Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing

Background: In the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization. Results: To demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination events. Most private SNPs (that is, a, variant found in only one strain in this set) selected for validation by Sanger sequencing were confirmed, although rare falsepositive SNPs were associated with variable nucleotide tandem repeats. Conclusions: The high-throughput, multiplexing capability, and accuracy of this system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation. By interrogating nearly every base of the genome, rare polymorphisms can be reliably discovered, thus facilitating high-resolution strain tracking and strengthening forensic attribution

An efficient and high-throughput approach for experimental validation of novel human gene predictions

AbstractA highly automated RT-PCR-based approach has been established to validate novel human gene predictions with no prior experimental evidence of mRNA splicing (ab initio predictions). Ab initio gene predictions were selected for high-throughput validation using predicted protein classification, sequence similarity to other genomes, colocalization with an MPSS tag, or microarray expression. Initial microarray prioritization followed by RT-PCR validation was the most efficient combination, resulting in approximately 35% of the ab initio predictions being validated by RT-PCR. Of the 7252 novel genes that were prioritized and processed, 796 constituted real transcripts. In addition, high-throughput RACE successfully extended the 5′ and/or 3′ ends of >60% of RT-PCR-validated genes. Reevaluation of these transcripts produced 574 novel transcripts using RefSeq as a reference. RT-PCR sequencing in combination with RACE on ab initio gene predictions could be used to define the transcriptome across all species

Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR® Yfiler® PCR amplification kit

The Y-chromosomal short tandem repeat (Y-STR) polymorphisms included in the AmpFlSTR® Yfiler® polymerase chain reaction amplification kit have become widely used for forensic and evolutionary applications where a reliable knowledge on mutation properties is necessary for correct data interpretation. Therefore, we investigated the 17 Yfiler Y-STRs in 1,730–1,764 DNA-confirmed father–son pairs per locus and found 84 sequence-confirmed mutations among the 29,792 meiotic transfers covered. Of the 84 mutations, 83 (98.8%) were single-repeat changes and one (1.2%) was a double-repeat change (ratio, 1:0.01), as well as 43 (51.2%) were repeat gains and 41 (48.8%) repeat losses (ratio, 1:0.95). Medians from Bayesian estimation of locus-specific mutation rates ranged from 0.0003 for DYS448 to 0.0074 for DYS458, with a median rate across all 17 Y-STRs of 0.0025. The mean age (at the time of son’s birth) of fathers with mutations was with 34.40 (±11.63) years higher than that of fathers without ones at 30.32 (±10.22) years, a difference that is highly statistically significant (p < 0.001). A Poisson-based modeling revealed that the Y-STR mutation rate increased with increasing father’s age on a statistically significant level (α = 0.0294, 2.5% quantile = 0.0001). From combining our data with those previously published, considering all together 135,212 meiotic events and 331 mutations, we conclude for the Yfiler Y-STRs that (1) none had a mutation rate of >1%, 12 had mutation rates of >0.1% and four of <0.1%, (2) single-repeat changes were strongly favored over multiple-repeat ones for all loci but 1 and (3) considerable variation existed among loci in the ratio of repeat gains versus losses. Our finding of three Y-STR mutations in one father–son pair (and two pairs with two mutations each) has consequences for determining the threshold of allelic differences to conclude exclusion constellations in future applications of Y-STRs in paternity testing and pedigree analyses

Purification, characterization and localization of an endogenous lectin from quail intestine

Bibliography: p. 94-104.Soluble extracts of quail intestinal mucosa contain a lectin activity specific for chicken and rabbit, trypsinized?? glutaraldehyde-fixed erythrocytes. This lectin activity could be totally inhibited by lac­tose and mucin. Quail lectin was first purified by affinity chromato­graphy on either asialofetuin-Sepharose or mucin-Sepharose, followed by DEAE-Sepharose chromatography. Silver stains of SOS-PAGE revealed a single 14.5 kDaprotein. Gel filtration on Sephadex G-200 also yielded an apparent molecular weight of 14.5 kDa. suggesting the lectin is in a monomeric form. Isoelectric focusing of purified quail gut lectin resulted in its separation into a major band at pl 6.2 and four trace bands at 6.25, 6.27, 6.38 and 6.45. Immunochemical localization of quail gut lectin in the intestine was carried out with polyclonal anti­body raised in rabbits and tested for specificity on Western blots. Immunohistochemical staining showed that the lectin is present in the secretion coating the mucosal surface of the small bowel and in goblet cells. Lectin could be purified from all sections of intestine and localized to goblet cells in all cases

Studies of the role(s) of galectin in quail intestinal mucin organization and secretion

Bibliography: p. 251-271.A program of studies was undertaken to explore the role(s) played by galectins at the mucosal surface. The first phase of this project involved purification and characterization of intestinal galectins from rabbit, rat, and quail. A family of galectins were found in mammalian intestinal tissues, however, only a single galectin was identified from the quail. In the second stage of this project, quail intestinal mucin was purified and the biochemical and immunological properties were characterized. Quail mucin was purified by two sequential isopycnic density-gradient centrifugations in CsCl followed by gel filtration chromatography. Purified quail mucin was then characterized by SDS-PAGE, amino acid and carbohydrate composition analyses, immuno-cross reactivity with mammalian mucin antibodies and sensitivity to disulphide-reduction, H2O2 , and trypsin. Quail mucin was revealed to be high molecular weight polymer composed of large heterogeneous glycoprotein monomers and a 110 kD 'link' protein, held together by disulphide bonds. Quail mucin was further identified as a putative ligand for quail galectin in binding studies and through immunohistochemical co-localization in goblet cells. The quail galectin binds quail mucin through the B-galactose residues on the carbohydrate side chains of quail mucin as shown by hapten inhibition studies. A putative membrane receptor of quail galectin on the brush border membrane of the quail small intestine was also detected by radioactive ligand binding assays. The regulation of mucin secretion from the quail small intestine was investigated using in vitro organ culture. Mucin secretion was enhanced by cholera toxin and regulated in a similar fashion to mammalian mucin; being stimulated by cholinergic but not adrenergic stimuli and being sensitive to the activation of protein kinase C and changes in Ca2 + levels. Quail galectin seems to serve as an adhesion molecule for extracellular mucus gel adherence to the epithelial surfaces and maybe also serve as a control for mucin release, which is evidenced by the following discoveries: (1) lactose could release both quail galectin and quail mucin from intestinal tissue; (2) blocking of galectin binding to the mucosal surfaces with antiserum against quail galectin could induce quail mucin release; and (3) binding the mucosal receptors for quail galectin with plant lectins could specifically reduce the rate of baseline secretion of quail mucin. However, lactose-induced mucin release was independent of the muscarinic receptor, protein kinase C and Ca2 +, suggesting that quail galectin may control mucin release through a unique pathway

Pdx1 inactivation restricted to the intestinal epithelium in mice alters duodenal gene expression in enterocytes and enteroendocrine cells

Null mutant mice lacking the transcription factor pancreatic and duodenal homeobox 1 (Pdx1) are apancreatic and survive only a few days after birth. The role of Pdx1 in regulating intestinal gene expression has therefore yet to be determined in viable mice with normal pancreatic development. We hypothesized that conditional inactivation of Pdx1 restricted to the intestinal epithelium would alter intestinal gene expression and cell differentiation. Pdx1flox/flox;VilCre mice with intestine-specific Pdx1 inactivation were generated by crossing a transgenic mouse strain expressing Cre recombinase, driven by a mouse villin 1 gene promoter fragment, with a mutant mouse strain homozygous for loxP site-flanked Pdx1. Pdx1 protein is undetectable in all epithelial cells in the intestinal epithelium of Pdx1flox/flox;VilCre mice. Goblet cell number and mRNA abundance for mucin 3 and mucin 13 genes in the proximal small intestine are comparable between Pdx1flox/flox;VilCre and control mice. Similarly, Paneth cell number and expression of Paneth cell-related genes Defa1, Defcr-rs1, and Mmp7 in the proximal small intestine remain statistically unchanged by Pdx1 inactivation. Although the number of enteroendocrine cells expressing chromogranin A/B, gastric inhibitory polypeptide (Gip), or somatostatin (Sst) is unaffected in the Pdx1flox/flox;VilCre mice, mRNA abundance for Gip and Sst is significantly reduced in the proximal small intestine. Conditional Pdx1 inactivation attenuates intestinal alkaline phosphatase (IAP) activity in the duodenal epithelium, consistent with an average 91% decrease in expression of the mouse enterocyte IAP gene, alkaline phosphatase 3 (a novel Pdx1 target candidate), in the proximal small intestine following Pdx1 inactivation. We conclude that Pdx1 is necessary for patterning appropriate gene expression in enterocytes and enteroendocrine cells of the proximal small intestine