Cloning of the uvrD gene of E. coli and identification of the product

The uvrD gene has been cloned from Escherichia coli chromosomal DNA into phage lambda, cosmid, and low-copy-number plasmid vectors. Comparison of the proteins encoded by the cloned fragments with those encoded by fragments in which the uvrD gene is inactivated by transposon insertion or by deletion shows that the uvrD gene product is a protein of Mr = 73000.     

Mapping of the plasmid (pLQ3) from Achromobacter and cloning of its cephalosporinase gene in Escherichia coli

We have constructed a physical map of the plasmid pLQ3 which was originally isolated from Achromobacter and which codes for a beta-lactamase. The enzyme specified by pLQ3 is expressed in Escherichia coli and is unusual in that it is a cephalosporinase, an enzyme usually coded for by chromosome. Plasmid pLQ3 is 12.4 kb in length and has a unique Bam HI site and two BglII sites. From a BamHI + BglII double digest of pLQ3, we have constructed a "shortened" plasmid, pLQ10, in which a 2.96-kb fragment is deleted. We have constructed a clone, pLQ22, in which a 3.27-kb fragment of pLQ3, carrying the beta-lactamase gene, is inserted into the BamHI site of pACYC184. By "comparative mapping" of single and multiple digests of each of these plasmids, we have been able to locate the cleavage sites for PstI, which makes seven cuts in pLQ3.     

Physical map of the nrdA-nrdB-ftsB-glpT region of the chromosomal DNA of Escherichia coli

Seven pLC plasmids (pLC 3-46, 8-12, 8-24, 8-29, 14-12, 19-24 and 42-17) which complemented nrdA, nrdB, ftsB and/or glpT mutations of Escherichia coli were analyzed. A restriction map of each plasmid was constructed and restriction fragments were subcloned into pBR322. A physical map of approx. a 15 X 10(6) Mr segment of the chromosomal DNA was deduced from the overlapping region of the pLC plasmids. The pLC plasmids and newly constructed plasmids were examined for the ability to rescue the mutations. The complementation tests defined the location of the genes in the 15 X 10(6) Mr segment in the following order: nrdA-nrdB-ftsB-glpT. Functional nrdAB and ftsB genes were located in the 3.1 X 10(6) Mr EcoRI-PstI fragment.     

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.     

Molecular cloning of replication and incompatibility regions from the R-plasmid R6K.

The construction of a physical map of R6K DNA on the basis of specific cleavage of R6K DNA by HindIII and EcoRI restriction endonucleases allowed us to determine the location of the R6K replication and drug resistance regions. Molecular cloning techniques were used to dissect the replication and incompatibility functions of R6K. This R-plasmid possesses two origins of replication, α and β, separated by a stretch of 3900 nucleotides. A region close to ori α. controls the copy number of the composite replicon. Inverted duplications which are 100 to 200 nucleotides long are found at the positions of ori α and ori β, respectively. A 1400-nucleotide long sequence within the region bounded by the inverted duplications and separate from the origins and the control region is involved in the R6K self-replication and replication under conditions of polymerase I deprivation. This region also contains some of the incompatibility genes of R6K. The sequentially asymmetrically bidirectional mode of R6K replication is due to the existence of a replication termination site. This terminator is located outside the sequences bounded by the inverted duplications and is not essential for plasmid DNA replication.     

Genomic landscape and evolutionary dynamics of mariner transposable elements within the Drosophila genus

Background

The mariner family of transposable elements is one of the most widespread in the Metazoa. It is subdivided into several subfamilies that do not mirror the phylogeny of these species, suggesting an ancient diversification. Previous hybridization and PCR studies allowed a partial survey of mariner diversity in the Metazoa. In this work, we used a comparative genomics approach to access the genus-wide diversity and evolution of mariner transposable elements in twenty Drosophila sequenced genomes.

Results

We identified 36 different mariner lineages belonging to six distinct subfamilies, including a subfamily not described previously. Wide variation in lineage abundance and copy number were observed among species and among mariner lineages, suggesting continuous turn-over. Most mariner lineages are inactive and contain a high proportion of damaged copies. We showed that, in addition to substitutions that rapidly inactivate copies, internal deletion is a major mechanism contributing to element decay and the generation of non-autonomous sublineages. Hence, 23% of copies correspond to several Miniature Inverted-repeat Transposable Elements (MITE) sublineages, the first ever described in Drosophila for mariner. In the most successful MITEs, internal deletion is often associated with internal rearrangement, which sheds light on the process of MITE origin. The estimation of the transposition rates over time revealed that all lineages followed a similar progression consisting of a rapid amplification burst followed by a rapid decrease in transposition. We detected some instances of multiple or ongoing transposition bursts. Different amplification times were observed for mariner lineages shared by different species, a finding best explained by either horizontal transmission or a reactivation process. Different lineages within one species have also amplified at different times, corresponding to successive invasions. Finally, we detected a preference for insertion into short TA-rich regions, which appears to be specific to some subfamilies.

Conclusions

This analysis is the first comprehensive survey of this family of transposable elements at a genus scale. It provides precise measures of the different evolutionary processes that were hypothesized previously for this family based on PCR data analysis. mariner lineages were observed at almost all “life cycle” stages: recent amplification, subsequent decay and potential (re)-invasion or invasion of genomes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-727) contains supplementary material, which is available to authorized users.     

The Trypanosoma cruzi L1Tc and NARTc non-LTR retrotransposons show relative site specificity for insertion

The trypanosomatid protozoan Trypanosoma cruzi contains long autonomous (L1Tc) and short nonautonomous (NARTc) non-long terminal repeat retrotransposons. NARTc (0.25 kb) probably derived from L1Tc (4.9 kb) by 3'-deletion. It has been proposed that their apparent random distribution in the genome is related to the L1Tc-encoded apurinic/apyrimidinic endonuclease (APE) activity, which repairs modified residues. To address this question we used the T. cruzi (CL-Brener strain) genome data to analyze the distribution of all the L1Tc/NARTc elements present in contigs larger than 10 kb. This data set, which represents 0.91x sequence coverage of the haploid nuclear genome ( approximately 55 Mb), contains 419 elements, including 112 full-length L1Tc elements (14 of which are potentially functional) and 84 full-length NARTc. Approximately half of the full-length elements are flanked by a target site duplication, most of them (87%) are 12 bp long. Statistical analyses of sequences flanking the full-length elements show the same highly conserved pattern upstream of both the L1Tc and NARTc retrotransposons. The two most conserved residues are a guanine and an adenine, which flank the site where first-strand cleavage is performed by the element-encoded endonuclease activity. This analysis clearly indicates that the L1Tc and NARTc elements display relative site specificity for insertion, which suggests that the APE activity is not responsible for first-strand cleavage of the target site.     

The NemaGENETAG initiative: large scale transposon insertion gene-tagging in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

The nematode Caenorhabditis elegans is a widely appreciated, powerful platform in which to study important biological mechanisms related to human health. More than 65% of human disease genes have homologues in the C. elegans genome, and essential aspects of mammalian cell biology, neurobiology and development are faithfully recapitulated in this organism. The EU-funded NemaGENETAG project was initiated with the aim to develop cutting-edge tools and resources that will facilitate modelling of human pathologies in C. elegans, and advance our understanding of animal development and physiology. The main objective of the project involves the generation and evaluation of a large collection of transposon-tagged mutants. In the process of achieving this objective the NemaGENETAG consortium also endeavours to optimize and automate existing transposon-mediated mutagenesis methodologies based on the Mos1 transposable element, in addition to developing alternatives using other transposon systems. The final product of this initiative—a comprehensive collection of transposon-tagged alleles—together with the acquisition of efficient transposon-based tools for mutagenesis and transgenesis in C. elegans, should yield a wealth of information on gene function, immediately relevant to key biological processes and to pharmaceutical research and development.     

Frequency and functional activity of Th17, Tc17 and other T-cell subsets in Systemic Lupus Erythematosus

To compare frequency and functional activity of peripheral blood (PB) Th(c)17, Th(c)1 and Treg cells and the amount of type 2 cytokines mRNA we recruited SLE patients in active (n = 15) and inactive disease (n = 19) and healthy age- and gender-matched controls (n = 15). The study of Th(c)17, Th(c)1 and Treg cells was done by flow cytometry and cytokine mRNA by real-time PCR. Compared to NC, SLE patients present an increased proportion of Th(c)17 cells, but with lower amounts of IL-17 per cell and also a decreased frequency of Treg, but with increased production of TGF-β and FoxP3 mRNA. Ιn active compared to inactive SLE, there is a marked decreased in frequency of Th(c)1 cells, an increased production of type 2 cytokines mRNA and a distinct functional profile of Th(c)17 cells. Our findings suggest a functional disequilibrium of T-cell subsets in SLE which may contribute to the inflammatory process and disease pathogenesis.