S. cerevisiae

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?
Geiser JR, van Tuinen D, Brockerhoff SE, Neff MM, Davis TN.
Calmodulin is a small Ca(2+)-binding protein proposed to act as the intracellular Ca2+ receptor that translates Ca2+ signals into cellular responses . We have constructed mutant yeast calmodulins in which the Ca(2+)-binding loops have been altered by site-directed mutagenesis . Each of the mutant proteins has a dramatically reduced affinity for Ca2+; one does not bind detectable levels of 45Ca2+ either during gel filtration or when bound to a solid support . Furthermore, none of the mutant proteins change conformation even in the presence of high Ca2+ concentrations . Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well . In contrast, yeast strains deleted for the calmodulin gene are not viable . Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca2+.

Science, 1991 Jun 14, 252(5012), 1553 - 6
Mediation of the attachment or fusion step in vesicular transport by the GTP-binding Ypt1 protein; Segev N; The function of the guanosine triphosphate (GTP)-binding protein Ypt1 in regulating vesicular traffic was studied in a cell-free system that reconstitutes transport from the endoplasmic reticulum to the Golgi . Blocking the Ypt1 protein activity resulted in accumulation of vesicles that act as an intermediate passing between the two compartments . The Ypt1 protein was found on the outer side of these vesicles . The transport process is completed by fusion of these vesicles with the acceptor compartment, and Ypt1 protein activity was needed for this step . Thus, a specific GTP-binding protein is required for either attachment or fusion (or both) of secretory vesicles with the acceptor compartment during protein secretion.

Nature, 1991 Jun 13, 351(6327), 583 - 6
Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis; Chen MS et al.; Dynamin was discovered in bovine brain tissue as a nucleotide-sensitive microtubule-binding protein of relative molecular mass 100,000 . It was found to cross-link microtubules into highly ordered bundles, and appeared to have a role in intermicrotubule sliding in vitro . Cloning and sequencing of rat brain dynamin complementary DNA identified an N-terminal region of about 300 amino acids which contained the three consensus elements characteristic of GTP-binding proteins . Extensive homology was found between this domain and the mammalian Mx proteins which are involved in interferon-induced viral resistance, and with the product of the VPS1 locus in Saccharomyces cerevisiae, which has been implicated both in membrane protein sorting, and in meiotic spindle pole separation . Dynamin-containing microtubule bundles were not observed in an immunofluorescence study of cultured mammalian cells, but a role for a GTP-requiring protein in intermicrotubule sliding during mitosis in plants has been reported . We report here that Drosophila melanogaster contains multiple tissue-specific and developmentally-regulated forms of dynamin, which are products of the shibire locus previously implicated in endocytic protein sorting.

Science, 1991 Jun 7, 252(5011), 1427 - 30
CREB: a Ca(2+)-regulated transcription factor phosphorylated by calmodulin-dependent kinases; Sheng M et al.; The mechanism by which Ca2+ mediates gene induction in response to membrane depolarization was investigated . The adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) was shown to function as a Ca(2+)-regulated transcription factor and as a substrate for depolarization-activated Ca(2+)-calmodulin-dependent protein kinases (CaM kinases) I and II . CREB residue Ser133 was the major site of phosphorylation by the CaM kinases in vitro and of phosphorylation after membrane depolarization in vivo . Mutation of Ser133 impaired the ability of CREB to respond to Ca2+ . These results suggest that CaM kinases may transduce electrical signals to the nucleus and that CREB functions to integrate Ca2+ and cAMP signals.

Biochemistry, 1991 Jun 4, 30(22), 5329 - 34
Identification of electrostatic interactions that determine the phosphorylation site specificity of the cAMP-dependent protein kinase; Gibbs CS et al.; "Charged-to alanine" scanning mutagenesis of the catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase (C1) identified three glutamate residues, E171, E214, and E274, that are involved in the recognition of a peptide substrate, kemptide (Leu1Arg2Arg3Ala4Ser5Leu6Gly7) . These glutamate residues are conserved or conservatively substituted with asparate in the serine/threonine protein kinases that have a requirement for basic residues on the N-terminal side of their phosphorylation sites . Alanine replacement mutants in C1 were subjected to kinetic analysis using alanine-substituted peptides as substrates . The additivity or nonadditivity of the effects of the alanine substitutions on the catalytic efficiency (kcat/Km) was analyzed . This allowed the identification of electrostatic interactions between the three glutamate residues in the enzyme and the two arginine residues present in the peptide substrate . The data suggest that E171 interacts with Arg2 in the substrate and that E214 and E274 both interact with Arg3 . This may be a general method for identifying simple intermolecular interactions involving proteins when there is no three-dimensional structure available of the complex of interacting species . The identification of these interactions provides the potential for rational protein engineering of enzymes with alternative specificities.

Eur J Biochem, 1991 Jun 1, 198(2), 477 - 84
Behavior of cysteine mutants of human lysozyme in de novo synthesis and in vivo secretion; Omura F et al.; To investigate the mechanism of disulfide-bond-coupled de novo folding of human lysozyme, we have constructed 23 mutant enzymes in which cysteine residue(s) were replaced by alanine(s) . The mutant genes were translated in vitro in a system composed of rabbit reticulocyte lysate, canine pancreatic microsomal vesicles and oxidized glutathione . This system allows the formation of intramolecular disulfide bonds in translation products translocated into the microsomal lumen . The mobilities of the translation products were analyzed by SDS/PAGE in nonreducing conditions . Some mutant lysozymes were found to form a compact conformation with native-like mobility in the presence of SDS . The de novo formation of the SDS-resistant compact conformation of each mutant correlated well with its efficiency of secretion by Saccharomyces cerevisiae . Our results suggest that the de novo synthesized products reflect the conformational states in vivo to some extent, and that the formation of SDS-resistant compact conformation can be regarded as a necessary condition for allowing lysozyme to be secreted . In addition, the analysis of a mutant C116A (Cys116----Ala) under different oxidative conditions suggests two distinct pathways for the disulfide-bond-coupled formation of the compact conformation.

Mol Cell Biol, 1991 Jun, 11(6), 3217 - 28
Complex formation by positive and negative translational regulators of GCN4; Cigan AM et al.; GCN4 is a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae whose expression is regulated by amino-acid availability at the translational level . GCD1 and GCD2 are negative regulators required for the repression of GCN4 translation under nonstarvation conditions that is mediated by upstream open reading frames (uORFs) in the leader of GCN4 mRNA . GCD factors are thought to be antagonized by the positive regulators GCN1, GCN2 and GCN3 in amino acid-starved cells to allow for increased GCN4 protein synthesis . Previous genetic studies suggested that GCD1, GCD2, and GCN3 have closely related functions in the regulation of GCN4 expression that involve translation initiation factor 2 (eIF-2) . In agreement with these predictions, we show that GCD1, GCD2, and GCN3 are integral components of a high-molecular-weight complex of approximately 600,000 Da . The three proteins copurified through several biochemical fractionation steps and could be coimmunoprecipitated by using antibodies against GCD1 or GCD2 . Interestingly, a portion of the eIF-2 present in cell extracts also cofractionated and coimmunoprecipitated with these regulatory proteins but was dissociated from the GCD1/GCD2/GCN3 complex by 0.5 M KCl . Incubation of a temperature-sensitive gcdl-101 mutant at the restrictive temperature led to a rapid reduction in the average size and quantity of polysomes, plus an accumulation of inactive 80S ribosomal couples; in addition, excess amounts of eIF-2 alpha, GCD1, GCD2, and GCN3 were found comigrating with free 40S ribosomal subunits . These results suggest that GCD1 is required for an essential function involving eIF-2 at a late step in the translation initiation cycle . We propose that lowering the function of this high-molecular-weight complex, or of eIF-2 itself, in amino acid-starved cells leads to reduced ribosomal recognition of the uORFs and increased translation initiation at the GCN4 start codon . Our results provide new insights into how general initiation factors can be regulated to affect gene-specific translational control.

Trends Biochem Sci, 1991 Jun, 16(6), 208 - 13
Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics; Brodsky FM et al.; Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat shock protein hsc70 control receptor-mediated pathways of intracellular transport . The clathrin light chains are major regulatory elements in these processes . These polypeptides consist of linear arrays of functional domains with distinctive sequence motifs . Comparison of unicellular and multicellular eukaryotes reveals differences in the numbers of clathrin light chains and in the functional domains they contain.

Biochem J, 1991 Jun 1, 276 ( Pt 2), 433 - 8
Structural properties of long- and short-chain alcohol dehydrogenases . Contribution of NAD+ to stability; Ribas De Pouplana L et al.; Structural studies were undertaken on long-chain and short-chain alcohol dehydrogenases (from horse liver and Drosophila respectively) . Far-u.v . c.d . measurements were used to estimate the secondary structure contents of the enzymes . For the horse liver enzyme, the results agree well with the X-ray data; for the Drosophila enzyme (for which a crystal structure is not yet available), the results are in good agreement with those obtained by applying a range of structure-prediction procedures to the amino acid sequence of this enzyme . The conformational stabilities of the two enzymes were investigated by studying the unfolding brought about by guanidinium chloride (GdnHCl) by using activity and c.d . measurements . The unfolding of the Drosophila enzyme was analysed in terms of a two-state model; the presence of the substrate NAD+ leads to considerable protection against unfolding . By contrast, the unfolding of the horse liver enzyme shows a plateau effect at intermediate concentrations of GdnHCl, indicating that a two-state model is not appropriate in this case . NAD+ affords little, if any, protection against unfolding for the horse liver enzyme.

Arch Biochem Biophys, 1991 Jun, 287(2), 276 - 82
Structural and functional effects of mutations altering the subunit interface of mitochondrial malate dehydrogenase; Steffan JS et al.; Among highly conserved residues in eucaryotic mitochondrial malate dehydrogenases are those with roles in maintaining the interactions between identical monomeric subunits that form the dimeric enzymes . The contributions of two of these residues, Asp-43 and His-46, to structural stability and catalytic function were investigated by construction of mutant enzymes containing Asn-43 and Leu-46 substitutions using in vitro mutagenesis of the Saccharomyces cerevisiae gene (MDH1) encoding mitochondrial malate dehydrogenase . The mutant enzymes were expressed in and purified from a yeast strain containing a disruption of the chromosomal MDH1 locus . The enzyme containing the H46L substitution, as compared to the wild type enzyme, exhibits a dramatic shift in the pH profile for catalysis toward an optimum at low pH values . This shift corresponds with an increased stability of the dimeric form of the mutant enzyme, suggesting that His-46 may be the residue responsible for the previously described pH-dependent dissociation of mitochondrial malate dehydrogenase . The D43N substitution results in a mutant enzyme that is essentially inactive in in vitro assays and that tends to aggregate at pH 7.5, the optimal pH for catalysis for the dimeric wild type enzyme.

Curr Opin Cell Biol, 1991 Jun, 3(3), 461 - 6
RNA polymerase III transcription; Wolffe AP; Remarkable progress has been made in defining the functional significance of the protein-DNA interactions involved in transcription complex formation on yeast tRNA and 5S RNA genes . This new information leads to a re-evaluation of how the class III gene transcription machinery operates.

Protein Eng, 1991 Jun, 4(5), 519 - 29
The solution structure of a leucine-zipper motif peptide; Saudek V et al.; We report the complete structure determination of a 34 residue synthetic peptide with the amino acid sequence of the dimerization domain (leucine zipper) of GCN4 . A high resolution structure in solution was obtained by 1H-NMR studies and distance geometry calculations followed by restrained energy minimization . A set of 20 final structures was obtained with an average root mean square deviation of 1.3 A for the backbone atoms (excluding the first and the last two residues) . The structure contains an uninterrupted helix . A comparison with a structure previously determined for a larger peptide containing both the DNA-binding region (basic region) and the leucine-zipper motif shows the structural independence of the leucine-zipper domain from the contiguous DNA binding region.

Bratisl Lek Listy, 1991 Jun, 92(6), 283 - 90
{Modulatory effect of glucans on the function of murine macrophages, NK-cells and lymphocytes}; Macela A et al.; The particulate glucan (G1), soluble glucan preparations (G2 to G5, and G7) isolated from Saccharomyces cerevisiae, and glucomanan prepared from culture fluid after cultivation of Candida utilis (G6) were tested for their immunomodulatory activity in vivo and in vitro . In tests in vivo three soluble glucans (G3, G4, and G7) injected s.c . to mice in the dose of 10 mg/kg increased the cytotoxic activity of peritoneal macrophages . The influence of glucans on natural killer cells was without significance . The lymphoproliferative reaction of spleen cells to polyclonal mitogens was inhibited by all the preparations used with the exception of soluble glucan G2 . The mitogenic effect of the preparations, co-stimulatory tests and direct cytotoxicity to cells of cell lines used in cytotoxicity assays were assessed in vitro . The transformation index of glucans in the study was increased according to the glucan and dose tested . Inhibition of the lymphoproliferative reaction measured by the co-stimulatory test for optimal concentration of Concanavalin A occurred in a wide range of doses for the preparations G1 to G6 . The preparation G7 increased the incorporation of 3HTdR under the same conditions . The use of a suboptimal concentration of Concanavalin A revealed co-stimulatory activity of all the preparations tested . Assessment of the cytotoxic activity of peritoneal macrophages and of the activity of natural killer cells induced in vitro was complicated by the direct cytotoxicity of particulate glucan and soluble glucan G5 (carboxymethylglucan) for target cells (YAC 1, and YAC 1 and K 562 resp.).

FASEB J, 1991 Jun, 5(9), 2258 - 66
Regulation of eukaryotic phospholipid metabolism; Kent C et al.; Phospholipids have diverse and critical roles in cellular metabolism and function . Questions about the mechanisms of regulation of phospholipid synthesis are being investigated with a variety of systems and approaches . For example, the yeast Saccharomyces cerevisiae is an organism in which both biochemical and genetic analyses are used . Biochemical approaches have yielded considerable information on the regulatory properties of enzymes of phospholipid biosynthesis . Studies of the activity of purified phosphatidylserine synthase have suggested how that enzyme is influenced by membrane phospholipids in the cell . The enzyme that regulates mammalian phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase, is also influenced by phospholipids . In addition, the activity of this enzyme often correlates with its translocation to membranes . The location of such enzymes in the cell is of particular interest in light of the possibility that the enzymatic reactions may be efficiently coupled in vivo . Techniques to render cultured cells permeable to phosphorylated molecules indicated that the enzymes of phosphatidylcholine biosynthesis may exist in an organized compartment so that the precursors of phosphatidylcholine are efficiently channeled through the pathway . To ask how phospholipids are transported in the cell, a combined biochemical and genetic approach has been used . These studies have revealed that the phosphatidylinositol/phosphatidylcholine transfer protein, considered to mediate intracellular phospholipid transfer, is a critical component of the secretory pathway for proteins . These results have allowed formulation of a number of new questions on the regulation of phospholipid metabolism and its relationship to general membrane processes.

Rev Biol Trop, 1991 Jun, 39(1), 177 - 80
{Use of the plasma coagulation method for ultrastructural analysis of biological specimens}; Hernandez F et al.; Biological particulate specimens, including Saccharomyces cerevisiae yeast, bovine spermatozoa and human blood cells (normal erythrocytes and leukemic cells) were processed for scanning and transmission electron microscopy using the coagulated plasma technique . The specimens were suspended in frozen and thawed plasma; later, coagulation was induced by adding CaCl2 . The clot was cut into small pieces and processed as tissue fragments . The technique is an useful tool when processing biological particulate specimens for electron microscopy.

Virology, 1991 Jun, 182(2), 682 - 9
The TMV movement protein: role of the C-terminal 73 amino acids in subcellular localization and function; Berna A et al.; The role of the C-terminal one-third of the tobacco mosaic virus (TMV) 30-kDa movement protein (MP) on its subcellular localization and on virus spread was investigated . We have constructed eight cDNAs encoding MPs with variable size deletions from the C-terminal end . Expression of the truncated proteins was verified in recombinant yeast using an antiserum directed to a synthetic peptide corresponding to 21 amino acids near the N-terminal end of the MP . In transgenic tobacco plants, MP from which more than 55 amino acids were deleted no longer accumulated in the cell wall fraction of a cellular extract, where the complete MP accumulates . Dye diffusion studies showed that both unmodified and modified MPs that accumulate in the cell wall fraction are able to alter plasmodesmatal size exclusion limits . Biological function of the modified MPs was tested in the transgenic plants with the TMV thermosensitive mutant Ls1 and a TMV genomic RNA transcript lacking a functional MP . There was a correlation between the cell wall localization of the modified MPs and its ability to potentiate virus spread . The results presented here demonstrate the dispensability of the C-terminal 55 amino acids of the MP in its subcellular localization in tobacco plants and its role in virus movement . Moreover, our results show that a stretch of 19 amino acids (195 to 213) is essential for localization of the MP to the cell wall fraction of plant cells.

Curr Opin Genet Dev, 1991 Jun, 1(1), 62 - 8
Applications of polymerase chain reaction methods in genome mapping; Nelson DL; Important new polymerase chain reaction-based techniques have been developed to assist in genome analysis . Applications range from genetic and physical mapping of DNA to sequence analysis . The polymerase chain reaction has played a significant role in increasing the feasibility of many aspects of genome analysis and positional cloning.

Curr Opin Cell Biol, 1991 Jun, 3(3), 502 - 7
Retrotransposition mechanisms; Boeke JD et al.; Recent developments in the area of the transposition mechanisms used by retrotransposons and related retroviral pathways are discussed . In particular, advances in the areas of retrotransposon gene expression, virus-like particle assembly, reverse transcription, and integration are reviewed.

Genomics, 1991 Jun, 10(2), 481 - 5
Genomic analysis of the 67-kDa laminin receptor in normal and pathological tissues: circumstantial evidence for retroposon features; Bignon C et al.; We have cloned two cDNAs for the human 67-kDa laminin receptor (LR) . In the present report we show that these clones hybridize to many restriction fragments in Southern experiments in human . This particular pattern is accounted for by the presence of up to 16 and 21 copies of the laminin receptor gene per haploid genome in human and mouse, respectively . In contrast, a single gene copy is found in chicken . Chromosomal localization reveals four main loci: LAMRP1, laminin receptor pseudogene 1 (Chr 3); LAMRP2, laminin receptor pseudogene 2 (Chr 12); LAMRP3, laminin receptor pseudogene 3 (Chr 14); LAMRP4, laminin receptor pseudogene 4 (Chr X) . Comparison of our experimental results to the known features of processed retropseudogenes enabled us to conclude that the LR gene belongs to a retroposon family in mammals.

Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 4606 - 10
The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo; Hochstrasser M et al.; The substrates of ubiquitin-dependent proteolytic pathways include both damaged or otherwise abnormal proteins and undamaged proteins that are naturally short-lived . Few specific examples of the latter class have been identified, however . Previous work has shown that the cell type-specific MAT alpha 2 repressor of the yeast Saccharomyces cerevisiae is an extremely short-lived protein . We now demonstrate that alpha 2 is conjugated to ubiquitin in vivo . More than one lysine residue of alpha 2 can be joined to ubiquitin, and some of the ubiquitin moieties form a Lys48-linked multiubiquitin chain . Overexpression of degradation-impaired ubiquitin variants was used to show that at least a significant fraction of alpha 2 degradation is dependent on its ubiquitination.

Mol Cell Biol, 1991 Jun, 11(6), 3020 - 6
Molecular cloning and expression of the type 1 and type 2 murine receptors for tumor necrosis factor; Goodwin RG et al.; Clones encoding the type 1 (p80) and type 2 (p60) forms of the murine receptors for tumor necrosis factor (TNF) were isolated by cross-hybridization using probes derived from the cloned human TNF receptors . Each of the murine receptors shows strong sequence homology to the corresponding human receptor (approximately 65% amino acid identity) throughout the molecule but only modest homology, limited to ligand-binding domains, between themselves . The ligand-binding characteristics of the recombinant murine receptors mirror those of the human homologs: both receptor types bind TNF-alpha and -beta with multiple affinity classes, and the ligands cross-compete . Analysis of the murine transcripts encoding these receptors revealed the presence of RNAs for one or both forms of the receptors in all cells examined . It was also demonstrated that for both types of human TNF receptor, variably sized transcripts are observed in different cells . The murine cDNAs were further used to determine the chromosomal locations of the TNF receptor genes . They are not linked, in contrast to the ligands, and map to chromosomes 4 (type 1) and 6 (type 2).

Enzyme Microb Technol, 1991 Jun, 13(6), 508 - 11
A method for the preparation of coimmobilizates by adhesion using polyethylenimine; D'Souza SF et al.; A method has been described for obtaining coimmobilizates by the simultaneous binding of glucose oxidase to the cell and the enzyme-bound cell to cotton thread through adhesion using polyethylenimine (PEI) . Glucose oxidase was found to adsorb onto PEI-coated yeast cells from a water suspension . The desorption observed at higher ionic strength could be obviated by cross-linking with 2% glutaraldehyde for 2 min . The enzyme-bound yeast cells could then be immobilized by adhesion on cotton thread . The coimmobilizate could be reused for over 10 batches without appreciable loss in activity.

J Chromatogr, 1991 May 31, 566(2), 435 - 43
Radio-detection high-performance liquid chromatographic enzyme assay for inhibitors of fungal sterol delta 14-reductase; Steel CC; An enzyme assay for inhibitors of fungal sterol delta 14-reductase employing isocratic reversed-phase high-performance liquid chromatography is described . A Hypersil 5-microns octadecylsilyl (ODS) column (250 mm x 4.6 mm I.D.) was used and a mobile phase consisting of methanol-water-ethanol (86:4:10, v/v) was pumped at a flow-rate of 1.5 ml/min . Typical analysis times were 15 min . Using {4-14C}ignosterol as a substrate and an enzyme preparation from Saccharomyces cerevisiae, this method was used to compare the inhibition of sterol delta 14-reductase by the fungicides fenpropidin and fenpropimorph with three N-substituted 8-azadecaline compounds.

J Biol Chem, 1991 May 25, 266(15), 9508 - 14
Aggregates of oligo(dG) bind and inhibit topoisomerase II activity and induce formation of large networks; Chung IK et al.; DNA cleavage by eukaryotic type II DNA topoisomerase (EC 5.99.1.3) was strongly inhibited by an oligonucleotide containing 10 dGua residues . Catalytic activities of topoisomerase II, as measured by relaxation and decatenation reactions, were also inhibited by oligo(dG)10 . Inhibition was specific to oligo(dG)10; other oligonucleotides, nucleotides, or single-stranded DNAs tested did not influence the activity of topoisomerase II . Oligo(dG)10 did not inhibit other activities such as restriction enzymes . Although the enzyme neither binds nor cleaves oligo(dG)10, inhibition can be explained by the finding that topoisomerase II binds tightly with aggregated oligo(dG) structures (estimated to contain between 20 and 30 molecules of monomeric oligo(dG)10) that form spontaneously prior to addition of enzyme . These aggregated oligo(dG)-topoisomerase complexes are large networks that can be pelleted by a 20-min centrifugation step in a Microfuge . Western blotting with a monoclonal antibody confirmed that topoisomerase II is trapped in these pellets . The ability of the enzyme to form large DNA-protein networks could be a biochemical mechanism by which topoisomerase II might promote or participate in chromosome condensation in vivo prior to mitosis.

Cell, 1991 May 17, 65(4), 691 - 9
Human D-type cyclin; Xiong Y et al.; A cDNA library prepared from a human glioblastoma cell line has been introduced into a budding yeast strain that lacks CLN1 and CLN2 and is conditionally deficient for CLN3 function . We rescued a gene that we call cyclin D1 . It is related to A-, B-, and CLN-type cyclins, but appears to define a new subclass within the cyclin gene family . Transcription of the cyclin D1 gene gives rise to two major transcripts through alternative polyadenylation . The cyclin D1 gene transcript and its 34 kd product are both abundant in the glioblastoma cell line of origin.

J Biol Chem, 1991 May 15, 266(14), 8923 - 31
Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interactions; Gibbs CS et al.; A systematic mutagenesis strategy was used to identify the functional regions and residues of a protein kinase . Clusters of the charged amino acids in the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase, were systematically mutated to alanine, producing a set of mutations that encompassed the entire molecule . Residues indispensable for enzyme activity were identified by testing the ability of the mutants to function in vivo . Active mutants were assayed in vitro, and mutants with reduced specific activity were subsequently analyzed by steady-state kinetics to determine the effects of the mutation on kcat and on Km for MgATP and for a peptide substrate . Specific residues and regions of the enzyme were identified that are likely to be important in catalysis and in binding of MgATP, functions that are common to all protein kinases . Additional regions were identified that are likely to be important in binding a peptide substrate, the recognition of which is likely to be specific to the serine/threonine protein kinases that have a requirement for basic residues around the target hydroxyamino acid . The properties of mutants defective in substrate recognition were consistent with an ordered sequential reaction mechanism . This represents the first comprehensive analysis of a protein kinase by a rational mutagenesis strategy.

Biochem J, 1991 May 15, 276 ( Pt 1), 53 - 6
Topography of very-long-chain-fatty-acid-activating activity in peroxisomes from rat liver; Lageweg W et al.; We have investigated the localization of palmitoyl-CoA (hexadecanoyl-CoA) synthetase (EC 6.2.1.3) and cerotoyl-CoA (hexacosanoyl-CoA) synthetase in peroxisomes isolated from rat liver . Palmitoyl-CoA and cerotoyl-CoA synthetases, like acyl-CoA: dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42), are present in the peroxisomal membrane . Trypsin treatment of intact peroxisomes led to the disappearance of both palmitoyl-CoA and cerotoyl-CoA synthetase activities but had little, if any, effect on L-alpha-hydroxy-acid oxidase (EC 1.1.3.15), D-amino acid oxidase (EC 1.4.3.3) or acyl-CoA:dihydroxyacetone phosphate acyltransferase . The latter three enzymes were inactivated if the trypsin treatment was preceeded by disruption of the peroxisomes by sonication . These results show that the active site, or at least domains essential for the activity of cerotoyl-CoA synthetase, like that of palmitoyl-CoA synthetase, is located on the cytosolic face of the peroxisomal membrane.

Arch Biochem Biophys, 1991 May 15, 287(1), 85 - 90
Purification and properties of a 4-nitrophenylphosphatase from Aspergillus niger; Versaw WK et al.; A 4-nitrophenylphosphatase (EC 3.1.3.41) was identified in extracts of Aspergillus niger . The production of this activity was decreased by growth on a phosphate-limiting medium and was greatest in a medium supplemented with corn steep liquor . The phosphatase activity was purified by hydrophobic, ion-exchange, and molecular sieve chromatography . The purified enzyme has a native size of approximately 80,000, polypeptide subunits with sizes of 37,000 upon denaturation, and a pI of 4.6 . The activity was optimal at pH 8.0 and was stimulated by Mg2+ and to a lesser extent by Mn2+ but was inhibited by Zn2+ and Ca2+ . The enzyme was highly specific for 4-nitrophenyl phosphate as substrate, having a Km of 0.77 mM and a turnover number of 108 s-1 . The purified enzyme did not hydrolyze any of 22 sugar phosphates, mononucleotides, or other phosphocompounds tested . A small, but reproducible, amount of activity was measured using 5'-DNA phosphate as a substrate . Although some similarities exist to three previously characterized 4-nitrophenylphosphatases from Saccharomyces cerevisiae, the enzyme from A . niger is distinctly different from at least two of these activities.

Biochemistry, 1991 May 14, 30(19), 4706 - 10
Effective concentrations of amino acid side chains in an unfolded protein; Muthukrishnan K et al.; Preferential interactions between chain segments are studied in unfolded cytochrome c . The method takes advantage of heme ligation in the unfolded protein, a feature unique to proteins with covalently attached heme . The approach allows estimation of the effective concentration of one polypeptide chain segment relative to another, and is successful in detecting differences for peptide chain segments separated by different numbers of residues in the linear sequence . The method uses proton NMR spectroscopy to monitor displacement of the histidine heme ligands by imidazole as guanidine hydrochloride unfolded cytochrome c is titrated with deuterated imidazole . When the imidazole concentration exceeds the effective (local) concentration of histidine ligands, the protein ligands are displaced by deuterated imidazole . On displacement, the histidine ring proton resonances move from the paramagnetic region of the spectrum to the diamagnetic region . Titrations have been carried out for members of the mitochondrial cytochrome c family that contain different numbers of histidine residues . These include cytochromes c from tuna (2), yeast iso-2 (3), and yeast iso-1-MS (4) . At high imidazole concentration, the number of proton resonances that appear in the histidine ring C2H region of the NMR spectrum is one less than the number of histidine residues in the protein . So one histidine, probably His-18, remains as a heme ligand . The effective local concentrations of histidines-26, -33, and -39 relative to the heme (position 14-17) are estimated to be (3-16) X 10(-3) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biochem, 1991 May 29-Jun 12, 104(1-2), 155 - 62
Multiple functional enhancer motifs of rat ribosomal gene; Jacob ST et al.; Previous studies from this laboratory have characterized a 174 bp enhancer element which is located 2 kb upstream of the initiation site . Half of the enhancer action is controlled by a 37 bp element at the 3' end of the 174 bp region . We now report that a 43 bp adjacent domain which is located upstream of the 37 bp element constitutes an additional motif of the rDNA enhancer . When the plasmid consisting of the 43 bp DNA upstream of the rDNA core promoter was transcribed in a fractionated rat tumor cell extract (fraction DE-B), transcription of rDNA was augmented 4 fold . Electrphoretic mobility shift and DNAase I footprinting analyses showed that the purified 37 bp enhancer (E1)-binding protein, (E1BF) not only interacted with the enhancer motif E1 but also interacted with the neighbouring 43 bp enhancer domain E2 . The specificity of the binding was demonstrated by competition with unlabelled 37 bp and 43 bp fragment and lack of competition with nonspecific DNAs in the mobility shift assay . These studies have shown that a single pol I transcription factor can bind to multiple enhancer domains with no significant sequence homologies and such multiple interactions may result in maximal transcription of ribosomal gene from the core promoter.

Nature, 1991 May 9, 351(6322), 158 - 61
Dependence of Ypt1 and Sec4 membrane attachment on Bet2; Rossi G et al.; Many small GTP-binding proteins are synthesized as soluble proteins that are post-translationally modified as a prerequisite for membrane attachment . Ypt1 and Sec4 are homologous Raslike GTP-binding proteins that have been proposed to regulate the specificity of vesicular traffic at different stages of the secretory pathway by cycling on and off membranes . Here we show that BET2, initially identified as a gene required for transport from endoplasmic reticulum to Golgi apparatus in yeast, encodes a factor that is needed for the membrane attachment of Ypt1 and Sec4 . DNA sequence analysis has revealed that Bet2 is homologous to Dpr1 (Ram1), an essential component of a protein prenyltransferase that modifies Ras, enabling it to attach to membranes . We propose that Bet2 modifies Ypt1 and Sec4 in an analogous manner.

FEBS Lett, 1991 May 6, 282(2), 425 - 30
Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency; Galanis M et al.; We describe a novel method for enhancing protein import into mitochondria, by tandemly duplicating the N-terminal cleavable leader peptide using a gene manipulation strategy . The import into isolated yeast mitochondria of passenger proteins (yeast mitochondrial ATP synthase subunits 8 and 9 and some mutagenised derivatives) that show little or no import when endowed with one such leader (that of Neurospora crassa mitochondrial ATP synthase subunit 9) is remarkably improved when the leader is tandemly duplicated . The import of these chimaeric proteins bearing a double leader is so rapid that a series of partially processed precursor intermediates accumulates inside the mitochondria before the final proteolytic release of leader sequences from the passenger proteins . It is considered that the duplicated leader greatly accelerates delivery of the import precursors to outer membrane receptor elements and the associated translocation systems, thereby enhancing precursor uptake into mitochondria.

J Biol Chem, 1991 May 5, 266(13), 8015 - 9
All internal promoter elements of Neurospora crassa 5 S rRNA and tRNA genes, including the A boxes, are functionally gene-specific; Shi YG et al.; The internal control elements of Neurospora crassa 5 S genes include an A box and a C box as in Xenopus and Saccharomyces cerevisiae, plus a novel element, the Ribo box at position +18 to +34 . The Ribo box is also found in the 40 S rRNA promoter and a ribosomal protein gene but is absent from tRNA genes in N . crassa . The 5 S A box diverges from the tRNA A box consensus at two positions . We tested whether replacement of the 5 S A box with a tRNALeu A box sequence would increase 5 S gene transcription in vitro or would remove the requirement for the Ribo box . The 5 S gene with the tRNALeu A box was transcribed poorly, and the Ribo box and the C box are still required for transcription . We tested the function of the Ribo box and 5 S A box in a tRNA-like transcription unit by constructing hybrids between a 5 S gene and a tRNALeu gene . In the tRNA-like context, the 5 S A box supported a lower level of transcription than the tRNA A box, and the Ribo box was not required at all . Therefore, in N . crassa, all of the 5 S internal control elements are gene-specific . In particular, the 5 S and tRNA A box sequences are not functionally interchangeable and may bind different transcription factors . Transcription of the hybrids was initiated at the 5 S initiation site, suggesting that the mechanism of initiation site selection is the same in the 5 S and tRNA genes . Competition experiments with the tRNA B box suggested that the N . crassa 5 S and tRNA genes require at least one common transcription factor such as TFIIIC.

Nature, 1991 May 2, 351(6321), 65 - 8
Early aspects of Caenorhabditis elegans sex determination and dosage compensation are regulated by a zinc-finger protein; Nonet ML et al.; The sdc-1 gene acts at an early step in the regulatory hierarchy that controls the choice of sexual fate in Caenorhabditis elegans . It functions at a point before the control of sex determination and X-chromosome dosage compensation diverge . Here we report that sdc-1 encodes a protein of 1,203 amino acids containing seven zinc fingers . This protein motif in combination with other genetic and molecular information suggests that sdc-1 is likely to function as an embryonic transcription factor regulating downstream genes involved specifically in the sex determination and dosage compensation pathways, or regulating other genes involved in the coordinate control of both processes . These results enhance our general understanding of sex determination strategies, which are already known to involve transcriptional regulation and alternative RNA splicing in Drosophila melanogaster, DNA rearrangements in Saccharomyces cerevisiae, and transcriptional regulation in mammals.

Biochim Biophys Acta, 1991 May 2, 1089(1), 95 - 102
Molecular cloning and sequence analysis of cDNAs for five major subunits of human proteasomes (multi-catalytic proteinase complexes); Tamura T et al.; Proteasomes are multicatalytic proteinase complexes consisting of a set of non-identical polypeptide components . Of these multiple components, the nucleotide sequences of five major subunits (named HC2, HC3, HC5, HC8 and HC9) of human proteasomes have been determined from recombinant cDNA clones by screening a human HepG2 hepatoblastoma cell cDNA library with rat proteasome cDNAs isolated previously as probes . The polypeptides deduced from their nucleotide sequences consisted of 263, 234, 241, 255 and 261 amino acid residues with calculated molecular weights of 29,554, 25,897, 26,487, 28,431 and 29,482, respectively, which are encoded by single independent genes . The primary structures of these subunits of human proteasomes closely resemble those of their rat counterparts and show considerably high inter-subunit homology, although the homology of HC5 is relatively low . These findings, together with the structural similarities of other eukaryotic proteasomes including those of Drosophila and yeast (Saccharomyces cerevisiae) support and extend the previously proposed concept that eukaryotic proteasome genes form a multi-gene family with the same evolutionary origin.

Somat Cell Mol Genet, 1991 May, 17(3), 311 - 22
Isolation of a human cDNA encoding amidophosphoribosyltransferase and functional complementation of a CHO Ade-A mutant deficient in this activity; Barton JW et al.; We report here the isolation of a human cDNA encoding the first step in de novo purine biosynthesis, amidophosphoribosyltransferase (PRAT) . The human PRAT cDNA was isolated by complementation of a Saccharomyces cerevisiae ade4 mutant deficient in PRAT enzymatic activity . The identity of the isolated cDNA, designated pAdeA-3, was confirmed by several independent methods . Genomic DNA sequences homologous to pAdeA-3 show coordinate segregation with the hypoxanthine nutritional requirement in Chinese hamster ovary (CHO) cell Ade-A-human hybrids, segregants of these hybrids, and irradiation reduction hybrids . The PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT enzyme deficiency in CHO Ade-A mutants . This correction was monitored by both cell-free PRAT assays and in vivo phosphoribosylformylglycinamide (FGAR) accumulation studies . FGAR accumulation is a classic method for assessment of the early steps of purine nucleotide biosynthesis . Two of the isolated transformants, designated PRAT-1 and PRAT-2, exhibited 22% and 53%, respectively, of wild-type CHO K1 PRAT enzymatic activity using a cell-free enzyme assay . These same two transformants plus an additional transformant, designated PRAT-13, showed FGAR accumulations of 150%, 260%, and 140%, respectively, compared to the levels of accumulation seen in CHO K1 . Transformants PRAT-1 and PRAT-2 both contained a mRNA species recognized by the PRAT cDNA of identical size to a mRNA species in human fibroblasts homologous to the PRAT cDNA . This observation, along with the functionality of the cDNA in both yeast and CHO cells deficient in PRAT activity, suggests the isolated cDNA is full length.

Biochem J, 1991 May 1, 275 ( Pt 3), 775 - 9
Evidence that rat liver pyruvate dehydrogenase kinase activator protein is a pyruvate dehydrogenase kinase; Mistry SC et al.; It is shown here that rat liver pyruvate dehydrogenase (PDH) kinase activator protein (KAP) catalyses ATP-dependent inactivation and {32P}phosphorylation of pig heart PDHE1 and of yeast (Saccharomyces cerevisiae) PDH complex devoid of PDH kinase activity, that fluorosulphonylbenzoyladenosine inactivates rat liver KAP and the intrinsic PDH kinase of rat liver PDH complex, and that KAP, like PDH kinase, is inactivated by thiol-reactive reagents . It is concluded that KAP is a free PDH kinase.

Biochem J, 1991 May 1, 275 ( Pt 3), 767 - 73
Alkylation of glyceraldehyde-3-phosphate dehydrogenase with haloacetylphosphonates . An unusual pH-dependence; Li YK et al.; Two new alkylating reagents, chloro- and bromo-acetylphosphonate, were found to be very effective thiol-blocking reagents . The pH-dependence of the reaction of BAP with 2,4-dinitrothiophenol (25 degrees C, I 0.5) shows a tailing bell-shaped curve (with a plateau at high pH) characteristic of two ionizing groups: the thiol group (pKa 3.2) and the phosphonate group (pKa2 4.6) . The rate constant for the reaction of the monoanionic inhibitor with dinitrothiophenolate (k2 = 7 M-1.s-1) is 120 times larger than that of the dianionic species . The haloacetylphosphonates were found to be irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase from a variety of sources . They react with the active-site thiol group (Cys-149) and are half-site reagents with yeast glyceraldehyde-3-phosphate dehydrogenase . Thus, when two of the identical four subunits are modified the enzyme is catalytically inactive . The effects of pH (7-10), 2H2O and NAD+ on the reaction with the yeast enzyme were examined in detail . NAD+ enhances the alkylation rates . The second-order rate constant does not show a simple sigmoidal dependence on pH but rather a tailing bell-shaped curve (pKa 7.0 and 8.4) qualitatively similar to that obtained with dinitrothiophenol . There is no significant solvent isotope effect on the limiting rate constants and a normal isotope effect on the two pKa values . The results are consistent with the more reactive enzyme species containing a thiolate and an acidic group that may either donate a proton to the dianionic haloacetylphosphonate or orient the inhibitor.

Mol Cell Biol, 1991 May, 11(5), 2905 - 8
Is 20S RNA naked?
Widner WR, Matsumoto Y, Wickner RB.
The 20S RNA of Saccharomyces cerevisiae is a single-stranded, circular RNA virus . A previous study suggested that this RNA is part of a 32S ribonucleoprotein particle, being associated with multiple copies of a 23-kilodalton protein . We show here that this protein is, in fact, the chromosome-encoded heat shock protein Hsp26 . Furthermore, it is apparently not associated with 20S RNA and plays no obvious role in the life cycle of the virus.

Biotechniques, 1991 May, 10(5), 616 - 25
High-sensitivity DNA detection with a laser-excited confocal fluorescence gel scanner; Quesada MA et al.; A high-sensitivity, laser-excited confocal fluorescence gel scanner has been developed and applied to the detection of fluorescently labeled DNA . An argon ion laser (1-10 mW at 488 nm) is focused in the gel with a high-numerical aperture microscope objective . The laser-excited fluorescence is gathered by the objective and focused on a confocal spatial filter, followed by a spectral filter and photodetector . The gel is placed on a computer-controlled scan stage, and the scanned image of the gel fluorescence is stored and analyzed in a computer . This scanner has been used to detect DNA separated on sequencing gels, agarose mapping gels and pulsed field gels . Sanger sequencing gels were run on M13mp18 DNA using a fluoresceinated primer . The 400-microns-thick gels, loaded with 30 fmol of DNA fragments in 3-mm lanes, were scanned at 78-microns resolution . The high resolution of our scanner coupled with image processing allows us to read up to approximately 300 bases in four adjacent sequencing lanes . The minimum band size that could be detected and read was approximately 200 microns . This instrument has a limiting detection sensitivity of approximately 10 amol of fluorescein-labeled DNA in a 1 x 3-mm band . In applications to agarose mapping gels, we have exploited the fact that DNA can be prestained with ethidium homodimer, followed by electrophoresis and fluorescence detection to achieve picogram sensitivity . We have also developed methods using both ethidium homodimer and thiazole orange staining which permit two-color detection of DNA in one lane.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosoma, 1991 May, 100(4), 267 - 77
Replication analysis of plasmid DNAs injected into Drosophila embryos; Roth GE; From a "shotgun" collection of DNA fragments, isolated from Drosophila melanogaster, we selected sequences that function as autonomously replicating sequences (ARS) in the yeast Saccharomyces cerevisiae . To investigate the replicative potential of such sequences in Drosophila, five of these ARS elements and also the Adh gene of D . melanogaster, which has been described earlier to have ARS function in yeast, were microinjected into developing Drosophila eggs and analysed after reisolation from first instar larvae . As an assay for DNA replication, we determined the sensitivity of recovered plasmid DNA to restriction enzymes that discriminate between adenine methylation and non-methylation . Within the limits of detection our results show that none of the plasmids replicated two or more rounds . However, a fraction of all injected plasmid DNAs, including vector DNA, seems to replicate once . The same result was obtained for a DNA sequence from mouse that had been reported to have replication origin function in mouse tissue culture cells . We excluded the possibility that methylation of the plasmids is the reason for their inability to replicate . These results demonstrate that homologous and heterologous DNA sequences that drive replication of plasmids in cells of other species are not sufficient to fulfil this function in Drosophila embryos.

Arch Biochem Biophys, 1991 May 1, 286(2), 441 - 7
Fluorescent labeling of the nucleotide site in cytosolic rat liver phosphoenolpyruvate carboxykinase; Rojas MC et al.; Reaction of rat liver phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) with the alkylating fluorescent probe N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS), results in complete loss of enzymatic activity . One mole of the fluorescent reagent is incorporated per mole of the inactivated enzyme . When the modification is carried out in the presence of GDPMn, the enzyme retains 97% of its activity with almost no incorporation of label . The specificity of the reaction is further supported by the detection of a unique fluorescent peptide from the trypsin-treated modified enzyme . Fluorescence emission of enzyme-bound AEDANS shows a broad band centered at 470 nm and presents a monoexponential decay with a lifetime of 19 ns . These data indicate that the probe-binding site is considerably less polar than water and similar in polarity to ethanol . Anisotropy determinations give evidence for restricted rotational freedom for AEDANS bound to the rat carboxykinase, while acrylamide quenching studies reveal limited accessibility to the probe site . The results are consistent with specific labeling of rat liver phosphoenolpyruvate carboxykinase at or near the GDP site . The characteristics of the nucleotide-binding sites of rat liver and yeast (ATP) phosphoenolpyruvate carboxykinase are compared.

Trends Biochem Sci, 1991 May, 16(5), 187 - 90
Who's on first? The U1 snRNP-5' splice site interaction and splicing; Rosbash M et al.; U1 small nuclear ribonucleoprotein (snRNP) is important for pre-mRNA splicing both in yeast (Saccharomyces cerevisiae) and mammalian systems . The RNA component of U1 snRNP, U1 snRNA, interacts by base pairing with pre-mRNA 5' splice sites . This article examines recent evidence suggesting that U1 snRNP is important for an early step in spliceosome assembly rather than a late step that contributes to the specificity of 5' splice-site cleavage.

Trends Biochem Sci, 1991 May, 16(5), 173 - 7
The TPR snap helix: a novel protein repeat motif from mitosis to transcription; Goebl M et al.; The recently discovered TPR gene family encodes a diverse group of proteins that function in mitosis, transcription, splicing, protein import and neurogenesis . These multi-domain proteins all contain tandemly arranged repeats of a 34-amino acid motif that are presumed to form helix-turn structures, each with a 'knob' and 'hole', acting as helix-associating domains.

Yeast, 1991 May-Jun, 7(4), 413 - 24
The complete sequence of the unit YCR59, situated between CRY1 and MAT, reveals two long open reading frames, which cover 91% of the 10.1 kb segment; Jia Y et al.; We have entirely sequenced YCR59, which is a 10.1 kb segment of the right arm of chromosome III, and is part of the clone E5F from the Newlon collection . The segment contains two long open reading frames (ORFs): YCR591 which starts in the adjacent fragment H9G (situated towards CRY1 and the centromere), and continues with 1833 codons in YCR59 . The second ORFYCR592 is 1226 codons long and encoded entirely within YCR59 . The two ORFs represent 91% of the total length of the segment . Excellent agreement in both location and length is found between the ORFs YCR591 and YCR592 and the transcripts 86 and 87 respectively in the Yoshikawa and Isono (1990) map of chromosome III . The two ORFs correspond to new genes and show no significant similarity with any known genes.

J Inorg Biochem, 1991 May 1, 42(2), 97 - 103
Binding of mercury(II) to protein thiol groups: a study of proteinase K and carboxypeptidase Y; Bagger S et al.; Chemically modified enzymes have been prepared by incorporating an -Hg-L group into proteinase K and carboxypeptidase Y at the thiol groups of Cys-73 and Cys-341, respectively (L = CN- or I-) . The -S-Hg-13CN group has been applied as a spectroscopic label for carbon-13 NMR spectroscopy.

Plant Cell, 1991 May, 3(5), 531 - 40
The Arabidopsis functional homolog of the p34cdc2 protein kinase; Ferreira PC et al.; The p34cdc2 protein kinase is a key component of the eukaryotic cell cycle, which is required for G1 to S-phase transition and for entry into mitosis . Using a 380-base pair DNA fragment obtained by polymerase chain reaction amplification from an Arabidopsis thaliana flower cDNA library as a probe, we isolated and sequenced a cdc2-homologous cDNA from Arabidopsis . The encoded polypeptide has extensive homology with cdc2-like kinases . Furthermore, when expressed in a CDC28ts Saccharomyces strain, it partially restores the capacity to grow at 36 degrees C, indicating that the plant cDNA is a functional homolog of the p34cdc2 kinase . Genomic hybridization demonstrated that there is one copy of the cdc2 gene per Arabidopsis haploid genome . Using RNA gel blot analysis, we found that cdc2 mRNA is present in all plant organs.

Genes Dev, 1991 May, 5(5), 773 - 85
Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs; Shannon KW et al.; U4 and U6 small nuclear RNAs are associated by an extensive base-pairing interaction that must be disrupted and reformed with each round of splicing . U4 mutations within the U4/U6 interaction domain destabilize the complex in vitro and cause a cold-sensitive phenotype in vivo . Restabilization of the U4/U6 helix by dominant (gain-of-function), compensatory mutations in U6 results in wild-type growth . Cold-insensitive growth can also be restored by two classes of recessive (loss-of-function) suppressors: (1) mutations in PRP24, which we show to be a U6-specific binding protein of the RNP-consensus family; and (2) mutations in U6, which lie outside the interaction domain and identify putative PRP24-binding sites . Destabilization of the U4/U6 helix causes the accumulation of a PRP24/U4/U6 complex, which is undetectable in wild-type cells . The loss-of-function suppressor mutations inhibit the binding of PRP24 to U6, and thus presumably promote the release of PRP24 from the PRP24/U4/U6 complex and the reformation of the base-paired U4/U6 snRNP . We propose that the PRP24/U4/U6 complex is normally a highly transient intermediate in the spliceosome cycle and that PRP24 promotes the reannealing of U6 with U4.

Bioessays, 1991 May, 13(5), 243 - 51
Fragile X-linked mental retardation and the difficulties of reverse genetics; Jordan BR; Fragile X-linked mental retardation is an enigmatic inheritable syndrome in which severe mental retardation, a cytogenetically detectable fragile site at Xq27.3 (FraX) and a number of dysmorphic features are associated . Genetic analysis shows that the mode of inheritance is more complex than a straightforward X-linked recessive trait and probably involves a two-step process for which several models have been proposed . Early attempts at 'cloning the fragile site' provided several DNA segments lying in its general vicinity, and large scale DNA mapping methods were extensively applied in an effort to generate maps including this region . These efforts were complemented by more focussed methods such as microdissection; together these approaches have now provided a number of DNA segments within a 5 cM interval around FraX, and with the help of these new probes the site is indeed being cloned . Unravelling the nature of the sequence(s) responsible for the mental retardation syndrome will probably take some time, however.

Biochemistry, 1991 Apr 30, 30(17), 4212 - 22
Sequential assignments of the 1H NMR resonances of Zn(II)2 and 113Cd(II)2 derivatives of the DNA-binding domain of the GAL4 transcription factor reveal a novel structural motif for specific DNA recognition; Pan T et al.; The DNA-binding domain of the GAL4 transcription factor, consisting of the 62 N-terminal amino acid residues and denoted GAL4(62*), contains a novel Zn(II)2Cys6 or Cd(II)2Cys6 binuclear cluster {Pan, T., & Coleman, J . E . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 2077} . Specific DNA recognition requires residues located within as well as C terminal to this binuclear cluster . 1H NMR sequential assignments have been carried out on Zn(II)2- and 113Cd(II)2GAL4(62*) by using DQF-COSY, relayed COSY, double-relayed COSY, and NOESY . The ligands of the two tetrahedral metal-binding sites have been identified as Cys11, Cys14, Cys21, and Cys31 to one metal ion and Cys28, Cys38, Cys21, and Cys31 to the other metal ion with Cys21 and Cys31 as ligands shared between the two metal ions . No alpha-helices can be found within the GAL4(62*) structure, which consists of a series of turns to accommodate the metal cluster, followed by irregular loops and turns from residues 42 to 60, the "specificity region", whose sequence contributes importantly to specific DNA recognition . Long-distance NOE's are observed between residues forming the binuclear cluster and several residues within the specificity region, indicating that the latter is folded compactly onto the metal cluster . The requirement of the Zn(II)2Cys6 binuclear cluster and the specificity region for binding to DNA reveals GAL4 as a member of a class of specific DNA-binding proteins using a new structural motif for the recognition of specific DNA sequences . Specific DNA binding by this class of proteins is achieved by use of turns and loops that enclose a Zn(II)2Cys6 binuclear cluster, instead of alpha-helices or beta-strands as observed in specific DNA-binding proteins described previously.

Biochemistry, 1991 Apr 23, 30(16), 4068 - 71
Secondary 15N isotope effects on the reactions catalyzed by alcohol and formate dehydrogenases; Rotberg NS et al.; Secondary 15N isotope effects at the N-1 position of 3-acetylpyridine adenine dinucleotide have been determined, by using the internal competition technique, for horse liver alcohol dehydrogenase (LADH) with cyclohexanol as a substrate and yeast formate dehydrogenase (FDH) with formate as a substrate . On the basis of less precise previous measurements of these 15N isotope effects, the nicotinamide ring of NAD has been suggested to adopt a boat conformation with carbonium ion character at C-4 during hydride transfer {Cook, P . F., Oppenheimer, N . J . & Cleland, W . W . (1981) Biochemistry 20, 1817} . If this mechanism were valid, as N-1 becomes pyramidal an 15N isotope effect of up to 2-3% would be observed . In the present study the equilibrium 15N isotope effect for the reaction catalyzed by LADH was measured as 1.0042 +/- 0.0007 . The kinetic 15N isotope effect for LADH catalysis was 0.9989 +/- 0.0006 for cyclohexanol oxidation and 0.997 +/- 0.002 for cyclohexanone reduction . The kinetic 15N isotope effect for FDH catalysis was 1.004 +/- 0.001 . These values suggest that a significant 15N kinetic isotope effect is not associated with hydride transfer for LADH and FDH . Thus, in contrast with the deformation mechanism previously postulated, the pyridine ring of the nucleotide apparently remains planar during these dehydrogenase reactions.

FEBS Lett, 1991 Apr 22, 282(1), 189 - 92
Identification of nuclear factor IV/Ku autoantigen in a human 2D-gel protein database . Modification of the large subunit depends on cellular proliferation; Stuiver MH et al.; Nuclear Factor IV (NFIV) is a heterodimeric DNA-binding protein from HeLa cells, recognizing molecular ends and is identical to the autoantigenic target Ku . We have identified the two NFIV/Ku subunits, by comigration, in the 2D-gel database of transformed human amnion cell (AMA) proteins . We observed that the large subunit of NFIV/Ku consists of at least 3 charge variants that correspond to SSP IEFs 5705 (81.2 kDa, pI 5.74), 6707 (81.2 kDa, pI 5.67) and 6706 (81.9 kDa, pI 5.60) in the AMA catalogue . The relative amounts of the 2 major variants (IEFs 5705 and 6707) was dependent on the state of cell proliferation . Inhibition of DNA-synthesis by hydroxyurea also changed the relative levels of the variants, whereas aphidicolin or a thymidine block had no effect . These results suggest a possible role for NFIV/Ku in DNA replication.

Biochem J, 1991 Apr 15, 275 ( Pt 2), 541 - 3
Purification and characterization of truncated ribonuclease inhibitor; Hofsteenge J et al.; A recombinant pig ribonuclease inhibitor (delta r-RI) lacking 90 or 93 N-terminal amino acid residues was isolated from a preparation of recombinant inhibitor . The kinetic parameters for the inhibition of ribonuclease A by delta r-RI were determined and found to be only slightly altered in comparison with the full-length inhibitor . The deletion did, however, affect the surface properties of RI . The results are discussed in relation to those obtained by Lee & Vallee {(1990) Proc . Natl . Acad . Sci . U.S.A . 87, 1879-1883}.

Proc Natl Acad Sci U S A, 1991 Apr 15, 88(8), 3125 - 9
Antiestrogen can establish nonproductive receptor complexes and alter chromatin structure at target enhancers; Pham TA et al.; We describe in this report experiments in vivo that demonstrate that antiestrogens promote DNA binding of the estrogen receptor without efficiently inducing transcription . When the receptor is modified to carry a foreign unregulated transactivation domain, transcription can be induced efficiently by both estrogen and antiestrogens . Under apparent saturation conditions, antihormone-receptor complexes binding to responsive enhancer elements elicit only a low level of transcription . In addition, we show that both estrogen and an antiestrogen, nafoxidine, effect very similar alterations in chromatin structure at a responsive promoter . These results indicate that in vivo steroid receptor action can be regulated subsequent to the DNA binding step, by regulating interactions with the target transcriptional machinery . In this regard, antihormones can function by establishing receptor-DNA complexes that are transcriptionally nonproductive.

Proc Natl Acad Sci U S A, 1991 Apr 15, 88(8), 3102 - 5
Localization of the estrogen-binding site of alpha-fetoprotein in the chimeric human-rat proteins; Nishi S et al.; Rat alpha-fetoprotein (AFP) has been demonstrated to bind estrogen, whereas human AFP lacks the activity . We constructed four chimeric molecules from cDNAs encoding these AFPs with the use of two restriction sites common to them and expressed them as well as rat and human AFP cDNA in yeast . The recombinant molecules were purified, characterized, and found to have the predicted structures . Analyses of estrogen binding indicated that a rat AFP sequence composed of residues 423-506 that contains 31 rat-specific amino acids is essential for the activity.

Eur J Biochem, 1991 Apr 10, 197(1), 81 - 91
The roles of ADP2- and Mg2+ in control steps of phosphoglycerate kinase; Graham HC et al.; 1H-NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide, ADP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ADP binding to the enzyme . A preliminary study of adenosine binding to phosphoglycerate kinase was made in order to be sure of the nature of the adenine site . From the change in chemical shifts of the 'basic patch' histidine resonances (His62, 167 and 170), the nucleotide C8-H, C2-H and C1'-H resonances and resonances 40 and 41 (assigned to Thr373 and Thr375 in the hydrophobic, i.e . catalytic, site), it is apparent that there are at least two ADP binding sites on the enzyme: one at the hydrophobic (catalytic) site and one at the electrostatic site . A comparison of the results for ADP and ATP reveals differences due to the differential binding of the phosphate groups . The presence of Mg2+ results in further differences being observed . The data suggest that the primary binding site of ADP, in the absence of Mg2+, involves electrostatic interactions between the diphosphate chain of the substrate and the 'basic patch' region of the N-terminal domain . In the presence of greater than or equal to 1:1 ratio of Mg2+/ADP, however, the primary binding site involves predominantly hydrophobic interactions between the adenosine moiety and the catalytic site, with secondary binding occurring at the electrostatic site . Addition of Mg2+, therefore, tends to reduce the affinity of the electrostatic site (presumably by competing for ADP) . It is suggested that alpha-helix XII, including residues 372, 373 and 375, moves differentially on binding ADP, Mg ADP, ATP or Mg . ATP, consistent with Mg2+ assisting the transfer of the gamma-phosphate of ATP to 3-phosphoglycerate during catalysis.

FEBS Lett, 1991 Apr 9, 281(1-2), 223 - 6
Polypeptide-metal cluster connectivities in Cd(II) GAL4; Gadhavi PL et al.; Two-dimensional 1H-113Cd correlation NMR spectra have been used to determine the polypeptide/metal cluster connectivities in Cd(II) GAL4 . The results show that the protein contains a two metal ion cluster where Cys-11 and Cys-28 are the bridging ligands.

Biochemistry, 1991 Apr 9, 30(14), 3365 - 71
ADR1a, a zinc finger peptide, exists in two folded conformations; Xu RX et al.; Two-dimensional NMR (2DNMR) studies of several different zinc finger peptides have yielded a picture of the three-dimensional structure of this small DNA-binding motif . Details of the differences among fingers with different sequences may provide some insight into how these domains interact with DNA . Toward this end, we have reanalyzed the 2DNMR spectra of the C-terminal zinc finger sequence from the yeast transcriptional factor ADR1 . Although this was the sequence on which our original report describing the overall fold of zinc fingers was based, complete spectral assignments (reported here) were needed to compare this sequence in detail with that of ADR1b, for which we have reported an atomic level structure . In the process of analyzing the spectra of ADR1a and a mutant of ADR1a, it was noted that the peptides give two sets of NMR lines, indicating that this sequence, unlike the other ADR1 zinc finger sequence, exists in two slowly interconverting folded conformations in solution . Residues that exhibit peak doubling are located in the Cys loop, the alpha-helix, and the extreme C-terminus of the peptide . Differences in NOEs observed for the two forms indicate that there are detectable conformational differences in the Zn2+ cluster and in the fingertip region . This conformational flexibility, which has not been observed for other zinc finger peptides, may stem from the presence of an additional residue between the histidine ligands (His-X4-His versus His-X3-His).

J Biol Chem, 1991 Apr 5, 266(10), 6456 - 61
Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme; Taniyama Y et al.; Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M . (1990) J . Biol . Chem . 265, 7570-7575) . Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91 . The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding . Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M . (1988) Biochem . Biophys . Res . Commun . 152, 962-967) . The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities . Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule . The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond . These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.

Nature, 1991 Apr 4, 350(6317), 436 - 8
A mediator required for activation of RNA polymerase II transcription in vitro; Flanagan PM et al.; Activator proteins bind to enhancer DNA elements and stimulate the initiation of transcription . It has been proposed that activators contact general initiation factors at a promoter, and evidence for such direct interaction has been obtained . Studies of transcription in vitro, however, have suggested that activators might function through an intermediary molecule(s) distinct from the general factors . In the first of these studies, we exploited the finding that one activator could inhibit transcription stimulated by a second activator (activator interference or 'squelching') . This inhibition, which is attributed to competition between the activators for a common target factor, could not be relieved by addition of a large excess of general initiation factors, suggesting that the target for which activators compete is distinct from these factors . Similar conclusions came from the observation that TFIID's expressed from cloned genes fail to replace partially purified 'natural' TFIID fractions in supporting activation, evidently because they lacked some component present in the impure fractions . While these lines of evidence for a novel 'mediator' of activation were negative, we also showed that a partially purified fraction from yeast would reverse activator interference . This positive effect of a presumptive mediator provided an assay for its activity, but its role in activation was still only inferred . We now present direct evidence for a mediator which is required for stimulation of transcription in vitro by the activators GAL4-VP16 and GCN4, but which has no effect on transcription in the absence of activator protein.

Biochim Biophys Acta, 1991 Apr 2, 1063(2), 197 - 202
Protein-catalyzed transport of ether phospholipids; Szolderits G et al.; The protein-catalyzed transfer of alkenylacyl-, alkylacyl-, or diacyl-glycerophosphocholines, carrying a pyrenedecanoyl residue as a fluorogenic acyl chain, was studied using unilamellar bilayer vesicles as donor and acceptor membranes in a fluorescence assay . Specific phospholipid transfer proteins, such as phosphatidylinositol transfer protein from yeast and phosphatidylcholine transfer protein from bovine liver showed higher transfer rates with ether lipid substrates . Transfer rates for alkylacyl- and alkenylacyl-glycerophosphocholine as compared to the diacyl analog were rather similar in the presence of non-specific lipid transfer proteins from maize or from bovine liver, respectively . When vesicles of fluorogenic compounds were titrated with the yeast phosphatidylinositol transfer protein, only a 15-20% higher binding affinity for alkenylacyl- and alkylacyl-glycerophosphocholine than for diacyl-glycerophosphocholine was observed . Thus the marked differences of transfer rates measured with this transfer protein cannot be attributed to different binding affinities for the respective phospholipid subclasses . A possible explanation for differences in transfer rates could be differences in the organization of the phospholipid subclasses at the hydrophobic/hydrophilic interface of bilayer membranes.

Mutat Res, 1991 Apr, 247(2), 213 - 9
Properties and applications of human DNA repair genes; Thompson LH; The importance of understanding DNA repair processes is discussed in terms of the origins of human cancer . Several human repair genes have been mapped to specific human chromosomes using somatic cell hybrids . It is noteworthy that 3 of these genes lie in the same region of chromosome 19: genes ERCC1 and ERCC2, which are involved in nucleotide excision repair, and XRCC1, which is involved in the repair of strand breaks . The genes XRCC1 and ERCC2 were cloned from cosmid libraries prepared from DNA transformants of the CHO mutants EM9 and UV5, respectively . Analysis of the cDNA sequence of ERCC2 showed that the protein encoded by this gene is highly homologous (73%) to the RAD3 repair protein in the yeast Saccharomyces cerevisiae . Thus, the known properties of RAD3 combined with the high homology provide the first insight about the biochemical role of a human repair protein involved in the incision step of nucleotide excision repair . So far XRCC1 is the only cloned mammalian gene involved in repairing damage from ionizing radiation . The UV5 mutant line was also applied to problems in environmental mutagenesis by introducing the mouse cytochrome P(3)450 (P450IA2 subfamily) gene for metabolic activation of aromatic amines . We show in a rapid differential cytotoxicity assay with 2 compounds found in cooked beef (IQ, 2-amino-3-methylimidazo{4,5-f}quinoline and PhIP, 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine) that this gene is efficiently expressed in the transformed UV5P3 cells . Reversion of the repair deficiency in these cells will give a matched pair of cell lines that are metabolically proficient and repair deficient . Such lines will provide a rapid assay for genotoxic heterocyclic amines requiring activation.

Genes Dev, 1991 Apr, 5(4), 629 - 41
A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family; Strauss EJ et al.; We have isolated a cold-sensitive mutant of Saccharomyces cerevisiae in which the first step of mRNA splicing is inhibited . The growth and splicing defects are recessive and cosegregate, thus defining a single essential gene (PRP28) . The wild-type PRP28 gene was cloned, and sequence analysis reveals extensive homology to a family of proteins that are thought to function as ATP-dependent RNA helicases . The cold sensitivity is caused by a glycine-to-glutamic acid change in a conserved sequence motif . Interestingly, double mutants containing conditional alleles of PRP28 and PRP24, which encodes a U6 snRNA-binding protein, are inviable . In addition, a suppressor of prp28-1 is a mutant allele of PRP8, which encodes a U5 protein, thus linking PRP28 with U5 . These data are consistent with a scenario in which PRP28 acts to unwind the U4/U6 base-pairing interaction in the U4/U6/U5 snRNP, facilitating the first covalent step of splicing.

Genes Dev, 1991 Apr, 5(4), 549 - 60
A suppressor of a centromere DNA mutation encodes a putative protein kinase (MCK1); Shero JH et al.; A new approach to identify genes involved in Saccharomyces cerevisiae kinetochore function is discussed . A genetic screen was designed to recover extragenic dosage suppressors of a CEN DNA mutation . This method identified two suppressors, designated MCK1 and CMS2 . Increased dosage of MCK1 specifically suppressed two similar CEN DNA mutations in CDEIII, but not comparably defective CEN DNA mutations in CDEI or CDEII . A strain containing a null allele of MCK1 was viable under standard growth conditions, had a cold-sensitive phenotype (conditional lethality at 11 degrees C), and grew slowly on Benomyl (a microtubule-destabilizing drug) . Furthermore, when grown at 18 degrees C or in the presence of Benomyl, the null mutant exhibited a dramatic increase in the rate of mitotic chromosome loss . The allele-specific suppression and chromosome instability phenotypes suggest that MCK1 plays a role in mitotic chromosome segregation specific to CDEIII function . The MCK1 gene encodes a putative protein-serine/threonine kinase, which suggests a possible role for the MCK1 protein in regulating the activity of centromere-binding proteins by phosphorylation . MCK1 was identified and cloned independently for its involvement in the induction of meiosis and is identical to a gene that encodes a phosphotyrosyl protein with protein kinase activity.

Mol Cell Biol, 1991 Apr, 11(4), 2162 - 8
In vitro studies of the binding of the ARGR proteins to the ARG5,6 promoter; Dubois E et al.; ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae . We show here that they are also required in vitro to observe a protein-DNA complex with the promoter of the ARG5,6 gene . The specific binding of ARGR proteins in vitro is stimulated by arginine . Antibodies raised against a synthetic MCM1 polypeptide retard the migration of ARGR-DNA complex on gel mobility shift assays . This result suggests that MCM1 could be an additional regulatory element of arginine metabolism.

Int J Biol Macromol, 1991 Apr, 13(2), 97 - 100
Mapping of isozymic differences in enolase; Lebioda L et al.; The existence of the isozymes of non-regulatory enzymes often has been linked to their interaction with other macromolecules . Enolase, a non-regulatory enzyme, has three isozymes for which sequences have been determined in two or more vertebrate species . The positions in the enolase sequences that differ between the isozymes were mapped in the 3-D structure of the enzyme . The positions in a given isozymic form which were not conserved in different species were considered to be resulting from the neutral drift of sequences and rejected . Also, the residues with no accessible surface were rejected . Three areas with relatively high densities of isozymic substitutions were found . We consider them as the likely sites of contact with other macromolecules.

Curr Genet, 1991 Apr, 19(4), 309 - 12
Short dispersed repeats localized in spacer regions of Chlamydomonas reinhardtii mitochondrial DNA; Boer PH et al.; In the mtDNA of Chlamydomonas reinhardtii, a unicellular green alga, we have identified a set of short repeated sequences up to 65 nucleotides long, each of which contains the palindromic consensus motif CTCGG(N4-14)CCGAG . Most of these repeated elements are localized in spacer regions that flank the transcribed coding regions of C . reinhardtii mtDNA . These algal mitochondrial repeats have features reminiscent of short repeats in some fungal mtDNAs, such as GC clusters in Saccharomyces cerevisiae and PstI palindromes in Neurospora crassa . The location of these elements suggests that they could play a role in gene expression, e.g., post-transcriptional processing, in C . reinhardtii mitochondria.

J Cell Biol, 1991 Apr, 113(1), 1 - 12
The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs; Lee WC et al.; We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences . We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein . p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast . Using antibodies against p67 we have cloned the gene for this protein . The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo . Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats . We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted . We have also shown that NSR1 (p67) is required for normal cell growth.

J Biotechnol, 1991 Apr, 18(1-2), 153 - 60
On-line determination of glucose in biotechnological processes: comparison between FIA and an in situ enzyme electrode; Filippini C et al.; Two different analysis techniques for on-line monitoring of glucose in biotechnological processes have been tested: an in situ enzyme electrode and a flow injection analysis system (FIA) . The measuring ranges, detection limits, response times and the reliabilities of each system have been compared during monitoring of batch and continuous cultures of Saccharomyces cerevisiae.

Biotechnology (N Y), 1991 Apr, 9(4), 378 - 81
Efficient KEX2-like processing of a glucoamylase-interleukin-6 fusion protein by Aspergillus nidulans and secretion of mature interleukin-6; Contreras R et al.; We have designed an expression vector for the secretion of human interleukin-6 (hIL-6) in which the mature protein is fused through a spacer peptide, containing a KEX-2 like protein processing signal, to the entire Aspergillus niger glucoamylase (glaA) gene . Transformation of Aspergillus nidulans with this vector results in fungal strains secreting equimolar amounts of the glucoamylase and IL-6 proteins . The KEX2-type processing signal, Lys-Arg, is recognized and cleaved efficiently by an enzyme present in A . nidulans resulting in the secretion of an authentic mature hIL-6 protein at levels of up to 5 mg/l.

Eur J Biochem, 1991 Mar 28, 196(3), 735 - 42
Determination of disulfide bridges in natural and recombinant insect defensin A; Lepage P et al.; The primary-structure comparison of natural insect defensin A from Phormia terranovae and recombinant insect defensin A from Saccharomyces cerevisiae has been accomplished using a combination of Edman degradation and liquid secondary ion mass spectrometry . The natural and recombinant proteins have the same primary structure with identical disulfide-bond designations (formula; see text) as determined from the peptides obtained after thermolysin digestion . The combined use of Edman degradation and mass spectometry allowed the disulfide-bridge structure to be determined with a total of only 40 micrograms (9.9 nmol) natural peptide . Mass spectrometry provides a rapid means of disulfide-bridge verification, requiring not more than 20 micrograms recombinant insect defensin A, which is compatible with use in batch analysis.

Cell, 1991 Mar 22, 64(6), 1155 - 61
Extensive 3'-overhanging, single-stranded DNA associated with the meiosis-specific double-strand breaks at the ARG4 recombination initiation site; Sun H et al.; Meiosis-specific double-strand breaks occur at the initiation site for meiotic gene conversion in the yeast ARG4 gene . Here we show that the break fragments end in extensive 3'-overhanging, single-stranded tails . The single-stranded tails very in length, generating a gradient of single-strandedness that parallels the gradient of gene conversion frequencies in ARG4 . In strains carrying a rad50S mutation, which blocks meiotic recombination, the extensive single-stranded tails do not form, suggesting that their generation is an obligatory step in meiotic recombination . Using the rad50S mutant, we have mapped the site of the ARG4 break to a small region within the genetically defined recombination initiation site . These results strongly support the double-strand break model of meiotic recombination.

Cell, 1991 Mar 22, 64(6), 1135 - 43
A functional interaction between the C-terminal domain of RNA polymerase II and the negative regulator SIN1; Peterson CL et al.; The C-terminal domain (CTD) of the largest subunit of yeast RNA polymerase II contains 26-27 tandem copies of a conserved heptapeptide of unknown function . Yeast strains whose CTD contains ten heptamers are viable but defective for transcription of the INO1 gene and cold sensitive for growth . Deletion of the SIN1 gene, which codes for a DNA-binding protein that negatively regulates HO transcription, restores INO1 transcription and reduces the cold sensitivity of such strains . A SIN1 deletion suppresses the lethality of a CTD with nine heptamer repeats but not with seven repeats . These observations indicate a functional relationship between SIN1 and the CTD: the CTD might remove SIN1 from DNA, or removal of SIN1 may be a prerequisite for function of the CTD . The SWI1, SWI2, and SWI3 genes, whose products activate HO transcription by antagonizing SIN1, are also required for INO1 transcription and may assist the CTD . In addition, an intact CTD binds nonspecifically to DNA in vitro.

Biochemistry, 1991 Mar 19, 30(11), 2823 - 7
Inhibition of enolase: the crystal structures of enolase-Ca2(+)- 2-phosphoglycerate and enolase-Zn2(+)-phosphoglycolate complexes at 2.2-A resolution; Lebioda L et al.; Enolase is a metalloenzyme which catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP) . Mg2+ and Zn2+ are cofactors which strongly bind and activate the enzyme . Ca2+ also binds strongly but does not produce activity . Phosphoglycolate (PG) is a competitive inhibitor of enolase . The structures of two inhibitory ternary complexes: yeast enolase-Ca2(+)-PGA and yeast enolase-Zn2(+)-PG, were determined by X-ray diffraction to 2.2-A resolution and were refined by crystallographic least-squares to R = 14.8% and 15.7%, respectively, with good geometries of the models . These structures are compared with the structure of the precatalytic ternary complex enolase-Mg2(+)-PGA/PEP (Lebioda & Stec, 1991) . In the complex enolase-Ca2(+)-PGA, the PGA molecule coordinates to the Ca2+ ion with the hydroxyl group, as in the precatalytic complex . The conformation of the PGA molecule is however different . In the active complex, the organic part of the PGA molecule is planar, similar to the product . In the inhibitory complex, the carboxylic group is in an orthonormal conformation . In the inhibitory complex enolase-Zn2(+)-PG, the PG molecule coordinates with the carboxylic group in a monodentate mode . In both inhibitory complexes, the conformational changes in flexible loops, which were observed in the precatalytic complex, do not take place . The lack of catalytic metal ion binding suggests that these conformational changes are necessary for the formation of the catalytic metal ion binding site.

Biochemistry, 1991 Mar 19, 30(11), 2817 - 22
Mechanism of enolase: the crystal structure of enolase-Mg2(+)-2-phosphoglycerate/phosphoenolpyruvate complex at 2.2-A resolution; Lebioda L et al.; Enolase in the presence of Mg2+ catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP) and the reverse reaction, the hydration of PEP to PGA . The structure of the ternary complex yeast enolase-Mg2(+)-PGA/PEP has been determined by X-ray diffraction and refined by crystallographic restrained least-squares to an R = 16.9% for those data with I/sigma (I) greater than or equal to 2 to 2.2-A resolution with a good geometry of the model . The structure indicates the substrate molecule in the active site has its hydroxyl group coordinated to the Mg2+ ion . The carboxylic group interacts with the side chains of His373 and Lys396 . The phosphate group is H-bonded to the guanidinium group of Arg374 . A water molecule H-bonded to the carboxylic groups of Glu168 and Glu211 is located at a 2.6-A distance from carbon-2 of the substrate in the direction of its proton . We propose that this cluster functions as the base abstracting the proton in the catalytic process . The proton is probably transferred, first to the water molecule, then to Glu168, and further to the substrate hydroxyl to form a water molecule . Some analogy is apparent between the initial stages of the enolase reverse reaction, the hydration of PEP, and the proteolytic mechanism of the metallohydrolases carboxypeptidase A and thermolysin . The substrate/product binding is accompanied by large movements of loops Ser36-His43 and Ser158-Gly162 . The role of these conformational changes is not clear at this time.

Biochem Biophys Res Commun, 1991 Mar 15, 175(2), 492 - 9
Spectroscopic studies of the DNA binding site of the GAL4 "zinc finger" protein; Hansen A et al.; The yeast GAL4 protein, a transcriptional activator of genes involved in galactose metabolism, binds as a dimer to several closely related seventeen base pair upstream activation sequences (UASGs) that are nearly symmetric about a central dT-dA base pair . A previous study of a GAL4-UASG complex (Carey, M., Kakidani, H., Leatherwood, J., Mostashari, F . and Ptashne, M . (1989) J . Mol . Biol . 209, 423-432) elucidated a pattern of contacts consistent with the protein partially wrapping itself around the helical cylinder, assuming a B-form conformation for the DNA . Alternatively, both monomers could sit on one face of the cylinder if the DNA exists in an underwound conformation such as A-form . Spectroscopic studies that distinguish between these models are reported here . Oligonucleotides containing the consensus UASG or a nine base pair "half site" both exhibit circular dichroism (CD) spectra characteristic of B-form DNA . Two-dimensional NMR studies of the half-site also indicate a B-form conformation . When a GAL4 protein fragment containing the entire DNA-binding and dimerization domains (amino acids 1-140) is bound to the UASG, the CD spectrum above 240 nm changes only slightly, and not in a manner consistent with DNA unwinding . Our studies suggest that the UASG does not adopt an unusual underwound conformation in the absence or presence of the GAL4 protein, and favor the model in which the dimer partially wraps around the helix cylinder.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2113 - 7
A second maternally expressed Drosophila gene encodes a putative RNA helicase of the "DEAD box" family; de Valoir T et al.; Recently, a family of proteins containing the conserved motif Asp-Glu-Ala-Asp, the "DEAD box" proteins, has been identified . This family is typified by the eukaryotic translation initiation factor eIF4A, and its members are believed to share the functional property of ATP-dependent RNA unwinding . One of the previously identified members of this family (vasa) is the product of a maternally expressed gene from Drosophila melanogaster that is known to play a role in the formation of the embryonic body plan . We report here the isolation of a Drosophila gene that has an mRNA expression pattern somewhat similar to that of vasa and also encodes a DEAD box protein . We have termed this gene ME31B to reflect its maternal (ovarian germ-line) expression and its location within the 31B chromosome region . Comparisons with the other members of this family reveal that although ME31B is most like the protein Tif1/Tif2, which probably represents the Saccharomyces cerevisiae version of eIF4A, it is unlikely that ME31B represents the Drosophila eIF4A protein per se . A search for mutations in the ME31B gene has established that the P element which causes the female-sterile mutation flipper lies in the 3' flank of the ME31B gene.

J Biol Chem, 1991 Mar 15, 266(8), 5286 - 90
Analysis of naturally occurring and site-directed mutations in the argininosuccinate lyase gene; Barbosa P et al.; Argininosuccinic aciduria is an inborn error of metabolism due to the genetic deficiency of argininosuccinate lyase . In order to determine the molecular basis for the disease, RNA isolated from cultured skin fibroblasts derived from four unrelated patients was reverse-transcribed and amplified using the polymerase chain reaction and the products were cloned and sequenced . Three single base missense mutations were identified: Arg111----Trp, Gln286----Arg, and Arg193----Gln . One single base amber mutation was identified at Gln454 . One mutation involved a 13-base pair deletion within exon 13, and it was noted that the majority of the mature RNA derived from this allele was deleted for the entire exon rather than containing the exon with the 13 bases deleted . A final mutation was observed in which exon 2 was deleted from the mature RNA . The molecular basis for this deletion was not determined . Of the eight potential mutations present in the four cell lines studied, six mutations were identified and further data indicate that the remaining two unidentified mutations were different from those identified . Two site-directed mutations were created in the cDNA, Lys51----Asn and His89----Gln, and these were expressed in yeast . The Lys51 mutation caused an approximate 2-fold reduction in activity and the His89 mutation resulted in an approximate 10-fold reduction in activity . The combination of determination of naturally occurring mutations and the study of the effect of site-directed mutations on the activity of argininosuccinate lyase provide insight into the amino acid residues critical to the function of the enzyme.

Eur J Biochem, 1991 Mar 14, 196(2), 519 - 23
Long-chain-acyl-CoA synthetase and very-long-chain-acyl-CoA synthetase activities in peroxisomes and microsomes from rat liver . An enzymological study; Lageweg W et al.; We have investigated the palmitic acid (C16:0) and cerotic acid (C26:0) activating activities in rat-liver microsomes and peroxisomes . The activation of the two fatty acids showed similar dependencies on ATP and coenzyme A, reflected in about equal apparent Km values both in microsomes and peroxisomes . In microsomes and peroxisomes similar apparent Km values for palmitic acid were found (15 microM and 22.8 microM, respectively), whereas apparent Km values for cerotic acid were 8.4 microM and 1.0 microM in microsomes and peroxisomes, respectively . The activation of cerotic acid was found to be inhibited to a progressively greater extent by increasing concentrations of 1-pyrenedecanoic acid (P10) as compared to the activation of palmitic acid, both in microsomes and peroxisomes . The inhibition by P10 of palmitic acid activation and cerotic acid activation was non-competitive in both organelles . From the observation that P10 activation is not affected by palmitic acid and cerotic acid, we conclude that P10 is activated by a distinct enzyme . Furthermore, our results are in accordance with earlier suggestions that activation of cerotic acid is brought about by an enzyme distinct from the palmitoyl-CoA synthetase.

Science, 1991 Mar 8, 251(4998), 1236 - 9
Isolation of sequences that span the fragile X and identification of a fragile X-related CpG island; Heitz D et al.; Yeast artificial chromosomes (YACs) were obtained from a 550-kilobase region that contains three probes previously mapped as very close to the locus of the fragile X syndrome . These YACs spanned the fragile site in Xq27.3 as shown by fluorescent in situ hybridization . An internal 200-kilobase segment contained four chromosomal breakpoints generated by induction of fragile X expression . A single CpG island was identified in the cloned region between markers DXS463 and DXS465 that appears methylated in mentally retarded fragile X males, but not in nonexpressing male carriers of the mutation nor in normal males . This CpG island may indicate the presence of a gene involved in the clinical phenotype of the syndrome.

Anal Biochem, 1991 Mar 2, 193(2), 287 - 91
Radiochemical assay of adenylosuccinase: demonstration of parallel loss of activity toward both adenylosuccinate and succinylaminoimidazole carboxamide ribotide in liver of patients with the enzyme defect; Van den Bergh F et al.; A radiochemical assay for adenylosuccinase, an enzyme which intervenes twice in the biosynthesis of adenine nucleotides, has been developed . The two substrates of the enzyme, succinylaminoimidazole carboxamide ribotide (SAICAR) and adenylosuccinate (S-AMP), were synthesized in radioactive form by incubating {2,3-14C}fumarate and, respectively, AICAR and AMP with partially purified adenylosuccinase from yeast . Enzyme activities were determined by measuring the release of labeled fumarate after its separation from the substrate by chromatography on polyethyleneimine thin-layer plates . The ratio of the activity of adenylosuccinase measured with SAICAR compared to that with S-AMP was about 1 in crude extracts of rat liver and muscle and around 0.5 in human liver . In rat and human liver, but not in rat muscle, 20 to 40% of both activities of adenylosuccinase were lost after freezing at -80 degrees C followed by thawing . In the liver of patients with adenylosuccinase deficiency, in whom the deficiency had hitherto been measured only with S-AMP, the activity of the enzyme toward S-AMP and SAICAR was found to be lost in parallel . This is in accordance with the finding that both SAICA-riboside and succinyladenosine accumulate in adenylosuccinase-deficient patients.

Parasite Immunol, 1991 Mar, 13(2), 137 - 45
Administration of beta-glucan following Leishmania major infection suppresses disease progression in mice; Goldman R et al.; The potential of beta-glucan (glucan) to suppress the progression of lesions caused by virulent strains of Leishmania major in genetically susceptible BALB/c mice when administered post challenge was evaluated . Glucan particles (glucanp) prepared from Saccharomyces cerevisiae were injected i.v . at 7-day intervals starting 7 days after parasite challenge . Four injections gave a more rapid and a higher extent of suppression than 1, 2 or 3 injections . Mice receiving only parasites, a glucose solution, starch particles or glucanp by the i.p . route showed a progressive increase in footpad thickness and developed ulcerating lesions . An alkali solubilized glucan (glucanas) was injected (50 micrograms, 200 micrograms and 400 micrograms/mouse) 4 times at 4 day intervals either i.v . or i.p . starting four days post parasite challenge . Glucanas injection by either route blocked lesion development; the 50 micrograms treatment had already substantial effects and 400 micrograms in the i.p . route prevented even the initial stages of lesion formation . Touch prints from the lesion area and from the liver of mice receiving 200 micrograms glucanas were amastigote free . The anti Leishmania antibody titre of glucanas treated mice was lower and their sera recognized fewer antigens than that of control Leishmania bearing mice.

Mol Biochem Parasitol, 1991 Mar, 45(1), 159 - 70
Immunogenicity of recombinant Plasmodium falciparum SERA proteins in rodents; Barr PJ et al.; We have expressed defined regions of the serine-repeat antigen (SERA) of the Honduras-1 strain of Plasmodium falciparum in the yeast Saccharomyces cerevisiae . Amino-terminal domains of the natural SERA protein have been shown previously to be targets for parasite-inhibitory murine monoclonal antibodies . Two recombinant SERA antigens were selected for purification and immunological analysis . The first (SERA 1), corresponding to amino acids 24-285 of the natural SERA precursor, was expressed by the ubiquitin fusion method . This allowed for in vivo cleavage by endogenous yeast ubiquitin hydrolase, and subsequent isolation of the mature polypeptide . The second, larger protein (SERA N), encompassing amino acids 24-506, was expressed at only low levels using this system, but could be isolated in high yields when fused to human gamma-interferon (gamma-IFN) . Each purified protein was used to immunize mice with either Freund's adjuvant or a muramyl tripeptide adjuvant that has been used in humans . Sera from immunized mice were shown to be capable of in vitro inhibition of invasion of erythrocytes by the Honduras-1 strain of P . falciparum . The results suggest that a recombinant SERA antigen may be an effective component of a candidate malaria vaccine.

EMBO J, 1991 Mar, 10(3), 607 - 15
Transcription-induced nucleosome 'splitting': an underlying structure for DNase I sensitive chromatin; Lee MS et al.; Utilizing yeast strains containing promoter mutations, we demonstrate that transcription of the HSP82 gene causes nucleosomes toward the 3'-end to become DNase I sensitive and 'split' into structures that exhibit a 'half-nucleosomal' cleavage periodicity . Splitting occurs even when only a few RNA polymerase II molecules are engaged in basal level transcription or during the first round of induced transcription . The split nucleosomal structure survives nuclear isolation suggesting that it may be stabilized by post-translational modifications or non-histone proteins, and may require DNA replication for reversal to a whole nucleosomal structure . Split nucleosomes represent a structure for DNase I sensitive chromatin and are probably of common occurrence but difficult to detect experimentally . We suggest that transient positive supercoils downstream of traversing RNA polymerase lead to nucleosome splitting.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1593 - 6
Eliminated chromatin of Ascaris contains a gene that encodes a putative ribosomal protein; Etter A et al.; Chromatin diminution in the nematodes Parascaris equorum and Ascaris lumbricoides leads to the formation of somatic cells that contain less DNA than the germ-line cells . We present molecular evidence for the coding potential of germ-line-specific DNA . We report on a cDNA clone that codes for a putative ribosomal protein (ALEP-1, for A . lumbricoides eliminated protein 1) . That the corresponding gene is located in the eliminated portion of the genome indicates a difference in germ-line and somatic ribosomes of A . lumbricoides and P . equorum . Elimination of the ALEP-1 gene from all somatic cells in its fully active state may represent an alternative way to gene regulation.

Mol Cell Biol, 1991 Mar, 11(3), 1480 - 7
An "attenuator domain" is sandwiched by two distinct transactivation domains in the transcription factor C/EBP; Pei DQ et al.; C/EBP is a rat liver DNA-binding protein which can act as a transcription factor . Its N-terminal portion contains three distinct domains . The first domain (amino acids 1 to 107) appears to be a highly potent transactivator . The second domain (amino acids 107 to 170) does not appear to exhibit either activation or repression activity . This domain is defined as an "attenuator domain" because its presence under four different sequence contexts reproducibly decreases the effect of transactivation of C/EBP . The third domain (amino acids 171 to 245) is a relatively weaker transactivator with a striking proline-rich motif . Deletional analysis of this third domain has shown that a 45-amino-acid region is sufficient for transactivation . This region (amino acids 171 to 215) contains 12 proline, 6 histidine, and mainly hydrophobic or noncharged amino acids . Further mutational analysis of a highly conserved proline-octamer region within this domain indicates that a specific proline content is not crucial for transactivation.

Mol Cell Biol, 1991 Mar, 11(3), 1222 - 31
A unique pathway of double-strand break repair operates in tandemly repeated genes; Ozenberger BA et al.; The RAD52 gene product of the yeast Saccharomyces cerevisiae is required for most spontaneous recombination and almost all double-strand break (DSB) repair . In contrast to recombination elsewhere in the genome, recombination in the ribosomal DNA (rDNA) array is RAD52 independent . To determine the fate of a DSB in the rDNA gene array, a cut site for the HO endonuclease was inserted into the rDNA in a strain containing an inducible HO gene . DSBs were efficiently repaired at this site, even in the absence of the RAD52 gene product . Efficient RAD52-independent DSB repair was also observed at another tandem gene array, CUP1, consisting of 18 repeat units . However, in a smaller CUP1 array, consisting of only three units, most DSBs (ca . 80%) were not repaired and resulted in cell death . All RAD52-independent DSB repair events examined resulted in the loss of one or more repeat units . We propose a model for DSB repair in repeated sequences involving the generation of single-stranded tails followed by reannealing.

J Cell Biol, 1991 Mar, 112(6), 1117 - 31
Sec15 protein, an essential component of the exocytotic apparatus, is associated with the plasma membrane and with a soluble 19.5S particle; Bowser R et al.; SEC15 encodes a 116-kD protein that is essential for vesicular traffic from the Golgi apparatus to the cell surface in yeast . Although the sequence predicts a largely hydrophilic protein, a portion (23%) of Sec15p is found in association with the plasma membrane . The remainder is not associated with a membrane but is found in a 19.5S particle which is not dissociated by 0.5 M NaCl . Sec15p may attach directly to the plasma membrane since it is not found on the Golgi apparatus nor on the secretory vesicle precursors to the plasma membrane . Loss of function of most of the late-acting sec gene products does not alter the distribution of Sec15p . However, the sec8-9 mutation and to a lesser extent the sec10-2 mutation result in a shift of Sec15p to the plasma membrane, suggesting a role for these gene products in the regulation of the Sec15p membrane attachment/detachment processes . Depletion of Sec15p by repression of synthesis indicates that the plasma membrane bound pool is the most stable . During the course of these studies we have found that two activities associated with the yeast Golgi apparatus, Kex2 endopeptidase and GDPase, are in separable subcompartments.

Arch Biochem Biophys, 1991 Mar, 285(2), 365 - 70
Complete amino acid sequence of the type III isozyme of rat hexokinase, deduced from the cloned cDNA; Schwab DA et al.; Clones containing cDNA coding for the Type III isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were isolated from a library prepared in lambda gt10 with rat liver mRNA . Three clones were characterized . Their composite sequence includes the entire coding region for Type III hexokinase, 3' untranslated sequence extending into the polyadenylated region, and 80 bp of 5' untranslated sequence . Extensive similarity in sequence of N- and C-terminal halves of the enzyme, previously seen with the Type I isozyme, is consistent with the view that these 100-kDa mammalian hexokinases are the evolutionary result of duplication and fusion of a gene coding for an ancestral hexokinase having a molecular weight of approximately 50 kDa . Extensive similarities are seen between sequences of the Type I and III isozymes, and those reported for mammalian glucokinase (also called Type IV hexokinase) and for the hexokinase and glucokinase of yeast . Residues thought to be involved in catalytic function are highly conserved in all of these enzymes . Based on a quantitative comparison of sequence similarities, it is concluded that the 50-kDa mammalian glucokinase is more closely related to the 100-kDa mammalian enzymes than it is to the 50-kDa enzymes from yeast . One interpretation of this might be that the mammalian glucokinase arose by resplitting of the gene coding for the 100-kDa mammalian hexokinases.

J Virol, 1991 Mar, 65(3), 1578 - 83
Characterization of murine polyclonal antisera and monoclonal antibodies generated against intact and denatured human papillomavirus type 1 virions; Yaegashi N et al.; Human papillomavirus type 1 (HPV1) virions, both as intact virion particles (IVP) and as detergent-denatured virions (DDV), were used to prepare polyclonal antisera and monoclonal antibodies (MAbs) in BALB/c mice . Anti-IVP antiserum contained type-specific HPV1 L2-reactive antibodies and no detectable HPV1 L1-reactive antibodies . Anti-IVP MAbs recognized a linear epitope between L2 amino acids 102 and 108 (PIDVVDP) . Anti-DDV antiserum contained type-specific HPV1 L1-reactive and HPV1 L2-reactive antibodies . An anti-DDV MAb recognized a linear epitope between L1 amino acids 127 and 133 (AENPTNY) . HPV1a L1- and L2-encoded polypeptides expressed in Saccharomyces cerevisiae and by in vitro translation were equivalent in size to the major and minor virion capsid proteins, respectively.

Trends Genet, 1991 Mar, 7(3), 95 - 9
G1-specific cyclins: in search of an S-phase-promoting factor; Reed SI; In budding yeast, Saccharomyces cerevisiae, the two principal cell cycle transitions, from G1 to S phase and from G2 to M phase, are controlled by the same protein from G2 to M phase, are controlled by the same protein kinase, CDC28, a homolog of the cdc2 protein kinase in fission yeast and other organisms . The G1 to S phase activity of the kinase is associated with accumulation of a novel family of G1 cyclins, distinct from cyclins that are required to activate the kinase for G2 to M phase functions . It remains to be determined whether G1 cyclins with similar functions exist in higher cells.

Agents Actions, 1991 Mar, 32(3-4), 277 - 82
Drugs effects on superoxide generation and chemiluminescence response of human leukocytes; Pascual C et al.; Luminol-enhanced chemiluminescence was used to determine the effects of diethyldithiocarbamate, dipyridamole, catechin and verapamil on the generation of reactive oxygen species in human leukocytes, and on superoxide generated by chemiluminescence of the hypoxanthine xanthine-oxidase reaction . These agents reduced the luminol enhanced chemiluminescence response of activated leukocytes, most probably by inhibiting the superoxide generation reaction . On the other hand, citrate and diethylcarbamazine, produced a slight increase of the luminol enhanced chemiluminescence of leukocytes.

J Biol Chem, 1991 Feb 25, 266(6), 3380 - 2
Alteration of high and low spin equilibrium by a single mutation of amino acid 209 in mouse cytochromes P450; Iwasaki M et al.; The identities of the amino acid at position 209 are most critical in determining specific coumarin 7- and steroid 15 alpha-hydroxylase activity in P450coh and P450(15)alpha, respectively . This system, therefore, provides us with an excellent model to study the structural basis for P450 specificity as a monooxygenase . We expressed in Saccharomyces cerevisiae a series of the mutated P450s in which residue 209 was substituted with the various amino acids and characterized the spectral property and hydroxylase activity of these mutated P450s . The positioning of a hydrophobic residue including Phe, Leu, and Val at position 209 resulted in shifting the P450 to the high-spin state, while a charged amino acid such as Lys or Asp produced the low-spin form . Moreover, a P450 with Asn or Gly in this position exhibited spectra indicating a mixture of the high- and low-spin forms . This spin alteration, depending upon the hydrophobicity and size of residue at position 209, indicates that this position is likely to reside close to the sixth axial ligand on the distal surface of the heme in these P450s . This proximity of residue 209 to the ligand may explain the critical role of this residue in determining the hydroxylase specificity and activity of these P450s.

J Biol Chem, 1991 Feb 25, 266(6), 3372 - 5
Probing the role of lysines and arginines in the catalytic function of cytochrome P450d by site-directed mutagenesis . Interaction with NADPH-cytochrome P450 reductase; Shimizu T et al.; To identify amino acids of cytochrome P450d (P450d) which participate in the interaction with NADPH-cytochrome P450 reductase, we changed conserved ionic amino acids of P450d to others by site-directed mutagenesis . Turnover numbers (0.032-0.008 min-1) of purified mutants Lys94-Glu, Lys99-Glu, Lys105-Glu, Lys440-Glu, Lys453-Glu, Arg455-Glu, and Lys463-Glu toward 7-ethoxycoumarin were much lower than that (0.380 min-1) of the wild type at 25 degrees C . Reduction rates (less than 0.054 s-1) of the heme of all mutants (0.1 microM) in the presence of NADPH and the reductase (0.3 microM) were much lower than that (5.9 s-1) of the wild type . Furthermore, a turnover number (0.042 min-1) of a microsomal triple mutant (Arg135-Leu + Arg136-Leu + Arg137-Leu) of a conserved Arg cluster was much lower than that (0.674 min-1) of the wild type at 37 degrees C . Thus, we suggest that Lys94, Lys99, Lys105, Lys440, Lys453, Arg455, Lys463, and perhaps the Arg cluster Arg135-Arg136-Arg137 of P450d will participate in the intermolecular electron transfer process by forming ionic bridges between the two proteins and/or by orienting appropriate geometry for electron transfer on the interfacial surface between the two proteins.

Cell, 1991 Feb 22, 64(4), 789 - 800
Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein; Cleves AE et al.; SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function . We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes . One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement . The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.

Biochem J, 1991 Feb 15, 274 ( Pt 1), 207 - 17
Electron-transfer steps involved in the reactivity of Hansenula anomala flavocytochrome b2 as deduced from deuterium isotope effects and simulation studies; Capeillere-Blandin C; The L-lactate-flavocytochrome b2-ferricyanide electron-transfer system from the yeast Hansenula anomala was investigated by rapid-reaction techniques . The kinetics of reduction of oxidized flavocytochrome b2 by L-lactate and L-{2H}lactate were biphasic both for flavin and haem prosthetic groups and at all concentrations tested . The first-order rate constants of the rapid and slow phases depended upon substrate concentrations, a saturation behaviour being exhibited . Substitution of the C alpha-H atom by 2H was found to cause appreciable changes in the rate constants for the initial reduction of flavin and haem (phase I), which were respectively about 3-fold and 2-fold less than with L-lactate . In contrast, no significant isotope effect was noted on the apparent reduction rate constants of the slow phase, phase II . Under steady-state conditions an isotope effect of 2.0 was found on the overall electron transfer from L-lactate to ferricyanide . These transient reduction results were discussed in terms of a kinetic model implying intra- and inter-protomer electron exchanges between flavin and haem b2, all of which have been experimentally described . Computer simulations indicate that the reaction scheme provides a reasonable explanation of the fast-reduction phase, phase I (in absence of acceptor) . The pseudo-first-order rate constant for oxidation of reduced haem b2 in flavocytochrome b2 increased with increasing ferricyanide concentration in a hyperbolic fashion . The limiting value at infinite ferricyanide concentration, which was attributed to the intramolecular electron-transfer rate from ferroflavocytochrome b2 to the iron of ferricyanide within a complex, was 920 +/- 50 s-1 at pH 7.0 and 5 degrees C . Stopped-flow and rapid-freezing measurements showed haem b2 and flavin to be 90 and 44% oxidized respectively under steady-state conditions in presence of ferricyanide . Simulation studies were carried out to check the participation of the proposed reduction sequence in the overall catalytic reaction together with the role of reduced haem b2 (Hr) and flavin semiquinone (Fsq) as electron donors to ferricyanide . When the rate of the intramolecular electron-transfer exchange between Fsq and ferricyanide was adjusted to 200 s-1, simulated data accounted for molar activities defined under various conditions of L-lactate, {2H}lactate and ferricyanide concentrations . Simulation studies were extended to data obtained using cytochrome c as acceptor and reaction catalysed by Saccharomyces cerevisiae flavocytochrome b2 . The differences in reactivity observed for Hr and Fsq with ferricyanide and cytochrome c were discussed in terms of redox potentials, electrostatic interactions, distances and accessibility of the participating groups.

J Biol Chem, 1991 Feb 15, 266(5), 3052 - 9
Purification and characterization of Ku-2, an octamer-binding protein related to the autoantigen Ku; May G et al.; The octamer motif (ATTTGCAT) is an important regulatory element in eukaryotic gene expression . A previously unidentified protein that recognizes this motif has been isolated from the human B cell line, Daudi . The protein, which we term Ku-2, bears a close resemblance to the DNA-binding autoantigen Ku . Like Ku, it is a heterodimer with subunits of 83 and 72 kDa; antisera raised against either subunit of Ku cross-react with Ku-2 . Two peptides have been sequenced and show a strong similarity to regions in the corresponding subunits of Ku . The sequences are not identical, however, suggesting that Ku-2 may be a B cell homologue of Ku . Both Ku and Ku-2 bind to the termini of DNA duplexes, but Ku-2 also binds to an internal octamer motif . It is not known whether Ku shares the latter property or whether the octamer binding is a consequence of sequence differences between the two proteins . Ku-2 does not react with antisera against the POU domain of the octamer-binding protein Oct-2, indicating that the DNA binding domains of the two proteins are dissimilar despite the ability of both to bind to the octamer motif . We discuss the evidence for the existence of a family of octamer-binding proteins related to Ku.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1090 - 4
Inhibition of the N-end rule pathway in living cells; Baker RT et al.; The N-end rule relates the metabolic stability of a protein to the identify of its amino-terminal residue . Previous work, using amino acid derivatives such as dipeptides to inhibit N-end rule-mediated protein degradation in an extract from mammalian reticulocytes, has demonstrated the existence of specific N-end-recognizing proteins in this in vitro system . We now show that these nontoxic amino acid derivatives, when added to growing cells of the yeast Saccharomyces cerevisiae, are able to inhibit the degradation of proteins by the N-end rule pathway in vivo . Moreover, this inhibition is shown to be selective for the two distinct classes of destabilizing amino-terminal residues in substrates of the N-end rule pathway.

Biochem J, 1991 Feb 15, 274 ( Pt 1), 145 - 52
Identity between palmitoyl-CoA synthetase and arachidonoyl-CoA synthetase in human platelet?
Bakken AM, Farstad M, Holmsen H.
Apparent Km values have been determined for the substrates ATP, CoA and fatty acids for the long-chain acyl-CoA synthetase (EC 6.2.1.3) reaction in lysates of human blood platelets . The apparent Km for ATP was higher for saturated fatty acids (C12:0 to C18:0) than for unsaturated acids (C18:1 to C22:6) . Other apparent Km values were very similar for all long-chain fatty acids tested . Palmitic acid inhibited the formation of {14C}arachidonoyl-CoA, and arachidonic acid inhibited the formation of {14C}palmitoyl-CoA, with {14C}arachidonate or {14C}palmitate respectively as substrate . After chromatography of Triton X-100-extracted platelet protein in several systems (hydroxyapatite, DEAE-Sepharose, Sephacryl S-200 HR, CoA-Sepharose, Sephadex G-100 and AcA 34), both arachidonoyl-CoA synthetase and palmitoyl-CoA synthetase activities were eluted together in the various protein peaks, and with approximately the same ratio of activities in all peaks . After some purification steps (DEAE-Sepharose and Sephacryl S-200 HR), the acyl-CoA synthetase activity was up to 37 nmol/min per mg of protein with {14C}palmitate as substrate, and up to 116 nmol/min per mg of protein with {14C}arachidonate as substrate . The purification was respectively about 8- and 10-fold . The results indicate that palmitoyl-CoA (or unspecific) synthetase and arachidonoyl-CoA (or specific) synthetase are in fact the same enzyme, in agreement with previously reported results from this laboratory.

Eur J Biochem, 1991 Feb 14, 195(3), 699 - 705
Synthesis of myristoyl-carba(dethia)-coenzyme A and S-(3-oxohexadecyl)-coenzyme A, two potent inhibitors of myristoyl-CoA:protein N-myristoyltransferase; Wagner AP et al.; 1 . Two non-hydrolysable analogues of myristoyl-coenzyme A were synthesised and spectroscopically characterized . Myristoyl-carba(dethia)coenzyme A was prepared in a multistep synthesis starting from tridecyl vinyl ketone . S-(3-Oxohexadecyl)-coenzyme A was synthesised from 3-oxohexadecyl chloride by direct condensation with coenzyme A . 2 . Both analogues were strong competitive inhibitors of N-myristoyltransferase from yeast . Ki values of 0.3 and 0.25 microM were determined for myristoyl-carba(dethia)-coenzyme A and S-(3-oxohexadecyl)-coenzyme A, respectively.

Biochem Biophys Res Commun, 1991 Feb 14, 174(3), 1232 - 8
The precursor of mitochondrial aspartate aminotransferase is imported into mitochondria faster than the homologous cytosolic isoenzyme with the same presequence attached; Hartmann CM et al.; Mitochondrial and cytosolic aspartate aminotransferase (AspAT) are homologous proteins with identically folded polypeptide chains . The cDNAs of the two isoenzymes of chicken were used to express the following proteins in yeast: the precursor of mitochondrial AspAT, mature mitochondrial AspAT, and two chimeric proteins in one of which (pc) the presequence of the precursor was attached to the entire cytosolic isoenzyme and in the other one (pmc) the N-terminal segment (amino acid residues -22 to 23) of the precursor was linked to the slightly truncated cytosolic isoenzyme (residues 34 to 412) . All presequence containing proteins were imported into the mitochondria and processed to the mature form whereas mature mitochondrial AspAT remained in the cytosol . The rate of import of the authentic precursor was four times faster than that of the chimeric proteins pc and pmc, t1/2 for importation at 29 degrees C being 3, 13 and 14 min, respectively . Apparently, the mature moiety of the precursor of mitochondrial AspAT promotes importation.

Biochemistry, 1991 Feb 12, 30(6), 1490 - 6
Ligand binding studies of engineered cytochrome P-450d wild type, proximal mutants, and distal mutants; Shimizu T et al.; Interactions of various axial ligands with cytochrome P-450d wild type, proximal mutants (Lys453Glu, Ile460Ser), and putative distal mutants (Glu318Asp, Thr319Ala, Thr322Ala) expressed in yeast were studied with optical absorption spectroscopy . P-450d wild type and all five mutants were purified essentially as the high-spin form, but the putative distal mutants contained about 5% low-spin form . Bindings of metyrapone and 4-phenylimidazole to the wild type and all mutants formed nitrogen-bound low-spin forms . In contrast, binding of 2-phenylimidazole to the wild type and most of mutants formed oxygen-bond low-spin forms except for the mutant Glu318Asp in which the nitrogen-bound low-spin form was formed . By analogy with the distal structure of P-450cam, it was thus suggested that Glu318 of P-450d, which corresponds with Asp251 of P-450cam, somehow interacts with 2-phenylimidazole over the heme plane . Addition of 1-butanol and acetanilide, a substrate of P-450d, to the wild type and mutants caused the spin change to the low-spin form . The order of dissociation constants of these oxygen ligands to P-450d was wild type greater than proximal mutants greater than putative distal mutants . Spectral analyses showed that the binding of acetanilide is the same as that of another substrate, 7-ethoxycoumarin, in the putative distal mutants but is not the same in the wild type and proximal mutants . From these findings together with other spectral data, it was suggested that the region from Glu318 to Thr322 is located at the distal region of the heme in membrane-bound P-450d as suggested from the X-ray crystal structure of water-soluble P-450cam and amino acid alignments of P-450s.

Nucleic Acids Res, 1991 Feb 11, 19(3), 605 - 9
The rate and specificity of a group I ribozyme are inversely affected by choice of monovalent salt; Partono S et al.; The fifth intron of the COB gene of yeast mitochondria splices autocatalytically . The rate of splicing is increased by high concentrations of monovalent salts, but the choice of both cation and anion is significant: The smaller the cation in solution, the faster the reaction (the rate in K+ greater than NH4+ greater than Na+ greater than Li+) . Chloride, bromide, iodide and acetate salts enhance autocatalytic processing, but sulfate salts do not and fluoride salts are inhibitory . The choice of monovalent salt affects the KM of the intron for guanosine nucleotide, implying an alteration in the affinity of the RNA for that substrate . Under optimal conditions (1M KCl, 50 mM MgCl2) the catalytic efficiency of this intron exceeds that reported for the ribosomal intron from Tetrahymena, but several side reactions occur, including guanosine-addition within the downstream exon . The site of addition resembles the 5' splice junction, but selection of this site does not involve the internal guide sequence of the intron.

Cell, 1991 Feb 8, 64(3), 533 - 44
Activation domains of stably bound GAL4 derivatives alleviate repression of promoters by nucleosomes; Workman JL et al.; GAL4 derivatives containing an activation domain alleviated repression of a promoter during nucleosome assembly . A GAL4 derivative lacking an activation domain stably bound the promoter during nucleosome assembly but was not sufficient to preserve promoter function . The activation domain of GAL4 derivatives was essential for preserving promoter function, and thus the transcriptional stimulatory activity attributable to these activation domains increased dramatically during nucleosome assembly . Furthermore, promoter-bound activation domains allowed the formation of preinitiation complexes after nucleosome assembly . Finally, GAL4 derivatives containing activation domains significantly stimulated transcription through bacterially produced yeast TFIID only from nucleosome-assembled templates . These data indicate that acidic activation domains stimulate transcription by enhancing the ability of basal transcription factors to compete with nucleosomes for occupancy of the promoter.

Nature, 1991 Feb 7, 349(6309), 494 - 9
PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome; Schwer B et al.; The assembly of the spliceosome is an ATP-dependent process . The splicing factor PRP16 contains variations of several motifs that define the eIF-4A-like ATP-dependent RNA helicase family . The protein has now been purified and shown to exhibit RNA-dependent ATPase activity . PRP16 is required specifically for the second catalytic step of the splicing reaction in vitro . This function requires ATP binding and/or hydrolysis, which appears to be concomitant with release of the protein from the spliceosome . PRP16 may be the prototype for a set of splicing factors which use ATP to drive a cycle of conformational changes.

Klin Wochenschr, 1991 Feb 6, 69(3), 105 - 11
Phospholipid transfer proteins: from lipid monolayers to cells; Wirtz KW; Eukaryotic cells contain phospholipid transfer proteins that act as carriers of phospholipids between membranes . In mammalian tissues three transfer proteins with different specificities have been identified: the phosphatidylcholine transfer protein (PC-TP), the phosphatidylinositol transfer protein (PI-TP) and the non-specific lipid transfer protein (nsL-TP) that transfers all common diacyl-phospholipids and cholesterol . Properties of these transfer proteins have been discussed with a special emphasis on the lipid binding site of bovine liver PC-TP . Application of photoactivatable and fluorescent analogues of PC have indicated that PC-TP contains specific and independent hydrophobic binding sites for the sn-1- and sn-2-fatty acyl chains . Because these sites have different properties, PC-TP can discriminate between positional isomers of PC and displays a distinct preference for those molecular species that carry a polyunsaturated fatty acid chain at the sn-2-position . Recent studies on bovine brain PI-TP have strongly suggested that this protein may be well-suited to maintain the levels of PI in natural membranes . Besides this proposed role, evidence has become available from studies on Swiss mouse 3T3 fibroblasts that, apart from its occurrence in cytosol, PI-TP is present in nuclei.

Biochemistry, 1991 Feb 5, 30(5), 1310 - 7
Solution structure of the basic region from the transcriptional activator GCN4; Saudek V et al.; The structure of the basic region (i.e., the region responsible for sequence-specific binding to DNA) of the transcriptional activator GCN4 was studied . Two peptide fragments containing either the basic region alone (residues 240-280) or the basic and the dimerization leucine zipper domains (220-280) were synthesized and investigated by nuclear magnetic resonance and circular dichroic spectroscopy . The basic region in the absence of DNA appears as a mobile flexible segment folded into a loose helix . The helical stability increases upon addition of trifluoroethanol and/or lowering of the temperature . Dimerization via the leucine zipper does not affect the three-dimensional structure of the basic region . Possible consequences for the binding to DNA are discussed.

J Biol Chem, 1991 Feb 5, 266(4), 2606 - 14
Human ADP-ribosylation factors . A functionally conserved family of GTP-binding proteins; Kahn RA et al.; A new member, hARF4, of the ADP-ribosylation factor (ARF) family, a subset of the superfamily of regulatory GTP-binding proteins, has been cloned from a cDNA expression library . Two other human ARF cDNA sequences, designated human ARF1 and ARF3, have been reported previously and are 96% identical in amino acid sequence . A human ARF1 cDNA, significantly longer than previously described clones, was obtained, by cross-species hybridization using a bovine ARF1 cDNA probe . Bovine ARF1p and human ARF1p are 100% identical while each is only 80% identical to hARF4p . Thus, hARF4p is the most divergent of the mammalian ARF proteins identified . Northern blot analysis revealed the expression of at least three different ARF messages in human placenta and adrenal carcinoma cells . Both hARF1 and hARF4 encode GTP-binding proteins with predicted molecular masses of 20,000-21,000 Da . Biochemical analysis of the purified recombinant proteins revealed a high degree of conservation of nucleotide binding properties and in vitro ARF activities . ARF is an essential gene in the yeast, Saccharomyces cerevisiae, and is encoded by two genes . Expression of either hARF1p or hARF4p in yeast was found to rescue the lethal double mutant, arf1-arf2-, thus demonstrating the functional conservation of ARF functions between yeast and man . The combination of in vivo and in vitro assays for ARF function provides a specific and unambiguous means of determining bona fide ARF proteins from divergent species from among the rapidly increasing number of structurally related, small molecular weight GTP-binding proteins.

Yeast, 1991 Feb, 7(2), 105 - 17
On the dependence of spontaneous mutation rates on the functional state of genes; Korogodin VI et al.; Spontaneous mutation of some genes was studied in haploid adenine and leucine auxotrophic yeast Saccharomyces . It was shown that a decrease in the amount of adenine (from 500 to 0 mg l-1) or leucine (from 300 to 0.3 mg l-1) in the medium, simultaneously with the transition from repression to derepression of the biosynthesis of these metabolites, resulted in a 15- to 150-fold increase in the reversion rate of genes ade 2 and leu2, respectively, for different strains . At the same time the mutation rate of suppressor genes varied relatively little (up to five-fold), and that of gene lys did not change at all . It was also demonstrated (on gene leu2) that the mutation rate is determined by the composition of the nutrient medium at the time of the S-phase of the cell cycle and it does not depend on the cultivation conditions during the presynthetic period . We discuss the hypothesis that derepressed genes mutate with a significantly higher rate than genes in the repressed state.

Proc Natl Acad Sci U S A, 1991 Feb 1, 88(3), 718 - 22
Negative supercoiling of DNA facilitates an interaction between transcription factor IID and the fibroin gene promoter; Mizutani M et al.; Transcription of the fibroin gene can be reconstituted with partially purified components from HeLa cells . Transcription factors IIB, IID, and IIE and RNA polymerase II are required for accurate initiation of transcription . Linear and relaxed closed circular DNA show a similar level of template activity . However, transcription of closed circular DNA is stimulated when negative supercoils are introduced by the addition of DNA topoisomerase II and supercoiling factor purified from the posterior silk gland of Bombyx mori . Dissection of transcription into pre- and postinitiation steps by the use of Sarkosyl reveals that DNA supercoiling promotes formation of a preinitiation complex . Furthermore, order of addition experiments suggest that DNA supercoiling facilitates a functional binding of transcription factor IID to the promoter.

Mol Cell Biol, 1991 Feb, 11(2), 954 - 62
Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization; Dang CV et al.; The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells . Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein . Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site . Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected . Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos . In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells . The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently . The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus . Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed . Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c-Myc protein . In contrast, the cytoplasmic marker was translocated into the nucleus when the GCN4 leucine zippers were present on both the cytoplasmic marker and a nuclear protein, presumably as a result of leucine zipper interaction . These results suggest that c-Myc function requires hetero-oligomerization to an as yet undefined factor.

Mol Cell Biol, 1991 Feb, 11(2), 1069 - 79
The role of RAP1 in the regulation of the MAT alpha locus; Giesman D et al.; The RAP1 gene of Saccharomyces cerevisiae encodes an abundant DNA-binding protein, also known as GRF1, TBA, or TUF, that binds to many sites in the yeast genome in vitro . These sites define a consensus sequence, {sequence: see text}, and deletion analyses of genes that contain this sequence have implicated the involvement of RAP1 in numerous cellular processes, including gene activation and repression . The MAT alpha locus, required for determination of the alpha cell type in yeast cells, contains a RAP1 binding site; this site coincides with the MAT alpha upstream activating sequence (UAS) and is necessary for expression of the two genes encoded by the MAT alpha locus, MAT alpha 1 and MAT alpha 2 . We show that the MAT alpha UAS is sufficient to activate transcription from a promoterless gene fusion of the yeast CYC1 upstream region and the lacZ gene . Constructs containing only the MAT alpha UAS generated elevated levels of beta-galactosidase activity which were indistinguishable from those of constructs containing the entire MAT alpha intergenic region . Further, the MAT alpha UAS has an intrinsic polarity of transcriptional activation; transcription of CYC1-lacZ was six- to sevenfold higher when the UAS was oriented in the direction normally associated with MAT alpha 2 transcription . Point mutations in the MAT alpha UAS that reduce MAT alpha expression three- to fivefold resulted in a bi-mating phenotype, while a mutation that reduced MAT alpha expression still further resulted in an a-mating phenotype . We isolated plasmids from a high-copy-number yeast library that suppressed the bi-mating defect of point mutations in the MAT alpha UAS, and the most effective dosage suppressor contained the gene encoding RAP1 . A temperature-sensitive rap1 mutant bi-mates at the semipermissive temperature . Double mutants at rap1 and mat alpha mate exclusively as a cells, at all temperatures, and do not express detectable levels of MAT alpha RNA . These data provide evidence that the RAP1 gene product functions at the MAT alpha UAS in vivo.

Biochem Genet, 1991 Feb, 29(1-2), 13 - 28
Regulation of nuclear histone acetyltransferase by nucleic acids, histone.DNA complex, and chromatin; Wong LJ et al.; Nuclear histone acetyltransferase is found to be inhibited by various nucleic acids and components . Of the adenosine phosphates, the order of inhibitory potency is ATP greater than ADP greater than AMP . Among the nucleoside triphosphates, GTP seems to be the best inhibitor, followed by ATP, CTP, and UTP . Deoxymononucleotides have the same order of inhibition potential as their ribonucleotide counterparts, with inhibition constants in the low millimolar range . Oligonucleotides and polynucleotides are much better inhibitors than mononucleotides . The inhibition constants of the DNA molecules are size dependent . Molecules larger than 40 base pairs have inhibition constants less than 18 micrograms/ml, whereas molecules with decreasing numbers of base pairs have increasing magnitudes of inhibition constants . However, acetyltransferase has a lower affinity for free DNA molecules than for DNA.histone complexes as revealed by its interaction with DNA-Sepharose and histone.DNA-Sepharose columns . Furthermore, native chromatin depleted of endogenous histone acetyltransferase activity shows no inhibitory effect on the enzyme . Yet heated chromatin not only loses substrate activity but also becomes an inhibitor for the enzyme . Since unmodified sea urchin sperm chromatin has been shown to be a potent acetyltransferase inhibitor, it seems possible that DNA.histone complexes may be the true inhibitory species and that the conformational states of such complexes may serve as a regulatory mechanism in the control of the enzyme activity.

Proc Natl Acad Sci U S A, 1991 Feb 1, 88(3), 999 - 1003
Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains; Vallee BL et al.; We now recognize three distinct motifs of DNA-binding zinc proteins: (i) zinc fingers, (ii) zinc clusters, and (iii) zinc twists . Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses of DNA-binding zinc proteins have not been available to serve as standards of reference for the zinc binding sites of these families of proteins . Those of the DNA-binding domains of the fungal transcription factor GAL4 and the rat glucocorticoid receptor are the first to have been determined . Both proteins contain two zinc binding sites, and in both, cysteine residues are the sole zinc ligands . In GAL4, two zinc atoms are bound to six cysteine residues which form a "zinc cluster" akin to that of metallothionein; the distance between the two zinc atoms of GAL4 is approximately 3.5 A . In the glucocorticoid receptor, each zinc atom is bound to four cysteine residues; the interatomic zinc-zinc distance is approximately 13 A, and in this instance, a "zinc twist" is represented by a helical DNA recognition site located between the two zinc atoms . Zinc clusters and zinc twists are here recognized as two distinctive motifs in DNA-binding proteins containing multiple zinc atoms . For native "zinc fingers," structural data do not exist as yet; consequently, the interatomic distances between zinc atoms are not known . As further structural data become available, the structural and functional significance of these different motifs in their binding to DNA and other proteins participating in the transmission of the genetic message will become apparent.

Proc Natl Acad Sci U S A, 1991 Feb 1, 88(3), 936 - 40
Single-step selection for Ty1 element retrotransposition; Curcio MJ et al.; The yeast retrotransposon Ty1 has been tagged with a reporter gene that allows selection of RNA-mediated transposition events and is applicable to the study of retroelements in other organisms . The reporter gene is a yeast HIS3 gene interrupted by an artificial intron (AI) in the antisense orientation . The HIS3AI sequences were inserted into a Ty1 element such that the intron is on the sense strand of the Ty1 element; therefore, splicing and retrotransposition of marked Ty1 transcripts can give rise to His+ cells . Fusion of the Ty1-H3mHIS3AI element to the inducible GAL1 promoter resulted in a high frequency of histidine prototrophs upon galactose induction . Moreover, spontaneous His+ revertants derived from strains containing genomic TymHIS3AI elements are a result of retrotransposition . By using this assay, we estimated the Ty1 transposition rate to be between 3 x 10(-7) and 1 x 10(-5) transpositions per Ty1 element per generation . Variations in the transposition rate of individual Ty1 elements are correlated with the relative abundance of their transcripts.

Proc Natl Acad Sci U S A, 1991 Feb 1, 88(3), 931 - 5
cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera; Aris JP et al.; We have isolated a 1.1-kilobase cDNA clone that encodes human fibrillarin by screening a hepatoma library in parallel with DNA probes derived from the fibrillarin genes of Saccharomyces cerevisiae (NOP1) and Xenopus laevis . RNA blot analysis indicates that the corresponding mRNA is approximately 1300 nucleotides in length . Human fibrillarin expressed in vitro migrates on SDS gels as a 36-kDa protein that is specifically immunoprecipitated by antisera from humans with scleroderma autoimmune disease . Human fibrillarin contains an amino-terminal repetitive domain approximately 75-80 amino acids in length that is rich in glycine and arginine residues and is similar to amino-terminal domains in the yeast and Xenopus fibrillarins . The occurrence of a putative RNA-binding domain and an RNP consensus sequence within the protein is consistent with the association of fibrillarin with small nucleolar RNAs . Protein sequence alignments show that 67% of amino acids from human fibrillarin are identical to those in yeast fibrillarin and that 81% are identical to those in Xenopus fibrillarin . This identity suggests the evolutionary conservation of an important function early in the pathway for ribosome biosynthesis.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1991 Feb, 13(1), 33 - 8
{Purification and characterization of ribosome-associated histone acetyltransferase from rabbit reticulocytes}; Qui C; Histone acetyltransferases from the ribosomes of rabbit reticulocytes were purified more than 100 fold . The procedures of purification included the following: acetyltransferases were released from ribosomes by 0.5 mol/L KCl treatment, then isolated by sedimentation through buffered sucrose containing 0.5 mol/L KCl . They were partially purified from the high salt washing fraction by sequential chromatography on histone-sepharose 4B, DEAE-cellulose 52 and phosphocellulose P11 columns and by nondenaturing polyacrylamide gel electrophoresis . Three main active bands were observed on 7.5% polyacrylamide gels under non-denaturing conditions with molecular weights of about 120,000, 70,000 and 40,000 respectively . The optimal pH for acetyltransferase activity was 7.5-8.0, and 1 mmol/L MgCl2 and 25 mmol/L KCl were required for activity . 70% of the activity was inhibited by 100 mmol/L MgCl2 . The Michealis constants derived from double-reciprocal plots of the acetyltransferases for histone and acetyl coenzyme A were 1.2 mumol/L and 18-20 mumol/L respectively.

Jpn J Genet, 1991 Feb, 66(1), 77 - 83
Suppression of the cr-1 mutation in Neurospora crassa; Kore-eda S et al.; We have cloned a DNA fragment, which hybridized with the adenylate cyclase gene (CYR1) of Saccharomyces cerevisiae, from genomic DNA libraries of Neurospora crassa . The cr-1 mutation was able to be suppressed by introducing this DNA fragment on a cosmid vector, judging from recovery of the adenylate cyclase activity and the abnormal morphology.

Mol Cell Biol, 1991 Feb, 11(2), 935 - 44
Characterization of Neurospora CPC1, a bZIP DNA-binding protein that does not require aligned heptad leucines for dimerization; Paluh JL et al.; CPC1 is the transcriptional activator of amino acid biosynthetic genes of Neurospora crassa . CPC1 function in vivo was abolished upon deletion of segments of cpc-1 corresponding to the presumed transcription activation domain, the DNA-binding and dimerization domains, or a 52-residue connector segment of CPC1 . A truncated CPC1 polypeptide containing only the carboxy-terminal 57-residue segment of CPC1 was sufficient to form homodimers that bound DNA . However, deletion of the segment of cpc-1 corresponding to the connector segment in the full-length CPC1 polypeptide abolished DNA binding . Removal of a segment of cpc-1 corresponding to the GIn-rich region of CPC1 reduced in vivo function only slightly . The homologous transcription activator of Saccharomyces cerevisiae, GCN4, did not substitute for CPC1 in N . crassa . Chimeric CPC1-GCN4 polypeptides that contained the GCN4 transcriptional activation domain or the domain of GCN4 that corresponds to the essential 52-residue connector segment of CPC1, functioned with reduced efficiency . However, a chimeric polypeptide containing the GCN4 DNA-binding and dimerization domains in place of those of CPC1 functioned essentially as well as wild-type CPC1 . The basic and dimerization domains of CPC1 were characterized by introducing deletions or site-directed amino acid replacements . The basic region was required for DNA binding but not for dimerization . CPC1 has a short dimerization domain containing heptad residues Leu-1, Leu-2, Trp-3, and His-4 . When Val was substituted for Leu-1 or Leu-2, CPC1 was fully active, but when Val replaced Trp-3, dimerization and DNA binding were prevented . DNA band shift analyses with CPC1 heterodimers demonstrated that CPC1 does not require aligned heptad leucine residues for dimerization . Replacement of two charged residues located between Leu-1 and Leu-2 of CPC1 abolished dimerization and DNA binding.

Mol Cell Biol, 1991 Feb, 11(2), 928 - 34
cpc-1, the general regulatory gene for genes of amino acid biosynthesis in Neurospora crassa, is differentially expressed during the asexual life cycle; Ebbole DJ et al.; CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae . We examined binding by CPC1 synthesized in vitro and by CPC1 present in N . crassa whole-cell extracts . CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting . Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed . Analyses of N . crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently . CPC1 levels could be increased at any time by imposing amino acid starvation . Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.

Proc Natl Acad Sci U S A, 1991 Feb 1, 88(3), 849 - 53
Cloning of murine ferrochelatase; Brenner DA et al.; Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) catalyzes the last step in the heme biosynthetic pathway, the chelation of ferrous iron and protoporphyrin to form heme . The activity of ferrochelatase is deficient in the inherited disease protoporphyria . In this study, murine ferrochelatase cDNAs were obtained by screening cDNA libraries with an oligonucleotide probe . The derived amino acid sequence of murine ferrochelatase has 47% identity with the recently cloned Saccharomyces cerevisiae ferrochelatase, but it is not significantly similar to other published sequences . Results of Southern blotting are consistent with a single murine ferrochelatase gene, while Northern blotting demonstrates two ferrochelatase transcripts in all tissues examined . The ferrochelatase protein and mRNAs have different relative concentrations in different tissues . The cloning of murine ferrochelatase cDNAs provides the basis for future studies on ferrochelatase gene expression and on the identification of the molecular defect in protoporphyria.

J Exp Med, 1991 Feb 1, 173(2), 423 - 7
Autoantibodies against the multicatalytic proteinase in patients with systemic lupus erythematosus; Arribas J et al.; Sera from patients with systemic lupus erythematosus contain specific autoantibodies directed against different polypeptide components of the multicatalytic proteinase (also known as proteasome or prosome) . These human autoantibodies, in contrast to polyclonal antibodies obtained in rabbits against the purified enzyme, recognize highly conserved epitopes of the multicatalytic proteinase polypeptides from yeast to human.

Biochimie, 1991 Feb-Mar, 73(2-3), 257 - 67
Biochemical studies of homologous and nonhomologous recombination in human cells; Fishel R et al.; Purified and partially purified protein fractions from human cells have been developed that promote homologous and nonhomologous recombination reactions in vitro . Homologous pairing of model DNA substrates is catalyzed by the homologous pairing protein HPP-1 in a magnesium-dependent, ATP-independent reaction that requires stoichiometric amounts of the protein . Addition of the human single-strand binding (SSB) holoprotein complex hRP-A reduces the requirement of HPP-1 in the reaction up to 20-fold . Although the combination of homologous pairing and SSB activities is similar to the bacterial strand-exchange process, the numbers, size, and requirements of the human reaction appear to preclude any detailed comparisons . We have used Z-DNA affinity chromatography as a major step in isolation of human recombination proteins and found that the activities appear to elute as a complex form in approximate multiples of 500 kDa . Associated with the homologous recombination complex is a potent blunt-end ligation activity that appears to mimic the nonhomologous joining functions that are frequently seen following transfection of DNA into mammalian cells . A simple scheme for the association of homologous and nonhomologous recombination functions in mammalian cells is discussed.

Agric Biol Chem, 1991 Feb, 55(2), 471 - 7
Cloning and nucleotide sequences of the complementary and genomic DNAs for the alkaline protease from Acremonium chrysogenum; Isogai T et al.; Complementary DNA encoding Ac . chrysogenum alkaline protease (Alp) was isolated from the Ac . chrysogenum ATCC11550 cDNA library by express-blot assay . The genomic DNAs encoding Ac . chrysogenum Alp were isolated from the Ac . chrysogenum genomic DNA library using the cloned cDNA as a probe . The 3150 nucleotides of the gene were sequenced . The prepro-Alp consists of 402 amino acids and two intervening sequences are found within the coding region . The amino acid sequence of Ac . chrysogenum Alp has 57% homology to that of Aspergillus oryzae Alp . The entire cDNA, encoding Ac . chrysogenum Alp, when introducing into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium.

Biochim Biophys Acta, 1991 Jan 30, 1061(2), 279 - 86
Characterization of pore-forming activity in liver mitochondria from Anguilla anguilla . Two porins in mitochondria?
De Pinto V, Zara V, Benz R, Gnoni GV, Palmieri F.
A fast purification procedure for the isolation and purification of eukaryotic porin (De Pinto et al., (1987) Biochim . Biophys . Acta 905, 499-502) was applied to liver mitochondria of the fish Anguilla anguilla . A protein preparation was obtained which formed slightly anionically selective pores in reconstitution experiments with lipid bilayer membranes . The distribution of single-channel conductances had two maxima of 2.4 nS and 4.0 nS in 1 M KCl . Sodium dodecylsulfate electrophoretograms of the protein preparation showed the presence of two bands of very similar electrophoretic mobility (32 and 32.5 kDa) . Both bands cross-reacted with antibodies raised against purified bovine heart porin and with antibodies raised against the 19 amino acids N-terminal end of human porin . No cross-reactivity was observed with antibodies against yeast porin . The peptide maps of the two bands showed slight differences . The possibility of the presence of two different porins in liver mitochondria of Anguilla anguilla is discussed . An extensive immunological comparison of different mitochondrial porins is presented.

FEBS Lett, 1991 Jan 28, 278(2), 247 - 51
Nature of primary product(s) of D-glucose 6-phosphate dehydrogenase reaction . 13C and 31P NMR study; Jarori GK et al.; Glucose 6-phosphate dehydrogenase catalyzes the oxidation of glucose 6-phosphate, resulting in the formation of 6-phosphogluconolactone . As this compound is unstable, it has not been characterized directly . NMR provides a way to directly monitor all components of a reaction and study their structure . Here we report some results on the glucose 6-phosphate dehydrogenase reaction using 31P and 13C-NMR . Our results indicate that two different lactones, namely gamma (1-4) and delta (1-5) 6-phosphogluconolactones, are formed as products in this reaction . This is in contrast to an earlier suggestion that glucose 6-phosphate dehydrogenase produces only the delta-lactone . On the basis of these results, a new mechanisms for dehydrogenation of the sugar phosphate is proposed.

FEBS Lett, 1991 Jan 28, 278(2), 217 - 21
Cloning and sequencing of the gene encoding the large (alpha-) subunit of the proteasome from Thermoplasma acidophilum; Zwickl P et al.; The gene encoding the alpha-subunit of the proteasome from the archaebacterium Thermoplasma acidophilum was cloned and sequenced . The gene encodes for a polypeptide with 233 amino acid residues and a calculated molecular weight of 25870 . Sequence similarity of the alpha-subunit with the Saccharomyces cerevisiae wild-type suppressor gene scll+ encoded polypeptide, which is probably identical with the subunit YC7-alpha of the yeast proteasome, lends support to a putative role of proteasomes in the regulation of gene expression . The significant sequence similarity to the various subunits of eukaryotic proteasomes make it likely that proteasomal proteins are encoded by one gene family of ancient origin.

Biochim Biophys Acta, 1991 Jan 23, 1073(1), 195 - 9
Interaction of nitrofurans with glutathione reductase; Cenas NK et al.; Nitrofurans inhibit the oxidation of NADPH by glutathione, catalyzed by yeast glutathione reductase (EC 1.6.4.2) . acting as uncompetitive incomplete inhibitors for NADPH and glutathione . The quinoline-substituted nitrofurans were the most effective inhibitors . These compounds increased the turnover numbers of enzyme at fixed concentrations of reduced glutathione, in the reverse reaction of glutathione reductase, but in most cases diminished the affinity of the enzyme for NAD+ . Nitrofurans are weak one-electron oxidants of glutathione reductase . Their reactivity is close to that of p-quinones possessing the analoguous one-electron reduction potential (Cenas, N.K., Rakauskiene, G.A . and Kulys, J.J . (1989) Biochim . Biophys . Acta 973, 399-404), and reaction is stimulated by NADP+ . It is assumed, that nitrofurans bind to the 'regulative' site of glutathione reductase (Karplus, P.A., Pai, E.F . and Schulz, G.E . (1989) Eur . J . Biochem . 178, 693-703).

J Chromatogr, 1991 Jan 18, 563(1), 11 - 21
Development of post-column enzymic reactors with immobilized alcohol oxidase for use in the high-performance liquid chromatographic assay of alcohols with electrochemical detection; Tagliaro F et al.; The development of a very sensitive, direct injection high-performance liquid chromatographic method, using a post-column reactor with immobilized alcohol oxidase, was undertaken with the aim of determining methanol and ethanol levels in microlitre volumes of biological samples . After reversed-phase chromatography to separate methanol and ethanol, the analytes were enzymically converted into the respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, which could be measured via electrochemical oxidation at a platinum electrode . Some problems were encountered in the development of solid-phase enzymic reactors, using a delicate enzyme, that is prone to lose activity, such as alcohol oxidase . Owing to the slightly alkaline pH required for the optimum activity of alcohol oxidase, polymeric columns seemed to be preferable for the chromatography . HEMA copolymer was chosen as the stationary phase, but the methanol and ethanol peaks eluted close together and posed severe problems of limiting post-column band spreading . Reactors based on coarse supports for enzyme immobilization gave unacceptable band spreading, causing the methanol and ethanol peaks to overlap . On the other hand high-performance liquid chromatographic packings maintained the efficiency of the chromatographic separation, quite independently of the reactor volume . Polymeric supports proved superior to silicas in maintaining the enzyme activity . However, relevant changes in the enzyme substrate specificity were observed after immobilization.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 340 - 4
Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans; Smeekens SP et al.; PC2 and furin are two recently identified members of a class of mammalian proteins homologous to the yeast precursor processing protease kex2 and the bacterial subtillisins . We have used the polymerase chain reaction to identify and clone a cDNA (PC3) from the mouse AtT20 anterior pituitary cell line that represents an additional member of this growing family of mammalian proteases . PC3 encodes a 753-residue protein that begins with a signal peptide and contains a 292-residue domain closely related to the catalytic modules of PC2, furin, and kex2 . Within this region 58%, 65%, and 50% of the amino acids of PC3 are identical to those of the aligned PC2, furin, and kex2 sequences, respectively, and the catalytically important Asp, His, and Ser residues are all conserved . On Northern blots, PC3 hybridizes to two transcripts of 3 and 5 kilobases . Tissue distribution studies indicate that both PC2 and PC3 are expressed in a variety of neuroendocrine tissues, including pancreatic islets and brain, but are not expressed in liver, kidney, skeletal muscle, and spleen . The high degree of similarity of PC3, PC2, and furin suggests that they are all members of a superfamily of mammalian proteases that are involved in the processing of prohormones and/or other protein precursors . In contrast to furin, PC3, like PC2, lacks a hydrophobic transmembrane anchor, but it has a potential C-terminal amphipathic helical segment similar to the putative membrane anchor of carboxypeptidase H . These and other differences suggest that these proteins carry out compartmentalized proteolysis within cells, such as processing within regulated versus constitutive secretory pathways.

Cell, 1991 Jan 11, 64(1), 137 - 48
Chromosome assembly in vitro: topoisomerase II is required for condensation; Adachi Y et al.; The role of topoisomerase II (topo II) in chromosome condensation was studied in a mitotic extract derived from Xenopus eggs by specific immunodepletion . HeLa nuclei, which have a high complement of endogenous topo II, are converted to mitotic chromosomes in the topo II-depleted extract equally well as in the control . Chicken erythrocyte nuclei, however, which have a very low content of topo II, do not convert to condensed chromosomes in the depleted extract, although their condensation is normal upon addition of purified topo II . Dosage experiments support the possible notion of a structural involvement of topo II in chromosome condensation . In the topo II-depleted extract the erythrocyte nuclei progress to precondensation chromosomes, which lack the nuclear membrane-lamina complex and consist of a cluster of swollen chromatids.

J Mol Biol, 1991 Jan 5, 217(1), 11 - 3
Crystallization and preliminary X-ray diffraction studies of a MAT alpha 2-DNA complex; Wolberger C et al.; Crystals have been obtained of the DNA-binding domain of the yeast MAT alpha 2 repressor bound to a 21 base-pair DNA site . The crystals are grown from polyethylene glycol and CaCl2 and form in space group P2(1) with a = 60.1 A, b = 39.4 A, c = 68.7 A and beta = 98 degrees . They diffract to 2.9 A resolution and contain one protein-DNA complex in the crystallographic asymmetric unit.

Anal Biochem, 1991 Jan, 192(1), 32 - 8
An angle-variable three-dimensional pulsed field gel electrophoresis system; Kolble K et al.; A three-dimensional pulsed field electrophoretic method based on the simultaneous application of fixed and cyclically alternating polarity fields at a right angle is described . Requiring only minimal electronic hardware it provides highly homogeneous field conditions over a large gel area and the versatility to vary the pulse vector angle . The electrophoretic parameters critical to achieve fast high resolution separation over a wide range of molecular sizes have been optimized and applied to megabase-size chromosomal DNA molecules . The empirical relationships between pulse time, field strength conditions, and resolution limits derived allow selection of coordinated experimental conditions for the separation of specific DNA size ranges.

Mol Endocrinol, 1991 Jan, 5(1), 111 - 22
Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2; Seidah NG et al.; Using a 796-basepair cDNA fragment obtained from a mouse pituitary library we have screened two mouse insulinoma libraries and isolated a full-length cDNA clone (2516 basepairs; 753 amino acids), designated mPC1 . The cDNA sequence of mPC1 codes for a protein containing 753 amino acids and three potential N-glycosylation sites . This cDNA encodes a putative novel subtilisin-like proteinase, exhibiting within its presumed catalytic domain 64%, 55%, and 47% amino acid sequence identity to the recently characterized candidate prohormone convertases human Furin, mouse PC2, and yeast Kex2 gene products, respectively . An identical sequence to mPC1 was derived from a cDNA library of mouse corticotroph AtT-20 tumor cells . An ArgGlyAsp tripeptide identical to the recognition sequence of integrins was observed in the structures of the mammalian PC1, PC2, and Furin . In situ hybridization results demonstrated a distinct localization of the mPC1 and mPC2 transcripts in pituitary and brain . Thus, whereas both mPC1 and mPC2 are found in the intermediate lobe of the pituitary, only mPC1 is easily detected in the anterior lobe . In extrahypothalamic regions of the brain, including cortex, hippocampus, thalamus, and spinal cord, mPC2 transcripts predominate over mPC1 . Both mRNAs are found in only a fraction of hypothalamic neurons, with greater abundance of mPC1 over mPC2 in the supraoptic nucleus . The genes coding for mPC1 and mPC2 map to the murine chromosomes 13 (band 13c) and 2 (2F3-2H2 region), respectively.

J Biochem (Tokyo), 1991 Jan, 109(1), 55 - 60
Studies on the flavin-binding region of old yellow enzyme with an active site probe, 8-fluoro-8-demethyl FMN; Fujii S et al.; The brewer's yeast old yellow enzyme (OYE) was reconstituted with 8-fluoro-8-demethyl FMN (8F-FMN) . The reconstituted enzyme exhibited absorption maxima at 355 and 450 nm in the visible region . This reconstituted enzyme underwent no further spectral changes, showing no evidence of modification in the flavin moiety . However, when the reconstituted enzyme was subjected to specific limited proteolysis with bovine alpha-chymotrypsin, gradual spectral changes were observed with disappearance of the 355- and 450-nm bands accompanied by the appearance of a new band at 496 nm . Identical spectral changes were observed when the proteolytically cleaved OYE (nicked OYE) was reconstituted with 8F-FMN . The process associated with these spectral changes was found to be unimolecular by kinetic analysis . Reverse-phase HPLC analysis revealed that these spectral changes resulted from covalent bond formation between 8F-FMN and the protein moiety after the proteolytic cleavage of the protein into 14K and 34K fragments . The reverse-phase HPLC monitored at 490 nm showed that the chromophore with 496 nm absorption maximum was covalently attached to the 14K fragment . The amino acid sequence analysis of the flavinylated 14K fragment together with that of the 14K fragment of native OYE indicated that the N-terminal leucine of the 14K fragment is the site of flavinylation . These findings imply that the amino group of the N-terminal leucine of the 14K fragment became available as the result of proteolysis and that this amino group nucleophilically attacked the 8-position of 8F-FMN, forming a covalent bond between the flavin moiety and the 14K fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Res, 1991 Jan 1, 51(1), 301 - 9
Expression and inducibility of drug-metabolizing enzymes in novel murine liver epithelial cell lines and their ability to activate procarcinogens; Paolini M et al.; Four novel nontransformed epithelial cell lines, isolated from fetal or adult mouse liver, were tested: (a) to determine the profile of xenobiotic metabolizing enzymes; (b) to evaluate the inducibility of the polysubstrate (cytochrome P-450-dependent) monooxygenase system by various classes of inducers; and (c) to assess the capacity of the cells to metabolize structurally different procarcinogens . With regard to the phase I pathway, the cells expressed various P-450 (class IA, IA2, IIB, IIE1, IIIA) and flavin adenine dinucleotide-containing monooxygenase-dependent bio-transformation enzyme activities at levels (in lines C2.8 and C6) comparable with those present in murine adult liver preparations . The expression of various P-450s was demonstrated also by immunoprecipitation assays using rabbit polyclonal antibodies . For the phase II pathway, cells expressed substantial levels of glutathione S-transferase, glutathione S-epoxide transferase, and UDP-glucuronosyltransferase . Low expression of epoxide hydrolase was observed . Induction of P-450 function by sodium phenobarbital, beta-naphthoflavone, isosafrole, ethanol, and pregnenolone 16 alpha-carbonitrile, monitored using specific P-450-linked activities, was considerably elevated (over 5-fold in class IIB with the C2.8 and C6 cell lines) . The most competent C2.8 and C6 cell lines were able to activate benzo(a)pyrene, cyclophosphamide, dimethylnitrosamine, diethylstilbestrol, and 2-naphthylamine as shown by the significantly increased frequencies of mitotic gene conversion, mitotic crossing-over, and point {reverse} mutation in the diploid D7 strain of Saccharomyces cerevisiae after 4 {cyclophosphamide}, 24 {benzo(a)pyrene,2-naphthylamine, dimethylnitrosamine} or 48 {diethylstilbestrol}, h of exposure in the presence of 3 x 10(6) cells/flask . The degree of conservation and the inducibility of representative oxidative and postoxidative reactions in the novel epithelial cell lines C2.8 and C6, together with their ability to activate a wide spectrum of procarcinogens, offers a means to study the potential of chemicals for inducing DNA damage in short-term genotoxicity testing . In addition the cells may be suitable for analyzing the metabolic disposition of compounds and the multistage process of carcinogenesis.

Mol Cell Biol, 1991 Jan, 11(1), 564 - 7
Proline-independent binding of PUT3 transcriptional activator protein detected by footprinting in vivo; Axelrod JD et al.; The PUT3 gene product is a transcriptional activator required for expression of the enzymes of the proline utilization pathway . Using two methods of footprinting in vivo, we have determined that PUT3 protein is poised at the promoters of the genes encoding these enzymes and that proline-mediated induction modulates the activity of constitutively bound PUT3.

Mol Cell Biol, 1991 Jan, 11(1), 486 - 96
Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control; Abastado JP et al.; GCN4 encodes a transcriptional activator of amino acid-biosynthetic genes in Saccharomyces cerevisiae that is regulated at the translational level by upstream open reading frames (uORFs) in its mRNA leader . uORF4 (counting from the 5' end) is sufficient to repress GCN4 under nonstarvation conditions; uORF1 is required to overcome the inhibitory effect of uORF4 and stimulate GCN4 translation in amino acid-starved cells . Insertions of sequences with the potential to form secondary structure around uORF4 abolish derepression, indicating that ribosomes reach GCN4 by traversing uORF4 sequences rather than by binding internally to the GCN4 start site . By showing that wild-type regulation occurred even when uORF4 was elongated to overlap GCN4 by 130 nucleotides, we provide strong evidence that those ribosomes which translate GCN4 do so by ignoring the uORF4 AUG start codon . This conclusion is in accord with the fact that translation of a uORF4-lacZ fusion was lower in a derepressed gcd1 mutant than in a nonderepressible gcn2 strain . We also show that increasing the distance between uORF1 and uORF4 to the wild-type spacing that separates uORF1 from GCN4 specifically impaired the ability of uORF1 to derepress GCN4 translation . As expected, this alteration led to increased uORF4-lacZ translation in gcd1 cells . Our results suggest that under starvation conditions, a substantial fraction of ribosomes that translate uORF1 fail to reassemble the factors needed for reinitiation by the time they scan to uORF4, but become competent to reinitiate after scanning the additional sequences to GCN4 . Under nonstarvation conditions, ribosomes would recover more rapidly from uORF1 translation, causing them all to reinitiate at uORF4 rather than at GCN4.

Appl Biochem Biotechnol, 1991 Spring, 28-29, 445 - 56
Bioconversion of a L-carnitin precursor in a one- or two-phase system; Bare G et al.; The ability of the yeast Saccharomyces cerevisiae to bioconvert stereo-selectively octyl-4-chloroacetoacetate (OCA) into the corresponding chiral alcohol, precursor of L-carnitin, an important physiological agent, was investigated . In a monophasic system with free cells, more than 90% of OCA (0.018 M) bioconversion have been reached after 6 h (enantiomeric excess for the R form, eeR:97%) . Immobilized cells in alginate beads were less efficient in conversion of OCA than free cells . In a two-phase system with free cells, the level of reduction of OCA (0.018 M) reached 85% after 48 h . With a medium containing a higher OCA concentration (0.270 M), 41% of this product were bioconverted after the same period . On the other hand, immobilized cells did not show any significant bioconversion of OCA in two-phase reactors . The limiting factor of these reactors in the regeneration of the cofactors involved in the OCA reduction.

Int J Cancer Suppl, 1991, 6, 18 - 9
Organization of the human HLA-class-II region; Hanson I et al.; A detailed map of the class-II region of the human MHC has been established by pulsed-field-gel-electrophoresis (PFGE) mapping and cloning in yeast-artificial-chromosome (YAC) vectors . The map revealed CpG islands, which are frequently associated with genes, in the gaps between the known HLA-class-II genes . Using cosmid walking and chromosome jumping, we have cloned 3 of these islands and have identified 5 novel genes, named RINGI-5 . This brief report summarizes our present knowledge of these genes.

Biochem J, 1991 Jan 1, 273(Pt 1), 225 - 8
Sequence of the beta-subunit of the phosphatidylinositol-specific phospholipase C-directed GTP-binding protein from squid (Loligo forbesi) photoreceptors; Ryba NJ et al.; The beta-subunit (G-beta) of the squid (Loligo forbesi) visual GTP-binding protein (G-protein), thought to be associated with a phosphatidylinositol-specific phospholipase C, has been identified and the sequence of the protein determined from its cDNA . The predicted polypeptide has a very marked sequence similarity with its mammalian counterparts (80-90% identity) . Squid G-beta also has somewhat lower similarity to the yeast protein STE4 (approx . 40% identity) . The role of G-beta in signal transduction is discussed in the light of its pronounced structural conservation.

Proc Natl Acad Sci U S A, 1991 Jan 1, 88(1), 224 - 8
Synergistic transcriptional enhancement does not depend on the number of acidic activation domains bound to the promoter; Oliviero S et al.; Many eukaryotic transcriptional activator proteins contain a DNA-binding domain that interacts with specific promoter sequences and an acidic activation region that is required to stimulate transcription . Transcriptional enhancement by such activator proteins is often synergistic and promiscuous; promoters containing multiple binding sites for an individual protein or even for unrelated proteins can be 10-100 times more active than promoters with single sites . It has been suggested that such synergy reflects a nonlinear response of the basic transcription machinery to the number and/or quality of acidic activation regions . Here, we determine the transcriptional activity of Jun-Fos heterodimers containing one or two GCN4 acidic activation regions on promoters containing one or two Ap-1 target sites . Surprisingly, heterodimers with one or two acidic regions activate transcription with similar efficiency and are equally synergistic (10- to 15-fold) on promoters containing two target sites . Thus, transcriptional synergy does not depend on the number of acidic activation regions but rather on the number of proteins bound to the promoter . This suggests that synergy is mediated either by cooperative DNA binding or by alternative mechanisms in which the DNA-binding domain plays a more direct role in transcription (e.g., changes in DNA structure, nucleosome displacement, or direct interactions with the transcriptional machinery).

Yi Chuan Xue Bao, 1991, 18(2), 175 - 84
{The effects of upstream region of SUC2 gene on its expression}; Xie KW et al.; A series of deletions were made at upstream region of SUC2 gene with the direction from about -900 bp to the initiation codon . The DNA fragments, which contain SUC22 gene and its deleted upstream region, were inserted into multicopy plasmid . After transforming resulted plasmid into SUC strain, the invertase activities produced by the transformants were determined . Under glucose repressing condition, the glycosylated invertase produced by transformants with deletion from -636 bp to -179 bp of SUC2 gene were gradually increased . The transformants with deletion down to -223 bp and -179 bp could produce about 100 times higher glycosylated invertase activity as compared to wild type . Under glucose derepressing condition, the glycosylated invertase produced by transformants with deletion from -395 bp to -179 bp of SUC2 gene were only slightly more than that produced under glucose repressing condition . Under either glucose repressing or derepressing condition, the transformants with deletion at -89 bp and -41 bp produced only a little of glycosylated invertase, while they produced remarkably higher nonglycosylated invertase activity.

Eur J Biochem, 1991 Jan 1, 195(1), 109 - 13
Irreversible transitions in the 6-phosphofructokinase/fructose 1,6-bisphosphatase cycle; Schellenberger W et al.; The dynamics of the fructose 6-phosphate fructose-1,6-bisphosphate cycle operating in an open and homogeneous system reconstituted from purified enzymes was extensively studied . In addition to 6-phosphofructokinase and fructose-1,6-bisphosphatase, pyruvate kinase, adenylate kinae and glucose-6-phosphate isomerase were involved . In that multi-enzyme system, the main source of non-linearity is the reciprocal effect of AMP on the activities of 6-phosphofructokinase and fructose-1,6-bisphosphatase . Depending upon the experimental parameter values, stable attractors, various types of multiple states and sustained oscillations were shown to occur . In the present report we show that irreversible transitions are also likely to occur for realistic operating conditions . Two parameters of the system, that is the adenylate energy charge of the influx and the fructose-1,6-bisphosphatase maximal activity, are potential candidates to provoke such irreversible transitions from one steady state to the other: (a) when varying the maximal activity of fructose-1,6-bisphosphatase, the system can jump irreversibly from a low to a high stable steady state, and (b) when the adenylate energy charge of the influx is the changing parameter, irreversible transitions occur from a high stable steady state to a stable oscillatory state (limit cycle motion) . This behavior can be predicted by constructing the loci of limit points and Hopf bifurcation points.

Enzyme, 1991, 45(5-6), 271 - 84
Mammalian neural and endocrine pro-protein and pro-hormone convertases belonging to the subtilisin family of serine proteinases; Seidah NG et al.; Conversion of pro-hormones and precursor proteins into biologically active peptides and proteins involves the concerted action of a number of convertases and post-translation modification enzymes . The identification of the yeast convertase kexin as a prototype processing enzyme led to the discovery of the mammalian convertase designated furin, PC1 and PC2 . Whereas furin is ubiquitously expressed, PC1 and PC2 are found only in endocrine and neural tissues and cell lines . In man and mouse, the genes coding for furin, PC1 and PC2 reside on three different chromosomes . The analysis of the intracellular processing of PC1 and PC2 and the removal of their pro-segment is presented, together with a summary of the cleavage specificity of these enzymes for precursors such as pro-opiomelanocortin (POMC) and human pro-renin . The distinct tissue distribution of PC1 and PC2 and their coregulation with POMC in the pituitary neurointermediate lobe adds credence to their physiological role as convertases involved in the tissue-specific processing of precursor proteins.

Enzyme, 1991, 45(5-6), 257 - 70
Furin: the prototype mammalian subtilisin-like proprotein-processing enzyme . Endoproteolytic cleavage at paired basic residues of proproteins of the eukaryotic secretory pathway; Van de Ven WJ et al.; Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues . Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae . Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway . Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2 . We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases . Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated . Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor.

Enzyme, 1991, 45(5-6), 239 - 43
Proteolytic processing and regulation; Neurath H; Many proteins, particularly proteolytic enzymes, protein hormones and neuropeptides are synthesized as inactive precursors that undergo posttranslational processing by proteolytic enzymes . The roots of current knowledge go back to the early observations of the activation of zymogens . A major advance followed the discovery of the polypreprotein, pre-pro-opiomelanolcortin and of proinsulin, and the characterization of the mammalian processing prohormone enzyme as members of the multidomain yeast kexin family . More recent applications of methods of molecular biology have greatly advanced our understanding of the nature and mode of action of these proteases.

Folia Microbiol (Praha), 1991, 36(1), 92 - 6
The use of some fluorescent stains for studying morphogenesis of micromycetes; Frank V; The use of some classical fluorochromes and optical brighteners in the fluorescence microscopy of micromycetes was investigated . Of the 16 compounds tested on slide cultures of Trichoderma viride 3 were too toxic, whereas the other stained primarily hyphae with various intensity . Reproductive structures did not stain or stained only weakly . With respect to vital staining the optical brightener Blankophor RKH exhibited most favorable properties . It did not inhibit either the growth or sporulation and stained intensively hyphae, septa and growth apices in particular . It also induced intensive fluorescence of a growing yeast culture.

Nucleic Acids Symp Ser, 1991, (24), 37 - 9
Further improvement in protecting method for the oligonucleotide synthesis in terms of a cellulose acetate derivative as a polymer support; Kamaike K et al.; For further improvement in the investigation to utilize a cellulose acetate derivative as a novel type of polymer-support for the synthesis of oligonucleotides, the investigations on utilizing another spacer; on protecting groups for O6-position of guanosine unit, ribothymidine, and pseudouridine; and on a novel protecting group for the introduction of phosphate function at 5'-terminal position, targeting the syntheses of 13-mer, ApApGpGpApApApApUpUpApUpG, 11-mer, pCpUpCpGpUpCpCpApCpCpA, and 12-mer, UpCpCpGpGprTp- psipCpGpApUpU, found in the partial structures of a yeast tRNA(Ala), will be described in detail.

Biomed Biochim Acta, 1991, 50(4-6), 403 - 12
Processing of mitochondrial precursor proteins; Arretz M et al.; The mitochondrial processing enzyme consists of two components, the mitochondrial processing peptidase (MPP) and processing enhancing protein (PEP) . MPP and PEP act cooperatively in proteolytic processing of mitochondrial precursor proteins . Most of the mitochondrial precursors possess aminoterminal presequences (also called "targeting sequences" or "signal sequences"), that do not display a common motif and that show only limited similarities of the cleavage sites . The mitochondrial processing peptidase is a metal-dependent endoprotease, sensitive to sulfhydryl-modifying reagents and appears to belong to a new class of proteases . MPP and PEP, together with the core 1 and core 2 proteins of the respiratory complex III, form a new protein family.

New Biol, 1991 Jan, 3(1), 18 - 26
E1a revisited: the case for multiple cooperative trans-activation domains; Braithwaite AW et al.; Products encoded in the E1a oncogene of adenoviruses are required to activate transcription of all viral early genes and some cellular genes . A current interpretation of experimental data supports the hypothesis that this "trans-activation" is mediated solely by a block of amino acids known as conserved domain 3, which is unique to the largest E1a protein, while the remaining E1a protein sequences contain discrete domains required for functions other than trans-activation . However, there is also considerable evidence inconsistent with this simple model of E1a structure and function . Both of the major E1a proteins appear to participate in trans-activation by three different types of interaction with cellular transcription factors and other regulatory proteins . In this review we attempt to rationalize the experimental data and provide a more integrated view of E1a structure and function.

Biomed Pharmacother, 1991, 45(10), 451 - 4
Study of the effects on DNA of electromagnetic fields using clamped homogeneous electric field gel electrophoresis; Novelli G et al.; A clamped homogeneous electric field (CHEF) electrophoresis allowing the separation of DNA molecules in the range of 200 to 3000 kb in size was used to study the biological effects of electric and magnetic fields (EMFs) . The results obtained did not show any detectable genomic damage on Saccharomyces cerevisiae.

Biomed Biochim Acta, 1991, 50(4-6), 459 - 64
Sequence comparison among subunits of multicatalytic proteinase; Sorimachi H et al.; The cDNAs for a number of multicatalytic proteinase (MCP) subunits have been cloned, characterized, and their primary structures have been determined . The mechanism for how MCP demonstrates its multicatalytic nature, especially protease activities, however, is still obscure, since no sequences similar to known protease sequences can be found in the sequences of MCP subunits thus far determined . To explain this fact, we propose a structural model for MCP: MCP consists of two classes of subunits, structural and catalytic, and the structural subunits constitute a "test-tube"-like container in which the other catalytic subunits sit and react with substrate . Most of the observations thus far obtained can be explained easily by this hypothesis, although various other possibilities are not excluded.

Fortschr Ophthalmol, 1991, 88(6), 671 - 6
{Experimental studies of proliferative vitreoretinopathy}; Kain HL; A new experimental rabbit model was developed to investigate vitreoretinal proliferation (PVR) . PVR was initiated by injection of zymosan A from Saccharomyces cerevisiae into the vitreous . The experiments were performed in two groups . In group A zymosan was injected into the normal vitreous; in group B zymosan was injected after the vitreous body had been degraded by the previous injection of hyaluronidase . In group A only moderate phagozytotic activity was found up to the 5 h day . However, in group B excessive invasion of macrophages was observed within 20 h and phagozytotic activity increased markedly . This was confirmed by an increase of enzymatic activity of beta-n-acetyl-glucoseaminidase in the anterior chamber and in the vitreous space . Transmission electron microscopy revealed characteristic morphology in zymosan A, which can apparently only be digested very slowly in the phagocytes . Therefore, macrophages could be traced during their transformation into fibroblastlike cells forming the vitreoretinal membranes.

Zentralbl Mikrobiol, 1991, 146(7-8), 557 - 62
{Suitability of support materials for the fixation of microfungi}; Menzel G et al.; Saccharomyces cerevisiae Muller-Thurgau F and Aspergillus spec . NH, a producer of glucoamylase, were cultivated in the presence of various supporting materials (three microspherical zeolitic particles, alpha-alumina and foam corundum) . The supports were microscopically tested to find out whether they are settled by microfungi or not . Whereas the yeast cells lay on the supports only loosely the hyphae of Aspergillus grew around the supports and with the exception of foam corundum enclosed them in mycelial spheres . This phenomenon may favour the separation of fungal biomass in biotechnological processes.

DNA Seq, 1991, 2(3), 181 - 91
Nucleotide sequence of murine PCNA: interspecies comparison of the cDNA and the 5' flanking region of the gene; Shipman-Appasamy PM et al.; Proliferating cell nuclear antigen (PCNA) RNA levels are regulated by transcription as well as changes in stability, in growing cells . We have cloned the murine PCNA cDNA and a fragment of the murine PCNA gene flanking the transcription initiation site . Comparison of the murine deduced amino acid sequence with the PCNA sequence from rat, human, Drosophila, Saccharomyces cerevisiae, and higher plants, reveals extensive homology between species . The homology is likely to be related to the fundamental role of PCNA as an auxiliary protein for DNA replication . Consensus sequences for transcriptional regulatory factors identified within 520 bp 5' of the cap site of the murine PCNA gene include: an inverted CCAAT site, an enhancer core element (EBP-1), three cAMP-response elements (CRE-BP), one AP-2 site, three Sp1 sites, and two octamer sequences . The first 20 bp of the transcriptional unit are homologous to an initiator element, which may direct transcription from RNA polymerase II in the absence of a TATAA box . The consensus elements in the murine PCNA gene are similar in sequence and/or location to elements identified in the genes for human, Drosophilia, and yeast PCNA.

Methods Enzymol, 1991, 194, 508 - 19
Epitope tagging and protein surveillance; Kolodziej PA et al.; The epitope tagging approach offers advantages of economy, universality, and precision over the use of antibodies raised directly against a protein of interest . The latter strategy promises a potentially greater diversity of reagents and obviates the need to modify the protein, but it may not yield sufficiently high-affinity, abundant, or specific antibodies . The major uncertainty in an epitope-tagging strategy, namely, the ability of the altered protein to function in vivo, is readily resolved in yeast by testing complementation of a null allele by the modified gene . Modification of the protein is easily accomplished by addition of the epitope coding sequence to the gene via oligonucleotide-mediated site-directed mutagenesis . The uniqueness of the epitope in the genome and the use of the monoclonal antibody assure a high-affinity, specific, and abundant antibody . Unrelated but identically modified proteins can be immunoprecipitated and affinity purified under the same conditions . Only extraction conditions and possibly a simple initial fractionation step need vary . Moreover, otherwise identical but differentially tagged proteins can be separated . Even proteins completely defective in an essential in vivo function can be purified and studied . Finally, polypeptides coprecipitating with the protein of interest are normally difficult to distinguish from those merely cross-reactive with the antibody used . As an alternative to defining a complex of proteins using a battery of antibodies, complexes are defined as a set of immunoprecipitable polypeptides present only in extracts containing the modified protein.

Comp Biochem Physiol C, 1991, 100(1-2), 111 - 3
Interaction with functional membrane proteins--a common mechanism of toxicity for lipophilic environmental chemicals?
Ahlers J, Cascorbi I, Foret M, Gies A, Kohler M, Pauli W, Rosick E.
1 . The effects of several phenols, anilines and aliphatic alcohols on yeast plasma membrane H(+)-ATPase and purine transport system as well as on Na+, K(+)-ATPase and adenosine uptake by Chinese hamster ovary cells (CHO) were investigated . 2 . In all cases an inhibition was observed, which could be correlated with the octanol/water partition coefficients of the substances tested, thus making quantitative structure-activity predictions possible . 3 . The observed effects correlated well with the influence of the chemicals on cell growth . 4 . The results suggest a common mechanism of toxicity by the action of hydrophobic xenobiotics on biomembranes.

Virchows Arch B Cell Pathol Incl Mol Pathol, 1991, 60(1), 21 - 6
Effect of methimazole-induced hypothyroidism on alveolar macrophages; Liu WK et al.; Chemically induced hypothyroidism changes the functions of rat alveolar macrophages . Treatment of female rats with an anti-thyroid drug, methimazole (1% aqueous solution in drinking water for 6 weeks) significantly (p less than 0.05) reduced the ability of alveolar macrophages (MAM) to phagocytose and kill the yeast, Saccharomyces cerevisiae . Undigested yeasts were observed in phagolysosomes within MAM using transmission electron microscopy . The activities of the lysosomal enzymes, acid phosphatase and beta-glucuronidase, and the Fc receptor binding ability for immunoglobulin G, were lowered in MAM when compared with control macrophages (CAM) . MAM also produced less tumor necrosis factor under the stimulation of lipopolysaccharide.

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