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In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage lambda transposon delivery system.

TFIID comprises the TATA box-binding protein and a set of highly conserved associated factors (TAF(II)s). yTAF(II)145, the core subunit of the yeast TAF(II) complex, is dispensable for transcription of most yeast genes but specifically required for progression through G1/S. Here we show that transcription of G1 and certain B-type cyclin genes is dependent upon yTAF(II)145. At high cell density or following nutrient deprivation, yeast cells cease division, enter a G0-like state, and terminate transcription of most genes. In this stationary phase, we find that the levels of yTAF(II)145, several other yTAF(II)s, and TBP are drastically reduced. Collectively, our results indicate that yTAF(II)145 and other TFIID components have a specialized role in transcriptional regulation of cell cycle progression and growth control.

The general transcription factor TFIID is composed of the TATA-box-binding protein (TBP) and a set of TBP-associated factors (TAFIIs). In vitro, TAFIIs are required for activated transcription, and have been proposed to be obligatory targets of transcriptional activator proteins (activators)2. The function of TAFIIs has not been investigated systematically in vivo. A Saccharomyces cerevisiae TAFII complex (yTAFII complex) has been identified that shares functional and structural similarities with higher eukaryotic TFIID. In particular, most yTAFIIs are the homologue of a higher eukaryotic TAFII. Here we report that inactivation or depletion of six different yTAFIIs, including the core yTAFII, that contacts TBP, does not compromise transcriptional activation. We conclude that in vivo, activated transcription of many genes can occur in the absence of functional yTAFIIS, and that in these instances another transcription component(s) must be the target of the activator.

Background: Transcription by RNA polymerase II in eukaryotic cells requires the ordered assembly of general transcription factors on the promoter to form a preinitiation complex. Transcriptional activator proteins (activators) stimulate transcription by increasing the rate and/or extent of preinitiation complex assembly. We have shown previously that acidic activators increase the stable association of TFIIB on the promoter, a process we refer to as 'recruitment'. In this study, we provide evidence that diverse activators facilitate TFIIB assembly by a related mechanism. We then investigate the activator-mediated assembly of TFIIB with regard to two aspects of transcription: the distance-dependence of activator function, and reinitiation., Results: We have previously described amino-acid-substitution mutants of TFIIB that are able to support an activator-independent basal level of transcription but do not respond to acidic activators. We now show that these mutants also do not respond to other classes of activators. We demonstrate that this defect is due to a failure of the activators to recruit the mutant TFIIB to the promoter. Activators often lose activity as their distance from the initiation site is increased. We show that this impaired transcriptional activity correlates with a decrease in TFIIB recruitment. Finally, we find that following the initiation of transcription, TFIIB dissociates from the promoter, requiring the activator-mediated reassembly of TFIIB in the preinitiation complex for each new round of transcription., Conclusion: We have provided evidence that diverse activators recruit TFIIB to the promoter by a related mechanism. This central step in transcriptional activation is sensitive to promoter architecture, and is required for each new round of transcription.

A total of 295 children (127 White boys, 15 Black boys, 133 White girls, and 20 Black girls) participated in reactivity examinations in 1987 (all were in 3rd grade; age, M = 9.1 years), 1988, 1989, 1991, 1992 and 1993 (all were in 9th grade; age, M = 15.1 years). An analysis of residualized reactivity change values indicated consistent and significant ethnicity effects (Blacks greater than Whites) for systolic and diastolic blood pressure and for heart rate. Gender effects were also apparent for both systolic and diastolic blood pressure (boys greater than girls). These data suggest that the transition from childhood to adolescence is associated with a significant pattern of ethnic differences in reactivity, although the association of this pattern with the development of cardiovascular risk and disease remains to be ascertained.

In higher eukaryotes the RNA polymerase II transcription factor TFIID is composed of a TATA-box-binding protein (TBP) and a set of tightly bound polypeptides, designated TBP-associated factors (TAFIIS). One or more TAFIIS are coactivators that are required for activated but not basal transcription. The eukaryotic transcription machinery is highly conserved and it is therefore puzzling that TAFIIS have not been identified in yeast. Here we use TBP as a protein-affinity ligand to isolate from yeast a multisubunit complex that is required specifically for activated transcription by RNA polymerase II. Microsequence analysis and cloning of two subunits of this complex reveal that they are the homologues of known mammalian and Drosophila TAFIIS. The genes encoding these two yeast TAFIIS are essential, suggesting that activated transcription is required for viability of Saccharomyces cerevisiae.

Investigations suggesting that the order of obtaining resting and cardiovascular reactivity measurements moderates values have provided inconsistent results and have not analyzed data from children; the generalizability of results is uncertain. In this investigation, all children enrolled in the eighth-grade classrooms of the public schools of an entire county (n = 451) participated in standardized reactivity assessments. The order of resting and reactivity measurements was randomized by examination day (a total of 19 days). Analyses indicated that all comparisons of order effects on mean resting blood pressure and heart rate, as well as reactivity (both change from resting and absolute values and both mean and maximal values), were nonsignificant. Results indicate that measurement order is not always a necessary consideration in studies of reactivity; the conditions under which measurement order is a consideration requires clarification.

We have determined that ARS121 is an efficient origin of replication on chromosome X of Saccharomyces cerevisiae. This origin is comprised of at least three distinct functional domains. One of these domains is the ARS121 core sequence (approximately 35 bp-long), which is essential for origin activity. This essential core contains an 11 bp sequence resembling (2 bp mismatch) the ARS consensus. Another important domain is an enhancer of DNA replication, which binds the OBF1 protein. The third domain, ATR (A/T-rich, approximately 72 bp), is auxiliary and works in either orientation, but only when located 3' to the essential core. When fused to the ARS121 core both the enhancer and the ATR domain act synergistically to enhance the activity of the origin. Furthermore, when fused to the essential core sequences of heterologous ARSs, ARS1 and ARS307, the auxiliary domains also appeared to stimulate synergistically origin function. These results suggest that (i) in order to elicit maximal origin activity all three domains have to interact and (ii) activation of the essential core sequences at different origins of replication may share a common mechanism.

Blood pressure and heart rate were measured at rest and during a television video game in 399 healthy children. At the first assessment, children were in third grade and at the 2-year follow-up, they were in fifth grade. Children were classified according to race (black/white), gender (boy/girl), body mass index (light/heavy), and video game performance (low score/high score). Correlational analysis indicated that irrespective of the classification variable, absolute reactivity values, e.g., a heart rate of 110 bpm, tended to be more stable than change reactivity values, e.g., a heart rate change of 10 bpm. The greater stability of absolute values was most pronounced with systolic blood pressure. Absolute values were at least as stable as resting values and frequently demonstrated greater stability. Mean and maximal reactivities were correlated comparably. We conclude that children's reactivity is a stable characteristic and that, unless indicated by a priori hypotheses, the use of absolute values is preferable to the use of change values.

After measuring blood pressure and heart rate at rest and during a video game procedure in 477 children enrolled in 3rd grade, 434 (91%) children had these measurements repeated a year later in 4th grade. Black children demonstrated greater blood pressure and heart rate reactivity than White children in both years, and an increase in heart rate reactivity from 3rd to 4th grade. Gender effects were inconsistent. Systolic and diastolic blood pressures during the video game were more highly correlated from year-to-year than were the resting measures. Regression analysis indicated that systolic reactivity was significantly related to subsequent systolic pressure at rest, particularly among Black girls. Diastolic reactivity was associated with subsequent resting diastolic pressure only among White children. Associations between reactivity and future blood pressure were independent of initial resting blood pressure. This study suggests that cardiovascular reactivity to psychological stress may be one important factor in future level of blood pressure and that the increased heart rate reactivity of Black children may be associated with the prevalence of hypertension among Black adults.

In this study, 395 healthy children had blood pressure and heart rate measured both at rest and during a stressful video game. Identical measurement procedures were used for three annual examinations. Consistently, black children demonstrated significantly greater pressor responses than white children. In view of the greater prevalence of hypertension among black adults than white adults, these data suggest that blacks exhibit hemodynamic irregularities well before the onset or diagnosis of hypertension.

Ninety-two healthy children provided self-reports of their physical activity. Self-reports were based upon posters depicting children performing typical childhood activities and were validated by measured oxygen uptake during maximal cycle ergometry. Results indicated that when children were classified as sedentary or active by their poster selection, active children had significantly greater oxygen uptake than sedentary children. These results, while preliminary, are the first to indicate that children's self-reports can provide a valid index of maximal oxygen uptake, the 'gold standard' of aerobic fitness.

Our data indicated that self-reports of consumption of salty foods by children and adolescents were associated with 24-hour urinary sodium excretion. Specifically, youths 10 to 18 years of age who selected a poster depicting high-sodium foods excreted significantly more sodium than youths who selected a poster depicting low-sodium foods. Future research is needed to refine simplified self-report measures, to corroborate the validity of the measures, and to extend the studies to other samples, e.g., younger children.

In order to determine the effects of upright posture on the electrophysiologic properties of the human heart, 12 patients underwent electrophysiologic studies in the supine and upright positions. Compared to supine, the upright position significantly reduced basic cycle length from 818 +/- 111 to 680 +/- 141 msec, sinoatrial conduction time from 186 +/- 94 to 135 +/- 56 msec, corrected sinoatrial recovery time from 206 +/- 104 to 108 +/- 55 msec, interatrial conduction time from 76 +/- 17 to 70 +/- 16 msec, and the AV nodal conduction time (AH interval) from 88 +/- 19 to 78 +/- 14 msec (all p less than 0.05). Right intra-atrial and His-Purkinje (HV interval) conduction times were not altered. When the subjects assumed the upright position, the effective refractory periods of the right atrium, atrioventricular node, and right ventricle decreased significantly at basic and paced cycle lengths. For the group as a whole, the upright posture did not significantly augment repetitive responses to atrial and ventricular extrastimuli.