Cloning, sequencing, and expression of an endoglucanase gene from the rumen anaerobic fungus Neocallimastix frontalis MCH3

A cDNA clone encoding an endo-1,4-beta-glucanase from a rumen fungus, Neocallimastix frontalis MCH3, was isolated. The nucleotide sequence showed that the gene, celA, encoded a multidomain enzyme containing a family 5 catalytic domain and a reiterated sequence that is involved in the association of a multienzyme complex, the cellulosome. The enzyme expressed in Escherichia coli showed the highest activity against carboxymethylcellulose at 40 degrees C and pH 8.5.     

Cloning and characterization of the gene encoding a new DNA methyltransferase from Neisseria gonorrhoeae

A HindIII fragment of N. gonorrhoeae MS11 DNA coding for DNA methyltransferase (MTase) activity was cloned and expressed in E. coli AP1-200-9 cells. The sequence of 4681 bp was determined, and its analysis revealed two open reading frames (ORFs) sharing some similarity with known DNA MTases. ORF1 encodes an active N4mC MTase (M.NgoMV). The enzyme modifies only one strand of double stranded DNA and preferentially recognises the sequence GCCHR although it is able to methylate other sites. The exact recognition sequence cannot be precisely defined due to a relaxed specificity. The second ORF shows high homology to 5mC Mtases, but we were unable to demonstrate DNA methylating activity of its product either in vivo or in vitro.     

Cloning and characterization of a eukaryotic pantothenate kinase gene (panK) from Aspergillus nidulans

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway. The PanK gene from Escherichia coli (coaA) has been previously cloned and the enzyme biochemically characterized; highly related genes exist in other prokaryotes. We isolated a PanK cDNA clone from the eukaryotic fungus Aspergillus nidulans by functional complementation of a temperature-sensitive E. coli PanK mutant. The cDNA clone allowed the isolation of the genomic clone and the characterization of the A. nidulans gene designated panK. The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is expressed constitutively. The amino acid sequence of A. nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and bore little resemblance to its bacterial counterpart, whereas a highly related protein was detected in the genome of Saccharomyces cerevisiae. In contrast to E. coli PanK (bPanK), which is regulated by CoA and to a lesser extent by its thioesters, aPanK activity was selectively and potently inhibited by acetyl-CoA. Acetyl-CoA inhibition of aPanK was competitive with respect to ATP. Thus, the eukaryotic PanK has a distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart.     

Cloning and characterization of an ecdysone receptor cDNA from Locusta migratoria

It has been suggested that some mitochondrial genes are important in cellular senescence. In order to identify the mitochondrial genes that are involved in cellular senescence, we have constructed a cDNA library from senescent human vascular endothelial cells and isolated 86 senescence-specific cDNA clones by differential screening. Among the clones, we identified four distinct mitochondrial genes including NADH dehydrogenase subunit 2 (ND2), ND3, ATPase 6 and 16S ribosomal RNA. We then compared the levels of expression of these genes in young and senescent cells by using two endothelial and two fibroblast cell strains. Northern blot and slot blot hybridization confirmed that the expression levels of ND3, ATPase 6 and 16S rRNA were elevated in senescent cells of all four strains. The expression level of ND2 was also elevated during cellular senescence in three of the four strains. Because mitochondria are actively involved in oxidative phosphorylation and respiratory functions, the altered expression levels of these genes may participate in aging processes.     

Cloning and biochemical characterization of an anionic peroxidase from Zea mays

We have isolated, cloned and characterized a cDNA from Zea mays L., denoted ZmAP1, coding for an anionic peroxidase. The open reading frame of ZmAP1 starting 72 residues from the 5' end of the cDNA predicts a 37,778 dalton protein of 356 amino acid residues. The protein has high similarity to other peroxidases and contains two peroxidase motifs that carry two highly conserved histidines in the active center. We expressed recombinant ZmAP1 protein in E. coli as a fusion with maltose-binding protein. The fusion protein was biochemically active after addition of hemin to the apoprotein. The maize peroxidase ZmAP1 has a pH optimum at pH 4.0 and a Km of 0.2 mM for the substrate 2,2'-azino-bis-(3-ethyl-benzothiazolin-6-sulfonic acid) at this pH. In maize seedlings the ZmAP1 gene is expressed predominantly in roots, the mesocotyl, the coleoptile and to a lower extent in the node, whereas no expression in the primary leaf was found. In situ hybridization shows that the expression of ZmAP1 in the young maize root is confined to the epidermis, hypodermis and the pericycle.     

Cloning and characterization of a rhoGAP homolog from Dictyostelium discoideum

Small GTPases interact with a variety of proteins that affect nucleotide binding and cleavage. GTPase activating proteins (GAPs) are one class of these proteins that act by accelerating the intrinsic GTPase rate resulting in the formation of the biologically inactive GDP-bound form of the GTPase. For the Rho subfamily of GTPases, there is a growing number of proteins with rhoGAP activity that are identifiable by a homologous region of about 150 amino acids. We have exploited this homology using the polymerase chain reaction to clone the first rhoGAP homolog, called DdRacGAP, from the slime mold Dictyostelium discoideum. The GAP domain of DdRacGAP (amino acids 1-212), when expressed and purified from Escherichia coli, is active on both Dictyostelium and human Rho family GTPases but not human Ras. The full-length protein is 1356 amino acids in length and has several interesting homologies in addition to the GAP domain, including an SH3 domain, a dbl homology domain, and a pleckstrin homology domain.     

Purification and characterization of a feruloyl esterase from the fungus Penicillium expansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and rho-coumaric acid from methyl esters of the acids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->3)-O-beta- D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and O-[5-O-((E)-rho-coumaroyl)-alpha-L-arabinofuranosyl]- (1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (PAXX). The esterase was purified 360-fold in successive steps involving ultrafiltration and column chromatography by gel filtration, anion exchange and hydrophobic interaction. These chromatographic methods separated the phenolic acid esterase from alpha-L-arabinofuranosidase, pectate and pectin lyase, polygalacturonase, xylanase and beta-D-xylosidase activities. The phenolic acid esterase had an apparent mass of 65 kDa under non-denaturing conditions and a mass of 57.5 kDa under denaturing conditions. Optimal pH and temperature were 5.6 and 37 degrees C, respectively and the metal ions Cu2+ and Fe3+ at concentrations of 5 mmol 1-1 inhibited feruloyl esterase activity by 95% and 44%, respectively, at the optimum pH and temperature. The apparent Km and Vmax of the purified feruloyl esterase for methyl ferulate at pH 5.6 and 37 degrees C were 2.6 mmol 1-1 and 27.1 mumol min-1 mg-1. The corresponding constants of rho-coumaroyl esterase for methyl coumarate were 2.9 mmol 1-1 and 18.6 mumol min-1 mg-1.     

Cloning and characterization of a gene cluster from Streptomyces cyanogenus S136 probably involved in landomycin biosynthesis

From a cosmid library of Streptomyces cyanogenus S136, DNA fragments encompassing approximately 35 kb of the presumed landomycin biosynthetic gene cluster were identified and sequenced, revealing 32 open reading frames most of which could be assigned through data base comparison.     

Cloning and sequence analysis of a gene encoding an erythrocyte binding protein from Plasmodium cynomolgi

We compared the metabolic response of a methionine(Met)-dependent (P60) human glioma cell line with that of a Met-independent variant (P60H) when cultured in a homocysteine (Hcy) medium and exposed to N2O. In Hcy medium (without Met), remethylation of Hcy in P60H cells was enhanced and supported growth, whereas remethylation was low in P60 cells, which failed to thrive under these conditions. Both cell types seemed to contain adequate amounts of folates and total cobalamin (Cbl). P60 cells showed increased total and methylcobalamin (CH3Cbl) content after the shift to a Hcy medium, but the high, stable level of CH3Cbl detected in P60H cells was not attained. Further metabolic differences were induced by N2O exposure, which markedly reduced Met-synthase activity in cell-free extracts in both cell lines and completely blocked intact-cell Hcy remethylation in P60, whereas Hcy remethylation was only partly inhibited in P60H cells cultured in Met medium. The residual Hcy remethylation in P60H cells may be related to only a moderate depletion of CH3Cbl. The resulting high CH3Cbl level relative to Met-synthase activity during N2O exposure was even higher in Hcy medium. These findings in P60H cells probably reflect increased provision of Cbl to support Hcy remethylation under metabolic strain. The inability of P60 to furnish CH3Cbl to the enzyme may explain both the Met-dependent phenotype and the increased sensitivity of Hcy remethylation to N2O exposure in these cells.     

Purification and characterization of diacylglycerol acyltransferase from the lipid body fraction of an oleaginous fungus

BACKGROUND AND PURPOSE: Ischemic cerebral infarction (CI) is a serious complication of acute myocardial infarction (MI). Little information exists on CI after thrombolytic therapy for MI. METHODS: Of 3924 MI patients treated with recombinant tissue plasminogen activator (rt-PA) and heparin, 29 (0.7%) developed CI after treatment. All CI patients had detailed neurological evaluations, and 27 (93%) had CT scans centrally reviewed. RESULTS: Age range was 40 to 74 years (mean, 60 years); 25 patients (86%) were men, and 22 (76%) were white. The electrocardiographic location of MI was anterior in 22 (76%) and nonanterior in 7 (24%). Five CIs occurred within 6 hours, 4 between 6 to 24 hours, 8 during the remainder of the first week, 10 during the second week, and 2 others distributed over the 4 weeks after study entry. Six of 29 CIs did not involve the cerebral cortex; 9 patients (31%) had multiple CIs. Of 28 CIs thought to be embolic in origin, 17 showed strong evidence for at least one cardiac abnormality (mural clot, wall-motion abnormality, aneurysm, or atrial fibrillation) known to be associated more specifically with embolism than MI. Eight of 27 CIs (30%) with CT scans had hemorrhagic transformation of varying degrees; 5 were symptomatic. CONCLUSIONS: The time of occurrence and sites of CI after rt-PA and heparin therapy for acute MI are similar to those reported during the prethrombolytic era.     

Cloning of a Streptococcus sobrinus oligo-isomaltosaccharide synthase gene and characterization of its product

A Streptococcus sobrinus gene coding for a glucosyltransferase (GTF)-S was cloned into Escherichia coli, using the bacteriophage lambda L47.1 and the plasmid vector pACYC184. The MD124 clone obtained expressed a 155 kDa GTF-S which did not react with any antisera against GTF-S1, -S2 and -I enzymes. The recombinant enzyme (designated rGTF-S3) was homogeneously purified from the MD124 cell-extract and characterized. The purified rGTF-S3 synthesized primer-independently alpha-1,6-linked linear oligosaccharides from sucrose. The dependence upon the sucrose concentration was diphasic, and the respective Km values were 1.3 and 25 mM. The properties except the Km values were similar to those of oligo-isomaltosaccharide synthase from S. sobrinus AHT.     

Cloning, sequencing, and characterization of the recA gene from Rhodopseudomonas viridis and construction of a recA strain

A recombination-deficient strain of the phototrophic bacterium Rhodopseudomonas viridis was constructed for the homologous expression of modified photosynthetic reaction center genes. The R. viridis recA gene was cloned and subsequently deleted from the R. viridis genome. The cloned R. viridis recA gene shows high identity to known recA genes and was able to complement the Rec- phenotype of a Rhizobium meliloti recA strain. The constructed R. viridis recA strain showed the general Rec- phenotype, i.e., increased sensitivity to DNA damage and severely impaired recombination ability. The latter property of this strain will be of advantage in particular for expression of modified, nonfunctional photosynthetic reaction centers which are not as yet available.     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new sigma factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34 kDa with strong similarity to the RpoH (sigma32) factors from other bacterial species. It was not possible to inactivate the R. capsulatus rpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5' ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a sigma factor-specific antibody revealed the accumulation of a protein of about 34 kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R. capsulatus cultures.     

Cloning, characterization, and transcriptional analysis of a gene encoding an alpha-crystallin-related, small heat shock protein from the thermophilic cyanobacterium Synechococcus vulcanus

hspA, a gene encoding a 16-kDa heat-induced protein from the thermophilic cyanobacterium Synechococcus vulcanus, has been cloned and sequenced. The deduced amino acid sequence of the gene product showed significant homology to sequences of the family of alpha-crystallin-related, small heat shock proteins. A monocistronic mRNA of hspA increased transiently in response to heat shock. The heat shock induction occurred at a vegetative promoter but without the CIRCE (controlling inverted repeat of chaperone expression) element.     

Cloning and characterization of mycovirus double-stranded RNA from the plant pathogenic fungus, Fusarium solani f. sp. robiniae

A mycovirus (named FusoV) from the phytopathogenic fungus, Fusarium solani f. sp. robiniae SUF704, has two kinds of double-stranded (ds) RNA genomes, designated M1 and M2. The cDNAs were constructed from FusoV genomic dsRNAs. The sequences of M1 and M2 cDNAs comprised 1645 and 1445bp, respectively. Sequence analysis showed that each dsRNA had a single long open reading frame (ORF) on only one of the strands. M1 ORF encodes a 519-amino acid residue polypeptide with a predicted molecular mass of 60 kDa. RNA-dependent RNA polymerase-conserved motifs were identified in the predicted amino acid sequence, and the polymerase synthesized dsRNA in vitro. The M2 ORF encodes a polypeptide of 413 amino acid residues with a predicted molecular mass of 44 kDa. The predicted amino acid sequence contained the sequence corresponding to those found in the purified 44-kDa capsid protein of FusoV.     

Cloning and characterization of the Arabidopsis thaliana lupeol synthase gene

A 2274 bp Arabidopsis thaliana cDNA was isolated that encodes a protein 57% identical to cycloartenol synthase from the same organism. The expressed recombinant protein encodes lupeol synthase, which converts oxidosqualene to the triterpene lupeol as the major product. Lupeol synthase is a multifunctional enzyme that forms other triterpene alcohols, including beta-amyrin, as minor products. Sequence analysis suggests that lupeol synthase diverged from cycloartenol synthase after plants diverged from fungi and animals. This evolutionary order is the reason that fungi and animals do not make lupeol.