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PubMed1991

Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2.

The TrfA proteins, encoded by the broad host range plasmid RK2, are required for replication of this plasmid in a variety of Gram-negative bacteria. Two TrfA proteins, 33 and 44 kDa in molecular mass (designated TrfA-33 and TrfA-44, respectively), are expressed from the trfA gene of RK2 through the use of two alternative in-frame start codons within the same open reading frame. The two proteins have been purified from Escherichia coli to near homogeneity as a mixture of wild-type TrfA-44/33, as TrfA-33 alone and as a functional variant form of TrfA-44, designated TrfA-44(98L), which contains a leucine in place of the TrfA-33 methionine start codon. Cross-linking experiments demonstrated that TrfA-33 can multimerize in solution. By using gel mobility shift and DNase I footprinting techniques the binding properties of TrfA-33, TrfA-44(98L), and TrfA-44/33 to the origin of replication of plasmid RK2 were analyzed. All three protein preparations were able to bind very specifically to the cluster of five direct repeats (iterons) contained in the minimal origin of replication. Each protein preparation produced a ladder of TrfA/minimal oriV complexes of decreasing electrophoretic mobility. The DNase I protection pattern on the five iterons was identical for all three protein preparations and extended from the beginning of the first iteron to 5 base pairs upstream of the fifth iteron. Studies on the affinity of the proteins for DNA fragments containing one, two, or all five iterons of the origin revealed a strong preference of TrfA protein for DNA containing at least two iterons. To study the stability of TrfA.DNA complexes, association and dissociation rates of TrfA-33 and DNA fragments with one, two, or five iterons were measured. This analysis showed that unlike complexes involving two or five iterons the TrfA/one iteron complexes were highly unstable, suggesting some form of cooperativity between proteins or iterons in the formation of stable complexes and/or the requirement of specific sequences bordering the iterons at the RK2 origin of replication for the stabilization of TrfA/DNA complexes.

Bacterial Proteins↗

PubMed2006

The intraleader AUG nucleotide sequence context is important for equine arteritis virus replication.

The 5(-terminal leader sequence of the equine arteritis virus (EAV) genome contains an open reading frame (ORF) with an AUG codon in a suboptimal context for initiation of protein synthesis. To investigate the significance of this intraleader ORF (ILO), an expression plasmid was generated carrying a DNA copy of the subgenomic mRNA7 behind a T7 promoter. Capped RNA transcribed from this construct was shown to direct, in an in vitro translation system, the synthesis of leader peptide as well as N protein. Site-directed mutations aimed to either optimize or weaken the sequence context of the ILO start codon affected leader peptide synthesis as predicted; no peptide was detected when the initiation codon was incapacitated. Translation of the downstream N gene was inversely affected by leader peptide production, consistent with a ribosomal leaky scanning mechanism. To investigate the role of the leader peptide in the EAV replication life cycle we generated, using an infectious EAV cDNA clone, two mutant viruses in one of which the ILO start codon was in an optimal Kozak context for translation initiation while in the other the codon was again incapacitated. Surprisingly, both mutant viruses were equally viable and exhibited similar phenotypes in BHK-21 cells. However, their replication kinetics and viral yields were reduced relative to that of the wild-type parental virus, as were their plaque sizes. Importantly, the mutations introduced into the viruses appeared to be rapidly and precisely repaired upon passaging. Already after one viral passage a significant fraction of the viruses had regained the wild-type sequence as well as its phenotype. The results demonstrate that EAV replication is not dependent on the synthesis of the intraleader peptide. Rather, the leader peptide does not seem to have any function in the EAV life cycle. As we discuss, the available data indicate that the ILO 5( nucleotide sequence per se, not its functioning in translation initiation, is of critical importance for EAV replication.

Animals↗

PubMed1988

Enrichment for 5'-TG termini: a method for subcloning structural genes into expression vectors.

We describe a method for creating a population of randomly digested, blunt-ended DNA fragments with 5'-TG... 3'-AC... (TG) at their termini. When these fragments were ligated to a blunt-ended vector that contains ...CATA-3' ...GTAT-5' at its termini, as high as 84% of the clones obtained after transformation contained plasmids with a reconstructed Nde I site ... CATATG... ...GTATAC.... When the DNA vector is prepared from an appropriate plasmid [Gross et al., Mol. Cell. Biol. 5 (1985) 1015-1024; Kotewicz et al., Gene 35 (1985) 249-258], the ATG within the restriction site corresponds to a start codon positioned downstream from a strong ribosome-binding site and controllable promoter. If a gene has been digested to the TG contained within its authentic initiation codon, the endogenous translation-initiation control sequences are deleted and expression can be controlled using the plasmid-derived promoter. In addition, a gene digested to other in-frame TGs can potentially express proteins with altered N termini. Using this method, we have placed the structural gene of SP6 RNA polymerase, trimmed precisely to its authentic start codon, under the control of the tac promoter.

Base Sequence↗

PubMed1991

Cloning and characterization of the MboII restriction-modification system.

The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.

Amino Acid Sequence↗

PubMed2006

RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation.

Osteoclast differentiation is tightly regulated by receptor activator of NF-kappaB ligand (RANKL) signaling. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase is highly expressed in osteoclast cells and plays an important role in degradation of extracellular matrix; however, the molecular mechanisms that regulate MMP-9 gene expression are unknown. In this study, we demonstrate that RANKL signaling induces MMP-9 gene expression in osteoclast precursor cells. We further show that RANKL regulates MMP-9 gene expression through TRAF6 but not TRAF2. Interestingly, blockade of p38 MAPK activity by pharmacological inhibitor, SB203580 increases MMP-9 activity whereas ERK1/2 inhibitor, PD98059 decreases RANKL induced MMP-9 activity in RAW264.7 cells. These data suggest that RANKL differentially regulates MMP-9 expression through p38 and ERK signaling pathways during osteoclast differentiation. Transient expression of MMP-9 gene (+1 to -1174 bp relative to ATG start codon) promoter-luciferase reporter plasmids in RAW264.7 cells and RANKL stimulation showed significant increase (20-fold) of MMP-9 gene promoter activity; however, there is no significant change with respect to +1 bp to -446 bp promoter region and empty vector transfected cells. These results indicated that MMP-9 promoter sequence from -446 bp to -1174 bp relative to start codon is responsive to RANKL stimulation. Sequence analysis of the mouse MMP-9 gene promoter region further identified the presence of binding motif (-1123 bp to -1153 bp) for the nuclear factor of activated T cells 1 (NFATc1) transcription factor. Inhibition of NFATc1 using siRNA and VIVIT peptide inhibitor significantly decreased RANKL stimulation of MMP-9 activity. We further confirm by oligonucleotide pull-down assay that RANKL stimuli enhanced NFATc1 binding to MMP-9 gene promoter element. In addition, over-expression of constitutively active NFAT in RAW264.7 cells markedly increased (5-fold) MMP-9 gene promoter activity in the absence of RANKL. Taken together, our results suggest that RANKL signals through TRAF6 and that NFATc1 is a downstream effector of RANKL signaling to modulate MMP-9 gene expression during osteoclast differentiation.

Animals↗

PubMed1980

Conformational alteration of mRNA structure and the posttranscriptional regulation of erythromycin-induced drug resistance.

The DNA sequence of the ermC gene of plasmid pE194 is presented. This determinant is responsible for erythromycin-induced resistance to the macrolide-lincosamide-streptogramin B group of antibiotics and specifies a 29,000 dalton inducible protein. The locations of the ermC promoter, as well as that of a probable transcriptional terminator, are established both from the sequence and by transcription mapping. The sequence contains an open reading frame sufficient to encode the previously identified 29,000 dalton ermC protein. Between the promoter and the putative ATG start codon is a 141 base pair leader sequence, within which several regulatory (constitutive) mutations have been mapped and sequenced. The leader has a second open reading frame, sufficient to encode a 19 amino acid peptide. It is suggested that induction by erythromycin involves a shift between alternative ribosome-bound mRNA conformations, so that the ribosome binding sequence and the start codon for synthesis of the 29K protein are unmasked in the presence of inducer. Possible active and inactive folded configuration of the leader sequence are presented, as well as the effects on these configurations of regulatory mutations.

Bacillus subtilis↗

PubMed1993

Nucleotide sequence of the 7 K gene of Helenium virus S.

The sequence of 380 nucleotides upstream of the start codon of the open reading frame (ORF) of the coat protein of the carlavirus Helenium virus (HelVS) was determined from cloned cDNA. This portion of the viral RNA contained one complete ORF encoding a protein of 7 K which had homology with similar sized proteins from other members of the carla- and potexvirus groups. Sequence data was also obtained beyond the start codon of the 7 K ORF into what was presumed to be the 12 K protein gene of the previously identified triple gene block of carla- and potexviruses. Significant sequence similarity of 46.3% and 41.6% was evident at the amino acid level with the equivalent regions of the 12 K proteins of potato virus S, Andean strain (PVSA) and potato virus M (PVM), respectively, with 25.4% similarity detected with the equivalent region of potato virus X (PVX).

Amino Acid Sequence↗

PubMed1996

Molecular analysis of the Rhodobacter capsulatus B10 porin (porCa) gene; purification and biochemical characterisation of the porin protein.

The pore-forming outer-membrane protein from Rhodobacter (R.) capsulatus (wild-type B10 strain) was isolated and purified under non-denaturing conditions. The monomer unit of the isolated porin has a molecular mass of about 28 kDa, as judged by SDS-PAGE, whereas the native protein migrates at 75 kDa. This suggests that the native porin from R. capsulatus B10 exists in a trimeric form. The N-terminal amino acid sequence was used to design an oligonucleotide which was utilised to screen a pBluescript library containing EcoRI fragments of R. capsulatus B10 DNA. A 5.3-kb DNA fragment, which included the entire structural porin gene (named porCa) and its flanking regions, was identified. A 945-bp open reading frame, coding for a mature protein of 295 amino acid residues (molecular mass 30,586 Da) plus a presequence of 20 amino acids, was found. The directly determined sequence of the amino-terminus and of four tryptic peptides of the purified porin matched perfectly with the deduced amino acid sequence. Northern blot analysis showed that the porin gene encodes an RNA transcript of 1050 nucleotides. In addition, there is no differential response in terms of either the size or abundance of the mRNA under different environmental conditions. Primer extension experiments confirmed a putative promoter upstream of the porin gene; and localised the RNA transcription start site 73 bp upstream of the ATG start codon, which is close to the putative promoter (-10/-35). As shown using a plasmid-borne porin-lacZ gene fusion, [in R. capsulatus] expression of the lacZ gene under the control of the porCa promoter is not regulated under the two different environmental conditions tested. The promoter of the porin gene was localised within 305 bp upstream of the ATG start codon. A model of the 3-D structure of porin B10 was deduced by comparative modelling with the R. capsulatus 37b4 and Rhodopseudomonas blastica porin crystallographic structures using the ProMod program on the Swiss-Model protein modelling e-mail Server. Analysis of the B10 sequence and comparison of a model of the B10 porin structure with the crystallographic structure of porin from the capsuleless strain 37B4 has revealed some important differences at the level of the protein surface, the pore and the putative ligand binding site.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence and transcript analysis of three photosystem II genes from the cyanobacterium Synechococcus sp. PCC7942.

The genome of the cyanobacterium Synechococcus sp. PCC7942 contains two genes encoding the D2 polypeptide of photosystem II (PSII), which are designated here as psbDI and psbDII. The psbDI gene, like the psbD gene of plant chloroplasts, is cotranscribed with and overlaps the open reading frame of the psbC gene, encoding the PSII protein CP43. The psbDII gene is not linked to psbC, and appears to be transcribed as a monocistronic message. The two psbD genes encode identical polypeptides of 352 amino acids, which are 86% conserved with the D2 polypeptide of spinach. In plants, the translational start codon of the psbC gene has been reported to be an ATG codon 50 bp upstream from the end of the psbD gene. This triplet is not present in the psbDI sequence of Synechococcus sp., but is replaced by ACG, a codon which is very unlikely to initiate translation. Translation of the psbC gene may begin at a GTG codon which overlap the psbDI open reading frame by 14 bp and is preceded by a block of homology to the 3' end of the 16S ribosomal RNA, a potential ribosome-binding site. There are only two bp differences between the sequences of the two psbD genes; one of these results in substitution in psbDII of GCG for the presumed GTG start codon in psbDI.

Amino Acid Sequence↗

PubMed1998

Analysis of activin A gene expression in human bone marrow stromal cells.

Activin A, a member of the TGF-beta superfamily, plays roles in differentiation and development, including hematopoiesis. Our previous studies indicated that the expression of activin A by human bone marrow cells and monocytes is highly regulated by inflammatory cytokines and glucocorticoids. The present study was undertaken to investigate the regulation of activin A gene expression in the human bone marrow stromal cell lines L87/4 and HS-5, as well as in primary stromal cells. Northern blots demonstrated that, like primary stromal cells, the cell lines expressed four activin A RNA transcripts (6.4, 4.0, 2.8, and 1.6 kb), although distribution of the RNA among the four sizes varied. The locations of the 5' ends of the RNAs were investigated by Northern blots and RNase protection assays. The results identified a transcription start site at 212 nucleotides upstream of the translation start codon. In addition, luciferase expression assays of a series of deletion constructs were used to identify regulatory sequences upstream of the activin A gene. A 58 bp upstream sequence exhibits promoter activity. However, severalfold higher expression requires a positive element consisting of an additional 71 bp of the upstream region. Promoter activity was also identified between 2.5 and 3.6 kb upstream of the start codon. These findings suggest that expression of activin A at the transcriptional level follows complex patterns of regulation.

Activins↗

PubMed2003

Tetracycline-aptamer-mediated translational regulation in yeast.

We describe post-transcriptional gene regulation in yeast based on direct RNA-ligand interaction. Tetracycline-dependent translational regulation could be imposed via specific aptamers inserted at two different positions in the 5' untranslated region (5'UTR). Translation in vivo was suppressed up to ninefold upon addition of tetracycline. Repression via an aptamer located near the start codon (cap-distal) in the 5'UTR was more effective than repression via a cap-proximal position. On the other hand, suppression in a cell-free system reached maximally 50-fold and was most effective via a cap-proximal aptamer. Examination of the kinetics of tetracycline-dependent translational inhibition in vitro revealed that preincubation of tetracycline and mRNA before starting translation led not only to the fastest onset of inhibition but also the most effective repression. The differences between the behaviour of the regulatory system in vivo and in vitro are likely to be related to distinct properties of mRNP structure and mRNA accessibility in intact cells as opposed to cell-extracts. Tetracycline-dependent regulation was also observed after insertion of an uORF sequence upstream of the aptamer, indicating that our system also targets reinitiating ribosomes. Polysomal gradient analyses provided insight into the mechanism of regulation. Cap-proximal insertion inhibits binding of the 43S complex to the cap structure whereas start-codon-proximal aptamers interfere with formation of the 80S ribosome, probably by blocking the scanning preinitiation complex.

5' Untranslated Regions↗

PubMed1984

DNA sequence of the Escherichia coli gene, gnd, for 6-phosphogluconate dehydrogenase.

Expression of gnd of Escherichia coli, which encodes 6-phosphogluconate dehydrogenase, an enzyme of the hexose monophosphate shunt, is subject to growth rate-dependent regulation and is gene dosage-dependent: the level of the enzyme increases in direct proportion to the cellular growth rate at both low and high gene copy numbers. We have determined the nucleotide sequence of gnd and flanking control regions, the 5'-end of in vivo gnd mRNA, and the start codon of the structural gene. Analysis of the sequence indicated that: (i) the gnd promoter is typical of other E. coli promoters and the structural gene is followed by a rho-independent transcription termination signal; (ii) the 56-nucleotide leader of gnd mRNA does not contain a rho-independent transcription termination signal, so growth rate-dependent regulation of 6-phosphogluconate dehydrogenase level is not carried out by an attenuation mechanism analogous to the one that controls expression of the E. coli ampC gene; (iii) the codon composition of the structural gene resembles that of other highly expressed E. coli genes and thus is not responsible for the regulation either; (iv) the structural gene is preceded at an optimal distance by a strong Shine-Dalgarno (SD) sequence, AGGAG ; (v) the leader region of the mRNA contains regions of dyad symmetry that have the potential to sequester the SD sequence and the start codon. This latter feature of the gene suggests that growth rate-dependent regulation may involve regulation of translation initiation frequency.

Amino Acid Sequence↗

PubMed2000

Regulation of mouse delta-opioid receptor gene transcription: involvement of the transcription factors AP-1 and AP-2.

The aim of this study was to examine the effects of the phorbol ester O-tetradecanoylphorbol 13-acetate (TPA) and forskolin on delta-opioid receptor gene transcription. Treatment of NG108-15 cells with TPA (100 nM) for 48 h increased delta-opioid receptor mRNA levels, whereas different concentrations of forskolin induced a transient down-regulation of mRNA 5 h after treatment, followed by increased mRNA levels after 48 h. Reporter gene assays in transiently transfected NG108-15 cells in combination with electrophoretic mobility shift assays indicate that the increase of delta-opioid receptor mRNA after stimulation with TPA is mediated by transcription factor AP-1, which binds 355 bp upstream of the start codon within the gene promoter. The forskolin-induced mRNA increase is mediated neither by a cyclic AMP-response element nor indirectly by AP-1 up-regulation. Reporter gene assays, mutational analysis, and electrophoretic mobility shift assays revealed that delta-opioid receptor gene regulation by forskolin is mediated by transcription factor AP-2, which binds to an element 157 bp upstream of the start codon.

1-Methyl-3-isobutylxanthine↗

PubMed2003

YACOP: Enhanced gene prediction obtained by a combination of existing methods.

The performance of gene-predicting tools varies considerably if evaluated with respect to the parameters sensitivity and specificity or their capability to identify the correct start codon. We were interested to validate tools for gene prediction and to implement a metatool named YACOP, which combines existing tools and has a higher performance. YACOP parses and combines the output of the three gene-predicting systems Criticia, Glimmer and ZCURVE. It outperforms each of the programs tested with its high sensitivity and specificity values combined with a larger number of correctly predicted gene starts. Performance of YACOP and the gene-finding programs was tested by comparing their output with a carefully selected set of annotated genomes. We found that the problem of identifying genes in prokaryotic genomes by means of computational analysis was solved satisfactorily. In contrast, the correct localization of the start codon still appeared to be a problem, as in all cases under test at least 7.8% and up to 32.3% of the positions given in the annotations differed from the locus predicted by any of the programs tested. YACOP can be downloaded from http://www.g2l.bio.uni-goettingen.de.

Algorithms↗

PubMed2006

Characterization of MobR, the 3-hydroxybenzoate-responsive transcriptional regulator for the 3-hydroxybenzoate hydroxylase gene of Comamonas testosteroni KH122-3s.

Comamonas testosteroni KH122-3s is an aerobic soil bacterium that utilizes 3-hydroxybenzoate as a sole carbon and energy source. In this strain, 3-hydroxybenzoate hydroxylase (MobA) acts on the initial step of the degradation to produce 3,4-dihydroxybenzoate, which is subsequently subjected to the meta-cleavage pathway leading to tricarboxylic acid cycle intermediates. Gene walking analysis of the upstream region of mobA revealed an open reading frame (mobR) that encodes a transcriptional regulator of the MarR family. Here, we report that MobR negatively regulates the expression of mobA, and that the repression is relieved by binding of 3-hydroxybenzoate, the substrate for MobA. A primer extension experiment was performed to determine the transcription start site for mobA and identified it at 83 bp upstream of the mobA start codon, accompanied by a typical sigma70-type promoter. The mobR gene was expressed in Escherichia coli cells and the recombinant product was purified to homogeneity. Gel mobility-shift assays and DNase I footprinting analyses indicated that MobR binds as a homodimer to an imperfect inverted repeat within the mobA-mobR intergenic region, with an apparent dissociation constant of 11.5(+/- 0.5) nM. The operator site is located between the start codon and the promoter region for mobA, suggesting that MobR functions as a transcriptional repressor for mobA expression. The results of effector-binding assays indicated that MobR, but not its isomers 4-hydroxybenzoate and salicylate, is released from the operator site by the addition of 3-hydroxybenzoate. This dissociation process is highly cooperative, with a Hill coefficient of approximately 2. In addition, CD spectroscopic studies demonstrated that MobR adopts two conformational states corresponding to the effector-bound and unbound forms. These results suggest that the MobR dimer possesses at least two effector-binding sites, and that the effector binding to MobR induces an allosteric conformational change required for dissociation of the protein-DNA complex.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1996

TransTerm: a database of translational signals.

The TransTerm database of sequence contexts of stop and start codons has been expanded to include approximately 50% more species than last year's release. It now contains 148 organisms and >39 500 coding sequences; it is now available on the World Wide Web. The database includes: (i) initiation and termination sequence contexts organized by species; (ii) summary parameters about the individual sequences (sequence length, GC%, GC3, Nc, CAI) in addition to tables of base frequencies for each species' stop and start codon sequence context; (iii) species codon usage tables; and (iv) summary tables of stop signal frequency.

Animals↗

PubMed1989

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation.

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.

Animals↗

PubMed1991

Different promoters of SHV-2 and SHV-2a beta-lactamase lead to diverse levels of cefotaxime resistance in their bacterial producers.

Clinical Klebsiella pneumoniae isolates as well as Escherichia coli transformants producing the beta-lactamases SHV-2 or SHV-2a demonstrate MIC values for cefotaxime of 4 mg l-1 or 64 to greater than 128 mg l-1, respectively. The beta-lactamases differ by one possibly insignificant amino acid exchange at position number 10 of the mature protein; their kinetic parameters are rather similar. The 5' untranslated regions of both corresponding genes show no homology starting 74 nucleotides upstream to the start codon. Hybridization of intragenically annealing oligonucleotides to dot-blotted serial dilutions of total cellular RNA from E. coli transformants harbouring these genes cloned into the same vector plasmid gave a positive signal down to 1.2 micrograms (SHV-2) and 0.32 to 0.16 micrograms (SHV-2a), indicating a four to eight times higher amount of specific transcript in the case of SHV-2a. By primer extension analysis and S1 nuclease digestion the starting point to transcription was located 100 nucleotides (SHV-2) and 50 nucleotides (SHV-2a) in front of the start codon. No other transcripts of different length could be detected after prolonged exposure. Northern blot analysis demonstrated the length of the beta-lactamase mRNA to be about 1.6 kb in both cases, thus comprising a potential open reading frame downstream of the two enzymes' genes. Selective PCR amplification of both promoter regions and of the structural gene of SHV-2 and subsequent combined cloning of each of the promoters and the SHV-2 gene into pBGS19 using a BamHI restriction site introduced by three point mutations into the cloned sequences was employed to transforms E. coli DH5 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗