Mapping and characterization of transcriptional pause sites in the early genetic region of bacteriophage T7.

During transcription of DNA templates in vitro, Escherichia coli RNA polymerase pauses at certain sequences before resuming elongation. Previous studies have established that some pausing events are brought about by the formation of RNA hairpin structures in the nascent transcript; however, it is not known whether this is an invariant and causal relationship. We have mapped and characterized almost 200 distinct pause sites located within the early region of bacteriophage T7 DNA using a collection of T7 deletion mutant DNAs and taking advantage of a procedure that permits synchronous transcription from the T7 A1 promoter. The pausing pattern is sensitive both to the overall concentration of nucleotide substrates and to the relative concentrations of the four nucleotides. The apparent Ks value for a particular nucleoside triphosphate can vary over a 500-fold range depending on the nucleotide sequence, and pausing at some sites can be induced by modest reductions in substrate concentrations. However, pausing is not solely a consequence of substrate limitation. Pausing at certain sites is caused by some feature of the template or of the transcript itself. Substitution of inosine triphosphate (ITP) for GTP during transcription strongly affects the pattern and strength of pausing events, suggesting that base-pairing interactions involving the RNA strand are important for some pausing events. Other pauses are determined by sequences downstream from the elongation site that have not yet been transcribed, and pausing at these sites is generally insensitive to substitution of IMP for GMP in the nascent transcript. Pausing at one particular site on T7 DNA is strongly enhanced by the presence of E. coli gene nusA protein. These results confirm that there are multiple classes of sites that lead to transcriptional pausing, and provide a collection of sites for further study. Using selected pause sites in the early region of T7 DNA, we have tried to evaluate the possible roles of primary sequence, base composition and secondary structure in pausing. Computer analysis was used to compare primary sequences and potential RNA hairpin structures in transcripts for pauses known to share similar biochemical properties. We see no correlation of pause sites with regions of particular base composition or with specific primary sequences. While some pauses are correlated with the potential to form stable RNA hairpins just upstream from the growing point of the RNA chain, there is not a strict one-to-one relationship between predicted RNA hairpins and the location of pause sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Homeodomain proteins: what governs their ability to recognize specific DNA sequences?

Deformed (Dfd) and Ultrabithorax (Ubx) are homeodomain proteins from Drosophila melanogaster that exert regulatory effects on gene expression by binding to specific target sites in the fly genome using a helix-turn-helix (HTH) motif. The recognition helices of these two proteins are almost identical and the DNA sequences they recognize are similar, containing a conserved TAAT core sequence flanked by a somewhat variable sequence. Yet the in vivo functions of the two proteins are quite different. We have used the homeodomains of these two proteins and in vitro selected DNA binding sites to characterize the structural details of homeodomain binding to DNA and to understand the basis for the differences in sequence specificity between homeodomains with similar recognition helices. We have employed hydroxyl radical cleavage of DNA to study the positioning of the proteins on the binding sites and have analyzed the effects of missing nucleosides and purine methylation on homeodomain binding. Our results indicate that the positioning of the Ubx and Dfd homeodomains on their binding sites is consistent with reported structures of other homeodomain/DNA complexes. Dfd and Ubx bind to DNA with the recognition helix in the major groove 3' to the TAAT core sequence and the N-terminal arm in the adjacent minor groove. However, we observe striking differences between the two homeodomains in their specific interactions with DNA. Missing nucleosides within the selected binding sites have differential effects on protein binding, which are dependent on the identity of the homeodomain. Differences at the 3' end of the binding site on the top strand indicate that the N-terminal arm of a homeodomain is capable of distinguishing an A.T base-pair from T.A in the minor groove. Specific orientation of the N-terminal arm within the binding site appears to vary between the homeodomains and influences the interaction of the recognition helix with the major groove.

Isolation and properties of transcribing ternary complexes of Escherichia coli RNA polymerase positioned at a single template base.

We have studied the conditions needed for the formation of stable ternary complexes by Escherichia coli RNA polymerase using a procedure in which elongation by the majority of active enzyme molecules is halted at a specific template base. Stable complexes of this sort, containing RNA chains as short as 15 nucleotides, have been formed from three different promoter sites (T7 A1, lambda PL, and E. coli rrnB P1) using di- and trinucleotides as primers in reactions limited by the presence of only three of the nucleoside triphosphate substrates. The resulting ternary complexes can be stored for at least five days without loss in activity, and provide useful reagents and substrates for studies of the properties of RNA polymerases engaged in chain elongation and termination. At all three promoter sites abortive initiation, leading to synthesis and release of oligomers up to ten nucleotides, competes with productive initiation, leading to the formation of stable elongating complexes. Thus the relative instability of ternary RNA polymerase complexes bearing transcripts shorter than ten nucleotides may be a general feature of the transcription initiation reaction.

Acceptability of mobile mammography among community-dwelling older women.

OBJECTIVE: To test the acceptability of mobile mammography among community-dwelling older women and to identify factors predictive of mobile mammography acceptance. DESIGN: Case series. SETTING: Twelve community meal sites sponsored by the City of Los Angeles Area on Aging. PARTICIPANTS: Two hundred fifty-five volunteers aged 60 to 84 years who attended community meal sites. INTERVENTION: On-site mammography offered to women who had not had a mammogram within the last year. MEASUREMENTS: Mammography acceptance rates, reasons for accepting or declining the mammogram, and breast cancer knowledge, beliefs, and intentions. MAIN RESULTS: One hundred seven of the 255 (42%) women were ineligible because they had received mammograms within the last year. Of the 148 women eligible, 57% accepted the mammograms and 43% declined; moreover, 20 of the 42 (48%) women who had not had a mammogram within the last 5 years or who never had a mammogram also accepted on-site mammography in the mobile van. Variables identified as predictive of mammogram acceptance included Asian American status, not being an HMO member, being married, a reported willingness to accept a screening mammogram if recommended by a physician, and previous mammogram screening history. CONCLUSION: Mobile mammography is acceptable to many older community-dwelling women. Although mobile mammography does not eliminate all barriers that inhibit a woman from receiving a mammogram, it may substantially increase screening for some groups.

Life-sustaining treatment decisions for nursing home residents: who discusses, who decides and what is decided?

OBJECTIVE: To evaluate whether nursing home residents and their families reported discussions about life-sustaining treatment with their physicians, the relationship between such discussions and orders to limit therapy, and predictors of physician-patient communication about life-sustaining treatment. DESIGN: Cross-sectional interviews and retrospective chart abstraction. SETTING: Three regions: West Coast, New England, Western. SAMPLE: A total of 413 nursing home residents, 363 family/surrogate interviews, and 192 resident interviews. MAIN OUTCOME MEASURES: Measured were (1) physician-resident communication about life-sustaining treatment and (2) presence of an advance directive or do not resuscitate (DNR) order in resident's chart. RESULTS: Seventy-four percent of residents had DNR orders, and 32% had advance directives; only 29% of residents reported discussions about life-sustaining treatment. Of residents with DNR orders who could have participated in discussions about life-sustaining treatment, nearly half reported they had not discussed CPR with their caregivers. Older age, longer duration of time living in nursing home, location in a New England nursing home, physician-family member discussion, and the presence of an advance directive in the medical chart were positively associated with having DNR orders. Physician-resident discussion was not associated with having a DNR order. For the subsample of interviewed residents, age and a diagnosis of cognitive impairment were negatively associated with a physician-resident discussion about life-sustaining treatment, whereas the likelihood of having a discussion increased with increasing numbers of medical diagnoses. CONCLUSIONS: Chart orders to limit therapy are common, but physician-resident discussions about life-sustaining treatments are not. Far more family members than residents report such discussions with the resident's physicians.

Overcoming feelings of powerlessness in "aging" researchers: a primer on statistical power in analysis of variance designs.

A general rationale and specific procedures for examining the statistical power characteristics of psychology-of-aging empirical studies are provided. First, 4 basic ingredients of statistical hypothesis testing are reviewed. Then, 2 measures of effect size are introduced (standardized mean differences and the proportion of variation accounted for by the effect of interest), and methods are given for estimating these measures from already-completed studies. Power and sample size formulas, examples, and discussion are provided for common comparison-of-means designs, including independent samples I-factor and factorial analysis of variance (ANOVA) design, analysis of covariance designs, repeated measures (correlated samples) ANOVA designs, and split-plot (combined between- and within-subjects) ANOVA designs. Because of past conceptual differences, special attention is given to the power associated with statistical interactions, and cautions about applying the various procedures are indicated. Illustrative power estimations also are applied to a published study from the literature. It is argued that psychology-of-aging researchers will be both better informed consumers of what they read and more "empowered" with respect to what they research by understanding the important roles played by power and sample size in statistical hypothesis testing.

Childhood lumbosacral plexus neuropathy.

Primary lumbosacral plexus neuropathy without underlying disease is a well-defined syndrome characterized by pain, weakness, and atrophy in the distribution of the lumbosacral plexus. It generally affects adults and rarely occurs in children. We report 2 children with primary lumbosacral plexus neuropathy evaluated at our institution and review the literature. This syndrome, despite initially severe symptoms, appears to be benign with a favorable prognosis in children.

Patterns of health promotion programs for older adults in local health departments.

Questionnaires were sent to all 2561 members of the National Association of County Health Officers in 1996; only 492 (19.2%) responded. Almost all departments have health promotion or disease prevention programs available to older adults, but only three-fifths specifically target older adults with these programs. Most of those programs are clinical preventive services, including immunizations.

RegRand: statistical software for the multiple-baseline design.

RegRand (Version 1.0) is Macintosh-based software that enables a researcher to conduct a non-parametric statistical analysis of the data from Koehler and Levin's (1998) recently described regulated randomization single-case multiple-baseline design. Regulated randomization design and analysis principles are reviewed in relation to an educational research application and a step-by-step illustration of them in relation to the RegRand program is presented.