Detection of femtomolar quantities of the ganglioside GM1 on thin-layer chromatography plates by native choleratoxin and labeled antisera

After separation of gangliosides by thin-layer chromatography, femtomolar quantities of G_M1 were detected by incubating the plate with native choleratoxin, followed by anticholeratoxin and species-specific labeled antiserum. Only stable reagents were involved when antiserum labeled with horseradish peroxidase was used. Native choleratoxin rather than iodinelabeled toxin ensured good reproducibility of the method.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi)

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3- crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.      

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

Evidence for gene conversion in the amylase multigene family of Drosophila pseudoobscura

The alpha-amylase (Amy) multigene family in Drosophila pseudoobscura is located on the third chromosome, which is polymorphic for more than 40 inverted gene arrangements. The number of copies in this family ranges from one to three, depending on the arrangement in question. A previous study of the three Amy genes from the Standard (ST) arrangement suggested either that duplicated copies (Amy2 and Amy3) are functionally constrained or that they are undergoing gene conversion with Amy1. In order to elucidate further the pattern of molecular evolution in this family, we cloned and sequenced four additional Amy genes, two from the Santa Cruz (SC) and two from the Chiricahua (CH) gene arrangement. Of the two alternatives, only the hypothesis of gene conversion is supported by the sequence analysis. The homogenization effect of gene conversion has been strongest in SC, whose copies differ by only two nucleotides, less noticeable in ST, and negligible in the CH. Furthermore, the action of gene conversion is apparently localized, occurring only in the coding region. Interestingly, these results concur with the findings of other workers for the duplicated Amy genes in the Drosophila melanogaster group. Thus, the occurrence of gene conversion in the Amy multigene family seems to be a common feature in the Drosophila species studied so far.      

Evaluation of a real-time PCR kit for detecting Escherichia coli O157 in bovine fecal samples

A commercially available real-time, rapid PCR test was evaluated for its ability to detect Escherichia coli O157. Both the sensitivity and specificity of the assay were 99% for isolates in pure culture. The assay detected 1 CFU of E. coli O157:H7 g(-1) in artificially inoculated bovine feces following enrichment.     

Prion gene sequence variation within diverse groups of U.S. sheep,beef cattle,and deer

Prions are proteins that play a central role in transmissible spongiform encephalopathies in a variety of mammals. Among the most notable prion disorders in ungulates are scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer. Single nucleotide polymorphisms in the sheep prion gene (PRNP) have been correlated with susceptibility to natural scrapie in some populations. Similar correlations have not been reported in cattle or deer; however, characterization of PRNP nucleotide diversity in those species is incomplete. This report describes nucleotide sequence variation and frequency estimates for the PRNP locus within diverse groups of U.S. sheep, U.S. beef cattle, and free-ranging deer (Odocoileus virginianus and O. hemionus from Wyoming). DNA segments corresponding to the complete prion coding sequence and a 596-bp portion of the PRNP promoter region were amplified and sequenced from DNA panels with 90 sheep, 96 cattle, and 94 deer. Each panel was designed to contain the most diverse germplasm available from their respective populations to facilitate polymorphism detection. Sequence comparisons identified a total of 86 polymorphisms. Previously unreported polymorphisms were identified in sheep (9), cattle (13), and deer (32). The number of individuals sampled within each population was sufficient to detect more than 95% of all alleles present at a frequency greater than 0.02. The estimation of PRNP allele and genotype frequencies within these diverse groups of sheep, cattle, and deer provides a framework for designing accurate genotype assays for use in genetic epidemiology, allele management, and disease control.     

Animal-to-Animal Variation in Fecal Microbial Diversity among Beef Cattle

The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.The gastrointestinal tracts (GIT) of beef cattle are colonized by microorganisms that profoundly impact animal physiology, nutrition, health, and productivity (5). The GIT microbiota potentially impact food safety via pathogen shedding (13) by interacting with organisms such as Salmonella and competing for resources in the GIT. Cattle intestinal microbiota also play an important role in methane emissions, with U.S. beef cattle alone contributing an estimated 3.87 million metric tons of methane into the environment each year, both from rumen and large-intestine fermentations (7). Although the bovine fecal microbiota have been well characterized using culture-based methods, these techniques are necessarily limited to characterizing bacteria that can be grown in the laboratory. Culture-independent methods can reveal community members that are recalcitrant to culture. Only a handful of deep-sequencing studies have been done using culture-independent 16S rRNA-based methods (1, 11, 12, 14), all with dairy cattle, which have a fundamentally different diet and metabolism from beef cattle. Despite the potential contributions of the beef cattle GIT microbiota to animal health, food safety, and global warming, these communities remain poorly characterized. With the advent of pyrosequencing technology, researchers now have the tools to characterize these important communities. Pyrosequencing will allow rapid characterization of large-sample data sets (1). However, the taxonomic information generated by rapid sequencing is approximate by necessity (9), and full-length 16S-rRNA sequencing remains the “gold standard” method. Accordingly, we have characterized fecal bacteria from six feedlot cattle by full-length capillary sequence analysis of 11,171 16S rRNA gene clones (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Bacterial diversity of six feedlot beef cattle. Gray bars represent the percentages of all 16S sequences that were assigned to each taxonomy. Colored dots represent the percentages of 16S sequences from each library that were assigned to each taxonomic group. Asterisks indicate unclassified members of the named taxon. Panel A shows the data for the first 99% of all the sequences. Panel B shows the data for the remaining 1% of sequences. Note differences in scales for panels A and B.Rectal grab fecal samples (n = 6) were collected according to institutional animal care guidelines. All animals were female cross-bred MARCIII beef heifers, 6 to 8 months of age, 214 to 241 kg, housed in the same feedlot pen for 2 months prior to fecal collection, and fed the same typical feedlot beef production growing rations consisting of 61.6% corn silage (41.3% dry matter), 15.2% alfalfa hay, 20.9% corn, and 2.3% liquid supplement.Total fecal DNA was isolated from homogenized samples using MoBio UltraClean fecal kit (Carlsbad, CA). PCR was performed using 27F and 1392R primers (11). Amplification consisted of 25 cycles, with an annealing temperature of 55°C. Amplicons from three reactions per sample were pooled (8), cloned using the Invitrogen TOPO TA cloning kit (Carlsbad, CA), and sequenced bidirectionally with M13 primers using an ABI 3700 sequencer (17). Low-quality and chimeric sequences (6) were excluded from further analysis. Distance matrices were compiled from ClustalW alignments (18) in PHYLIP (4). Pairwise estimates of shared richness were calculated using EstimateS, version 8.2 (R. K. Colwell; http://purl.oclc.org/estimates). DOTUR (16) was used to identify operational taxonomic units (OTUs) and to generate rarefaction curves (Fig. ​(Fig.2),2), richness and evenness estimates, and Shannon''s and Simpson''s diversity indices (Table ​(Table1).1). A 97% similarity cutoff and an 85% similarity cutoff for estimating OTUs were used to approximate species and class-level designations (15). Taxonomies were assigned to one member of each OTU using the RDP “classifier” tool (19), and the RDP taxonomic information was used for Fig. ​Fig.11 and ​and3.3. Common bovine taxa were identified based on inclusion in all three U.S. culture-independent studies (this study and references 1 and 11).Open in a separate windowFIG. 2.Rarefaction curves for six feedlot beef cattle. OTUs were assigned at the 85% DNA sequence similarity level. For comparison purposes, all six curves were truncated after 1,321 sequences.Open in a separate windowFIG. 3.Phylum-level distribution of bacterial sequences from six beef feedlot cattle. Asterisks indicate unclassified members of the named taxon.

TABLE 1.

Richness and diversity indices for 6 beef feedlot cattle

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.