Published Gene Sequences Research Articles

Prevalence and clinical significance of SEN virus infection in patients with non A-E hepatitis and volunteer blood donors in Shanghai

To explore the prevalence of SEN virus (SENV) in patients with non A-E hepatitis and volunteer blood donors in Shanghai. According to the published gene sequences, primers from the conserved region were designed. Then, the prevalence of SEN virus in 30 samples from healthy voluntary blood donors and 30 samples from patients with non A-E hepatitis were detected by nested-PCR of SENV-D/H. Some PCR products were cloned and sequenced. The specificity of genotype-specific PCR was confirmed by sequencing, the SENV DNA was detected in 53.3% of the patients with non A-E hepatitis and 10% of the blood donors. The prevalence of SENV-D/H viremia was significantly higher in patients with non A-E hepatitis than in blood donors (P = 0.0002). SENV-H subtype and SENV-D subtype were found in 2 and 1 samples, respectively from blood donors. SENV-H subtype, SENV D subtype, mixed SENV-D and SENV-H subtype were found in 8, 6 and 2 samples, respectively, from patients with non A-E hepatitis. The gene type of SENV in patients with non A-E hepatitis and blood donors in shanghai is D or H subtype, and transfusion is not the only transmitting form of SENV.

Protein-sequence polymorphisms and post-translational modifications in proteins from human saliva using top-down Fourier-transform ion cyclotron resonance mass spectrometry

Single nucleotide polymorphisms (SNPs) can result in protein-sequence polymorphisms (PSPs) when codon translations are altered. Both top-down and bottom-up proteomics strategies can identify PSPs, but only if databases and software are used with this in mind. A 14,319 Da protein from human saliva was characterized using the top-down approach on a hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometer equipped for both collisionally activated (CAD) and electron-capture (ECD) dissociation. Sequence tags identified the protein as Cystatin SN, and defined the N-terminal signal peptide cleavage site, as well as two disulfide bonds, in agreement with previous studies. The mass of the intact protein (<5 ppm error) deviated from that calculated from the published gene sequence by 16.031 Da, and, based on CAD and ECD fragment ion assignments, it was concluded that the isoform of the protein analyzed carried a PSP at residue 11 such that the Pro translated from the genome was in fact Leu/Ile. An independently determined SNP (rs2070856) subsequently confirmed the genetic basis of the mass spectral interpretation and defined the residue as Leu. In another example, the PRP3 protein with mass ∼10,999 Da was found to be an isomeric/isobaric mixture of the reported sequence with PSPs D4N or D50N (rs1049112). Both CAD and ECD datasets support two phosphorylation sites at residues Ser8 and Ser22, rather than Ser17. In the context of discovery proteomics, previously undefined PSPs and PTMs will only be detected if the logic of data processing strategies considers their presence in an unbiased fashion.

High-resolution phylogenetic analysis of hepatitis C virus adaptation and its relationship to disease progression.

Hepatitis C virus (HCV) persists in the majority of those infected despite host immune responses. Evidence has accrued that selectively fixed mutations in the envelope genes (E1 and E2) are associated with viral persistence, particularly those that occur within the first hypervariable region of E2 (HVR1). However, the individual amino acid residues under selection have not been identified, nor have their selection pressures been measured, despite the importance of this information for understanding disease pathogenesis and for vaccine design. We performed a high-resolution analysis of published gene sequence data from individuals undergoing acute HCV infection, employing two phylogenetic methods to determine site-specific selection pressures. Strikingly, we found a statistically significant association between the number of sites selected and disease outcome, with the fewest selected sites in fulminant HCV cases and the greatest number of selected sites in rapid progressors, reflecting the duration and intensity of the arms race between host and virus. Moreover, sites outside the HVR1 appear to play a major role in viral evolution and pathogenesis, although there was no association between viral persistence and specific mutations in E1 and E2. Our analysis therefore allows fine dissection of immune selection pressures, which may be more diverse than previously thought. Such analyses could play a similarly informative role in studies of other persistent virus infections, such as human immunodeficiency virus.

Prevalence of a newly identified SEN virus in China.

To establish nested-PCR methods for the detection of SENV-D and SENV-H and to investigate the epidemiology of SEN virus in China. According to published gene sequences, primers from the conserved region were designed. Then, 135 samples from healthy voluntary blood donors and 242 samples from patients with various forms of liver disease were detected by nested-PCR of SENV-D/H. Some PCR products were cloned and sequenced. By sequencing, the specificity of genotype-specific PCR was confirmed. SENV-D/H DNA was detected in 31% of the blood donors, which was higher than those in America and Italy (2%), and in Japan and Taiwan (15-20%). The prevalence of SENV-D/H viremia was significantly higher in patients with hepatitis B and hepatitis C than in blood donors (59-85% vs 31%, P<0.05). The prevalence among patients with non-A-E hepatitis was significantly higher than among blood donors (68% vs 31%, P<0.01), and equivalent to that among patients with hepatitis B and C. Nested-PCR with genotype-specific primers could serve as a useful SENV screening assay. SENV has the same transmission modes as HBV and HCV. The high prevalence in patients with non-A-E hepatitis may attribute to the transmission modes, and SENV may not serve as the causative agents.

Identification of Genes Up-Regulated in Urothelial Tumors: The 67-kd Laminin Receptor and Tumor-Associated Trypsin Inhibitor

Studies investigating changes in gene expression in urothelial carcinoma have generally compared tumors of different stages and grades but comparisons between low-grade, noninvasive tumors and normal urothelium are needed to identify genes involved in early tumor development. We isolated the urothelium from a low-grade tumor and corresponding normal mucosa by laser capture microdissection on frozen sections. The RNA extracted was amplified to generate suppressive subtractive cDNA libraries. Random sequencing of cDNA clones identified approximately 100 unique species. Of these 83% were known genes, 15% had homology to genes with an unknown function in humans, and 2% did not show homology to any published gene sequence. Two of the known genes, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), had previously been associated with metastatic progression in many tumor types, although 67LR has not been investigated in urothelial tumors. Immunolabeling of the original tissue with antibodies against these two genes confirmed overexpression, validating our strategy: 67LR was not expressed in the normal urothelium but was present in the tumor, whereas TATI expression was confined to umbrella cells in the normal urothelium, but extended to all cell layers in the tumor. We investigated both markers further in a separate series of tumors of different stages and grades. TATI was more consistently overexpressed than 67LR in all tumor grades and stages. Levels of secreted TATI were significantly higher in urine samples from patients with tumors compared to controls. Our strategy, combining laser capture microdissection and cDNA library construction, has identified genes that may be involved in the early phases of urothelial tumor development rather than with disease progression, highlighting the importance of comparing tumor with normal rather than just tumors of different stages and grades.

Identification of a Novel Testicular Orphan Receptor-4 (TR4)-associated Protein as Repressor for the Selective Suppression of TR4-mediated Transactivation

Although many co-activators have been identified for various nuclear receptors, relatively fewer co-repressors have been isolated and characterized. Here we report the identification of a novel testicular orphan nuclear receptor-4 (TR4)-associated protein (TRA16) that is mainly localized in the nucleus of cells as a repressor to suppress TR4-mediated transactivation. The suppression of TR4-mediated transactivation is selective because TRA16 shows only a slight influence on the transactivation of androgen receptor, glucocorticoid receptor, and progesterone receptor. Sequence analysis shows that TRA16 is a novel gene with 139 amino acids in an open reading frame with a molecular mass of 16 kDa, which did not match any published gene sequences. Mammalian two-hybrid system and co-immunoprecipitation assays both demonstrate that TRA16 can interact strongly with TR4. The electrophoretic mobility shift assay suggests that TRA16 may suppress TR4-mediated transactivation via decreased binding between the TR4 protein and the TR4 response element on the target gene(s). Furthermore, TRA16 can also block the interaction between TR4 and TR4 ligand-binding domain through interacting with TR4-DNA-binding and ligand-binding domains. These unique suppression mechanisms suggest that TRA16 may function as a novel repressor to selectively suppress the TR4-mediated transactivation.

Glutamate dehydrogenase of Halobacterium salinarum: evidence that the gene sequence currently assigned to the NADP +-dependent enzyme is in fact that of the NAD +-dependent glutamate dehydrogenase

A GDH gene from Halobacterium salinarum has been cloned and sequenced and the publication assigns the sequence to the NADP +-glutamate dehydrogenase of this organism. We have expressed this gene in Escherichia coli and find that it encodes an NAD +-dependent glutamate dehydrogenase without activity towards NADP +. Further, peptide sequence from the two corresponding proteins supports the view that the deposited sequence is indeed that of the NAD +-dependent glutamate dehydrogenase. Sequence from the NAD +-dependent protein matches the published gene sequence, whereas sequence from the NADP + glutamate dehydrogenase does not.

Glutamate dehydrogenase of Halobacterium salinarum: evidence that the gene sequence currently assigned to the NADP+-dependent enzyme is in fact that of the NAD+-dependent glutamate dehydrogenase.

A GDH gene from Halobacterium salinarum has been cloned and sequenced and the publication assigns the sequence to the NADP+-glutamate dehydrogenase of this organism. We have expressed this gene in Escherichia coli and find that it encodes an NAD+-dependent glutamate dehydrogenase without activity towards NADP+. Further, peptide sequence from the two corresponding proteins supports the view that the deposited sequence is indeed that of the NAD+-dependent glutamate dehydrogenase. Sequence from the NAD+-dependent protein matches the published gene sequence, whereas sequence from the NADP+ glutamate dehydrogenase does not.

Characterization of Escherichia coli MoeB and Its Involvement in the Activation of Molybdopterin Synthase for the Biosynthesis of the Molybdenum Cofactor

Amino acid sequence comparisons of Escherichia coli MoeB suggested that the MoeB-dependent formation of a C-terminal thiocarboxylate on the MoaD subunit of molybdopterin synthase might resemble the ubiquitin-activating step in the ubiquitin-targeted degradation of proteins in eukaryotes. To determine the exact role of MoeB in molybdopterin biosynthesis, the protein was purified after homologous overexpression. Using purified proteins, we have demonstrated the ATP-dependent formation of a complex of MoeB and MoaD adenylate that is stable to gel filtration. Mass spectrometry of the complex revealed a peak of a molecular mass of 9,073 Da, the expected mass of MoaD adenylate. However, unlike the ubiquitin activation reaction, the formation of a thioester intermediate between MoeB and MoaD could not be observed. There was also no evidence for a MoeB-bound sulfur during the sulfuration of MoaD. Amino acid substitutions were generated in every cysteine residue in MoeB. All of these exhibited activity comparable to the wild type, with the exception of mutations in cysteine residues located in putative Zn-binding motifs. For these cysteines, loss of activity correlated with loss of metal binding.

Strain-based sequence variations and structure analysis of murine prostate specific spermine binding protein using mass spectrometry.

Mouse spermine binding protein (SBP) has been characterized using mass spectrometry, including its localization within the prostate, sequence verification, and its posttranslational modifications. MALDI (matrix-assisted laser desorption/ionization) mass spectrometry was employed for localization of proteins expressed by different lobes of the mouse prostate obtained after tissue blotting on a polyethylene membrane. The mass spectra showed complex protein profiles that were different for each lobe of the prostate. The prostate-specific spermine binding protein (SBP), primarily identified by its in-source decay fragment ion signals, was found predominantly expressed by the ventral lobe of the prostate. The MALDI in-source decay measurements combined with nanoESI (nanoelectrospay ionization) MS/MS measurements obtained after specific proteolysis of SBP, allowed the exact positioning of a single N-linked carbohydrate group, and the identification of a pyroglutamate residue at the sequence N-terminus. The N-linked carbohydrate component was further investigated and the general pattern of the N-linked carbohydrate identified. The presence of a disulfide bridge between cysteine78 and cysteine124 was also established. The full sequence characterization of SBP showed several strain-based sequence differences when compared to the published gene sequence.

Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9.

The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp). The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain. This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP. The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC. However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT). In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9. This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323. Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found. In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence. We also discuss these results in comparison with those from recombinant ACT expressed in E. coli strain K12 XL1-Blue. The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Molecular Evolution near a Two-locus Balanced Polymorphism

Balancing selection at one locus can increase the amount of selectively neutral variation within neighboring genomic regions. Discrete phenotypic polymorphisms studied in natural populations are frequently determined by sets of interacting genes instead of alternative alleles at single loci. We extend coalescent theory to investigate balancing selection on combinations of linked genes. We find that variation at neutral sites is increased across a much larger genomic region relative to the single-locus models: the entire region lying between the two loci in balanced combination is affected to some degree. Epistatic selection maintains these high levels of neutral variation because it directly opposes the homogenizing effect of recombination. The results of the theory are discussed in relation to published gene sequence data, primarily from Drosophila.

Molecular analysis of the virulence determinants of Clostridium perfringens associated with foal diarrhoea

During an epidemiological study of foal diarrhoea, over half of the cases yielded Clostridium perfringens which was significantly associated with disease (Netherwood et al., 1996b). However, the association could not be accounted for by enterotoxigenic isolates which had a low prevalence (Netherwood et al., 1997). Nonetheless, we have hypothesized that the association may be caused by a pathogenic sub-population which would be significantly more common amongst C. perfringens-positive cases compared with C. perfringens-positive healthy controls if it acted as a pathogen when present. Conversely, if foal diarrhoea caused by C. perfringens was dependent on a predisposing factor, then such an association might not be evident. As a first step to determine if a molecular marker was more frequently to be found in C. perfringens-positive cases than controls, we have genotyped the study isolates (up to five per foal) by polymerase chain reaction (PCR) based on the published gene sequences for the major lethal toxins alpha, beta, epsilon and iota as well as for theta toxin, large and small sialidases, hyaluronidase and virulence regulation. Isolates of major toxin types B, C, D and E, or isolates which were untypeable, were isolated from less than 15% of C. perfringens-positive foals and these were not associated with diarrhoea nor were they more commonly found in C. perfringens-positive cases. Isolates of type A were found in more than 90% of all C. perfringens-positive foals. A number of different genotypes were identified by their different patterns of gene possession but types without any of the genes for theta toxin, large and small sialidases, hyaluronidase and virulence regulation were found in only 10% of positive foals. Only type A isolates with all of these genes were associated with diarrhoea overall but they were not more commonly isolated from C. perfringens-positive cases than controls. In conclusion, genotyping by the sequenced virulence genes did not identify a marker for a sub-population of C. perfringens which may be acting more frequently as a pathogen when present.

Detection and identification of ruminant and porcine pestiviruses by nested amplification of 5′ untranslated cDNA regions

Based on published gene sequences of bovine viral diarrhoea virus (BVDV) type I and classical swine fever virus (CSFV), genus- and species-specific primers were designed to detect and identify pestivirus cDNA sequences in a nested polymerase chain reaction (PCR). The PCR primers were validated using cDNA synthesized from 146 pestivirus isolates, comprising representatives of all four so far described genotypes (BVDV type I, BVDV type II, CSFV and border disease virus), as well as others of uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 cattle isolates, including 5 typed antigenically as BVDV type II were amplified by the internal BVDV-specific primers, but not the CSFV-specific primers. The same result was found for other BVDV type I and II viruses isolated from sheep and pigs. Seventy-seven CSF viruses were amplified by their respective internal primers. Available information strongly indicate that 4 CSF viruses also amplified by the BVDV-specific primers had been contaminated with BVDV in cell cultures. Border disease viruses were mostly not detected by the BVDV-specific primers, but were detected weakly by the CSFV-specific primer pair. Using carrier RNA for extraction of viral RNA, the sensitivity of detection of the single and nested PCR was, respectively, 5 and 50 times higher than obtained with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified three earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples.

Association between fulminant hepatic failure and a strain of GBV virus C

The GB virus C (GBV-C) and the hepatitis G virus (HGV) have been detected in patients with acute indeterminant hepatitis and post-transfusion hepatitis. However, the role of the new hepatitis viruses in the aetiology of fulminant hepatitis is little understood. We investigated the presence of GBV-C/HGV in patients with fulminant hepatic failure. Serum samples from 22 German patients with fulminant hepatic failure and 106 symptom-free blood donors (controls) were studied for presence of GBV-C RNA by seminested reverse transcriptase PCR. Primer sequences were derived from the published gene sequences of the conserved NS3 region of the GBV-C prototype and the published isolates. Nucleotide and amino acid sequences of GBV-C-positive isolates, the control RNA, and the published HGV and GBV-C prototype sequences were compared by multiple sequence alignment. We also compared the GBV-C sequences of virus-positive patients who had fulminant hepatic failure with those of 19 patients with chronic hepatitis from our centre. In addition, we searched databases and published papers for further GBV-C helicase sequences in patients with non-fulminant hepatitis. GBV-C RNA was detected in 11 (50%) of the 22 patients with fulminant hepatic failure and in five (4.7%) of 106 control-group blood donors. Among the patients with fulminant hepatic failure, six of seven with fulminant hepatitis B and five of ten with fulminant non-A-E hepatitis were positive for GBV-C RNA. Analysis of nucleic acid sequences showed six mutations at defined positions in all 11 patients with fulminant hepatic failure who were positive for GBV-C. None of these mutations were found in the five GBV-C-positive control-group blood donors. Of the six nucleotide changes, four caused no amino acid changes, whereas two mutations at position 100 (G to T) and 102 (T to C) led to an alanine to serine change in the predicted translation product. However, comparison with GBV-C sequences of patients with non-fulminant hepatitis showed that this amino acid mutation was not specific for fulminant hepatic failure. The sequence-motif containing the six nucleotide mutations detected in all patients with fulminant hepatic failure was found in only two of 19 German patients with chronic hepatitis from our centre, and in only one of 88 GBV-C sequences from non-fulminant patients reported by others. The frequency of GBV-C RNA is higher in fulminant hepatic failure than in any other group of patients with hepatitis, particularly in patients with fulminant hepatitis B or fulminant non-A-E hepatitis. A specific strain of GBV-C may occur in serum of German patients with fulminant hepatic failure.

Metallothionein isoform analysis by liquid chromatography-electrospray ionisation mass spectrometry.

Metallothionein (MT) is a class of small metal-binding proteins [ l ] whose mass can vary from under 6OOO to over 7000 daltons depending on a) variation in non-conserved amino acid regions b) the degree of metal saturation and c) the species of metal bound. Many mammalian species have several functioning genes of MT and accurate mass values of the corresponding protein isoforms can be deduced from published gene sequences. However, the translated product of each gene has yet to be confirmed in purified MT protein samples because the isoforms can be difficult to separate by conventional chromatography or electrophoresis. In the case of sheep which have at least 4 functioning MT genes [2], the deduced N-terminally acetylated apoprotein masses are 5993 (MT-la), 6023 (MT-lb), 6027 (MT-lc) and 6070 (MT-2). The objective of this work was to study the proteins by high accuracy, high resolution electrospray mass spectrometry and to confirm their predicted mass values. Metallothionein was purified from the liver of a gray-faced sheep which had been injected with Zn. For LC-MS studies, an LDC 4100 MS gradient HPLC system with 2 x 125 mm C,, column and UV detector was coupled to a MAT 900 mass spectrometer fitted with a PATRIC detector and using an electrospray ionisation source (Finnigan Mat GmbH). Separations were performed using a 2-step linear gradient of 030% acetonimle with 0.1% TFA over 5 min. followed by 30-40% acetonitrile with 0.1% TFA during the remaining 40 min. Flow rate was 300 pl/min. and sample injection was 20 p1 at a concentration of 1 pg/pL. Separated components were detected by UV monitoring at 214 nm prior to electrospray at 3 kV (1 pA) with a capillary temperature of 270'C. Mass spectra were collected over the range lo00 2300 amu using a resolution of 1400 at 5 sec/decade. For flow injection, the same MS parameters were used and samples were dissolved in either water/acetonitrile (1/1) or water/acetonitrile (4/6) + 0.1% TFA. They were then analysed using a 5 FL loop injection with a flow rate of 30 pUmin. Analysis of sheep MT-I proteins by on-line HPLCESI-MS revealed components with distinct mass profiles whose chromatograms can be seen in Fig. 1. Mass spectra and derived deconvoluted spectra of the components resolved by chromatography confirmed the presence of proteins whose masses were within a fraction of predicted values for MT-la, -1b and -1c (Fig. 1, inset table). Indeed, the relative abundance of these isoforms was similar to that reported for the corresponding MT mRNA in sheep liver [3] and the translation of all 3 MT-1 proteins is consistent with the evidence for isoform heterogeneity obtained using capillary electrophoresis techniques [4]. A further minor isoform of mass 5951 was detected in MT-1 (Fig. 1). which is consistent with the presence of a form of deacetylated MT la. This may have physiological relevance since the rate of protein degradation may be influenced by the degree of deacetylation. The study of MT-1 by flow injection ESI-MS at acid and neutral pH gave rise to complex spectra (Fig. 2). A repetitive pattern with a repeat interval of 63-64 amu can be seen, with components at neutral pH being of higher mass than those at low pH. This pattern was caused by a varying degree of Zn or Cu dissociation from MT isoforms depending on the combined effect of pH and electrospray conditions.

Crystal Structure of S-Adenosylmethionine Synthetase

Crystal Structure of S-Adenosylmethionine Synthetase

Highly bioluminescent Streptococcus thermophilus strain for the detection of dairy-relevant antibiotics in milk

Inefficient translational initiation is often the cause of poor foreign gene expression in gram-positive organisms. The expression of bacterial luciferase (lux) genes in Streptococcus thermophilus (bioluminescence) was improved by addressing this problem in two ways; by ribosome-binding site (RBS) replacement, and by enhancing lux RBS access by polymerase chain reaction modification either alone or combined with translational coupling to a truncated upstream open- reading frame (orf') having its own RBS. Lactococcal expression signals were employed for plasmid-based lux expression. The same constructs were used to monitor bioluminescence in Lactococcus lactis, as well as two non-lactic bacterial strains, for comparison. High lux expression was achieved in all four organisms with a heterodimeric thermostable enzyme. Surprisingly, where ready access to the lux RBS was predicted, translational coupling to the lactococcal orf remained a prerequisite for detectable lux expression in L. lactis. In contrast, high bioluminescence in S. thermophilus was independent of coupling. Consistent with these observations, inspection of published gene sequences suggests that RBS "strength" may be a more important factor in translation in S. thermophilus than in L. lactis. Using reduced light production in highly bioluminescent S. thermophilus as an indicator of biocide presence in milk, test times could be significantly shortened compared with a commercial test utilizing the related non-bioluminescent strain. lux genes appear to be sensitive, exponential-phase reporters of gene activity in S. thermophilus, an organism with molecular biology and genetics that remain largely unstudied.

Application of two microsatellite sequences in wheat storage proteins as molecular markers

In eukaryotes, tandem arrays of simple-sequence repeat sequences can find applications as highly variable and multi-allelic PCR-based genetic markers. In hexaploid bread wheat, a large-genome inbreeding species with low levels of RFLP, di- and trinucleotide tandem repeats were found in 22 published gene sequences, two of which were converted to PCR-based markers. These were shown to be genome-specific and displayed high levels of variation. These characteristics make them especially suitable for intervarietal breeding applications.

Detection of verotoxigenic Escherichia coli in bull semen using the polymerase chain reaction

Oligonucleotide primers used in a polymerase chain reaction (PCR) protocol detected the verotoxin 2 (VT2) gene in E. coli present in experimentally contaminated bull semen. The VT2 (Shiga-like toxin II [SLT-II]) primers targeted a 346-bp fragment of the gene coding for the A subunit of the toxin. PCR products, corresponding to the VT2 gene sequence, were amplified from template E. coli nucleic acid extracted from 18-h broth culture and from E. coli in contaminated semen in the undiluted state, diluted in egg yolk-Tris and diluted in milk. The sensitivity of the assay to detect E. coli was determined to be 1 pg of nucleic acid, and as few as 10–20 E. coli organisms/ml could be detected in raw and diluted semen. Preliminary confirmation of the PCR product was accomplished by slot blot hybridization to a radiolabeled specific oligoprobe. Sequencing of the PCR products identifying VT2 gene sequence revealed 99.7% homology with published gene sequences for VT2. This study demonstrates the feasibility of applying PCR technology for the detection of E. coli in bovine semen. This technique may find wide application for the detection of other pathogens that may be present in semen.