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H Atomi

Publications and source records attributed to H Atomi.

At least 37 records · Page 2Linked to original sources
PubMed1998

A regulatory factor, Fil1p, involved in derepression of the isocitrate lyase gene in Saccharomyces cerevisiae--a possible mitochondrial protein necessary for protein synthesis in mitochondria.

A mutant was isolated that failed to derepress the 5' upstream region of Candida tropicalis isocitrate lyase gene (UPR-ICL)-mediated gene expression in acetate medium, and the gene (FIL1) that complemented this mutation was isolated. The fil1 null mutant in which FIL1 is disrupted (deltafil1 strain) could not grow on acetate or ethanol, and the derepression of the isocitrate lyase encoded by ICL1 in Saccharomyces cerevisiae was also defected. The amino acid sequence of Fil1p (230 amino acids) showed similarity to ribosome recycling factors (RRFs) of prokaryotes. Compared to prokaryotic RRFs, Fil1p had an N-terminal 46-amino-acid extension which was shown to be able to function as a mitochondrial-targeting sequence. The subcellular fractionation of the deltafil1 strain showed that protein constituents of the mitochondrial fraction of the deltafil1 strain differed from those of the wild-type strain, but resembled those of chloramphenicol-treated cells or rho(o) cells. The specific activity of cytochrome c oxidase, was severely decreased in deltafil1, rho(o) and chloramphenicol-treated cells compared with wild-type cells, while enzymatic levels of mitochondrial NAD+-linked isocitrate dehydrogenase, which is encoded by nuclear DNA, were not affected. These results suggest that Fillp is necessary for protein synthesis in mitochondria of S. cerevisiae. Furthermore, cells treated with antimycin A, along with chloramphenicol-treated, rho(o), and deltafil1 cells, showed deficiency in derepression of isocitrate lyase. Northern-blot analysis showed that this can be ascribed to no increase in transcription of ICL1 and FBP1 encoding fructose 1,6-bisphosphatase. The results indicate the presence of a communication pathway between mitochondria and the nucleus which represses expression of genes encoding the key enzymes of the glyoxylate cycle and gluconeogenic pathway when there is a deficiency in the mitochondrial respiratory chain.

Amino Acid Sequence↗
PubMed1998

Peroxisomal and mitochondrial carnitine acetyltransferase isozymes of the n-alkane-assimilating yeast, Candida tropicalis, occurred by alternative initiation of translation from the transcripts of a single gene.

Carnitine acetyltransferase (CAT; EC 2.3.1.7) is localized in two subcellular organelles, peroxisomes and mitochondria, in an n-alkane-assimilating yeast, Candida tropicalis. The isozymes are synthesized from the first and second ATG codon of the open reading frame of one gene, CtCAT. Primer extension analysis and RNase protection assay (RPA) revealed that multiple transcription initiation sites were found upstream of the first ATG codon. 5' ends could not be detected between the first and second ATG codons. These results suggested that the peroxisomal CAT of C. tropicalis, initiating at the second AUG codon of the transcripts, was synthesized by a translational readthrough of the first AUG codon of the open reading frame. When CtCAT was introduced into the other yeast, Saccharomyces cerevisiae, 5' ends of transcripts and the protein products were similar to those observed in C. tropicalis. This suggested that the transcripts harbored sufficient information to bring about alternative initiation of translation in both yeasts. Using S. cerevisiae as the host cell, introduction of mutations into the sequence near the first AUG codon or a deletion in the region between the first and second AUG codons resulted in an increased ratio of translation from the first AUG codon, although initiation sites of transcription did not change. Moreover, replacing the 5' leader sequence to that of C. tropicalis isocitrate lyase promoter (UPR-ICL) eliminated the product initiating at the second AUG codon. The transcript from these cells was shorter than those detected from the native CtCAT-harboring cells. From these results, it was strongly suggested that peroxisomal and mitochondrial CAT isozymes occurred by the alternative initiation of translation mainly dependent on the structure and sequence context of the region from the 5' end to the second AUG codon, and not the insufficient length of the 5' leader.

Amino Acid Sequence↗
PubMed1998

Functional expression of a mammalian acetylcholinesterase in Pichia pastoris: comparison to acetylcholinesterase, expressed and reconstituted from Escherichia coli.

The mature rat brain acetylcholinesterase gene (T subunit, AChE) was subcloned downstream of the temperature-inducible lambda promoter PL and fused to the signal peptide of the OmpA protein. Three different expression vectors were constructed: (i) pCompmA containing the mature AChE, (ii) pComp delta TA containing a truncated AChE and (iii) pComp delta TAH containing the truncated AChE C-terminal fused to a 6xHis-tag. With all expression vectors the overexpression of AChE in Escherichia coli resulted mainly in cytoplasmic inclusion bodies (IB). However, some activity was found in the periplasmic space. The inclusion bodies were refolded in vitro, yielding up to 1.42 U/mg IB of active AChE. The refolded AChE was partially purified (approx. 300-fold) by affinity chromatography with a specific activity of approx. 250 U/mg. Removing the cysteine residue near the C-terminus (truncated AChE, delta TAChE) assuming to affect the refolding, did not increase the amount of active enzyme obtained after refolding. Purification of denatured delta TAChE-6xHis prior to refolding by Ni-NTA-chromatography increased the refolding efficiency by a factor of 1.5. Functional expression and secretion of rat brain acetylcholinesterase into the medium was achieved in Pichia pastoris. By optimizing the culture conditions, 100 mU/ml AChE in the medium was produced. In this work we are describing the functional expression of a mammalian AChE in a microbial host in good yields for the first time. The physico-chemical properties of both, the bacterial and yeast expressed AChE were compared with those of the native AChE. The properties of the yeast expressed AChE and the native AChE were similar, whereas the E. coli expressed enzyme was found to be less stable and had different inhibition properties.

Acetylcholinesterase↗
PubMed1998

High-level expression of the thermoalkalophilic lipase from Bacillus thermocatenulatus in Escherichia coli.

An efficient expression system for the previously only weakly expressed thermophilic lipase BTL2 (Bacillus thermocatenulatus lipase 2) was developed for the production of large amounts of lipase in Escherichia coli. Therefore, the gene was subcloned in the pCYT-EXP1 (pT1) expression vector downstream of the temperature-inducible lambda promoter PL. Three different expression vectors were constructed: (i) pT1-BTL2 containing the mature lipase gene, (ii) pT1-preBTL2 containing the prelipase gene and (iii) pT1-OmpABTL2 containing the mature lipase gene fused to the signal peptide of the OmpA protein, the major outer membrane protein of E. coli. With pT1-BTL2 and pT1-preBTL2, comparable expression levels of 7000-9000 U/g cells were obtained independently of the E. coli host. In contrast, with E. coli JM105 harbouring pT1-OmpABTL2, 660,000 soluble lipase U/g cells was produced, whereas, with E. coli DH5 alpha and BL321, production levels of 30,000 U/g cells were achieved. However, most of the lipase remained insoluble but active after cell breakage because of the unprocessed OmpA signal peptide. A simple cholate extraction followed by proteinase K cleavage and ultrafiltration allowed the isolation of 1.15 x 10(6) units of 90% pure mature lipase/wet cells.

Bacillus↗
PubMed1998

Genetic evaluation of physiological functions of thiolase isoenzymes in the n-alkalane-assimilating yeast Candida tropicalis.

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1a delta/t1bdelta null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3a delta/t3b delta null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C10 to C13). All thiolase activities in this yeast disappeared with the ct-t1a delta/t1bdelta and ct-t3a delta/t3bdelta null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bdelta null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal beta-oxidation. In addition to its essential contribution to the beta-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the beta-oxidation of a C4 substrate butyrate.

Acetyl-CoA C-Acetyltransferase↗
PubMed1997

Gene analysis of an NADP-linked isocitrate dehydrogenase localized in peroxisomes of the n-alkane-assimilating yeast Candida tropicalis.

In n-alkane-utilizing yeast, Candida tropicalis, two NADP-linked isocitrate dehydrogenase (NADP-IDH) isozymes are present, one in mitochondria (Mt-NADP-IDH) and the other in peroxisomes (Ps-NADP-IDH). Here we report the isolation, sequencing, and expression of the gene encoding Ps-NADP-IDH (CtIDP2), distinct from the Mt-NADP-IDH gene (CtIDP1). Based on the N-terminal amino acid sequence of purified Ps-NADP-IDH, a cDNA fragment specific for Ps-NADP-IDH was obtained by the 5'-RACE method. Using this fragment as a probe, the genomic CtIDP2 gene was isolated. Nucleotide sequence analysis of CtIDP2 disclosed that the region encoding CtIdp2p had a length of 1233 bp, corresponding to 411 amino acid residues. The deduced N-terminal amino acid sequence matched the results obtained from the purified protein. When this CtIDP2 was expressed in Saccharomyces cerevisiae using the C. tropicalis isocitrate lyase gene promoter (UPR-ICL), high intracellular NADP-IDH activity was observed. Comparison of amino acid sequences and phylogenetic tree analysis with NADP-IDH enzymes from all reported eukaryotic sources revealed that mammalian mitochondrial NADP-IDHs formed a cluster, as did plant NADP-IDHs. CtIdp2p and other yeast NADP-IDHs were not included in these clusters and seemed to diverge at an early stage from all other enzymes of higher eukaryotes. Ps-NADP-IDH had no typical C-terminal peroxisomal targeting signal and no processing was demonstrated at the N-terminus. However, we could find a region near the N-terminus of the protein with high similarity to both the putative N-terminal peroxisomal targeting signal sequence of Fox3p of S. cerevisiae and an internal region of Pox4p of C. tropicalis. The results of northern blot analysis indicated that the biosynthesis of CtIdp2p was induced in a medium containing alkanes as a carbon source, where profuse proliferation of peroxisomes is observed.

Alkanes↗
PubMed1997

Derepression of gene expression mediated by the 5' upstream region of the isocitrate lyase gene of Candida tropicalis is controlled by two distinct regulatory pathways in Saccharomyces cerevisiae.

The 5' upstream region of the gene encoding isocitrate lyase of Candida tropicalis (UPR-ICL) is functional as a promoter in Saccharomyces cerevisiae, and it is regulated by carbon source; the expression of the gene is repressed when cells are grown on glucose, while it increases to a higher level in acetate-grown cells. Therefore, we have investigated regions in UPR-ICL responsible for gene expression in glucose-grown and acetate-grown cells. In glucose-grown cells, a deletion of the region between -801 and -569 (region G1) significantly decreased gene expression compared with that observed with the complete UPR-ICL. The region from -421 to -379 (region G2) also repressed gene expression in glucose-grown cells. In acetate-grown cells, two regions were found to strongly enhance gene expression, one between -728 and -569 (region A1) and the other between -370 and -356 (region A2). Whereas region A2 contained a sequence motif similar to the carbon-source-responsive element (CSRE), which mediates regulation by carbon source of S. cerevisiae ICL1, region A1 did not show similarity to any reported cis-acting elements. Deletion mutants of UPR-ICL containing only one of these regions showed that each region could independently activate gene expression to a similar level when the cells were grown on acetate. The influences of null mutations in the MIG1, SNF1 and CAT8 genes on regulation of UPR-ICL-mediated gene expression were examined. Expression of the ICL gene with full-length UPR-ICL increased about tenfold in mig1 cells grown on glucose, while little difference was observed in acetate-grown cells. The effects of snf1 and cat8 mutations were different between region-A1-mediated and region-A2-mediated gene expression in acetate-grown cells. Region-A2-mediated expression decreased 95% and 86% in snf1 and cat8 cells, respectively, while region-A1-mediated expression decreased 72% in snf1 cells and was not affected by the cat8 mutation. This finding indicates that region-A1-mediated gene expression is regulated by a pathway independent of CAT8, which is necessary for derepression of CSRE-mediated gene expression in S. cerevisiae.

Base Sequence↗
PubMed1997

Analysis of carbon source-regulated gene expression by the upstream region of the Candida tropicalis malate synthase gene in Saccharomyces cerevisiae.

We investigated the regulation of expression of a gene encoding malate synthase (MS) of an n-alkane-utilizable yeast Candida tropicalis in the yeast Saccharomyces cerevisiae, where its expression is highly induced by acetate. By comparing levels of gene expression in cells grown on glucose, acetate, lactate, and oleic acid, we found that the increase in gene expression was due to a glucose repression-derepression mechanism. In order to obtain information concerning the regulation of the gene expression, a fusion gene which consists of the 5'-upstream region of MS-2 (UPR-MS-2) and the lacZ gene (encoding Escherichia coli beta-galactosidase), was introduced into S. cerevisiae, and beta-galactosidase activities were measured with cells grown on glucose or acetate. Deletion analysis of UPR-MS-2 revealed that the region between -777 and -448 (against the translation initiation codon) enhanced the level of gene expression in both glucose- and acetate-grown cells. In this region, sequences which resemble binding sites of Rap1p/Grf1p/Tufp, a global transcription activator, were found at seven locations and one was found for another pleiotropic activator Abf1p. The result also suggested the presence of multiple upstream repression sequences (URSs), which function specifically in glucose-grown cells, in the region between -368 and -126. In the repressing region, there were three tandem C(A/T)CTCCC sequences and also a putative binding site of Mig1p, a transcriptional repressor which mediates glucose repression of several other genes. When MIG1 gene of S. cerevisiae was disrupted, the expression of the UPR-MS-2-lacZ gene in glucose-grown cells increased approx. 10-fold. Furthermore, the effect of deletion of a putative Mig1p binding site was abolished in the MIG1-disrupted strain, suggesting Mig1p binds to this site and brings about glucose repression. When the SNF1 gene was disrupted, the high level gene expression observed in acetate-grown cells bearing UPR-MS-2 was abolished. This indicated that derepression of UPR-MS-2 -mediated gene expression was dependent on Snf1p, as is the case of genes encoding isocitrate lyase and gluconeogenic enzymes in S. cerevisiae.

Acetates↗
PubMed1997

Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae.

Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5'-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast lambdaEMBL3 library. The nucleotide sequence of the gene encoding C. tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPR-ICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae.

Amino Acid Sequence↗
PubMed1997

Immunochemically distinct NADP-linked isocitrate dehydrogenase isozymes in mitochondria and peroxisomes of Candida tropicalis.

Although peroxisomal localization of NADP-linked isocitrate dehydrogenase (Idp) was first demonstrated in Candida tropicalis, the mitochondrial isozyme has not been found in this yeast. Here we report that the presence of mitochondrial Idp in the yeast was demonstrated by screening for its gene with a DNA probe containing conserved sequences of Idps from various organisms. The nucleotide sequence of the gene (CtIDP1) revealed a 1,290-bp open reading frame corresponding to a 430-amino-acid protein with a high similarity to previously reported Idps. Overexpression of CtIDP1 in Saccharomyces cerevisiae gave a high intracellular Idp activity, and the purified recombinant Idp was shown to be a homodimer with a subunit molecular mass of approximately 44 kDa, different from that of peroxisomal Idp (45 kDa) previously purified from C. tropicalis. Western blot analysis of the subcellular fractions from acetate-grown C. tropicalis with polyclonal antibodies raised against the recombinant CtIdp1 showed that the CtIdp1 in C. tropicalis was localized in mitochondria but not in peroxisomes. Similar levels of CtIDP1 mRNA and its protein product were detected in cells grown on glucose, acetate, and n-alkane, although a slight decrease was observed in n-alkane-grown cells. From these results, CtIdp1 was demonstrated to be mitochondrial Idp. The properties of mitochondrial Idp and peroxisomal Idp isozymes were proven to be similar, but they were immunochemically distinct, suggesting the presence of another gene responsible for peroxisomal Idp in C. tropicalis.

Amino Acid Sequence↗
PubMed1997

Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae.

We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form.

Aspergillus↗
PubMed1997

Expression of acetoacetyl-CoA thiolase isozyme genes of n-alkane-assimilating yeast, Candida tropicalis: isozymes in two intracellular compartments are derived from the same genes.

In the n-alkane-assimilating yeast Candida tropicalis, there are two isozymes of acetoacetyl-CoA thiolase, peroxisomal acetoacetyl-CoA thiolase (peroxisomal Thiolase I), and cytosolic acetoacetyl-CoA thiolase (cytosolic Thiolase I). We have previously isolated two genes (CT-T1A and CT-T1B) which encode Thiolase I. In order to compare the expressed products of Thiolase I isozyme-encoding genes in C. tropicalis, cytosolic Thiolase I was first purified from glucose-grown C. tropicalis in which the proliferation of peroxisomes and the expression of peroxisomal Thiolase I were repressed. Cytosolic Thiolase I was virtually identical to peroxisomal Thiolase I in molecular mass, kinetic and immunochemical properties, and primary structure at the N-terminus. Amino acid sequence analysis revealed that cytosolic Thiolase I was the mixture of products of two genes (CT-T1A and CT-T1B), as in the case of the peroxisomal enzyme. CT-T1A and CT-T1B were expressed independently in the yeast Saccharomyces cerevisiae and the recombinant proteins were purified. Recombinant Thiolase IA and IB exhibited practically identical enzymatic properties to cytosolic and peroxisomal Thiolase Is from C. tropicalis. These results revealed that cytosolic Thiolase I and peroxisomal Thiolase I were encoded not by different genes, but by the same genes (CT-T1A and CT-T1B) and are present as a mixture of products expressed by both genes, although their subcellular localizations are different.

Acetyl-CoA C-Acetyltransferase↗
PubMed1997

Recombinant thermostable cycloinulo-oligosaccharide fructanotransferase produced by Saccharomyces cerevisiae.

A truncated fragment of the cycloinulo-oligosaccharide fructanotransferase (CFTase) gene of Bacillus circulans MCI-2554 was fused to the prepro secretion sequence of the alpha-factor and expressed in Saccharomyces cerevisiae under the control of the 5' upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL). Efficiently secreted recombinant CFTase protein (yeast CFTase) was purified. Yeast CFTase consisted of three protein molecules, each of which had CFTase activity (yeast CFTase 1 [116 kDa], yeast CFTase 2 [117 kDa], and yeast CFTase 3 [116 kDa]). Yeast CFTase 2 was the major product of the expression system employed and was shown to be N glycosylated by endoglycosidase H treatment. Yeast CFTase 1 was N glycosylated but had a short truncation at its N terminus, while yeast CFTase 3 did not contain an N-glycosylated carbohydrate chain(s). Yeast CFTase 2 showed an optimum pH, an optimum temperature, and a pH stability similar to those of CFTase purified from B. circulans but exhibited a significant increase in thermostability. Production of yeast CFTase by the strain which had two copies of the CFTase gene integrated into its chromosomes reached 391 U per liter of culture at 120 h, which corresponded to 8.40 mg of protein per liter, by shake-flask cultivation.

Amino Acid Sequence↗
PubMed1997

Construction of a starch-utilizing yeast by cell surface engineering.

We have engineered the cell surface of the yeast Saccharomyces cerevisiae by anchoring active glucoamylase protein on the cell wall, and we have endowed the yeast cells with the ability to utilize starch directly as the sole carbon source. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The constructed plasmid containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The glucoamylase activity as not detected in the culture medium, but it was detected in the cell pellet fraction. The glucoamylase protein transferred to the soluble fraction from the cell wall fraction after glucanase treatment but not after sodium dodecyl sulfate treatment, indicating the covalent binding of the fusion protein to the cell wall. Display of the fused protein was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. The transformant cells could surely grow on starch as the sole carbon source. These results showed that the glucoamylase was anchored on the cell wall and displayed as its active form. This is the first example of an application of cell surface engineering to utilize and improve the metabolic ability of cells.

Cell Membrane↗
PubMed1996

Peroxisomal and mitochondrial carnitine acetyltransferases of the n-alkane-assimilating yeast Candida Tropicalis. Analysis of gene structure and translation products.

A genomic DNA clone encoding carnitine acetyltransferases (EC 2.3.1.7), localized in two subcellular organelles, peroxisomes and mitochondria of an n-alkane-assimilating yeast Candida tropicalis, was isolated from the yeast lambda EMBL library using a carnitine acetyltransferase CDNA probe. Nucleotide sequence analysis disclosed that the open reading frame was 1881 bp, corresponding to 627 amino acids with a molecular mass of 70760 Da. Comparison of the predicted amino acid sequence of the C. tropicalis enzyme with that of Saccharomyces cerevisiae mitochondrial matrix carnitine acetyltransferase revealed 46.3% identity. It was noticeable that the C. tropicalis enzymes had amino acid sequences similar to both proposed mitochondrial and peroxisomal targeting signals. When the C. tropicalis gene was expressed in S. cerevisiae using its own 5'-upstream region, a 12-fold increase in activity was observed. Western blot analysis revealed the presence of two major proteins whose sizes corresponded to the peroxisomal and mitochondrial proteins detected in C. tropicalis. This suggested that peroxisomal and mitochondrial carnitine acetyltransferases were encoded by one gene, as suggested for the S. cerevisiae enzyme. Furthermore, we have separated and purified these enzymes from peroxisomes and mitochondria of C. tropicalis, and analyzed the amino-terminal amino acid sequences of each. The amino-terminal sequence of the mitochondrial enzyme suggested that a signal sequence had been cleaved during translocation into mitochondria. Concerning the peroxisomal enzyme, the evidence obtained indicated that in vivo the translation was initiated at the second methionine of the open reading frame.

Alkanes↗
PubMed1996

Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. molecular cloning, nucleotide sequence, purification and some properties.

An expression library was generated by partial Sau3A digestion of genomic DNA from the thermophile Bacillus thermocatenulatus and cloning of DNA fragments in pUC18 in Escherichia coli DH5alpha. Screening for lipase activity identified a 4.5 kb insert in pUC18 which directed the production of lipase in E. coli DH5alpha. A subclone with a 2.2 kb insert was sequenced. The lipase gene codes for a mature lipase of 388 amino acid residues, corresponding to a molecular weight of 43 kDa. As in other Bacillus lipases, an Ala replaces the first Gly in the conserved pentapeptide Gly-X-Ser-X-Gly found in most lipases. The region upstream of the lipase gene contains a Bacillus promoter which directs the expression of lipase in E. coli DH5alpha. The expressed lipase was isolated and purified 312-fold to homogeneity. N-terminal sequencing of the purified lipase revealed a correct cleavage of the preprotein in E. coli DH5alpha. Maximum activity was found at pH 8.0-9.0 with tributyrin and olive oil as substrates and at 60-70 degrees C with p-NPP and olive oil as substrates. The lipase showed high stability at pH 9.0-11.0 and towards various detergents and organic solvents.

Amino Acid Sequence↗
PubMed1996

A novel heterologous gene expression system in Saccharomyces cerevisiae using the isocitrate lyase gene promoter from Candida tropicalis.

We have found that the upstream region of the isocitrate lyase gene (UPR-ICL) from the n-alkane-utilizing yeast Candida tropicalis was functional in Saccharomyces cerevisiae as a novel promoter with nonfermentable carbon sources, such as oleic acid, acetate, ethanol, and glycerol/lactate. The expression of two foreign genes coding for beta-galactosidase from Escherichia coli (LacZ) and glutamate decarboxylase from rat brain was carried out under the control of UPR-ICL. Expression of LacZ was repressed by glucose and enhanced over 300-fold by acetate. When an expression vector pWI3 containing multicloning sites between UPR-ICL and the transcriptional terminator of the isocitrate lyase gene (TERM-ICL) was used, the smaller isoform of glutamate decarboxylase (GAD65) was highly produced in a soluble and active form. These results demonstrate that the novel expression system using UPR-ICL and TERM-ICL from C. tropicalis is useful for the production of heterologous proteins in S. cerevisiae.

Animals↗