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J W Roberts

Publications and source records attributed to J W Roberts.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1988

Activation of protease-constitutive recA proteins of Escherichia coli by all of the common nucleoside triphosphates.

To understand why the RecA proteins of the protease-constitutive recA1202 and recA1211 mutants show very high protease activities in vivo without the usual need for DNA damage (E. S. Tessman and P. Peterson, J. Bacteriol. 163:677-687, 1985), we examined the activation of the mutant proteins by nucleoside triphosphates (NTPs) in vitro. In vivo, the mutant protease activities are resistant to inhibition by cytidine plus guanosine (C + G) in the growth medium, in contrast to the activities of weaker mutants, such as recA441, which are sensitive to C + G inhibition. We found that RecA1202 and RecA1211 proteins, in contrast to RecA+, can use natural NTPs other than ATP and dATP as cofactors in the cleavage of LexA repressor. The effectiveness of NTPs in promoting LexA cleavage by RecA1202 and RecA1211 proteins decreased in roughly the following order: dATP greater than ATP greater than UTP greater than ATP-gamma S greater than dCTP greater than CTP greater than dGTP greater than GTP greater than TTP. These mutant proteins showed higher affinities for ATP and single-stranded DNA and higher repressor cleavage activities than RecA+ protein. With the various effectors (single-stranded DNA or NTPs), the RecA1202 protein always showed more activity than RecA1211 in the cleavage of LexA repressor in vitro, which is consistent with the greater activity of the recA1202 mutant in vivo. The results explain, in part, why some recA mutants have unusually high constitutive RecA protease activity and why that activity is more or less resistant to C + G inhibition.

Adenosine Triphosphate↗
PubMed1988

SOS-like induction in Bacillus subtilis: induction of the RecA protein analog and a damage-inducible operon by DNA damage in Rec+ and DNA repair-deficient strains.

We quantitated the induction of the Bacillus subtilis Rec protein (the analog of Escherichia coli RecA protein) and the B. subtilis din-22 operon (representative of a set of DNA damage-inducible operons in B. subtilis) following DNA damage in Rec+ and DNA repair-deficient strains. After exposure to mitomycin C or UV irradiation, each of four distinct rec (recA1, recB2, recE4, and recM13) mutations reduced to the same extent the rates of both Rec protein induction (determined by densitometric scanning of immunoblot transfers) and din-22 operon induction (determined by assaying beta-galactosidase activity in din-22::Tn917-lacZ fusion strains). The induction deficiencies in recA1 and recE4 strains were partially complemented by the E. coli RecA protein, which was expressed on a plasmid in B. subtilis; the E. coli RecA protein had no effect on either induction event in Rec+, recB2, or recM13 strains. These results suggest that (i) the expression of both the B. subtilis Rec protein and the din-22 operon share a common regulatory component, (ii) the recA1 and recE4 mutations affect the regulation and/or activity of the B. subtilis Rec protein, and (iii) an SOS regulatory system like the E. coli system is highly conserved in B. subtilis. We also showed that the basal level of B. subtilis Rec protein is about 4,500 molecules per cell and that maximum induction by DNA damage causes an approximately fivefold increase in the rate of Rec protein accumulation.

Bacillus subtilis↗
PubMed1988

Gallium-67 uptake by a benign adrenocortical adenoma.

A 55-yr-old man presented with an atypical relapsing meningitis and was found to have intense unilateral adrenal uptake by 67Ga imaging. Computed tomography showed a 4-cm right adrenal mass which was hypointense on the T1-weighted images and mildly hyperintense on the T2-weighted images of a magnetic resonance (MR) scan. At surgery, a coincidental benign adrenocortical adenoma was found. Because 67Ga uptake is usually associated with inflammatory or malignant lesions and malignant adrenal lesions are hyperintense on T2-weighted MR images, these findings contributed to diagnostic uncertainty in this patient. Thus, a nonhyperfunctional adrenocortical adenoma may be associated with abnormal 67Ga uptake and atypical MR findings.

Adenoma↗
PubMed1987

Bacteriophage 82 gene Q and Q protein. Sequence, overproduction, and activity as a transcription antiterminator in vitro.

Phage 82 gene Q encodes a phage-specific positive regulator of late gene expression, thought, by analogy to the corresponding gene of phage lambda, to be a transcription antiterminator. We have cloned and sequenced the phage 82 gene Q and have overproduced and purified the 82 Q protein. We also have identified and sequenced DNA containing the phage 82 late gene promoter and terminator. We show that purified 82 Q protein is active and specific for DNA containing the 82 late gene promoter in a well defined in vitro transcription reaction: RNA polymerase initiating at the phage 82 late gene promoter and modified by 82 Q protein reads through a downstream transcriptional terminator. We used T1 RNase mapping to confirm that the putative readthrough RNA made in the presence of 82 Q protein is in fact an elongation product of the shorter RNA.

Amino Acid Sequence↗
PubMed1987

Management of nonpalpable breast abnormalities.

From January 1982 to June 1986, 444 patients had localization of 500 nonpalpable mammographically suspicious lesions using the Kopans hook wire technique. Four hundred ninety-nine biopsies were performed in 443 patients. Cancer was identified in 12% of the biopsies performed for a suspicious mass or density and in 20% of biopsies performed for suspicious calcifications. Carcinoma was identified in a total of 72 biopsies (14%) performed in 65 patients; 82% of the malignant lesions were invasive. All lesions were small; 76% of the cancers were 1.0 cm or less in diameter. Sixty-two axillary dissections were performed of which seven (11%) had positive nodes. Advantages of preoperative needle localization include precise localization of the lesion, a small incision, and removal of a small amount of breast tissue with no cosmetic deformity. Outpatient biopsy of these lesions can be easily performed under local anesthesia. Identification and treatment of these small preclinical cancers should lead to improved survival from breast cancer.

Adult↗
PubMed1987

Transcription antitermination by phage lambda gene Q protein requires a DNA segment spanning the RNA start site.

The gene Q protein of phage lambda is a transcription antiterminator that modifies RNA polymerase near the phage late gene promoter and thereby causes antitermination at distant sites. To define the site of action of Q protein, we have reconstructed the regulatory system on plasmids that allow the intracellular concentration of Q protein to be regulated, and that allow the effect of Q protein on transcription from variant promoter segments to be measured in vivo and in vitro. We show that DNA sequences essential for Q protein-mediated antitermination span the RNA start site, but do not extend beyond nucleotide 18 of the late RNA coding region. We also show that the modification that permits antitermination persists while RNA polymerase passes at least two terminators in vivo and in vitro.

Bacteriophage lambda↗
PubMed1986

Primary carcinoma of the gallbladder.

Carcinoma of the gallbladder is a rapidly progressive disease with a dismal prognosis. This very poor prognosis justifies an aggressive surgical approach for all potentially resectable lesions in stage II to stage V disease. Chemotherapy appears to be of some benefit, and its use for treatment of patients with early lesions is justified by the currently poor survival statistics. Radiotherapy at this point seems primarily to provide palliative relief.

Adult↗
PubMed1986

Carcinoma of the extrahepatic bile ducts.

Carcinoma of the extrahepatic bile ducts is relatively uncommon. Although these tumors usually grow slowly, the patient's prognosis is poor. This article reviews the records of patients with a diagnosis of cancer of the extrahepatic biliary tract treated surgically at Scott and White Memorial Hospital from 1960 to 1984. The pertinent history, physical findings, laboratory data, type of operation, adjuvant therapy, and survival were collected and recorded.

Adult↗
PubMed1986

Insertion and/or deletion of many repeated DNA sequences in human and higher ape evolution.

The total numbers of copies of two repeat families, L1 (Kpn I) and Alu, have been measured in the DNA of four higher apes by an accurate titration method. The number of members of the Alu family repeats in the four genomes are as follows: human, 910,000; chimpanzee, 330,000; gorilla, 410,000; orangutan, 580,000. For the Kpn I family (3'-ward higher frequency region) the number of copies in these genomes are as follows: human, 107,000; chimpanzee, 51,000; gorilla, 64,000; orangutan, 84,000. Thermal stability measurements show that, although the families of repeats are moderately divergent in sequence, little net sequence change has occurred during the evolution of the higher apes. Most or all of the members of these families of repeats are interspersed throughout the genome. Therefore, a large number of events of insertion and/or deletion of these DNA sequences has occurred during higher primate evolution.

Animals↗
PubMed1985

Purification of a RecA protein analogue from Bacillus subtilis.

We have identified in Bacillus subtilis an analogue of the Escherichia coli RecA protein. Its activities suggest that it has a corresponding role in general genetic recombination and in regulation of SOS (DNA repair) functions. The B. subtilis protein (B. subtilis Rec) has a Mr of 42,000 and cross-reacts with antisera raised against E. coli RecA protein. Its level is significantly reduced in the recombination-deficient recE4 mutant. B. subtilis Rec is induced 10- to 20-fold in rec+ strains following treatment with mitomycin C, whereas it is not induced in the recombination-deficient mutants recE4, recE45, and recA1. We have purified B. subtilis Rec about 2000-fold to near homogeneity and we describe its activities. It catalyzes DNA-dependent hydrolysis of dATP at a rate comparable to that of E. coli RecA protein. However, B. subtilis Rec has a negligible ATPase activity, although ATP effectively inhibits dATP hydrolysis. In the presence of dATP, B. subtilis Rec catalyzes DNA strand transfer, assayed by the conversion of phi X174 linear duplex DNA and homologous circular single-stranded DNA to replicative form II (circular double-stranded DNA with a discontinuity in one strand). ATP does not support strand transfer by this protein. B. subtilis Rec catalyzes proteolytic cleavage of E. coli LexA repressor in a reaction that requires single-stranded DNA and nucleoside triphosphate. This result suggests that an SOS regulatory system like the E. coli system is present in B. subtilis. The B. subtilis enzyme does not promote any detectable cleavage of the E. coli bacteriophage lambda repressor.

Adenosine Triphosphatases↗