Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. 1977.
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Cloning, Molecular↗
Biomedical subjects
F Bolivar
Publications and source records attributed to F Bolivar.
The role of penicillin amidases in nature and in industry.
Penicillin amidase (PA) is the enzyme used commercially for the production of semisynthetic penicillins. During the past decade, a detailed picture of the structure and regulation of the gene encoding this enzyme has emerged, revealing a variety of interesting features that are unique among microorganisms. Clues to the biological role of this enzyme have been provided, as well as new strategies for the commercial production and utilization of PA.
Escherichia coli↗
Design and construction of expression plasmid vectors in Escherichia coli.
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Base Sequence↗
The ribonucleoside diphosphate reductase gene (nrdA) of Escherichia coli carries a repetitive extragenic palindromic (REP) sequence in its 3' structural terminus.
A computer search for repeated sequences led us to identify five REP (repetitive extragenic palindromic) sequences in the 3'-terminal region of the Escherichia coli ribonucleoside diphosphate reductase gene (nrdA). These REP sequences are located within a putative duplicated DNA region, the first of them being part of the carboxy-terminal coding region of the nrdA gene. This is the first report of a REP sequence within a structural gene and also the first example of a REP sequence apparently generated by DNA duplication.
Amino Acid Sequence↗
Amino acid sequence analysis of the glutamate synthase enzyme from Escherichia coli K-12.
The amino acid sequence for the two subunits of the glutamate synthase of Escherichia coli K-12 was compared to the protein sequences compiled in the National Biomedical Research Foundation databank. Similarities were detected between the small glutamate synthase subunit and three members of the flavin-containing pyridine nucleotide-disulphide oxidoreductase superfamily, and also with three members of a lactate dehydrogenase family. Two segments in this glutamate synthase subunit showed similarity to regions previously proposed as part of dinucleotide-binding sites in some members of these two families. Similarity can be extended if the predicted secondary structure is considered. Based on these data, residues 148-260 and 289-409 in the small GOGAT subunit are proposed as dinucleotide-binding regions. Comparison of the amino acid sequence of the large glutamate synthase subunit with the glutamine phosphoribosylamine:pyrophosphate phosphoribosyltransferases of B. subtilis and E. coli revealed a significant similarity between the amino termini of these three enzymes. In these last two amidotransferases, the glutamine-binding site has been located in their amino-terminal region. The comparison with a second group of glutamine amidotransferases did not show any significant global similarity with the large glutamate synthase subunit. However, this polypeptide contains a small segment that shares similarity with a 13-amino acid segment proposed as part of the glutamine-binding site in this second group of amidotransferases.
4-Hydroxybenzoate-3-Monooxygenase↗
Identification of a functional promoter for the Escherichia coli gdhA gene and its regulation.
Glutamate dehydrogenase (GDH) catalyzes the synthesis of L-glutamate from 2-oxoglutarate and ammonia. The complete nucleotide sequence of the Escherichia coli gdhA gene, as well as its 5' and 3' flanking regions have been previously reported [Valle et al., Gene 23 (1983) 199-209; 27 (1984) 193-199]. In this paper we present data on the GDH specific activities using both excess and limiting concentrations of ammonia as nitrogen sources. Evidence is presented on the regulation of the mRNA levels for this enzyme by the ammonia concentration in the growth medium. We have identified a single and apparently invariant transcript for several metabolic growth conditions. We also report the identification of a functional promoter and the corresponding transcription start point under several growth conditions. Finally, possible regulatory sequences located at the 5' flanking region of the gdhA gene are discussed.
Base Sequence↗
Cloning, sequencing, and expression in Ficoll-generated minicells of an Escherichia coli heat-stable enterotoxin gene.
The gene encoding a heat-stable enterotoxin of Escherichia coli was cloned as a 960-bp fragment from a plasmid isolated from a Mexican strain of human origin. Deoxyribonucleotide sequencing unveiled a 216-bp open reading frame similar to that of a previously sequenced ST-toxin gene. The gene is preceded by a proposed binding site for the cAMP-mediated positive regulator (CAP) that is part of a 23-bp inverted repeat. The proposed CAP site is followed by a 6A, 1T, and 6A deoxyribonucleotides. Minicells containing the toxin gene, which were isolated from Ficoll gradients, shown to preserve the localization of intracellular and periplasmic enzymes, allowed the detection of a biosynthetically radiolabeled polypeptide with an apparent Mr 8400. The data suggest that the enterotoxin genes estA2, estA3, and estA4 are very similar, even in clinical strains isolated from distinct geographical locations; that the transcription of heat-stable enterotoxin genes is controlled by the cAMP-mediated positive regulatory system, and that the heat-stable enterotoxins are initially synthesized as 72 amino acid precursors to yield the extracellular active 18-19 amino acid polypeptides.
Amino Acid Sequence↗
The plasmid, pBR322.
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Amino Acid Sequence↗
Determination of the nucleotide sequence for the glutamate synthase structural genes of Escherichia coli K-12.
We have determined the complete nucleotide sequence of a 6.3-kb chromosomal HpaI-EcoRI fragment, that contains the structural genes for both the large and small subunits of the Escherichia coli K-12 glutamate synthase (GOGAT) enzyme, as well as the 5'- and 3'-flanking and intercistronic DNA regions. The Mrs of the two subunits, as deduced from the nucleotide (nt) sequence, were estimated as 166,208 and 52,246. Partial amino acid sequence of the GOGAT enzyme revealed that the large subunit starts with a cysteine residue that is probably generated by a proteolytic cleavage. Northern blotting experiments revealed a transcript of approximately 7300 nt, that at least contains the cistrons for both subunits. A transcriptional start point and a functional promoter were identified in the 5' DNA flanking region of the large subunit gene. The messenger RNA nontranslated leader region has 120 nt and shares identity with the leader regions of E. coli ribosomal operons, in particular around the so-called boxA sequence implicated in antitermination. Other possible regulatory sequences are described.
Base Sequence↗
Deletion of a repetitive extragenic palindromic (REP) sequence downstream from the structural gene of Escherichia coli glutamate dehydrogenase affects the stability of its mRNA.
A deletion that removes one repetitive extragenic palindromic sequence downstream of the structural gene of Escherichia coli glutamate dehydrogenase, reduces twofold the half-life of gdhA mRNA.
Chromosome Deletion↗
Plasmid vector pBR322 and its special-purpose derivatives--a review.
The plasmid pBR322 was one of the first EK2 multipurpose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of recombinant DNA molecules in Escherichia coli. This 4363-bp DNA molecule has been extensively used as a cloning vehicle because of its simplicity and the availability of its nucleotide sequence. The widespread use of pBR322 has prompted numerous studies into its molecular structure and function. These studies revealed two features that detract from the plasmid's effectiveness as a cloning vector: plasmid instability in the absence of selection and, the lack of a direct selection scheme for recombinant DNA molecules. Several vectors based on pBR322 have been constructed to overcome these limitations and to extend the vector's versatility to accommodate special cloning purposes. The objective of this review is to provide a survey of these derivative vectors and to summarize information currently available on pBR322.
Base Sequence↗
Characterization of the regulatory region of the Escherichia coli penicillin acylase structural gene.
Penicillin acylase is utilized in the enzymatic production of semisynthetic penicillins. The enzyme is composed of two different subunits that originate from a common precursor. The partial nucleotide (nt) sequence of the structural gene has been published. This paper reports the nt sequence of the regulatory region of this gene, the identification of a functional promoter, the transcriptional start point, and the description of possible regulatory regions.
Amidohydrolases↗
A common precursor for the two subunits of the penicillin acylase from Escherichia coli ATCC11105.
Penicillin acylase (PA) is an industrial enzyme that is used to convert penicillin G into a precursor for semisynthetic penicillins. We have cloned a segment of DNA that codes for the two subunits required for PA activity. We also report the nucleotide sequence of a DNA fragment that codes for (i) the small subunit, (ii) the N-terminal region of the large subunit and (iii) a putative connecting peptide. These results confirm the existence of a common precursor for both peptides.
Amidohydrolases↗
Repetitive extragenic palindromic (REP) sequences in the Escherichia coli gdhA gene.
Deletions of the 3' flanking DNA region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, have been produced on a plasmid that carries the complete gdhA gene. Those deletions include part of the repetitive extragenic palindromic (REP) sequences proposed by Stern et al. [Cell 37 (1984) 1015-1026], as a novel and major feature of the bacterial genome. The effect of these deletions on the final GDH level in the cell, has been determined. A broader compilation, analysis and alternative functions of the REP sequences, is also presented.
Chromosome Deletion↗
Construction and characterization of new cloning vehicles. VII. Construction of plasmid pBR327par, a completely sequenced, stable derivative of pBR327 containing the par locus of pSC101.
In vitro recombinant DNA experiments, using plasmid pBR327 and a DNA fragment derived from plasmid pSC101 containing the par region, resulted in the construction of plasmid pBR327par. This new cloning vehicle has all the cloning properties of the parental plasmid, and is more stable than pBR327. Since the nucleotide sequence of the par region has been determined, this new vector is completely characterized. Some features of the sequence with possible functional significance are discussed.
Base Sequence↗
Complete nucleotide sequence of the glutamate dehydrogenase gene from Escherichia coli K-12.
A 2.3-kb PstI- ClaI chromosomal DNA segment, carrying the complete coding region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, has been sequenced. The complete amino acid sequence (447 residues) of the GDH monomer has been deduced, and comparisons are made with reported amino acid sequences of GDH from other organisms.
Amino Acid Sequence↗
Nucleotide sequence of the promoter and amino-terminal coding region of the glutamate dehydrogenase structural gene of Escherichia coli.
A 610-bp DNA fragment carrying the promoter and amino-terminal coding regions of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli has been sequenced. The amino-terminal sequence of the enzyme was also determined to help localize the transcriptional and translational signals for this gene. Three possible promoters and a CRP binding site were identified by concensus criteria. The sequence of 102 amino acids at the amino terminus of the enzyme is compared with the amino acid sequence from other GDH enzymes.
Animals↗
Structural organization of the genes that encode two glutamate synthase subunits of Escherichia coli.
Plasmid pRSP20, a recombinant plasmid isolated from the Clarke-Carbon Escherichia coli gene bank, contains the two genes coding for the subunits of glutamate synthase (GOGAT). We have constructed several derivatives of pRSP20, and analyzed the direction of transcription and genetic organization of these genes. Unexpectedly, we have found that although they are tightly linked and are transcribed in the same direction, each of them has its own promoter.
Chromosome Mapping↗