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Background— Endothelial dysfunction predisposes to vascular injury in association with hypertension. Endothelin (ET-1) is a potent vasoactive peptide that is synthesized and released by the vascular endothelium and is a marker of endothelial function. Chromogranin A (CHGA) regulates the storage and release of catecholamines and may have direct actions on the microvasculature. CHGA , a candidate gene for intermediate phenotypes that contribute to hypertension, shows a pattern of single nucleotide polymorphism variations that alter the expression and function of this gene both in vivo and in vitro. Methods and Results— In a study of twins (n=238 pairs), plasma ET-1 was 58±5% ( P G ]; for the CHGA precursor, ρ G =0.318±0.105; P =0.0032). We therefore hypothesized that variation in the CHGA gene may influence ET-1 secretion. Carriers of the CHGA promoter −988G, −462A, and −89A minor alleles showed significantly higher mean plasma ET-1 than their major allele homozygote counterparts ( P =0.02, P =0.006, P =0.03, respectively). Analysis of a linkage disequilibrium block that spans these 3 single nucleotide polymorphisms showed a significant association between the GATACA haplotype and plasma ET-1 ( P =0.0075). In cultured human umbilical vein endothelial cells, CHGA caused dose-dependent secretion of ET-1 over a brief ( Conclusions— These results suggest that common, heritable variation in expression of the human CHGA gene influences endothelial ET-1 secretion in vivo, explained by a CHGA stimulus/ET-1 secretion coupling in endothelial cells in vitro. The findings document a previously unsuspected interaction between the sympathochromaffin system and the endothelium and suggest novel genetic and cell biological approaches to the prediction, diagnosis, and mechanism of endothelial dysfunction in human disease.

BACKGROUND C-reactive protein (CRP) both reflects and participates in inflammation, and its circulating concentration marks cardiovascular risk. Here we sought to understand the role of heredity in determining CRP secretion. METHODS CRP, as well as multiple facets of the metabolic syndrome, were measured in a series of 229 twins, both monozygotic (MZ) and dizygotic (DZ), to estimate trait heritability (h2). Single nucleotide polymorphism (SNP) genotyping was done at adrenergic pathway loci. Haplotypes were inferred from genotypes by likelihood methods. Association of CRP with hypertension and the metabolic syndrome was studied in a larger series of 732 individuals, including 79 with hypertension. RESULTS MZ and DZ twin variance components indicated substantial h2 for CRP, at approximately 56 +/- 7% (P < 0.001). CRP was significantly associated (P < 0.05) with multiple features of the metabolic syndrome in twins, including body mass index (BMI), blood pressure (BP), leptin and lipids. In established hypertension, elevated CRP was associated with increased BP, BMI, insulin, HOMA (index of insulin resistance), leptin, triglycerides and norepinephrine. Twin correlations indicated pleiotropy (shared genetic determination) for CRP with BMI (P = 0.0002), leptin (P < 0.001), triglycerides (P = 0.002) and systolic blood pressure (SBP) (P = 0.042). Approximately 9800 genotypes (43 genetic variants at 17 loci) were scored within catecholaminergic pathways: biosynthetic, receptor and signal transduction. Plasma CRP concentration in twins was predicted by polymorphisms at three loci in physiological series within the catecholamine biosynthetic/beta-adrenergic pathway: TH (tyrosine hydroxylase), ADRB1 (beta1-adrenergic receptor) and ADRB2 (beta2-adrenergic receptor). In the TH promoter, common allelic variation accounted for up to approximately 6.6% of CRP inter-individual variance. At ADRB1, variation at Gly389Arg predicted approximately 2.8% of CRP, while ADRB2 promoter variants T-47C and T-20C also contributed. Particular haplotypes and diplotypes at TH and ADRB1 also predicted CRP, though typically no better than single SNPs alone. Epistasis (gene-by-gene interaction) was demonstrated for particular combinations of TH and ADRB2 alleles, consistent with their actions in a pathway in series. In an illustration of pleiotropy, not only CRP but also plasma triglycerides were predicted by polymorphisms at TH (P = 0.0053) and ADRB2 (P = 0.027). CONCLUSIONS CRP secretion is substantially heritable in humans, demonstrating pleiotropy (shared genetic determination) with other features of the metabolic syndrome, such as BMI, triglycerides or BP. Multiple, common genetic variants in the catecholaminergic/beta-adrenergic pathway contribute to CRP, and these variants (especially at TH and ADRB2) seem to interact (epistasis) to influence the trait. The results uncover novel pathophysiological links between the adrenergic system and inflammation, and suggest new strategies to probe the role and actions of inflammation within this setting.

Background When confronted with a stressor, animals react with several physiological and behavioral responses. Although sustained or repeated stress can result in severe deleterious physiological effects, the causes of stress in free-ranging animals are yet poorly documented. In our study, we aimed at identifying the main factors affecting stress levels in free-ranging wolves (Canis lupus). Methodology/Principal Findings We used fecal cortisol metabolites (FCM) as an index of stress, after validating the method for its application in wolves. We analyzed a total of 450 fecal samples from eleven wolf packs belonging to three protected populations, in Italy (Abruzzo), France (Mercantour), and the United States (Yellowstone). We collected samples during two consecutive winters in each study area. We found no relationship between FCM concentrations and age, sex or social status of individuals. At the group level, our results suggest that breeding pair permanency and the loss of pack members through processes different from dispersal may importantly impact stress levels in wolves. We measured higher FCM levels in comparatively small packs living in sympatry with a population of free-ranging dogs. Lastly, our results indicate that FCM concentrations are associated with endoparasitic infections of individuals. Conclusions/Significance In social mammals sharing strong bonds among group members, the death of one or several members of the group most likely induces important stress in the remainder of the social unit. The potential impact of social and territorial stability on stress levels should be further investigated in free-ranging populations, especially in highly social and in territorial species. As persistent or repeated stressors may facilitate or induce pathologies and physiological alterations that can affect survival and fitness, we advocate considering the potential impact of anthropogenic causes of stress in management and conservation programs regarding wolves and other wildlife

The foraging and feeding ecology of gray wolves is an essential component to understanding the role that top carnivores play in shaping the structure and function of terrestrial ecosystems. In Yellowstone National Park (YNP), predation studies on a highly visible, reintroduced population of wolves are increasing our understanding of this aspect of wolf ecology. Wolves in YNP feed primarily on elk, despite the presence of other ungulate species. Patterns of prey selection and kill rates in winter have varied seasonally each year from 1995 to 2004 and changed in recent years as the wolf population has become established. Wolves select elk based on their vulnerability as a result of age, sex, and season and therefore kill primarily calves, old cows, and bulls that have been weakened by winter. Summer scat analysis reveals an increased variety in diet compared with observed winter diets, including other ungulate species, rodents, and vegetation. Wolves in YNP hunt in packs and, upon a successful kill, share in the evisceration and consumption of highly nutritious organs first, followed by major muscle tissue, and eventually bone and hide. Wolves are adapted to a feast-or-famine foraging pattern, and YNP packs typically kill and consume an elk every 2-3 d. However, wolves in YNP have gone without fresh meat for several weeks by scavenging off old carcasses that consist mostly of bone and hide. As patterns of wolf density, prey density, weather, and vulnerability of prey change, in comparison with the conditions of the study period described here, we predict that there will also be significant changes in wolf predation patterns and feeding behavior.

DNA sequence information is being deposited into public databases at a rapid rate, largely through worldwide genomic efforts to sequence the human genome as well as the genomes of other species. This information has enabled the identification of over a million of naturally occurring genetic variations in humans, in particular, single-nucleotide polymorphisms (SNPs). SNPs have become a focal point in the study of the genetic basis of multifactorial diseases and traits and human evolutionary history. In this article, we will outline bioinformatic and molecular approaches for using available sequence information and technology to identify SNPs in the human population. These approaches may be extended to study SNPs in other organisms as well as other types of polymorphisms. Keywords: SNPs; polymorphism discovery; haplotype determination; SNP Databases

Few studies have examined to what extent genetic variants of the β 2 -adrenoceptor (ADRB2) are involved in the development of hypertension with age, although β 2 -adrenergic receptor responsiveness declines in older subjects. To investigate this, 10 common single-nucleotide polymorphisms (SNPs) in the promoter and coding regions of the ADRB2 gene were genotyped in an unrelated population consisting of 2 ethnic groups: European American (EA; n=610) and African American (AA; n=420). ADRB2 haplotypes were estimated by expectation maximization (EM) algorithm–based methods. In the general population for EAs and AAs, the variants of the ADRB2 gene, including the individual SNPs and their haplotypes, were not associated with hypertension. However, there was a significant interaction between age and one of the common haplotypes (haplotype 1) in EAs ( P =0.01). Haplotype 1 was associated with protection against hypertension in young (≤50 years of age) but not in old (>50 years of age) EAs (odds ratio, 0.5; 95% confidence interval, 0.27 to 0.91; P =0.02). This age-specific effect was further supported by the observations that young subjects carrying ≥1 copy of haplotype 1 had significantly lower diastolic blood pressure and nearly 2-fold higher ADRB2 binding density than the noncarriers ( P P P

alpha2-Adrenergic blockade responses suggest adrenergic dysfunction in hypertension. alpha2-Blockade is also used to treat autonomic dysfunction. However, pharmacokinetic determinants of yohimbine disposition are not understood.We evaluated alpha2-blockade with intravenous yohimbine in 172 individuals. Specific cytochrome P450 (CYP) isoform-mediated metabolism was investigated. Results were evaluated by ANOVA and by maximum likelihood analysis for bimodality of response distributions.Yohimbine metabolism to 11-hydroxy-yohimbine displayed greater than 1000-fold variability, with 17 individuals showing no metabolism. Nonmetabolizers differed from others in ethnicity but not in age, sex, body habitus, blood pressure, heart rate, or family history of hypertension. Bimodality of metabolism was suggested by frequency histogram, as well as maximum likelihood and cluster analysis. Among ethnic groups, subjects of European ancestry had the highest frequency of nonmetabolism. In vitro oxidation suggested that the major route of metabolism (lowest Michaelis-Menten constant and greatest intrinsic clearance) was likely via CYP2D6 to 11-hydroxy-yohimbine. In vivo genotypes at both CYP2D6 and CYP3A4 were necessary to predict metabolism (overall F = 3.03, P =.005); an interaction of alleles at these 2 loci (interaction F = 3.05, P =.033) suggested an epistatic effect on drug metabolism in vivo. Nonmetabolizers had greater activation of sympathetic nervous system activity. Yohimbine increased blood pressure, an effect mediated hemodynamically by elevation of cardiac output rather than systemic vascular resistance. Blood pressure and cardiac output responses did not differ by metabolizer group.We conclude that heterogeneous, bimodally distributed yohimbine metabolism depends on common genetic variation in both CYP2D6 and CYP3A4 and contributes to differences in sympathetic neuronal response to alpha2-blockade. These results have implications for both diagnostic and therapeutic uses of this alpha2-antagonist.

Bovet claims that Smith et al. (D.W. Smith, R.O. Peterson, T.D. Drummer, and D.S. Sheputis, 1991. Can. J. Zool. 69: 2178–2182) and Dyck and MacArthur (A.P. Dyck and R.A. MacArthur, 1992. Can. J. Zool. 70: 1668–1672) analyzed their data on activity and body temperature of beavers (Castor canadensis) incorrectly because they ignored the likelihood that northern beavers in winter have activity rhythms with periods >24 h. The analysis by Smith et al. was in fact appropriate for demonstrating seasonal changes in body temperature and its correlation with activity. These are important issues because researchers have obtained conflicting results and because seasonal changes in activity and body temperature have implications for colony energetics in the context of food-hoarding behavior and social organization.

The microbial rhodopsins (MR) are homologous to putative chaperone and retinal-binding proteins of fungi. These proteins comprise a coherent family that we have termed the MR family. We have used modeling techniques to predict the structure of one of the putative yeast chaperone proteins, YRO2, based on homology with bacteriorhodopsins (BR). Availability of the structure allowed depiction of conserved residues that are likely to be of functional significance. The results lead us to predict an extracellular protein folding function and a transmembrane proton transport pathway. We suggest that protein folding is energized by a novel mechanism involving the proton motive force. We further show that MR family proteins are distantly related to a family of fungal, animal and plant proteins that include the human lysosomal cystine transporter (LCT) of man (cystinosin), mutations in which cause cystinosis. Sequence and phylogenetic analyses of both the MR family and the LCT family are reported. Proteins in both families are of the same approximate size, exhibit seven putative transmembrane α-helical spanners (TMSs) and show limited sequence similarity. We show that the LCT family arose by an internal gene duplication event and that TMSs 1–3 are homologous to TMSs 5–7. Although the same could not be demonstrated statistically for MR family members, homology with the LCT family suggests (but does not prove) a common evolutionary pathway. Thus, TMSs 1–3 and 5–7 in both LCT and MR family members may share a common origin, accounting for their shared structural features.

The evolutionary relationship of Dictyostelium discoideum to the yeasts, fungi, plants, and animals is considered on the basis of physiological, morphological and molecular characteristics. Previous analyses of five proteins indicated that Dictyostelium diverged after the yeasts but before the metazoan radiation. However, analyses of the small ribosomal subunit RNA indicated divergence prior to the yeasts. We have extended the molecular phylogenetic analyses to six more proteins and find consistent evidence for a more recent common ancestor with metazoans than yeast. A consensus phylogeny generated from these new results by both distance matrix and parsimony analyses establishes Dictyostelum's place in evolution between the yeasts Saccharomyces cerevisiae and Schizzosaccharomyces pombe and the worm Caenorhabditis elegans.

Despite Yellowstone National Park's (YNP) long history and well studied large mammals and vegetation, beavers (Castor canadensis), an important ecosystem driver, have received relatively little study. We summarize population surveys of beavers that began in 1921 and continued up to the present. The first surveys (1921 and 1923) were from the ground and conducted in a limited area in the northern portion (northern range; NR) of the park. Twenty-five colonies were found and beavers were considered abundant and using aspen (Populus tremuloides) and willow (Salix spp.) as a food source and building material. A follow up survey in 1953 found 6 NR sites, but none of the earlier sites from the 1920s were active and no aspen use was reported. Some locations were reported from the park interior. A limited ground survey was conducted in 1979–80. In 1988–89 and 1994 two incomplete, mostly ground surveys were conducted and estimated 71 and 44 colonies, respectively, in YNP. In 1996, 1998, 1999, 2001, 2003, 2005, 2007 and 2009 complete, park-wide aerial surveys were conducted and active colonies ranged from 44 (1996) to 127 (2007) with an increasing trend. Therefore, in a period of about 90 years (1920s—2000s) the beaver population in the northern portion of the park appears to have declined then increased probably because of a willow recovery.

Publisher Summary This chapter discusses the current and future roles of supercomputers and parallel processing platforms in the informatics needs of genome projects. Supercomputers currently are used mainly for protein structure analysis and protein-small molecule interactions, particularly for molecular dynamics and energy minimization calculations and for structure refinement and model building. The genome projects aim to delineate genetic and physical maps of the total DNA complement of a given organism, ultimately yielding the total nucleotide sequence of this DNA. In fulfilling these objectives, they generate enormous amounts of information. A detailed explanation of various parallel processing platforms is presented in the chapter. These include the following: (1) Parallel-Computer Architectures, (2) MIMD Architectures, and (3) MIMD Distributed-Memory Architectures.

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.

The coupling of replication to the cell cycle and cell growth involves events that occur at oriC. Immediately after initiation, there is an eclipse phase during which reinitiation from the newly synthesized origins is prevented. GATC sites in oriC remain in a hemimethylated state longer than other sites because of their association with the outer membrane, which prevents DnaA from binding and activating additional rounds of initiation. After the origins are methylated and released from the outer membrane, the concentration of newly synthesized DnaA and the activation of oriC by transcription from the nearby mioC and gid promoters determine when the next rounds of replication initiate. If growth rate is reduced, the synthesis of (p)ppGpp will increase, and this will lead to a decrease in dnaA, mioC, and gid transcription. On the other hand, if growth rate is increased by access to a tasty meal, synthesis of (p)ppGpp will decrease, expression of dnaA, mioC, and gid genes will increase, and a shortening of the interinitiation time will result. The participation of all these control features ensures rapid and precise coordination of DNA replication with cell growth.

A test was devised for exploring the question of whether it will be possible to identify genes in large-scale genome studies solely by sequence comparison with current sequence collections. To this end, a facsimile data set was constructed by dividing GenBank Release 56 randomly into two halves, one to serve as a reference set and the other intended to simulate raw data anticipated from large genome sequence projects. All supplementary information and identifying marks were removed from the test set after assignment of random identification numbers to each entry and their encryption. Because noncoding intervening sequences (introns) are underrepresented in GenBank, a program that introduced (simulated) introns into mRNA and prokaryotic sequences was devised. In a further attempt to make the problem of identification more realistic, random base substitutions and single-base deletions were also incorporated. The randomly ordered entries were concatenated, along with random intergenic flanking sequences, into a single long "chromosome" 33 Mb in length and then cut into "cosmids" 50-100 kb long. The chopping process was conducted in such a way that terminal overlaps would allow the order of the entries in the chromosome to be reconstituted. Finally, the sequences of a substantial fraction of the cosmids were converted to their complements. Preliminary searching of 10 test cosmids revealed that more than two-thirds of the entries in the test set should be readily identifiable by type of gene product solely on the basis of comparison with the reference set. These preliminary results suggest that existing computer regimens and sequence collections would be able to identify the majority of eukaryotic genes in any new raw data set, the existence of introns not withstanding. Moreover, the analysis can be conducted in pace with the data collection so that the search results and summary identifications will be instantly available to the research community at large.

Incidents are described of Bison (Bison bison) in Yellowstone National Park mauling and possibly killing a young Elk (Cervus elaphus) calf, chasing wolves (Canis lupus) off Elk they had just killed or were killing, and keeping the wolves away for extended periods. During one of the latter cases, the Bison knocked a wolf-wounded Elk down. Bison were also seen approaching wolves that were resting and sleeping, rousting them, following them to new resting places and repeating this behavior. These behaviors might represent some type of generalized hyper-defensiveness that functions as an anti-predator strategy.

With gray wolves restored to Yellowstone National Park, this ecosystem once again supports the full native array of large ungulates and their attendant large carnivores. We consider the possible ecological implications of wolf restoration in the context of another national park, Isle Royale, where wolves restored themselves a half-century ago. At Isle Royale, where resident mammals are relatively few, wolves completely eliminated coyotes and went on to influence moose population dynamics, which had implications for forest growth and composition. At Yellowstone, we predict that wolf restoration will have similar effects to a degree, reducing elk and coyote density. As at Isle Royale, Yellowstone plant communities will be affected, as will mesocarnivores, but to what degree is as yet undetermined. At Yellowstone, ecosystem response to the arrival of the wolf will take decades to unfold, and we argue that comprehensive ecological research and monitoring should be an essential long-term component of ...

We studied wolf (Canis lupus) predation on elk (Cervus elaphus) in Yellowstone National Park from 17 March to 15 April 1997 (severe winter conditions) and from 2 to 31 March 1998 (mild winter conditions) 2-3 years after wolves were reintroduced to the park. Elk composed 91% of 117 kills. Data comparisons for 1997 versus 1998 were: hunting success rate, 26% versus 15%; kill rate, 17.1 kg/wolf/day versus 6.1; percent of kill consumed in first day 7 versus 86; percent femur marrow fat of adult kills, 27 versus 70; calf:adult ratios of kills, 2:33 versus 17:23; sex ratio of kills, 14M:19F versus 17M:6F; mean age of elk killed, males 6.1 years, females 15.2 versus males, 4.8, females 13.0. Winter severity influenced the wolf-elk relationship more than the naivete of the elk herd to predation by wolves.

Beavers (Castor canadensis) were studied on two Lake Superior islands; one (Stockton) had black bears (Ursus americanus), while the other (Outer) did not. Both islands were colonized by beavers during the late 1940s or early 1950s in the absence of bears, and bears immigrated to Stockton Island in the 1970s. Active beaver colonies on Outer Island increased during the study whereas they declined on Stockton Island. Bears ate five beavers in live-traps and disturbed twelve other sets. Six of 90 bear scats found had beaver hair in them, and 18 of 26 beaver lodges were dug into by bears. Compared to Outer Island, beavers on Stockton Island cut fewer food trees relative to their availability, and cut few trees >30 m from water, whereas beavers on Outer Island traveled >200 m inland to forage. Mainland studies have not reported such intense beaver/bear interactions, nor is the behavioral or population response of beavers as significant where they are preyed upon by wolves (Canis lupus), a more typical beaver predator.

Construction of deletion derivative plasmids and cloning of restriction fragments from plasmids containing the Salmonella typhimurium origin of replication (ori) were used to locate the functional origin to within a DNA fragment of 296 base pairs between the genes uncB and asn. The nucleotide sequence of the S. typhimurium ori region was determined and compared with the Escherichia coli ori sequence. In the 296-base pair fragment, 85.8% of the bases are conserved between the two species. A nearly equal number of transition and transversion type differences, with no insertions or deletions, occurs between the two bacterial origins, such that the relatively high percentage (adenine plus thymine) of 59.5% is conserved. The 296-base pair fragment contains 14 GATC sequences, all of which are conserved. The high frequency of occurrence of GATC, which is the site of methylation under control of the dam gene, may explain in part why the bacterial ori region appears to be so highly conserved. A large number of secondary structures are possible. One such structure, with a "cloverleaf," is favored by ori nucleotide sequence comparisons and leads to potential novel macromolecular interactions.

DNA containing the Escherichia coli dam gene and sequences upstream from this gene were cloned from the Clarke-Carbon plasmids pLC29-47 and pLC13-42. Promoter activity was localized using pKO expression vectors and galactokinase assays to two regions, one 1650-2100 bp and the other beyond 2400 bp upstream of the dam gene. No promoter activity was detected immediately in front of this gene; plasmid pDam118, from which the nucleotide sequence of the dam gene was determined, is shown to contain the pBR322 promoter for the primer RNA from the pBR322 rep region present on a 76 bp Sau3A fragment inserted upstream of the dam gene in the correct orientation for dam expression. The nucleotide sequence upstream of dam has been determined. An open reading frame (ORF) is present between the nearest promoter region and the dam gene. Codon usage and base frequency analysis indicate that this is expressed as a protein of predicted size 46 kDa. A protein of size close to 46 kDa is expressed from this region, detected using minicell analysis. No function has been determined for this protein, and no significant homology exist between it and sequences in the PIR protein or GenBank DNA databases. This unidentified reading frame (URF) is termed urf-74.3, since it is an URF located at 74.3 min on the E. coli chromosome. Sequence comparisons between the regions upstream of urf-74.3 and the aroB gene show that the aroB gene is located immediately upstream of urf-74.3, and that the promoter activity nearest to dam is found within the aroB structural gene. This activity is relatively weak (about 15% of that of the E. coli gal operon promoter). The promoter activity detected beyond 2400 bp upstream of dam is likely to be that of the aroB gene, and is 3 to 4 times stronger than that found within the aroB gene. Three potential DnaA binding sites, each with homology of 8 of 9 bp, are present, two in the aroB promoter region and one just upstream of the dam gene. Expression through the site adjacent to the dam gene is enhanced 2- to 4-fold in dnaA mutants at 38 degrees C. Restriction site comparisons map these regions precisely on the Clarke-Carbon plasmids pLC13-42 and pLC29-47, and show that the E. coli ponA (mrcA) gene resides about 6 kb upstream of aroB.

Novel RNA polymerase activities (termed type II reaction) can be found in toluene-treated Escherichia coli with Ca2+, Fe2+, or endogenously bound cations, probably Mg2+. These activities are distinguishable from the well characterized DNA-dependent RNA polymerase (type I reaction) by: (i) their divalent cation requirements, i.e., the classical enzyme is activated by exogenously added Mn2+, Mg2+, or Co2+ ions; (ii) their relative resistance to inhibition by actinomycin D, rifampicin, and streptolydigin; (iii) their selective synthesis of low molecular weight RNA; (iv) their sensitivity to inhibition by arabinonucleoside 5′-triphosphates or deoxyribonucleoside 5′-triphosphates; and (v) the strict requirement for ATP in Ca2+ and bound cation-activated reactions. The Ca2+-activated and endogenous RNA polymerase activities are inhibited by orthophosphate. The properties of the type II RNA polymerase(s) are compared with those of polynucleotide phosphorylase, the dnaG gene product, and the RNA polymerase described by Ohasa and Tsugita.

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of Escherichia coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas renitiation was not inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid ((RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initation of DNA replication (origin-RNA). Reinitiation at 30 degrees C was not inhibited by streptolydigin in a stretolydigin-sensitive dnaA muntant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 muntant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribounucleotide polymerization event wheareas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events that occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.

Arginine deiminase (EC 3.5.3.6) from Mycoplasma arthritidis is a dimeric enzyme. Velocity centrifugation in 6 M guanidine HCl and peptide mapping of the BrCN fragments suggest that the subunits are identical. The reaction of one out of four sulfhydryl groups with 0.3 mM 5,5′-dithiobis-(2-nitrobenzoic acid) has a half-life of about 30 min in 2 M guanidine HCl at 15°, pH 8. The enzyme is irreversibly inhibited by 1 mM formamidinium ion within 1 min. Inactivation by this affinity label is resolvable into two concurrent first-order reactions in the presence of guanidinium ion; the fraction of enzyme which reacts at the faster rate is about 50%. These results are interpreted as evidence for two catalytic subunits which differ in conformation.

The sedimentation properties of membrane-associated folded chromosomes prepared from Escherichia coli TAU-bar at 0 to 4 C were studied. Utilizing a modification of the procedure of Stonington and Pettijohn (1971), quantitative yields of membrane-associated folded chromosomes may be obtained. Folded chromosomes remained associated with the cell envelope during their replication and after completion of residual synthesis in the absence of required amino acids, as demonstrated by sedimentation velocities and the presence of high levels of cosedimenting protein. Membrane-associated folded chromosomes isolated from amino acid-starved cells sedimented more rapidly than membrane-associated folded chromosomes isolated from exponentially growing cells.

Arginine deiminase (EC 3.5.3.6) catalyzes the hydrolysis of arginine to ammonia and citrulline. This reaction is postulated to occur in three steps: (1) formation of the Michaelis complex, (2) the formation of an amidino-enzyme intermediate and liberation of ammonia, and (3) the rate-determining step, hydrolysis of the amidino-enzyme. The enzymic reaction is accelerated 5-fold by 0.2 M imidazole. This striking effect is expected for the amidino-enzyme mechanism but otherwise is difficult to explain. The putative amidino-enzyme intermediate can be demonstrated by quenching the [14C]arginine-arginine deiminase reaction at low pH. Under these conditions, 0.5 equivalents of 14C label per mol enzyme dimer were covalently bound.

E. coli mutants deficient in DNA polymerase I, in DNA polymerases I and II, or in DNA polymerase III, can efficiently and completely execute excision repair and post-replication repair of UV-damaged DNA at 43° when assayed by alkaline sucrose gradients. Repair by cells deficient in polymerase I and in polymerases I and II is inhibited by 1-β-D-arabinofuranosylcytosine at 43°, whereas that by cells deficient in polymerase III is insensitive to the inhibitor. When both DNA polymerases I and III are deficient, both excision repair and post-replication repair are greatly reduced at 43°, and the residual repair capability is inhibited by 1-β-D-arabinofuranosylcytosine. Very little dark repair is observed in cells deficient in DNA polymerases I, II, and III, and the DNA is extensively degraded. These results suggest that either DNA polymerase I or DNA polymerase III is required for complete and efficient repair, and that when both DNA polymerases I and III are deficient, DNA polymerase II mediates a limited, incomplete dark repair of UV-damaged DNA. DNA polymerases I and III thus appear to be important enzymes in both DNA replication and DNA dark repair.

Chain elongation during E. coli DNA replication proceeds in three distinct stages. Participatory roles of the three E. coli DNA polymerases in each of these stages has been determined.

Escherichia coli DNA polymerases II and III have been extensively studied in vitro when activated with Mg2+. The Mn2+-activated polymerization reactions are considered here, and shown to differ from the Mg2+-activated reactions. The Mn2+-activated DNA polymerase II reaction requires K+ or spermidine, and the effects of monovalent cation and polyamine are additive. In contrast, the Mg2+-activated reaction does not require, but is stimulated by, K+ or spermidine, in a non-additive manner. Under optimal conditions, DNA polymerase II is activated better with Mn2+ than it is with Mg2+, suggesting a physiological role for the Mn2+-activated enzyme. The observed preference for Mn2+ over Mg2+ in reaction kinetics and at high DNA template concentrations suggest that Mg2+ may preferentially activate the associated exonuclease activity. At 29 degrees C, the Mn2+-activated DNA polymerase III reaction is stimulated by K+ and inhibited by ethanol or phosphatidylethanolamine. In contrast, the latter compounds and Triton X-100 increase the initial rate of the Mg2+-activated reaction, whereas K+ inhibits this reaction at all concentrations. The K+ inhibition is reduced at low Mg concentrations when Mn2+ is also present. After stimulating the initial reaction rate, ethanol causes a rapid decrease in the rate of the Mg2+-activated reaction during incubation at 20 degrees C. At 27 degrees C, all surface-active compounds inhibit the Mg2+-activated reaction. Preincubation of the enzyme at 30 degrees C or below with DNA template and divalent cation increases the initial reaction rate, suggesting that formation of an enzyme-divalent cation-DNA template complex occurs as the first step in DNA polymerase III catalysis. The apparent Km at 21 degrees C for gapped calf thymus DNA was 25 muM with Mn2+ and 125 muM with Mg2+ for DNA polymerase III, and 18 muM at 30 degrees C for DNA polymerase II with either Mn2+ or Mg2+. Reactions with poly[d(A-T)] were enhanced by Mn2+ relative to Mg2+, and activity with poly(rA)-poly(dT) was Mn2+ dependent for both enzymes.

The states available to a light particle in models for gases and gas mixtures are investigated. The models are characterized by atoms (molecules) which mutually interact as hard spheres and which interact with a light particle via a short ranged repulsive interaction. The trends in the densities of states for the light particle, the highest energy for localization, and the number of localized states are pursued as a function of the physical parameters—the particle densities of the constituents, the scattering strengths, the packing fraction, and the relative atomic sizes—using a variational technique and numerical computations. Mean field effects and those due to fluctuations may be isolated. It is noted that these type of calculations provide a context within which experiments, such as those for ortho‐positronium annihilation rates in gases, may be analyzed.

The DNA of a strain of the pleuropneumonia-like organism Mycoplasma laidlawii B, adapted to grow in a semi-defined medium, was density-labeled for various periods of time by substituting 5-bromodeoxyuridine for thymidine in the growth medium. Analysis of extracted DNA fragments by CsCl equilibrium sedimentation demonstrated semi-conservative replication, proceeding sequentially along the chromosome. The kinetics of formation of density-labeled DNA showed that 5-bromodeoxyuridine probably effects a premature initiation of a round of replication. By lysing the cells directly in the centrifuge tube, the M. laidlawii B chromosome was isolated in one piece in the CsCl gradient. Comparison of the buoyant-density distribution of the intact chromosomes with that of very mildly sheared chromosomes (shear yielding pieces at least 1 8 to 1 4 the size of the total chromosome) eliminated nearly all dispersive replication mechanisms and suggested that replication probably proceeds from only a few growing points. The buoyant-density distributions of intact chromosomes isolated after different times of density labeling are shown to be consistent with theory. Some physicochemical properties of the hybrid (density-labeled) and normal M. laidlawii B DNA are presented. These properties, and similarities and differences of DNA replication in M. laidlawii B and in Escherichia coli, are discussed.