Development of a SNP genotyping panel for genetic monitoring of the laboratory mouse

We have developed a genotyping system for detecting genetic contamination in the laboratory mouse based on assaying single-nucleotide polymorphism (SNP) markers positioned on all autosomes and the X chromosome. This system provides a fast, reliable, and cost-effective way for genetic monitoring, while maintaining a very high degree of confidence. We describe the allelic distribution of 235 SNPs in 48 mouse strains, thereby creating a database of polymorphisms useful for genotyping purposes. The SNP markers used in this study were chosen from publicly available SNP databases. Four genotyping methods were evaluated, and dynamic two-tube allele-specific PCR assays were developed for each marker and tested on a set of 48 inbred mouse strains. The minimal number of assays sufficient to distinguish groups consisting of different numbers of mouse strains was estimated, and a panel of 28 SNPs sufficient to distinguish virtually all of the inbred strains tested was selected. Amplifluor SNP detection assays were developed for these markers and tested on an extended list of 96 strains. This panel was used as a genetic quality control approach to monitor the genotypes of nearly 300 inbred, wild-derived, congenic, consomic, and recombinant inbred strains maintained at The Jackson Laboratory. We have concluded that this marker panel is sufficient for genetic contamination monitoring in colonies containing a large number of genetically diverse mouse strains and that reduced versions of the panel could be implemented in facilities housing a lower number of strains.     

Identification and Expression of a <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> Collagenase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Extracellular protein profiles were compared for broth-grown cultures of Burkholderia pseudomallei and its avirulent close relative Burkholderia thailandensis. A number of protein bands were present in the B. pseudomallei profile but absent or less abundant in the B. thailandensis profile. Four such prominent secreted proteins were identified by using N-terminal sequencing coupled to searches of the B. pseudomallei genome sequence database. The genes for two proteins with similarity to carbohydrate-binding proteins, and a further protein homologous to known bacterial collagenases, were present in both B. pseudomallei and B. thailandensis. The putative collagenase gene was cloned and expressed as a fusion protein in Escherichia coli. Cell lysates from Escherichia coli containing the recombinant protein exhibited detectable gelatinase and collagenase activities.     

The incidence of Helicobacter pylori acquisition in children of a Canadian First Nations community and the potential for parent-to-child transmission

BACKGROUND AND AIMS: We have previously reported that Wasagamack, a Canadian First Nations community has a seroprevalence rate of Helicobacter pylori of 95% and a prevalence rate among children aged 0-12 years as measured by stool antigen testing of 56%. We aimed to determine the rate of infection acquisition and possible modes of transmission of childhood Helicobacter pylori infection in this Canadian First Nations community. METHODS: Children who were previously negative for H. pylori by stool antigen testing in August 1999 were eligible for enrollment in August 2000; 50 (77%) eligible children underwent stool collection. H. pylori stool antigen status was tested using the Premier Platinum HpSA test. Drinking water samples, maternal saliva, breast milk, local berries and flies were tested by three complementary H. pylori-specific PCR assays. Soothers or bottle nipples, collected from 16 children whose H. pylori stool antigen status was determined, were bathed in sterile water and this water was tested by PCR. RESULTS: Stool was positive for H. pylori in 16% (8/ 50) of children retested. Five had no other siblings infected and three had infected siblings. The mothers of all children infected were positive for H. pylori. The median age of newly infected children was 6 years (range 1-13 years). By PCR, 78% (18/23) mothers' saliva samples, 69% (11/16) soother water samples and 9% (1/11) water samples from infected homes tested positive. All of 24 sequenced PCR-produced DNA fragments from samples showed 99% homology with that from ATCC type strain H. pylori. CONCLUSIONS: The rate of childhood H. pylori acquisition was 16% over 1 year, and was not dependent on number of siblings infected. The finding of homologous H. pylori DNA in saliva and in soother water suggests the possibility of human to human transmission, particularly via an oral-oral route. Thus, there is the potential for further investigations in this population and other endemic communities that are directed at prevention of infection transmission via this modality.     

Structure and functional analysis of the fungal galectin CGL2

Recognition of and discrimination between potential glyco-substrates is central to the function of galectins. Here we dissect the fundamental parameters responsible for such selectivity by the fungal representative, CGL2. The 2.1 A crystal structure of CGL2 and five substrate complexes reveal that this prototype galectin achieves increased substrate specificity by accommodating substituted oligosaccharides of the mammalian blood group A/B type in an extended binding cleft. Kinetic studies on wild-type and mutant CGL2 proteins demonstrate that the tetrameric organization is essential for functionality. The geometric constraints due to the orthogonal orientation of the four binding sites have important consequences on substrate binding and selectivity.     

Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.     

A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase

A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins.     

The Tat protein export pathway

The Tat (twin-arginine translocation) system is a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a characteristic sequence motif, which includes consecutive arginine residues. Here, we review recent progress on the characterization of the Tat system and critically discuss the structure and operation of this major new bacterial protein export pathway.     

Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins

The survival of Escherichia coli following treatment with a low dose (1-3 mM) of hydrogen peroxide (H(2)O(2)) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H(2)O(2)-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair. To make this determination, we examined mode-one killing of DeltarecA cells carrying plasmid-borne recA (P(tac)-recA(+)) and constitutively expressing a fully induced level of wild-type RecA protein when SOS genes other than recA are non-inducible in a lexA3 (Ind(-)) genetic background or inducible in a lexA(+) background. At a H(2)O(2) dose resulting in maximal killing, DeltarecA lexA3 (Ind(-)) cells with P(tac)-recA(+) show 40-fold greater survival than lexA3 (Ind(-)) cells with chromosomal recA having a low, non-induced level of RecA protein. However, they still show 10- to 15-fold lower survival than wild-type cells and DeltarecA lexA(+) cells with P(tac)-recA(+). To determine if the inducible RuvA protein stimulates survival, we examined a ruvA60 mutant that is defective for the repair of UV-induced DNA damage. This mutant also shows 10- to 15-fold lower survival than wild-type cells. We conclude that while induction of RecA protein has a pronounced stimulatory effect on the recombinational repair of H(2)O(2)-induced oxidative DNA damage, the induction of other SOS proteins such as RuvA is essential for wild-type repair.