Cloning restriction fragments that promote expression of a gene in Bacillus subtilis

Plasmid pPL603 (3.1 megadaltons) specifies neomycin resistance in Bacillus subtilis and contains a structural gene for chloramphenicol acetyltransferase. Cells harboring the plasmid cannot grow on solid media containing 10 microgram of chloramphenicol per ml. Cloning EcoRI (or EcoRI)-generated fragments of deoxyribonucleic acid from several sources into the single EcoRI site in plasmid pPL603, with subsequent selection of transformants of media containing 10 micrograms of chloramphenicol per ml, permits the identification of restriction fragments that promote expression of the chloramphenicol acetyltransferase gene.     

Adenoviral proteins mimic nutrient/growth signals to activate the mTOR pathway for viral replication

Like tumor cells, DNA viruses have had to evolve mechanisms that uncouple cellular replication from the many intra- and extracellular factors that normally control it. Here we show that adenovirus encodes two proteins that activate the mammalian target of rapamycin (mTOR) for viral replication, even under nutrient/growth factor-limiting conditions. E4-ORF1 mimics growth factor signaling by activating PI3-kinase, resulting in increased Rheb.GTP loading and mTOR activation. E4-ORF4 is redundant with glucose in stimulating mTOR, does not affect Rheb.GTP levels and is the major mechanism whereby adenovirus activates mTOR in quiescent primary cells. We demonstrate that mTOR is activated through a mechanism that is dependent on the E4-ORF4 protein phosphatase 2A-binding domain. We also show that mTOR activation is required for efficient S-phase entry, independently of E2F activation, in adenovirus-infected quiescent primary cells. These data reveal that adenovirus has evolved proteins that activate the mTOR pathway, irrespective of the cellular microenvironment, and which play a requisite role in viral replication.     

Additive effect of TAp63 deficiency on the adrenocortical dysplasia (acd) phenotype

Adrenocortical dysplasia (acd) is a spontaneous autosomal recessive mouse mutation exhibiting caudal truncation, vertebral segmentation defects, hydronephrosis, limb hypoplasia, and perinatal lethality. Acd encodes TPP1, a component of the shelterin complex that maintains telomere integrity, and consequently acd mutant mice have telomere dysfunction and genomic instability. We previously showed that apoptosis is the primary mechanism causing the acd skeletal phenotype, and that p53 deficiency rescues the skeletal defects of the acd phenotype but has no effect on the perinatal lethality. The Trp63 gene encodes multiple isoforms, which play a role in proliferation, apoptosis, and stem/progenitor cell maintenance. Different p63 isoforms exhibit both proapoptotic (TAp63) and antiapoptotic (ΔNp63) functions. We hypothesized that deficiency of proapoptotic TAp63 isoforms might rescue the acd skeletal phenotype, similar to our previous observations with deficiency of p53. Mice heterozygous for a null allele of TAp63 were crossed to heterozygous acd mice to determine the effect of TAp63 deficiency on the acd mutant phenotype. In contrast to our results with the acd?×?p53 cross, skeletal anomalies were not rescued by deficiency of TAp63. In fact, the limb and vertebral anomalies observed in double-mutant embryos were more severe than those of embryos with the acd mutation alone, demonstrating a dose-dependent effect. These studies suggest that TAp63 isoforms do not facilitate p53-like apoptosis during development in response to acd-mediated telomere dysfunction and are consistent with the proposed roles of TAp63 in maintaining genomic stability.     

Outcome of the first electron microscopy validation task force meeting

This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.     

Summer Redox Dynamics in a Eutrophic Reservoir and Sensitivity to a Summer’s End Drawdown Event

Ecosystems - In eutrophic lakes and reservoirs, reduced mixing during stratified conditions limits oxygen (O2) supply to the hypolimnion (that is, bottom waters). In the absence of an O2 supply,...     

Population genetics and relatedness in a critically endangered island raptor, Ridgway’s Hawk Buteo ridgwayi

Many island avian populations are of conservation interest because they have a higher risk of extinction than mainland populations. Susceptibility of island birds to extinction is primarily related to human induced change through habitat loss, persecution, and introduction of exotic species, in combination with genetic factors. We used microsatellite profiles from 11 loci to assess genetic diversity and relatedness in the critically endangered hawk Buteo ridgwayi endemic to the island of Hispaniola in the Caribbean. Using samples collected between 2005 and 2009, our results revealed a relatively high level of heterozygosity, evidence of a recent genetic bottleneck, and the occurrence of inbreeding within the population. Pair relatedness analysis found 4 of 7 sampled breeding pairs to be related similar to that of first cousin or greater. Pedigree estimates indicated that up to 18 % of potential pairings would be between individuals with relatedness values similar to that of half-sibling. We discuss our findings in the context of conservation genetic management suggesting both carefully managed translocations and the initiation of a captive population as a safeguard of the remaining genetic diversity.     

The Genome Sequence of Trypanosoma brucei gambiense,Causative Agent of Chronic Human African Trypanosomiasis

Background

Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans.

Methods and Findings

An improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.

Conclusions

This study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.