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Biomedical subjects

J F Gardner

Publications and source records attributed to J F Gardner.

At least 55 records · Page 3Linked to original sources
PubMed1992

Compliance and quality in residential life. Quality, organization design, and standards.

Regulations and voluntary standards within the context of organizational design and mission as well as leadership in management for quality were discussed. Rules and regulations are closely associated with the structure and mission of the organization. Altering them may require fundamental redesign of organizational structure and purpose. Likewise, alterations in organizational design can change the need for rules and regulations. In addition, standards do not automatically yield quality. Rather, they define expected levels of performance from individuals, programs, and the organization. The senior management of organizations is responsible for exercising the leadership in managing for quality.

Community Mental Health Services↗
PubMed1991

Isolation and footprint analysis of the Escherichia coli thr leader paused transcription complex.

The E. coli thr operon leader region contains a cluster of transcription pause sites upstream of the attenuator. In this report, we determine the exact sites of pausing and analyze the structure of the ternary complex by footprint techniques. Under synchronized transcription initiation conditions in vitro, three closely-spaced transcription pause sites were identified. These pause sites appeared downstream of the first region of dyad symmetry, which encodes an RNA hairpin in the transcript, and occurred at positions G112, G114 and A116 of the thr leader RNA. The results showed that the half-life of the thr paused complexes at G112 and G114 could be enhanced by limiting the concentration of the nucleoside triphosphate GTP in the transcription reactions. In addition, the half-life of the paused complexes was shown to increase in the presence of NusA protein. The thr leader complex that paused immediately before residues G112 and G114 of the nascent transcript was isolated and its structure was analyzed with enzymatic and chemical cleavage reagents. The footprinting studies using DNase I showed that there were approximately 35 nucleotides on both strands of the DNA that were protected by RNA polymerase from DNase I cleavage. The DNA segment protected by RNA polymerase is approximately 19 nucleotides upstream and 14 nucleotides downstream of the pause sites. The results from hydroxyl radical footprints also showed a similar pattern of protection at the transcription pause sites. However, no significant differences in the footprinting patterns were observed in the presence or absence of NusA protein.

Base Sequence↗
PubMed1991

A genetic analysis of Xis and FIS interactions with their binding sites in bacteriophage lambda.

The bacteriophage P22-based challenge-phage system was used to study the binding of Xis and FIS to their sites in attP of bacteriophage lambda. Challenge phages were constructed that contained the X1, X2, and F sites within the P22 Pant promoter, which is required for expression of antirepressor. If Xis and FIS bind to these sites in vivo, they repress transcription from Pant, allowing lysogenization to occur. Challenge phages carrying the XIX2F region in either orientation exhibited lysogenization dependent on both Xis and FIS. Neither Xis nor FIS was capable of functioning by itself as an efficient repressor in this system. This was the first time challenge phages have been constructed that require two different proteins bound simultaneously to act as a repressor. Mutations in the X1, X2, and F sites that inhibit Xis and FIS from binding were isolated by selecting mutant phages that still expressed antirepressor synthesis in the presence of Xis and FIS. DNA sequence analysis of the mutants revealed 38 unique mutations, including single-base-pair substitutions, multiple-base-pair changes, deletions, and insertions throughout the entire X1, X2, and F regions. Some of the mutations verified the importance of certain bases within the proposed consensus sequences for Xis and FIS, while others provided evidence that the DNA sequence outside of the proposed binding sites may affect the binding of the individual proteins or the cooperativity between them.

Attachment Sites, Microbiological↗
PubMed1991

Genetic analysis of Escherichia coli integration host factor interactions with its bacteriophage lambda H' recognition site.

The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda. We constructed challenge phages that carried H' inserts in both orientations within the P22 Pant promoter, which is required for antirepressor synthesis. We found that IHF repressed expression of Pant from either challenge phage when expressed from an inducible Ptac promoter on a plasmid vector. Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF. Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IHF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site. Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specified in the consensus sequence, and in the dA + dT-rich region that flanks the consensus region of the site. Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt IHF binding to the mutated site.

Bacterial Proteins↗
PubMed1990

A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage lambda.

Triple-base changes were made in each of the five Integrase (Int) arm-type binding sites of bacteriophage lambda. These triple changes, called ten mutants, were compared with single-base changes (hen mutants) for their effects on integrative and excisive recombination. The presence of ten or hen mutations in the P1, P'2, or P'3 sites inhibited integration, but the ten P'3 mutant was 10-fold more defective than the analogous hen mutant. The results with these mutants suggest that the P1, P'2, P'3, and possibly the P'1 sites are required for integration. In wild-type E. coli, the ten P'1 mutant reduced the frequency of excision 5-fold, whereas the hen P'1 mutant had no effect. The presence of ten mutations in the P2, P'1, or P'2 sites inhibited lambda excision in an E. coli strain deficient in the production of FIS, while hen mutations in the P2 and P'2 sites had little or no effect. The results with the ten mutants suggest that the P2, P'1, and P'2 sites are required for excision. The differences in the severity of the effects between the ten and hen mutations may be due to the inability of cooperative interactions among Int, IHF, Xis, and FIS to overcome the disruption of Int binding to sites with triple-base changes compared to sites with single-base changes.

Attachment Sites, Microbiological↗
PubMed1990

Transcription termination by bacteriophage T7 RNA polymerase at rho-independent terminators.

We have investigated the mechanism of transcription termination by T7 RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. The thr attenuator comprises the following two distinct structural elements: a G + C-rich inverted repeat, which encodes an RNA hairpin structure, and A + T-rich regions, one of which contains a continuous sequence of template deoxyadenosine residues within which the transcription terminates. Fourteen attenuator variants were analyzed and we find that not only the hairpin structure itself but also its sequence influences termination. Furthermore, the formation of a hairpin in the RNA encoded by the A + T-rich regions of the attenuator is not mandatory for termination. A series of seven deletion variants that successively shorten the deoxyadenosine tract in the attenuator template were also analyzed. Results from these experiments indicate that complete readthrough occurs when there are four or fewer deoxyadenosine residues. With 5 template deoxyadenosine residues there is 5% termination increasing to 32% with 8 deoxyadenosines, the value produced by the wild-type attenuator. In addition, a comparison with E. coli RNA polymerase shows that T7 RNA polymerase requires a more perfect region of dyad symmetry and a longer deoxyadenosine tract than does the bacterial enzyme to terminate with maximum efficiency.

Base Composition↗
PubMed1990

Genetic analysis of bacteriophage lambda integrase interactions with arm-type attachment site sequences.

The bacteriophage P22-based challenge phage system was used to study lambda integrase (Int) protein binding to its arm-type recognition sequences in the bacteriophage lambda attachment site. Challenge phages were constructed that carried inserts containing either the contiguous P'123 arm-type sites or the single P'1 site within the P22 phage promoter, Pant, which is required for expression of antirepressor. If Int protein binds to these sequences in vivo, it represses transcription from Pant. We found that Int repressed Pant in phages carrying the P'123 sites more efficiently than those carrying only the P'1 site, suggesting that the protein binds cooperatively at the three adjacent sites. The Int protein from a related lambdoid phage, HK022, also repressed transcription by binding to the same arm-type sites. Mutations in the P'123 or P'1 sites that impair Int binding were isolated by selecting mutant phages that express antirepressor in the presence of Int. DNA sequence analyses showed that most of the mutants in the challenge phages carrying the P'123 sites contained multiple changes and that two mutants contained only single-base-pair changes at positions that are completely conserved among all arm-type sites. Thirty-five mutants were isolated and analyzed from phages containing only the P'1 site. Most mutants contained single-nucleotide changes, and mutations were isolated at 8 of the 10 positions of the site, suggesting that most if not all base pairs in the conserved recognition sequence are involved in Int binding.

Bacteriophage lambda↗
PubMed1989

Transcription termination directed by heteroduplex thr attenuator templates. Evidence that the transcript stem and loop structure is the termination signal.

We have constructed both homoduplex and heteroduplex restriction fragment templates that contained mutations in the G + C-rich region of dyad symmetry in the Escherichia coli thr operon attenuator and have studied their termination properties in an in vitro transcription system. We found that the DNA sequence of the template strand determines the transcription termination efficiency. We also constructed two double mutant heteroduplex templates to examine the role of possible complementary base pairs between the nontranscribed DNA strand and the nascent RNA in the transcription bubble. There was no indication that the potential DNA-RNA interactions in the G + C-rich region are involved in transcription termination. Taken together, our results are consistent with and extend those obtained by Ryan and Chamberlin (Ryan, T., and Chamberlin, J. (1983) J. Biol. Chem. 258, 4690-4693) for the trp operon attenuator and argue strongly against models that propose that DNA hairpin formation in the nontranscribed DNA strand or pairing of the nascent RNA with the nontranscribed DNA strand are important for transcription termination.

Base Sequence↗
PubMed1989

The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination.

Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.

Attachment Sites, Microbiological↗
PubMed1988

Contributions of RNA secondary structure and length of the thymidine tract to transcription termination at the thr operon attenuator.

The thr operon of Escherichia coli is regulated by an attenuation mechanism in which regulated transcription termination occurs in response to the levels of charged tRNAthr and tRNAile. Transcription of the thr operon regulatory region in vitro produces a 162-base transcript that is terminated efficiently at the attenuator. The attenuator sequence is similar to other rho-independent terminators. It contains a G + C region of dyad symmetry followed by a run of 9 A + T residues. We have characterized in detail the sequence requirements for efficient transcription termination in vitro. Using a set of point mutations in the G + C region of dyad symmetry of the thr attenuator, we have characterized the effects of these mutations on the efficiency of transcription termination. The efficiency was reduced in all of the mutants analyzed with the greatest effect being an approximate 20% decrease in termination. In some instances the electrophoretic mobilities of the terminated transcripts on 8% polyacrylamide, 8 M urea gels were shifted substantially relative to the wild type-terminated transcript, but the sites of transcription termination were altered by only a few base pairs. We also constructed a set of deletions removing consecutive thymidines which follow the G + C-rich region of dyad symmetry. Removal of 1 or 3 of the 9 thymidine residues had no effect on termination efficiency in vitro or in vivo. Removal of four to six thymidines caused a linear decrease in the efficiency of termination. When only one or two thymidines were present in the template, termination was completely abolished. These results indicate that both the integrity of the RNA stem and the length of the consecutive thymidine residues are important signals recognized by RNA polymerase during transcription of the thr operon regulatory region.

Bacteriophage lambda↗
PubMed1987

Specificity of the attenuation response of the threonine operon of Escherichia coli is determined by the threonine and isoleucine codons in the leader transcript.

Expression of the threonine (thr) operon enzymes of Escherichia coli is regulated by an attenuation mechanism. The regulatory portion of the operon contains a region coding for a leader peptide that contains consecutive threonine and isoleucine codons. It is thought that translation of the leader peptide controls the frequency of transcription termination at the attenuator site. Using oligonucleotide-directed site-specific mutagenesis we have altered the putative control codons of the leader peptide coding region. In two of the mutants the threonine and isoleucine codons were changed to produce peptides containing histidine and tyrosine codons. Both mutants showed loss of regulation by threonine and isoleucine. A hisT mutation, which leads to an undermodification of tRNA(His), increased thr operon expression in the mutants threefold but did not affect expression of the wild-type thr operon. Two other mutants were constructed that contained two histidine codons early in the leader peptide. Expression in both of these mutants was unaltered by the presence of the hisT allele or by the addition of threonine and isoleucine to the growth medium. In addition, a wild-type strain containing a temperature-sensitive threonyl-tRNA synthetase mutation showed increased thr operon expression at the non-permissive temperature, whereas none of the mutants showed any change. Taken together these data indicate that the specificity of the attenuation response is effected by specific control codons within the thr leader peptide coding region. We have also directly demonstrated thr leader peptide synthesis in vitro using a plasmid encoding the wild-type thr leader region to direct the synthesis of a peptide of the appropriate molecular weight when labeled with [3H]threonine but not with [3H]histidine or [3H]tyrosine. Conversely, when extracts were incubated with templates containing the mutated DNAs, peptides were labeled that showed patterns consistent with the expected amino acid compositions. These data indicate that the thr leader RNA is translated into the predicted leader peptide.

Codon↗
PubMed1987

Role of homology in site-specific recombination of bacteriophage lambda: evidence against joining of cohesive ends.

Bacteriophage lambda integration and excision take place at specific loci called attachment sites. Earlier work has shown that efficient recombination requires the identical sequence to be present in both attachment sites throughout the seven-base-pair region between the points of strand exchange. A plausible model for the role of homology postulates that Int, the site-specific recombinase, makes double-strand breaks at attachment sites such that each broken end has a short single-strand protrusion. Recombination would then depend upon the capacity of these protrusions to form Watson-Crick helices--i.e., to anneal--a process that might require perfect complementarity between the cohesive ends. To test this model, we have studied Int-promoted crosses in which one attachment site is a heteroduplex. Specifically, we constructed sites in which the seven-base-pair region between the points of strand exchange contains one or more noncomplementary pairs. The double-strand break and annealing mechanism predicts that crosses with these heteroduplex sites should yield one completed recombinant and one broken site. We find that such nonreciprocal recombination is uncommon and that the typical outcome of crosses involving a heteroduplex site is a reciprocal recombinant in which both products are resealed. Moreover, the occasional appearance of nonreciprocal products can be explained by our finding that Int can cleave heteroduplex attachment sites after recombination is completed. Taken together, our data strongly indicate that bacteriophage lambda recombination does not proceed by the homology-dependent annealing of cohesive ends; acceptable alternatives for the role of homology are discussed.

Bacteriophage lambda↗
PubMed1986

Mutational analysis of integrase arm-type binding sites of bacteriophage lambda. Integration and excision involve distinct interactions of integrase with arm-type sites.

Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome (attP) and the Escherichia coli genome (attB) results in a prophage flanked by the hybrid recombinant sites attL and attR. Each att site contains sequences to which proteins involved in recombination bind. Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int "arm-type" binding sites located within attP, attL and attR. Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int. Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm-type sites within attP are required for efficient integrative recombination. Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm-type sites in attL and attR. These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm-type sites.

Attachment Sites, Microbiological↗
PubMed1986

Role of Escherichia coli IHF protein in lambda site-specific recombination. A mutational analysis of binding sites.

The phage lambda attachment site, attP, contains three binding sites for an Escherichia coli protein, IHF, that is needed for efficient integrative recombination. We have used synthetic oligodeoxyribonucleotides to direct multiple base changes at each of these three sites. Alteration by two base-pairs of the consensus sequence for the leftmost binding site specifically interferes with IHF binding to that site and modestly depresses recombination in vitro. For each of the three binding sites, alteration of the consensus sequence by four base-pairs strongly depresses recombination in vitro, indicating that all three sites are important for attP function. The mutated attP sites are also depressed for recombination in vivo but some of the mutants are less affected than they are in vitro. The disparity between effects in vivo and in vitro for some mutants but not others suggests that the three binding sites are not functionally equivalent and that at some sites additional E. coli factors may replace or assist IHF. The non-equivalence of the three IHF sites is also indicated by the behavior of prophage attachment sites carrying mutations in the binding sites.

Attachment Sites, Microbiological↗
PubMed1986

Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome.

The gene for resistance to erythromycin and clindamycin, which is carried on the conjugative Bacteroides plasmid, pBF4, has been shown previously to be part of an element (Tn4351) that transposes in Escherichia coli. We have now introduced Tn4351 into Bacteroides uniformis 0061 on the following two suicide vectors: (i) the broad-host-range IncP plasmid R751 (R751::Tn4351) and (ii) pSS-2, a chimeric plasmid which contains 33 kilobases of pBF4 (including Tn4351) cloned into the IncQ plasmid RSF1010 and which is mobilized by R751. When E. coli HB101, carrying either R751::Tn4351 or R751 and pSS-2, was mated with B. uniformis under aerobic conditions, Emr transconjugants were detected at a frequency of 10(-6) to 10(-5) (R751::Tn4351) or 10(-8) to 10(-6) (R751 and pSS-2). In matings involving pSS-2, all Emr transconjugants contained simple insertions of Tn4351 in the chromosome, whereas in matings involving R751::Tn4351, about half of the Emr transconjugants had R751 cointegrated with Tn4351 in the chromosome. Of the Emr transconjugants, 13% were auxotrophs. Bacteroides spp. which had R751 cointegrated with Tn4351 in the chromosome did not transfer R751 or Tn4351 to E. coli HB101 or to isogenic B. uniformis, nor did the intergrated R751 mobilize pE5-2, an E. coli-Bacteroides shuttle vector that contains a transfer origin that is recognized by R751.

Bacteroides↗
PubMed1986

Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications.

The manner in which integration host factor (IHF) regulates lambda site-specific recombination has been analyzed by examining the behavior of both wild-type and mutant DNAs in integrative and excisive recombination as well as in protein binding. While integrative recombination of an attP with two base changes in the H1 site required 8-fold more IHF than did wild type, binding to this site was lowered at least 500-fold, suggestive of cooperative interactions. A mutant attP with nine base changes did not integrate at all in vitro, with the defect being less severe in vivo. IHF inhibition of excisive recombination was relieved by both mutations in vitro and in vivo. These results imply that occupancy of the H1 site is critical for determining the direction of recombination. It is proposed that IHF inhibition of excision provides a monitor of the strength of the induction stimulus and the nutritional state of the cell; this would allow the prophage to excise selectively in conditions which favor successful completion of the lytic cycle.

Bacterial Proteins↗