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PubMed1997

Nested DNA inversion of Campylobacter fetus S-layer genes is recA dependent.

Wild-type strains of Campylobacter fetus are covered by a monomolecular array of surface layer proteins (SLPs) critical for virulence. Each cell possesses eight SLP gene cassettes, tightly clustered in the genome, that encode SLPs of 97 to 149 kDa. Variation of SLP expression occurs by a mechanism of nested DNA rearrangement that involves the inversion of a 6.2-kb sapA promoter-containing element alone or together with one or more flanking SLP gene cassettes. The presence of extensive regions of identity flanking the 5' and 3' ends of each SLP gene cassette and of a Chi-like recognition sequence within the 5' region of identity suggests that rearrangement of SLP gene cassettes may occur by a generalized (RecA-dependent) homologous recombination pathway. To explore this possibility, we cloned C. fetus recA and created mutant strains by marker rescue, in which recA is disrupted in either S+ or S- strains. These mutants then were assessed for their abilities to alter SLP expression either in the presence or absence of a complementary shuttle plasmid harboring native recA. In contrast to all previously reported programmed DNA inversion systems, inversion in C. fetus is recA dependent.

Bacterial Outer Membrane Proteins↗
PubMed2005

Multiclonal pattern of Jaagsiekte sheep retrovirus integration sites in ovine pulmonary adenocarcinoma.

Insertional mutagenesis and envelope (Env)-mediated oncogenesis are hypothesized mechanisms by which Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma (OPA). Twenty-eight JSRV integration sites in lung tumors (LTs) from four sheep with OPA were cloned and sequenced by a multiple step gene walking technique. Using nested PCR, clonal expansion of these integration sites could be detected, if at all, only in the localized regions of LT from which the integration sites were derived. One sheep had a viral integration site in a sequence with 85 and 81% identity, respectively, over 100 bp to exon 2 of the human and mouse receptor protein tyrosine phosphatase gamma genes. Clonal integration of Jaagsiekte sheep retrovirus in this gene was demonstrated by nested PCR and Southern blot hybridization in the DNA sample from which the integration site was cloned, but not in other LT or kidney DNA samples from the same sheep. OPA may develop from multiple independent oncogenic events and a role for insertional mutagenesis cannot be ruled out.

Animals↗
PubMed1998

Nested clade analyses of phylogeographic data: testing hypotheses about gene flow and population history.

Since the 1920s, population geneticists have had measures that describe how genetic variation is distributed spatially within a species' geographical range. Modern genetic survey techniques frequently yield information on the evolutionary relationships among the alleles or haplotypes as well as information on allele frequencies and their spatial distributions. This evolutionary information is often expressed in the form of an estimated haplotype or allele tree. Traditional statistics of population structure, such as F statistics, do not make use of evolutionary genealogical information, so it is necessary to develop new statistical estimators and tests that explicitly incorporate information from the haplotype tree. One such technique is to use the haplotype tree to define a nested series of branches (clades), thereby allowing an evolutionary nested analysis of the spatial distribution of genetic variation. Such a nested analysis can be performed regarding the geographical sampling locations either as categorical or continuous variables (i.e. some measure of spatial distance). It is shown that such nested phylogeographical analyses have more power to detect geographical associations than traditional, nonhistorical analyses and, as a consequence, allow a broader range of gene-flow parameters to be estimated in a precise fashion. More importantly, such nested analyses can discriminate between phylogeographical associations due to recurrent but restricted gene flow vs. historical events operating at the population level (e.g. past fragmentation, colonization, or range expansion events). Restricted gene flow and historical events can be intertwined, and the cladistic analyses can reconstruct their temporal juxtapositions, thereby yielding great insight into both the evolutionary history and population structure of the species. Examples are given that illustrate these properties, concentrating on the detection of range expansion events.

Alleles↗
PubMedDate not supplied

Comparison of one-step RT-PCR and a nested PCR for the detection of canine distemper virus in clinical samples.

OBJECTIVE: To develop a rapid and sensitive method for the detection of canine distemper virus (CDV) by nested PCR using clinical specimens. DESIGN: A nested PCR was developed, compared to a one-step RT-PCR and validated. PROCEDURE: Two sets of specific primers for a one-step RT-PCR and a nested PCR, targeting a 640 bp fragment and a 297 bp fragment, respectively, were selected from the highly conserved region of the nucleocapsid protein (NP) gene of CDV. The nested PCR and the one-step RT-PCR were used to amplify a part of the CDV NP gene of a CDV vaccinal strain and samples of urine, blood, nasal discharge and saliva from 29 dogs suspected of suffering CD. RESULTS: Both the one-step RT-PCR and the nested PCR reacted with the CDV vaccinal strain, but not with canine parvovirus. The expected 640 bp fragment of the NP gene was detected in 11/22 (50.0%) blood, 10/20 (50.0%) urine, 5/25 (20.0%) saliva and 6/27 (22.2%) nasal swab samples by one-step RT-PCR, whereas the nested PCR amplified an expected 297 bp fragment of the NP gene in 18/22 (81.8%) blood, 15/20 (75.0%) urine, 14/25 (56%) saliva and 19/27 (70.3%) nasal swab samples. CONCLUSION: The nested PCR detected CDV in blood, urine, nasal swab and saliva more frequently than did the one-step RT-PCR. Therefore, this assay should be a useful aid to antemortem diagnosis of CDV infections in dogs.

Animals↗
PubMed2002

The transcription factor basic transcription element-binding protein 1 is a direct thyroid hormone response gene in the frog Xenopus laevis.

Several genes have been identified that are activated or repressed by thyroid hormone in tadpole tissues during metamorphosis of the frog Xenopus laevis. One rapidly and strongly induced gene encodes the Xenopus homolog of basic transcription element-binding protein 1 (xBTEB1), an SP1-related transcription factor. xBTEB1 has similar DNA-binding activity and transcriptional activation properties as mammalian BTEB1. The thyroid hormone-dependent regulation of xBTEB1 was investigated using a modified electrophoretic mobility shift assay to scan genomic DNA for receptor-binding sites. Due to the tetraploid X. laevis genome, xBTEB1 is duplicated, and thyroid hormone regulates both copies. A consensus thyroid hormone response element (TRE) lies far upstream of the transcriptional start site of both genes. The TRE is nested within a 200-bp region of high sequence conservation between these two genes that duplicated millions of years ago. The TRE acts as a strong response element in transfection assays using a heterologous promoter or its native context. Thus, one of the earliest thyroid hormone-induced genes in tadpoles is a transcription factor regulated through an evolutionarily conserved TRE. xBTEB1 is predicted to play an important role in downstream gene regulation leading to the growth and remodeling of tissues at metamorphosis.

Animals↗
PubMed1993

Developmental and tissue-specific expression of a tomato anionic peroxidase (tap1) gene by a minimal promoter, with wound and pathogen induction by an additional 5'-flanking region.

The tomato anionic peroxidase genes (tap1 and tap2) are induced by wounding and pathogen attack. The 5'-flanking region of tap1 confers wound- and pathogen-inducible beta-glucuronidase (GUS) expression in tobacco plants transformed with a tap1/GUS chimeric fusion gene construct. A series of nested 5' promoter deletions in the tap1/GUS fusion gene construct was created, and introduced into tobacco protoplasts via polyethylene glycol-mediated DNA transfer. A -202 construct (where the transcriptional start site is denoted +1) and larger tap1 promoter constructs showed constitutive GUS expression. A 2-fold increase in GUS expression over the high constitutive levels was observed with -358 bp and larger tap1 constructs when protoplasts were incubated with elicitor preparations from Verticillium albo-atrum. In tobacco plants transformed with the tap1 promoter deletion/GUS fusion gene constructs, wounding caused induction of GUS expression by 20 h that increased 6- to 18-fold by 72 h. The region between -202 and -358 of the tap1 promoter conferred wound responsiveness. GUS was also found to be expressed in the epidermis and trichomes in the aerial parts of transgenic plants. High-level GUS expression was observed in the nodal region of stems that was associated with the leaf traces. GUS that was absent in very young flower buds was found in the subsequent developmental stages in the pistils, ovaries and anthers. The developmentally regulated tissue-specific expression of GUS was found with all constructs containing the -202 and larger promoters whereas wound and pathogen induction required -358 or larger promoter. These results suggest that the tap1 gene, which was heretofore thought to be expressed only upon wounding or pathogen attack, plays a role in normal developmental processes of the plant and this gene acquired additional 5'-flanking promoter for the purpose of responding to wounding and fungal attack.

Base Sequence↗
PubMed2006

The detection and prevalence of Clostridium botulinum in pig intestinal samples.

To determine the prevalence of Clostridium botulinum types A, B, E, and/or F in pig intestinal samples, different PCR-based methods were compared using artificially contaminated faeces. The methods included a multiplex PCR targeted to botulinum neurotoxin genes A, B, E, and F or a nested PCR targeted to toxin genes B, E, and F, combined with different pre-enrichment protocols and forms of templates. A method including the two-step enrichment followed by DNA extraction and multiplex PCR yielded the highest number of positives. This assay protocol was employed to investigate 100 pig intestinal samples. The sample materials studied included colon wall, intestinal content, and mucus peeled from the colon wall. Three pigs (3%) were positive for C. botulinum type B, and no other toxinotypes were detected in any sample. The number of positive samples was higher when colon wall or peeled mucus was analyzed compared to the intestinal content. C. botulinum was isolated from two PCR-positive samples and confirmed to be type B by PCR. Both isolates were shown to be proteolytic and thus to represent C. botulinum group I.

Animals↗
PubMed2006

Spatial population genetic structure and limited dispersal in a Rocky Mountain alpine stream insect.

Using the mitochondrial cytochrome oxidase I (COI) gene, we assessed the phylogeographic structure of Prosimulium neomacropyga, a black fly (Simuliidae) whose distribution in the US Southern Rockies ecoregion is limited to alpine tundra streams. Given high habitat specificity, lack of hydrological connection between streams, and a terrestrial environment restrictive to insect flight, we hypothesized limited gene flow. A spatially nested sampling design showed that grouping populations according to high-elevation 'islands' of alpine tundra (which typically include headwater streams of > 1 watershed) explained a significant proportion of genetic variation while grouping streams according to major watershed (across islands) did not. Nested clade analysis and isolation-by-distance (IBD) relationships further implicated limited ongoing gene flow within but not among the isolated alpine islands. IBD was strong among five streams within an individual island using each of four alternative models of pairwise landscape connectivity for flying insects. Results of all landscape models were positively correlated, suggesting that straight-line distance is an acceptable surrogate for presumably more biologically meaningful connectivity measures in this system. IBD was significantly weaker across the entire study area, comprised of three separate islands. Overall, population structure was significant with F(ST) = 0.38, suggesting limited dispersal across a small spatial extent.

Animals↗
PubMed2005

Diazotrophic diversity and distribution in the tropical and subtropical Atlantic Ocean.

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3 degrees N and 56.6 to 18.5 degrees W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as gamma-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and gamma-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30 degrees C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30 degrees C, more often in waters with temperature of <26 degrees C, and were sometimes recovered from waters with detectable nitrate concentrations.

Atlantic Ocean↗
PubMed2004

Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags.

We report on the development of a new PCR technique for the isolation of genomic fragments that flank known DNA sequences. This technique, single oligonucleotide nested (SON)-PCR, relies on only two amplification reactions with two or three nested sequence-specific primers. It allows the isolation of DNA regions located on either side of a known DNA sequence, with high specificity. DNA products of 2 kb in size can be generated that all contain one copy of the same primer at both ends. Sequence analysis of these products indicates that the binding of the primers to non-specific DNA sites mainly depends on their overall complementarity to the target sequence. Moreover, analysis shows that short extensions of the primers can occur during the first amplification reaction and that a 2-bp overlap between subsequent primers can target their annealing to their predecessor's sequence. Ninety percent of the DNA products larger than 0.5 kb correspond to fragments of interest and we obtained successful results with various templates and primer sets. SON-PCR therefore seems a very efficient and widely applicable method for the rapid identification of large unknown DNA regions. Based on available expressed sequence tags, this technique was applied to isolate the palH and pacC genes of the phytopathogenic fungus Botrytis cinerea, with their 5' or 3' flanking regions.

Base Sequence↗
PubMed2005

Prenatal fetal sex diagnosis by detecting amelogenin gene in maternal plasma.

OBJECTIVES: To provide a new, reliable noninvasive method for fetal sex determination. METHODS: Fetal sex was detected in 32 early pregnant women by identifying the amelogenin gene in maternal plasma using nested PCR analysis. First, the 122/128 bp of X-Y homologous region containing 6 bp deletions in the intron 3 of amelogenin gene in X chromosome was amplified, and then the nested PCR was carried out, whose 3' end of the upstream primer is just located in the deletion region. The fetus was male or female, depending on whether it had the 89-bp nested PCR product or not. RESULTS: The 89 bp of nested PCR product was detected in 19 plasma samples obtained from pregnant women, deducing they bear the male fetus and the remaining pregnant women bear female. When compared with the birth outcome, two samples were pseudo-positive. The coincidence was 93.8%. This method had high sensitivity that even trace amount of target fetal DNA (10 pg) could be detected. CONCLUSIONS: This conventional nested PCR analysis of amelogenin gene promises to be a reliable method for noninvasive fetal sex determination at early pregnancy using maternal plasma DNA.

Amelogenin↗
PubMed2003

Nested PCR assays for detection of Blastomyces dermatitidis DNA in paraffin-embedded canine tissue.

A Blastomyces dermatitidis nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family ONYGENACEAE: We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively. The B. dermatitidis PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy. Sequencing revealed that all PCR products were homologous to the B. dermatitidis WI-1 adhesin gene. No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma. The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively. Only six products were identified as being identical to B. dermatitidis 18S rDNA; they were exclusively obtained from specimens positive by the B. dermatitidis nested PCR. For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific H. capsulatum product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the B. dermatitidis PCR assay. In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of B. dermatitidis in canine tissue.

Animals↗
PubMed2000

Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1.

Pallial and subpallial morphological subdivisions of the developing chicken telencephalon were examined by means of gene markers, compared with their expression pattern in the mouse. Nested expression domains of the genes Dlx-2 and Nkx-2.1, plus Pax-6-expressing migrated cells, are characteristic for the mouse subpallium. The genes Pax-6, Tbr-1, and Emx-1 are expressed in the pallium. The pallio-subpallial boundary lies at the interface between the Tbr-1 and Dlx-2 expression domains. Differences in the expression topography of Tbr-1 and Emx-1 suggest the existence of a novel "ventral pallium" subdivision, which is an Emx-1-negative pallial territory intercalated between the striatum and the lateral pallium. Its derivatives in the mouse belong to the claustroamygdaloid complex. Chicken genes homologous to these mouse genes are expressed in topologically comparable patterns during development. The avian subpallium, called "paleostriatum," shows nested Dlx-2 and Nkx-2.1 domains and migrated Pax-6-positive neurons; the avian pallium expresses Pax-6, Tbr-1, and Emx-1 and also contains a distinct Emx-1-negative ventral pallium, formed by the massive domain confusingly called "neostriatum." These expression patterns extend into the septum and the archistriatum, as they do into the mouse septum and amygdala, suggesting that the concepts of pallium and subpallium can be extended to these areas. The similarity of such molecular profiles in the mouse and chicken pallium and subpallium points to common sets of causal determinants. These may underlie similar histogenetic specification processes and field homologies, including some comparable connectivity patterns.

Age Factors↗
PubMed2006

Detection of Babesia bigemina infection in strains of Rhipicephalus (Boophilus) microplus collected from outbreaks in south Texas.

The sudden death of several cattle infested experimentally with Rhipicephalus (Boophilus) microplus led to a clinical investigation into the reasons for the unexpected mortality. Microscopic evidence for Babesia bigemina infection was found in blood smears from the affected animals and a PCR assay was designed to detect the presence of B. bigemina and Babesia bovis in all R. microplus strains received and propagated at the laboratory. The assay utilizes a nested PCR approach with the first PCR amplifying a well-conserved segment from the Babesia 18S ribosomal RNA gene followed by a nested PCR with Babesia species-specific primers and annealing temperatures enabling amplification of the 18S ribosomal RNA gene fragment specific to either B. bigemina or B. bovis. DNA from groups of 50 larvae was extracted using a rapid DNA preparation protocol, which consisted of grinding the frozen tick larvae in PCR buffer and boiling the mixture for 5min. The assay sensitivity allowed for the detection of the equivalent of a single infected tick larva. R. microplus eggs were also analyzed, but yolk protein viscosity created inconsistent results with the crush and boil DNA isolation protocol, necessitating the use of a more extensive proteinase K digestion-based DNA purification method. We detected the presence of B. bigemina in all strains of R. microplus currently reared at the laboratory and 4 of 26 strains collected from infestation outbreaks in Texas by the U.S. Cattle Fever Tick Eradication Program.

Animals↗
PubMed1995

Amplification of DNA polymerase gene fragments from viruses infecting microalgae.

Nested PCR with three highly degenerate primers was used for amplification and identification of DNA polymerase (pol) genes from viruses which infect three genera of microalgae. Group-specific primers (AVS1 and AVS2) were designed on the basis of inferred amino acid sequences unique to the DNA pol genes of viruses (PBCV-1 and NY-2A) that infect an endosymbiotic Chlorella-like alga (Chlorophyceae) and a virus (MpV-SP1) which infects the photosynthetic flagellate Micromonas pusilla (Prasinophyceae). In addition, a nested primer (POL) was designed on the basis of the highly conserved amino acid sequence YGDTDS found in most B-family (alpha-like) DNA pol genes. These primers were used to amplify DNA from the three viruses, PBCV-1, NY-2A, and MpV-SP1, for which the primers were designed, as well as eight clonal isolates of genetically distinct viruses which infect M. pusilla and others which infect Chrysochromulina spp. (Prymnesiophyceae), suggesting that these are a group of related viruses. In contrast, no product resulted from using DNA from viruses which infect the marine brown algae Ectocarpus siliculosis and Feldmannia sp. (Phaeophyceae), suggesting that these viruses may not be closely related to those that infect microalgae. These primers were also used to amplify DNA from natural virus communities. Our results indicate that nested PCR, even under low-stringency conditions, can be used as a rapid method to verify the presence in seawater of a group of related viruses which infect microalgae. Sequence analysis of these fragments should provide information on the genetic diversity and potentially the phyletic relationships among these viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2002

Mannose-binding lectin and the prognosis of fulminant hepatic failure caused by HBV infection.

BACKGROUND/AIMS: The mannose-binding lectin (MBL) gene was reported to play an important role in determining the clinical outcome of persistent hepatitis B virus (HBV) infection. We investigated serum MBL concentrations and MBL gene mutations to determine whether they were related to the prognosis of patients with fulminant hepatic failure (FHF) caused by HBV infection. METHODS: We investigated serum MBL concentrations and MBL gene mutations in 43 HBV-infected Japanese patients with FHF and 260 HBsAg-negative healthy controls. Serum MBL concentrations were measured by an enzyme-linked immunosorbent assay, and mutations in the MBL gene were analysed by nested PCR and direct DNA sequencing. RESULTS: Only a mutation in codon 54 of the MBL gene was found. The frequency of this mutation in nonsurvivors (40%, 8/20) was higher than in survivors (13%, 3/23), and the difference was slightly significant (p = 0.043). The H allele frequency in survivors (70.5%, 31/44) was higher than in nonsurvivors (39.5%, 15/38) (p = 0.0048). Because of these factors the mean serum MBL concentration in survivors, 1.61 ,micro/ml (range 0.3-3.86), was significantly higher than in nonsurvivors, 0.79 microg/ml (range 0.04-1.51) (p < 0.0001). The likelihood ratio for nonsurvival was 0 for over 2.0 microg/ml, 0.67 for 1.0-2.0 microg/ml, and 2.24 for 0-1.0 microg/ml. CONCLUSIONS: The mutation in codon 54 of the MBL gene tended to be higher in nonsurvivors than in survivors. The H allele frequency (high producing allele in H/Y) in survivors was higher than that in nonsurvivors. High levels of serum MBL correlated with the survival of patients with FHF due to HBV infection. Serum MBL may be useful as a predictive factor for the survival of patients with FHF caused by HBV.

Adult↗
PubMed2000

A novel composite locus of Arabidopsis encoding two polypeptides with metabolically related but distinct functions in lysine catabolism.

Both plants and animals catabolize lysine via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide. We recently demonstrated that Arabidopsis plants possess not only a bifunctional LKR/SDH but in addition a monofunctional SDH enzyme. We also speculated that these two enzymes may be controlled by a single gene (G. Tang et al. Plant Cell, 1997, 9, 1305-1316). By expressing several epitope-tagged and GUS reporter constructs, we demonstrate in the present study that the Arabidopsis monofunctional SDH is encoded by a distinct gene, which is, however, nested entirely within the coding and 3' non-coding regions of the larger bifunctional LKR/SDH gene. The entire open reading frame of the monofunctional SDH gene, as well as some components of its promoter, are also parts of the translated coding sequence of the bifunctional LKR/SDH gene. These special structural characteristics, combined with the fact that the two genes encode simultaneously two metabolically related but distinct enzymes, render the LKR/SDH locus a novel type of a composite locus. Not all plant species possess an active monofunctional SDH gene and the production of this enzyme is correlated with an increased flux of lysine catabolism. Taken together, our results suggest that the composite LKR/SDH locus serves to control an efficient, highly regulated flux of lysine catabolism

Amino Acid Sequence↗