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PubMed1994

Nested PCR optimized for detection of Bordetella pertussis in clinical nasopharyngeal samples.

Several genes and sequences in Bordetella pertussis have been used as targets in diagnostic PCR assays. A previously developed single-step PCR assay for the detection of B. pertussis was based on an insertion sequence, IS480, that is present in about 70 to 80 copies in each genome. The diagnostic sensitivity, specificity, and reliability of this assay with aspirated and heat-treated samples from the nasopharynx of patients and their contacts was improved by the use of a nested PCR configuration. The nested PCR assay produced a 205-bp fragment with all of the 115 B. pertussis strains tested and was negative with all strains belonging to other Bordetella species (n = 44) as well as other bacteria commonly found in the upper respiratory tract (n = 115). The diagnostic value of the assay was verified by giving positive results for B. pertussis in all the 51 nasopharyngeal aspirates from culture-positive patients. The assay also detected 18 positive aspirates from a total of 196 culture-negative patients. A confirmatory cleavage of the 205-bp nested PCR product by MvaI gave in all cases two bands of 88 and 117 bp. In conclusion, this newly developed nested PCR assay was shown to be reasonably fast and uncomplicated, with an optimal sensitivity and a high degree of specificity for the diagnosis of B. pertussis in aspirated nasopharyngeal samples processed simply by heat treatment. The detection level in the nested PCR was about 10 bacteria per ml, or seven to eight insertion sequence copies per 10 microliters of boiled sample.

Base Sequence↗

PubMed2004

Dissemination of the highly expressed Bx7 glutenin subunit (Glu-B1al allele) in wheat as revealed by novel PCR markers and RP-HPLC.

Increased expression of the high molecular weight glutenin subunit (HMW-GS) Bx7 is associated with improved dough strength of wheat (Triticum aestivum L.) flour. Several cultivars and landraces of widely different genetic backgrounds from around the world have now been found to contain this so-called 'over-expressing' allelic form of the Bx7 subunit encoded by Glu-B1al. Using three methods of identification, SDS-PAGE, RP-HPLC and PCR marker analysis, as well as pedigree information, we have traced the distribution and source of this allele from a Uruguayan landrace, Americano 44D, in the mid-nineteenth century. Results are supported by knowledge of the movement of wheat lines with migrants. All cultivars possessing the Glu-B1al allele can be identified by the following attributes: (1) the elution of the By sub-unit peak before the Dx sub-unit peak by RP-HPLC, (2) high expression levels of Bx7 (>39% Mol% Bx), (3) a 43 bp insertion in the matrix-attachment region (MAR) upstream of the gene promoter relative to Bx7 and an 18 bp nucleotide duplication in the coding region of the gene. Evidence is presented indicating that these 18 and 43 bp sequence insertions are not causal for the high expression levels of Bx7 as they were also found to be present in a small number of hexaploid species, including Chinese Spring, and species expressing Glu-B1ak and Glu-B1a alleles. In addition, these sequence inserts were found in different isolates of the tetraploid wheat, T. turgidum, indicating that these insertion/deletion events occurred prior to hexaploidization.

Base Sequence↗

PubMed2003

AmpC cephalosporinase hyperproduction in Acinetobacter baumannii clinical strains.

OBJECTIVE: To compare the genetic environments of ampC genes in different Acinetobacter baumannii isolates showing different levels of beta-lactam resistance. METHODS: The patterns of beta-lactam resistance and beta-lactamase production were investigated for 42 A. baumannii clinical strains. The MICs of various beta-lactams were determined in the presence or absence of the class C cephalosporinase inhibitor, cloxacillin (500 mg/L). The ampC gene and its 5' adjacent sequence were analysed by PCR and DNA sequencing. An RT-PCR method was developed to evaluate ampC transcript levels. RESULTS: Strains fell into three resistance groups: first, strains with a ceftazidime MIC < or =8 mg/L (20 strains, 47.6%); secondly, strains with a ceftazidime MIC 32 mg/L, which was reduced four-fold in the presence of cloxacillin (eight strains, 19%); and thirdly, strains with a ceftazidime MIC > or =256 mg/L, which did not decrease in the presence of cloxacillin (14 strains, 33.4%). In all of the resistant isolates (groups II and III), but not in any of the ceftazidime-susceptible isolates (group I), a 1180 bp insert showing all the characteristics of an insertion sequence was detected upstream from the ampC gene. Isolates having this insert overexpress ampC, according to RT-PCR experiments. CONCLUSION: Presence of an insertion sequence upstream of ampC in A. baumannii clinical isolates, possibly including a strong promoter, has the potential to cause over-expression of AmpC, resulting in high-level ceftazidime resistance.

5' Untranslated Regions↗

PubMed1994

Development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides.

It has recently been shown that cowpea plants can be infected with a cowpea mosaic virus (CPMV) chimera containing an antigenic site from foot-and-mouth disease virus (Usha et al., Virology 197, 366-374, 1993). Analysis of progeny RNA produced during such an infection has revealed that the inserted sequence is rapidly lost during serial passaging, probably by a process of homologous recombination. Using the information gained from this analysis, we have redesigned the chimeras in such a way that they are now genetically stable. The modified constructs have been used to obtain large quantities of purified virus particles expressing epitopes derived from human rhinovirus 14 (HRV-14) and human immunodeficiency virus type 1 (HIV-1). The chimeric virus particles possess the antigenic properties of the inserted sequence and, in the case of the HRV-14-derived construct, it has been shown that the inserted epitope is immunogenic in rabbits. These results demonstrate that CPMV can be used as a high-yielding system for the presentation of foreign peptide sequences.

Amino Acid Sequence↗

PubMed1989

Nucleotide sequence and insertional inactivation of a Bacillus subtilis gene that affects cell division, sporulation, and temperature sensitivity.

Located at 135 degrees on the Bacillus subtilis genetic map are several genes suspected to be involved in cell division and sporulation. Previously isolated mutations mapping at 135 degrees include the tms-12 mutation and mutations in the B. subtilis homologs of the Escherichia coli cell division genes ftsA and ftsZ. Previously, we cloned and sequenced the B. subtilis ftsA and ftsZ genes that are present on an 11-kilobase-pair EcoRI fragment and found that the gene products and organization of these two genes are conserved between the two bacterial species. We have since found that the mutation in the temperature-sensitive filamenting tms-12 mutant maps upstream of the ftsA gene on the same 11-kilobase-pair EcoRI fragment in a gene we designated dds. Sequence analysis of the dds gene and four other open reading frames upstream of ftsA revealed no significant homology to other known genes. It was found that the dds gene is not absolutely essential for viability since the dds gene could be insertionally inactivated. The dds null mutants grew slowly, were filamentous, and exhibited a reduced level of sporulation. Additionally, these mutants were extremely temperature sensitive and were unable to form colonies at 37 degrees C. Another insertion, which resulted in the elimination of 103 C-terminal residues, resulted in a temperature-sensitive phenotype less severe than that in the dds null mutant and similar to that in the known tms-12 mutant. The tms-12 mutation was cloned and sequenced, revealing a nonsense codon that was predicted to result in an amber fragment that was about 65% of the wild-type size (elimination of 93 C-terminal residues).

Amino Acid Sequence↗

PubMed1995

cDNA cloning of thyroid hormone receptor beta for the Japanese flounder.

cDNA encoding the beta type of thyroid hormone receptor (THR) was cloned from a lambda gt10 library prepared from the whole bodies of metamorphosing flounder (Paralichthys olivaceus). The deduced amino acid sequence of the flounder THR beta (fTHR beta 1) showed higher homologies to the known THR beta s of other vertebrate animals than to THR alpha s, including flounder THR alpha s. Unlike any other THR, fTHR beta 1 possessed an insertion sequence composed of nine amino acids in the region prior to the hormone-binding domain. PCR analyses suggested the presence of transcripts for another THR beta (fTHR beta 2) that had a longer insertion sequence than fTHR beta 1. The analyses further suggested that the cDNA sequences of the two flounder THR beta s most likely shared a constant sequence except for a 60-base additional sequence found only in fTHR beta 2. The flounder genome possessed a single gene for both fTHR beta 1 and beta 2, suggesting that the two THR beta variants are produced from the same gene through an alternative splicing system.

Amino Acid Sequence↗

PubMed1981

Analysis of mutations affecting Ty-mediated gene expression in Saccharomyces cerevisiae.

Yeast translocatable, Ty, elements can cause constitutive synthesis of the glucose-repressible alcohol dehydrogenase (ADHII) when inserted upstream from the 5' end of the structural gene, ADR2. These insertion mutations, ADR3c, are unstable and give rise to secondary ADHII- mutations. The majority of such mutants, adr3, can be attributed to excision of the insertion sequence, leaving behind a single copy of the delta-sequence which occurs as a direct repeat at the ends of the Ty elements. A few adr3 mutants appear to be generated by DNA-rearrangements in the vicinity of the Ty insertion. The occurrence of recessive mutants, tye, which are unlinked to ADR2 indicates that the constitutive expression of ADR2 caused by the Ty insertions requires the function of trans-acting genes. These results support the idea that regulation of Ty-linked ADR2 is actively mediated by the insertion sequence and is probably not due to a mere disruption of the wild-type controlling site.

Alcohol Oxidoreductases↗

PubMed1999

IS1634, a novel insertion element creating long, variable-length direct repeats which is specific for Mycoplasma mycoides subsp. mycoides small-colony type.

A new insertion sequence, IS1634, has been identified in Mycoplasma mycoides subsp. mycoides small-colony type (SC). IS1634 shows structural and functional similarities to IS1549 of Mycobacterium smegmatis and with it seems to form a new class or family of insertion sequences. IS1634 has a size of 1,872 bp, including two 13-bp terminal inverted repeats. It contains an open reading frame (ORF) encoding a product of 533 amino acids which shows similarity to the transposase of IS1549 and to a lesser extent to the transposases of IS elements of the IS4 family. IS1634 is present at about 30 copies in the genome of all 22 different field strains of M. mycoides subsp. mycoides SC tested. Characteristic of IS1634 are the long and variable-length direct repeats at the sites of insertion which were found to reach up to about 500 bp. IS1634 is specific to M. mycoides subsp. mycoides SC and is not present in any of the other members of the M. mycoides cluster. Neither was it found in other closely related Mycoplasma species of ruminants.

Cloning, Molecular↗

PubMed1994

Alternative splicing of bovine terminal deoxynucleotidyl transferase cDNA.

Two cDNA fragments of terminal deoxynucleotidyl transferase, which contained new insertion sequences, were found from bovine thymus cDNA. One of the clones encoded 18 new amino acids and the other encoded 9 new amino acids. In the results of genomic structure analysis around the new insertion sequences, alternative splicing of the bovine terminal deoxynucleotidyl transferase gene was suggested.

Alternative Splicing↗

PubMed1994

Molecular cloning and sequence analysis of yps-3, a yeast-phase-specific gene in the dimorphic fungal pathogen Histoplasma capsulatum.

Genes specifically expressed in the parasitic yeast phase of Histoplasma capsulatum have been cloned to clarify the mechanisms underlying both pathogenesis and morphogenesis in this human dimorphic fungal pathogen. Previous studies have determined that the yeast-phase-specific gene, yps-3, is expressed differentially in two non-isogenic strains which differ in their thermotolerance and virulence. We have cloned the yps-3 homologues from the high virulence (G217B) and low virulence (Downs) strains, and obtained a partial cDNA clone representing the expressed gene from H. capsulatum G217B. The Downs clone harbours a 287 bp insertion sequence that disrupts a long ORF defined by the yps-3 G217B cDNA. Although the insertion sequence contains features reminiscent of mobile genetic elements, including 15 bp direct repeats of flanking sequence, it is not randomly distributed in the H. capsulatum genome. S1 nuclease analysis was utilized to map the 5' end of the expressed yps-3 gene in G217B to potential regulatory regions which are largely homologous in both strains. This finding may point to a deficiency in a temperature inducible regulatory protein in the low virulence, temperature-sensitive Downs strain. The nucleotide sequence of the yps-3 gene and the predicted amino acid sequence of its product represents the first report of phase-specific gene and protein sequences in this widely distributed fungal pathogen. Further analysis of the product encoded by the yps-3 gene may provide significant insight into the pathogenic repertoire of H. capsulatum.

Amino Acid Sequence↗

PubMed1997

Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination.

One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of approximately 6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.

Alleles↗

PubMed2003

Identification of genetic loci required for capsular expression in Vibrio vulnificus.

Transposon mutagenesis of an encapsulated, virulent strain of Vibrio vulnificus 1003(O) led to the identification of four genetic regions that are essential to capsular polysaccharide (CPS) expression and virulence. Of the four regions, three are believed to be part of a capsule gene locus comprised of biosynthesis, polymerization, and transport genes clustered on a single chromosomal fragment. Genes indicating a Wzy-dependent system of polymerization and transmembrane export are present, suggesting that the CPS of V. vulnificus is lipid linked. The fourth region, while it contains a gene essential for CPS expression, is characteristic of an integron-gene cassette region, similar to the super integron of V. cholerae. It is not believed to be part of a CPS gene locus and is located in a region of the chromosome separate from the putative CPS loci. It is comprised of open reading frames (ORFs) carrying genes of unknown function surrounded by direct repeats. This region also contains IS492, an insertion sequence located numerous times throughout a region of the genome, demonstrating a restriction fragment length polymorphism among an encapsulated and nonencapsulated morphotype of V. vulnificus. Collectively, 22 ORFs were recognized: 13 capsule synthesis genes, 4 insertion sequences, 1 truncated biosynthesis gene, and 4 genes of unknown function. This study has led to the identification of previously unrecognized genetic loci that may help to increase the understanding of capsular genetics and antigenic diversity among V. vulnificus strains.

Animals↗

PubMed1981

Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis.

A 44-megadalton plasmid associated with virulence and Ca2+ dependence from Yersinia enterocolitica 8081 was compared at the molecular level with a 47-megadalton plasmid associated with Ca2+ dependence from Yersinia pestis EV76. The plasmids were found to share 55% deoxyribonucleic acid sequence homology distributed over approximately 80% of the plasmid genomes. One region in which the plasmids differed was found to contain sequences concerned with essential plasmid functions. Forty-five mutants of Y. pestis were isolated which had spontaneously acquired the ability to grow on calcium-free medium (Ca2+ independence). Of these mutants, 21 were cured of their 47-megadalton plasmid, whereas the remaining had either suffered a major deletion of the plasmid or had a 2.2-kilobase insertion located in one of two adjacent BamHI restriction fragments encompassing approximately 9 kilobases. The inserted sequence was found at numerous sites on the Y. pestis chromosome and on all three plasmids in the strain and may represent a Y. pestis insertion sequence element.

Base Sequence↗

PubMedDate not supplied

Molecular fingerprinting of Salmonella typhimurium by IS200-typing as a tool for epidemiological and evolutionary studies.

The aim of this work was to develop and evaluate a molecular typing strategy for Salmonella based on hybridization of chromosomal DNA with two different probes derived from insertion sequence IS200. Probe IS200-TT was specifically constructed for this study as a trimer of a 112 pb TaqI-TaqI fragment of IS200. Among several restriction enzymes evaluated, two were selected: EcoRI, which cuts the insertion sequence in two pieces, each one complementary to one of the probes used, and PstI, a restriction enzyme with no recognition site into IS200. With several combinations of these restrictions enzymes and probes, 43 Salmonella typhimurium strains were analyzed for copy number and location of IS200, as well as reproducibility and stability of the patterns. IS200 types have been shown to be stable, both in strains isolated from different patients implicated in the same salmonellosis outbreak and in strains isolated from the same patient at different times or from different specimens. The discriminatory power of the method has been 0.91 to 0.94. As a comparison, S. typhimurium strains were also ribotyped. Discriminatory power of the ribotypes oscillated between 0.44 and 0.55, depending on the enzyme used, and achieved a 0.78 value when the information obtained with two restriction enzymes was combined. Moreover, IS200 typing was able to differentiate among a group of S. typhimurium strains which were identical by ribotype and enzymatic electrophoretic mobility. These results enable us to conclude that, for the stability, reproducibility and discriminatory power of the patterns generated, IS200 probes can be a very useful tool in the molecular typing of S. typhimurium.

Bacterial Typing Techniques↗

PubMed1986

Myeloma mutant with a novel 3' flanking region: loss of normal sequence and insertion of repetitive elements leads to decreased transcription but normal processing of the alpha heavy-chain gene products.

We isolated and characterized LP1.2, a mouse myeloma mutant with a deletion of at least 4 kilobases (kb) immediately 3' of the alpha gene and introduction of at least 5 kb of novel (nonimmunoglobulin) sequence in its place. A 6.2-kb genomic EcoRI fragment from the mutated allele was cloned, and a subfragment was sequenced. The deletion begins 11 base pairs (bp) beyond the normal site of cleavage and polyadenylation for the secreted form of alpha mRNA. A short direct repeat, eight copies of the 17-mer GCCT ATAGAAGTAAGGA, is located at the junction of the alpha and novel sequences. The first 4 bp of the 17-mer are identical to the last 4 bp of the alpha sequence. Novel sequences downstream of the direct repeats in LP1.2 include a low-copy-number sequence flanked by two distinct, highly repetitive elements. The low-copy-number portion of the novel sequence appears on a single 30-kb EcoRI fragment in several myelomas and in liver DNA; one copy of this fragment has rearranged in cell line W3129, and this allele has rearranged a second time in LP1.2. LP1.2 contains low levels of apparently normal alpha protein and mRNA. The S1 nuclease protection of nuclear and cytoplasmic RNAs shows that cleavage and polyadenylation are efficient and accurate and that they occur without the accumulation of aberrant transcripts. Alpha transcription in isolated nuclei is decreased sevenfold in LP1.2 relative to its parent, which accounts for the low steady-state levels of cytoplasmic alpha mRNA and protein in LP1.2. Decreased alpha transcription could result either from the deletion of a positive regulator in the 3' flanking region or from the introduction of novel sequences which exert a negative effect.

Alleles↗

PubMed1997

Identification of two sequence elements associated with the gene encoding the 24-kDa crystalline component in Bacillus thuringiensis ssp. fukuokaensis: an example of transposable element archaeology.

A 6.5-kb fragment of plasmid DNA from Bacillus thuringiensis (Bt) ssp. fukuokaensis that encodes a 24-kDa crystalline component was analyzed to identify additional open reading frames (orfs). A novel Bt IS240-like element was found upstream of this gene and is considered to be a vestige of a once active insertion sequence due to a stop codon that interrupts the long orf encoding the putative transposase. This element was bounded by 17-bp terminal inverted repeats that defined the length of the insertion sequence as 802 bp. Further upstream of this element two tandem overlapping and out of phase open reading frames (orfX and orfY) were identified which represent the first example of an IS150-like element in Bt containing both orfs. orfX and orfY are not bounded by terminal inverted repeats but are associated with a gene encoding a putative site-specific recombinase of a type found in Staphylococcus aureus Class II transposons but not previously in Bt.

Amino Acid Sequence↗

PubMed1979

The transforming gene of Moloney murine sarcoma virus.

A cleavage map of the Moloney murine sarcoma viral DNA was constructed and compared with that of a spontaneously occurring deletion mutant. By restriction enzyme analysis, it was shown that a region encompassing over 40% of the viral information was not essential for transformation or rescue of the deletion mutant. The transforming region was further localised by analysis of the transforming activity in tissue culture of isolated restriction fragments of linear duoble-stranded sarcoma viral DNA. In each case, DNA fragments that retained transforming activity preserved the cell-derived insertion sequences of the viral genome. Moreover, such transformants invariably expressed RNA specific to this region. By these two approaches, it was possible to demonstrate that the transforming region of the viral genome begins very near or within the cell-derived insertion sequences. Thus, the transforming gene of this mammalian sarcoma virus originates from within the mouse cell genome.

Cell Transformation, Viral↗

PubMed2005

Engineering the genome of Grapevine virus A into a vector for expression of proteins in herbaceous plants.

Grapevine virus A (GVA), a species of the genus Vitivirus, consists of a approximately 7.4 kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). In addition to grape varieties, GVA infects Nicotiana benthamiana plants and protoplasts. We engineered the genome of GVA as a vector that includes duplication of homologous sequences that contain the promoter of the movement protein (MP) sgRNA, supplemented by enzymatic restriction sites to be used as a convenient tool for transient expression of foreign genes from an individual sgRNA. The resulting vector was able to infect and to move in N. benthamiana plants in a manner similar to the wild-type GVA, but it was not stable and the inserted sequence was lost from the genome. Replacing the duplicated promoter with a GVA-MP promoter derived from a distantly related isolate of GVA improved the stability of the inserted sequence. The resulting vector was successfully used to express the reporter gene beta-glucuronidase (GUS) and the coat protein gene of Citrus tristeza virus in inoculated N. benthamiana plants. Development of a useful GVA vector is expected to find a use as a biotechnological tool for improvement of grapevines and it may enable vine breeders to bypass obstacles involved in genetic manipulation of perennial and fruiting plants.

Binding Sites↗