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J W Roberts

Publications and source records attributed to J W Roberts.

At least 19 recordsLinked to original sources
PubMed1992

The phage lambda gene Q transcription antiterminator binds DNA in the late gene promoter as it modifies RNA polymerase.

The bacteriophage lambda gene Q transcription antiterminator modifies RNA polymerase during an extended pause in elongation at nt +16 and +17 of the phage late gene promoter transcript. We show here that Q binds a specific DNA site between the -10 and -35 elements of the promoter as it interacts with the enzyme. We show that the pause must reflect a specialized elongation structure that is receptive to modification by Q, because Q does not bind to RNA polymerase stopped artificially after transcribing 16 nt of mutant DNA that does not encode the natural pause. Footprinting shows that RNA polymerase in the paused complex makes distinctive interactions with DNA in the region where Q binds; binding of Q, in turn, changes the footprint both at the Q-binding site and in the transcription bubble. Binding of Q to the paused transcription complex is stabilized by the transcription factor NusA, as expected from the dependence of lambda Q-mediated antitermination on NusA.

Bacteriophage lambda↗
PubMed1992

What are the contraindications for laparoscopic cholecystectomy?

Acute cholecystitis, morbid obesity, and previous upper abdominal surgery have been reported as relative contraindications to laparoscopic cholecystectomy. An analysis of 706 laparoscopic cholecystectomies performed at our institution was undertaken to determine if these relative contraindications led to increased morbidity, an increased rate of conversion to the open technique, or longer operating time. One hundred ninety-seven patients demonstrated one or more relative contraindications to laparoscopic cholecystectomy. Morbidity was not increased in patients with these risk factors, but conversion to open cholecystectomy was required in a greater percentage of patients with acute cholecystitis. We favor an attempt at laparoscopic cholecystectomy in patients with these risk factors; however, they should be counseled as to the increased risk of conversion to open cholecystectomy in the presence of acute cholecystitis.

Abdomen↗
PubMed1992

Evidence of genetic heterogeneity among the nondystrophic myotonias.

Recent in vitro electrophysiologic studies have demonstrated abnormal sodium channel gating in muscle from patients with Thomsen's disease and have called the chloride hypothesis into question. Abnormal sodium channel function, like myotonia, is a feature common to Thomsen's disease and several myotonias that are genetically linked to a chromosome-17q sodium channel locus. We present a pedigree segregating an allele for Thomsen's disease that is unlinked to this sodium channel locus, thus constituting evidence of genetic heterogeneity among the nondystrophic myotonias.

Child↗
PubMed1991

Rho-dependent transcription termination. Characterization of the requirement for cytidine in the nascent transcript.

By substituting template segments encoding AU-rich, GU-rich, and CA-rich transcripts for natural sequences upstream of the phage lambda rho-dependent tR1 termination site, we demonstrate that cytidines are required in the upstream RNA for rho-dependent termination to occur. These results are extended through in vitro mutagenesis of a template encoding an inactive AU-rich upstream sequence: certain mutant templates encoding new cytidines are able to activate rho-dependent termination. Cytidines must be dispersed over a region of the transcript in order for rho to be activated, although no specific pattern of cytidines appears to be required. The results show that no local clustering or regular spacing of cytidines is necessary and that cytidines are not used as a "ruler" to determine the location of termination sites. Rho is somewhat sensitive to the relative positions of cytidines since slightly different nascent transcripts activate rho termination activity to various degrees. A model is presented in which hexameric rho binds 78 nucleotides of contiguous RNA in a primary site, such that each monomer interacts with at least one cytidine somewhere in the 13 nucleotides allotted to the monomeric primary site.

Bacteriophage lambda↗
PubMed1991

Open versus laparoscopic cholecystectomy. A comparison of postoperative pulmonary function.

Upper abdominal surgery is associated with characteristic changes in pulmonary function which increase the risk of lower lobe atelectasis. Sixteen patients undergoing open cholecystectomy and 20 patients undergoing laparoscopic cholecystectomy were prospectively evaluated by pulmonary function tests (forced vital capacity [FVC], forced expiratory volume [FEV-1], and forced expiratory flow [FEF] 25% to 75%) before operation and on the morning after surgery to determine if the laparoscopic technique lessens the pulmonary risk. Fraction of the baseline pulmonary function was calculated by dividing the postoperative pulmonary function by the preoperative pulmonary function and multiplying by 100%. Postoperative FVC measured 52% of preoperative function for open cholecystectomy and 73% for laparoscopic cholecystectomy (p = 0.002). Postoperative FEV-1 measured 53% of baseline function for open cholecystectomy and 72% for laparoscopic cholecystectomy (p = 0.006). Postoperative FEF 25% to 75% measured 53% for open cholecystectomy and 81% for laparoscopic cholecystectomy (p = 0.07). It is concluded that laparoscopic cholecystectomy offers improved pulmonary function compared to the open technique.

Adult↗
PubMed1991

Characterization of the late-gene regulatory region of phage 21.

A segment of Escherichia coli bacteriophage 21 DNA encoding the late-gene regulator, Q21, and the late-gene leader RNA segment was sequenced; its structure is similar to those of the related phages lambda and 82. The leader RNA is about 45 nucleotides long and consists essentially entirely of sequences encoding the p-independent terminator that is the putative target of the antitermination activity of Q21. Like the corresponding regions of lambda and 82, the 21 late-gene promoter segment encodes an early transcription pause in vitro, at about nucleotide 18, during which Q21 presumably acts to modify RNA polymerase. The 21 Q gene, cloned in isolation, is active on the late-gene leader segment in trans, and its purified product is active as an antiterminator in vitro; Q21 represents a third late-gene antiterminator, in addition to those of lambda and 82. There is little evident similarity in the primary sequences of the three Q genes.

Amino Acid Sequence↗
PubMed1991

Paramyotonia congenita and hyperkalemic periodic paralysis map to the same sodium-channel gene locus.

Paramyotonia congenita (PC), an autosomal dominant muscle disease, shares some clinical and electrophysiological similarities with another myotonic muscle disorder, hyperkalemic periodic paralysis (HYPP). However, clinical and electrophysiologic differences allow differentiation of the two disorders. The HYPP locus was recently shown to be linked to a skeletal muscle sodium-channel gene probe. We now report that PC maps to the same locus (LOD score 4.4, theta = 0 at assumed penetrance of .95). These linkage results, coupled with physiological data demonstrating abnormal sodium-channel function in patients with PC, implicate a sodium-channel gene as an important candidate for the site of mutation responsible for PC. Furthermore, this is strong evidence for the hypothesis that PC and HYPP are allelic disorders.

DNA Probes↗
PubMed1990

Heterogeneous initiation due to slippage at the bacteriophage 82 late gene promoter in vitro.

RNAs synthesized in vitro by purified Escherichia coli RNA polymerase from a bacteriophage 82 promoter are heterogeneous at the 5' end. We show that this heterogeneity results from variable addition of extra adenine residues, allowed by slippage of the initial oligonucleotide pppAAA-OH against its DNA template sequence TTT. Slippage backward by one base allows another A to be added, giving pppAAAA-OH, and this cycle can continue more than 20 times before it is ended by incorporation of UMP encoded by the fourth template base A. Slippage is abolished by mutation of the TTT template sequence to TGT and is sensitive to the concentrations of UTP and ATP in the reaction mixture. Analysis of deletions, substitutions, and point mutants implies that the slippage reaction requires only the existence of TTT at the initiation site of the template strand, although changes in neighboring nucleotides slightly affect its efficiency.

Adenosine Triphosphate↗
PubMed1990

Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication.

The SOS genes of Escherichia coli, which include many DNA repair genes, are induced by DNA damage. Although the central biochemical event in induction, activation of RecA protein through binding of single-stranded DNA and ATP to promote cleavage of the LexA repressor, is known, the cellular event that provides this activation following DNA damage has not been well understood. We provide evidence here that the major pathway of induction after damage by a typical agent, ultraviolet light, requires an active replication fork; this result supports the model that DNA replication leaves gaps where elongation stops at damage-induced lesions, and thus provides the single-stranded DNA that activates RecA protein. In order to detect quantitatively the immediate product of the inducing signal, activated RecA protein, we have designed an assay to measure the rate of disappearance of intact LexA repressor. With this assay, we have studied the early phase of the induction process. LexA cleavage is detectable within minutes after DNA damage and occurs in the absence of protein synthesis. By following the reaccumulation of LexA in the cell, we detect repair of DNA and the disappearance of the inducing signal. Using this assay, we have measured the LexA content of wild-type and various mutant cells, characterized the kinetics and conditions for development of the inducing signal after various inducing treatments and, finally, have shown the requirement for DNA replication in SOS induction by ultraviolet light.

Bacterial Proteins↗
PubMed1990

Naloxone in septic shock.

Treatment of septic shock is a persistent dilemma. The clinical use of agents such as naloxone has resulted in variable success. Because the dosage and timing of these agents are considered critical factors in their efficacy, we investigated both dosage and timing of naloxone. Thirteen consecutive patients with documented septic shock and resistance to a one-liter fluid challenge underwent invasive hemodynamic monitoring and the administration of naloxone by initial bolus of 0.03 mg/kg followed by infusion at a rate of 0.2 mg/kg.h over one hour. During the one-hour observation period, iv fluid administration, concomitant pressor agents, and respirator values were constant. After infusion, adjustments in fluid administration, respirator status, and pressor agents were made as required by the clinical situation. A significant increase in mean arterial pressure (MAP) over baseline (60 +/- 3 mm Hg) was noted at 5 min (77 +/- 6 mm Hg, p less than .005) and at 30 min (73 +/- 6 mm Hg, p less than .025). Similarly, a significant increase in systolic arterial pressure was noted over prenaloxone levels (89 +/- 3 mm Hg) at 5 min (114 +/- 6 mm Hg, p less than .001), 30 min (107 +/- 8 mm Hg, p less than .05), and at one hour (106 +/- 8 mm Hg, p less than .05). There was a moderate nonsignificant increase in cardiac index, pulmonary capillary wedge pressure, and systemic vascular resistance. No side-effects to naloxone were noted in our group. No effect on survival could be demonstrated. We found no overall effect on mortality. However, by its increase of MAP, naloxone may serve as a temporizing agent during the treatment of critically ill patients with septic shock.

Adult↗
PubMed1989

Specificity and mechanism of antitermination by Q proteins of bacteriophages lambda and 82.

Lambdoid phage late gene operons are positively regulated by genome-specific antiterminator proteins encoded by the Q gene of each phage. In this paper, we compare the activity of phage lambda and phage 82 Q proteins. Q82-mediated antitermination, like that of Q lambda, involves a transcription pause during which the regulator can modify RNA polymerase. We show that the activities of both Q82 and Q lambda are genome-specific in chasing RNA polymerase out of the early pause sites and in mediating antitermination. Finally, we show that the length of the RNA in the paused complex, or the exact position of the pause, affects the efficiency with which Q82 chases RNA polymerase out of the pause.

Bacterial Proteins↗
PubMed1989

Sequences required for antitermination by phage 82 Q protein.

The gene Q antiterminator proteins of phages lambda and 82 modify RNA polymerase at sites (named qut) that are close to, and apparently inseparable from the promoters themselves. Modification occurs while RNA polymerase has paused close to the start site, at nucleotide 16 for lambda, and nucleotides 15 and 25 for phage 82. We present a deletion analysis of the phage 82 qut site that identifies sequences required for pausing and shows that these sequences also are required for efficient Q function in vivo and in vitro. We show (1) that deletions as close as +5 to the RNA start site retain some ability to be modified by Q82, suggesting that part of the qut site is in the non-transcribed region of the promoter; (2) that NusA protein is required for activity of Q82 on certain qut82 site deletions, whereas it only modestly stimulates antitermination from the native qut82 site; and (3) that qut82 is active only on RNA polymerase that initiates at the qut-associated promoter, and not on RNA polymerase that initiates upstream and passes through an otherwise active qut82 site.

Bacterial Proteins↗
PubMed1989

Early transcribed sequences affect termination efficiency of Escherichia coli RNA polymerase.

We have constructed novel transcription templates in which we have fused the late gene promoters of Escherichia coli phages lambda and 82 upstream from three different rho-independent transcription terminators. Using an in vitro transcription assay and an in vivo galactokinase expression assay, we find that the initial portion of the transcribed region significantly affects the efficiency of some downstream terminators. We have identified, by deletion, substitution and point mutation analysis, sequences responsible for these increased levels of factor-independent readthrough. Since these important sequences occur within about 30 nucleotides of the RNA start site, we suggest that the initial portion of the transcript can affect termination efficiency.

Bacteriophages↗
PubMed1989

Gene Q antiterminator proteins of Escherichia coli phages 82 and lambda suppress pausing by RNA polymerase at a rho-dependent terminator and at other sites.

The Q genes of phages lambda and 82 encode transcription antiterminators that are active in vitro in a purified transcription system. Transcription termination is thought to involve two distinct steps: pausing of the transcription complex at the terminator and release of enzyme and RNA; either or both steps might be inhibited by Q protein. We show that Q-modified RNA polymerase pauses much less efficiently than does unmodified enzyme at the natural pause sites of a rho-dependent terminator as well as at other pause sites. This changed behavior can account for the termination properties of Q-modified RNA polymerase and reflects a fundamental alteration of the elongation properties of the enzyme.

Bacteriophage lambda↗
PubMed1989

Upstream regulatory sequences of the yeast RNR2 gene include a repression sequence and an activation site that binds the RAP1 protein.

The small subunit of ribonucleotide reductase in Saccharomyces cerevisiae (RNR2) was induced 3- to 20-fold by a variety of DNA-damaging agents. Induction of the RNR2 transcript by at least one of these agents, methyl methanesulfonate, did not require protein synthesis. To identify sequences involved in the regulation of RNR2, we introduced deletions upstream of the transcription start site. Sequences required for induction were contained within a 200-base-pair region that could confer methyl methanesulfonate inducibility on the heterologous CYC1 promoter. This region contained a repression sequence and at least two positive activation sites. One of these activation sites bound RAP1, a protein known to associate with mating-type silencers and the upstream activation sequences of a number of genes. The behavior of deletions of the repression sequence suggests that induction of RNR2 may occur, at least in part, through relief of repression.

Base Sequence↗