Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes.

The mutation Gly467-->Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka congruent with 10(13).M(-1)) than the Glu enzyme (Ka congruent with 10(12).M(-1)). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (Ka congruent with 10(4).M(-1)) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.     

Systems properties of proteins encoded by imprinted genes

Genomically imprinted genes show parentally fixed mono-allelic expression and are important for the mammalian development. Dysregulation of genomic imprinting leads to several complex pathological conditions. Though the genetic and epigenetic regulation of imprinted genes has been well studied, their protein aspects are largely ignored. Here, we systematically studied a sub-network centered on proteins encoded by imprinted genes within human interactome. Using concepts of network biology, we uncover a highly connected, transitive and central network module of imprinted gene-products and their interacting partners (IGPN). The network is enriched in development, metabolism and cell cycle related functions and its malfunctioning ascribes error intolerance to human interactome network. Further, detailed analysis revealed that its higher centrality is determined by ‘date’ interactions among the proteins belonging to different functional classes than the ‘party’ interactions within the same functional class. Interestingly, a significant proportion of this network genetically associates with disease phenotypes. Moreover, the network comprises of gene-sets that are upregulated in leukemia, psychosis, obesity/diabetes and downregulated in autism. We conclude that imprinted gene-products are part of a functionally and topologically important module of human interactome and errors in this sub-network are intolerant to, otherwise robust, human interactome. The findings might also shed light on how imprinted genes, which are rather very few, coordinate at protein level to pleiotropically regulate growth and metabolism during embryonic and post-natal development.     

Glucoamylases: structural and biotechnological aspects

Glucoamylases, one of the main types of enzymes involved in starch hydrolysis, are exo-acting enzymes that release consecutive glucose units from the non-reducing ends of starch molecules. Glucoamylases are microbial enzymes, present in bacteria, archaea, and fungi but not in plants and animals. Structurally, they are classified in family 15 of glycoside hydrolases and characterised by the invariable presence of a catalytic domain with (α/α)₆-fold, often bound to a non-catalytic domain of diverse origin and function. Fungal glucoamylases are biotechnologically very important as they are used industrially in large amounts and have been extensively studied during the past 30 years. Prokaryotic glucoamylases are of biotechnological relevance for being generally thermophilic enzymes, active at elevated temperatures.     

Primary structures and catalytic properties of isoenzymes encoded by the two 4-coumarate: CoA ligase genes in parsley

We have determined the primary structures of two 4-coumarate: CoA ligase (4CL) isoenzymes in parsley (Petroselinum crispum) by sequencing near full-length cDNAs corresponding to the two 4CL genes, Pc4CL-1 and Pc4CL-2, present in this plant. Comparison of the cDNA and genomic nucleotide sequences showed that each 4CL gene is organized in five exons separated by introns of varying lengths. The positions of introns are the same in both genes and 97-99% of the corresponding nucleotide sequences are identical. The two isoenzymes, which are nearly identical in their primary structures, were separated by ion-exchange chromatography, and were found to be indistinguishable with regard to substrate specificity. Assignment to Pc4CL-1 and Pc4CL-2 was achieved by comparison with catalytically active 4CL proteins, isolated from Escherichia coli cells which had been transformed with plasmids harboring the corresponding cDNAs.     

Human acetylcholinesterase and butyrylcholinesterase are encoded by two distinct genes

1. Various hybridization approaches were employed to investigate structural and chromosomal interrelationships between the human cholinesterase genes CHE and ACHE encoding the polymorphic, closely related, and coordinately regulated enzymes having butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities. 2. Homologous cosmid recombination with a 190-base pair 5' fragment from BuChEcDNA resulted in the isolation of four overlapping cosmid clones, apparently derived from a single gene with several introns. The Cosmid CHEDNA included a 700-base pair fragment known to be expressed at the 3' end of BuChEcDNA from nervous system tumors and which has been mapped by in situ hybridization to the unique 3q26-ter position. In contrast, cosmid CHEDNA did not hybridize with full-length AChEcDNA, proving that the complete CHE gene does not include AChE-encoding sequences either in exons or in its introns. 3. The chromosomal origin of BuChE-encoding sequences was further examined by two unrelated gene mapping approaches. Filter hybridization with DNA from human/hamster hybrid cell lines revealed BuChEcDNA-hybridizing sequences only in cell lines including human chromosome 3. However, three BuChEcDNA-homologous sequences were observed at chromosomal positions 3q21, 3q26-ter, and 16q21 by a highly stringent in situ hybridization protocol, including washes at high temperature and low salt. 4. These findings stress the selectivity of cosmid recombination and chromosome blots, raise the possibility of individual differences in BuChEcDNA-hybridizing sequences, and present an example for a family highly similar proteins encoded by distinct, nonhomologous genes.     

Characterization of embryo globulins encoded by the maizeGlb genes

Two of the most abundant proteins in maize embryos are saline-soluble, water-insoluble globulins. One is aM _r 63,000 protein encoded by theGlbl gene and the other is aM _r 45,000 component encoded by theGlb2 gene. Both proteins accumulate to high levels during embryo development and are rapidly degraded during the early stages of seed germination. Amino acid composition analysis indicates that these proteins may serve as storage reserves to provide sources of nitrogen and carbon to the germinating embryo. Amino-terminal sequence analysis of the finalGlb1 gene product, GLB1, and its immediate precursor, GLB1′, indicates that the latter is proteolytically cleaved near the amino terminus to form GLB1. In addition to these biochemical studies, we describe the identification of a novel maize variant which lacks the protein product of theGlb2 gene. This contribution from the University of Illinois Agricultural Experiment Station was supported by grants from The Standard Oil Corporation, a wholly owned subsidiary of BP America, Inc., and the U.S. Department of Agriculture (No. 88-37262-3427).     

Structural characterization of antigens encoded by rabbitRLA-11 histocompatibility genes

Products of rabbitRLA-11 histocompatibility genes were isolated from rabbit lymphoid tumor cells (RL-5) and characterized. The tumor cells were grown in culture with radioactive amino acids and were lysed by treatment with the detergent NP-40. Glycoprotein molecules were isolated by affinity chromatography using immobilized lentil lectin. Chromatography on purified sheep anti-rabbit beta-2-microglobulin yielded a 43,000 dalton glycoprotein fraction, designated RLA-11gp, which was noncovalently associated with beta-2-microglobulin. N-terminal amino acid sequence analysis using radiochemical methods allowed assignment of 28 of the N-terminal 35 residues. The data revealed 89% homology of the RLA-11gp N-terminus with that of the human HLA-B7 and 82% with the mouse H-2K^b histocompatibility antigens. Comparison of the RLA-11 gp N-terminal sequence data obtained in this work to sequence data reported for major histocompatibility complex antigens of other species revealed no amino acid substitutions unique to the rabbit antigens.     

Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes.

A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional beta-galactosidase. Deletion and complementation analysis showed that the coding region for beta-galactosidase was located on a 5.8-kb SalI-BamHI fragment. Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively. The L. lactis beta-galactosidase was overproduced in E. coli by using a lambda pL expression system. Two new proteins with M(r)s of 75,000 and 36,000 appeared upon induction of PL. The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional beta-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of beta-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral beta-galactosidase gene.     

Nisin biosynthesis genes are encoded by a novel conjugative transposon

Summary Genes for biosynthesis of the lactococcal peptide antibiotic nisin were shown to be encoded by a novel chromosomally located transposon Tn5301. The element is 70 kb in size and lacks inverted repeats at its termini. Although a copy of the insertion sequence IS904 is located near to one end, this did not appear to be involved in the transposition process. The integrated element is flanked by the directly repeated sequence 5-TTTTTG-3. Analysis of ten independent transconjugants revealed that Tn5301 integration is site-specific; two chromosomal targets were identified and shown to have some sequence homology. The element shares features with the Tn916 family of conjugative transposons and with Tn554 but is also exhibits some unique properties. Tn5301 is thus considered to be the prototype of a novel class of conjugative transposon.     

Expression of proteins encoded by foreign genes in Saccharomyces cerevisiae

The yeast, Saccharomyces cerevisiae is currently used for the production of recombinant DNA-generated proteins derived from a variety of eukaryotic organisms. The applications of a yeast-based technology in the production of proteins for pharmaceutical and industrial purposes is discussed including current methods for introducing recombinant genes into yeast and strategies for maximizing their expression.     

Tumour genes in plants: T-DNA encoded cytokinin biosynthesis

Gene 4 from the T-region of Ti plasmids is responsible for cytokinin effects in crown gall cells; we investigated whether it codes for an enzyme of hormone biosynthesis. In a first set of experiments, gene 4 from octopine plasmid pTiAch5 and nopaline plasmid pTiC58 was expressed in Escherichia coli, and the gene products were identified by reaction with antiserum raised against a decapeptide derived from the DNA sequence of the gene. Extracts from cells expressing the gene contained high isopentenyl-transferase activity catalyzing the formation of N6-(delta2-isopentenyl)adenosine from 5'-AMP and delta2-isopentenylpyrophosphate. The cytokinin was identified by sequential h.p.l.c. chromatography and mass spectrometry. In a second set of experiments it was shown that crown gall cells contained isopentenyltransferase activity and a protein of mol. wt. 27 000 which was identified as the product of gene 4 by reaction with the antiserum. Isopentenyltransferase activity was specifically inhibited by the antiserum. No comparable enzyme activity or immunoreactive protein was detected in cytokinin-autotrophic, T-DNA free tobacco cells. The results establish that gene 4 from the T-region of octopine and nopaline Ti plasmids codes for an enzyme of cytokinin biosynthesis.     

Identification of proteins encoded by Epstein-Barr virus trans-activator genes.

Specific antisera were generated to characterize Epstein-Barr virus proteins reported to have trans-activating properties. Open reading frame BRLF1 was found to be expressed in two modifications in vivo, with molecular sizes ranging from 94 to 98 kilodaltons (kDa) depending on the cell line, whereas only one protein (Raji cells, 96 kDa) was detected by in vitro translation. Open reading frame BZLF1 encoded polypeptides of 38 and 35 kDa and additional smaller forms. A BZLF1-encoded 30-kDa protein could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be shown for the proteins derived from reading frames BZLF1 and BMLF1. BMLF1 expression gave a heterogeneous protein pattern, with molecular sizes between 45 and 70 kDa, including a predominant 60-kDa protein detected in different B-cell lines.     

Multiple xylanases of Cellulomona fimi are encoded by distinct genes

Cellulomonas fimi genomic DNA encoding xylanase activity has been cloned and expressed in Escherichia coli. As judged by DNA hybridization and restriction analysis, twelve xylanase-positive clones carried a minimum of four different xylanase (xyn) genes. The encoded enzymes were devoid of cellulase activity but three of the four bound to Avicel.     

Lipophilic proteins encoded by mitochondrial and nuclear genes in Neurospora crassa.

Mitochondrial proteins soluble in neutral chloroform-methanol (2:1) were separated from lipids by ether precipitation and resolved by Sephadex G-200 filtration in the presence of dodecylsulfate into two major fractions eluting in the excluded region (peak I) and in a region of an apparent molecular weight 8000 (peak II). Residual phospholipids are found only in peak II. Peak I consists of several aggregated small polypeptides of molecular weights around 8000, which can be disaggregated by mild oxidation with performic acid. Cycloheximide stimulates almost two-fold incorporation of radioactive phenylalanine into peak I proteins but inhibits labelling of peak II proteins by 95%. Chloramphenicol and ethidium bromide inhibit the synthesis of peak I proteins by 70% and 95% respectively, but do not affect labelling of peak II proteins. At least 30% of the translation products of mitochondrial DNA in vitro behave like peak I proteins: they are soluble in organic solvents, they aggregate in dodecylsulfate buffer after removal of phospholipids and they contain species of molecular weights around 8000 that disaggregate upon oxidation. The data strongly suggest that the proteins of peak I are encoded by mitochondrial genes and synthesized on mitochondrial ribosomes, whereas the proteins of peak II are encoded by nuclear genes and synthesized on cytoplasmic ribosomes. Both groups of lipophilic proteins are very similar in their molecular weights, but the mitochondrially coded peak I proteins have the unique property of forming large heat-stable aggregates in the presence of dodecylsulfate.     

Identification and localization of proteins encoded by two DIF-inducible genes of Dictyostelium

We show that pDd56 and pDd63, two related DIF-inducible genes of Dictyostelium, respectively encode the ST310 and ST430 polypeptides identified by Morrissey, Devine, and Loomis (1984, Dev. Biol. 103, 414-424). We localize the two proteins by immunoelectron microscopy to the extracellular matrix surrounding the stalk cells and the stalk tube. Coupled with their predicted amino acid sequence and biochemical properties, this suggests that they are structural proteins of the stalk.     

Starch fermentation by recombinant saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from lipomyces kononenkoae and saccharomycopsis fibuligera

Lipomyces kononenkoae and Saccharomycopsis fibuligera possess highly efficient alpha-amylase and/or glucoamylase activities that enable both of these yeasts to utilize raw starch as a carbon source. Eight constructs containing the L. kononenkoae alpha-amylase genes (LKA1 and LKA2), and the S. fibuligera alpha-amylase (SFA1) and glucoamylase (SFG1) genes were prepared. The first set of constructs comprised four single gene cassettes each containing one of the individual amylase coding sequences (LKA1, LKA2, SFA1 or SFG1) under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator, while the second set comprised two single cassettes containing SFA1 and SFG1 linked to their respective native promoters and terminators. The third set of constructs consisted of two double-gene cassettes, one containing LKA1 plus LKA2 under the control of the PGK1 promoter and terminator, and the other SFA1 plus SFG1 controlled by their respective native promoters and terminators. These constructs were transformed into a laboratory strain Saccharomyces cerevisiae (Sigma1278b). Southern-blot analysis confirmed the stable integration of the different gene constructs into the S. cerevisiae genome and plate assays revealed amylolytic activity. The strain expressing LKA1 and LKA2 resulted in the highest levels of alpha-amylase activity in liquid media. This strain was also the most efficient at starch utilization in batch fermentations, utilizing 80% of the available starch and producing 0.61g/100 mL of ethanol after 6 days of fermentation. The strain expressing SFG1 under the control of the PGK1 expression cassette gave the highest levels of glucoamylase activity. It was shown that the co-expression of these heterologous alpha-amylase and glucoamylase genes enhance starch degradation additively in S. cerevisiae. This study has resulted in progress towards laying the foundation for the possible development of efficient starch-degrading S. cerevisiae strains that could eventually be used in consolidated bioprocessing, and in the brewing, whisky, and biofuel industries.