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J F Gardner

Publications and source records attributed to J F Gardner.

At least 73 records · Page 4Linked to original sources
PubMed1985

Identification and characterization of mutants affecting transcription termination at the threonine operon attenuator.

Mutations that map in or delete the attenuator of the threonine (thr) operon of Escherichia coli were isolated and characterized. These mutations disrupt or delete the transcription termination structure encoded by the attenuator leading to increased transcriptional readthrough into the thr operon structural genes. Most of the base substitutions and single base-pair insertions and deletions map in the G + C-rich region of dyad symmetry in the attenuator and decrease the calculated stabilities of the attenuator RNA secondary structures to similar extents (from -30.8 kcal/mol to approximately -21 kcal/mol). Most of the mutants showed a three- to fourfold increase in homoserine dehydrogenase (thrA gene product) synthesis relative to the wild-type parent strain. The mutation in one mutant (thrL153 + G) lowered the calculated stability of the RNA secondary structure only slightly (from -30.8 to 27.8 kcal/mol) but the mutant still exhibited high levels of homoserine dehydrogenase synthesis. In addition, three base substitution mutants (thrL135U, thrL139A and thrL156U) showed only slightly (1.5 to 2-fold) elevated levels of homoserine dehydrogenase activity, even though the calculated stabilities of the attenuator RNA secondary structures were reduced as much as most of the other mutants. Two of the mutations (thrL135U and thrL156U) mapped in the G + C-rich-A + T-rich junction of the attenuator. The third mutation (thrL139A) creates an A X C pair in the center of the G + C-rich region of the attenuator stem. The results obtained for these mutants show that the stability of the RNA secondary structure does not always correlate with the efficiency of transcription termination. Finally, analysis of the base changes in the substitution mutations showed that the mutational changes do not appear to be random.

Base Sequence↗
PubMed1985

Extent of sequence homology required for bacteriophage lambda site-specific recombination.

Bacteriophage lambda integration and excision occur by reciprocal recombination within a 15-base homologous core region present in the recombining attachment (att) sites. Strand exchange within the core occurs at precise nucleotide positions, which define an overlap region in which the products of recombination contain DNA strands derived from different parents. In order to define the role of sequence homology during recombination we have constructed point mutations within the core and assayed their effects in vivo and in vitro on site-specific recombination. Two of the mutations are located at position -3 of the core, which is one base-pair outside of the overlap region where strand exchange occurs. These mutations do not affect integrative or excisive recombination, thereby suggesting that homology outside the overlap region is not required for recombination. Two other mutations are located at position -2 of the core, which is one base-pair within the overlap region. These mutations show severely depressed integrative and excisive recombination activities in vitro and in vivo when recombined against wild-type att sites. However, the -2 mutations show normal recombination activity when recombined against att sites containing the homologous mutation, thereby suggesting that homology-dependent DNA interactions are required within the overlap region for effective recombination. In vitro recombination between homoduplex attP sites and heteroduplex attB sites demonstrated that the DNA interactions require only one strand of the attB overlap region to be homologous to attP in order to promote recombination.

Attachment Sites, Microbiological↗
PubMed1985

A genetic enrichment for mutations constructed by oligodeoxynucleotide-directed mutagenesis.

A genetic enrichment procedure for mutations constructed by oligodeoxynucleotide(oligo)-directed mutagenesis of DNA cloned in M13mp vectors is described. The procedure uses an M13 vector that contains the cloned target DNA and amber (am) mutations within the phage genes I and II. This vector cannot replicate in a suppressor-free (sup degrees) bacterial strain. A gapped heteroduplex is formed by annealing portions of a complementary (-)strand containing wild-type copies of genes I and II to the am-containing template (+)strand. The oligo is annealed to the single-stranded (ss) region and the remaining gaps and nicks are repaired enzymatically to form a closed circular heteroduplex structure. By transfecting the DNA into a sup degrees host we promote the propagation of heteroduplexes with the oligo-containing (-)strand since only this construction contains the wild-type copies of genes I and II. This procedure eliminates the need for any physical separation of the covalently closed circular DNA that contains the oligo from the ss template. Using this technique we have constructed 17 point mutations with mutation frequencies ranging from 2-20% for single base changes and from 0.3-9% for multiple base changes. In addition, we found that the mutation frequencies were affected by the state of DNA methylation in the (+) and (-)strands.

Coliphages↗
PubMed1985

Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element.

We constructed a shuttle vector, pE5-2, which can replicate in both Bacteroides spp. and Escherichia coli. pE5-2 contains a cryptic Bacteroides plasmid (pB8-51), a 3.8-kilobase (kb) EcoRI-D fragment from the 41-kb Bacteroides fragilis plasmid pBF4, and RSF1010, an IncQ E. coli plasmid. pE5-2 was mobilized by R751, an IncP E. coli plasmid, between E. coli strains with a frequency of 5 X 10(-2) to 3.8 X 10(-1) transconjugants per recipient. R751 also mobilized pE5-2 from E. coli donors to Bacteroides uniformis 0061RT and Bacteroides thetaiotaomicron 5482 with a frequency of 0.9 X 10(-6) to 2.5 X 10(-6). The Bacteroides transconjugants contained only pE5-2 and were resistant to clindamycin and erythromycin. Thus, the gene for clindamycin and erythromycin resistance must be located within the Eco RI-D fragment of BF4. A second recombinant plasmid, pSS-2, which contained 33 kb of pBF4 (including the EcoRI-D fragment and contiguous regions) could also be mobilized by R751 between E. coli strains. In some transconjugants, a 5.5-kb (+/- 0.3 kb) segment of the pBF4 portion of pSS2 was inserted into one of several sites on R751. In some other transconjugants this same 5.5-kb segment was integrated into the E. coli chromosome. This segment could transfer a second time onto R751. Transfer was RecA independent. The transferred segment included the entire EcoRI-D fragment, and thus the clindamycin-erythromycin resistance determinant, from pBF4.

Bacteroides↗
PubMed1984

Specificity and kinetics defining the interaction between a murine monoclonal autoantibody and DNA.

Interactions between a murine monoclonal anti-DNA autoantibody (BV17-45) and DNA were examined by direct binding and competitive radioimmunoassays. Binding isotherms constructed by titration of purified BV17-45 with a series of distinct 32P-labeled double-stranded DNA ([32P]dsDNA) fragments were super-impossible, suggesting: 1) BV17-45/[32P]dsDNA binding is independent of dsDNA size using fragments greater than or equal to 192 base pairs in length, and 2) BV17-45 does not exhibit stringent sequence specificity. Single-stranded DNA-specific monoclonal antibody BV04-01 did not react with [32P]dsDNA, confirming its duplex character. In competition experiments, BV17-45 cross-reacted with phage (phi X174, M13) RF AND VIRION DNAS AT PICOMOLAR concentrations. Selectivity for B-form DNA was suggested by the ability of poly(dA) . poly(dT), but not other helical duplex forms, to block BV17-45/[32P] dsDNA binding. Among the four deoxyribohomopolymers, only deoxyadenylic acid polymers completely inhibited BV17-45/[32P]dsDNA complex formation. [32P]dsDNA binding was relatively insensitive to ionic strength, suggesting minimal contribution of electrostatic forces to the binding free energy. Measured BV17-45/[32P]dsDNA association and dissociation rate constants (4 degrees C) were 7.4 X 10(6) M-1 s-1 and 9.2 X 10(-5) s-1, respectively, yielding a functional affinity of 8 X 10(10) M-1. Results are discussed in terms of the relative contribution of B-DNA structural and substructural determinants to the mechanism of BV17-45 recognition.

Antibodies↗
PubMed1984

DNA interactions during bacteriophage lambda site-specific recombination.

Extensive research on site-specific recombination has provided many details, particularly with respect to the protein-DNA interactions. However, very little is known about the molecular mechanism of recombination during synapsis and strand exchange. Presumably, these steps of recombination involve various forms of DNA-DNA, DNA-protein, and protein-protein interactions. One stage at which DNA-DNA interactions may be occurring is at the level of synapsis where the recombining DNAs are juxtaposed. In this paper we have presented evidence that homology-dependent DNA interactions do occur within the overlap region before strand exchange. This interaction is presumably at the synaptic stage of recombination. Furthermore, we have demonstrated that the homology-dependent interactions require that only one strand of attB have homology to attP. Another stage in recombination at which DNA-DNA interactions could occur is during strand exchange where complementary strands from the recombining parents are paired and resealed. We have also presented evidence that homology-dependent DNA interactions occur during strand exchange prior to the resealing of the strands and that disruption of this interaction results in nonreciprocal recombination. Taken together, these results suggest that DNA-DNA interactions during reciprocal site-specific recombination occur during at least two stages in the reaction.

Bacteriophage lambda↗
PubMed1983

Use of randomly cloned DNA fragments for identification of Bacteroides thetaiotaomicron.

Randomly cloned fragments of DNA from Bacteroides thetaiotaomicron were used as hybridization probes for differentiation of B. thetaiotaomicron from closely related Bacteroides species. HindIII digestion fragments of DNA from B. thetaiotaomicron (type strain) were inserted into plasmid pBR322 and labeled with [alpha-32P]dCTP by nick translation. These labeled plasmids were screened for hybridization to HindIII digests of chromosomal DNA from type strains of the following human colonic Bacteroides species: B. thetaiotaomicron, Bacteroides ovatus, reference strain 3452-A (formerly part of B. distasonis), Bacteroides uniformis, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides eggerthii, and reference strain B5-21 (formerly B. fragilis subsp. a). Two of the five cloned fragments hybridized only to DNA from B. thetaiotaomicron. Each of these two fragments hybridized to the same DNA restriction fragment in five strains of B. thetaiotaomicron other than the strain from which the DNA was cloned. One of the cloned fragments (pBT2) was further tested for specificity by determining its ability to hybridize to DNA from 65 additional strains of colonic Bacteroides.

Bacteroides↗
PubMed1982

Initiation, pausing, and termination of transcription in the threonine operon regulatory region of Escherichia coli.

The 6 S leader RNA transcript from the Escherichia coli threonine operon controlling region was synthesized in vitro using purified RNA polymerase and restriction fragment DNA templates. The terminated leader transcript was analyzed by RNase T1 digestion followed by electrophoresis on 20% polyacrylamide, 8 M urea gels. Oligonucleotides of 7, 8, 13, 15, and 35 bases in length were detected and correlated with the known DNA sequence. The kinetics of RNase T1 digestion indicated that the RNA forms extensive secondary structure, especially at the 3'-terminus of the transcript. The sites of transcription initiation were determined by labeling the 5'-end of the transcript with [gamma-32P]ATP or -GTP followed by direct RNA sequencing. The DNA sequence preceding the initiation site shows homology with the equivalent regions of other bacterial and bacteriophage promoters. The transcription termination sites were determined by mapping of the RNase T1 oligonucleotides arising from the 3'-terminus of the transcript. Comparison of the mobilities of the 3'-oligonucleotides with the mobilities of standards on 20% polyacrylamide, 8 M urea gels indicated that the RNA contains a heterogeneous 3'-terminus. The two predominant oligonucleotides were CU7 and CU8. The 3'-terminus of the transcript also contains a region of dyad symmetry immediately preceding a stretch of uridine residues, characteristic of other rho-independent transcripts. In addition, kinetic studies indicated that RNA polymerase pauses approximately 50 base pairs upstream from the site of termination. The pause site appears to be immediately distal to another region of dyad symmetry.

Amino Acid Sequence↗
PubMed1982

Attenuation regulation in the thr operon of Escherichia coli K-12: molecular cloning and transcription of the controlling region.

Recombinant plasmids were constructed which carry defined regions of the threonine (thr) operon regulatory region of Escherichia coli. In vitro transcription experiments utilizing plasmid or restriction fragment templates showed that two major RNA transcripts, which differ in length by one to a few bases, are transcribed from this region. The approximate length of the transcripts is 150 to 170 bases, and the site(s) of termination is near or within the thr attenuator. The efficiency of termination at the thr operon attenuator in vitro is approximately 90%. A regulatory mutation, thr79-20, which is a G-C insertion in the attenuator, reduces the frequency of transcription termination to 75%. In addition, in vivo RNA transcripts were identified which hybridize to the thr operon regulatory region. These transcripts appeared to be identical to the two major in vitro transcripts as judged by their mobilities on 8% polyacrylamide-8 M urea gels. This result indicates that the thr operon regulatory region is transcribed in vivo and that termination occurs near or within the thr attenuator.

Cloning, Molecular↗
PubMed1981

Secondary lambda attachment site in the threonine operon attenuator of Escherichia coli.

We have determined the nucleotide sequence of a secondary gamma attachment (att) site which overlaps the Escherichia coli threonine (thr) operon attenuator. The secondary att site shows uninterrupted homology (8 out of 15) with the 15 base-pair "common core" sequence found in gamma and at the primary bacterial attachment site. These 8 base paired also overlap the thr operon attenuator. Comparison of the secondary att site with the flanking prophage sites shows that the crossover site for gamma integration lies within the -7 to 0 region of the core. Sequences on both sides of the core show no obvious homology with analogous sequences of the gamma or primary bacterial att sites. The core sequences of the left prophage att site is completely homologous to the wild-type core and also shows the same 8-base pair overlap with the thr operon attenuator. The position of the thr operon attenuator, relative to the left prophage att site, indicates that ribonucleic acid transcripts, initiated at a gamma promoter, are terminated efficiently at the thr attenuator. It is also possible that this prophage att site is able to undergo int dependent site-specific recombination which with another nearby secondary att site. Evidence is also presented which suggests that a base or sequence to the left of position -6 in the core is necessary fo excisive recombination.

Attachment Sites, Microbiological↗
PubMed1980

RsaI: a new sequence-specific endonuclease activity from Rhodopseudomonas sphaeroides.

A new type II sequence-specific endonuclease, RsaI, has been identified from Rhodopseudomonas sphaeroides strain 28/5. An RsaI purification scheme that yields enzyme which is free of contaminating exonuclease and phosphatase activities after a single column fractionation has been developed. The enzyme recognized the tetranucleotide sequence 5'-GTAC-3' and cleaved between the T and A, thereby generating flush ends. RsaI should be extremely useful in deoxyribonucleic acid sequencing experiments.

Base Sequence↗
PubMed1979

Regulation of the threonine operon: tandem threonine and isoleucine codons in the control region and translational control of transcription termination.

The DNA sequence of 178 base pairs preceding the first structural gene of the threonine operon of Escherichia coli has been determined. A region of perfect 2-fold rotational symmetry, involving 28 base pairs, precedes the first structural gene. The structural similarity of this sequence to known RNA polymerase termination sites suggests that this region is the termination site of the threonine operon leader RNA. Moreover a mutation (thr 79-20), which confers a depressed, constitutive phenotype, was sequenced and found to be a G.C insertion in the putative terminator. A potential coding region for a 21-amino acid leader peptide ends approximately 18 base pairs before the terminator. This peptide contains eight threonine and four isoleucine codons. Eleven of these codons are in tandem. A model for threonine operon regulation, involving alternative secondary RNA structures and translation of leader RNA, is discussed.

Base Sequence↗
PubMed1979

Characterization of a site-specific restriction endonuclease from Rhodopseudomonas sphaeroides.

A type II restriction endonuclease, RshI, has been partially purified from photoheterotrophically grown Rhodopseudomonas sphaeroides strain 2.4.1. The enzyme preparation, after a single DE-52 column fractionation, is free of 5' exonuclease and phosphatase activities but contains a trace of 3' exonuclease activity. Based upon deoxyribonucleic acid (DNA) sequencing data in the vicinity of the enzyme-promoted cleavage of pBR322 DNA, we have concluded that RshI probably recognizes the palinodromic hexanucleotide sequence 5'-CGATCG-3' and cleaves between the T and C. lambda cI857 DNA contains three RshI sites, two of which lie in the replaceable region. The plasmid pBR322, which carries resistances to ampicillin and tetracycline, contains a single RshI site in the ampicillin resistance determinant. Insertion of DNA into the RshI site of pBR322 results in loss of ampicillin resistance but retention of tetracycline resistance, thereby providing a convenient screening procedure for recombinant plasmids.

Base Sequence↗
PubMed1978

Skin disinfection.

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Disinfection↗
PubMed1975

Operator-promoter functions in the threonine operon of Escherichia coli.

The prophage curing properties of secondary-site lysogens of coliphage lambda have been studied. The site of integration in the original lysogen (L79) is within the ooerator-promoter region of the thr operon. As a result, expression of the thr enzymes is reduced, and the strain is a leaky threonine auxotroph. Heat pulse curing of strain L79 and a thr+ lysogenic revertant (L79-20) showed that heat pulse curing of both lysogens was int and xis dependent and occurred by correct excisions of the prophage. The heat pulse curing restored strain L79 to prototrophy whereas strain L79-20 synthesized the thr enzymes constitutively and at high levels. This indicates that the reversion mutation in strain L79-20 occurred outside of the prophage and within the operator-promoter region of the thr operon. In contrast, spontaneous curing of both lysogens occurred by both correct and incorrect excisions. Spontaneously cured derivatives of strain L79-20 gave rise to three classes of regulatory mutants affecting operator and promoter functions to the thr operon.

Cell-Free System↗