Cloning and characterization of theddsAgene encoding decaprenyl diphosphate synthase fromRhodobacter capsulatusB10

2006 ◽  
Vol 52 (12) ◽  
pp. 1141-1147 ◽  
Author(s):  
Xinyi Liu ◽  
Haizhen Wu ◽  
Jiang Ye ◽  
Qinsheng Yuan ◽  
Huizhan Zhang
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Open Reading Frame ◽  
High Similarity ◽  
Reading Frame ◽  
Endogenous Production ◽  
Genome Library

A decaprenyl diphosphate synthase gene (ddsA, GenBank accession No. DQ191802) was cloned from Rhodobacter capsulatus B10 by constructing and screening the genome library. An open reading frame of 1002 bp was revealed from sequence analysis. The deduced polypeptide consisted of 333 amino acids residues with an molecular mass of about 37 kDa. The DdsA protein contained the conserved amino acid sequence (DDXXD) of E-type polyprenyl diphosphate synthase and showed high similarity to others. In contrast, DdsA showed only 39% identity to a solanesyl diphosphate synthase cloned from R. capsulatus SB1003. DdsA was expressed successfully in Escherichia coli. Assaying the enzyme in vivo found it made E.coli synthesize UQ-10 in addition to the endogenous production UQ-8.Key words: ubiquinone, polyprenyl diphosphate synthase, gene expression, Rhodobacter capsulatus.

scholarly journals Functional Characterization of Alanine Racemase fromSchizosaccharomyces pombe: a Eucaryotic Counterpart to Bacterial Alanine Racemase

2001 ◽  
Vol 183 (7) ◽  
pp. 2226-2233 ◽  
Author(s):  
Takuma Uo ◽  
Tohru Yoshimura ◽  
Naotaka Tanaka ◽  
Kaoru Takegawa ◽  
Nobuyoshi Esaki
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Source ◽  
Functional Characterization ◽  
Open Reading Frame ◽  
Alanine Racemase ◽  
Sole Nitrogen Source ◽  
Recombinant Yeast ◽  
Reading Frame ◽  
Putative Protein

ABSTRACT Schizosaccharomyces pombe has an open reading frame, which we named alr1 +, encoding a putative protein similar to bacterial alanine racemase. We cloned thealr1 + gene in Escherichia coli and purified the gene product (Alr1p), with an M rof 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparentKm and V max values as follows: for l-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for d-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but l-serine and l-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that ofl-alanine, respectively. S. pombe usesd-alanine as a sole nitrogen source, but deletion of thealr1 + gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway forl-alanine coupled with racemization plays a major role in the catabolism of d-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses l-alanine but notd-alanine as a sole nitrogen source. Moreover,d-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1 + gene enabled S. cerevisiae to grow efficiently ond-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of d-alanine.

scholarly journals Characterization of late gene expression factors lef-9 and lef-8 from Bombyx mori nucleopolyhedrovirus

2002 ◽  
Vol 83 (8) ◽  
pp. 2015-2023 ◽  
Author(s):  
Asha Acharya ◽  
Karumathil P. Gopinathan
Keyword(s):  
Gene Expression ◽  
Bombyx Mori ◽  
Late Gene ◽  
Late Gene Expression ◽  
Reading Frame ◽  
Early Transcription ◽  
A Site ◽  
Translational Termination

Late gene expression factors, LEF-4, LEF-8, LEF-9 and P47 constitute the primary components of the Autographa californica multinucleocapsid polyhedrovirus (AcMNPV)-encoded RNA polymerase, which initiates transcription from late and very late promoters. Here, characterization of lef-9 and lef-8, which encode their corresponding counterparts, from Bombyx mori NPV is reported. Transcription of lef-9 initiated at two independent sites: from a GCACT sequence located at −38 nt and a CTCTT sequence located at −50 nt, with respect to the +1 ATG of the open reading frame. The 3′ end of the transcript was mapped to a site 17 nt downstream of a canonical polyadenylation signal located 7 nt downstream of the first of the two tandem translational termination codons. Maximum synthesis of LEF-9 was seen from 36 h post-infection (p.i.). The transcription of lef-8 initiated early in infection from a GTGCAAT sequence that differed in the corresponding region from its AcMNPV counterpart (GCGCAGT), with consequent elimination of the consensus early transcription start site motif (underlined). Peak levels of lef-8 transcripts were attained by 24 h p.i. Immunocopurification analyses suggested that there was an association between LEF-8 and LEF-9 in vivo.

scholarly journals Isolation and Characterization of IS1409, an Insertion Element of 4-Chlorobenzoate-DegradingArthrobacter sp. Strain TM1, and Development of a System for Transposon Mutagenesis

2001 ◽  
Vol 183 (12) ◽  
pp. 3729-3736 ◽  
Author(s):  
Karl-Heinz Gartemann ◽  
Rudolf Eichenlaub
Keyword(s):  
Resistance Genes ◽  
Micrococcus Luteus ◽  
Transposon Mutagenesis ◽  
Open Reading Frame ◽  
Insertion Element ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
Insertion Elements

ABSTRACT A new insertion element of 1,449 bp with 25-bp perfect terminal repeats, designated IS1409, was identified in the chromosome of 4-chlorobenzoate-degrading Arthrobactersp. strain TM1 NCIB12013. Upon insertion, IS1409 causes a target duplication of 8 bp. IS1409 carries only a single open reading frame of 435 codons encoding the transposase TnpA. Both TnpA and the overall organization of IS1409are highly similar to those of some related insertion elements of the ISL3 group (J. Mahillon and M. Chandler, Microbiol. Mol. Biol. Rev. 62:725–774, 1998). IS1409 was also found in other 4-chlorobenzoate-degrading Arthrobacter strains and Micrococcus luteus. Based on IS1409, a series of transposons carrying resistance genes for chloramphenicol and gentamicin were constructed. These transposons were used to demonstrate transposition events in vivo and to mutagenizeArthrobacter sp. strains.

scholarly journals Characterization of the Escherichia coli gene encoding a new member of the short-chain dehydrogenase/reductase (SDR) family.

1997 ◽  
Vol 44 (1) ◽  
pp. 153-157 ◽  
Author(s):  
A Sirko ◽  
A Wegleńska ◽  
M Hryniewicz ◽  
D M Hulanicka
Keyword(s):  
Escherichia Coli ◽  
Short Chain ◽  
Chromosomal Dna ◽  
Large Protein ◽  
Gene Encoding ◽  
Reading Frame ◽  
Large Protein Family ◽  
Dna Fragment

The nucleotide sequence of a chromosomal DNA fragment located upstream from the cysPTWAM operon of Escherichia coli was established. Sequence analysis indicates the presence of an open reading frame which has been designated ucpA (upstream cys P). The potential protein products exhibits strong sequence homology to the members of a large protein family, short-chain dehydrogenases/reductases. Involvement of Crp, FruR and IHF in the regulation of ucpA transcription in vivo was demonstrated.

scholarly journals In Vitro Identification of Rns-Regulated Genes

2002 ◽  
Vol 184 (4) ◽  
pp. 1196-1199 ◽  
Author(s):  
George P. Munson ◽  
Lisa G. Holcomb ◽  
Heather L. Alexander ◽  
June R. Scott
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Binding Sites ◽  
Genomic Dna ◽  
Maltose Binding Protein ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Fusion Analysis

ABSTRACT To identify Rns-regulated genes, a maltose binding protein (MBP)-Rns fusion protein was used to isolate DNA fragments from enterotoxigenic Escherichia coli genomic DNA that carry Rns binding sites. In vivo transcription fusion analysis shows that Rns positively regulates the expression of the open reading frame of yiiS, which lies immediately downstream of one MBP-Rns-bound fragment.

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