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PubMed1981

DNA sequence of the rat growth hormone gene: location of the 5' terminus of the growth hormone mRNA and identification of an internal transposon-like element.

The present communication describes the molecular cloning and DNA sequence determination of the rat growth hormone (rGH) gene. The rGH gene was cloned on an 11 kilobase EcoRI fragment of total rat DNA; it has four intervening sequences which correspond in position to those of the human growth hormone (hGH) gene. One of the intervening sequences in the rGH gene contains a possible transposable element: a 200 base pair direct repeat that is itself flanked by an exact 15 base pair direct repeat. The DNA sequence was used to estimate the location of the 5' end of the mature growth hormone mRNA. By S1 nuclease mapping it was located approximately 25 bases "downstream" from a TATAAA sequence presumed to play a role in initiation of transcription of the rGH gene.

Animals↗
PubMed2004

Natural genetic variation caused by transposable elements in humans.

Transposons and transposon-like repetitive elements collectively occupy 44% of the human genome sequence. In an effort to measure the levels of genetic variation that are caused by human transposons, we have developed a new method to broadly detect transposon insertion polymorphisms of all kinds in humans. We began by identifying 606,093 insertion and deletion (indel) polymorphisms in the genomes of diverse humans. We then screened these polymorphisms to detect indels that were caused by de novo transposon insertions. Our method was highly efficient and led to the identification of 605 nonredundant transposon insertion polymorphisms in 36 diverse humans. We estimate that this represents 25-35% of approximately 2075 common transposon polymorphisms in human populations. Because we identified all transposon insertion polymorphisms with a single method, we could evaluate the relative levels of variation that were caused by each transposon class. The average human in our study was estimated to harbor 1283 Alu insertion polymorphisms, 180 L1 polymorphisms, 56 SVA polymorphisms, and 17 polymorphisms related to other forms of mobilized DNA. Overall, our study provides significant steps toward (i) measuring the genetic variation that is caused by transposon insertions in humans and (ii) identifying the transposon copies that produce this variation.

DNA Transposable Elements↗
PubMed1989

Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis.

The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported. This is the first characterised example of a mycobacterial insertion element. IS900 consists of 1451bp of which 66% is G + C. It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity. A single open reading frame (ORF 1197) coding for 399 amino acids is predicted. This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2). It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family. IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases.

Amino Acid Sequence↗
PubMed2007

Random segment deletion based on IS31831 and Cre/loxP excision system in Corynebacterium glutamicum.

A simple and random genome deletion method combining insertion sequence (IS) element IS31831 and the Cre/loxP excision system generated 42 Corynebacterium glutamicum mutants (0.2-186 kb). A total of 393.6 kb (11.9% of C. glutamicum R genome) coding for 331 genes was confirmed to be nonessential under standard laboratory conditions. The deletion strains, generated using only two vectors, varied not only in their lengths but also the location of the deletion along the C. glutamicum R genome. By comparing and analyzing the generated deletion strains, identification of nonessential genes, the roles of genes of hitherto unknown function, and gene-gene interactions can be easily and efficiently determined.

Blotting, Southern↗
PubMed1995

Identification in the human genome of mobile elements spread by DNA-mediated transposition.

We have identified in the human genome two families of mobile elements possessing the sequence characteristics of transposons that move directly from DNA to DNA rather than requiring the reverse transcription of an RNA intermediate. One type of element is closely related to the autonomous transposable element, mariner, and comprises a coding region for a transposase protein flanked by short terminal inverted repeat sequences (TIRs) of 31 or 32 bp. Elements of the second type form a family of short interspersed repetitive elements (SINEs) that are composed simply of two 37 bp TIRs surrounding six unique bps. The TIRs of the human mariner family are identical in all but one position to those of the SINE family, suggesting that the inverted-repeat SINEs represent non-autonomous transposable elements dependent on mariner-type transposase for mobility. Evidence for the mobility of both types of element is provided by examples of their integration into other repeat sequences and by the comparison of orthologous sites in cattle and human genomes. This evidence also shows that these elements have been active in DNA-mediated transposition at some point in the mammalian lineage. Therefore, it appears that the process of DNA-mediated transposition has occurred in mammalian cells and that its maximal cis-requirements are contained in the 80 bp consensus sequence of the human inverted-repeat SINE family.

Amino Acid Sequence↗
PubMed2005

Characterization of clonal complexity in tuberculosis by mycobacterial interspersed repetitive unit-variable-number tandem repeat typing.

In recent years, the application of molecular tools has shown us that clonal complexity in infection by Mycobacterium tuberculosis is not anecdotal. Exogenous reinfections, mixed infections, compartmentalization, and microevolution are different aspects of this issue. The detection and characterization of clonal variants of M. tuberculosis by standard genotyping methods is laborious and frequently requires expertise. Our aim was to evaluate a new genotyping PCR-based method for M. tuberculosis, mycobacterial interspersed repetitive unit-variable-number tandem repeat typing (MIRU-VNTR), as a potential tool to simplify and optimize the clonal analysis of tuberculosis. MIRU-VNTR was able to detect mixed clonal variants in vitro, even for clones at low ratios (1:99). This technique was prospectively applied to search for cases infected by more than one clone. Clonal variants within the same host were detected in 3 out of 115 cases (2.6%), including cases with clones which were indistinguishable by restriction fragment length polymorphism or spoligotyping. In one case, coinfecting clonal variants differed in antibiotic susceptibilities. MIRU-VNTR was applied to cases with proven polyclonal infection, and it succeeded in detecting the coinfecting strains and proved useful in confirming cases of compartmentalized infection. MIRU-VNTR is a simple, rapid, and sensitive method which could facilitate and optimize the identification and characterization of clonal complexity in M. tuberculosis infection.

DNA Transposable Elements↗
PubMed1995

Insertion of an Alu sequence in the Ca(2+)-sensing receptor gene in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.

Missense mutations in the calcium-sensing receptor (CaR) gene have previously been identified in patients with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). We studied family members of a Nova Scotian deme expressing both FHH and NSHPT and found, by PCR amplification of CaR gene exons, that FHH individuals were heterozygous and NSHPT individuals were homozygous for an abnormally large exon 7. This is due to an insertion at codon 877 of an Alu-repetitive element of the predicted-variant/human-specific-1 subfamily. It is in the opposite orientation to the CaR gene and contains an exceptionally long poly(A) tract. Stop signals are introduced in all reading frames within the Alu sequence, leading to a predicted shortened mutant CaR protein. The loss of the majority of the CaR carboxyl-terminal intracellular domain would dramatically impair its signal transduction capability. Identification of the specific mutation responsible for the FHH/NSHPT phenotype in this community will allow rapid testing of at-risk individuals.

Base Sequence↗
PubMed1980

[Use of a transposon determining tetracycline resistance (Tn10) in genetic studies of cholera vibrios].

Introduction of tetracycline-resistance transposon Tn10 into Rts1 (Km), a plasmid which is temperature-sensitive for DNA replication, made it possible to construct pNS10 (Km Tc) plasmid used as a vehicle for transferring Tn10 from Escherichia coli to cholera vibrio V. El Tor 218 as a result of intergeneric crosses. While being translocated from the plasmid genome to the chromosome of cholera vibrio transconjugants, Tn10 was able of insertion into structural genes thus leading to the formation of auxotrophic mutants. Ability of Tn10 to promote chromosomal rearrangements of the regions linked to Tn-element insertion site was used for identification of some structural genes in the neighbourhood of Tn-element insertion site.

Chromosome Mapping↗
PubMed1999

Abr1, a transposon-like element in the genome of the cultivated mushroom Agaricus bisporus (Lange) Imbach.

A 300-bp repetitive element was found in the genome of the white button mushroom, Agaricus bisporus, and designated Abr1. It is present in approximately 15 copies per haploid genome in the commercial strain Horst U1. Analysis of seven copies showed 89 to 97% sequence identity. The repeat has features typical of class II transposons (i.e., terminal inverted repeats, subterminal repeats, and a target site duplication of 7 bp). The latter shows a consensus sequence. When used as probe on Southern blots, Abr1 identifies relatively little variation within traditional and present-day commercial strains, indicating that most strains are identical or have a common origin. In contrast to these cultivars, high variation is found among field-collected strains. Furthermore, a remarkable difference in copy numbers of Abr1 was found between A. bisporus isolates with a secondarily homothallic life cycle and those with a heterothallic life cycle. Abr1 is a type II transposon not previously reported in basidiomycetes and appears to be useful for the identification of strains within the species A. bisporus.

Agaricus↗
PubMed2006

SXT-related integrating conjugative element in New World Vibrio cholerae.

SXT-related integrating conjugative elements (ICEs) became prevalent in Asian Vibrio cholerae populations after V. cholerae O139 emerged. Here, we describe an SXT-related ICE, ICEVchMex1, in a Mexican environmental V. cholerae isolate. Identification of ICEVchMex1 represents the first description of an SXT-related ICE in the Western Hemisphere. The significant differences between the SXT and ICEVchMex1 genomes suggest that these ICEs have evolved independently.

Conjugation, Genetic↗
PubMed1999

Genome scanning in Haemophilus influenzae for identification of essential genes.

We have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames of Haemophilus influenzae by PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented.

Blotting, Southern↗
PubMed1982

Nucleotide sequences of the murine retrovirus Friend SFFVp long terminal repeats: identification of a structure with extensive dyad symmetry 5' to the TATA box.

The two long terminal repeats (LTRs) of the integrated provirus of the polycythemia strain of the Friend spleen focus forming virus have been sequenced. Each of the identical LTRs is 514 nucleotides long and together confer on the provirus the features of a transposable element. They are terminated by perfect 11 base-pair inverted repeats, and the entire provirus is flanked by an apparent duplication of host DNA four nucleotides long. The assumed transcription regulatory sequences (Hogness-Goldberg box, CAAT box, polyadenylation signal) can be identified within the LTRs, as well as a region of an imperfect inverted repeat which extends from approximately 140 to 270 nucleotides 5' from the point of transcription initiation. This sequence may form a hairpin-type structure which might have some function in the promotion of transcription.

Animals↗
PubMed1993

Construction of a retrotransposition indicator sequence using a neomycin resistance-encoding gene containing a functional intron.

An intron from a Syrian hamster gene was inserted into a neo gene such that splicing of the neo gene mRNA results in the synthesis of active aminoglycoside phosphotransferase. The unspliced construct is inactive in Escherichia coli, but confers resistance to G418 when transfected into mouse and hamster cells. This selectable marker is designed to aid in the cloning and identification of genomic integration sites following retrotransposition.

Animals↗
PubMed1999

A gene involved in quinate metabolism is specific to one DNA homology group of Xanthomonas campestris.

A gene involved in quinate metabolism was cloned from Xanthomonas campestris pv. juglandis strain C5. The gene, qumA, located on a 4. 2-kb KpnI-EcoRV fragment in plasmid pQM38, conferred quinate metabolic activity to X. c. pv. celebensis. Tn3-spice insertional analyses further located the qumA gene on a region of about 3.0 kb within pQM38. Nucleotide sequencing of this 3.0-kb fragment reveals that the coding region of qumA is 2373 bp, the deduced amino acid sequence of which closely resembles a pyrrolo-quinoline quinone-dependent quinate dehydrogenase of Acinetobacter calcoaceticus. A 0.7 kb SalI-PstI fragment internal to qumA was used as a probe to hybridize against total genomic DNA from 43 pathovars of X. campestris. The fragment hybridized only to total genomic DNA from the four pathovars of DNA homology group 6, X. c. pv. celebensis, X. c. pv. corylina, X. c. pv. juglandis and X. c. pv. pruni, and from X. c. pv. carotae, which belongs to DNA homology group 5. This 0.7 kb fragment was also used as a probe to hybridize BamHI-digested total genomic DNAs from the four pathovars of DNA homology group 6 and X. c. pv. carotae. The restriction fragment length polymorphism pattern of DNA homology group 6 was different from that of X. c. pv. carotae. The probe hybridized to a 5.7-kb BamHI fragment in all four pathovars of group 6 and to a 6.1-kb BamHI fragment in three of four pathovars. It hybridized only to a 9. 9-kb BamHI fragment in X. c. pv. carotae. Quinate metabolism has previously been reported as a phenotypic property specific to X. campestris DNA homology group 6. Accordingly, a combination of the quinate metabolism phenotypic test and Southern hybridization using a qumA-derived probe will be very useful in the identification of pathovars in DNA homology group 6.

Alcohol Oxidoreductases↗
PubMed1986

Construction and properties of Tn917-lac, a transposon derivative that mediates transcriptional gene fusions in Bacillus subtilis.

A derivative of Tn917 was constructed, referred to as Tn917-lac, which is capable of generating fusions that connect the transcripts of Bacillus subtilis chromosomal genes to the coding sequence of the lacZ gene of Escherichia coli. Two independent insertions of Tn917-lac into the gltA gene and one insertion into the trpE gene (in the trpEDCFBA operon) of B. subtilis were studied in detail, and the results confirmed that Tn917-lac-mediated transcriptional fusions produce levels of beta-galactosidase that reflect accurately the regulated expression of interrupted genes. To facilitate these studies, a procedure was developed that permits the analysis of Tn917-lac-mediated fusions in partial diploids where insertional mutations are complemented by an intact copy of the interrupted genes. Tn917 is known to function efficiently in bacteria representing three quite different Gram-positive genera (Streptococcus, Bacillus, and Staphylococcus) and is known to display a relatively high degree of randomness in its insertions into bacterial genomes, making it likely that Tn917-lac will be useful for the identification and study of many kinds of regulated genes in a wide range of Gram-positive species.

Bacillus subtilis↗
PubMed2001

Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp. facilitates TnphoA mutagenesis.

Burkholderia pseudomallei and Burkholderia mallei are pathogens responsible for disease in both humans and animals. Burkholderia thailandensis, while phylogenetically similar, is considered avirulent in comparison. These three species exhibit phosphatase activity when grown on media containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (XP). Tn5-OT182 mutagenesis has been utilized to isolate mutants of B. pseudomallei and B. thailandensis unable to hydrolyse XP. Sequence analysis of these mutants revealed an ORF of 1734 nucleotides demonstrating a high degree of homology to the acpA gene product of Francisella tularensis. PCR primers were designed based on the B. pseudomallei acpA gene sequence and were used to amplify an acpA homologue from B. mallei. The predicted amino acid sequence of B. pseudomallei AcpA differed from those of the predicted B. thailandensis AcpA and B. mallei AcpA by 15 and 3 amino acids, respectively. Allelic exchange was used to construct DeltaacpA mutants in each of these Burkholderia spp. These mutants were shown to be devoid of phosphatase activity and have subsequently allowed for the implementation of phoA fusion transposon mutagenesis systems. Two such systems have been successfully utilized in Burkholderia spp. for the identification of several genes encoding exported proteins.

Acid Phosphatase↗