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PubMed2003

Identification and characterization of a novel enhancer for the human MCT-1 oncogene promoter.

Cloning and characterization of the promoter region for the MCT-1 oncogene is described. We used luciferase assays to identify cis-acting elements responsible for human MCT-1 promoter function. The MCT-1 promoter is TATA-less with a consensus initiator element located at the transcription start site and facilitated by two Sp1 sites that directs basal transcription. Deletion of a region of the MCT-1 promoter (-133 to -122) resulted in significant decrease in luciferase activity, suggesting that this region contains a positive cis-acting element. Using mobility shift assays with a 26-mer oligonucleotide, which contains this fragment and its flanking regions, we demonstrated the presence of sequence-specific DNA-binding protein in both Jurkat and Hela nuclear extracts that we designated as LMBF (for lymphoid MCT-1 binding factor). This 26-mer oligonucleotide containing the LMBF binding site is required for maximum transcriptional activity of the MCT-1 promoter. Although the 26-mer oligonucleotide contains a sequence with strong homology to a heat-shock factor consensus, competitive electrophoretic mobility shift assay (EMSA) analysis demonstrated that the binding protein is not a known member of heat shock family. Furthermore, this sequence when placed in reverse orientation downstream of the luciferase gene was able to enhance luciferase activity driven by a minimal promoter. These data are consistent with this sequence behaving as an enhancer. Finally, Southwestern blot analysis revealed a 96-kDa protein capable of binding a probe containing the LMBF binding site.

5' Flanking Region↗
PubMed2004

Pathogenicity islands in bacterial pathogenesis.

In this review, we focus on a group of mobile genetic elements designated pathogenicity islands (PAI). These elements play a pivotal role in the virulence of bacterial pathogens of humans and are also essential for virulence in pathogens of animals and plants. Characteristic molecular features of PAI of important human pathogens and their role in pathogenesis are described. The availability of a large number of genome sequences of pathogenic bacteria and their benign relatives currently offers a unique opportunity for the identification of novel pathogen-specific genomic islands. However, this knowledge has to be complemented by improved model systems for the analysis of virulence functions of bacterial pathogens. PAI apparently have been acquired during the speciation of pathogens from their nonpathogenic or environmental ancestors. The acquisition of PAI not only is an ancient evolutionary event that led to the appearance of bacterial pathogens on a timescale of millions of years but also may represent a mechanism that contributes to the appearance of new pathogens within a human life span. The acquisition of knowledge about PAI, their structure, their mobility, and the pathogenicity factors they encode not only is helpful in gaining a better understanding of bacterial evolution and interactions of pathogens with eukaryotic host cells but also may have important practical implications such as providing delivery systems for vaccination, tools for cell biology, and tools for the development of new strategies for therapy of bacterial infections.

Bacteria↗
PubMed1992

The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase.

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5' termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3'-5' exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.

Amino Acid Sequence↗
PubMed1995

Segmented structure of protein sequences and early evolution of genome by combinatorial fusion of DNA elements.

A theory of an early stage of genome evolution by combinatorial fusion of circular DNA units is suggested, based on protein sequence "fossil" evidence. The evidence includes preference of protein sequence lengths for certain sizes--multiples of 123 aa for eukaryotes and multiples of 152 aa for prokaryotes. At the DNA level these sizes correspond to 350-450 base pairs--the known optimal range for DNA ring closure. The methionine residues repeatedly appear along the sequences with the same period of about 120 aa (in eukaryotes), presumably marking the sites of insertion of the early genes--rings of protein-coding DNA. No torsional constraint in this DNA results in very sharp estimate of the helical periodicity of the early DNA, indistinguishable from the experimental mean value for extant DNA. According to the combinatorial fusion theory, based on the above evidence, in the pregenomic, prerecombinational stage the genes and the noncoding sequences existed in form of autonomously replicating DNA rings of close to standard size, randomly segregating between dividing cells, like modern plasmids do. In the recombinational early genomic stage the rings started to fuse, forming larger DNA molecules consisting of several unit genes connected in various combinations and forming long protein-coding sequences (combinatorial fusion). This process, which involved, perhaps, noncoding sequences as well, eventually resulted in the formation of large genomes. The dispersed circular DNA--or, rather, evolutionarily advanced derivatives thereof--may still exist in the form of various mobile DNA elements.

Biological Evolution↗
PubMed2026

Long-read sequencing reveals putatively mobilizable resistance genes and multi-drug resistance plasmids underestimated by short-read metagenomics.

While shotgun metagenomics is often used to profile antibiotic resistome in gut microbial communities, few studies have investigated if the choice of sequencing platform and assembly strategy affect what mobile genetic elements and antimicrobial resistance genes are recovered. In this study, we compared three platforms (Illumina, Oxford Nanopore, and PacBio HiFi) and seven assembly strategies on gut metagenomes from cattle, pig, and human as case studies. Long-read assemblies recovered 5- to 7-fold more plasmid sequence than Illumina in cattle and pig (mean 17.0 Mb vs. 3.1 Mb), while Illumina performed comparably in the less diverse human gut where high per-species coverage enabled effective short-read plasmid assembly. Long reads also detected more resistance genes on plasmid contigs. Hybrid assembly results depended on the algorithm: scaffolding-based OPERA-MS preserved long-read contiguity and recovered more plasmid-borne resistance genes, while the short-read-centric metaSPAdes hybrid mode produced fragmented assemblies. After collapsing haplotype redundancy, PacBio HiFi identified 2 and 49 unique multi-drug resistance plasmid lineages in cattle and pig, respectively. On the other hand, only 2 and 4 were identified from Illumina. Long reads also placed far more ARGs in a putative mobilization context (50-73%) compared to 14-21% for short reads. Platform and assembly strategy are thus key variables in mobilome and resistome characterization and should be accounted for in antimicrobial resistance surveillance.

Animals↗
PubMed2001

Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens.

Previous studies have demonstrated that a proportion of Staphylococcus aureus isolates from bovine mastitis coproduce toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin C (SEC). In this study, molecular genetic analysis of one such strain, RF122, revealed the presence of a 15,891-bp putative pathogenicity island (SaPIbov) encoding the genes for TSST (tst), the SEC bovine variant (sec-bovine), and a gene (sel) which encodes an enterotoxin-like protein. The island contains 21 open reading frames specifying hypothetical proteins longer than 60 amino acids including an integrase-like gene. The element is bordered by 74-bp direct repeats at the left and right junctions, and the integration site lies adjacent to the 3' end of the GMP synthase gene (gmps) in the S. aureus chromosome. SaPIbov contains a central region of sequence identity with the previously characterized tst pathogenicity island SaPI1 (J. A. Lindsay et al., Mol. Microbiol. 29:527-543, 1998). A closely related strain, RF120, of the same multilocus enzyme electrophoretic type, random amplified polymorphic DNA type, and ribotype, does not contain the island, implying that the element is mobile and that a recent insertion/deletion event has taken place. TSST and TSST/SEC-deficient mutants of S. aureus strain RF122 were constructed by allele replacement. In vitro bovine Vbeta-specific lymphocyte expansion analysis by culture supernatants of wild-type strains and of tst and sec-bovine allele replacement mutants revealed that TSST stimulates BTB13-specific T cells whereas SEC-bovine stimulates BTB93-specific T cells. This suggests that the presence of SaPIbov may contribute to modulation of the bovine immune response.

Animals↗
PubMed1996

A widespread transposable element masks expression of a yeast copper transport gene.

The trace element copper (Cu) is essential for cell growth. In this report we describe the identification of a new component of the high-affinity Cu transport machinery in yeast, encoded by the CTR3 gene. Ctr3p is a small intracellular cysteine-rich integral membrane protein that restores high-affinity Cu uptake, Cu, Zn superoxide dismutase activity, ferrous iron transport, and respiratory proficiency to strains lacking the CTR1 (Cu transporter 1) gene. In most commonly used Saccharomyces cerevisiae laboratory strains, expression of CTR3 is abolished by a Ty2 transposon insertion that separates the CTR3 promoter from the transcriptional start sites by 6 kb. In strains that do not possess a Ty2 transposon at the CTR3 locus, expression of CTR3 is repressed by copper and activated by copper starvation. In such strains inactivation of both CTR1 and CTR3 is required to generate lethal copper-deficient phenotypes. Although Ctr1p and Ctr3p can function independently in copper transport, the expression of both proteins provides maximal copper uptake and growth rate under copper-limiting conditions. These results underscore the importance of mobile DNA elements in the alteration of gene function and phenotypic variation.

Amino Acid Sequence↗
PubMed2026

Characterizing the ecological niche of insertion sequences within prokaryotic genomes.

Insertion sequences (ISs) are widespread prokaryotic transposable elements, often regarded as genomic parasites that primarily cause deleterious mutations. However, they can also promote adaptive changes. These antagonistic properties make their overall impact on prokaryotic evolution difficult to grasp. Here, we address this challenge by leveraging the framework of transposon ecology to analyze IS occurrences across and within 30 499 prokaryotic genomes. Combining phylogenomics with multi-scale genomic analysis, quantitative ecology, and mathematical modeling, we provide evidence that although genomes generally provide sufficient resources for IS coexistence, universal mechanisms shape their occurrence and chromosomal distribution across genomes. These include (i) the preferential localization of ISs within highly variable and GC-heterogeneous chromosomal regions of genomic plasticity, which act as the primary reservoir of IS niches; (ii) a linear scaling between IS abundance and niche size, with an average of $5.4$ additional accessible insertion sites per IS; (iii) a dependence of IS occurrence on the presence of other ISs, suggesting a form of group behavior; (iv) the accumulation of AT-rich sequences in both coding and noncoding regions up to 100 kb around ISs, indicative of ecological isolation; and (v) the spatial partitioning of mobile genetic elements around ISs, reminiscent of ecological niche differentiation. Besides these general principles, we also uncover niche specificities associated with particular IS families, hinting at regulatory mechanisms that modulate IS activity. Altogether, this comprehensive transposon ecology approach offers new insights and avenues for understanding IS-host interactions and genome evolution, moving beyond traditional host-centric perspectives.

DNA Transposable Elements↗
PubMed1997

Common themes in microbial pathogenicity revisited.

Bacterial pathogens employ a number of genetic strategies to cause infection and, occasionally, disease in their hosts. Many of these virulence factors and their regulatory elements can be divided into a smaller number of groups based on the conservation of similar mechanisms. These common themes are found throughout bacterial virulence factors. For example, there are only a few general types of toxins, despite a large number of host targets. Similarly, there are only a few conserved ways to build the bacterial pilus and nonpilus adhesins used by pathogens to adhere to host substrates. Bacterial entry into host cells (invasion) is a complex mechanism. However, several common invasion themes exist in diverse microorganisms. Similarly, once inside a host cell, pathogens have a limited number of ways to ensure their survival, whether remaining within a host vacuole or by escaping into the cytoplasm. Avoidance of the host immune defenses is key to the success of a pathogen. Several common themes again are employed, including antigenic variation, camouflage by binding host molecules, and enzymatic degradation of host immune components. Most virulence factors are found on the bacterial surface or secreted into their immediate environment, yet virulence factors operate through a relatively small number of microbial secretion systems. The expression of bacterial pathogenicity is dependent upon complex regulatory circuits. However, pathogens use only a small number of biochemical families to express distinct functional factors at the appropriate time that causes infection. Finally, virulence factors maintained on mobile genetic elements and pathogenicity islands ensure that new strains of pathogens evolve constantly. Comprehension of these common themes in microbial pathogenicity is critical to the understanding and study of bacterial virulence mechanisms and to the development of new "anti-virulence" agents, which are so desperately needed to replace antibiotics.

Bacteria↗
PubMed1987

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover.

Amino Acid Sequence↗
PubMed1997

Segmented structure of separate and transposable DNA and RNA elements as suggested by their size distributions.

A collection of about 1000 different eukaryotic and prokaryotic DNA mobile and separate elements is compiled from literature-transposons, plasmids, extrachromosomal circular DNA, insertion sequences, as well as viral genomes and separate genome segments. Only small elements are collected, upto 2000 base pairs. Analysis of the sequence length distributions of the elements reveals that certain sizes are clearly preferred, namely those which correspond to multiples of about 345 bp in eukaryotes and multiples of about 210 bp in prokaryotes. This provides additional evidence in support of the theory (1) that segmented structure is characteristic of not only protein-coding sequences (2) but rather of genomes in general. In particular, it confirms the prediction (1) that mobile and separate elements would also be segmented.

Animals↗
PubMed2003

Ah receptor regulation of mouse Cyp1B1 is additionally modulated by a second novel complex that forms at two AhR response elements.

Cyp1B1 is expressed constitutively in many extrahepatic cells and is induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). An enhancer region (AhER-810 to -1075 of the mouse Cyp1B1 promoter), which mediates aryl hydrocarbon receptor (AhR) regulation of transcription, contains three consensus XRE sequences (designated XRE1, XRE4, and XRE5) and a central Ebox. XRE5 is essential for both basal and induced activity in C3H10T1/2 cells. AhR/ARNT binding to XRE1, XRE4, and the Ebox complex function in combination to support the AhR/ARNT complex at XRE5. The identical 12 base cores of XRE1 and XRE4 differ from the core of XRE5 by two bases outside of the consensus XRE. These sites bind a constitutive complex slightly smaller than AhR/ARNT (anomalous complex; anC), which is not formed at XRE5 or six Cyp1A1 XREs. Exchange of these bases (m3 mutations) restores selective AhR/ARNT binding at XRE1/XRE4 and introduces anC binding at XRE5. The activities of multimeric XRE1 and XRE5 luciferase reporters responded in parallel to the extent of AhR/ARNT binding. The consensus anC binding sequence ((C/T)GCG(C/T)GCGC(C/A)GC) overlaps the XRE1/XRE4 AhR/ARNT element. Gel mobility analyses show that anC binds to XRE1/XRE4 under basal conditions, while AhR/ARNT partially displaces anC following TCDD induction. Selective depletion of anC with biotin-oligonucleotides increases AhR/ARNT binding. M3-mutations at, respectively, XRE1 and XRE4 of the AhER sequence, had opposite effects on luciferase reporters. Activities increased for the XRE1 mutation and decreased for the XRE4 mutation, but also depended on the level of AhR transfected into AhR -/- fibroblasts. AnC compete with AhR at XRE1 while playing an activating role at XRE4. This positive effect of constitutive anC binding at XRE4 may contribute to the characteristic basal Cyp1B1 expression in embryo fibroblasts, which is mediated by low constitutive activities of AhR.

Animals↗
PubMed2004

Flytrap, a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster.

Flytrap is a web-enabled relational database of transposable element insertions in Drosophila melanogaster. A green fluorescent protein (GFP) artificial exon carried by a transposable P-element is mobilized and inserted into a host gene intron creating a GFP fusion protein. The sequence of the tagged gene is determined by sequencing inverse-PCR products derived from genomic DNA. Flytrap contains two principle data types: micrographs of protein localization and a cellular component ontology, based on rules derived from the Gene Ontology consortium (http://www.geneontology.org), describing protein localization. Flytrap also has links to gene information contained in Flybase (http:// flybase.bio.indiana.edu). The system is designed to accept submissions of micrographs and descriptions from any type of tissue (e.g. wing imaginal disk, ovary) and at any stage of development. Insertion lines can be searched using a number of queries, including Berkeley Drosophila Genome Project (BDGP) numbers and protein localization. In addition, Flytrap provides online order forms linked to each insertion line so that users may request any line generated from this project. Flytrap may be accessed from the homepage at http://flytrap.med. yale.edu.

Animals↗
PubMed2006

Lysophosphatidic acid induces early growth response gene 1 expression in vascular smooth muscle cells: CRE and SRE mediate the transcription.

OBJECTIVE: Lysophosphatidic acid (LPA), one component of oxidized low-density lipoprotein, is a potent bioactive phospholipid. Early growth response gene-1 (Egr-1), an important transcription factor, regulates expression of an array of genes involved in vascular diseases. Whether and how LPA regulates the transcriptional machinery of Egr-1 gene is unknown and is addressed in this study. METHOD AND RESULTS: We found that LPA markedly induces Egr-1 mRNA and protein in aortic smooth muscle cells (SMCs). RNA stability and nuclear run-on assays reveal that LPA-induced Egr-1 gene expression is controlled at the transcriptional level. Reporter gene analyses have shown that the -141 to +20 nt region of the Egr-1 promoter contains regulatory elements. Electrophoretic mobility shift assays reveal that the DNA-binding activities of both CREB and SRF to the CRE and SRE motifs of the Egr-1 promoter are markedly elevated in response to LPA. The increased binding activity depends on the phosphorylation of CREB and SRF. Luciferase assays of a series of deleted or mutated Egr-1 promoter-reporter gene constructs, along with dominant negative CREB transfection analysis revealed that the 2 CRE sites and the 2 proximal SRE sites in the Egr-1 promoter are required for maximal LPA-induced Egr-1 gene expression. CONCLUSIONS: Our data reveal that LPA regulates Egr-1 expression via transcription factors CREB and SRF. These results establish a novel role for CREB in mediating LPA-induced gene expression. Our results imply that elevated LPA levels may, through activation of Egr-1, which regulates an array of atherogenic genes, exacerbate atheromatous lesions.

Animals↗
PubMed2003

Alterations in the expression of transcription factors and the reduced folate carrier as a novel mechanism of antifolate resistance in human leukemia cells.

The human reduced folate carrier (hRFC) is the dominant transporter mediating the uptake of reduced folate cofactors and antifolate anticancer drugs. Defective antifolate uptake due to inactivating mutations in the hRFC gene is an established mechanism of drug resistance in various tumor cells. However, while antifolate transport is frequently impaired, either no or only a single hRFC allele is inactivated, suggesting that additional mechanism(s) of resistance are operative. Here we studied the relationship between the expression and function of transcription factors and antifolate resistance in transport-defective leukemia cells that poorly express or completely lack RFC mRNA. Stable transfection with a hRFC expression construct resulted in restoration of normal RFC mRNA expression and nearly wild type drug sensitivity in these antifolate-resistant cells. The loss of RFC gene expression prompted us to explore transcription factor binding to the hRFC promoter. The hRFC promoter contains an upstream GC-box and a downstream cAMP-response element (CRE)/AP-1-like element. Electrophoretic mobility shift assays and oligonucleotide competition revealed a substantial loss of nuclear factor binding to CRE and GC-box in these drug-resistant cell lines. Consistently, antibody-mediated supershift analysis showed a marked decrease in the binding of CRE-binding protein 1 (CREB-1) and specificity protein 1 (Sp1) to CRE and GC-box, respectively. Western blot analysis revealed undetectable expression of CREB-1, decreased ATF-1 levels, parental Sp1 levels, and increased levels of the short Sp3 isoforms, recently shown to repress hRFC gene expression. Transient transfections into these antifolate-resistant cells demonstrated a marked loss of GC-box-dependent, and CRE-driven reporter gene activities and introduction of CREB-1 or Sp1 expression constructs resulted in restoration of hRFC mRNA expression. These results establish a novel mechanism of antifolate resistance that is based on altered expression and function of transcription factors resulting in transcriptional silencing of the hRFC promoter.

Alleles↗
PubMed2004

Evolution of the MAT locus and its Ho endonuclease in yeast species.

The genetics of the mating-type (MAT) locus have been studied extensively in Saccharomyces cerevisiae, but relatively little is known about how this complex system evolved. We compared the organization of MAT and mating-type-like (MTL) loci in nine species spanning the hemiascomycete phylogenetic tree. We inferred that the system evolved in a two-step process in which silent HMR/HML cassettes appeared, followed by acquisition of the Ho endonuclease from a mobile genetic element. Ho-mediated switching between an active MAT locus and silent cassettes exists only in the Saccharomyces sensu stricto group and their closest relatives: Candida glabrata, Kluyveromyces delphensis, and Saccharomyces castellii. We identified C. glabrata MTL1 as the ortholog of the MAT locus of K. delphensis and show that switching between C. glabrata MTL1a and MTL1alpha genotypes occurs in vivo. The more distantly related species Kluyveromyces lactis has silent cassettes but switches mating type without the aid of Ho endonuclease. Very distantly related species such as Candida albicans and Yarrowia lipolytica do not have silent cassettes. In Pichia angusta, a homothallic species, we found MATalpha2, MATalpha1, and MATa1 genes adjacent to each other on the same chromosome. Although some continuity in the chromosomal location of the MAT locus can be traced throughout hemiascomycete evolution and even to Neurospora, the gene content of the locus has changed with the loss of an HMG domain gene (MATa2) from the MATa idiomorph shortly after HO was recruited.

Deoxyribonucleases, Type II Site-Specific↗
PubMed2006

Characterization of integrons and tetracycline resistance determinants in Aeromonas spp. isolated from South African aquaculture systems.

An increasing incidence of multidrug resistance amongst Aeromonas spp. isolates, which are both fish pathogens and emerging opportunistic human pathogens, has been observed worldwide. This can be attributed to the horizontal transfer of mobile genetic elements, viz.: plasmids and class 1 integrons. The antimicrobial susceptibilities of 37 Aeromonas spp. isolates, from tilapia, trout and koi aquaculture systems, were determined by disc-diffusion testing. The plasmid content of each isolate was examined using the alkaline lysis protocol. Tet determinant type was determined by amplification using two degenerate primer sets and subsequent HaeIII restriction. The presence of integrons was determined by PCR amplification of three integrase genes, as well as gene cassettes, and the qacEDelta1-sulI region. Thirty-seven Aeromonas spp. isolates were differentiated into six species by aroA PCR-RFLP, i.e., A. veronii biovar sobria, A. hydrophila, A. encheleia, A. ichtiosoma, A. salmonicida, and A. media. High levels of resistance to tetracycline (78.3%), amoxicillin (89.2%), and augmentin (86.5%) were observed. Decreased susceptibility to erythromycin was observed for 67.6% of isolates. Although 45.9% of isolates displayed nalidixic acid resistance, majority of isolates were susceptible to the fluoroquinolones. The MAR index ranged from 0.12 to 0.59, with majority of isolates indicating high-risk contamination originating from humans or animals where antibiotics are often used. Plasmids were detected in 21 isolates, with 14 of the isolates displaying multiple plasmid profiles. Single and multiple class A family Tet determinants were observed in 27% and 48.7% of isolates, respectively, with Tet A being the most prevalent Tet determinant type. Class 1 integron and related structures were amplified and carried different combinations of the antibiotic resistance gene cassettes ant(3'')Ia, aac(6')Ia, dhfr1, oxa2a and/or pse1. Class 2 integrons were also amplified, but the associated resistance cassettes could not be identified. Integrons and Tet determinants were carried by 68.4% of isolates bearing plasmids, although it was not a strict association. These plasmids could potentially mobilize the integrons and Tet determinants, thus transferring antimicrobial resistance to other water-borne bacteria or possible human pathogens. The identification of a diversity of resistance genes in the absence of antibiotic selective pressure in Aeromonas spp. from aquaculture systems highlights the risk of these bacteria serving as a reservoir of resistance genes, which may be transferred to other bacteria in the aquaculture environment.

Aeromonas↗
PubMed2004

Lig4 and rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila.

Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w(hd) P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w(hd) P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females was not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w(hd) excision. PCR analysis indicated that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2-8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. Our data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4.

Alleles↗