RESTRICTION-ENDONUCLEASE ANALYSIS OF MITOCHONDRIAL-DNA OF MUNTJACS AND RELATED DEER

Twelve restriction endonucleases were employed to analyze the mitochondrial DNA of four species of muntjacs and two related species of deer: red muntjac (M. muntjak), Gongshan muntjac (M. gongshanensis), black muntjac (M. crinifrons), Chinese muntjac (M. reevesi), tufted deer (Elaphodus cephalophus), and forest musk deer (Moschus berezovskii). A total of 170 restriction fragments were detected among the samples. Fragments data were used to calculate the genetic distance (i.e. percent sequence divergency) among species, which in turn were used to construct a phylogenetic tree and to estimate divergency times. Our analysis indicates that the black muntjac and the Gongshan muntjac are most closely related, and that they are closely realted to the red muntjac and the Chinese muntjac. Additionally, the tufted deer is genetically closer to muntjacs than the musk deer is.

Assessment of visibility restriction mechanisms for RFID data Discovery Services

RFID is a technology that enables the automated capture of observations of uniquely identified physical objects as they move through supply chains. Discovery Services provide links to repositories that have traceability information about specific physical objects. Each supply chain party publishes records to a Discovery Service to create such links and also specifies access control policies to restrict who has visibility of link information, since it is commercially sensitive and could reveal inventory levels, flow patterns, trading relationships, etc. The requirement of being able to share information on a need-to-know basis, e.g. within the specific chain of custody of an individual object, poses a particular challenge for authorization and access control, because in many supply chain situations the information owner might not have sufficient knowledge about all the companies who should be authorized to view the information, because the path taken by an individual physical object only emerges over time, rather than being fully pre-determined at the time of manufacture. This led us to consider novel approaches to delegate trust and to control access to information. This paper presents an assessment of visibility restriction mechanisms for Discovery Services capable of handling emergent object paths. We compare three approaches: enumerated access control (EAC), chain-of-communication tokens (CCT), and chain-of-trust assertions (CTA). A cost model was developed to estimate the additional cost of restricting visibility in a baseline traceability system and the estimates were used to compare the approaches and to discuss the trade-offs. © 2012 IEEE.

Identification of Cj1051c as a Major determinant for the restriction barrier of Campylobacter jejuni StrainNCTC11168

Campylobacter jejuni is a leading cause of human diarrheal illness in the world, and research on it has benefitted greatly by the completion of several genome sequences and the development of molecular biology tools. However, many hurdles remain for a full understanding of this unique bacterial pathogen. One of the most commonly used strains for genetic work with C. jejuni is NCTC11168. While this strain is readily transformable with DNA for genomic recombination, transformation with plasmids is problematic. In this study, we have identified a determinant of this to be cj1051c, predicted to encode a restriction-modification type IIG enzyme. Knockout mutagenesis of this gene resulted in a strain with a 1,000-fold-enhanced transformation efficiency with a plasmid purified from a C. jejuni host. Additionally, this mutation conferred the ability to be transformed by plasmids isolated from an Escherichia coli host. Sequence analysis suggested a high level of variability of the specificity domain between strains and that this gene may be subject to phase variation. We provide evidence that cj1051c is active in NCTC11168 and behaves as expected for a type IIG enzyme. The identification of this determinant provides a greater understanding of the molecular biology of C. jejuni as well as a tool for plasmid work with strain NCTC11168. © 2012, American Society for Microbiology.

Soil microbial community analysis using terminal restriction fragment length polymorphisms

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e. g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e. g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.

Phylogenetic relationships of five pika species (genus Ochotona) based on mitochondrial DNA restriction maps

Restriction site mapping of mitochondrial DNA (mtDNA) with 16 restriction endonucleases was used to examine the phylogenetic relationships of Ochotona cansus, O. huangensis, O. thibetana, O. curzoniae and O. erythrotis. A 1-kb length variation between O. erythrotis of subgenus Pika and other four species of subgenus Ochotona was observed, which may be a useful genetic marker for identifying the two subgenera. The phylogenetic tree constructed using PAUP based on 61 phylogenetically informative sites suggests that O. erythrotis diverged first, followed by O. cansus, while O. curzoniae and O. huangensis are sister taxa related to O. thibetana, The results indicate that both O. cansus and O. huangensis should be treated as independent species. If the base substitution rate of pikas mtDNA was 2% per million years, then the divergence time of the two subgenera, Pika and Ochotana, is about 8.8 Ma ago of late Miocence, middle Bao-dian of Chinese mammalian age, and the divergence of the four species in subgenus Ochotona would have occurred about 2.5 - 4.2 Ma ago, Yushean of Chinese mammalian age. This calculation appears to be substantiated by the fossil record.

Effects of Aging and Moderate Calorie Restriction on the Reproductive Axis of the Male Rhesus Macaque (Macaca mulatta)

Calorie restriction (CR) has been established as the only non-genetic method of altering longevity and attenuating biological changes associated with aging. This nutritional paradigm has been effective in nematodes, flies, rodents, dogs and possibly non-human primates. Its long history notwithstanding, little is known regarding the exact mechanism(s) of CR action or its potential impact on the hypothalamic-pituitary-gonadal (HPG) axis. The objectives of this project were to: 1) analyze neuroendocrine changes to the HPG axis that occur with aging and 2) evaluate the effects of moderate CR on reproductive function in male rhesus macaques. Pituitary gene expression profiling, semi-quantitative RT-PCR (sqRT-PCR) and immunohistochemistry showed circadian clock mechanism components present in three age categories of macaques, demonstrated age differences in expression for Per2, indicated differential expression of Per2 and Bmal1 at opposing time points and revealed daily rhythmic expression of REV-ERBα protein. These data indicate the ability of the macaque pituitary to express core-clock genes, their protein products, and to do so in a 24-hour rhythm. Young Adult CON and CR pituitary gene expression profiles detected potential differential expression in <150 probesets. A decline in>TSHR and CGA was detected in CR macaques as measured by sqRT-PCR. Other genes investigated showed no diet-induced changes. Young Adult CON and CR testicular gene expression profiles detected potential differential expression in <300 probesets although mRNA expression was not altered based on sqRT-PCR and real-time RT-PCR. Age-related>and/or diet-induced changes in HSD17β3, INSL3, CSNK1E and CGA were observed in a separate experiment with CGA in Old Adult CR subjects returning to youthful levels. Semen samples were collected from Young Adult CON and CR macaques. Normal spermiogram measures, ZP-binding, AR assay and SCSA^® were conducted and indicated no differences between CON and CR-treated animals. Both groups exhibited similar daily testosterone profiles with no differences in mean or maximum levels; however, daily minimum testosterone levels were lower in CON animals. It appears that moderate CR had limited impact on neuroendocrine or reproductive function in male rhesus macaques based on our selected endpoints. Thus, advantageous CR health benefits can be achieved without obvious negative consequences to the HPG axis.