ABSTRACT
The human organic anion transporter 1 (hOAT1) plays a key role in the secretion of an array of potentially toxic organic anions including many clinically important drugs. Here we report on the genomic cloning of hOAT1. A human genomic library was used for screening of a PAC (P1 artificial chromosome) clone applying PCR techniques. Sequencing of several restriction subclones and of a PCR-generated clone revealed that the hOAT1 gene spans 8.2 kb and is composed of 10 exons divided by 9 introns. RT-PCR studies in a human kidney specimen led to the detection of two new splice variants, hOAT1-3 and hOAT1-4, showing a 132-bp in-frame deletion. Using fluorescence in situ hybridization (FISH) we mapped the hOAT1 gene as a single signal to chromosome 11q13.1-q13.2. Additionally, 600 bp of the 5' flanking region was analyzed, illustrating the probable transcription start site at nt -280, a NF-kappaB-site at nt -397 and several putative transcription factor binding sites.
Subject(s)
Carrier Proteins/genetics , Anion Transport Proteins , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 11 , DNA , DNA Primers , Exons , Humans , In Situ Hybridization, Fluorescence , Introns , Molecular Sequence Data , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The 1410 bp DNA region (glnA) encoding glutamine synthetase I (GSI) from Streptomyces viridochromogenes was amplified by PCR, cloned and sequenced. The molecular mass of the deduced GSI protein (469 residues) was determined to be 50 kDa. The DNA region showed 90% nucleotide identity with the Streptomyces coelicolor A3(2) glnA gene, but no significant nucleotide sequence similarity with the glnII (GSII) gene of S. viridochromogenes. The chromosomal glnA and glnII genes of S. viridochromogenes were disrupted by site-specific mutagenesis. Neither glnA nor glnII single mutants required glutamine for growth and both were normal in their sporulation. Measurement of the GS activity in cultures grown with different nitrogen sources revealed that GSI (heat-stable) and GSII (heat-labile) were always expressed together, with GSI as the predominant activity. It could be proposed that GSI, but not GSII is inactivated by adenylylation under conditions of nitrogen excess. GSI and GSII activities are inhibited by amino acids and by nucleotides.
Subject(s)
Gene Expression Regulation, Bacterial , Glutamate-Ammonia Ligase/genetics , Organophosphorus Compounds/metabolism , Streptomyces/enzymology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Culture Media , DNA, Bacterial , Enzyme Stability , Exons , Gene Expression Regulation, Enzymologic , Glutamate-Ammonia Ligase/metabolism , Hot Temperature , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Restriction Mapping , Spores, Bacterial , Streptomyces/genetics , Streptomyces/growth & developmentABSTRACT
The coding region for human neutrophil short form procollagenase lacking the hemopexin like domain coding region was amplified by polymerase chain reaction. Recombinant short form procollagenase was expressed in E. coli and purified in a three step procedure. Renaturation of this proenzyme was carried out by an effective new method using Q-Sepharose chromatography. Treatment of short form procollagenase with mercurials resulted in active short form collagenase M(r) 21,000 and an intermediate product of M(r) 23,000. These two products were separated by hydroxamate affinity chromatography. The active, short form collagenase M(r) 21,000 is stable. Despite full proteolytic activity, it lacks type I collagen substrate specificity and forms the basis for crystallisation experiments.
