Promoter recognition as measured by binding of polymerase to nontemplate strand oligonucleotide.

In transcription initiation, the DNA strands must be separated to expose the template to RNA polymerase. As the closed initiation complex is converted to an open one, specific protein-DNA interactions involving bases of the nontemplate strand form and stabilize the promoter complex in the region of unwinding. Specific interaction between RNA polymerase and the promoter in Escherichia coli was detected and quantified as the binding affinity of nontemplate oligonucleotide sequences. The RNA polymerase subunit sigma factor 70 contacted the bases of the nontemplate DNA strand through its conserved region 2; a mutation that affected promoter function altered the binding affinity of the oligonucleotide to the enzyme.

Mechanism of intrinsic transcription termination and antitermination.

Gene expression is modulated by regulatory elements that influence transcription elongation by RNA polymerase: terminators that disrupt the elongation complex and release RNA, and regulators that overcome termination signals. RNA release from Escherichia coli RNA polymerase can be induced by a complementary oligonucleotide that replaces the upstream half of the RNA hairpin stem of intrinsic terminator transcripts, implying that RNA hairpins act by extracting RNA from the transcription complex. A transcription antiterminator inhibits this activity of oligonucleotides and therefore protects the elongation complex from destabilizing attacks on the emerging transcript. These effects illuminate the structure of the complex and the mechanism of transcription termination.

Determination of intrinsic transcription termination efficiency by RNA polymerase elongation rate.

Transcription terminators recognized by several RNA polymerases include a DNA segment encoding uridine-rich RNA and, for bacterial RNA polymerase, a hairpin loop located immediately upstream. Here, mutationally altered Escherichia coli RNA polymerase enzymes that have different termination efficiencies were used to show that the extent of transcription through the uridine-rich encoding segment is controlled by the substrate concentration of nucleoside triphosphate. This result implies that the rate of elongation determines the probability of transcript release. Moreover, the position of release sites suggests an important spatial relation between the RNA hairpin and the boundary of the terminator.

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.

Identification of the gene for the yeast ribonucleotide reductase small subunit and its inducibility by methyl methanesulfonate.

We have identified, cloned, and sequenced the gene for the small subunit of ribonucleotide diphosphate reductase of Saccharomyces cerevisiae. The protein and its transcript are induced about 10-fold by the alkylating agent methyl methanesulfonate, a result which suggests that the gene is induced by DNA damage.

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.

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.

Kinetics of RNA polymerase initiation and pausing at the lambda late gene promoter in vivo.

We have measured the kinetics of transcription initiation and pausing by Escherichia coli RNA polymerase (RNAP) at the bacteriophage lambda late promoter, pR's, in growing cells. RNAP initiating transcription from pR' pauses after transcribing 16 or 17 nucleotides, and escape from this pause could in theory be the rate-limiting step in promoter function. We tested this hypothesis by analyzing pausing and non-pausing variants of both the pR' promoter segment and a more active mutant version of pR'; we measured reporter gene expression and used KMnO4 footprinting to measure directly occupancy of the promoter and pause sites in growing cells. We find that RNAP paused at +16/+17 does not limit expression of pR'. However, RNAP paused at +16/+17 does limit expression from the more active promoter by impeding formation of open complex. Therefore, the activity of the late gene regulatory protein Q to suppress the early pause, in addition to its antitermination activity, is unlikely to be important in phage gene expression.

Deletion analysis of the lambda tR1 termination region. Effect of sequences near the transcript release sites, and the minimum length of rho-dependent transcripts.

In order to determine how much of the natural sequence is required for function of the lambda tR1 rho-dependent terminator, and to determine the minimum length required, we made two deletion series of a tR1 derivative that contains mostly foreign DNA, encoding C-rich RNA, substituted for the natural upstream sequences of tR1. We find the minimum transcript length to be 85 to 90 nucleotides, although an additional 10 to 25 nucleotides provide more efficient termination. Sequences as close as ten nucleotides to the release sites could be replaced without destroying termination, although termination efficiency was reduced by substitution of these proximal regions with DNA encoding the cytidine-rich RNA that is active at upstream sites. These results suggest that sequences proximal and distal to release sites have different functions and optimal structures for rho activation. We also show that two potential stem-loop structures in the tR1 region are not essential for terminator function, or for pausing at the release region. The results are consistent with the model that any pause site downstream of DNA encoding unstructured C-rich RNA is a potential rho-dependent terminator.

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.

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.

Domain organization of the Escherichia coli RNA polymerase sigma 70 subunit.

We used limited trypsin digestion to determine the domain organization of the Escherichia coli RNA polymerase sigma 70 subunit. Trypsin-resistant fragments containing sigma 70 conserved region 2 (sigma 70(2)), and carboxy-terminal fragments containing conserved regions 3 and 4 (sigma 70(3-4)) were identified by a combination of amino acid sequencing and mass spectrometry. The domains were studied for partial biochemical functions of sigma 70.sigma 70(2) bound core RNA polymerase competitively with intact sigma 70. In contrast to sigma 70(2) alone, the RNA polymerase holoenzyme formed with sigma 70(2) specifically bound a single-stranded DNA oligomer with a sequence corresponding to the non-template strand of the -10 promoter element (the Pribnow box). Sigma 70(2) also forms crystals that are suitable for X-ray analysis. Sigma 70(3-4) bound the T4 AsiA protein with high affinity. The epitope for T4 AsiA on sigma 70 was further localized to within sigma 70[551-608], comprising sigma conserved region 4.2.

Mitochondrial DNA sequences in the nuclear genome of Strongylocentrotus purpuratus.

Two sea urchin embryo complementary DNA clones representing mitochondrial 16 S ribosomal RNA and cytochrome oxidase subunit I messenger RNA have been characterized. The cloned cDNAs are colinear with sea urchin mitochondrial DNA, and their identification is based on cross-hybridization with known restriction fragments of human mitochondrial DNA, and on nucleotide sequence determinations. The mitochondrial cDNA clones also displayed an unexpected reaction with specific genomic DNA sequences in gel blot hybridizations. Genomic phage lambda recombinants containing sequences hybridizing with the mitochondrial clones were isolated and the arrangement of these sequences was determined. The genomic region studied contains a sequence homologous with the 3' end of the mitochondrial 16 S rRNA gene, flanked on one side by what is possibly a complete copy of the cytochrome oxidase subunit I gene, and on the other by a duplication of a fragment of this gene. The nucleotide sequence divergence between the mitochondrial and nuclear homologues of the cytochrome oxidase subunit I gene varies for different regions of the gene, from about 13% to 25%, while there is about 8% sequence divergence between nuclear and mitochondrial versions of the 3' 16S rRNA sequence. The structure of the genomic mitochondrial sequence homologues indicates that during sea urchin evolution there occurred a germ-line transposition of a fragment of the mitochondrial genome into the nuclear DNA, followed by rearrangements and single nucleotide substitutions.

Putting prevention into practice. Impact of a multifaceted physician education program on preventive services in the inner city.

BACKGROUND: Physicians' prevention practices often differ from guidelines published by national authorities. Effective preventive services are most needed in inner city settings that suffer disproportionately from preventable diseases. This study examined the impact of a multifaceted physician prevention education program on the provision of preventive services in an inner city municipal hospital. METHODS: The study used a controlled intervention comparative design at two inner city municipal hospitals--Harlem Hospital Center, New York, NY (intervention site) and Kings County Hospital, Brooklyn, NY (comparison site)--serving predominantly African-American patient populations. The intervention site received prototype materials for physicians, patients, and the office setting from the US Public Health Service's Put Prevention Into Practice campaign and a series of prevention lectures from November 1991 through April 1992. Change in physician prevention practices and knowledge was assessed by self-administered questionnaires and change in patients' reports of preventive services received was assessed by structured interviews. RESULTS: Physicians at Harlem Hospital Center reported a greater postintervention increase in prevention practices and demonstrated a greater increase in prevention knowledge in comparison with physicians at Kings County Hospital. Patients at Harlem Hospital Center reported receiving increased preventive services from physicians after the intervention, while patients at Kings County Hospital did not report any significant change in preventive services received. CONCLUSIONS: A multifaceted physician education program using prototype materials from the Put Prevention Into Practice campaign with prevention lectures significantly increased the prevention knowledge and practices reported by physicians and the preventive services reported received by patients at an inner city municipal hospital.

Persistence of human vascular endothelium in experimental human prostate cancer bone tumors.

Using the SCID-human model, we recently found that human circulating prostate cancer cells formed tumors in human bone but not mouse bone (Nemeth et al. Cancer Res 1999; 59: 1987-93). It is possible that this tissue preference was mediated by interaction between human tumor cells and human endothelial cells within the implanted bone tissue. We sought to determine the relative amounts of human and mouse vasculature within human bone implants and resulting prostate cancer bone tumors in the SCID-human model. Paraffin sections of plain bone implants or PC3 or LNCaP human bone tumors were double stained for factor VIII (all vessels) and human CD31 (human vessels) followed by fluorescent secondary reagents. At 4 weeks post implantation (when cancer cells are typically introduced), the vasculature within human bone fragments remained primarily human (84.5%), and this pattern persisted to at least 10 weeks (91.6% human). Injection of PC3 cells into the bone resulted in an increase in mouse-derived vessels, however the majority (58%) of the vessels remained human even after the formation of large bone tumors. LNCaP bone tumors were highly angiogenic, and there was a sharp decline in the proportion of vessels which were antigenically human (36.8%), suggesting recruitment of mouse endothelial cells during the angiogenic process. Nonetheless, the persistence of human vasculature suggests the SCID-human model can be used to study the interaction between bone-seeking tumor cells, such as prostate cancer, and human bone endothelium in vivo, and to test potential therapeutic strategies which may depend on the presence of human vessels.

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.

Cognitive deficits in patients with essential tremor.

OBJECTIVE: To assess cognitive and affective functioning in patients with essential tremor (ET). BACKGROUND: ET is traditionally thought to occur in isolation, without other neurologic abnormalities or cognitive changes. Recent evidence of gait disturbance and bradykinesia in these patients suggests that the neurologic abnormalities in ET may be more widespread than was once thought. Cognitive function in these patients has not been the subject of in-depth study. METHODS: Cognitive performance and mood were assessed in 18 consecutive patients with ET and 18 consecutive patients with PD who visited the neurosurgical clinic for surgical treatment of their symptoms. RESULTS: The patients with ET were found to have deficits on tests of verbal fluency, naming, mental set-shifting, verbal memory, and working memory, as well as higher levels of depression. In contrast to these areas of deficit, their performance was better than that of the normative sample on several tests of verbal and nonverbal conceptualization and reasoning. Tremor severity was not correlated with cognitive deficits. Patients with PD had deficits on the same tests that were impaired in the ET group and on tests of visuospatial processes. Direct comparison of the ET and PD groups showed greater impairment in facial perception in the PD group and greater impairment in verbal fluency and working memory in the ET group. CONCLUSION: Patients with ET have deficits in specific aspects of neuropsychological functioning, particularly those thought to rely on the integrity of the prefrontal cortex, which suggests involvement of frontocerebellar circuits in this disease.

End-of-dose dystonia in Parkinson's disease.

To evaluate the pathogenesis of end-of-dose dystonia in levodopa-treated patients with Parkinson's disease, we discontinued a steady-state optimal-dose levodopa infusion either abruptly or slowly. Although dystonic signs appeared sooner after sudden levodopa termination, in both situations dystonia emerged only when circulating drug levels had fallen to the same concentration and parkinsonian scores had declined by the same amount. Dystonia onset thus appears to reflect the degree, rather than the rate, of reduction in dopaminergic stimulation, and may involve the preferential interaction of dopamine with a receptor subpopulation that does not mediate its antiparkinsonian efficacy.

Catechol-O-methyltransferase inhibitor tolcapone prolongs levodopa/carbidopa action in parkinsonian patients.

The wearing-off phenomenon frequently complicates levodopa therapy of Parkinson's disease (PD). These response fluctuations appear when intrasynaptic dopamine concentrations begin to reflect the swings in levodopa availability that attend standard dosing regimens. Drugs that prolong the biologic half-life of levodopa and dopamine should thus prove beneficial. We administered levodopa/carbidopa in combination with single oral doses of tolcapone (Ro 40-7592), an inhibitor of catechol-O-methyltransferase, under controlled conditions to 10 PD patients with the wearing-off phenomenon. Tolcapone prolonged the antiparkinson response to levodopa/carbidopa by about 67% at several doses ranging from 50 to 400 mg (p < 0.05). There was no significant change in the peak levodopa effect on parkinsonian signs or in the severity of dyskinesias. No dose-limiting adverse effects occurred. Multiple daily dosing with tolcapone would thus be expected to safely reduce the wearing-off phenomenon associated with levodopa/carbidopa therapy.

A brief consideration of the SOS inducing signal.

SOS functions are induced in E. coli by treatments that damage cellular DNA or interrupt its synthesis. The biochemical basis of induction is activation of the specific proteolytic activity of recA protein, which then inactivates the lexA repressor. We discuss the development of the inducing signal in the cell.