Discontinuity and Limited Linkage in the Homologous Recombination System of a Hyperthermophilic Archaeon

Genetic transformation of Sulfolobus acidocaldarius by a multiply marked pyrE gene provided a high-resolution assay of homologous recombination in a hyperthermophilic archaeon. Analysis of 100 Pyr⁺ transformants revealed that this recombination system could transfer each of 23 nonselected base pair substitutions to the recipient chromosome along with the selected marker. In 30% of the recombinants, donor markers were transferred as multiple blocks. In at least 40% of the recombinants, donor markers separated by 5 or 6 bp segregated from each other, whereas similar markers separated by 2 bp did not segregate. Among intermarker intervals, the frequency of recombination tract endpoints varied 40-fold, but in contrast to other recombination systems, it did not correlate with the length of the interval. The average length of donor tracts (161 bp) and the frequent generation of multiple tracts seemed generally consistent with the genetic properties observed previously in S. acidocaldarius conjugation. The efficiency with which short intervals of diverged pyrE sequence were incorporated into the genome raises questions about the threat of ectopic recombination in Sulfolobus spp. mediated by this apparently efficient yet permissive system.All cells and some viruses encode systems of homologous recombination (HR) which support the successful replication of their genomes. In eukaryotic cells, HR systems repair double-strand breaks and ensure proper chromosome segregation during meiosis (1, 2, 17). Double-strand break repair by eukaryotic HR has been studied intensively in yeast, where it has been shown to cause the net transfer of a short section of sequence from the intact DNA to the broken DNA in a unilateral, i.e., nonreciprocal, manner. Outside this central zone, the flanking segments of the two DNAs may also be exchanged, generating a crossover. The relative yields of noncrossover versus crossover events vary in different situations, and this appears to reflect the different ways in which displacement loops formed by strand invasion are ultimately resolved (1, 17, 36).In bacteria, HR helps reassemble replication forks disrupted by encounters with various DNA lesions (6, 20, 27). For practical reasons, however, genetic assays of bacterial HR typically follow the process of replacing a segment of a recipient chromosome or plasmid with a corresponding (i.e., homologous) donor DNA segment introduced into the cell. This “ends-out” mode of HR underlies the classical techniques of genetic mapping and strain construction of Escherichia coli and other bacteria. It also occurs in natural populations and contributes to genome evolution, as indicated by the “mosaic” patterns of sequence polymorphisms documented in various E. coli lineages (28). Thus, the functional properties of the host HR system combine with those of the DNA transfer systems to influence the rate of genetic exchange and the nature (including the abundance and average length) of the homologous DNA segments incorporated as a result.The importance of HR for genetic exchange and genome stability raises questions about its role in microorganisms highly diverged from model microbial species and adapted to extreme environments. Many archaea meet these criteria, but HR has not been examined extensively in archaea, particularly with respect to functional properties in vivo. The archaeal homologues of the RecA and Rad51/Dmc1 proteins, termed “RadA,” share a distinct motif structure which more closely resembles the eukaryotic than the bacterial consensus (32). In vitro, the RadA proteins of various hyperthermophilic archaea have been shown to bind single-stranded DNA (ssDNA) preferentially, thereby forming nucleoprotein filaments. This binding has been reported to stimulate ATP hydrolysis, displacement loop formation, and strand exchange (19, 26, 34). Evidence for HR in vivo includes observations that DNA of Pyrococcus and related archaea fragmented by gamma irradiation reassembles quickly in vivo (8) and that Sulfolobus species can be genetically transformed by linear DNA (7, 16, 22). In other archaea (methanogens and extreme halophiles), small, nonreplicating, circular DNAs have been observed to integrate into recipient genomes by reciprocal crossovers (11), and deletion of the radA gene of the extreme halophile Haloferax volcanii has demonstrated that the protein is essential for HR in that species but not for viability (37).In Sulfolobus spp., which grow optimally at about pH 3 and 80°C (12), auxotrophic mutants provide sensitive assays of recombination at particular chromosomal loci. Several studies suggest that in Sulfolobus acidocaldarius, many of these events require only short DNA segments. In conjugation assays, for example, more than 90% of randomly chosen pairs of 5-fluoroorotic acid (FOA)-resistant S. acidocaldarius mutants generated Pyr⁺ recombinants (31), despite the fact that about 95% of such mutations normally arise in the 594-bp pyrE coding sequence (14). Furthermore, when pyrE mutations of known positions were tested in pairwise combinations, the relative yield of recombinants did not decrease significantly until the separation became less than about 50 bp, indicating that donor sequences were transferred to the recipient chromosome as small segments (18).Although these results reveal genetic consequences of conjugation in S. acidocaldarius, they do not clarify whether these consequences primarily reflect the DNA transfer process or, alternatively, subsequent HR. For example, transfer of short DNA fragments from a donor cell to a recipient cell seems able to explain both the facile resolution of very closely spaced pyrE mutations (31) and the inefficient replacement of large pyrE deletions (18) in S. acidocaldarius conjugation. However, other Sulfolobus spp. transfer relatively large conjugative plasmids between cells (33), so a similar transfer capability must be considered for S. acidocaldarius. In that case, the observed “short-patch” nature of the exchanges would reflect processing of longer intervals of transferred DNA by the HR system to yield short replacement tracts; this would resemble noncrossover events in eukaryotic double-strand break repair (1, 17, 36) or the patches of DNA incorporated by transformation of Helicobacter pylori (21, 25). An apparent failure of circular DNA containing a full-length pyrE gene with a promoter to integrate into the S. acidocaldarius genome by a single reciprocal crossover raises other questions. Despite protection of the circular DNA against the host restriction system and scoring by PCR within several generations of transformation, no sequences of the nonreplicating vector could be detected in any of a number of independent transformants (22). Possible explanations include an extremely high frequency of reciprocal crossovers, leading to stochastic elimination of the nonselectable vector sequences within a short time, or an intrinsically nonreciprocal mode of the initial recombination, which simply copied the functional pyrE sequence onto the host chromosome. While the mechanistic basis remains unresolved, the observations themselves combine with the presence of unusual DNA enzymes (10) and the absence of otherwise conserved DNA repair proteins (13) to suggest that HR and related DNA transactions of Sulfolobus spp. and other hyperthermophilic archaea may have unusual functional features.As an important step toward understanding Sulfolobus HR in molecular terms, we developed a quantitative assay to analyze individual recombination events in S. acidocaldarius to high resolution. The results show that this HR process transfers markers from a short donor DNA to the recipient genome to generate a diversity of configurations in which both the length and number of replacement tracts vary widely. In addition, the HR system exhibited limited genetic linkage and readily resolved certain markers spaced only 5 or 6 bp apart.     

高等植物中的磷酸烯醇式丙酮酸羧激酶

简要介绍了近年来有关高等植物中磷酸烯醇式丙酮酸羧激酶（ＰＥＰＣＫ）的研究进展,并讨论了此酶的结构、功能和调节等方面的问题。     

Changes in cognition and mortality in relation to exercise in late life: a population based study

Background

On average, cognition declines with age but this average hides considerable variability, including the chance of improvement. Here, we investigate how exercise is associated with cognitive change and mortality in older people and, particularly, whether exercise might paradoxically increase the risk of dementia by allowing people to live longer.

Methods and Principal Findings

In the Canadian Study of Health and Aging (CSHA), of 8403 people who had baseline cognition measured and exercise reported at CSHA-1, 2219 had died and 5376 were re-examined at CSHA-2. We used a parametric Markov chain model to estimate the probabilities of cognitive improvement, decline, and death, adjusted for age and education, from any cognitive state as measured by the Modified Mini-Mental State Examination. High exercisers (at least three times per week, at least as intense as walking, n = 3264) had more frequent stable or improved cognition (42.3%, 95% confidence interval: 40.6–44.0) over 5 years than did low/no exercisers (all other exercisers and non exercisers, n = 4331) (27.8% (95% CI 26.4–29.2)). The difference widened as baseline cognition worsened. The proportion whose cognition declined was higher amongst the high exercisers but was more similar between exercise groups (39.4% (95% CI 37.7–41.1) for high exercisers versus 34.8% (95% CI 33.4–36.2) otherwise). People who did not exercise were also more likely to die (37.5% (95% CI 36.0–39.0) versus 18.3% (95% CI 16.9–19.7)). Even so, exercise conferred its greatest mortality benefit to people with the highest baseline cognition.

Conclusions

Exercise is strongly associated with improving cognition. As the majority of mortality benefit of exercise is at the highest level of cognition, and declines as cognition declines, the net effect of exercise should be to improve cognition at the population level, even with more people living longer.     

Ecological reproductive isolation of coast and inland races of Mimulus guttatus

Adaptive divergence due to habitat differences is thought to play a major role in formation of new species. However it is rarely clear the extent to which individual reproductive isolating barriers related to habitat differentiation contribute to total isolation. Furthermore, it is often difficult to determine the specific environmental variables that drive the evolution of those ecological barriers, and the geographic scale at which habitat-mediated speciation occurs. Here, we address these questions through an analysis of the population structure and reproductive isolation between coastal perennial and inland annual forms of the yellow monkeyflower, Mimulus guttatus. We found substantial morphological and molecular genetic divergence among populations derived from coast and inland habitats. Reciprocal transplant experiments revealed nearly complete reproductive isolation between coast and inland populations mediated by selection against immigrants and flowering time differences, but not postzygotic isolation. Our results suggest that selection against immigrants is a function of adaptations to seasonal drought in inland habitat and to year round soil moisture and salt spray in coastal habitat. We conclude that the coast and inland populations collectively comprise distinct ecological races. Overall, this study suggests that adaptations to widespread habitats can lead to the formation of reproductively isolated species.     

Determination of the cyanide metabolite 2-aminothiazoline-4-carboxylic acid in urine and plasma by gas chromatography-mass spectrometry

The cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) is a promising biomarker for cyanide exposure because of its stability and the limitations of direct determination of cyanide and more abundant cyanide metabolites. A simple, sensitive, and specific method based on derivatization and subsequent gas chromatography-mass spectrometry (GC-MS) analysis was developed for the identification and quantification of ATCA in synthetic urine and swine plasma. The urine and plasma samples were spiked with an internal standard (ATCA-d(2)), diluted, and acidified. The resulting solution was subjected to solid phase extraction on a mixed-mode cation exchange column. After elution and evaporation of the solvent, a silylating agent was used to derivatize the ATCA. Quantification of the derivatized ATCA was accomplished on a gas chromatograph with a mass selective detector. The current method produced a coefficient of variation of less than 6% (intra- and interassay) for two sets of quality control (QC) standards and a detection limit of 25 ng/ml. The applicability of the method was evaluated by determination of elevated levels of ATCA in human urine of smokers in relation to non-smokers for both males and females.     

Differences in need for antihypertensive drugs among those aware and unaware of their hypertensive status: a cross sectional survey

Peripheral arterial disease (PAD) is a common, progressive manifestation of atherothrombotic vascular disease, which should be managed no different to cardiac disease. Indeed, there is growing evidence that PAD patients are a high risk group, although still relatively under-detected and under treated. This is despite the fact that PAD patients are an increased mortality rate comparable to those with pre-existing or established cardiovascular disease [myocardial infarction, stroke]. With a holistic approach to atherothrombotic vascular disease, our management of PAD can only get better.     

Changes in relative fitness and frailty across the adult lifespan: evidence from the Canadian National Population Health Survey

Background

The prevalence of frailty increases with age in older adults, but frailty is largely unreported for younger adults, where its associated risk is less clear. Furthermore, less is known about how frailty changes over time among younger adults. We estimated the prevalence and outcomes of frailty, in relation to accumulation of deficits, across the adult lifespan.

Methods

We analyzed data for community-dwelling respondents (age 15–102 years at baseline) to the longitudinal component of the National Population Health Survey, with seven two-year cycles, beginning 1994–1995. The outcomes were death, use of health services and change in health status, measured in terms of a Frailty Index constructed from 42 self-reported health variables.

Results

The sample consisted of 14 713 respondents (54.2% women). Vital status was known for more than 99% of the respondents. The prevalence of frailty increased with age, from 2.0% (95% confidence interval [CI] 1.7%–2.4%) among those younger than 30 years to 22.4% (95% CI 19.0%–25.8%) for those older than age 65, including 43.7% (95% CI 37.1%–50.8%) for those 85 and older. At all ages, the 160-month mortality rate was lower among relatively fit people than among those who were frail (e.g., 2% v. 16% at age 40; 42% v. 83% at age 75 or older). These relatively fit people tended to remain relatively fit over time. Relative to all other groups, a greater proportion of the most frail people used health services at baseline (28.3%, 95% CI 21.5%–35.5%) and at each follow-up cycle (26.7%, 95% CI 15.4%–28.0%).

Interpretation

Deficits accumulated with age across the adult spectrum. At all ages, a higher Frailty Index was associated with higher mortality and greater use of health care services. At younger ages, recovery to the relatively fittest state was common, but the chance of complete recovery declined with age.On average, health declines with age. Even so, at any given age the health status across a group of people varies. Variability in health status and in the risk for adverse outcomes for people of the same age is referred to as “frailty,” which typically has been studied among older adults.^1,2 Although frailty can be operationalized in different ways, in general, people who report having no health problems are more likely to be fit than people who report having many problems. Unsurprisingly, the chance of adverse outcomes — death, admission to a long-term care institution or to hospital, or worsening of health status — increases with the number of problems that the individual has.^3,4The antecedents of frailty appear to arise some time before old age,^5–9 although how frailty emerges as people age, whether it carries the same risk at all ages and the extent to which it fluctuates are less clear.^9,10 In the study reported here, we evaluated changes in relative fitness and frailty across the adult lifespan. Our objectives were to investigate the effect of age on the prevalence of relative fitness and frailty, the characteristics of people who were relatively fit in comparison with those who were frail across the adult lifespan, the effects of fitness and frailty on mortality in relation to age and sex, and the characteristics of people who maintained the highest levels of fitness across a decade relative to those who at any point reported any decline.     

Organ-distribution of the metabolite 2-aminothiazoline-4-carboxylic acid in a rat model following cyanide exposure

The reaction of cyanide (CN(-)) with cystine to produce 2-aminothiazoline-4-carboxylic acid (ATCA) is one of the independent detoxification pathways of cyanide in biological systems. In this report, in vivo production of ATCA and its distributions in plasma and organs were studied after a subcutaneous sublethal dose of 4?mg/kg body weight potassium cyanide (KCN) administration to rats. At this sublethal dose of KCN, ATCA concentration was not significantly increased in the plasma samples, however, it was found significantly increased in liver samples. These results suggested that ATCA might not be a good diagnostic biomarker in plasma for sublethal cyanide exposure; however, liver could serve as the right organ for the detection of ATCA in post-mortem examinations involving cyanide exposure in military, firefighting, industrial and forensic settings.