Endocrinology, 1991 Oct, 129(4), 2263 - 5 Identification of a somatostatin-14-generating propeptide converting enzyme as a member of the kex2/furin/PC family; Mackin RB et al.; A propeptide converting enzyme capable of producing somatostatin-14 has been identified and partially characterized from anglerfish pancreatic islets . Results from N-terminal protein sequence analysis indicate that this enzyme is a member of the kex2/furin/PC family . This observation provides strong corroborative evidence that the kex2/furin/PC protease family is involved in propeptide conversion . Comparison of the obtained protein sequence with the cDNA sequence of mammalian PC2 also suggests that the active enzyme is derived from a precursor by cleavage at a site containing four consecutive basic amino acids. Gene, 1991 Sep 30, 106(1), 129 - 33 Promoter structure and expression of the 3-phosphoglycerate kinase-encoding gene (pgk1) of Trichoderma reesei; Vanhanen S et al.; Transcription of the 3-phosphoglycerate kinase (PGK)-encoding gene (pgk1) of Trichoderma reesei results in two transcripts due to two main transcription start points (tsp) which are differentially regulated during the growth cycle . The nucleotide sequence of the promoter reveals a number of putative regulatory elements present also in the PGK promoter of Saccharomyces cerevisiae: a 20-nt long sequence similar to the CTTCC-repeat region of the upstream activating sequence UAS, the eukaryotic heat-shock consensus sequence, HSE, and a putative eukaryotic cAMP regulatory sequence . The functionality of the putative HSE sequence was examined, but no clear effect could be seen on the total amount of pgk1 mRNA at elevated temperatures nor on transcription initiation from the upstream tsp, preceded by the HSE sequence. Nucleic Acids Res, 1991 Sep 25, 19(18), 4891 - 4 Proposed secondary structure of eukaryotic U14 snRNA; Shanab GM et al.; U14 snRNA is a small nuclear RNA that plays a role in the processing of eukaryotic ribosomal RNA . We have investigated the folded structure of this snRNA species using comparative analysis of evolutionarily diverse U14 snRNA primary sequences coupled with nuclease digestion analysis of mouse U14 snRNA . Covariant nucleotide analysis of aligned mouse, rat, human, and yeast U14 snRNA primary sequences suggested a basic folding pattern in which the 5' and 3' termini of all U14 snRNAs were base-paired . Subsequent digestion of mouse U14 snRNA with mung bean (single-strand-specific), T2 (single-strand-preferential), and V1 (double-strand-specific) nucleases defined the major and minor cleavage sites for each nuclease . This digestion data was then utilized in concert with the comparative sequence analysis of aligned U14 snRNA primary sequences to refine the secondary structure model suggested by computer-predicted folding . The proposed secondary structure of U14 snRNA is comprised of three major hairpin/helical regions which includes the helix of base-paired 5' and 3' termini . Strict and semiconservative covariation of specific base-pairs within two of the three major helices, as well as nucleotide changes that strengthen or extend base-paired regions, support this folded conformation as the evolutionary conserved secondary structure for U14 snRNA. J Biol Chem, 1991 Sep 25, 266(27), 18179 - 87 Transactivation functions facilitate the disruption of chromatin structure by estrogen receptor derivatives in vivo; Pham TA et al.; The activation of gene transcription by nuclear receptors is invariably associated with alterations in chromatin structure at hormone-responsive elements of target genes . To identify the molecular functions underlying receptor-mediated chromatin structure alterations we have evaluated the effects of DNA binding and transactivation of estrogen receptor derivatives on the promoter chromatin structure of estrogen-responsive reporter minichromosomes in Saccharomyces cerevisiae . We report here that the DNase I-hypersensitive chromatin structure at the promoter region is not simply a consequence of estrogen receptor binding to estrogen-responsive elements but is greatly enhanced by transactivation functions . These chromatin structure alterations are dependent on the presence of more than one estrogen-responsive element as well as downstream promoter sequences and appear to be correlated with transcriptional competence of the promoter . Our results imply that a disruption of chromatin structure at promoters is associated with the establishment of active transcription complexes . Since RNA polymerase cannot initiate transcription on nucleosomal DNA in vitro (Lorch, Y., Lapointe, J.W., and Kornberg, R.D . (1987) Cell 49, 203-210) this local disruption of chromatin structure may represent a nucleosome-free window, allowing initiation to occur in vivo. Biochim Biophys Acta, 1991 Sep 10, 1068(1), 52 - 60 Transport of phosphatidylinositol to rat hepatocyte plasma membrane catalyzed by phosphatidylinositol transfer protein; Borror CA et al.; Plasma membrane sheets were isolated from fresh rat liver and characterized by electron microscopy and marker enzyme activities . Plasma membrane sheets were used as the acceptor membrane in the measure of transport of phosphatidyl{3H}inositol from small unilamellar phospholipid vesicles or rough endoplasmic reticulum donor membranes . Catalysis of this transport was achieved with phosphatidylinositol transfer protein purified from rat or bovine brain . Assays were designed to separate donor and acceptor membranes by density gradient centrifugation . Rates of transfer were directly proportional to incubation time and the amounts of transfer protein and plasma membrane sheet added . These results are discussed in terms of cellular phosphatidylinositol metabolism, membrane phospholipid composition, and vesicle trafficking in rat hepatocytes. Biochemistry, 1991 Sep 17, 30(37), 9030 - 4 DNA-induced increase in the alpha-helical content of C/EBP and GCN4; O'Neil KT et al.; Leucine zipper proteins comprise a recently identified class of DNA binding proteins that contain a bipartite structural motif consisting of a "leucine zipper" dimerization domain and a segment rich in basic residues responsible for DNA interaction . Protein fragments encompassing the zipper plus basic region domains (bZip) have previously been used to determine the conformational and dynamic properties of this motif . In the absence of DNA, the coiled-coil portion is alpha-helical and dimeric, whereas the basic region is flexible and partially disordered . Addition of DNA containing a specific recognition sequence induces a fully helical conformation in the basic regions of these fragments . However, the question remained whether the same conformational change would be observed in native bZip proteins where the basic regions might be stabilized in an alpha-helical conformation even in the absence of DNA, through interactions with portions of the protein not included in the bZip motif . We have now examined the DNA-induced conformational transition for an intact bZip protein, GCN4, and for the bZip fragment of C/EBP with two enhancers that are differentially symmetric . Our results are consistent with the induced helical fork model wherein the basic regions are largely flexible in the absence of DNA and become fully helical in the presence of the specific DNA recognition sequence. Eur J Biochem, 1991 Sep 15, 200(3), 643 - 9 Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I; Aho S et al.; Cellulases from Trichoderma reesei form an enzyme group with a common structural organization . Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD) . To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced . Five mAb were obtained against CBHI, ten against CBHII and eight against EGI . The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae . Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results . Specific antibodies were detected against the core and the CBD epitopes for all three cellulases . Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein . To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts . The mAb were used to quantitative the corresponding enzymes in T . reesei culture medium . Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions . Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes. Cell, 1991 Sep 6, 66(5), 1015 - 26 Transcriptional activation of CLN1, CLN2, and a putative new G1 cyclin (HCS26) by SWI4, a positive regulator of G1-specific transcription; Ogas J et al.; SWI4 of budding yeast codes for a component of a transcription factor (cell cycle box factor, or CCBF) necessary for G1-specific expression of HO . We show that SWI4 is essential for haploid cell viability at high temperature and in a/alpha cells at all temperatures: SWI4-deficient cells arrest as large unbudded cells . Eight high copy number plasmids were identified that allow swi4- strains to grow under nonpermissive conditions . Two carry G1 cyclin genes, CLN1 and CLN2; another carries HCS26, coding for a putative cyclin, a/alpha swi4- mutants exhibit 3- to 20-fold reductions in the levels of CLN1, CLN2, and HCS26 transcripts . The requirement of SWI4 for transcription appears to be direct: each gene contains sites similar to the CCBF-binding site; CCBF binds to the upstream region of HCS26 . We propose that SWI4 participates in a positive feedback loop by which CLN1, CLN2, and possibly HCS26 promote their own transcription in G1. Biochemistry, 1991 Sep 3, 30(35), 8684 - 90 Constraints on amino acid substitutions in the N-terminal helix of cytochrome c explored by random mutagenesis; Auld DS et al.; The interaction of the N- and C-terminal helices is a hallmark of the cytochrome c family . Oligodeoxyribonucleotide-directed random mutagenesis within the gene encoding the C102T protein variant of Saccharomyces cerevisiae iso-1-cytochrome c was used to generate a library of mutations at the evolutionary invariant residues Gly-6 and Phe-10 in the N-terminal helix . Transformation of this library (contained on a low-copy-number yeast shuttle phagemid) into a yeast strain lacking a functional cytochrome c, followed by selection for cytochrome c function, reveals that 4-10% of the 400 possible amino acid substitutions are compatible with function . DNA sequence analysis of phagemids isolated from transformants exhibiting the functional phenotype elucidates the requirements for a stable helical interface . Basic residues are not tolerated at position 6 or 10 . There is a broad volume constraint for amino acids at position 6 . The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effect alone is sufficient to promote helical association . There are severe constraints that limit the combinations consistent with function, but the number of functionally consistent combinations observed exemplifies the plasticity of proteins. J Protozool, 1991 Sep-Oct, 38(5), 495 - 501 Small GTP-binding proteins associated with secretory vesicles of Paramecium; Peterson JB; GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion . One group of GTP-binding proteins, 20-30 kDa, is related to the product of the ras proto-oncogene . In Saccharomyces cerevisiae, ras-like GTP-binding proteins regulate vesicular traffic in secretion . The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis . Using {alpha-32P}GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22-31 kDa) in extracts of Paramecium tetraurelia . Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa) . The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts . Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts . This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis. Mol Cell Biol, 1991 Sep, 11(9), 4483 - 9 Assessment of the transcriptional activation potential of the HMG chromosomal proteins; Landsman D et al.; Chromosomal proteins HMG-14, HMG-17, and HMG-1 are among the most abundant, ubiquitous, and evolutionarily conserved nonhistone proteins . Analysis of their structure reveals features which are similar to those of certain transcription factors . The distribution of charged amino acid residues along the polypeptide chains is asymmetric: positive charges are clustered toward the N-terminal region, while negative charges are clustered toward the C-terminal region . The residues in the C-terminal region have the potential to form alpha helices with negatively charged surfaces . The abilities of HMG-14, -17, and -1 to function as transcriptional activators were studied in Saccharomyces cerevisiae cells expressing LexA-HMG fusion proteins (human HMG-14 and -17 and rat HMG-1) which bind to reporter molecules containing the beta-galactosidase gene downstream from a lexA operator . Fusion constructs expressing deletion mutants of HMG-14, -17, and -1 were also tested . Analysis of binding to the lexA operator with in vitro-synthesized fusion proteins shows that there are more sites for HMG-14, -17, and -1 binding than for LexA binding and that only the fusion constructs which contain the C-terminal, acidic domains of HMG-17 bind the lexA operator specifically . None of the LexA-HMG fusion protein constructs elevate the level of beta-galactosidase activity in transfected yeast cells . Thus, although HMG-14, -17, and -1 are structurally similar to acidic transcriptional activators, these chromosomal proteins do not function as activators in this test system. Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7749 - 53 Separation of transcriptional activation and silencing functions of the RAP1-encoded repressor/activator protein 1: isolation of viable mutants affecting both silencing and telomere length; Sussel L et al.; The repressor/activator protein 1 (RAP1) binds to the upstream activating sites of many genes, the silencer elements flanking the unexpressed mating-type loci HMR and HML, and the poly(C1-3A) sequences at telomeres, suggesting that RAP1 might have three distinct regulatory functions . To determine the in vivo role of RAP1 in repression of the HMR silent locus, we developed a screen to isolate rap1 mutants specifically defective in silencing . Fifteen independent mutants defining four different rap1 alleles were isolated . These alleles are defective to different extents in repression of an HMR locus containing a mutated, but fully functional, silencer . All four alleles are missense mutations in only three codons within a small C-terminal region of the gene . These silencing-defective mutants have no apparent growth defects, indicating that expression of the large number of essential genes that have promoters containing RAP1-binding sites is normal . A transcriptional silencing function of RAP1 can therefore be genetically separated from its presumably essential activation functions . Surprisingly, three of the silencing-defective rap1 alleles have significantly longer telomeres, suggesting that the function of RAP1 in both transcriptional silencing and telomere-length regulation may be related . In addition, we have demonstrated that increased gene dosage of either SIR1 or SIR4, two other factors required for silencing, suppresses the silencing defect of the rap1 mutants . The properties of SIR4 dosage suppression suggest that SIR4 protein may interact directly with RAP1 at silencers. Mol Cell Biol, 1991 Sep, 11(9), 4350 - 5 In situ distinction between steroid receptor binding and transactivation at a target gene; McDonnell DP et al.; We have developed a DNA interference assay in the yeast Saccharomyces cerevisiae that is designed to indicate the intracellular DNA-binding status of the estrogen receptor . The assay utilizes a promoter containing multiple copies of a GAL4-estrogen receptor binding sequence . This element is designed so that either an estrogen receptor or a GAL4 molecule, but not both, can occupy it simultaneously . The assay is extremely sensitive, and at concentrations of estrogen receptor below that required for maximal transcriptional activation of its target estrogen response element, a quantitative inhibition of GAL4-mediated transcription is seen . Inhibition occurs thought the disruption of complex cooperative interactions among the GAL4 molecules in this reporter . The data obtained from our experiments show that at low concentrations of receptor, hormone is required to promote DNA binding . Overexpression of receptor leads to occupation of the estrogen receptor element in the absence of ligand . In contrast, this latter receptor form will not activate transcription . Our results are consistent with a two-step process for receptor activation . Ligand first causes dissociation of receptor from an inhibitory complex within the cell and produces a DNA-binding form . Second, it converts receptor to a transcriptionally competent form . With use of this yeast model system, these two steps can be distinguished in situ. J Virol, 1991 Sep, 65(9), 4609 - 18 Utilization of DNA recombination for the two-step replacement of growth factor sequences in the vaccinia virus genome; Spyropoulos DD et al.; An efficient procedure for the generation of sequence-specific alterations of the vaccinia virus genome was demonstrated . Homologous DNA recombination within cells infected with vaccinia virus was used for the deletion or replacement of promoter sequences of the viral growth factor gene by a procedure comparable to transplacement in Saccharomyces cerevisiae . This DNA replacement procedure can potentially be used to generate any sequence alteration within the vaccinia virus genome . Deletion of growth factor promoter sequences resulted in a dramatic reduction in growth factor gene transcription and protein synthesis . Replacement of growth factor promoter sequences with promoter sequences of the strong constitutive 40-kDa gene resulted in an increase in gene transcription and protein synthesis and an altered temporal pattern of expression . Virus containing mutations in the growth factor gene demonstrated different plaque morphologies on cell culture monolayers. J Leukoc Biol, 1991 Sep, 50(3), 229 - 39 Kinetics of phagocytosis and phagosome-lysosome fusion in hamster lung and peritoneal macrophages; Bizal CL et al.; The time course of phagocytosis and phagosome-lysosome fusion (PLF) in lung and peritoneal macrophages (LMs and PMs) was measured . Lysosomes in unelicited hamster LMs and PMs were labeled with lucifer yellow . Macrophages then phagocytized heat-killed Saccharomyces cerevisiae (yeast) and were evaluated at several time points for the degree to which yeast particles were adherent vs . internalized and for the presence or absence of PLF as based on the presence or absence of lucifer yellow in yeast-containing phagosomes . A three-compartment model (adherent, ingested, fused) of independent phagocytosis and PLF was developed; the number of yeast particles in each compartment was counted, and rate constants for ingestion and fusion were determined . Comparison of rate constants showed that ingestion was significantly faster in PMs (0.047 +/- 0.005 min-1) than in LMs (0.016 +/- 0.005 min-1) (mean +/- pooled SEM; P less than 0.001) . Similarly, PLF was significantly faster in PMs (0.109 +/- 0.013 min-1) than in LMs (0.046 +/- 0.013 min-1) (P less than 0.003). Curr Genet, 1991 Sep, 20(4), 349 - 51 Sequence of the nuclear ATP synthase subunit 9 gene of Podospora anserina: lack of similarity to the mitochondrial genome; Ridder R et al.; The nuclear gene coding for the mitochondrial subunit 9 of the F0F1-ATP synthase complex was isolated from a genomic library of Podospora anserina . Nucleotide sequencing revealed an open reading frame capable to code for 144 amino acids including an amino-terminal pre-sequence of 63 amino acid residues for mitochondrial import of the pre-proteolipid . The P . anserina proteolipid shows extensive sequence identity with the corresponding gene products of the related filamentous fungi Neurospora crassa, Aspergillus nidulans and Aspergillus niger . In contrast to the situation in Saccharomyces cerevisiae, N . crassa and A . nidulans, no sequence similarity of the ATP synthase subunit 9 gene to the mitochondrial genome of P . anserina could be detected . Thus, in P . anserina this gene appears to be exclusively encoded by the nuclear genome. Plant Cell, 1991 Sep, 3(9), 1025 - 35 The tobacco luminal binding protein is encoded by a multigene family; Denecke J et al.; We have cloned cDNAs of the tobacco homolog of the luminal binding protein (BiP) that has been described in other higher eukaryotes . In contrast to the mammalian and yeast protein, tobacco BiP is encoded by a multigene family . The gene products of all the cloned members of this family contain a carboxy-terminal His-Asp-Glu-Leu peptide that may form the signal for retention in the endoplasmic reticulum . Analysis of expression patterns revealed that BiP transcripts are predominantly present in tissues with high rates of cell divisions, in secretory tissues, and in cells treated with tunicamycin . We also show that a chimeric gene containing the coding region of one of the tobacco BiP genes is able to complement a mutation in the Saccharomyces cerevisiae BiP gene. Med Lav, 1991 Sep-Oct, 82(5), 439 - 45 {The evaluation of the presumed mutagenic activity of barium nitrate}; Monaco M et al.; Barium nitrate, which is used in industry in the production of green signal lights, to remove gases from vacuum tubes, and in the production of barium oxide, was assayed to assess the possible mutagenic effects using both the Ames test (S . typhimurium TA 1535, TA 1537, TA 1538, TA 97a, TA 98, TA 100, TA 102c), with and without metabolic activation with the plate incorporation assay and pre-incubation assay methods, and using the mitotic crossing over test, the mitotic genic conversion test, and the retromutation test in Saccharomyces cerevisiae, D7 strain, with and without metabolic activation . In the experimental conditions of the study, at various gradually increasing concentrations, barium nitrate gave negative results. Arzneimittelforschung, 1991 Sep, 41(9), 891 - 4 Interactions between calcium entry blocking drugs and carbonic anhydrase; Botre C et al.; The interactions between some of the most common calcium entry blocker drugs (CEB) and the enzyme carbonic anhydrase (CA) are studied in the present work by an electroanalytical approach . The study comprises drugs belonging to the classes of phenylalkylamines, dihydropyridines, benzothiazepines and piperazines . The evaluation of the potential inhibitory power towards CA was performed either by measuring the speed of CO2 diffusion taking place from a buffered solution of NaHCO3, or by monitoring the metabolic activity of yeast cells . The results obtained according to both of these procedures have shown that verapamil and gallopamil are endowed with a relevant inhibitory power on CA catalytic activity, whereas all the other compounds, tested in the same experimental conditions, did not show any effect on CA activity. Jinrui Idengaku Zasshi, 1991 Sep, 36(3), 229 - 43 Single DNA marker generated by "YAC-Alu PCR" that is end-specific; Tashiro H et al.; A simple strategy for the rapid preparation of an end-specific linking-DNA probe from the YAC-human chromosome 21 DNA recombinant clone and the characterization of this single DNA probe are described . Synthetic oligodeoxynucleotide primers, based on the consensus Alu sequence, and the Sup4 DNA fragment in the YAC arms were used to amplify end-specific DNA sequences by the polymerase chain reaction (PCR) for screening of the linking YAC recombinant clones ("YAC-Alu PCR") . Nucleotide sequencing of the product of PCR from human genomic DNA in a YAC insert confirmed the boundary between the vector and the insert and the presence of the 3'-end Alu-like structure . The probe R1, prepared by "YAC-Alu PCR" amplification, was assigned to chromosome 21 by Southern hybridization of somatic cell hybrid DNAs . In situ hybridization allowed localization of the R1 DNA probe to the human chromosome 21q21-q22.1 region . Thus, this approach has significant advantages not only for isolation of a single DNA probe specific for human chromosome 21 but also for the screening of YAC linking recombinant clones for mapping of the human genome. J Mol Evol, 1991 Sep, 33(3), 216 - 25 Essential factors determining codon usage in ubiquitin genes; Mita K et al.; Ubiquitin is ubiquitous in all eukaryotes and its amino acid sequence shows extreme conservation . Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers . The nucleotide sequences coding for 13 ubiquitin genes from 11 species reported so far have been compiled and analyzed . The G + C content of codon third base reveals a positive linear correlation with the genome G + C content of the corresponding species . The slope strongly suggests that the overall G + C content of codons of polyubiquitin genes clearly reflects the genome G + C content by AT/GC substitutions at the codon third position . The G + C content of ubiquitin codon third base also shows a positive linear correlation with the overall G + C content of coding regions of compiled genes, indicating the codon choices among synonymous codons reflect the average codon usage pattern of corresponding species . On the other hand, the monoubiquitin gene, which is different from the polyubiquitin gene in gene organization, gene expression, and function of the encoding protein, shows a different codon usage pattern compared with that of the polyubiquitin gene . From comparisons of the levels of synonymous substitutions among ubiquitin repeats and the homology of the amino acid sequence of the tail of monomeric ubiquitin genes, we propose that the molecular evolution of ubiquitin genes occurred as follows: Plural primitive ubiquitin sequences were dispersed on genome in ancestral eukaryotes . Some of them situated in a particular environment fused with the tail sequence to produce monomeric ubiquitin genes that were maintained across species . After divergence of species, polyubiquitin genes were formed by duplication of the other primitive ubiquitin sequences on different chromosomes . Differences in the environments in which ubiquitin genes are embedded reflect the differences in codon choice and in gene expression pattern between poly- and monomeric ubiquitin genes. Trends Biotechnol, 1991 Sep, 9(9), 303 - 9 Retroelement particles as purification, presentation and targeting vehicles; Kingsman AJ et al.; The manipulation of retrotransposon and retroviral particles to carry biologically active molecules is becoming feasible . In addition, recent experiments suggest that it may be possible to target these engineered particles to specific cell types . This has implications for gene therapy, biological drug delivery and vaccine design. Biotechnol Prog, 1991 Sep-Oct, 7(5), 455 - 61 Evaluation of immunoglobulins from plant cells; Hein MB et al.; Expression of cDNA constructs encoding full-length mouse immunoglobulin chains with their native leader sequences or fusion constructs substituting the native leader with a pre-pro sequence derived from Saccharomyces cerevisiae yielded blocked N-termini on the gamma chain or the correct amino terminal sequence on the mature kappa chain . Lectin binding assays revealed that assembled immunoglobulin complexes contained a glycosylated heavy chain . The attached glycan was resistant to digestion by endoglycosidase H and its lectin binding pattern was distinguishable from that of the mammalian glycan . The results indicated processing of the immunoglobulin carbohydrate in the tobacco Golgi to yield a complex oligosaccharide . Secretion of antibody by protoplasts isolated from regenerated transgenic plants or from suspension callus cells was demonstrated by pulse-chase labeling experiments . When purified, the tobacco-produced antibody was found to possess the antigen binding and catalytic properties of the murine monoclonal antibody . Kinetic parameters (Km, Ki, Vmax, and kcat) of the tobacco-derived antibody were comparable to those of the mouse-derived antibody . The results in general show that the endomembrane system of tobacco cells possesses cognate mechanisms for the recognition of diverse leader sequences . These signals can be used to initiate the assembly, processing, and secretion by plant cells of complex foreign proteins. Biochem Biophys Res Commun, 1991 Aug 30, 179(1), 314 - 8 The primary structure of rat ribosomal protein S28; Chan YL et al.; The amino acid sequence of the rat 40S ribosomal subunit protein S28 was deduced from the sequence of nucleotides in a recombinant cDNA . Ribosomal protein S28 has 69 amino acids and has a molecular weight of 7,836 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 8-10 copies of the S28 gene . The mRNA for S28 is about 450 nucleotides in length . Rat S28 is homologous to Saccharomyces cerevisiae S33. Biochemistry, 1991 Aug 27, 30(34), 8287 - 95 GTP hydrolysis mechanisms in ras p21 and in the ras-GAP complex studied by fluorescence measurements on tryptophan mutants; Antonny B et al.; We have substituted leucine 56 or tyrosine 64 of p21 ras with a tryptophan . The intrinsic fluorescence of this tryptophan was used as an internal conformational probe for time-resolved biochemical studies of the ras protein . The slow intrinsic GTPase, GDP/GTP exchange induced by the SDC25 "exchange factor", and the fast GTP hydrolysis induced by GAP were studied . Tryptophan fluorescence of mutated ras is very sensitive to magnesium binding, GDP/GTP exchange, and GTP hydrolysis (changes in tyrosine fluorescence of wild-type ras are also observed but with a lower sensitivity) . Nucleotide affinities, exchange kinetics, and intrinsic GTPase rates of the mutated ras could be measured by this method and were found to be close to those of wild-type ras . The SDC25 gene product enhances GDP/GTP exchange in both mutants . In both mutants, a slow fluorescence change follows the binding of GTP gamma S; its kinetics are close to those of the intrinsic GTPase, suggesting that a slow conformational change precedes the GTPase and is the rate-limiting step, as proposed by Neal et al . (1990) (Proc . Natl . Acad . Sci . U.S.A . 87, 3562-3565) . GAP interacts with both mutant ras proteins and accelerates the GTPase of (L56W)ras but not that of (Y64W)ras, suggesting a role for tyrosine 64 in GAP-induced GTP hydrolysis . However, GAP does not accelerate the slow conformational change following GTP gamma S binding in either of the mutated ras proteins . This suggests that the fast GAP-induced catalysis of GTP hydrolysis that is observed with (L56W)ras bypasses the slow conformational change associated with the intrinsic GTPase and therefore might proceed by a different mechanism. Biochim Biophys Acta, 1991 Aug 26, 1067(2), 139 - 44 Changes of the compositional asymmetry of phospholipids associated to the increment in the membrane surface potential; Cerbon J et al.; The contribution of phosphatidylinositol (PI) and phosphatidylserine (PS) to the outer negative membrane surface potential was studied in normal, PS-rich and PI-rich yeast cells . Under carefully defined conditions; PS and PE were quantified by using the non-penetrating chemical probe trinitrobenzenesulfonic acid (TNBS) and the PI by degradation with a specific phospholipase C . An asymmetric distribution of phospholipids in the plasma membrane with more PS (80-90%), PI (70-85%) and PE (70-85%) in the inner leaflet was found . When compared to normal cells there were 3-times more PI and 2-times more PS in the outer leaflet of the PI-rich and PS-rich cells . These values are consistent with the two-times increased surface potential in these cells . Interestingly, the contribution of PI was around twice the contribution of PS to the surface potential in the cells studied . When compared to normal cells there was a two-times increased accessibility of PS to TNBS in the PI-rich cells and the accessibility of PI to phospholipase C was also increased two-times in the PS-rich cells, while the proportion of derivatized PE was similar in all cells . Taking into account that the amount of PI is similar in normal cells and PS-rich cells and the amount of PS is similar in PI-rich cells and normal cells, a charge driven transbilayer transport of acidic phospholipids can be proposed. J Biol Chem, 1991 Aug 25, 266(24), 15679 - 83 Identification of kex2-related proteases in chromaffin granules by partial amino acid sequence analysis; Christie DL et al.; We have characterized glycoprotein H (GpH) from bovine adrenal medullary chromaffin granules . Two-dimensional gel electrophoresis was used to purify GpH from an insoluble fraction obtained following extraction of chromaffin granule membranes with lithium diiodosalicylate . The GpH material was recovered from two-dimensional gel spots by concentration and recovery on a one-dimensional gel followed by electro-blotting to a poly(vinylidene difluoride) membrane . This material was subjected to in situ tryptic digestion . The released peptides were purified by microbore high performance liquid chromatography and sequenced . The peptide sequences revealed extensive similarity to the mammalian kex2/subtilisin-related proteases (PC2 and PC3) which have been characterized recently by molecular cloning and sequence analysis (Smeekens, S . P., and Steiner, D . F . (1990) J . Biol . Chem . 265, 2997-3000; Smeekens, S . P., Avruch, A . S., LaMendola, J., Chan, S . J., and Steiner, D . F . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 340-344) . The sequence similarity included regions that contain residues equivalent to the aspartic acid and histidine residues which are involved in the active site of the subtilisin family of serine proteases . The sequence data revealed the presence of tryptic peptides derived from both PC2 and PC3 . NH2-terminal sequence analysis of GpH gave two sequences which were aligned with residues 110-121 of PC2 and PC3 . It is likely that these sequences represent the mature form of PC2 and PC3 in chromaffin granules . These forms would be generated by cleavage at a site which is conserved in mammalian kex2-related enzymes and which would result in the release of approximately 80-residue propeptides . It was concluded that the spot identified as GpH by two-dimensional gel electrophoresis contains the bovine counterparts of both PC2 and PC3 . The direct identification of these components in chromaffin granules supports their role in the processing of protein precursors. Nature, 1991 Aug 22, 352(6337), 725 - 8 Transport of cationic amino acids by the mouse ecotropic retrovirus receptor; Kim JW et al.; Susceptibility of rodent cells to infection by ecotropic murine leukaemia viruses (MuLV) is determined by binding of the virus envelope to a membrane receptor that has multiple membrane-spanning domains . Cells infected by ecotropic MuLV synthesize envelope protein, gp70, which binds to this receptor, thereby preventing additional infections . The consequences of envelope-MuLV receptor binding for the infected host cell have not been directly determined, partly because the cellular function of the MuLV receptor protein is unknown . Here we report a coincidence in the positions of the first eight putative membrane-spanning domains found in the virus receptor and in two related proteins, the arginine and histidine permeases of Saccharomyces cerevisiae (Fig . 1), but not in any other proteins identified by computer-based sequence comparison of the GenBank data base . Xenopus oocytes injected with receptor-encoding messenger RNA show increased uptake of L-arginine, L-lysine and L-ornithine . The transport properties and the expression pattern of the virus receptor behave in ways previously attributed to y+, the principal transporter of cationic L-amino acids in mammalian cells. Anal Biochem, 1991 Aug 15, 197(1), 19 - 24 Measurement of biological thiols and disulfides by high-performance liquid chromatography and electrochemical detection of silver mercaptide formation; Kuninori T et al.; A rapid and sensitive method is described for the measurement of picomole levels of the biological thiols glutathione, cysteine, penicillamine, cysteamine, and ergothioneine by a combination of high-performance liquid chromatography and electrochemical detection (ECD) . The compounds were separated isocratically on a reversed-phase C18 column by ion-pair chromatography with a mobile phase containing 5 mM acetic acid and 2.5 mM sodium 1-octanesulfonate . After chromatographic separation, the eluate was combined with silver nitrate dissolved in ammonium nitrate buffer at pH 10.5 . A platinum disc electrode was used at -0.1 V vs Ag/AgCl to detect the amount of silver ions that had been consumed by the reaction with thiols . For measurement of disulfide, S-sulfonation with sodium sulfite or electroreduction were used to cleave the disulfide, and the thiol anions produced were detected by HPLC-ECD as for the reduced forms . The method was used to assay thiols and disulfides in biological materials. Eur J Biochem, 1991 Aug 15, 200(1), 35 - 41 Interference with myosin subfragment-1 binding by site-directed mutagenesis of actin; Aspenstrom P et al.; Three N-terminal double mutants of beta-actin expressed in the yeast Saccharomyces cerevisiae have been characterized with respect to DNase-I interaction, N-terminal post-translational modification, polymerizability and myosin subfragment-1 binding . The results strongly support earlier suggestions that the acidic residues at the N-terminus of actin are part of the myosin-binding site, while they seem to be of no importance for the other aspects of actin biochemistry tested . The suitability of this expression system for production of recombinant actin in general is discussed. Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 6911 - 5 Isolation of a cDNA for HSF2: evidence for two heat shock factor genes in humans; Schuetz TJ et al.; The heat shock response is transcriptionally regulated by an evolutionarily conserved protein termed heat shock factor (HSF) . We report the purification to homogeneity and the partial peptide sequence of HSF from HeLa cells . The peptide sequence was used to isolate a human cDNA with a predicted open reading frame that has homology to the DNA binding domains of both Saccharomyces cerevisiae and Drosophila HSFs . The cDNA directs the synthesis of a protein that binds to the heat shock element with specificity identical to HeLa HSF and stimulates transcription from a heat shock promoter . The expressed protein cross-reacts with anti-HSF antibodies . Surprisingly, however, this cDNA does not encode all of the peptides obtained from purified HeLa HSF . These peptides are encoded by a distinct human cDNA, HSF1, described by Rabindran et al . {Rabindran, S . K., Giorgi, G., Clos, J . & Wu, C . (1991) Proc . Natl . Acad . Sci . USA 88, 6906-6910.} It therefore appears that there is a human heat shock factor gene family and that at least two separate but related HSF proteins regulate the stress response in humans. Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 6901 - 5 The leucine zipper symmetrically positions the adjacent basic regions for specific DNA binding; Pu WT et al.; The bZIP structural motif present in several eukaryotic transcription factors is defined by the leucine zipper, a coiled-coil dimerization interface, and an adjacent basic region that directly interacts with DNA . To examine the functional importance of the highly conserved spacing between the leucine zipper and the basic region, we have analyzed the DNA-binding ability of yeast GCN4 proteins containing amino acid insertions between these two subdomains . Proteins containing a surprisingly wide variety of seven-amino acid insertions, but none containing two-, four-, or six-amino acid insertions, are functional . However, heterodimers between wild-type GCN4 and functional derivatives containing seven amino acid insertions are unable to bind DNA . These observations provide strong experimental support for several aspects of the scissors grip and induced fork models for DNA-binding by bZIP proteins . Specifically, they demonstrate that continuous alpha-helices symmetrically diverging from the leucine zipper correctly position the two basic regions for specific binding to abutting DNA half-sites . In addition, the results indicate that GCN4 homodimers are primarily responsible for transcriptional activation in yeast cells. FEMS Microbiol Lett, 1991 Aug 15, 66(3), 313 - 8 The nucleotide sequence of Schwanniomyces occidentalis alpha-amylase gene; Wu FM et al.; The nucleotide sequence of the coding and regulatory regions of the alpha-amylase-encoding gene (AMY) of Schwanniomyces occidentalis has been determined . This sequence contained an open reading frame of 512 codons, from which a protein with Mr of 53,723 could be predicted . A putative signal sequence encoding for 25 amino acids was proposed for the 5' end of the open reading frame . Regulatory sequences, such as TATA box, CCAAT box and a signal sequence for polyadenylation and transcription termination could be found in the flanking regions of AMY gene . The deduced amino acid sequence also contained four common conserved regions characteristic of other alpha-amylase proteins. Science, 1991 Aug 2, 253(5019), 557 - 60 Identification of profilin as a novel pollen allergen; IgE autoreactivity in sensitized individuals; Valenta R et al.; A complementary DNA encoding a pollen allergen from white birch (Betula verrucosa) that was isolated from a pollen complementary DNA library with serum immunoglobulin E from a birch pollen-allergic individual revealed significant sequence homology to profilins . The recombinant protein showed high affinity to poly-L-proline . Immunoglobulin E antibodies from allergic individuals bound to natural and recombinant birch profilin and also to human profilin . In addition, birch and human profilin induced histamine release from blood basophils of profilin-allergic individuals, but not of individuals sensitized to other plant allergens . The structural similarity of conserved proteins might therefore be responsible for maintaining immunoglobulin E antibody titers in type I allergy. Mol Cell Biol, 1991 Aug, 11(8), 4135 - 46 A negative regulator of HO transcription, SIN1 (SPT2), is a nonspecific DNA-binding protein related to HMG1; Kruger W et al.; The SIN1 gene was initially identified because mutations in SIN1 bypass the need for SWI1 to activate transcription of the yeast HO gene . We show here that transcription of HO in swi1 sin1 cells efficiently utilizes the normal start site . We have cloned SIN1 and found that it is identical to the previously identified gene SPT2, mutations in which allow transcription from certain mutated regulatory regions . The predicted SIN1/SPT2 protein has a distinctive amino acid composition (45% charged residues, 25% basic and 20% acidic) and has similarity to the mammalian HMG1 protein, a nonhistone component of chromatin . We show that SIN1 is concentrated in the nucleus and binds to DNA with little or no sequence specificity in vitro . It thus exhibits properties of an HMG protein . Addition of random DNA segments to a test promoter alters regulation by SIN1 in a manner similar to addition of a segment from the HO upstream region . Functional analysis of certain SIN1 mutations suggests that SIN1 may be part of a multiprotein complex . On the basis of these results, we propose that SIN1 is a nonhistone component of chromatin which creates the proper context for transcription . Because sin1 mutants exhibit increased loss of chromosome III, SIN1 may also play a role in fidelity of chromosome segregation. EMBO J, 1991 Aug, 10(8), 2305 - 10 Affinity purification of transcription factor IIA from HeLa cell nuclear extracts; Usuda Y et al.; One of the general transcription factors, TFIIA, was purified to homogeneity from HeLa cell nuclear extracts by yeast TFIID affinity chromatography . Human TFIIA had a molecular weight of approximately 38 kd . It was able to associate with the complex formed by yeast TFIID and the TATA elements of the adenovirus E4 and ML promoters, and the HSP70 promoter . The association extended the protected region on each TATA element by yeast TFIID from DNase I digestion . Affinity-purified TFIIA was also able to stimulate transcription from the E4 and ML promoters in in vitro reconstituted systems. Bioessays, 1991 Aug, 13(8), 413 - 7 YACs and the C . elegans genome; Coulson A et al.; During the past decade, it has become apparent that it is within our grasp to understand fully the development and functioning of complex organisms . It is widely accepted that this undertaking must include the elucidation of the genetic blueprint - the genome sequence - of a number of model organisms . As a prelude to the determination of these sequences, clone-based physical maps of the genomes of a number of multicellular animals and plants are being constructed . Yeast artificial chromosome (YAC) vectors, by virtue of their relatively unbiased cloning capabilities and capacity to carry large inserts, have come to play a central role in the construction of these maps . The application of YACs to the physical map of the Caenorhabditis elegans genome has enabled cosmid clone 'islands' to be linked together in an efficient manner . The long-range continuity has improved the linkage between the genetic and physical maps, greatly increasing its utility . Since the genome can be represented by a relatively small number of YACs, it has been possible to make replica filters of genomically ordered YACs available to the community at large. Vet Immunol Immunopathol, 1991 Aug, 29(1-2), 127 - 38 Serum opsonic activity and neutrophil phagocytic capacity of newborn lambs before and 24-36 h after colostrum uptake; Bernadina WE et al.; Neutrophil (PMN) counts, immune complex (IC) uptake by PMN, and serum opsonising activity for promoting yeast uptake were used to evaluate infection clearing capacity in 16 lambs prior to colostrum feeding (two lambs fed bovine colostrum, 14 suckled lambs) and at 2 days of age . At 2 days of age lambs had more circulating PMN than they had prior to colostrum uptake (P less than 0.01) . Colostrum feeding caused a significant increase in the percent of lamb PMN phagocytosing IC, although at Day 2 the percent phagocytosis was significantly lower (32.2%) than for adult controls (90%) . Yeast opsonophagocytosis was greater when 24-36 h post-feeding serum was the source of opsonin than when pre-feeding serum was used (P less than 0.001) . When adult serum was the opsonin, yeast opsonophagocytosis was approximately twice the phagocytosis mediated by 24-36 h post-feeding serum . The peripheral neutrocytosis and the enhancement of opsonophagocytosis generated by absorption of either ovine or bovine colostrum did not differ . The results of this study suggest that the parameters evaluated may be used for indicating the presence (or absence) of passively acquired protective immunity. Protein Eng, 1991 Aug, 4(6), 649 - 59 Automated modeling of coiled coils: application to the GCN4 dimerization region; Nilges M et al.; A novel approach for the modeling of coiled coils through molecular dynamics is described and applied to the dimerization region of the yeast transcriptional activator GCN4 . Initially, a model is created consisting of C alpha atoms only, representing an idealized coiled coil with infinite pitch . Human bias in the placing of the other atoms is reduced by an automatic building procedure using simulated annealing with simple geometric restraints . The resulting all-atom model is then allowed to relax during a short molecular dynamics run using an empirical energy function and weak restraints which reflect the coiled coil assumption . These models are then further refined using unrestrained molecular dynamics in water . In this report we test the model-building procedure on the known dimerization region of catabolyte gene activator protein (CAP), part of which forms a coiled coil, and we predict the structure of the coiled coil dimerization region (the 'leucine zipper' domain) of GCN4 . Several models are built, starting from different arrangements of the C alpha atoms in the initial structures . The final structures show similar crossing angles of the coiled coil, although this was not used as a restraint in the calculation . The leucines adopt a ladder-like conformation around the 2-fold axis of the coiled coil . A number of electrostatic interactions could be identified which may contribute to the stability of the helical structure of the monomers and of the dimer. Curr Genet, 1991 Aug, 20(3), 219 - 24 Cloning, sequencing and expression of the Schwanniomyces occidentalis NADP-dependent glutamate dehydrogenase gene; De Zoysa PA et al.; The cloned NADP-specific glutamate dehydrogenase (GDH) genes of Aspergillus nidulans (gdhA) and Neurospora crassa (am) have been shown to hybridize under reduced stringency conditions to genomic sequences of the yeast Schwanniomyces occidentalis . Using 5' and 3' gene-specific probes, a unique 5.1 kb BclI restriction fragment that encompasses the entire Schwanniomyces sequence has been identified . A recombinant clone bearing the unique BclI fragment has been isolated from a pool of enriched clones in the yeast/E . coli shuttle vector pWH5 by colony hybridization . The identity of the plasmid clone was confirmed by functional complementation of the Saccharomyces cerevisiae gdh-1 mutation . The nucleotide sequence of the Schw . occidentalis GDH gene, which consists of 1380 nucleotides in a continuous reading frame of 459 amino acids, has been determined . The predicted amino acid sequence shows considerable homology with GDH proteins from other fungi and significant homology with all other available GDH sequences. Biochem Med Metab Biol, 1991 Aug, 46(1), 75 - 84 Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates . A model for D-{2-3H}glucose metabolism in human erythrocytes; Liemans V et al.; When D-{2-3H}glucose 6-phosphate mixed with the unlabeled ester is converted to D-{1-3H}fructose 6-phosphate and 3HOH in the phosphoglucoisomerase reaction and then to D-{1-3H}fructose 1,6-bisphosphate in the phosphofructokinase reaction, the specific radioactivity of the latter metabolite and the production of 3HOH relative to the total generation of tritiated end products are both inversely related to the concentration of phosphofructokinase . In human erythrocytes, the modeling of D-{2-3H}glucose metabolism, based on the activity of phosphoglucoisomerase in cell homogenates and on the steady-state content of D-glucose 6-phosphate and D-fructose 6-phosphate in intact cells, indicates that the back-and-forth interconversion of these esters is about five-times higher than the net glycolytic flux . Yet, the production of 3HOH from D-{2-3H}glucose is about 20% lower than the net glycolytic flux, as judged from the production of 3HOH from D-{5-3H}glucose . Thus, an incomplete detriation of D-{2-3H}glucose is not incompatible with an extensive interconversion of hexose 6-phosphates in the reaction catalyzed by phosphoglucoisomerase. Genomics, 1991 Aug, 10(4), 1079 - 82 Assignment of two of the translation initiation factor-4E (EIF4EL1 and EIF4EL2) genes to human chromosomes 4 and 20; Pelletier J et al.; The eukaryotic translation initiation factor (eIF-4E) has recently been cloned from human, mouse, and yeast . This polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis . We have designed oligonucleotide primers to the 3' untranslated region of the gene encoding eIF-4E and specifically amplified the human gene in human/rodent somatic cell hybrids using the polymerase chain reaction . By this method, one of the human eIF-4E genes (EIF4EL1, eukaryotic translation initiation factor 4E-like 1) has been mapped to human chromosome 4qter-4p15 . In addition, we have localized a second eIF-4E gene (EIF4EL2, eukaryotic translation initiation factor 4E-like 2) to human chromosome 20 by Southern blot analysis of mapping panels established from human/rodent somatic cell hybrids. J Cell Biol, 1991 Aug, 114(4), 663 - 70 Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER; d'Enfert C et al.; Sec12p is an integral membrane protein required in vivo and in vitro for the formation of transport vesicles generated from the ER . Vesicle budding and protein transport from ER membranes containing normal levels of Sec12p is inhibited in vitro by addition of microsomes isolated from a Sec12p-overproducing strain . Inhibition is attributable to titration of a limiting cytosolic protein . This limitation is overcome by addition of a highly enriched fraction of soluble Sar1p, a small GTP-binding protein, shown previously to be essential for protein transport from the ER and whose gene has been shown to interact genetically with sec12 . Furthermore, Sar1p binding to isolated membranes is enhanced at elevated levels of Sec12p . Sar1p-Sec12p interaction may regulate the initiation of vesicle budding from the ER. J Invest Dermatol, 1991 Aug, 97(2), 249 - 53 Southern analysis of the 230-kD bullous pemphigoid antigen gene in normal humans, animals, and patients with junctional epidermolysis bullosa; Amagai M et al.; To begin to characterize the 230-kD bullous pemphigoid antigen (BPA) gene, we performed Southern analysis on genomic DNA with probes derived from 7 kb of cDNA that spans most of the coding region of this hemidesmosomal plaque protein . When hybridized to a 1-kb fragment of this BPA cDNA, normal human genomic DNA digested with EcoRI, BamHI, PstI, HindIII, or EcoRV showed only a single band, which was unique for each enzyme, indicating a single human gene for BPA . To determine if a related gene exists in animals, we used probes covering the full 7 kb of cDNA for Southern analysis of genomic DNA from various vertebrates . A related gene was detected in other mammals (monkey, cow, dog, rabbit, mouse, and rat) but not in chicken, frog, or fish . Under these same hybridization conditions a probe for human beta-actin could detect an actin gene in all these species . Furthermore, immunofluorescence showed that an antibody raised against portions of the 230-kD BPA bound to the epidermal basement membrane of mammals but not that of a bird or amphibian . Finally, because most patients with junctional epidermolysis bullosa (JEB) have defective hemidesmosomes in ultrastructure, and probably function, we analyzed genomic DNA from these patients . No restriction fragment length polymorphisms (RFLP) were detected when the DNA from 11 normals and 8 JEB patients (representing 16 possible defective genes) was digested with BamHI, EcoRI, or PstI and hybridized to any part of the cDNA . These findings indicate that 1) there is a single BPA gene in humans; 2) a closely related gene exists in other mammals but not birds, amphibia, or fish; and 3) gross abnormalities of the BPA gene are not characteristic of JEB patients. Semin Cell Biol, 1991 Aug, 2(4), 233 - 41 From growth to cell cycle control; Ducommun B; How does a quiescent cell decide to re-enter the cell cycle and start replicating its DNA? What controls cell proliferation? These are fundamental questions that have to be solved in order to understand the mechanisms of oncogenesis . Some recent data have provided clues about how signal transduction pathways may be connected to the cell cycle . A protein kinase cascade starting from the membrane growth factor receptor is thought to be involved in transducing extracellular stimuli to the master switches of the cell cycle control machinery . The recently identified extracellular-signal regulated kinases (ERKs) appear to play an important role in this pathway . Expression of cyclins, which are regulatory subunits of the universal cell cycle oscillator cdc2, may also be controlled through this kinase cascade . The products of tumor suppressor genes Rb and p53 also play an important role in regulating cell proliferation by interfering with the cell cycle pathway . Here, I will review and discuss the importance of these different new results. Genes Dev, 1991 Aug, 5(8), 1430 - 8 U1 snRNP can influence 3'-splice site selection as well as 5'-splice site selection; Goguel V et al.; To address the mechanisms that underlie splice site selection and splice site partner assignment, we analyzed the splicing of yeast (Saccharomyces cerevisiae) transcripts containing splice site region duplications . When the 5'-splice site region was duplicated, both sites were utilized to the same extent, indicating little or no influence of proximity on 5'-splice site choice . However, the effect of a 5'-mutant site was greatly enhanced by the presence of an adjacent wild-type site, and this effect was reversed by the restoration of base-pairing with U1 snRNA . 3'-Splice site choice was apparently influenced by proximity, as the site closest to the 5'-splice site was greatly preferred . Studies with strains carrying some U1 snRNA mutations showed an increase in the use of the distal 3'-splice site, indicating a role for U1 snRNP in 3'-splice site selection . The data are compared with those from mammalian splice site choice experiments and suggest mechanisms that influence differential splice site choice as well as exon skipping. Mol Cell Biol, 1991 Aug, 11(8), 4022 - 35 The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis; Kubelik AR et al.; The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A . Collins, H . Bertrand, R.J . LaPolla, and A.M . Lambowitz, Mol . Gen . Genet . 177:73-84, 1979) . We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs) . A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis . The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S . Kappy and R.L . Metzenberg, J . Bacteriol . 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene . The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants . The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant . The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth . The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis. Curr Opin Cell Biol, 1991 Aug, 3(4), 615 - 20 Proteins involved in vesicular transport and membrane fusion; Waters MG et al.; In the past year, new information about proteins involved in vesicular transport has been plentiful . Particularly noteworthy are the complementary findings that Sec17p is required for vesicle consumption in endoplasmic reticulum-to-Golgi transport in yeast and that an analogous activity in mammalian cells, termed SNAP, is required for transport from the cis to the medial cisternae of the Golgi apparatus. J Interferon Res, 1991 Aug, 11(4), 207 - 13 Signal transduction and transcriptional regulation of interferon-alpha-stimulated genes; Williams BR; Interferon-alpha (IFN-alpha) stimulates the expression of a number of genes in a pathway that begins with binding to specific high-affinity plasma membrane receptors . All IFN-alpha-stimulated genes cloned thus far are characterized by the presence of a DNA element, termed Interferon-Stimulated Response Element (ISRE), usually in the 5' upstream region of the genes . The ISRE binds a nuclear factor(s) following IFN-receptor triggered signal transduction and provides a convenient assay for the rapid phase of IFN-alpha signal transduction . This phase utilizes a phospholipase A2-generated second messenger which modulates ISRE-binding factors . Expression cloning has resulted in the identification of two specific ISRE-binding proteins that are candidates as signal recipients . Further advances in our understanding of the molecular mechanisms of IFN action may come through the use of yeast genetics . The human p68 kinase expressed in yeast has a growth inhibitory phenotype and provides a useful alternative system for analyzing components of the IFN-stimulated pathways. Curr Opin Cell Biol, 1991 Aug, 3(4), 702 - 9 Uniporters and anion antiporters; Hebert DN et al.; The past year has seen a flurry of activity in the area of protein-mediated hexose uniport . Topics of interest covered here include: structure-function studies; the interaction of glucose carriers with glycolytic enzymes; regulation of cell surface glucose-carrier concentrations by insulin and the signalling mechanisms involved; and the role of the glucose-carrier isoform, GLUT2, in pancreatic beta-cell glucose-dependent insulin secretion . Nucleoside uniport and Glu-Asp antiport are also discussed briefly. Curr Opin Cell Biol, 1991 Aug, 3(4), 585 - 91 Recycling of proteins between the endoplasmic reticulum and Golgi complex; Pelham HR; Several lines of investigation have shown that protein transport from the endoplasmic reticulum to the Golgi is more complex than previously imagined . Dynamic sorting of both membrane and soluble proteins is now believed to occur on the cis side of the Golgi apparatus with some proteins returning to the endoplasmic reticulum while others travel onwards. Eur J Cell Biol, 1991 Aug, 55(2), 312 - 7 Ubiquitin-encoding mRNA and mRNA recognized by genes encoding ubiquitin-conjugating enzymes are differentially expressed in division-synchronized cultures of Chlamydomonas reinhardtii; von Kampen J et al.; Cells of Chlamydomonas reinhardtii were synchronized by a light/dark illumination cycle of 14:10 h . All cells divided within the first 2 h of the dark period, the synchronization index was calculated as 0.916 . RNA was isolated every 2 h and hybridized to 32P-labeled probes encoding (i) ubiquitin from Chlamydomonas reinhardtii (UBM) and (ii) two different ubiquitin-conjugating enzymes from Saccharomyces cerevisiae (UBC2 and UBC3) . Sequences with homology to yeast UBC2 and UBC3, which are required for sporulation/DNA repair and G1/S transition in yeast, respectively, were detected in C . reinhardtii . In the algae, the relative abundance of transcripts encoding ubiquitin fusion proteins and UBC2 homologues is most prominent at the end of the light phase and throughout the dark . The highest amount of a putative polyubiquitin encoding transcript was detected during the dark phase of the synchronized culture . A high amount of this transcript is also present during the 8th hour of the light phase which may imply that the transcription of polyubiquitin gene is not only restricted to stress conditions in C . reinhardtii . The relative abundance of transcripts with homology to UBC3 is most pronounced within the light period corresponding to G1 and S phases of the C . reinhardtii cell cycle. Eur J Biochem, 1991 Aug 1, 199(3), 553 - 60 Characterisation of the electron self-exchange rates in hexametaphosphate-cytochrome-c aggregates measured using high-resolution 1H-NMR spectroscopy; Concar DW et al.; 1H-NMR spectroscopy has been used to measure the rate of unimolecular electron exchange between cytochrome c molecules in protein aggregates stabilised by the addition of sodium hexametaphosphate . The average intracomplex electron exchange rate is measured from line broadening of hyperfine-shifted resonances of ferricytochrome c in an equimolar mixture of reduced and oxidised protein . The line-broadening due to electron exchange is significantly greater than that due to protein aggregation and reaches a maximum value between 1-2 mol hexametaphosphate/mol protein . Significantly the exchange-induced broadening is a first-order process and is directly proportional to the size of the cytochrome c oligomer . From the temperature dependence of exchange broadening the activation enthalpy was estimated to be 75.8 kJ mol-1 whereas the activation entropy was 295 J mol-1 K-1 for a dimer of cytochrome c at a hexametaphosphate/protein molar ratio of 1 . Both activation parameters decrease in magnitude as the order of the cytochrome c oligomer increases . The rates of intracomplex electron exchange in Saccharomyces cerevisiae iso-2 and Candida krusei cytochromes c are lower than that of the horse protein, implying that primary sequence plays a fundamental part in determining the rate of exchange . The relevance of these observations is discussed in terms of the function of cytochrome c. FEBS Lett, 1991 Jul 29, 286(1-2), 225 - 8 Autoimmune antigen Ku is enriched on oligonucleotide columns distinct from those containing the octamer binding protein DNA consensus sequence; Quinn JP et al.; During purification of the AP1 complex from the T cell line MLA144 we enriched for a complex which bound to an oligonucleotide column containing the AP1 DNA consensus sequence and co-eluted with a fraction required for AP1 binding activity . This complex although co-eluting with AP1 binding activity had previously been determined to be non-specific in its DNA binding properties . Further investigation determined that the complex was a heterodimer of 85 and 70 kDa which was antigenically related to the autoimmune antigen Ku . It is important to be aware of the abundance and avidity of the Ku complex to bind oligonucleotide columns when purifying sequence specific binding proteins. J Biol Chem, 1991 Jul 25, 266(21), 13495 - 8 In vitro identification of a soluble protein:geranylgeranyl transferase from rat tissues; Joly A et al.; The gamma subunit of mammalian trimeric G proteins has been shown previously to be modified in vivo on a cysteine residue situated at the carboxyl-terminal sequence-Cys-Ala-Ile-Leu-COOH by a 20-carbon prenyl moiety geranylgeranyl (Mumby, S . M., Casey, P . J., Gilman, A . G., Gutowski, S., and Sternweis, P . C . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 5873-5877; Yamane, H . K., Farnsworth, C . C., Xie, H., Howald, W., Fung, B . K-K., Clarke, S., Gelb, M . H., and Glomset, J . A . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 5866-5872) . A biotinylated peptide acceptor comprising the eight carboxyl-terminal amino acids of the gamma subunit and tritiated geranylgeranyl diphosphate were utilized to monitor a protein:prenyl transferase activity in rat organs of varying age . The transferase activity was dependent upon the presence of divalent metal ions and maximal activity was achieved with either 1 mM ZnCl2 or 20 mM MgCl2 . Activity was shown to be linear with respect to time, protein concentration, substrate concentration, and the pH optimum was 7.5 . Protein:geranylgeranyl transferase activity was detected in all rat organs studied with the highest specific activity in brain S100 . No activity was detected in the membrane fraction . The specific activity in brain, liver, kidney, and heart increased with age . Radioactivity incorporated into the peptide acceptor from both {1-3H}geranylgeranyl diphosphate and {5-3H}mevalonate by 21-day-old rat brain S100 was released by treatment with methyl iodide, and in both cases, analysis of the cleavage products by reversed phase high performance liquid chromatography showed a peak of radioactivity co-eluting with a geranylgeraniol standard which was well resolved from a farnesol standard . This indicated that the rat brain S100 contained not only the protein:geranylgeranyl transferase but also geranylgeranyl synthetase activity and that the peptide acceptor was specific for geranylgeranyl under the conditions tested. Eur J Biochem, 1991 Jul 15, 199(2), 459 - 66 Molecular species of cardiolipin in relation to other mitochondrial phospholipids . Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier? Schlame M, Beyer K, Hayer-Hartl M, Klingenberg M. Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae . In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine . To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin {Beyer, K . and Klingenberg, M . (1985) Biochemistry 24, 3821-3826} . This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours . Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin . Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy . The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation . ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier . However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin . It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment. Nature, 1991 Jul 11, 352(6331), 165 - 8 v-Src and EJ Ras alleviate repression of c-Jun by a cell-specific inhibitor; Baichwal VR et al.; The AP-1 family of transcription factors, which includes the proto-oncogene products c-Jun and c-Fos, controls the stimulation of cellular genes by growth factors and the expression of oncogenes, including src and ras . Transcriptional activation by c-Jun is regulated by a cell-type-specific inhibitor that represses the activity of a transcriptional activation domain (A1) of c-Jun by operating through the adjacent negative regulatory region (delta) . Here we show that cotransfection of the src or ras oncogene enhances the transcriptional activity of a GAL4:c-Jun hybrid that includes the delta-A1 region of c-Jun, suggesting that the DNA binding and dimerization domain of c-Jun is not required for stimulation by Src or Ras . Moreover, induction of c-Jun activity by Src and Ras occurs in cell lines containing the c-Jun inhibitor but not in a cell line lacking it . The region in c-Jun essential for the stimulatory action of these oncogenes maps to domain A1 . These findings suggest the existence of signal-transduction pathways that result in an increase in transcriptional activity of c-Jun and AP-1 by disrupting the c-Jun:inhibitor interaction. Biochem Pharmacol, 1991 Jul 5, 42(2), 295 - 302 Inhibition of long-chain acyl-CoA synthetase by the peroxisome proliferator perfluorodecanoic acid in rat hepatocytes; Vanden Heuvel JP et al.; Perfluorodecanoic acid (PFDA) is a potent peroxisome proliferator and is known to affect hepatic lipid metabolism in rats . The effects of PFDA on fatty acid utilization were examined in isolated rat hepatocyte suspensions and in rat liver mitochondria and microsomes . PFDA inhibited the oxidation of palmitic acid but not octanoic or pyruvic acids when hepatocytes were incubated with 1 mM PFDA . At this PFDA concentration the esterification of palmitic acid into triacylglycerols was also reduced . The activity of long-chain acyl-CoA synthetase (ACS), an enzyme essential for both oxidation and esterification of fatty acids, was reduced in hepatocytes incubated with 1 mM PFDA . Carnitine palmitoyltransferase (CPT), an important enzyme for the oxidation of long-chain fatty acids, was not altered in hepatocytes incubated with this PFDA concentration . In rat liver mitochondria, palmitate oxidation and ACS activity were reduced significantly (P less than 0.01) at a PFDA concentration that had no effect on CPT activity . The inhibition of ACS by PFDA was similar in liver mitochondria and microsome preparations . In mitochondria incubated with PFDA, the inhibition of ACS appears to be noncompetitive for the substrates palmitic acid and CoA . However, the ACS inhibition by PFDA appeared to be competitive for the ATP binding site of the enzyme . Several chain length perfluorinated fatty acids were examined for their ability to inhibit mitochondrial ACS . Short-chain perfluorinated fatty acids (perfluoroproprionic and -butyric acid) did not inhibit ACS activity . However, medium-chain perfluorinated acids (perfluorooctanoic, -ananoic and -decanoic acid) were found to be potent inhibitors of ACS in isolated mitochondria . Whether ACS inhibition is causally related to PFDA-induced peroxisome proliferation and altered lipid metabolism seen in vivo is yet to be determined. Biochemistry, 1991 Jul 2, 30(26), 6626 - 32 Effect of cysteine replacements at positions 13 and 50 on metallothionein structure; Cismowski MJ et al.; Recombinant wild-type and mutant Chinese hamster metallothioneins, purified from the yeast Saccharomyces cerevisiae, were analyzed for their chemical and spectroscopic properties . The mutant proteins contain cysteine to tyrosine replacements at positions 13 and 50 . Wild-type and mutant metallothioneins, in their cadmium-bound forms, all showed characteristic ultraviolet absorption spectra with shoulders at 245-250 nm due to cadmium-thiolate charge transfer . Upon acidification, these absorption shoulders were abolished . In all cases, two distinct titrations were seen, presumably corresponding to two independent cadmium binding domains in each of the proteins . Analysis of domain structures was performed both with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) and with the protease subtilisin . These studies indicated that both mutations affected domain structure by disrupting the normally tight protein clusters . Circular dichroism spectra obtained for wild-type and mutant metallothioneins showed unique structural rearrangements in mutants containing a cysteine-50 to tyrosine alteration . These data, along with previously obtained 113Cd NMR data, were incorporated into a model which can account for the in vivo and in vitro properties of these mutant proteins. Genes Dev, 1991 Jul, 5(7), 1285 - 98 Facilitated binding of GAL4 and heat shock factor to nucleosomal templates: differential function of DNA-binding domains; Taylor IC et al.; Regulatory factors must contend with chromatin structure to function . Although nucleosome structure and position on promoters can be important in determining factor access, the intrinsic ability of factors to bind to nucleosomal DNA might also play an essential regulatory role . We have used templates where nucleosomes were either randomly positioned or rotationally phased to demonstrate that two transcription factors, heat shock factor (HSF) and GAL4, differ significantly in their ability to bind to nucleosomes . GAL4 was able to bind to nucleosomal templates . Surprisingly, in contrast to its behavior on naked DNA, GAL4 bound better to multiple GAL4 sites than to a single GAL4 site on these templates . HSF alone was not able to bind to nucleosomal templates . HSF was able to bind to nucleosomal templates, however, when the TATA-binding factor TFIID was present . Consequently, binding to nucleosomal templates could be facilitated by adjacent binding of the same protein in the case of GAL4 but required binding of a second protein in the case of HSF . Taken together, these data demonstrate that regulatory factors differ in their inherent ability to bind to nucleosomal templates . These differences are likely to be important to the function of these factors in vivo. Genes Dev, 1991 Jul, 5(7), 1183 - 90 Cell cycle-specific expression of the SWI4 transcription factor is required for the cell cycle regulation of HO transcription; Breeden L et al.; Expression of the HO endonuclease triggers mating-type switching in Saccharomyces cerevisiae . Transcription of the HO gene is start-dependent and restricted to the late G1/early S phase of haploid mother cells . The HO promoter contains 10 copies of a cell cycle-regulated upstream activation sequence, which is activated by SWI4 and SWI6 . SWI4 mRNA levels vary at least 10-fold throughout the cell cycle and rise sharply just before the rise in HO mRNA levels . Constitutive synthesis of SWI4 mRNA leads to constitutive synthesis of HO mRNA . These data suggest that the cell cycle regulation of SWI4 mRNA is required for the tight cell cycle regulation of HO transcription . High-level constitutive synthesis of SWI4 also suppresses swi5 and swi6 mutations, suggesting that SWI4 is the predominant activator of HO transcription and that mutations in negative regulators of SWI4 could be isolated as suppressors of swi6 mutations . One recessive suppressor of swi6 (ssx1-1) that allowed high-level expression of SWI4 during alpha-factor arrest and constitutive expression of both SWI4 and HO after release from the arrest was isolated . This result suggests that SSX1 has a negative regulatory role in the cell-cycle regulation of SWI4 mRNA accumulation. Proc Natl Acad Sci U S A, 1991 Jul 1, 88(13), 5518 - 22 Molecular sequence accuracy and the analysis of protein coding regions; States DJ et al.; Molecular sequences, like all experimental data, have finite error rates . The impact of errors on the information content of molecular sequence data is dependent on the analytic paradigm used to interpret the data . We studied the impact of nucleic acid sequence errors on the ability to align predicted amino acid sequences with the sequences of related proteins . We found that with a simultaneous translation and alignment algorithm, identification of sequence homologies is resilient to the introduction of random errors . Proteins with greater than 30% sequence identity can be reliably recognized even in the presence of 1% frameshifting (insertion or deletion) error rates and 5% base substitution rates . Incorporation of prior knowledge about the location and characteristics of errors improves tolerance to error of amino acid sequence alignments . Similarly, inclusion of prior knowledge of biased codon utilization by yeast (Saccharomyces cerevisiae) allows reliable detection of correct reading frames in yeast sequences even in the presence of 5% substitution and 1% frameshift errors. Radiobiologiia, 1991 Jul-Aug, 31(4), 571 - 7 {A molecular version of a probability model of radiation injury of cells}; Glazunov AV et al.; A molecular version of cell inactivation probability model has been proposed . Formal parameters of the model are interpreted . It has been shown that yeast cell inactivation regularities can be explained by DNA double-strand breaks processing during the postirradiation cell division. Lipids, 1991 Jul, 26(7), 495 - 9 Effect of acylation stimulating protein on the triacylglycerol synthetic pathway of human adipose tissue; Yasruel Z et al.; Acylation stimulating protein (ASP) is a 14 kDa plasma protein which causes in vitro triacylglycerol synthesis in human adipocytes and fibroblasts to increase substantially . ASP was found to stimulate human adipose tissue microsomal glycerophosphate acyltransferase and diacylglycerol acyltransferase activities by 23% and 90%, respectively . However, phosphatidate phosphohydrolase activity showed no increase in activity, nor did microsomal acyl-CoA synthetase activity . Moreover, ASP did not decrease the apparent Km of diacylglycerol acyltransferase (DGAT), but rather increased its apparent Vmax suggesting direct interaction of ASP with DGAT. Arch Biochem Biophys, 1991 Jul, 288(1), 22 - 8 Characterization and molecular properties of 2-oxoglutarate decarboxylase from Euglena gracilis; Shigeoka S et al.; 2-Oxoglutarate decarboxylase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis . It had a molecular weight of 250,000 and consisted of four identical subunits of molecular weight 62,000 . The enzyme was specific for 2-oxoglutarate, but not for other 2-oxo acids such as pyruvate and oxalacetate . Thiamin pyrophosphate and MgCl2 were required for maximum activity . The Km values of the enzyme for 2-oxoglutarate, thiamin pyrophosphate, and MgCl2 were 330, 56, and 93 microM, respectively . 2-Mercaptoethanol and NADP+ augmented significantly the enzyme activity . The amino acid composition and amino acid sequence of the amino-terminal region of 2-oxoglutarate decarboxylase were determined . On ouchterlony double-immunodiffusion gels, the anti-2-oxoglutarate decarboxylase antibody gave sharp precipitin lines against the mitochondrial fraction of E . gracilis and the purified 2-oxoglutarate decarboxylase, but not against pyruvate decarboxylase from Saccharomyces cerevisiae . On Immunoblots of the crude extract of Euglena, the antibody recognized two polypeptides whose molecular weights were 62,000 and 65,000, respectively . The polypeptide with the molecular weight of 62,000 was found only in mitochondrial fractions . In vitro translation of Euglena polyadenylated RNA in a cell-free rabbit reticulocyte lysate system explained the formation of a single polypeptide with a molecular weight of 65,000, suggesting that a putative precursor of 2-oxoglutarate decarboxylase which is about 3000 larger than the subunit of the mature enzyme is synthesized in Euglena cells. Genomics, 1991 Jul, 10(3), 661 - 5 Rapid screening of a YAC library by pulsed-field gel Southern blot analysis of pooled YAC clones; Mendez MJ et al.; A new method for screening of YAC libraries is described . Individual YACs were pooled into groups of 384 clones and prepared as samples suitable for pulsed-field gel electrophoresis . A five hit human YAC library (Brownstein et al., 1989) containing approximately 60,000 clones was condensed into 150 such pools and chromosomal DNAs in each sample were separated on three pulsed field gels containing 50 samples each . Southern blots prepared from these gels were hybridized with probes of interest to identify pools containing homologous YACs . Further purification was performed using standard colony hybridization procedures . Twenty-one probes used thus far have identified 47 positive pools and corresponding YACs have been purified from 28 of these . Some significant advantages of this method include avoidance of DNA sequence analysis and primer generation prior to YAC screening and the ability to handle the entire library on three filters . The screening approach described here permits rapid isolation of YACs corresponding to unsequenced loci and will accelerate establishment of YAC contigs for large chromosomal segments. J Antibiot (Tokyo), 1991 Jul, 44(7), 716 - 22 Isolation and characterization of new allosamidins; Nishimoto Y et al.; Three of new allosamidins, termed glucoallosamidins A (5), B (6) and methyl-N-demethyl-allosamidin (4), were isolated as yeast chitinase inhibitors from the mycelium of Streptomyces sp . SA-684. Ann Hematol, 1991 Jul, 63(1), 45 - 8 Effects of suramin on phagocytes in vitro; Sipka S et al.; In the therapeutically important range (100-200 micrograms/ml), suramin was found to increase the phagocytic activity of human monocytes (measured by the uptake of Saccharomyces cerevisiae and sensitized sheep red blood cells) in vitro . Suramin itself was a chemotactic signal for monocytes and increased the chemiluminescence of neutrophil granulocytes . Suramin seems to act via the ATP-binding P2 receptors of human phagocytes. Mol Pharmacol, 1991 Jul, 40(1), 69 - 79 Separation of human liver microsomal tolbutamide hydroxylase and (S)-mephenytoin 4'-hydroxylase cytochrome P-450 enzymes; Srivastava PK et al.; Purification and immunoinhibition studies have suggested that the hydroxylations of (S)-mephenytoin and tolbutamide are catalyzed by rather similar forms of human liver cytochrome P-450 (P-450) . However, the two activities are not well correlated in vivo; sulfaphenzaole is a selective inhibitor of tolbutamide hydroxylation, and expression of P-450 2C10 cDNA in yeast yields a protein that hydroxylates tolbutamide but not (S)-mephenytoin . The P-450 2C8, 2C9, and 2C10 cDNAs have all been isolated, and their sequences are known to be closely related (greater than 80%) . Highly sensitive radiochromatographic assays were set up, and tolbutamide and (S)-mephenytoin hydroxylation activities were monitored during chromatography of human liver microsomal fractions . The two activities could be separated by chromatography, and proteins were purified to near-homogeneity that catalyzed either tolbutamide hydroxylation (P-450TB) or (S)-mephenytoin 4'-hydroxylation (P-450MP) but not both . Approximately 16 and 45% of the primary sequences of P-450TB and P-450MP, respectively, were determined by analysis of the tryptic peptides . The sequences of the P-450TB peptides matched those predicted by the P-450 2C9 and 2C10 cDNAs exactly; the P-450MP peptides showed two mismatches (of 219 residues) with the P-450 2C10 sequence . Proteins with the P-450 2C10 and P-450 2C9 sequences were expressed in Saccharomyces cerevisiae grown under different nutritional conditions, and both were found to be proficient in the hydroxylation of tolbutamide but not (S)-mephenytoin . We conclude, on the basis of this and previous work, that 1) P-450s 2C8, 2C9, and 2C10 all catalyze the hydroxylation of tolbutamide and 2) the protein involved in polymorphic (S)-mephenytoin 4'-hydroxylation is closely related to but distinct from P-450 2C8, 2C9, and 2C10. Biochem J, 1991 Jul 1, 277 ( Pt 1), 17 - 21 Kinetic studies on protoporphyrinogen oxidase inhibition by diphenyl ether herbicides; Camadro JM et al.; Diphenyl ethers (DPEs) and related herbicides are powerful inhibitors of protoporphyrinogen oxidase, an enzyme involved in the biosynthesis of haems and chlorophylls . The inhibition kinetics of protoporphyrinogen oxidase of various origins by four DPEs, (methyl)-5-{2-chloro-4-(trifluoromethyl)phenoxy}-2-nitrobenzoic acid (acifluorfen and its methyl ester, acifluorfen-methyl), methyl-5-{2-chloro-4-(trifluoromethyl) phenoxy}-2-chlorobenzoate (LS 820340) and methyl-5-{2-chloro-5-(trifluoromethyl)phenoxy}-2-nitrobenzoic acid (RH 5348), were studied . The inhibitions of the enzymes from maize (Zea mays) mitochondrial and etiochloroplastic membranes and mouse liver mitochondrial membranes were competitive with respect to the substrate, protoporphyrinogen IX, for all four molecules . The relative efficiencies of the inhibitors were: acifluorfen-methyl greater than LS 820340 much greater than RH 5348 greater than or equal to acifluorfen . The four molecules showed mixed-competitive type inhibition of the enzyme from yeast mitochondria where acifluorfen, a carboxylic acid, had the same inhibitory activity as its methyl ester, acifluorfen-methyl, and both were much greater than that of LS 820340 and RH 5348. Plant Cell, 1991 Jul, 3(7), 695 - 708 Different legumin protein domains act as vacuolar targeting signals; Saalbach G et al.; Legumin subunits are synthesized as precursor polypeptides and are transported into protein storage vacuoles in field bean cotyledons . We expressed a legumin subunit in yeast and found that in these cells it is also transported into the vacuoles . To elucidate vacuolar targeting information, we constructed gene fusions of different legumin propolypeptide segments with either yeast invertase or chloramphenicol acetyltransferase as reporters for analysis in yeast or plant cells, respectively . In yeast, increasing the length of the amino-terminal segment increased the portion of invertase directed to the vacuole . Only the complete legumin alpha chain (281 amino acids) directed over 90% to the vacuole . A short carboxy-terminal legumin segment (76 amino acids) fused to the carboxy terminus of invertase also efficiently targeted this fusion product to yeast vacuoles . With amino-terminal legumin-chloramphenicol acetyltransferase fusions expressed in tobacco seeds, efficient vacuolar targeting was obtained only with the complete alpha chain . We conclude that legumin contains multiple targeting information, probably formed by higher structures of relatively long peptide sequences. J Cell Sci, 1991 Jul, 99 ( Pt 3), 669 - 74 Conserved structural motifs in cyclins identified by sequence analysis; Nugent JH et al.; Cyclins, as regulatory subunits of the ubiquitous p34cdc2 protein kinase, act as key controlling elements of the eukaryotic cell cycle . We have examined published sequences of A- and B-type cyclins for both amino acid and secondary structure homologies . In particular, we sought regions of homology outside the recognised area of sequence conservation known as the "cyclin box', as well as conserved features predicted to lie at the protein surface . Our analysis demonstrates the existence of a number of islands of homology outside the cyclin box, and indicates candidate residues for phosphorylation . One of these, a motif containing the amino acids SPXXXE/D is also present in fission yeast p13suc1, another protein known to interact with p34cdc2 . This motif may define a possible p34cdc2 binding or phosphorylation site . A database search revealed that the CDC25 and SCD25 genes of the budding yeast Saccharomyces cerevisiae also contain some of the newly identified motifs, perhaps indicating a common regulatory or degradation pathway. Mol Biol (Mosk), 1991 Jul-Aug, 25(4), 1071 - 9 {Fractionation of eukaryotic DNA in a pulsating electrical field . I . Detection and properties of discrete DNA fragments}; Solovian VT et al.; The fractionation of eukaryotic DNA by field inversion gel electrophoresis results in the appearance of discrete DNA-fragments . The set of these fragments is similar to that of different eukaryotic representatives and consists of various chromosomal DNAs, unified by size . The physical properties of DNA-fragments suggest that they can form multimeric structures due to the presence of sticky ends flanking discrete fragments . We suppose that the set of discrete DNA-fragments results in a specific cleavage of intact nuclear DNA and can reflect different levels of chromatin structural organization. J Protozool, 1991 Jul-Aug, 38(4), 427 - 37 Glucan synthesis in Pneumocystis carinii; Williams DJ et al.; Rat-derived Pneumocystis carinii lysed with sodium deoxycholate catalysed the incorporation of uridine diphosphoglucose into an insoluble polymer . This enzyme activity was present in both the pellet and the supernatant when the P . carinii preparations were centrifuged . The polymer whose production was catalysed by the supernatant was examined by mass spectrometry and found to be an alpha 1----4 glucan, which is either unbranched or has relatively few branches . Polymer formation was completely inhibited by the addition of alpha amyloglucohydrolase to the supernatant . Polymer formation in the pellet of deoxycholate P . carinii preparations, unlike that in the supernatant, was partially resistant to alpha amyloglucohydrolase . The soluble glucan synthase activity in the supernatant was stable for more than 30 h at room temperature and was approximately 50 times more active on a cell-to-cell basis than the supernatant from deoxycholate preparations of the yeast Saccharomyces cerevisae. Acta Chem Scand, 1991 Jul, 45(6), 593 - 603 Coordination geometry of cadmium at the zinc and copper sites of superoxide dismutases: a study using perturbed angular correlation of gamma-rays from excited 111Cd; Bauer R et al.; 111Cd time-differential perturbed gamma-gamma angular correlation (PAC) has been used to investigate the Zn site in yeast and bovine copper and zinc-containing superoxide dismutases by substitution of the zinc ions with excited 111Cd(2+) ions . The PAC spectra obtained from the enzymes in aqueous solution reveal a single coordination geometry of 111Cd(2+) showing that the coordination of 111Cd(2+) to the Zn site in the two subunits is identical . Furthermore, the PAC spectra of the yeast and bovine enzymes show that the Zn sites are very similar in the two enzymes . The PAC experiments show a clear difference depending on whether the copper ion is in the oxidized or the reduced state . In the latter case the results resemble those obtained for derivatives with no metal ion at the Cu site . Hence the coordination geometry of the Zn site in these two situations must be similar, and it is very unlikely that the imidazole ring of His61 bridges the two metal ions in the reduced enzyme . The PAC spectrum of 111Cd(2+) ions at the Zn site with copper(II) ions at the Cu site is in agreement with that predicted by applying the angular overlap model (AOM) to the known crystal structure of the bovine enzyme, with known nuclear quadrupole interactions for the ligands involved . Furthermore results from experiments with copper in the reduced state show that reduction of the copper ion causes a significant change at the Zn site . An explanation for this conformational change has been proposed by computer modelling . The PAC experiments also show that it is possible to incorporate cadmium ions into the Cu site in the absence of copper ions, and the result has also been interpreted in terms of the AOM. Genetika, 1991 Jul, 27(7), 1174 - 9 {Genetic recombination and steroid metabolism in Drosophila}; Kamilova TA et al.; A double-species ecologo-genetical model, including Drosophila and yeast, has been used as a new methodological instrument for investigation of the physiological mechanism of recombination . Incubation of Drosophila females in the medium containing yeast of the strain mutant for ergosterol synthesis leads to suppression of temperature-induced crossing over . The mass-spectrum analysis of steroid fraction from Drosophila females has shown that incubation of the yeast medium without ergosterol results in arrest of ecdysterone synthesis . These data are explained by the absence of ecdysterone synthesis precursor in the fly organism . The endocrinal control of crossing over is discussed in the light of hormonal regulation of meiosis. Eur J Biochem, 1991 Jul 1, 199(1), 123 - 31 Core I protein of bovine ubiquinol-cytochrome-c reductase; an additional member of the mitochondrial-protein-processing family . Cloning of bovine core I and core II cDNAs and primary structure of the proteins; Gencic S et al.; Core I and core II proteins are the largest nuclear-encoded subunits of the mitochondrial ubiquinol-cytochrome-c reductase (bc1 complex) lacking redox prosthetic groups . cDNA clones of the two bovine core proteins have been isolated by the screening of lambda ZAP cDNA libraries either with an oligonucleotide probe based on the sequence of an internal peptide or with a polymerase-chain-reaction-amplified fragment . The core I precursor protein consists of 362 amino acids with a 34-amino-acid presequence typical for mitochondrial targeting signals . The mature protein migrates in SDS/polyacrylamide gels with an apparent molecular mass of 47 kDa, which does not correspond to the actual molecular mass of the protein of 35.8 kDa deduced from the cDNA sequence . The core II precursor protein is composed of 453 amino acids having a 14-amino-acid presequence as a targeting sequence . Comparison of the core I amino acid sequence with sequences of the newly discovered protein family {Schulte, U., Arretz, M., Schneider, H., Tropschug, M., Wachter E., Neupert, W . & Weiss, H . (1989) Nature 339, 147 - 149} comprising the processing enhancing protein (PEP), matrix processing peptidase (MPP), and core I and II proteins from Neurospora crassa and Saccharomyces cerevisiae, revealed a remarkable identity of 39% and a high similarity of 49% to N . crassa PEP, which in this fungus is identical to core I . Core II protein is only a distant relative of this protein family . Based on these sequence comparisons and data obtained by genomic Southern blots, we anticipate that the bovine core I subunit, like the N . crassa core I protein, is bifunctional, being responsible for the maintenance of electron transport and processing of proteins during their import into the mitochondrial matrix . The analysis of the primary structure of the two core proteins completes the set of primary structures of all subunits of bovine ubiquinol-cytochrome-c reductase. J Pharm Pharmacol, 1991 Jul, 43(7), 522 - 4 Toxicity of amphotericin B emulsion to cultured canine kidney cell monolayers; Lamb KA et al.; The effect of amphotericin B in an emulsion formulation on the integrity of monolayers of kidney cells has been studied . Whereas a conventional solubilized amphotericin formulation (Fungizone, Squibb) caused a loss in monolayer integrity at concentrations above 1 microgram mL-1, the emulsion formulation had no measurable effect on confluence at amphotericin concentrations up to 100 micrograms mL-1 . The emulsion retained a comparable antifungal activity to that of Fungizone against Saccharomyces cerevisiae in suspension culture . These results parallel the observed erythrocyte lysis data obtained previously using amphotericin B emulsions, and suggest that the emulsion formulation may have a lower toxicity and improved therapeutic potential over existing formulations. Curr Genet, 1991 Jul, 20(1-2), 17 - 23 Ubiquitin gene expression: response to environmental changes; Fraser J et al.; It has previously been shown that the yeast ubiquitin genes UBI1, 2 and 3 are strongly expressed during the log-phase of batch culture growth, whereas the UBI4 gene is weakly expressed . We found that heat shock, treatment with DNA-damaging agents, starvation, and the feeding of starved cells all transiently induced UBI4 . These results suggest that UBI4 is induced whenever a change in culture conditions dictates a dramatic shift in cellular metabolism, and that UBI4 expression returns to lower levels once cellular metabolism has adapted to the new conditions . In contrast, all of the treatments tested, except starvation, transiently repressed the UBI1, 2 and 3 genes . Although starvation also repressed UBI1, 2 and 3 its effect was not transient, and expression only recovered upon the addition of fresh media . These results, together with others presented here, suggest that high levels of UBI1, 2 and 3 expression are dependant upon ongoing cell growth, and that treatments which slow or stop growth repress their expression. Mol Biol Evol, 1991 Jul, 8(4), 545 - 58 The dipole moment of cytochrome c; Koppenol WH et al.; Vertebrate cytochromes c and the cytochromes c of insects and plants have, on average, dipole moments of 320 and 340 debye, respectively . The direction of the dipole vector with respect to the haem plane, at the solvent-accessible edge of which electron transfer presumably takes place, is conserved in these two groups--at 32 degrees +/- 7 degrees and 22 degrees +/- 10 degrees, respectively . The variation of dipole orientations and magnitudes observed in these species is compared with the results of a model in which charge distributions occur randomly . Since this model does not generate the observed charge asymmetries of the various cytochromes c, it is concluded that the dipole moment of cytochrome c is a feature that is evolutionarily conserved, apparently because it has an important influence on the interaction of this mobile electron carrier with its physiological electron donors and acceptors in the intermembrane space of mitochondria. J Cell Biol, 1991 Jul, 114(2), 219 - 29 Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles; Rexach MF et al.; The transport of pro-alpha-factor from the ER to the Golgi apparatus in gently lysed yeast spheroplasts is mediated by diffusible vesicles . These transport vesicles contain core-glycosylated pro-alpha-factor and are physically separable from donor ER and target Golgi compartments . The formation of diffusible vesicles from the ER requires ATP, Sec12p, Sec23p, and GTP hydrolysis . The vesicles produced are functionally distinct from the ER: they transfer pro-alpha-factor to the Golgi apparatus faster and more efficiently than the ER, they do not require Sec12p or Sec23p to complete transfer, and transfer is resistant to GTP gamma S . Targeting of vesicles to the Golgi apparatus requires Ypt1p and Sec18p . Fusion of vesicles that have targeted requires calcium and ATP. Nucleic Acids Res, 1991 Jun 25, 19(12), 3307 - 14 Structural requirements for selection of 5'- and 3' splice sites of group II introns; Wallasch C et al.; The group II intron bl1 in the gene for apocytochrome b in yeast mitochondrial DNA (COB) is self-splicing in vitro . It could recently be shown that self-splicing of this intron is fully reversible in vitro . In addition, intron integration is not restricted to parental exons, since the intron can also integrate into a foreign RNA . The position of insertion seems to be immediately 3' to a cryptic intron binding site 1 (IBS1) . We confirmed and extended these results by sequencing 26 individual RNAs with transposed introns after reverse transcription and PCR amplification . Results show that intron integration into authentic exons is generally correct, but that integration into a foreign RNA is often inaccurate, i.e . insertion is one nt downstream or upstream of the 3' end of IBS1 . This leads to the generation of 5' splice junctions of the new intron-harbouring 'preRNAs' with addition (or deletion) of a single A residue at the 3' end of IBS1 . To investigate which structures help to define the position of 5'- and 3' cleavage, preRNAs of i) these clones with aberrant 5' splice junctions and ii) preRNAs with artificial hairpins between domains 5 and 6 of the intron were spliced under different reaction conditions . Results obtained let us conclude that i) branchpoint dependent 5' cleavage is directed by the 5' terminal G residue of the intron and, ii) the first nucleotide(s) of the 3' exon play an important role in defining the 3' splice site. FEBS Lett, 1991 Jun 24, 284(2), 277 - 80 Characterization of PC2, a mammalian Kex2 homologue, following expression of the cDNA in microinjected Xenopus oocytes; Shennan KI et al.; A human insulinoma cDNA (PC2) that encodes a protein homologous to the Kex2/subtilisin-like proteinases has recently been described {1990, J . Biol . Chem . 265, 2997-3000} . In order to characterise the associated proteinase activity, mRNA encoding PC2 was synthesised in vitro and microinjected into Xenopus oocytes . The proteinase activity released into the media from oocytes microinjected with PC2 mRNA was assayed using small peptide fluorogenic substrates . Boc.Gln.Arg.Arg aminomethyl coumarin was hydrolysed in a Ca(2+)-dependent manner, but substrate analogues bearing a single basic aminoacid were not . The substrate specificity, inhibitor profile, and pH optimum of 5.5 were compatible with an involvement of PC2 in prohormone processing in mammalian cells. FEBS Lett, 1991 Jun 24, 284(2), 173 - 7 Change in charge of an unvaried heme contact residue does not cause a major change of conformation in cytochrome c; Thurgood AG et al.; The structure of the Ala38 variant of yeast iso-1-cytochrome c, in which the previously unchanged Arg38 has been replaced, has been characterised by NMR . The NMR data indicate that the structure of the Ala38 variant is very similar to that of the wild type protein . In particular, the heme environment and interactions of the heme macrocycle are shown to be preserved . Analysis of the chemical shift perturbations to the resonances of Ile35 is shown to be consistent with the change in charge at position 38 . The only significant area of conformational change detected was at residues 39 and 58, close to the site of modification . Therefore the redox potential change accompanying the modification {1988, Biochemistry 28, 3188-3197} appears to be a direct consequence of the altered side-chain of residue 38 and not a result of secondary conformational changes induced by the modification. Biochemistry, 1991 Jun 18, 30(24), 5826 - 32 Computer simulation and analysis of the reaction pathway of triosephosphate isomerase; Bash PA et al.; A theoretical approach designed for chemical reactions in the condensed phase is used to determine the energy along the reaction path of the enzyme triosephosphate isomerase . The calculations address the role of the enzyme in lowering the barrier to reaction and provide a decomposition into specific residue contributions . The results suggest that, although Lys-12 is most important, many other residues within 16 A of the substrate contribute and that histidine-95 as the imidazole/imidazolate pair could act as an acid/base catalyst. Biochemistry, 1991 Jun 18, 30(24), 5821 - 6 Structure of the triosephosphate isomerase-phosphoglycolohydroxamate complex: an analogue of the intermediate on the reaction pathway; Davenport RC et al.; The glycolytic enzyme triosephosphate isomerase (TIM) catalyzes the interconversion of the three-carbon sugars dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP) at a rate limited by the diffusion of substrate to the enzyme . We have solved the three-dimensional structure of TIM complexed with a reactive intermediate analogue, phosphoglycolohydroxamate (PGH), at 1.9-A resolution and have refined the structure to an R-factor of 18% . Analysis of the refined structure reveals the geometry of the active-site residues and the interactions they make with the inhibitor and, by analogy, the substrates . The structure is consistent with an acid-base mechanism in which the carboxylate of Glu-165 abstracts a proton from carbon while His-95 donates a proton to oxygen to form an enediol (or enediolate) intermediate . The conformation of the bound substrate stereoelectronically favors proton transfer from substrate carbon to the syn orbital of Glu-165 . The crystal structure suggests that His-95 is neutral rather than cationic in the ground state and therefore would have to function as an imidazole acid instead of the usual imidazolium . Lys-12 is oriented so as to polarize the substrate oxygens by hydrogen bonding and/or electrostatic interaction, providing stabilization for the charged transition state . Asn-10 may play a similar role. Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5159 - 62 Direct selection for sequences encoding proteases of known specificity; Smith TA et al.; We have developed a simple genetic selection that could be used to isolate eukaryotic cDNAs encoding proteases that cleave within a defined amino acid sequence . The selection was developed by using the transcription factor GAL4 from Saccharomyces cerevisiae as a selectable marker, a cloned protease from tobacco etch virus (TEV), and an 18-amino acid TEV protease target sequence . In yeast, TEV protease cleaves its target even when the target is fused to internal regions of the GAL4 protein . This cleavage separates the DNA binding domain from the transcription activation domain of GAL4, rendering it transcriptionally inactive . The proteolytic cleavage can be detected phenotypically by the inability of cells to metabolize galactose . Cells expressing the TEV protease can also be selected on the suicide substrate 2-deoxygalactose . DNA binding studies show that the TEV protease decreases the activity of the GAL4/target fusion protein . Because another protease target sequence of 55 amino acids can be inserted into GAL4 without any loss of transcriptional activity, this assay offers the opportunity to use high-efficiency cDNA cloning and expression vectors to select coding sequences of other proteases from various species . The assay could also be used to help define both target specificities and functional domains of proteases. J Biol Chem, 1991 Jun 15, 266(17), 11347 - 54 Identification of the DNA-binding domain of the FLP recombinase; Pan H et al.; We have subjected the FLP protein of the 2-micron plasmid to partial proteolysis by proteinase K and have found that FLP can be digested into two major proteinase K-resistant peptides of 21 and 13 kDa, respectively . The 21-kDa peptide contains a site-specific DNA-binding domain that binds to the FLP recognition target (FRT) site with an affinity similar to that observed for the native FLP protein . This peptide can induce DNA bending upon binding to a DNA fragment containing the FRT site, but the angle of the bend (approximately 24 degrees) is smaller in magnitude than that induced by the native FLP protein (60 degrees) . The additional DNA bending induced by the interaction between two native FLP molecules bound to the FRT site is not observed with the 21-kDa DNA-binding peptide . Amino-terminal sequencing has been used to map this peptide to an internal region of FLP that begins at residue Leu-148 . It is likely that the DNA-binding peptide includes the catalytic site of the FLP protein. Biochem J, 1991 Jun 15, 276 ( Pt 3), 833 - 6 Purification and N-terminal sequence of the p21rho GTPase-activating protein, rho GAP; Garrett MD et al.; Eukaryotic cells contain numerous small-molecular-mass GTP-binding proteins, but the processes that they regulate are not known . Different members of this protein family appear to be associated with specific GTPase-activating proteins (GAPs), and we have previously reported the identification of a cytoplasmic GAP (rho GAP) that stimulates the GTPase activity of p21rho but not of other small-molecular-mass GTP-binding proteins . We have now purified rho GAP 2000-fold from human spleen tissue using f.p.l.c . Electrotransfer of this 27.5 kDa protein on to an Immobilon-P transfer membrane followed by reconstitution of its enzymic activity confirmed its identity . Rho GAP was subjected to N-terminal sequence analysis and 15 amino acids were obtained . The sequence showed 53% identity with a region present in IRA1, a protein which stimulates the GTPase activity of RAS proteins in Saccharomyces cerevisiae . These results suggest that there is a family of sequence-related GAP proteins, which to date includes ras GAP and its yeast counterparts IRA1 and IRA2, rho GAP and the Neurofibromatosis gene product NF1. Eur J Biochem, 1991 Jun 15, 198(3), 767 - 73 Inhibitors of metabolic reactions . Scope and limitation of acyl-CoA-analogue CoA-thioethers; Lill U et al.; Substrate and intermediate analogue inhibitors of enzymes were prepared in which the thioester oxygen of acyl-CoA substrates is replaced by hydrogen with formation of CoA-thioethers . Experiments performed with ATP citrate lyase and S-(3,4-dicarboxy-3-hydroxybutyl)-CoA are consistent with citryl-CoA but not with citryl-enzyme being the direct precursor of the products acetyl-CoA and oxaloacetate . Consistent with these results, a previously described isotopic exchange between acetyl-CoA and {3H}CoASH, indicating the formation of an acetyl-enzyme in the reaction pathway, could not be confirmed . Substrate analogue CoA-thioethers of malate synthase are inhibitors endowed with the affinity of the substrates . Acetyl carboxylase and fatty acid synthetase are not inhibited by the substrate analogue S-ethyl-CoA; S-carboxyethyl-CoA, which could substitute for malonyl-CoA, is likewise not inhibitory . An explanation is proposed . Previously suggested roles of S-carboxymethyl-CoA, an acetyl-CoA-related inhibitor of citrate synthase, are discussed in the light of new experimental data . S-Acetyl, S-propionyl and S-carboxymethyl derivatives of 1,N6-etheno-CoA loose the high affinity of their CoA-counterparts to citrate synthase, probably because the ethylene group prevents proper binding to the enzyme. Cell, 1991 Jun 14, 65(6), 949 - 59 Can calmodulin function without binding calcium? Gei