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PubMed1995

Multilocus genomic mapping with intracisternal A-particle proviral oligonucleotide probes hybridized to mouse DNA in dried agarose gels.

Recently, oligonucleotide probes that detect intracisternal A-particle (IAP) gene subfamilies with a limited number of proviral copies have been shown to be useful multilocus markers. A procedure for hybridization of these probes has been developed and is described. In summary, the main features of the method are the following: (i) A pulse controller is used during agarose gel electrophoresis to improve resolution of restriction fragments in genomic DNA. (ii) Hybridization is performed in a dried gel. (iii) The hybridized gel is washed in tetramethylammonium chloride to eliminate differences in oligonucleotide composition on hybrid stability. Use of the procedure is demonstrated by genomic mapping of IAP loci in the AXB BXA recombinant inbred mouse strains, identification of hypomethylated loci in tumor cells, and detection of a transposed IAP provirus previously identified as the basis for a mutation at the agouti locus.

Animals↗
PubMed2006

Sequence analysis and quantification of transposase cDNAs of transposon TCp3.2 in Cydia pomonella larvae.

The Tc1-like transposable element TCp3.2 was previously found to be horizontally transferred from the genome of Cydia pomonella to the C. pomonella granulovirus (CpGV). In this study, the transcription of transposase genes of endogenous TCp3.2 copies in the insect host genome was investigated. Cloning and sequencing of cDNAs prepared from TCp3.2 transposase transcripts resulted in the identification of a 199-bp-long intron. Sequence heterogeneities among different cDNA clones suggested that multiple copies of the transposase are transcribed, but that a part of these copies encode a defective transposase. The actin gene of C. pomonella was cloned and sequenced, and used to standardise quantitative real time PCR on prepared cDNA of the TCp3.2 transposase. Comparison of cDNA levels of TCp3.2 transposase prepared from mock and CpGV-infected C. pomonella larvae did not provide evidence that CpGV infection influenced the transcription level of TCp3.2 transposase.

Amino Acid Sequence↗
PubMed1998

In vivo transposon mutagenesis in Haemophilus influenzae.

In order to devise an in vivo insertion mutagenesis scheme for Haemophilus influenzae, a novel set of transposons has been constructed. These are Tn10-based minitransposons carried on pACYC184- and pACYC177-based replicons, which are suitable for in vivo transposition in H. influenzae. The transposon delivery system was designed to contain an H. influenzae-specific uptake signal sequence which facilitates DNA transformation into H. influenzae. The following mini-Tn10 elements have been made suitable for specific tasks in H. influenzae: (i) Tn10d-cat, which can be used to generate chloramphenicol-selectable insertion mutations; (ii) Tn10d-bla, an ampicillin-selectable translational fusion system allowing the detection of membrane or secreted proteins; and (iii) Tn10d-lacZcat, a chloramphenicol-selectable lacZ transcriptional fusion system. For the rapid identification of the transposon insertions, a PCR fragment enrichment method was developed. This report demonstrates that this in vivo mutagenesis technique is a convenient tool for the analysis of biochemical and regulatory pathways in the human pathogen H. influenzae.

Chloramphenicol O-Acetyltransferase↗
PubMed2025

Genome-wide analysis of Enterococcus faecalis genes that facilitate interspecies competition with Lactobacillus crispatus.

Enterococci are opportunistic pathogens notorious for causing a variety of infections. While both Enterococcus faecalis and Lactobacillus crispatus are commensal residents of the vaginal tract, the molecular mechanisms that enable E. faecalis to take advantage of a vaginal biome with lower counts of lactobacilli to colonize the vaginal tract and induce aerobic vaginitis remain unknown. Here, we show that L. crispatus eradicates E. faecalis in a contact-independent manner. Using transposon sequencing to identify E. faecalis OG1RF transposon (Tn) mutants that are either under-represented or over-represented when co-cultured with L. crispatus, we found that Tn mutants with disruption in the dltABCD operon, that encodes the proteins responsible for the D-alanylation of teichoic acids, and OG1RF_11697 encoding for an uncharacterized hypothetical protein are more susceptible to killing by L. crispatus. Inversely, Tn mutants with disruption in ldh1, which encodes for L-lactate dehydrogenase, are more resistant to L. crispatus killing. Using the Galleria mellonella infection model, we show that co-injection of L. crispatus with E. faecalis OG1RF enhances larvae survival while this L. crispatus-mediated protection was lost in larvae co-infected with either L. crispatus and E. faecalisΔldh1 or Δldh1Δldh2 strains. Last, using RNA sequencing to identify E. faecalis genes that are differently expressed in the presence of L. crispatus, we found major changes in the expression of genes associated with glycerophospholipid metabolism, central metabolism, and general stress responses. The findings in this study provide insights into how E. faecalis mitigate assaults by L. crispatus.IMPORTANCEEnterococcus faecalis is an opportunistic pathogen notorious for causing a multitude of infections. As vaginal commensals, E. faecalis must interact with Lactobacillus crispatus, but how E. faecalis overcomes or mitigate assaults by L. crispatus killing remains unknown. We show that L. crispatus eradicates E. faecalis temporally in a contact-independent manner. Using high-throughput molecular approaches, we identified genetic determinants that enable E. faecalis to compete with L. crispatus. This study represents an important first step for the identification of adaptive genetic traits required for enterococci to tolerate assaults by lactobacilli.

Enterococcus faecalis↗
PubMedDate not supplied

Specificity of modified Drosophila mariner transposons in the identification of Leishmania genes.

Genetic manipulation of the protozoan Leishmania has led to a better understanding of the survival and development of these pathogens within their hosts. The association of the Leishmania genome sequencing information with the ability of transposons to introduce or destroy phenotypes allows a global perspective on the role and importance of genes in cellular pathways. Herein we report the construction and testing of mariner transposable elements carrying the neomycin phosphotransferase, green fluorescent protein, or beta-glucuronidase genes as reporters for translational fusion events. We demonstrate that the expression of the reporter genes will occur only when the genes are inserted in-frame within predicted genes. Our results not only add to the mariner toolkit for gene manipulation but also strengthen the evidence that the mariner system is a reliable means for the study of gene expression in Leishmania.

Animals↗
PubMed1993

Promoter binding proteins of an active human L1 retrotransposon.

Human Long Interspersed elements (LINEs or L1Hs elements) are a highly repetitive class of DNA sequences which are dispersed by retrotransposition. Full length L1Hs transcripts and L1-encoded proteins have been shown by others to be present in NTera2D1 and other related teratocarcinoma cell lines. Using standard methods for the identification of DNA binding proteins we identified two proteins or complexes of proteins which specifically bind to the L1Hs promoter region and are qualitatively different between NTera2D1 and HeLa cell nuclear extracts. These proteins represent candidates for the factors controlling the differential expression of L1 sequences.

Base Sequence↗
PubMed1994

Screening of genomic DNA to identify mutations in the gene for Bruton's tyrosine kinase.

Mutations in the gene for Bruton's tyrosine kinase (Btk) are responsible for X-linked agammaglobulinemia (XLA). Thus far, mutations in this gene have been identified based on alterations in Southern or Northern blot analysis or cDNA sequence. To permit detection of mutations in genomic DNA, we designed PCR primers to flank each of the 19 exons of Btk with splice sites. Two overlapping PCR products were employed for exons longer than 230 base pairs. Single strand conformation polymorphism (SSCP) analysis was used to screen PCR products from 30 unrelated families presumed to carry a Btk mutation. It was possible to amplify DNA in every reaction from every patient, indicating that large deletions in Btk are uncommon. Twenty three different mutations were found in 25 unrelated families, including one family in whom DNA was available from a carrier but not an affected patient. Seven mutations were single base pair substitutions resulting in premature stop codons scattered throughout the gene. Small insertions or deletions causing frameshifts and secondary premature stop codons constituted an additional seven mutations. One patient had a point mutation in the start codon and one patient had a mutation in a splice donor site. Point mutations resulting in amino acid substitutions were seen in nine patients. Northern blot analysis of RNA from three patients with premature stop codons showed an absence of Btk transcript whereas four patients with amino acid substitutions had normal amounts of transcript of normal size. These studies document the considerable variability in the Btk mutations causing XLA and they demonstrate an approach that will be useful for carrier detection as well as mutation identification.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1989

Cloning of the altered mRNA stability (ams) gene of Escherichia coli K-12.

A temperature-sensitive mutation in the ams gene of Escherichia coli causes an increase in the chemical half-life of pulse-labeled RNA at the nonpermissive temperature. Using lambda clones containing DNA fragments from the 23- to 24-min region on the E. coli chromosome, we have isolated a 5.8-kilobase DNA fragment which, when present in a low-copy-number plasmid, complements the conditional lethality and increased mRNA stability associated with the ams-1 mutation. The approximate initiation site and the direction of transcription of the ams gene were determined from the size of truncated polypeptides produced by Tn1000 insertions and Bal 31 deletions. Overexpression of the ams locus by using a T7 RNA polymerase-promoter system permitted the identification of an ams-encoded polypeptide of 110 kilodaltons.

Blotting, Southern↗
PubMed2000

Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes.

Viridans streptococci, which include Streptococcus gordonii, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of S. gordonii Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn916 transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants of S. gordonii Challis were identified based on Southern hybridization with a Tn916-based probe and DNA sequences of the Tn916-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.

Biofilms↗
PubMed2004

PIF- and Pong-like transposable elements: distribution, evolution and relationship with Tourist-like miniature inverted-repeat transposable elements.

Miniature inverted-repeat transposable elements (MITEs) are short, nonautonomous DNA elements that are widespread and abundant in plant genomes. Most of the hundreds of thousands of MITEs identified to date have been divided into two major groups on the basis of shared structural and sequence characteristics: Tourist-like and Stowaway-like. Since MITEs have no coding capacity, they must rely on transposases encoded by other elements. Two active transposons, the maize P Instability Factor (PIF) and the rice Pong element, have recently been implicated as sources of transposase for Tourist-like MITEs. Here we report that PIF- and Pong-like elements are widespread, diverse, and abundant in eukaryotes with hundreds of element-associated transposases found in a variety of plant, animal, and fungal genomes. The availability of virtually the entire rice genome sequence facilitated the identification of all the PIF/Pong-like elements in this organism and permitted a comprehensive analysis of their relationship with Tourist-like MITEs. Taken together, our results indicate that PIF and Pong are founding members of a large eukaryotic transposon superfamily and that members of this superfamily are responsible for the origin and amplification of Tourist-like MITEs.

Amino Acid Sequence↗
PubMed2002

Identification and partial characterization of a new Ceratitis capitata-specific 44-bp pericentromeric repeat.

Tandem satellite DNA repeats are often associated with centromeres. In spite of their importance in the organization of the centromere, they do not seem to be broadly conserved among species and their role is still unclear. Here we report the identification of a new 44-bp tandem pericentromeric repeat from the medfly, Ceratitis capitata. The repeat is specific to this insect and is not found in any of the other closely related species tested. It localizes in four out of its five autosomes and in the X chromosome. It is organized in long arrays, interspersed by transposable elements and other less well-defined sequence motifs.

Amino Acid Sequence↗
PubMed1994

Identification of distinct domains for signaling and receptor interaction of the sensory rhodopsin I transducer, HtrI.

The phototaxis-deficient mutant of Halobacterium salinarium, Pho81, lacks both sensory rhodopsin I (SR-I) and its putative transducer protein HtrI, according to immunoblotting and spectroscopic criteria. From restriction analysis and selected DNA sequencing, we have determined that the SR-I- HtrI- phenotype results from an insertion of a 520-bp transposable element, ISH2, into the coding region of the SR-I apoprotein gene sopI and deletion of 11 kbp upstream of ISH2 including the first 164 bp of sopI and the entire htrI gene. SR-I and HtrI expression as well as full phototaxis sensitivity are restored by transformation with a halobacterial plasmid carrying the htrI-sopI gene pair and their upstream promoter region. An internal deletion of a portion of htrI encoding the putative methylation and signaling domains of HtrI (253 residues) prevents the restoration of phototaxis, providing further evidence for the role of HtrI as a transducer for SR-I. Analysis of flash-induced photochemical reactions of SR-I over a range of pH shows that the partially deleted HtrI maintains SR-I interactions sites responsible for modulation of the SR-I photocycle.

Antibodies, Bacterial↗
PubMed2001

Detection of Mycobacterium tuberculosis in clinical samples using insertion sequences IS6110 and IS990.

To detect Mycobacterium tuberculosis in clinical samples, we used the M. tuberculosis-complex specific insertion sequence IS990 as the target in a simple DIG-PCR ELISA assay, as this element is present as a single copy in all strains of M. tuberculosis we have examined to date. The IS990 test was compared with a similar PCR that utilizes IS6110 as target. For detection of PCR product, digoxigenin-11-dUTP (DIG-dUTP) was incorporated into the product. After amplification, the PCR product was hybridized with biotinylated capture probe, which was complementary to the inner part of the amplicon. The hybrid was captured onto streptavidin-coated microtiter plate and DIG-labeled PCR product was detected using a peroxidase-conjugated antibody to DIG. We evaluated DIG-PCR ELISA for the detection of M. tuberculosis DNA in 265 respiratory and non-respiratory specimens taken from patients with known and suspected tuberculosis disease or from controls. The sensitivity and specificity of both IS990-based test and IS6110-based test was 96.5% and 95.3% respectively, comparable to the sensitivity and specificity of the IS6110-based test. The results demonstrate that the IS990 PCR ELISA test is a rapid and sensitive tool for the detection and identification of M. tuberculosis in clinical samples, and may have advantages to the more widely used IS6110-based tests, particularly in areas where IS6110-negative strains are found.

DNA Transposable Elements↗
PubMed1996

Development of techniques for the genetic manipulation of the gliding bacteria Lysobacter enzymogenes and Lysobacter brunescens.

Lysobacter enzymogenes and Lysobacter brunescens are Gram-negative gliding bacteria that belong to the gamma subgroup of the proteobacteria. As a first step toward a molecular analysis of Lysobacter gliding motility, we developed techniques to genetically manipulate these bacteria. Cosmid pSUP106 of the broad host range incompatibility group Q (Inc Q) was introduced into L. enzymogenes and L. brunescens by conjugation and electroporation. pSUP106 replicated stably in both organisms and conferred antibiotic resistance. We also identified several other plasmids (pKT210, pH1JI) that functioned in L. enzymogenes and a transposon (mini-Tn5Sp) that functioned in L. brunescens. The identification of these tools allows genetic analysis of Lysobacter gliding motility, exoenzyme production, and production of antibiotics and other secondary metabolites.

Anti-Bacterial Agents↗
PubMed1998

Transposable elements and genome organization: a comprehensive survey of retrotransposons revealed by the complete Saccharomyces cerevisiae genome sequence.

We conducted a genome-wide survey of Saccharomyces cerevisiae retrotransposons and identified a total of 331 insertions, including 217 Ty1, 34 Ty2, 41 Ty3, 32 Ty4, and 7 Ty5 elements. Eighty-five percent of insertions were solo long terminal repeats (LTRs) or LTR fragments. Overall, retrotransposon sequences constitute >377 kb or 3.1% of the genome. Independent evolution of retrotransposon sequences was evidenced by the identification of a single-base pair insertion/deletion that distinguishes the highly similar Ty1 and Ty2 LTRs and the identification of a distinct Ty1 subfamily (Ty1'). Whereas Ty1, Ty2, and Ty5 LTRs displayed a broad range of sequence diversity (typically ranging from 70%-99% identity), Ty3 and Ty4 LTRs were highly similar within each element family (most sharing >96% nucleotide identity). Therefore, Ty3 and Ty4 may be more recent additions to the S. cerevisiae genome and perhaps entered through horizontal transfer or past polyploidization events. Distribution of Ty elements is distinctly nonrandom: 90% of Ty1, 82% of Ty2, 95% of Ty3, and 88% of Ty4 insertions were found within 750 bases of tRNA genes or other genes transcribed by RNA polymerase III. tRNA genes are the principle determinant of retrotransposon distribution, and there is, on average, 1.2 insertions per tRNA gene. Evidence for recombination was found near many Ty elements, particularly those not associated with tRNA gene targets. For these insertions, 5'- and 3'-flanking sequences were often duplicated and rearranged among multiple chromosomes, indicating that recombination between retrotransposons can influence genome organization. S. cerevisiae offers the first opportunity to view organizational and evolutionary trends among retrotransposons at the genome level, and we hope our compiled data will serve as a starting point for further investigation and for comparison to other, more complex genomes.

Base Sequence↗
PubMed1986

Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli.

The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430 omega APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430 omega APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).

Bacillus thuringiensis↗
PubMed2026

PAT: An Image Analysis Tool for Automated Scoring of Pollen in Alexander-Stained Anthers.

Quantitative pollen viability analysis is a critical but labor-intensive step in plant reproductive biology. Existing deep-learning Segment Anything Models (SAM) fail to reliably segment viable pollen in Alexander-stained anthers. To address this, we fine-tuned an existing Cellpose-SAM model for pollen segmentation. We integrated it into PAT (Pollen Analysis Tool), a cross-platform desktop application. PAT features instance segmentation with interactive quality control, an in-app model retraining module, and publication-ready statistical outputs. We deployed PAT in an EMS suppressor screen of semi-sterile Arabidopsis smg7-6 mutants, enabling efficient candidate prioritization for whole-genome sequencing and mapping of the candidate mutation. This screen led to the identification of a point mutation in CAP-D2 (capd2-2), a Condensin I subunit, that rescues the smg7-6 meiotic phenotype. Notably, mutation in a Condensin II subunits (CAP-D3 and CAP-H2) does not confer rescue. Further characterization suggests the capd2-2 allele is hypomorphic, showing no defects in vegetative growth, chromocenter compaction, or transposable element silencing. Collectively, we demonstrate that accessible AI tools have the potential to bridge gaps in plant phenotyping and accelerate the pace of biological discovery.

Alexander staining↗
PubMed2000

Evolutionary history of the human endogenous retrovirus family ERV9.

Several distinct families of endogenous retrovirus-like elements (ERVs) exist in the genomes of primates. Despite the important evolutionary consequences that carrying these intragenomic parasites may have for their hosts, our knowledge about their evolution is still scarce. A matter of particular interest is whether evolution of ERVs occurs via a master lineage or through several lineages coexisting over long periods of time. In this work, the paleogenomic approach has been applied to the study of the evolution of ERV9, one of the human endogenous retrovirus families mobilized during primate evolution. By searching the GenBank database with the first 676 bp of the ERV9 long terminal repeat, we identified 156 different element insertions into the human genome. These elements were grouped into 14 subfamilies based on several characteristic nucleotide differences. The age of each subfamily was roughly estimated based on the average sequence divergence of its members from the subfamily consensus sequence. Determination of the sequential order of diagnostic substitutions led to the identification of four distinct lineages, which retained their capacity of transposition over extended periods of evolution. Strong evidence for mosaic evolution of some of these lineages is presented. Taken altogether, the available data indicate that the possibility of ERV9 still being active in the human lineage can not be discarded.

Animals↗