Biomedical subjects

J F Gardner

Publications and source records attributed to J F Gardner.

At least 37 records · Page 2Linked to original sources

PubMed1997

Mutational analysis of protein binding sites involved in formation of the bacteriophage lambda attL complex.

Bacteriophage lambda site-specific recombination requires the formation of higher-order protein-DNA complexes to accomplish synapsis of the partner attachment (att) sites as well as for the regulation of the integration and excision reactions. The att sites are composed of a core region, the actual site of strand exchange, and flanking arm regions. The attL site consists of two core sites (C and C'), an integration host factor (IHF) binding site (H'), and three contiguous Int binding arm sites (P'1, P'2, and P'3). In this study, we employed bacteriophage P22 challenge phages to determine which protein binding sites participate in attL complex formation in vivo. The C', H', and P'1 sites were critical, because mutations in these sites severely disrupted formation of the attL complex. Mutations in the C and P'2 sites were less severe, and alteration of the P'3 site had no effect on complex formation. These results support a model in which IHF, bound to the H' site, bends the attL DNA so that the Int molecule bound to P'1 also interacts with the C' core site. This bridged complex, along with a second Int molecule bound to P'2, helps to stabilize the interaction of a third Int with the C core site. The results also indicate that nonspecific DNA binding is a significant component of the Int-core interactions and that the cooperativity of Int binding can overcome the effects of mutations in the individual arm sites and core sites.

Attachment Sites, Microbiological↗

PubMed1997

Personal outcomes as measures of quality.

The Council on Quality and Leadership in Supports for People With Disabilities (The Council), formerly known as The Accreditation Council, altered its definition of quality from "compliance with organizational process" to "responsiveness to people." Council representatives conducted focus group and individual meetings with people who have disabilities to identify priority outcomes they expect from services and supports. The 1993 Outcome Based Performance Measures was used in 447 interviews as part of 54 accreditation reviews. Staff analysis and factor analysis of the outcome interviews using a principle components extraction and varimax rotation resulted in 24 variables loading onto seven major factors (Identity, Autonomy, Affiliation, Attainment, Rights, Health, and Safeguards), which form the basis of The Council 1997 Personal Outcome Measures.

Humans↗

PubMed1996

Examining the contribution of a dA+dT element to the conformation of Escherichia coli integration host factor-DNA complexes.

DNA binding proteins that induce structural changes in DNA are common in both prokaryotes and eukaryotes. Integration host factor (IHF) is a multi-functional DNA binding and bending protein of Escherichia coli that can mediate protein-protein and protein-DNA interactions by bending DNA. Previously we have shown that the presence of a dA+dT element 5'-proximal to an IHF consensus sequence can affect the binding of IHF to a particular site. In this study the contribution of various sequence elements to the formation of IHF-DNA complexes was examined. We show that IHF bends DNA more when it binds to a site containing a dA+dT element upstream of its core consensus element than to a site lacking a dA+dT element. We demonstrate that IHF can be specifically crosslinked to DNA with binding sites either containing or lacking this dA+dT element. These results indicate the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF.

Attachment Sites, Microbiological↗

PubMed1996

Genetic analysis of the bacteriophage lambda attL nucleoprotein complex.

Site-specific recombination in bacteriophage lambda involves interactions among proteins required for integration and excision of DNA molecules. We have analyzed the elements required to form an in vivo nucleoprotein complex of integrase (Int) and integration host factor (IHF). Interaction of Int with the core (the site of strand exchange) is stabilized by the flanking arm region of attL. IHF, in addition to Int, is required for efficient Int-core binding. We used the in vivo attL binding assay to characterize several Int variants for their abilities to form stable attL complexes. Substitution of Int active site tyrosine 342 by phenylalanine had no effect on the ability of the protein to form attL complexes. Three other amino acids that are completely conserved in the integrase family of recombinases (arginine 212, histidine 308, and arginine 311) were separately substituted by glutamine, leucine, and histidine, respectively. In each case, the mutant protein was altered in its ability to form attL complexes while retaining its ability to bind to the lambda arm-type sites. We propose that, in addition to their role in catalysis, this triad of amino acids helps the Int protein to interact with the lambda core sites.

Bacterial Proteins↗

PubMed1995

The specific binding of Escherichia coli integration host factor involves both major and minor grooves of DNA.

The integration host factor (IHF) of Escherichia coli is a small, sequence-specific DNA-binding protein. The specific and nonspecific binding constants of IHF were estimated by gel-retardation assays. The equilibrium association constant of IHF for the H' site in lambda attP is 6.8 x 10(8) M-1 (Kd = 1.5 nM), and the nonspecific binding constant is 5.8 x 10(5) M-1 (Kd = 1.7 microM), giving a selectivity of approximately 1,000-fold for a specific site over random sequences. To study the molecular determinants specifying IHF binding, we used a series of 41 oligonucleotides containing adenine analogues that modified the surfaces of the major and minor grooves of the DNA. Many of the analogue substitutions within the previously defined consensus region caused decreased binding. Replacement with various analogues outside the consensus domain had little effect. Quantifying the binding constants for those sites with reduced affinities indicated an interaction with the minor groove within the consensus sequence. The binding constants of sites with 2-aminopurine and an inosine substitution within the same region suggest that IHF may also interact with the major groove. Thus, the specific interaction of IHF with its H' site likely involves interactions with both the minor and major grooves of the DNA.

2-Aminopurine↗

PubMed1995

Transcription termination at the thr attenuator. Evidence that the adenine residues upstream of the stem and loop structure are not required for termination.

The Escherichia coli thr operon attenuator has a structure similar to other Rho-independent terminators. The DNA sequence immediately 5' to the termination site is dG+dC-rich and contains a region of dyad symmetry that, when transcribed into RNA, encodes a hairpin structure in the transcript. It also contains a stretch of 9 consecutive dA-dT residues immediately distal to the region of dyad symmetry which encode uridine residues at the 3' end of the terminated transcript. In addition, the thr attenuator has a stretch of 6 dA-dT residues immediately upstream of the region of dyad symmetry which encode 6 adenines. These adenines could potentially pair with the distal uridines to form a hairpin structure extended by as much as 6 A-U base pairs. In this report we have examined the role of the upstream adenines in transcription termination. We used templates that specify mismatches or create new base pairs in the potential A-U secondary structure of the transcript as well as templates that delete segments of the A residues upstream of the hairpin. We conclude that A-U pairing is not required for efficient transcription termination at the thr attenuator. This conclusion is likely to apply to other Rho-independent terminators that contain hairpin-proximal dA-dT residues.

Amino Acid Sequence↗

PubMed1995

Selection of mutations altering specificity in restriction-modification enzymes using the bacteriophage P22 challenge-phage system.

A method for selecting mutants of site-specific DNA-binding proteins has been applied to the study of the EcoRI and RsrI restriction-modification enzymes. Catalytically inactive variants of both endonucleases are shown to function as pseudo-repressors in the bacteriophage P22 challenge-phage assay, and, upon further mutagenesis of the gene encoding R.EcoRI, a variant of that enzyme has been selected which appears to bind EcoRI-methylated GAATTC sequences to the exclusion of unmethylated sites: this specificity is the opposite of that belonging to the native enzyme. Variants of the EcoRI methylase have also been found that lack either catalytic activity or both binding and catalytic activities.

Bacteriophage P22↗

PubMed1994

Mapping the functional domains of bacteriophage lambda integrase protein.

Bacteriophage lambda encodes a site-specific recombination system that promotes the movement of the phage genome into and out of the host bacterial chromosome. The phage-encoded integrase (Int) is composed of 356 amino acid residues and carries out the required strand exchanges by means of a type I topoisomerase activity. Int also contains two distinct DNA-binding domains that interact with two different, specific sequences (arm-type and core-type sites) on DNA. In order to help understand the mechanism of site-specific recombination, we have used a genetic approach to isolate mutants defective in different steps in the recombination reaction. We developed a genetic screen for Int mutants that are defective in catalyzing excisive recombination in vivo. These mutants were screened for proficiency in binding to the P'123 arm-type sites using the bacteriophage P22 challenge-phage assays. In all, 78 such mutants were isolated and the mutational changes mapped and sequenced. These mutants have been further characterized (1) for their ability to bind the P'1 and P'123 arm-type sites and for their ability to form the attL complex in vivo, (2) for negative dominance in vitro, (3) for the presence of type I topoisomerase activity, and (4) for the ability to resolve artificially constructed recombination intermediates. We found that (1) residues in a stretch of 88 amino acids in the middle of the protein may be involved in Int-Int interactions, (2) a region around Arg212 is involved in the catalytic site, (3) residues near the carboxyl terminus play a role in enhancing Int binding to its arm-type sites, possibly by interacting with the small amino-terminal region that has been shown to be responsible for specific recognition of the arm-type sites, and (4) residues at the very carboxyl end of the protein may be involved in modulating the cleavage or religation activities of the Int protein.

Bacteriophage lambda↗

PubMed1994

Mutants of Escherichia coli integration host factor: DNA-binding and recombination properties.

Integration host factor (IHF) is a protein encoded by Escherichia coli, which was first discovered as a requirement for bacteriophage lambda site-specific recombination. In this study, we characterized mutants of IHF for their ability to bind to various IHF binding sites in vivo and to promote recombination of lambda in vitro. DNA-binding in vivo was monitored using the challenge-phage system. If IHF binds to its DNA-binding site that has been placed into the P(ant) region of bacteriophage P22, it acts as a repressor of the ant (antirepressor) gene, leading to the formation of lysogens of Salmonella typhimurium. If IHF cannot bind to its site, antirepressor is made leading to cell lysis. Challenge phages containing chimeras of different lambda IHF binding sites were constructed to test the contribution to the binding of a dA+dT-rich region, found in the sequence of the H' site but not in the H' site. In one case, the binding of mutant IHF proteins was enhanced by the presence of the dA+dT-rich region, indicating that IHF may be affected by neighboring bases and local DNA structure when it binds to its site. A subset of the mutant proteins retained the ability to form a looped attL complex in vivo, representing part of a higher-order protein-DNA complex (the 'intasome'). Additionally, this same subset of proteins also promoted the integration and excision of bacteriophage lambda in vitro. Thus, these mutant proteins not only retain their DNA-bending ability but make any protein-protein contacts necessary to form a recombination-proficient intasome.

Bacterial Proteins↗

PubMed1994

Determining the DNA sequence elements required for binding integration host factor to two different target sites.

Binding sites for the Escherichia coli protein integration host factor (IHF) include a set of conserved bases that can be summarized by the consensus sequence WATCAANNNNTTR (W is dA or dT, R is dA or dG, and N is any nucleotide). However, additional 5'-proximal bases, whose common feature is a high dA+dT content, are also thought to be required for binding at some sites. We examine the relative contribution of these two sequence elements to IHF binding to the H' and H1 sites in attP of bacteriophage lambda by using the bacteriophage P22-based challenge-phage system. IHF was unable to act as a repressor in the challenge-phage assay at H' sites containing the core consensus element but lacking the dA+dT-rich element. This indicates that both elements are required for IHF to bind to the H' site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site, which lacks an upstream dA+dT-rich region. Fifty mutants that decreased or eliminated IHF binding to the H1 site were isolated. Sequence analysis showed changes in the bases in the core consensus element only, further indicating that this determinant is sufficient for IHF binding to the H1 site. We found that placement of a dA+dT-rich element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT-rich element enhances IHF binding.

Bacterial Proteins↗

PubMed1993

Direct genetic selection for a specific RNA-protein interaction.

The decision between lytic and lysogenic development of temperate DNA bacteriophages is determined largely by transcriptional regulation through DNA-binding proteins. To determine whether a heterologous RNA-binding activity could control the developmental fate of a DNA bacteriophage, a derivative of P22 was constructed in which the chosen developmental pathway is regulated by an RNA-binding molecule interacting with its RNA target site located in a phage mRNA. In the example presented, lysogenic development of the phage relies upon R17 coat protein expression in the susceptible host cell and the availability of a suitable coat protein binding site encoded by the phage genome. Through the analysis of phage mutants that are able to grow lytically in susceptible cells that express the coat protein, additional insights were obtained regarding the specific interaction of the R17 coat protein with its RNA binding site. This study also suggests a novel and extremely sensitive strategy for selecting RNA-binding activities in vivo.

Bacteriophage P22↗

PubMed1993

Complementation of bacteriophage lambda integrase mutants: evidence for an intersubunit active site.

Site-specific recombination of bacteriophage lambda starts with the formation of higher-order protein--DNA complexes, called 'intasomes', and is followed by a series of steps, including the initial DNA cleavage, top-strand exchange, branch migration and bottom-strand exchange, to produce recombinant products. One of the intasomes formed during excisive recombination (the attL complex) is composed of the phage-encoded integrase (Int), integration host factor (IHF) and one of the recombination substrates, attL DNA. Int is the catalytic recombinase and has two different DNA binding domains. When IHF is present, Int binds to two types of sites in attL DNA, the three arm-type sites (P'123) and the core-type sites (B and C') where the reciprocal strand exchange takes place. The Tyr342 residue of Int serves as a nucleophile during strand cleavage and covalently attaches to the DNA through a phosphotyrosyl bond. In vitro complementation assays have been performed for strand cleavage using attL suicide substrates and mutant proteins containing amino acid substitutions at residues conserved in the integrase family of recombinases. We demonstrate that at least two Int monomers are required to form the catalytically-competent species that performs cleavage at the B site. It is likely that the active site is formed by two Int monomers.

Bacteriophage lambda↗

PubMed1993

Aortic pressure estimation with electro-mechanical circulatory assist devices.

An adaptive technique for the estimation of the time history of aortic pressure (from applied voltage and position feedback) has been designed, implemented, and bench tested using the Penn State Electric Ventricular Assist Device (EVAD). This method, known in the field of automatic control as a dynamic observer, utilizes gains which were determined using experimental data collected while the EVAD was running on a mock circulatory system. An adaptive scheme provides the observer with a method of changing its initial conditions on a stroke-by-stroke basis which improves observer performance. In both determining the feedback gains and developing the adaptation scheme, a range of beat rates and pressure loads was taken into account to yield satisfactory observer performance over a range of operating conditions. The observer was implemented, its performance was verified in vitro and results are reported. In the six experimental operating conditions, the beat rate ranged from 56-104 beats per minute (bpm) and the span of the mean systolic aortic pressure was 10.7-18.7 kPa (80-140 mmHg). For these cases, the mean deviation between the actual and estimated aortic pressure during the latter two-thirds of systole was 0.41 kPa (3.1 mmHg).

Algorithms↗

PubMed1993

The era of optimism, 1850-1870: a preliminary reappraisal.

Hervey B. Wilbur founded the Barre School, the first private residential school in the United States, in 1848. Samuel G. Howe began the Massachusetts School for Idiotic and Feeble Minded Youth, the first residential public school in the United States, in 1850. Correspondence of the period indicates educational results were less than expected and that strains of pessimism surfaced quite early. By the late 1850s, both Howe and Wilbur raised the possibility of custodial care. The primary source material suggests that the disillusionment with the residential facility as a vehicle for reform and habilitation reflected the decreased faith in moral education and remediation of problems associated with insanity, criminal behavior, and the poverty in the pre-Civil War period.

Child↗

PubMed1992

Transcription termination in vitro by bacteriophage T7 RNA polymerase. The role of sequence elements within and surrounding a rho-independent transcription terminator.

rho-Independent transcription terminators in Escherichia coli contain a dG+dC-rich dyad-symmetrical structure that encodes an RNA hairpin structure and an adjacent, downstream dA+dT-rich region which encodes uridines at the 3'-end of the transcript. In the threonine (thr) attenuator, there are at least six sequence segments in the DNA that might affect termination: the sequence upstream of the attenuator, the deoxythymidine-rich stretch immediately preceding the G+C-rich region, the G+C-rich region itself and its hairpin loop-encoding region, the deoxyadenosine tract following the G+C-rich region, and the following downstream sequence. Our previous studies (Jeng, S.-T., Gardner, J.F., and Gumport, R.I. (1990) J. Biol. Chem. 265, 3823-3830) indicate that both the stability and sequence of the RNA hairpin formed by the G+C-rich region and the length of the uridine tract encoded by the deoxyadenosine stretch influence the termination of T7 RNA polymerase in vitro. In this report, we demonstrate that the template deoxythymidine run upstream of the G+C-rich region, the loop-encoding segment, and the sequences upstream and downstream of the thr attenuator also affect termination. These results indicate that: 1) a deoxythymidine tract is not absolutely required for termination, but increasing the number of deoxythymidines from one to nine base pairs causes T7 RNA polymerase to terminate more efficiently; 2) a template with the natural loop sequence reversed results in a higher termination efficiency than one encoded by the the wild-type attenuator; 3) the termination of T7 RNA polymerase is affected by sequences both proximal and distal to the thr attenuator.

Base Sequence↗

PubMed1992

Integration host factor facilitates repression of the put operon in Salmonella typhimurium.

Transcriptional regulation of the put operon is mediated by a unique mechanism involving autogenous regulation by the PutA protein, a membrane-associated dehydrogenase. The 420-bp put control region contains the putP and putA promoters, multiple operator sites, multiple catabolite repression protein binding sites, and several potential integration host factor (IHF)-binding sites (ihf). In this study, we show that IHF facilitates repression of the put operon in vivo, and IHF binds specifically to two ihf sites in the put control region in vitro. DNA gyrase mutants that alter the degree of chromosomal supercoiling do not affect put regulation, indicating that the effect of IHF on put expression is in this case independent of supercoiling.

Amino Acid Sequence↗

PubMed1992

The isolation and characterization of mutants of the integration host factor (IHF) of Escherichia coli with altered, expanded DNA-binding specificities.

The integration host factor (IHF) of Escherichia coli is a small, basic protein that is required for lambda site-specific recombination and a variety of cellular processes. It is composed of two subunits, alpha and beta, that are encoded by the himA and hip (himD) genes, respectively. IHF is a sequence-specific DNA-binding protein and bends the DNA when it binds. We have used the bacteriophage P22-based challenge phage selection to isolate suppressor mutants with altered, expanded DNA binding specificities. The suppressors were isolated by selecting mutants that recognize variants of the phage lambda H'IHF recognition site. Two of the mutants recognize both the wild-type and a single variant site and contain amino acid substitutions at positions 64 (Pro to Leu) or 65 (Lys to Ser) of the alpha subunit. These substitutions are in a region of the protein that is predicted to contain a flexible arm that interacts with DNA. Three other mutants, which recognize the wild-type and a different variant site, contain amino acid substitutions at position 44 (Glu to Lys, Val or Gly) of the beta subunit. These substitutions are in the middle of a predicted beta-strand of the subunit. We discuss the possible mechanisms of suppression by the mutants in terms of a model of the IHF-DNA complex proposed by Yang and Nash [Cell, 57, 869-880 (1989)].

Amino Acid Sequence↗

PubMed1992

Characterization of the bacteriophage lambda excisionase (Xis) protein: the C-terminus is required for Xis-integrase cooperativity but not for DNA binding.

We have performed a mutational analysis of the xis gene of bacteriophage lambda. The Xis protein is 72 amino acids in length and required for excisive recombination. Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision. Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA. In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision. However, these three mutants were unable to bind DNA cooperatively with Int. Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites. This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.

Amino Acid Sequence↗