactors may exist for UAS1 and CYC7. Arch Biochem Biophys, 1992 Oct, 298(1), 91 - 5 Laser flash photolysis studies of electron transfer mechanisms in cytochromes: an aromatic residue at position 82 is not required for cytochrome c reduction by flavin semiquinones or electron transfer from cytochrome c to cytochrome oxidase; Hazzard JT et al.; The influence of an aromatic side chain at position 82 of yeast iso-1-cytochrome c on the kinetics of its electron transfer reactions has been investigated using laser flash photolysis methods to compare a series of site-specific mutant cytochromes in their reduction by free flavin semiquinone and in electron transfer from reduced cytochrome to bovine cytochrome c oxidase . Although small (approximately 10%) but significant differences are observed between some of the mutants (S82, Y82, I82) and wild-type (F82) or G82 cytochrome in the second-order rate constant for reduction by lumiflavin semiquinone, these do not correlate with side-chain aromaticity . In the reaction between the ferrocytochromes and cytochrome c oxidase, significantly larger deviations from exponentiality are found for those mutants having aliphatic residues at position 82 than for wild type or Y82 . We interpret the nonexponential behavior in terms of multiple orientations of the cytochromes within the oxidase binding site; the extent to which this occurs is apparently influenced by the character of the residue at position 82 . However, a comparison of the average rate constants for electron transfer to cytochrome oxidase for the various mutants reveals that all are closely comparable to WT, except for I82 which is significantly slower (approximately threefold) . These results, combined with those obtained previously from steady-state kinetic and thermodynamic measurements, suggest that the observed differences among the mutants are due to alterations in the mode of binding of the cytochrome to the oxidase, rather than to a specific requirement for the presence of an aromatic group at position 82. Biochem Cell Biol, 1992 Oct-Nov, 70(10-11), 1073 - 80 Mutational analysis of a DNA sequence involved in linking gene expression to the cell cycle; Andrews BJ et al.; Entry of budding yeast cells into the mitotic cell cycle requires the activity of a conserved regulatory kinase encoded by the CDC28 gene . The kinase is thought to trigger entry into the cell cycle or START, through association with a number of regulatory subunits known as G1 cyclins . A number of genes whose transcription is dependent on CDC28 and thus linked to START are controlled by two transcription factors, SWI4 and SWI6 . The genes controlled by SWI4 and SWI6 include two known G1 cyclins (CLN1 and CLN2), a putative new G1 cyclin (HCS26), and the HO gene whose product initiates cell type switching . SWI4 and SWI6 act through a repeated sequence element, SCB (SWI4,6-dependent cell cycle box), found 2-10 times in the upstream regulatory sequences of target genes . We have constructed a library of mutants in the SCB using doped oligonucleotide mutagenesis . All single base pair changes examined compromised the ability of the SCB to activate transcription in vivo . Analysis of the behaviour of the mutant SCBs in an in vitro DNA binding assay shows that the inability to activate transcription can be explained by reduced binding of SWI4 and SWI6 to the mutant SCBs . This analysis, together with a consideration of the SCBs found upstream of known SWI4,6-dependent genes, leads to the proposal of a revised consensus sequence for this important regulatory element. J Biol Chem, 1992 Sep 25, 267(27), 19705 - 9 Phosphorylation of tau protein by purified p34cdc28 and a related protein kinase from neurofilaments; Mawal-Dewan M et al.; It has been suggested that hyperphosphorylation of the tau protein in neurofibrillary tangles may be relevant to the etiology of Alzheimer's disease and that at least one of the hyperphosphorylated sites lies within a consensus sequence for the p34cdc2/cdc28 family of kinases . We describe a new method for large-scale purification of p34cdc28 kinase from Saccharomyces cerevisiae and show that the purified enzyme can phosphorylate bovine and human tau . Phosphorylation was greatly enhanced by the addition of basic and acidic substrate modulators . The effect of the substrate modulators differed both with the structures of the substrates and the modulators . Similar results were obtained with a kinase that could be purified from neurofilaments by p13suc1 affinity chromatography, a hallmark of p34cdc2/cdc28-type kinases . These results are consistent with the hypothesis that a kinase of this type is involved in tau phosphorylation in vivo and open the possibility that hyperphosphorylation in Alzheimer's disease may be controlled by substrate modulators. Science, 1992 Sep 25, 257(5078), 1958 - 61 Association of human cyclin E with a periodic G1-S phase protein kinase; Dulic V et al.; G1 cyclins control the G1 to S phase transition in the budding yeast, Saccharomyces cerevisiae . Cyclin E was discovered in the course of a screen for human complementary DNAs that rescue a deficiency of G1 cyclin function in budding yeast . The amounts of both the cyclin E protein and an associated protein kinase activity fluctuated periodically through the human cell cycle; both were maximal in late G1 and early S phases . Cyclin E-associated kinase activity was correlated with the appearance of complexes containing cyclin E and the cyclin-dependent kinase Cdk2 . Thus, the cyclin E-Cdk2 complex may constitute a human G1-S phase-specific regulatory protein kinase. Biochim Biophys Acta, 1992 Sep 23, 1159(2), 169 - 78 A study of hydrogen exchange of monoclonal antibodies: specificity of the antigen-binding induced conformational stabilization; Rizzo P et al.; Amide-hydrogen exchange of three anti-yeast iso-1-cytochrome-c IgG monoclonal antibodies and the Fab, prepared from one of them, were studied by infrared spectrophotometry in the presence and absence of the deuterated immunogen and evolutionarily related species (the deuterated immunogen contained a population of a dimer . Each subunit of the dimer appeared to bind to the antibodies in a manner similar to the monomer) . The number of hydrogens of the antibodies whose exchange was suppressed on binding to the immunogen was found to exceed that estimated for the residues shielded by the immunogen . Analysis of the data suggests that such suppression of hydrogen exchange occurs mainly for the Fab domains, but not for the Fc . One of the antibodies showed two distinct classes of amide-hydrogens . Class-1 hydrogens (approx . 36/site) exchange faster than class 2 (approx . 37/site) . The exchange of class-1 hydrogens was suppressed by binding to the immunogen, but not to the evolutionarily related species . The exchange of class-2 hydrogens was suppressed by binding to the evolutionarily related species, as well as to the immunogen . Thus, the suppression of exchange of class-1 hydrogens appears to occur by some kind of conformational stabilization, the mechanism of which differentiates between the deuterated immunogen and the evolutionarily related species . Evidence suggests that the trans-interactions of the Fab domains may modulate the hydrogen exchange . If it is assumed that the antigen-binding strengthens the trans-interactions in such a way that the exchange of the slower exchanging hydrogens is suppressed, this could explain the suppression of exchange of class-2 hydrogens. Gene, 1992 Sep 21, 119(1), 37 - 48 Bending-incompetent variants of Flp recombinase mediate strand transfer in half-site recombinations: role of DNA bending in recombination; Chen JW et al.; One key feature of the interaction of Flp recombinase with its target site (FRT) is the large bend introduced in the substrate as a result of protein binding . The extent of bending was found to depend on the phasing and spacing of the Flp monomers occupying the two Flp-binding elements (FBE) bordering the strand-exchange region (spacer) of the substrate . The relative mobilities of the Flp complexes formed by the two permuted substrate fragments, containing the FRT site near the end or in the middle, corresponded to a DNA bend of approx . 140 degrees when each of the two FBEs flanking the spacer was occupied by a protein monomer . The estimated bend angle was the same when the reference DNA fragment with the FRT site at the end was substituted by one with the site in the middle, but containing a 4-bp insertion within the spacer . We used a combination of wild-type Flp and Flp variants that were competent or incompetent in DNA bending, together with full, or half FRT sites, to ask whether bending is a conformational requirement for catalysis, namely cleavage and exchange of strands . We obtained the following results: in full-site (FRT) vs . full-site recombinations or in full-site vs . half-site (half FRT) recombinations, there was a large difference in the reactivity between Flp and a bending-incompetent Flp variant . This difference virtually disappeared when reactions were done with half-FRT sites . We conclude that bending is not a prerequisite for catalysis, but represents the manner in which the substrate accommodates the Flp protomer-protomer interactions that are pertinent to catalysis. Biochem Biophys Res Commun, 1992 Sep 16, 187(2), 1022 - 8 Molecular cloning of bovine actin-like protein, actin2; Tanaka T et al.; Actins are major cytoskeletal components and highly conserved in evolution . In mammals, there are six actin isoforms, a pair of which shows at least 93% identity in the amino acid sequence . We have cloned cDNA for a bovine protein that is distantly related to members of the mammalian actin isotypes . The predicted amino acid sequence (418 residues long, calculated molecular mass 47369) shows that this protein, which we have named actin2, exhibits 36% identity to mammalian actins and 60% identity to the yeast actin-like protein, act2 . We have concluded that actin2 defines a new class of mammalian actin-like proteins . It was also revealed that actin2 messenger RNA is expressed in a broad range of tissues. J Biol Chem, 1992 Sep 15, 267(26), 19011 - 6 Cloning of a functional cDNA for the rabbit ferritin mRNA repressor protein . Demonstration of a tissue-specific pattern of expression; Patino MM et al.; Ferritin synthesis is controlled at the translational level in response to cellular iron status . A component of this regulatory system is the ferritin repressor protein (FRP) which binds to the iron-responsive element (IRE) located at the 5' end of all known ferritin mRNAs, thus inhibiting its translation . Antibodies against purified FRP were raised in mouse and used to isolate an FRP cDNA from a rabbit liver cDNA library cloned in the expression vector lambda gt11 . The FRP cDNA encodes a 98.5-kilodalton protein which shares greater than 90% identity with IRE-binding proteins from other species . The FRP cDNA was placed under the transcriptional direction of the yeast GAL1 promoter . Yeast transformed with this gene express IRE-specific binding activity, illustrating the potential utility of yeast for the study of FRP structure/function . Analysis of FRP distribution in rabbit tissues shows that it is present in a variety of tissues . The levels of FRP differ dramatically from tissue to tissue, however . An examination of FRP mRNA levels and comparison to FRP protein suggest that synthesis of FRP is regulated transcriptionally and post-transcriptionally. Biochemistry, 1992 Sep 15, 31(36), 8488 - 94 Segmental motion in catalysis: investigation of a hydrogen bond critical for loop closure in the reaction of triosephosphate isomerase; Sampson NS et al.; A residue essential for proper closure of the active-site loop in the reaction catalyzed by triosephosphate isomerase is tyrosine-208, the hydroxyl group of which forms a hydrogen bond with the amide nitrogen of alanine-176, a component of the loop . Both residues are conserved, and mutagenesis of the tyrosine to phenylalanine results in a 2000-fold drop in the catalytic activity (kcat/Km) of the enzyme compared to the wild-type isomerase . The nature of the closure process has been elucidated from both viscosity dependence and primary isotope effects . The reaction catalyzed by the mutant enzyme shows a viscosity dependence using glycerol as the viscosogen . This dependence can be attributed to the rate-limiting motion of the active-site loop between the "open" and the "closed" conformations . Furthermore, a large primary isotope effect is observed with {1-2H}dihydroxyacetone phosphate as substrate {(kcat/Km)H/(kcat/Km)D = 6 +/- 1} . The range of isotopic experiments that were earlier used to delineate the energetics of the wild-type isomerase has provided the free energy profile of the mutant enzyme . Comparison of the energetics of the wild-type and mutant enzymes shows that only the transition states flanking the enediol intermediate have been substantially affected . The results suggest either that loop closure and deprotonation are coupled and occur in the same rate-limiting step or that these two processes happen sequentially but interdependently . This finding is consistent with structural information that indicates that the catalytic base glutamate-165 moves 2 A toward the substrate upon loop closure.(ABSTRACT TRUNCATED AT 250 WORDS) Nature, 1992 Sep 10, 359(6391), 154 - 5 A meiotic gene conversion gradient opposite to the direction of transcription; Malone RE et al.; Genetic recombination involves classical crossing-over and gene conversion (aberrant segregation) . In fungi that produce an ascus containing four spores, a gene conversion event is manifested as 3:1 or 1:3 (or more rarely 4:0 or 0:4) segregations, in contrast to the normal mendelian 2:2 segregation . Polarity is one of the properties of gene conversion; in almost all cases the frequency of conversion exhibits a gradient across the gene monitored . The frequency of conversion is usually independent of the specific allele used as a marker, but dependent on its location . An interpretation of conversion polarity is that it is caused by the existence of specific initiation sites for meiotic recombination, located at the high end of the polarity gradient . Here we show that the polarity gradient for the HIS2 gene of Saccharomyces cerevisiae is high at the 3' end of the gene, implying that the promoter of HIS2 is not the initiation site. Biochemistry, 1992 Sep 8, 31(35), 8291 - 9 Characterization of the deamidated forms of recombinant hirudin; Tuong A et al.; Recombinant hirudin variant rHV2-Lys 47 (MW = 6906.5) was intentionally deamidated by incubation in pH 9 phosphate buffer at 37 degrees C . Anion-exchange HPLC analysis showed that 11 forms could be generated . These were isolated and purified by combined anion-exchange and reversed-phase HPLC . Acid-catalyzed carboxyl methylation was used to introduce a mass shift of +15 amu per deamidated residue present in the molecule before analysis by liquid secondary ion mass spectrometry (LSIMS) . Methylation enhanced, in particular, the abundance of the sequence ions in the LSIMS spectra . This permitted the determination of both the number (three) and the localization of the deamidated residues: Asn 52, Asn 53, and a residue located in the N-terminal 1-39 domain . Complementary sequencing techniques proved that the latter residue was Asn 33 . Altogether four mono-, three di-, and four tri-deamidated forms were identified . The heterogeneity of the forms having identical deamidation positions but being chromatographically separable is thought to arise from the generation of alpha- and beta-aspartyl iso forms during the nonenzymatic deamidation process. Nature, 1992 Sep 3, 359(6390), 73 - 6 The Pas2 protein essential for peroxisome biogenesis is related to ubiquitin-conjugating enzymes; Wiebel FF et al.; In the yeast Saccharomyces cerevisiae, PAS genes are essential for the biogenesis and proliferation of peroxisomes . Recently, the first two genes, PAS1 (ref . 3) and PAS3 (ref . 4), have been characterized . Here we report the cloning and sequencing of the PAS2 gene . It encodes a new member of the ubiquitin-conjugating (UBC) protein family and is the first member associated with peroxisomes . The proposed function of the Pas2 protein as a UBC enzyme (UBC10) is supported by the fact that site-directed mutagenesis of a strictly conserved and functionally essential cysteine residue of UBC proteins leads to mutant Pas2 proteins unable to complement pas2 mutant strains . Ubiquitination of proteins is known to play an important part in DNA repair, sporulation, cell cycle control and degradation of abnormal proteins . We provide evidence for a crucial role of the ubiquitin-conjugation pathway in organelle formation. Am J Med, 1992 Sep, 93(3), 307 - 12 Autoantibodies in patients with primary pulmonary hypertension: association with anti-Ku; Isern RA et al.; PURPOSE: Patients with primary pulmonary hypertension (PPH) frequently have Raynaud's phenomenon, serum antinuclear antibodies (ANAs), and/or pulmonary vascular lesions similar to those seen in certain connective tissue diseases, especially scleroderma . A number of relatively disease-specific autoantibodies have been described in connective tissue diseases but have not been studied in patients with PPH . Therefore, sera from PPH patients were studied for a variety of autoantibodies, seeking a possible link between this pulmonary disorder and connective tissue diseases . PATIENTS AND METHODS: Sera from 31 patients with PPH and 24 with secondary pulmonary hypertension (SPH) were studied for the following autoantibodies: anti-centromere (indirect immunofluorescence of Hep-2 cells), anti-CENP-B by immunoblotting and enzyme immunoassay (EIA) using cloned CENP-B fusion protein, anti-topoisomerase I (Scl-70), anti-Ku using immunoblotting of affinity purified antigens, anti-cardiolipin using EIA, and anti-Ro (SS-A), La (SS-B), Sm, nRNP, Jo-1, PM-Scl, and Mi-2 by counter-current immunoelectrophoresis . RESULTS: Anti-Ku antibodies were found in 23% of patients with PPH, 4% with SPH, and none of 24 normal controls (PPH versus SPH, p = 0.06: PPH versus controls, p = 0.01) . Antibodies to CENP-B were found in one patient each with PPH and SPH, anti-topoisomerase I in one with SPH, and anti-Ro (SS-A) and La (SS-B) in one with PPH . Overall, 12 patients (39%) with PPH had Raynaud's phenomenon or positive ANA results, with 9 (29%) having more specific autoantibodies associated with connective tissue diseases . CONCLUSIONS: These results further suggest a link between at least a subgroup of patients with PPH and autoimmune connective tissue diseases, with anti-Ku antibodies being a possible new serologic marker. Genes Dev, 1992 Sep, 6(9), 1716 - 27 The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA; Wang W et al.; The assembly of activated RNA polymerase II (pol II) transcription complexes has been investigated by assaying whether pre-assembly of intermediate complexes reduces the extended time required for start-site melting . The results show that a closed complex requiring factors IIA, IID, and the acidic activator GAL4-AH forms in a rate-limiting step . This directs the templates into a productive assembly pathway . Factor TFIIB is then added rapidly, affording further protection against diversion into nonproductive pathways . These events are followed by a series of rapid steps in which the remaining general factors are assembled onto the template, which is then melted using the energy of ATP hydrolysis. J Cell Biol, 1992 Sep, 118(5), 1041 - 56 Sec8p and Sec15p are components of a plasma membrane-associated 19.5S particle that may function downstream of Sec4p to control exocytosis; Bowser R et al.; The SEC8 and SEC15 genes are essential for exocytosis in the yeast Saccharomyces cerevisiae and exhibit strong genetic interactions with SEC4, a gene of the ras superfamily . The SEC8 gene encodes a hydrophilic protein of 122 kD, while the temperature-sensitive sec8-9 allele encodes a protein prematurely truncated at 82 kD by an opal stop codon . The Sec8p sequence contains a 202 amino acid region that is 25% identical to the leucine rich domain of yeast adenylate cyclase that has been implicated in ras responsiveness . Fractionation, stability, and cross-linking studies indicate that Sec8p is a component of a 19.5S particle that also contains Sec15p . This particle is found both in the cytosol and peripherally associated with the plasma membrane, but it is not associated with secretory vesicles . Gel filtration studies suggest that a portion of Sec4p is in association with the Sec8p/Sec15p particle . We propose that this particle may function as a downstream effector of Sec4p, serving to direct the fusion of secretory vesicles with the plasma membrane. Gene, 1992 Sep 1, 118(1), 143 - 4 A convenient cloning vector containing the GAL4 DNA-binding domain; Raycroft L et al.; A DNA fragment encoding the yeast GAL4 DNA-binding domain (amino acids 3-147) was cloned into a convenient vector . This vector contains unique restriction sites at both the 5' and 3' ends and allows the generation of fusion proteins containing the GAL4 DNA-binding domain . These fusion proteins can be tested for their ability to activate transcription. Mol Cell Biol, 1992 Sep, 12(9), 4015 - 25 A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo; Morse RH et al.; Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo {R . T . Simpson, Nature (London) 343:387-389, 1990} . Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells . We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected . A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element . These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids . Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes . Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted . This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals. Mol Cell Biol, 1992 Sep, 12(9), 3750 - 6 Isolation of rsp-1, a novel cDNA capable of suppressing v-Ras transformation; Cutler ML et al.; Using an expression cloning assay, we have isolated a novel cDNA, referred to as rsp-1, which suppresses the v-Ras-transformed phenotype . When introduced into NIH 3T3 fibroblasts under the control of a metallothionein promoter, rsp-1 confers resistance to v-Ras, but not to v-Mos or v-Src, and inhibits growth of the cells . The rsp-1 cDNA contains a 831-bp open reading frame encoding a 277-amino-acid leucine-rich protein . The rsp-1 cDNA exhibits no significant homology to sequences in the DNA data bases . However, searches of the protein data bases revealed that it contains a series of leucine-based repeats which are homologous to the leucine repeats found in the regulatory region of the yeast adenylyl cyclase . rsp-1 specific RNA is detectable in a wide variety of cell lines and tissues, and the gene is conserved among eukaryotic species . These data suggest that rsp-1 plays a role in Ras signal transduction. EMBO J, 1992 Sep, 11(9), 3279 - 88 A novel gene, spp91-1, suppresses the splicing defect and the pre-mRNA nuclear export in the prp9-1 mutant; Chapon C et al.; Processing and export of nuclear pre-mRNA are believed to be competing processes in the nucleus . In order to identify factors which are involved in these processes, we isolated suppressors that relieve the growth defect of a prp9-1 temperature-sensitive mutant strain of Saccharomyces cerevisiae . The prp9-1 mutation was previously shown to abolish splicing and to target pre-mRNA to the cytoplasm . One of the suppressors, spp91-1, corrects the prp9-1 growth defect through partial restoration of splicing and by a complete reversion of the pre-mRNA escape phenotype . This suppressor is specific for two prp9 alleles and cannot substitute for PRP9 function . The mutant and wild-type alleles of SPP91 were cloned and sequenced . SPP91 encodes a novel protein essential for mitotic growth whose sequence contains motifs indicative of a nuclear localization . In vivo depletion of SPP91 in a prp9-1 genetic background is lethal and is associated with reduced amounts of spliced mRNA and accumulation of pre-mRNA . This observation strongly supports the hypothesis that SPP91 encodes a PRP factor . We suggest that spp91-1 increases pre-mRNA retention in the nucleus by improving the formation of the spliceosome and thereby allowing a larger proportion of the pre-mRNA molecules to be spliced. Res Virol, 1992 Sep-Oct, 143(5), 361 - 70 Processing of the minor capsid protein of the cauliflower mosaic virus requires a cysteine proteinase; Guidasci T et al.; The major capsid protein of the cauliflower mosaic virus (CaMV) is processed in vivo . The viral aspartic proteinase that catalyses this maturation has been characterized previously and is coded by the CaMV gene V . This virus has a second capsid protein, a minor component, encoded by gene III . This protein, P3, is also processed at its C-terminus in vivo . To determine whether P3 is matured by the CaMV proteinase P5, we expressed, in Saccharomyces cerevisiae, P3, P5 and a fusion protein P7-P4, containing potential sites of cleavage . P5 was found to be involved in maturation of P7-P4 but did not cleave P3 . The latter result was confirmed by experiments carried out with an in vitro translation system (the reticulocyte lysate) and with preparations of replication complexes purified from infected plants . Moreover, {N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leu cyl}-amido(4-guanido)butane, a specific inhibitor of cysteine proteinases, inhibited the maturation of P3, suggesting that the two CaMV capsid proteins are not processed by the same proteolytic event. Genetika, 1992 Sep, 28(9), 33 - 44 {Ploidy level in Streptomyces cerevisiae strain 15B-P4 strain}; Trubacheeva LIa et al.; Hybridization analysis of seven clones of Saccaromyces cerevisiae 15B-P4 strain, which were obtained from different cultures, was carried out . Yeast strains taken from the Peterhoff breeding stocks were kept in the laboratory of genetics at Biological Institute of Irkutsk University from 1970 . Increase in the number of prototrophic segregants was shown to by the result of aneuploidy (2n + 1) in the hybrids under study . Aneuploidy was revealed for the chromosomes I, III, IX, XII . Disomic clones of 15B-P4 strain were unstable. Ukr Biokhim Zh, 1992 Sep-Oct, 64(5), 42 - 7 {Pyruvate decarboxylase inactivation by interaction with substrate and molecular oxygen}; Vovk AI et al.; Pyruvate may promote the yeast pyruvate decarboxylase inactivation when affected by molecular oxygen . In the presence of pyruvate and O2 inactivation of enzyme increases with the initial substrate concentration increasing . pH-dependence of pyruvate decarboxylase inactivation under joint action of substrate and O2 has maximum in the region 6.9-7.5 . It is suggested that the influence of pyruvate and molecular oxygen is connected with the coenzyme-substrate complex oxidation in an active site of yeast pyruvate decarboxylase on the steps preceding the release of free acetaldehyde. Biochem Int, 1992 Sep, 27(6), 1073 - 81 Six-base deletion occurring in messages of human cytochrome P-450 in the CYP2C subfamily results in reduction of tolbutamide hydroxylase activity; Ohgiya S et al.; We isolated and expressed a clone, hPA6, possibly corresponding to the CYP2C9 cDNA . Compared with the other CYP2C9 cDNA clones, hPA6 showed a 6-nucleotide deletion near its middle . From the same cDNA library, we could also isolate another cDNA clone, named hPA22, which retained the 6 bases . For clarification of the effect of the 2-amino acid deletion resulting from the 6-base deletion on enzymatic activities, both clones were expressed in yeast . The expressed enzymes showed tolbutamide hydroxylase activities, and these activities were inhibited by antibodies against P-450-HM2, a probable CYP2C9 . The activity of the enzyme encoded by hPA6 was lower than that encoded by hPA22; thus the 2-amino acid deletion in the CYP2C9 reduced the enzymatic activity. Yeast, 1992 Sep, 8(9), 787 - 90 AFG1, a new member of the SEC18-NSF, PAS1, CDC48-VCP, TBP family of ATPases; Lee YJ et al.; We have sequenced a gene that encodes a 377 amino acid putative protein with an ATPase motif typical of the protein family including SEC18p (NSF = N-ethyl maleimide-sensitive fusion protein; vesicle-mediated endoplasmic reticulum to Golgi protein transfer), PAS1p (peroxisome assembly), CDC48p (VCP = valosin-containing protein; cell cycle) and TBP1 (Tat-binding protein) . This gene, AFG1 for ATPase family gene, also has substantial homology to these proteins outside the ATPase domain . AFG1 is located on chromosome V immediately centromere-proximal to MAK10. Mol Endocrinol, 1992 Sep, 6(9), 1441 - 50 Prohormone convertase-1 will process prorelaxin, a member of the insulin family of hormones; Marriott D et al.; Relaxin is a polypeptide hormone involved in remodeling of the birth canal during parturition . It is synthesized as a preprohormone precursor, which undergoes specific processing to form the mature two-chain disulfide-linked active species that is secreted by the cell . A major part of this processing requires endoproteolytic cleavage at specific pairs of basic amino acid residues, an event necessary for the maturation of a variety of important biologically active proteins, such as insulin and nerve growth factor . Human type 2 preprorelaxin was coexpressed in human kidney 293 cells with the candidate prohormone convertase-processing enzymes mPC1 or mPC2, both cloned from the mouse pituitary tumor AtT-20 cell line, or with the yeast kex2 alpha-mating factor-converting enzyme from Saccharomyces cerevisiae . Prorelaxin expressed alone in 293 cells was secreted into the culture medium unprocessed . Transient coexpression with mPC1 or kex2, but not with mPC2, resulted in the secretion of a low mol wt species with an electrophoretic mobility very similar, if not identical, to that of authentic mature relaxin purified from human placenta . This species was precipitable by monoclonal antibodies specific for relaxin and had a retention time on reverse phase HPLC comparable to that of relaxin . Its analysis by both electrospray and fast atom bombardment mass spectrometry generated mass data that were consistent only with mature relaxin . The basic residues required for mPC1-dependent cleavage of prorelaxin are defined by site-directed mutagenesis. Eur J Epidemiol, 1992 Sep, 8(5), 650 - 5 Long-term immunogenicity safety and efficacy of a recombinant hepatitis B vaccine in healthy adults; Dentico P et al.; Two hundred healthy adults seronegative for HBV markers received three 10 or 20 mcg injections of a vaccine formulated from HBsAg produced by a recombinant strain of the yeast Saccharomyces cerevisiae . The vaccine was administered intramuscularly at 0, 1, and 6 months in the deltoid region . The seroconversion rates, expressed in GMT/IU/1 were determined at 1, 2, 6, 7, 12, 24, 36 and 48 months following the initial injection . No severe or serious adverse reactions attributable to the HB vaccines were observed in any subject . The seroconversion rates following the 20 mcg dose of recombinant vaccine were always higher than those observed after the 10 mcg dose, but the differences were not statistically significant . Also the GMT values were lower after the 10 mcg dose of vaccine . Females showed a higher anti-HBs response than males; an age-dependent effect was observed in the anti-HBs response as regards both the percentage of responders and the antibody concentrations in the serum . No adverse reactions to the vaccine were observed . The rDNA vaccine did not induce a response to yeast-derived impurities and did not increase anti-yeast IgE antibody titres . The results of this study have shown that the Amgen rDNA vaccine is safe and clinically well tolerated, and that it provides protection against infection and disease . A vaccination dose of 20 mcg appears more advantageous for healthy adult subjects. Biophys J, 1992 Sep, 63(3), 689 - 99 Vibrationally enhanced tunneling as a mechanism for enzymatic hydrogen transfer; Bruno WJ et al.; We present a theory of enzymatic hydrogen transfer in which hydrogen tunneling is mediated by thermal fluctuations of the enzyme's active site . These fluctuations greatly increase the tunneling rate by shortening the distance the hydrogen must tunnel . The average tunneling distance is shown to decrease when heavier isotopes are substituted for the hydrogen or when the temperature is increased, leading to kinetic isotope effects (KIEs)--defined as the factor by which the reaction slows down when isotopically substituted substrates are used--that need be no larger than KIEs for nontunneling mechanisms . Within this theory we derive a simple KIE expression for vibrationally enhanced ground state tunneling that is able to fit the data for the bovine serum amine oxidase (BSAO) system, correctly predicting the large temperature dependence of the KIEs . Because the KIEs in this theory can resemble those for nontunneling dynamics, distinguishing the two possibilities requires careful measurements over a range of temperatures, as has been done for BSAO. Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 7958 - 62 Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells; Fearon ER et al.; We describe a strategy and reagents for study of protein-protein interactions in mammalian cells, termed the karyoplasmic interaction selection strategy (KISS) . With this strategy, specific protein-protein interactions are identified by reconstitution of the functional activity of the yeast transcriptional activator GAL4 and the resultant transcription of a GAL4-regulated reporter gene . Reconstitution of GAL4 function results from specific interaction between two chimeric proteins: one contains the DNA-binding domain of GAL4; the other contains a transcriptional activation domain . Transcription of the reporter gene occurs if the two chimeric proteins can form a complex that reconstitutes the DNA-binding and transcriptional activation functions of GAL4 . Using the KISS system, we demonstrate specific interactions for sequences from three different pairs of proteins that complex in the cytoplasm . In addition, we demonstrate that reporter genes encoding cell surface or drug-resistance markers can be specifically activated as a result of protein-protein interactions . With these selectable markers, the KISS system can be used to screen specialized cDNA libraries to identify novel protein interactions. Mutat Res, 1992 Sep, 275(3-6), 237 - 41 Structural dynamics of the mitochondrial compartment; Thorsness PE; The metabolic activities of mitochondria have been extensively characterized . However, there is much less known about the morphogenic changes of the mitochondrial compartment during growth, development and aging of the cell and the consequences of those structural changes on cellular metabolism . There is a growing body of evidence for interactions of mitochondria with cytoskeletal components and changes of mitochondrial structure during development and in response to changing environmental conditions . Segregation and recombination of mitochondrial genomes are also processes dependent upon the dynamic nature of the mitochondrial compartment . These regulatory and structural aspects of mitochondrial compartment dynamics will play an important role in the analysis of mitochondrial function and pathology. Biosci Biotechnol Biochem, 1992 Sep, 56(9), 1424 - 8 Structural and functional properties of hen egg-white lysozyme deamidated by protein engineering; Kato A et al.; The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique . The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl . The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation . The Gibbs free energy changes of unfolding (delta G) at 20 degrees C for N103D and N106D were almost the same as that of wild-type . On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation . The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme . These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability. Hum Mol Genet, 1992 Sep, 1(6), 417 - 25 Molecular dissection of the Prader-Willi/Angelman syndrome region (15q11-13) by YAC cloning and FISH analysis; Kuwano A et al.; Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation disorders associated with deletions of proximal 15q (q11-q13) of different parental origin . Yeast artificial chromosome (YAC) clones were isolated for 9 previously mapped DNA probes from this region, and for one newly derived marker, LS6-1 (D15S113) . A YAC contig of 1-1.5 Mb encompassing four markers (ML34, IR4-3R, PW71, and TD189-1) was constructed . Multi-color fluorescence in situ hybridization (FISH) analysis of interphase nuclei was combined with YAC contig information to provide the following order of markers: cen-IR39-ML34-IR4-3R-PW71-TD189-1-LS6++ +-1-TD3-21-GABRB3-IR10-1-CMW1-tel . FISH analysis was performed on 8 cases of PWS and 3 cases of AS, including 5 patients with normal karyotypes . All eleven patients were deleted for YACs in the interval from IR4-3R to GABRB3 . On the proximal side of the deletion interval, 10/10 breakpoints fell within a single ML34 YAC of 370 kb . On the distal side, 8/9 breakpoints fell within a single IR10-1 YAC of 200 kb . These results indicate a striking consistency in the location of the proximal and distal breakpoints in PWS and AS patients . FISH analysis on a previously reported case of familial AS confirmed a submicroscopic deletion including YACs corresponding to LS6-1, TD3-21 and GABRB3 and supports the separation of the PWS and AS critical regions . Since these three YACs do not overlap each other, the minimum size of the AS critical region is > or = 650 kb. J Gen Microbiol, 1992 Sep, 138 ( Pt 9), 1797 - 800 An acetate-sensitive mutant of Neurospora crassa deficient in acetyl-CoA hydrolase; Connerton IF et al.; The predicted amino acid sequence of the product of the acetate-inducible acu-8 gene of Neurospora crassa, previously of unknown function, has close homology to the recently published sequence of Saccharomyces cerevisiae acetyl-CoA hydrolase . An acu-8 mutant strain, previously characterized as acetate non-utilizing, shows strong growth-inhibition by acetate, but will use it as carbon source at low concentrations . The mutant was shown to be deficient in acetyl-CoA hydrolase and to accumulate acetyl-CoA when supplied with acetate . As in Saccharomyces, the Neurospora enzyme is acetate-inducible. Biofizika, 1992 Sep-Oct, 37(5), 910 - 9 {Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein}; Vlasova II et al.; A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label . The saturation curves of ESR spectra of the spin-labeled enzyme were studied . The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme . The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied . It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre). Biochem Int, 1992 Sep, 27(6), 1127 - 34 Dynamic structure of transfer RNA in solution monitored by reaction with hydroxyl radicals; Barciszewska MZ et al.; The dynamic structure of initiator and elongator tRNAs was analyzed using a very sensitive reaction with hydroxyl radicals . The main target for this reagent is the ribose moieties not buried (accessible) in the tertiary structure of the RNA molecule . At variable time, temperature and magnesium concentrations, some nucleoside residues of lupin initiator tRNA or yeast tRNA(Phe) become accessible or not depending on the actual tRNA conformation . The nucleotides in the thymidine stem of yeast tRNA(Phe) and amino acid stem in both tRNAs do not change their reactivity and conformation . Also the reactivities of the nucleosides of the anticodons are not changing . Our data clearly suggest that hydroxyl radicals can be very useful for the analysis of the tertiary structure of tRNA. Biochemistry, 1992 Sep 1, 31(34), 8043 - 54 Synthesis and properties of a conformationally restricted spin-labeled analog of ATP and its interaction with myosin and skeletal muscle; Alessi DR et al.; The synthesis is described of a spin-labeled analog of ATP, 2',3'-O-(1-oxy-2,2,6,6-tetramethyl-4-piperidylidene)adenosine 5'-triphosphate (SL-ATP) . The spin-label moiety is attached by two bonds to the ribose ring as a spiroketal and hence has restricted conformational mobility relative to the ribose moiety of ATP . The synthesis proceeds via an acid-catalyzed addition of adenosine 5'-monophosphate to 1-acetoxy-4-methoxy-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine in acetonitrile . The spiroketal product is pyrophosphorylated, and alkaline hydrolysis with concomitant aerial oxidation gives the required product . The spin-labeled moiety probably takes up two rapidly interconverting conformations with respect to the ribose ring on the basis of the 1H NMR spectra of its precursors and related uridine derivatives {Alessi et al . (1991) J . Chem . Soc., Perkin Trans.1,2243-2247} . SL-ATP is a substrate for myosin and actomyosin with similar kinetic parameters to ATP during triphosphatase activity . SL-ATP supports muscle contraction and permits relaxation of permeabilized rabbit skeletal muscle fibers . SL-ADP is a substrate for yeast 3-phosphoglycerate kinase, thus permitting regeneration of SL-ATP from SL-ADP within muscle fibers . Electron paramagnetic resonance (EPR) studies of SL-ADP bound to myosin filaments and to myofibrils show a degree of nanosecond motion independent of that of the protein, which may be due to conformational flexibility of the ribose moiety of ATP bound to myosin's active site . This nanosecond motion is more restricted in myofibrils than in myosin filaments, suggesting that the binding of actin affects the ribose binding site in myosin . EPR studies on SL-ADP bound to rigor cross-bridges in muscle fiber bundles showed the nucleotide to be highly oriented with respect to the fiber axis. Mycoses, 1992 Sep-Oct, 35(9-10), 215 - 20 A retrospective clinical comparison between antifungal treatment with liposomal amphotericin B (AmBisome) and conventional amphotericin B in transplant recipients; Tollemar J et al.; Invasive fungal infections are an important cause of morbidity and mortality in immunosuppressed bone marrow and solid organ transplant recipients . Treatment with amphotericin B, the drug of choice for these infections, is however often limited by toxicity . Ten transplant patients receiving a liposomal amphotericin B formulation (AmBisome) were compared to ten retrospective control patients given conventional amphotericin B . Each group included bone marrow (8), kidney (1), and liver transplant (1) recipients . Conventional amphotericin B treatment was instituted due to nine Candida infections, and one Aspergillus fumigatus infection . In the AmBisome group treatment was instituted due to eight Candida infections, one infection caused by Saccharomyces cerevisiae and in one case as prophylactic treatment . In the amphotericin B group, maximal daily doses ranged from 0.1 to 0.65 mg kg-1 and cumulative doses were 21-836 mg kg-1 and were given over 3-32 days . In the AmBisome group, maximal daily doses ranged from 0.9 to 2.3 mg kg-1 and cumulative doses ranged from 225 to 3525 mg kg-1 over 8-28 days . All patients in the amphotericin B group experienced severe toxicity, especially nephrotoxicity which in four cases caused withdrawal of the drug . In contrast, the only adverse reaction in the AmBisome group was cholestasis in one patient . Only three out of ten patients in the amphotericin B group responded to treatment, seven patients died and six patients still had evidence of invasive fungal infection at autopsy . In contrast, eight out of nine patients in the AmBisome group responded to treatment, and the patient that received prophylaxis had a successful course. Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 32 - 8 Affinity labeling of vertebrate oxidosqualene cyclases with a tritiated suicide substrate; Abe I et al.; Pig and rat liver oxidosqualene cyclase (OSC) enzymes were purified to homogeneity and showed single bands on SDS-polyacrylamide gel electrophoresis with molecular masses of 75 kDa (pig) and 78 kDa (rat) . Pig liver OSC was purified for the first time (441-fold with a yield of 39%) . Chemical affinity labeling of pure or crude preparations of the liver cyclases using the mechanism-based irreversible inhibitor of OSC, {3H}29-methylidene-2,3-oxidosqualene ({3H}29-MOS), showed a single radioactive band at 75 kDa (pig) and 78 kDa (rat) . Affinity labeling experiments were also performed with dog and human microsomal preparations and with yeast and plant cyclases . All of the vertebrate OSC enzymes were specifically labeled with {3H}29-MOS and gave a single band with molecular masses ranging from 70 to 80 kDa (rat, 78 kDa; dog, 73 kDa; pig, 75 kDa; and human, 73 kDa) . In contrast, yeast lanosterol cyclase and plant cycloartenol cyclase were not labeled, demonstrating subtle differences in the active sites of animal, plant, and fungal enzymes. FEBS Lett, 1992 Aug 31, 309(1), 51 - 5 Steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease; Bednarek J et al.; An adrenal-specific protein reacting with autoantibodies in the sera of patients with adult onset Addison's disease has been purified from human adrenal glands . The protein, mol.wt . 55K, has the biochemical characteristics of steroid 21-hydroxylase and reacts on Western blots with rabbit antibodies to recombinant 21-hydroxylase . Absorption of the native human 55K adrenal protein with human adrenal autoantibodies prevented the subsequent reaction of the 55K protein with rabbit antibodies to 21-hydroxylase in Western blot analysis . In addition, human adrenal autoantibodies reacted with recombinant 21-hydroxylase expressed in yeast . These data indicate that the adrenal specific enzyme steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison's disease. J Biol Chem, 1992 Aug 25, 267(24), 17208 - 15 Cloning and functional expression of Dfurin2, a subtilisin-like proprotein processing enzyme of Drosophila melanogaster with multiple repeats of a cysteine motif; Roebroek AJ et al.; Production of a variety of regulatory eukaryotic proteins, such as growth factors and polypeptide hormones, often involves endoproteolytic processing of proproteins at cleavage sites consisting of paired basic residues . The first known mammalian proprotein processing enzyme with such specificity is the human fur gene product furin . Structurally and functionally, furin is related to the subtilisin-like serine endoprotease kexin (EC 3.4.21.61) of yeast Saccharomyces cerevisiae; unlike kexin, it contains a cysteine-rich region with an unknown function . Here, we describe cloning and sequencing of a 5.8-kbp cDNA of the Dfur2 gene, a fur-like gene of Drosophila melanogaster, which we found expressed during various stages of development . This Dfur2 cDNA has an open reading frame for a 1680-residue protein, called Dfurin2 . Dfurin2 contains similar protein domains as mammalian furin, however, it has an extended amino-terminal region and its cysteine-rich region is much larger than that of mammalian furin . Because of this latter phenomenon, we were able to identify a particular cysteine motif that was repeated multiple times in Dfurin2 but present only twice in mammalian furin . Furthermore, we show that Dfur2 encodes an endoproteolytic enzyme with specificity for paired basic amino acid residues as, in cotransfection experiments, correct cleavage was demonstrated of the precursor of the von Willebrand factor but not of a cleavage mutant . Finally, Dfur2 was mapped to region 14C of the X chromosome of D . melanogaster. J Biol Chem, 1992 Aug 25, 267(24), 17201 - 7 Cloning and structural analysis of the gene for the regulatory subunit of cAMP-dependent protein kinase in Blastocladiella emersonii; Marques Mdo V et al.; We have isolated and characterized cDNA and genomic DNA clones encoding the regulatory subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii . Nucleotide sequence analysis has shown that the predicted protein comprises 403 amino acids with a calculated molecular mass of 44,263 Da and an overall 40% identity to mammalian RII subunits, including a serine in the phosphorylation site, which confirms the Blastocladiella protein as a type II regulatory subunit . The B . emersonii R gene presents two introns, one located in the 5'-noncoding region, whereas the other interrupts the coding region, just after the dimerization domain of the protein . The promoter region does not contain recognizable TATA or CCAAT sequences and is very GC rich, a characteristic shared by mammalian cAMP-dependent protein kinase subunit genes previously analyzed . S1 mapping and primer extension experiments revealed multiple transcription initiation sites . Several sequence motifs were identified in the 5'-flanking region which could be responsible for the regulation of this gene. Nucleic Acids Res, 1992 Aug 25, 20(16), 4355 - 61 Purification and characterization of a mammalian endo-exonuclease; Couture C et al.; An endo-exonuclease has been purified from cultured monkey (CV-1) cells . The enzyme which was purified to near homogeneity to be a 65 kDa monomeric protein . The single-strand DNase activity is endonucleolytic and nonprocessive, whereas the double-strand DNase activity is exonucleolytic and processive . The enzyme was also found to have RNase activity using poly-rA as substrate . The pH optimum for ss-DNase is 8 and for ds-DNase it is 7.5 . Both DNase activities require a divalent metal ion (Mg2+, Mn2+, Ca2+, Zn2+) for activity and exhibit the same kinetics of heat inactivation . The purified protein binds to and cleaves a synthetic Holliday junction substrate . The overall enzymatic characteristics of the mammalian protein are very similar to the putative recombination endo-exonucleases purified from Neurospora crassa, Aspergillus nidulans and Saccharomyces cerevisiae. Science, 1992 Aug 21, 257(5073), 1089 - 95 Human and Drosophila homeodomain proteins that enhance the DNA-binding activity of serum response factor; Grueneberg DA et al.; Cells with distinct developmental histories can respond differentially to identical signals, suggesting that signals are interpreted in a fashion that reflects a cell's identity . How this might occur is suggested by the observation that proteins of the homeodomain family, including a newly identified human protein, enhance the DNA-binding activity of serum response factor, a protein required for the induction of genes by growth and differentiation factors . Interaction with proteins of the serum response factor family may allow homeodomain proteins to specify the transcriptional response to inductive signals . Moreover, because the ability to enhance the binding of serum response factor to DNA residues within the homeodomain but is independent of homeodomain DNA-binding activity, this additional activity of the homeodomain may account for some of specificity of action of homeodomain proteins in development. Cell, 1992 Aug 21, 70(4), 595 - 607 The growth arrest-specific gene, gas1, is involved in growth suppression; Del Sal G et al.; This report describes the structure of the mRNA, the protein product, and the growth-regulating activity of one of the growth arrest-specific genes, gas1 . From the predicted amino acid sequence, in vitro translation of gas1 mRNA, and immunofluorescence of cells in culture, it appears that the gas1 protein is an integral plasma membrane protein whose expression is linked to growth arrest . When gas1 is overexpressed from a constitutive promoter in quiescent cells, the serum-induced transition from the G0 to the S phase of the cell cycle is inhibited without affecting the normal early serum response . Ectopic expression of the gas1 gene by microinjection in normal and transformed NIH 3T3 cell lines with the notable exception of SV40-transformed 3T3 cells leads to inhibition of DNA synthesis . Thus, gas1 appears to be one component of a negative circuit that governs growth suppression . Its effect is, however, abolished in SV40-transformed cells. J Mol Biol, 1992 Aug 20, 226(4), 913 - 6 Sequence of a second human KDEL receptor; Lewis MJ et al.; Retention of luminal endoplasmic reticulum (ER) proteins is mediated via a conserved carboxy-terminal tetrapeptide that serves as a signal for their retrieval from subsequent compartments of the secretory pathway . The signal is recognized by a receptor molecule that is believed to cycle between the Golgi apparatus and the ER . This receptor in Saccharomyces cerevisiae is encoded by the ERD2 gene, and a human cDNA homologue of the gene has been isolated . Binding of ligand by the product of this gene results in a shift of its steady-state location from Golgi to ER, suggesting that retrograde transport has been triggered . Here we report the identification of a related human protein with similar properties . This indicates that there are at least two distinct genes in humans that encode functional KDEL receptors. Biochim Biophys Acta, 1992 Aug 17, 1132(1), 35 - 42 Isolation and characterization of a cdc 2 cDNA from Dictyostelium discoideum; Michaelis C et al.; A cdc2 homologous sequence was amplified from Dictyostelium discoideum by the polymerase chain reaction and used to isolate several cDNA clones . The amino acid sequence encoded by these cDNAs exhibited approx . 60% identity to the Cdc2 proteins of other species . A cDNA containing the entire coding sequence complemented the temperature sensitive cdc28 mutant of Saccharomyces cerevisiae, although growth of the transformants was slow and limited . Southern blot analysis of restriction digests under high stringency conditions provided evidence that Dictyostelium contains a single cdc2 gene, although at lower stringency multiple fragments were detected, suggesting the existence of a cdc2 gene family . Northern blot analysis of RNA from different stages of Dictyostelium development showed that cdc2 mRNA levels increased during aggregation and then decreased to low levels by the pseudoplasmodial stage of development . By contrast, cdc2 mRNA levels remained relatively constant as cells passed from exponential growth to the stationary phase. J Biol Chem, 1992 Aug 15, 267(23), 16335 - 40 Consensus sequence for precursor processing at mono-arginyl sites . Evidence for the involvement of a Kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites; Nakayama K et al.; Many peptide hormones and neuropeptides are produced from larger, inactive precursors through endoproteolysis at sites usually marked by paired basic residues (primarily Lys-Arg and Arg-Arg), or occasionally by a monobasic residue (primarily Arg) . Based upon data concerning processing of prorenin and its mutants around the native Lys-Arg cleavage site expressed in mouse pituitary AtT-20 cells, we present the following sequence rules that govern mono-arginyl cleavages: (a) a basic residue at the fourth (position -4) or the sixth (position -6) residue upstream of the cleavage site is required, (b) at position -4, Arg is more favorable than Lys, and (c) at position 1, a hydrophobic aliphatic residue is not suitable . These rules are compatible with those proposed by comparison of precursor sequences around mono-arginyl cleavage sites . We also provide evidence that precursor cleavages at mono-arginyl and dibasic sites can be catalyzed by the same Kex2-like processing endoprotease, PC1/PC3. J Biol Chem, 1992 Aug 15, 267(23), 16094 - 9 Purification and characterization of furin, a Kex2-like processing endoprotease, produced in Chinese hamster ovary cells; Hatsuzawa K et al.; Furin, a mammalian homolog of the yeast Kex2 protease, is associated with Golgi membranes and is involved in cleavage of precursor proteins at sites marked by the Arg-X-Lys/Arg-Arg (RXK/RR) motif . We have recently shown that a furin mutant lacking the transmembrane domain can be secreted from cDNA-transfected cells with proteolytic activity for the fluorogenic peptide t-butoxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumarin-7- amide . In this study, we purified and characterized the recombinant furin from the conditioned medium of these cells . Furin was purified as a mixture of 83- and 81-kDa forms and a 96-kDa form . The differences in molecular mass were not due to differences in molecular mass were not due to differences in glycosylation . Moreover, all forms had the same NH2-terminal sequence beginning at the residue after the Arg-Ala-Lys-Arg sequence . These data suggest that the three different forms may be produced by differential COOH-terminal processing of a furin molecule and that mature furin may be autocatalytically produced . Both enzyme preparations showed a pH optimum at 7.0, required Ca2+ for the activity, and showed essentially the same inhibitor profile . These properties resembled those of the Kex2 protease . Both preparations efficiently cleaved fluorogenic peptides with an RXK/RR sequence and moderately cleaved a peptide with an RXXR sequence, but did not cleave dibasic peptides . The sequence requirements determined in vitro were compatible with those determined by expression studies in cultured cells . These data unequivocally demonstrate that furin is an endogenous cellular protease responsible for cleavage of precursor proteins mainly at RXK/RR sites. Anal Biochem, 1992 Aug 15, 205(1), 27 - 35 Selective photochemical modification by trichloroethanol of tryptophan residues in proteins with a high tyrosine-to-tryptophan ratio; Casas-Finet JR et al.; We present an improved procedure for the selective modification of tryptophan residues in proteins . A simple, low-cost set-up allows rapid tryptophan photoreaction upon ultraviolet irradiation in the presence of 2,2,2-trichloroethanol . This photochemical reaction is carried out under native conditions, occurs only in the excited state of tryptophan, and yields a single, as yet unidentified, photoproduct . Except for tyrosine, no reaction with other amino acid side chains are known . Stringent photoselection of tryptophan, ensuring that tyrosine residues are not affected, is achieved in situ without the need for an elaborate system of optical filters or lenses . Illumination with a medium-wave uv lamp of samples placed in disposable, dual pathlength, polystyrene fluorescence cuvettes allows treatment of small sample volumes (greater than or equal to 100 microliters) of various optical density . Chromophore accessibility in oligomeric assemblies or protein-nucleic acid complexes can be assessed by this reaction since the integrity of these structures is preserved . Moreover, this technique can be used to evaluate the involvement of tryptophan residues in catalytic or ligand binding processes. Anal Biochem, 1992 Aug 15, 205(1), 179 - 82 Epitope mapping by a method that requires no amino acid sequence information; Yuan J et al.; A simple, rapid method of epitope mapping has been developed that avoids the often cumbersome requirement of obtaining amino acid sequence information . The protein antigen is digested with various concentrations of carboxypeptidase into a nearly continuous series of polypeptides of different molecular weights, all containing a common N-terminus . The peptides are separated by polyacrylamide gel electrophoresis and then transferred to nitrocellulose paper . After developing the blot with the antibody to be mapped, a nearly continuous stain is observed extending from the position of the intact antigen to the molecular weight of the smallest N-terminal fragment still containing the antibody's epitope . By noting the molecular weight where the stain terminates, the position of the epitope relative to the N-terminus can be determined . Using this methodology, and taking special precautions to inhibit all endoproteinases in the carboxypeptidase preparation, the previously mapped epitopes of six nonoverlapping antibodies to the erythrocyte anion transporter were confirmed. Nature, 1992 Aug 13, 358(6387), 560 - 3 A small metalloribozyme with a two-step mechanism; Pan T et al.; An RNA molecule consisting of an asymmetric internal loop of six nucleotides can be rapidly and specifically cleaved by Pb2+ in the presence of Mg2+ . The 5' cleavage product terminates with a 3' phosphomonoester generated from a 2',3'-cyclic phosphodiester reaction intermediate . This two-step reaction mechanism resembles that of many protein ribonucleases but has not previously been observed for reactions catalysed by RNA. Biochemistry, 1992 Aug 11, 31(31), 7166 - 73 Structure of the bis divalent cation complex with phosphonoacetohydroxamate at the active site of enolase; Poyner RR et al.; Phosphonoacetohydroxamate (PhAH) is a tight-binding (Ki = 15 pM) inhibitor of enolase that is believed to mimic the aci-carboxylate form of the intermediate carbanion in the reaction {Anderson, V . E., Weiss, P . M., & Cleland, W . W . (1984) Biochemistry 23, 2779} . Electron paramagnetic resonance (EPR) spectroscopy of Mn2+ has been used to map sites of interaction of PhAH with the two divalent cations at the active site of enolase from bakers' yeast . EPR spectra of enolase-PhAH complexes containing two Mn2+ bound at the active site contain multiple fine structure transitions each with a 45-G 55Mn hyperfine spacing that is a characteristic of spin exchange coupled pairs of Mn2+ . Magnetically dilute complexes were obtained by preparation of specific Mg2+/Mn2+ hybrid complexes by manipulating the order of addition of the divalent metal species . Thus, Mn2+ was placed in the higher affinity site by addition of 1 equiv of Mn2+ to a solution of enolase and PhAH, followed by addition of 1 equiv of Mg2+ . Reversing the order of addition of Mg2+ and Mn2+ placed Mn2+ in the lower affinity site . Regiospecifically 17O-labeled forms of PhAH were prepared, and the binding of the functional groups on PhAH to Mn2+ at the two metal ion sites was determined from the presence or absence of 17O superhyperfine coupling in the EPR signals . The hydroxamate oxygen is a ligand of Mn2+ at the higher affinity site, a phosphonate oxygen is a ligand of Mn2+ at the lower affinity site, and the carbonyl oxygen is a mu-O bridge of the two metal ions.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1992 Aug 5, 267(22), 15993 - 9 Rat procathepsin B . Proteolytic processing to the mature form in vitro; Rowan AD et al.; Expression of rat procathepsin B in yeast led to the secretion of both the latent and mature forms of the enzyme . Culture in the presence of a cysteine proteinase inhibitor prevented this processing . We have expressed and purified a mutant form of rat procathepsin B whose active-site cysteine residue has been changed to a serine, and which also lacks the glycosylation site in the mature region of the protein . This non-active mutant protein was secreted essentially in an unprocessed form . The purified protein has been incubated with a variety of proteinases, and results indicate that cathepsins D and L, as well as mature cathepsin B itself, can produce a processed (single-chain) form of cathepsin B from this precursor . Amino-terminal sequencing of these processed forms has revealed that they are all elongated by a few residues with respect to the mature form found in vivo . The action of a combination of cathepsin B with dipeptidylpeptidase I produced a single-chain form of cathepsin B with the correct amino terminus . This work has also shown that the processing of procathepsin B to a single-chain form can be an autocatalytic process, in at least an intermolecular manner. J Biol Chem, 1992 Aug 5, 267(22), 15511 - 5 Genistein inhibits protein histidine kinase; Huang J et al.; Protein histidine kinase was prepared from whole cell extracts of the yeast, Saccharomyces cerevisiae . The enzyme was assayed using either histone H4 or a synthetic peptide corresponding to residues 70 to 102 of histone H4 as an in vitro substrate . With either substrate, both genistein and its solvent, dimethyl sulfoxide (Me2SO), inhibited protein histidine kinase . Me2SO alone gave a cooperative dose-response curve, with inhibition changing from almost zero below 10% Me2SO to 80% at 20% Me2SO with either substrate . Genistein gave a simple dose-response curve with 50% inhibition of protein histidine kinase at 110 microM genistein . In experiments with protein histidine kinase, genistein was a noncompetitive inhibitor with respect to ATP, histone H4 or the synthetic peptide, although, in the case of the synthetic peptide, the data were also consistent with competitive inhibition . These data gave Km values for both ATP and histone H4 of 15 microM, in satisfactory agreement with previously reported values (Huang, J., Wei, Y., Kim, Y., Osterberg, L., and Matthews, H . R . (1991) J . Biol . Chem . 266, 9023-9031) . The Km for the synthetic peptide was 80 microM . The KI values were 270 or 310 microM measured with histone H4 or the synthetic peptide as substrate, respectively . While these KI values are relatively high, relative to published KI values for genistein inhibition of protein tyrosine kinases, many reported experiments use genistein at concentrations where inhibition of protein histidine kinase occurs . It is possible that some of the observed effects of genistein in vivo may be due to inhibition of protein histidine kinase. J Biol Chem, 1992 Aug 5, 267(22), 15277 - 80 Phosphatidylethanolamine is the donor of the ethanolamine residue linking a glycosylphosphatidylinositol anchor to protein; Menon AK et al.; Numerous cell surface glycoproteins from eukaryotic organisms including African trypanosomes and budding yeast (Saccharomyces cerevisiae), are anchored to the lipid bilayer by a glycophospholipid, glycosylphosphatidylinositol, covalently linked to the carboxyl terminus of the protein via a phosphoethanolamine bridge . In this paper we describe metabolic labeling experiments aimed at identifying the biosynthetic origin of the ethanolamine residue in the phosphoethanolamine bridge . Using yeast mutants generated by disruption of the ethanolaminephosphotransferase (EPT1) and cholinephosphotransferase (CPT1) genes, we report data consistent with the proposal that the ethanolamine residue is derived from phosphatidylethanolamine. Biochemistry, 1992 Aug 4, 31(30), 6871 - 5 Minimum length of a sequence-specific DNA binding peptide; Talanian RV et al.; NMR experiments show that a stable complex can be formed between a 14-base-pair oligonucleotide and a disulfide-bonded dimer of a peptide containing 27 residues of the basic region of the yeast transcriptional activator GCN4; the complex is in slow exchange on the NMR time scale . In contrast, a nonspecific complex is in fast exchange on the NMR time scale . DNase I footprinting experiments show that dimers of peptides containing as few as 20 residues of GCN4 bind DNA with sequence specificity similar to that of the intact protein . Circular dichroism experiments suggest that specific binding involves only 15 residues, corresponding to residues 231-245 of GCN4, in an alpha-helical conformation . These results limit substantially the region of GCN4 involved in sequence-specific DNA contacts and provide a uniquely simple model for studying protein-DNA interactions in detail. J Gen Virol, 1992 Aug, 73 ( Pt 8), 2115 - 9 An N-proximal sequence of the alfalfa mosaic virus movement protein is necessary for association with cell walls in transgenic plants; Erny C et al.; We have made transgenic tobacco plants (Nicotiana tabacum, cv . Xanthi nc) expressing the movement protein (P3, 300 amino acids) of alfalfa mosaic virus (A1MV) and two N-terminally deleted proteins lacking respectively 12 and 77 amino acids of the P3 sequence (P3 delta{1-12} and P3 delta{1-77}) . The same proteins were expressed in recombinant yeast . By subcellular fractionation, the full-length P3 protein expressed by transgenic plants was found to be associated with cell walls as well as with cytoplasmic particulate material, as was the wild type movement protein expressed by A1MV-infected tobacco plants . P3 delta{1-12} behaved similarly but P3 delta{1-77} was found only in the cytoplasm . It thus appears that a polypeptide domain located between amino acids 13 and 77 of the P3 sequence is necessary for association of the protein with cell walls. J Cell Biol, 1992 Aug, 118(3), 607 - 17 A temperature-sensitive calmodulin mutant loses viability during mitosis; Davis TN; Although rare, a recessive temperature-sensitive calmodulin mutant has been isolated in Saccharomyces cerevisiae . The mutant carries two mutations in CMD1, isoleucine 100 is changed to asparagine and glutamic acid 104 is changed to valine . Neither mutation alone conferred temperature sensitivity . A single mutation that allowed production of an intact but defective protein was not identified . At the nonpermissive temperature, the temperature-sensitive mutant displayed multiple defects . Bud formation and growth was delayed, but this defect was not responsible for the temperature-sensitive lethality . Cells synchronized in G1 pro