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PubMed1989

High efficiency vectors for cosmid microcloning and genomic analysis.

We describe the construction and use of cosmid vectors designed for microcloning, gene isolation and genomic mapping starting from submicrogram amounts of eukaryotic DNA. These vectors contain (1) multiple cos sites to allow for simple and efficient cloning using non size-selected DNA; (2) bacteriophage T3 and T7 promoter sequences flanking the cloning site to allow for the synthesis of end-specific probes for chromosome walking; (3) a selectable gene for immediate gene transfer of cosmid DNA into mammalian cells; (4) recognition sequences for specific oligodeoxyribonucleotides to allow rapid restriction mapping; (5) unique NotI, SacII or SfiI sites flanking the cloning site to allow for removal of the cloned DNA insert from the vector. These cosmid vectors allow the construction of high quality genomic libraries in situations where the quantity of purified DNA is extremely limited, such as when using DNA prepared from purified mammalian chromosomes isolated by fluorescence-activated cell sorting.

Bacteriophages↗
PubMed1986

Chromosomal mapping of four different integration sites of Moloney murine leukemia virus including the locus for alpha 1(I) collagen in mouse.

Infection of mouse embryos with Moloney murine leukemia virus (M-MuLV) has yielded several mouse substrains with stable germ line integration of retroviral DNA at distinct chromosomal loci (Mov loci; Jaenisch et al., 1981). There is evidence that flanking DNA sequences can have an effect on virus expression and, conversely, inserted viral DNA may affect the expression of adjacent host genes. As part of our studies on the interaction of inserted M-MuLV with the mouse genome, we have chromosomally mapped four different Mov loci by hybridizing single-copy mouse sequences, flanking the proviral DNA, to interspecies somatic cell hybrids. Furthermore, these sequences were assigned regionally by in situ hybridization to mouse metaphase chromosomes. In Mov-13 mice, M-MuLV had inserted into the alpha 1(I) collagen gene leading to early embryonic death in homozygotes. We have assigned this locus to the distal region of chromosome 11. Thus, the alpha 1(I) collagen gene is part of an evolutionarily conserved linkage group with the homologous genes on human chromosome 17. Three other proviral integration sites were mapped to chromosome 1, bands BC (Mov-7), chromosome 11, bands BC (Mov-9), and chromosome 3, bands FG (Mov-10). The Mov-10-specific probe detects an EcoRI-specific restriction fragment length polymorphism, which can make this probe a useful genetic marker.

Animals↗
PubMed2002

Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements.

Sleeping Beauty (SB) is the most active Tc1/mariner-type transposable element in vertebrates, and is therefore a valuable vector for transposon mutagenesis in vertebrate models and for human gene therapy. We have analyzed factors affecting target site selection of SB in mammalian cells, by generating transposition events from extrachromosomal plasmids to chromosomes. In contrast to the local hopping observed when transposition is induced from a chromosomal context, mapping of 138 unique SB insertions on human chromosomes showed a fairly random genomic distribution, and a 35% occurrence of transposition into genes. Inspection of the DNA flanking the sites of element integration revealed significant differences from random DNA in both primary sequence and physical properties. The consensus sequence of SB target sites was found to be a palindromic AT-repeat, ATATATAT, in which the central TA is the canonical target site. We found however, that target site selection is determined primarily on the level of DNA structure, and not by specific base-pair interactions. Computational analyses revealed that insertion sites tend to have a bendable structure and a palindromic pattern of potential hydrogen-bonding sites in the major groove of the DNA. These features appear conserved in the Tc1/mariner family of transposons and in other, distantly related elements that share a common catalytic domain of the transposase, and integrate fairly randomly. No similar target site preference was found for non-randomly integrating elements. Our results suggest common factors influencing target site selection of a wide range of transposable elements.

Animals↗
PubMed1986

Consecutive A X T pairs can adopt a left-handed DNA structure.

The capacity of six sequences with different numbers and orientations of A.T pairs flanked by alternating C.G pairs to adopt left-handed structures was evaluated in recombinant plasmids. A series of synthetic oligodeoxynucleotides were cloned into the BamHI site of pRW790, a small plasmid (approximately 2 kilobases) prepared especially for conformational studies of this type. Supercoil relaxation studies by two-dimensional gel electrophoresis on topoisomers of each plasmid revealed the energetics and structures of the left-handed helices. Also, the presence of supercoil-induced altered DNA conformations within the inserts of topoisomer populations of the plasmids was detected by reaction with S1 nuclease followed by restriction mapping of the cleavage sites. We conclude that consecutive T.A base pairs, whether alternating (TATA) or contiguous (TTTT), can adopt a left-handed conformation (presumably Z) when flanked by reasonably short runs of alternating (C-G)n (n = 3-5). Thus, these results substantially broaden the range of DNA sequences that can adopt left-handed Z conformations.

Adenine↗
PubMed2006

Analysis of T-DNA insertion site distribution patterns in Arabidopsis thaliana reveals special features of genes without insertions.

Large collections of sequence-indexed T-DNA insertion mutants are invaluable resources for plant functional genomics. Flanking sequence tag (FST) data from these collections indicated that T-DNA insertions are not randomly distributed in the Arabidopsis thaliana genome and that there are still a fairly high number of annotated genes without T-DNA insertions. We have analyzed FST data from the FLAGdb, GABI-Kat, and SIGnAL mutant populations. The lack of detectable transcriptional activity and the absence of suitable restriction sites were among the reasons genes are not covered by insertions. Additionally, a refined analysis of FSTs to genes with annotated noncoding regions showed that transcription initiation and polyadenylation site regions of genes are favored targets for T-DNA integration. These findings have implications for the use of T-DNA in saturation mutagenesis and for our chances to find a useful knockout allele for every gene.

Arabidopsis↗
PubMed1980

An unusual RNA polymerase binding site in the immunity region of phage lambda.

RNA polymerase binds very tightly at a site called Brex in the lambda immunity region, to the left of the rex gene and about 600 nucleotides to the right of PL. The complex formed is resistant to 1 M NaCl in the absence of nucleotide triphosphate. While in vitro little or no transcription is observed from Brex, in vivo, when inserted in a plasmid vector which allows detection of its activity, it acts as an efficient promoter. We have mapped the site protected by RNA polymerase against DNase and determined its sequence which is abnormal compared that of an average promoter.

Bacteriophage lambda↗
PubMed1993

Linked and unlinked transposition of a genetically marked Dissociation element in transgenic tomato.

We have introduced a genetically marked Dissociation transposable element (Dsneo) into tomato. In the presence of Ac transposase, Dsneo excised from an integrated T-DNA and reinserted at numerous new sites in the tomato genome. The marker genes of Dsneo (NPTII) and the T-DNA (HPT) facilitated identification of plants bearing transposon excisions and insertions. To explore the feasibility of gene tagging strategies in tomato using Dsneo, we examined the genomic distribution of Dsneo receptor sites, relative to the location of the donor T-DNA locus. Restriction fragment length polymorphism mapping of transposed Dsneo elements was conducted in two tomato families, derived from independent primary transformants each bearing Dsneo within a T-DNA at a unique position in the genome. Transposition of Dsneo generated clusters of insertions that were positioned on several different tomato chromosomes. Dsneo insertions were often located on the same chromosome as the T-DNA donor site. However, no insertion showed tight linkage to the T-DNA. We consider the frequency and distance of Dsneo transposition observed in tomato to be well suited for transposon mutagenesis. Our study made use of a novel, stable allele of Ac (Ac3) that we discovered in transgenic tomato. We determined that the Ac3 element bears a deletion of the outermost 5 base pairs of the 5'-terminal inverted repeat. Though incapable of transposition itself, Ac3 retained the ability to mobilize Dsneo. We conclude that a dual element system, composed of the stable Ac3 trans-activator in combination with Dsneo, is an effective tool for transposon tagging experiments in tomato.

Alleles↗
PubMed1998

Mapping viral DNA specificity to the central region of integrase by using functional human immunodeficiency virus type 1/visna virus chimeric proteins.

We previously described the construction and analysis of the first set of functional chimeric lentivirus integrases, involving exchange of the N-terminal, central, and C-terminal regions of the human immunodeficiency virus type 1 (HIV-1) and visna virus integrase (IN) proteins. Based on those results, additional HIV-1/visna virus chimeric integrases were designed and purified. Each of the chimeric enzymes was functional in at least one oligonucleotide-based IN assay. Of a total of 12 chimeric IN proteins, 3 exhibit specific viral DNA processing, 9 catalyze insertion of viral DNA ends, 12 can reverse that reaction, and 11 are active for nonspecific alcoholysis. Functional data obtained with the processing assay indicate that the central region of the protein is responsible for viral DNA specificity. Target site selection for nonspecific alcoholysis again mapped to the central domain of IN, confirming our previous data indicating that this region can position nonviral DNA for nucleophilic attack. However, the chimeric proteins created patterns of viral DNA insertion distinct from that of either wild-type IN, suggesting that interactions between regions of IN influence target site selection for viral DNA integration. The results support a new model for the functional organization of IN in which viral DNA initially binds nonspecifically to the C-terminal portion of IN but the catalytic central region of the enzyme has a prominent role both in specific recognition of viral DNA ends and in positioning the host DNA for viral DNA integration.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the aacC2 gene, a gentamicin resistance determinant involved in a hospital epidemic of multiply resistant members of the family Enterobacteriaceae.

A gentamicin resistance determinant of a conjugative plasmid from Enterobacter cloacae was cloned on a 3.2-kilobase fragment in the PstI site of pBR322. Substrate profiles for eight aminoglycosides at three concentrations showed that the resistance was due to aminoglycoside-(3)-N-acetyltransferase isoenzyme II. Insertion mapping by the gamma-delta transposon revealed that the size of the gene was approximately 1 kilobase. Nucleotide sequencing of the aacC2 gene identified an open reading frame of 858 base pairs, preceded by a promoter and a ribosome-binding site. From these data the molecular mass of the protein was calculated to be 30.6 kilodaltons. A comparison of the nucleotide sequence of the aacC2 gene with those published for the aacC3 and aacC4 genes showed complete homology of the aacC2 gene and the presumed aacC3 gene. An internal restriction fragment of the gene used as a probe in colony hybridization demonstrated the presence of the aacC2 gene in 86% of 86 multiply resistant isolates of the family Enterobacteriaceae obtained during an 18-month hospital epidemic. This corroborates our earlier data on the enzyme identification by the susceptibility profiling method.

Autoradiography↗
PubMed2006

Identification of potential human oncogenes by mapping the common viral integration sites in avian nephroblastoma.

Gene deregulation is a frequent cause of malignant transformation. Alteration of the gene structure and/or expression leading to cellular transformation and tumor growth can be experimentally achieved by insertion of the retroviral genome into the host DNA. Retrovirus-containing host loci found repeatedly in clonal tumors are called common viral integration sites (cVIS). cVIS are located in genes or chromosomal regions whose alterations participate in cellular transformation. Here, we present the chicken model for the identification of oncogenes and tumor suppressor genes in solid tumors by mapping the cVIS. Using the combination of inverse PCR and long terminal repeat-rapid amplification of cDNA ends technique, we have analyzed 93 myeloblastosis-associated virus type 2-induced clonal nephroblastoma tumors in detail, and mapped >500 independent retroviral integration sites. Eighteen genomic loci were hit repeatedly and thus classified as cVIS, five of these genomic loci have previously been shown to be involved in malignant transformation of different human cell types. The expression levels of selected genes and their human orthologues have been assayed in chicken and selected human renal tumor samples, and their possible correlation with tumor development, has been suggested. We have found that genes associated with cVIS are frequently, but not in all cases, deregulated at the mRNA level as a result of proviral integration. Furthermore, the deregulation of their human orthologues has been observed in the samples of human pediatric renal tumors. Thus, the avian nephroblastoma is a valid source of cancer-associated genes. Moreover, the results bring deeper insight into the molecular background of tumorigenesis in distant species.

Animals↗
PubMed1985

Site-specific cleavage of left-handed DNA in pBR322 by lambda-tris(diphenylphenanthroline)cobalt(III).

The chiral complex tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), lambda-Co(DiP)3(3+), binds to and, with photoactivation, cleaves left-handed DNA helices, thereby providing a unique molecular probe for local DNA conformation. We have mapped the specific left-handed sites where lambda-Co(DiP)3(3+) cleaves in the plasmids pBR322 and pLP32, which is the derivative of pBR322 containing a Z-form d(C-G)16 insert. For pLP32, a primary cleavage is at the insert; for native pBR322, cleavage occurs at four discrete sites: 1.45, 2.3, 3.3, and 4.2 kilobase pairs. These sites correspond to segments of alternating purine-pyrimidines. Moreover, these positions map to the ends of the three distinct coding regions in pBR322: the tetracycline-resistance gene, the origin of replication, and either end of the ampicillin-resistance (beta-lactamase) gene. The locations of these left-handed segments suggest to us that Z-DNA might serve as a conformational punctuation mark to demarcate the ends of genes.

Base Sequence↗
PubMed1998

In situ analysis of a variant surface glycoprotein expression-site promoter region in Trypanosoma brucei.

In Trypanosoma brucei, the active variant surface glycoprotein genes (vsg) are located at telomeric expression sites (ES), whose expression is highly regulated during the life cycle. In the procyclic form, all ESs are repressed. In the bloodstream form, where antigenic variation occurs, only one of approximately 20 ESs is active at a given time. We have investigated chromatin structure and DNA sequence around the ES promoter to identify cis-acting regulatory regions. A marker gene, inserted 1 kb downstream of the ES promoter, was used as a specific probe to map the position of nuclease hypersensitive sites. A prominent hypersensitive site was detected within the core promoter. This site was present in both active and inactive ES promoters, suggesting that a protein complex is bound to the promoter irrespective of its transcriptional state. However, none of the regions showed differential nuclease sensitivity between active and inactive transcriptional states. A systematic deletion analysis of the sequences surrounding the active ES promoter in situ confirmed the absence of cis-regulatory elements. We find that only 70 bp within the ES promoter are necessary to support ES regulation. Analysis of the reporter activities in an inactive bloodstream-form ES revealed the existence of an intermediate promoter activity in some clones, but we never observed full activation of more than one ES. The vsg mRNA from this intermediate ES was expressed less efficiently.

Animals↗
PubMed1995

An improved TnMax mini-transposon system suitable for sequencing, shuttle mutagenesis and gene fusions.

A new collection of mini-transposons (mini-Tn) of the previously described TnMax series [Haas et al, Gene 130 (1993a) 23-31] has been constructed. The transposons (Tn) bear genes conferring resistance to either chloramphenicol (Cm) or kanamycin (Km). Each member of the new series (TnMax5-TnMax11) contains the general M13 forward (M13-FP) and reverse (M13-RP1) sequencing primers close to the inverted repeats (IR), facilitating the rapid and convenient determination of the DNA sequences flanking the transposon insertion site. Furthermore, the mini-Tn possess the infrequently occurring NotI sites, allowing the localization of genes on macro-restriction maps of bacterial species. Some derivatives contain promoterless trp-lacZ (TnMax11), xylE (TnMax10), phoA (TnMax6) or blaM (TnMax7, TnMax9) genes next to the IR, suitable for the generation of in vivo gene- and operon fusions to study gene regulation, protein export, or to determine the topology of proteins in bacterial membranes. A set of conjugative minimal plasmid vectors (pMin1, pMin2) are used to select for TnMax insertions into the cloned insert, rather than the vector sequences. Due to the small size of the mini-Tn, and a simple and efficient mutagenesis procedure, the TnMax system is a useful tool for targeting and sequencing of cloned genes in Escherichia coli, and especially for shuttle mutagenesis of bacterial species which cannot be targeted by direct transposition.

Base Sequence↗
PubMed1991

The chloroplast genome of the gymnosperm Pinus contorta: a physical map and a complete collection of overlapping clones.

Overlapping restriction fragments of chloroplast DNA from the conifer Pinus contorta were cloned. Out of a total of 49 clones, 33 comprise the minimum set required to represent the entire genome. Using the purified inserts of these clones as probes in filter hybridizations, all sites for the three restriction enzymes KpnI, HpaI and SacI in the P. contorta chloroplast genome were mapped. Heterologous filter hybridizations and sequence analysis of some of the P. contorta clones were used to determine the position of 15 genes on the restriction map. The size of the genome, which lacks an inverted repeat organization, was found to be approximately 121 kilobase pairs (kbp). Unusual features of this genome are a duplication of the psbA gene and the presence of two genes, gidA and frxC, which are not found in angiosperms. The genome appeared essentially colinear with that of Pinus radiata, for which a map has previously been published. Two different restriction fragment length polymorphisms were found to be produced by variable numbers of copies of 124 bp- and 150 bp-long, tandemly repeated elements.

Base Sequence↗
PubMed1995

The murine AIDS defective provirus acts as an insertional mutagen in its infected target B cells.

In susceptible mice, the murine AIDS (MAIDS) defective virus can induce marked expansion of its target cells, the majority of which belong to the B-cell lineage. This expansion, which appears to be critical for the development of the immunodeficiency syndrome, is initially polyclonal but becomes oligoclonal late in the disease, suggesting the involvement of a secondary genetic event(s) during this proliferation. To determine whether integration of the MAIDS defective provirus into particular regions of the cellular genome contributes to this oligoclonal expansion, we searched for common provirus integration sites in enlarged lymphoid organs of MAIDS mice. We identified two common proviral integration sites, Dis-1 and Dis-2, which were occupied by a defective provirus at frequencies of 20 and 13%, respectively. Our analysis revealed that the Dis-1 region corresponds to the Sfpil1 (Spi-1, PU.1) locus, which maps on chromosome 2, and encodes a transcription factor. Insertion of the MAIDS defective provirus into this region led to a two- to threefold increase in the expression of Sfpi1 RNA. The Dis-2 locus was found to map to mouse chromosome 11, between Hox2 and Scya. It appears to be a novel locus probably harboring a gene involved in B-cell proliferation. The present study indicates that the MAIDS defective provirus can act as an insertional mutagen, thus contributing to the oligoclonal expansion of infected cells. The detection of two common proviral integration sites, each of which targetted at a low frequency in diseased organs, suggests that the deregulation of a unique gene through provirus insertion is essential for neither proliferation of infected B cells nor development of the immunodeficiency syndrome.

Animals↗
PubMed1995

The distribution of the transposable element Bari-1 in the Drosophila melanogaster and Drosophila simulans genomes.

The distribution of the transposable element Bari-1 in D. melanogaster and D. simulans was examined by Southern blot analysis and by in situ hybridization in a large number of strains of different geographical origins and established at different times. Bari-1 copies mostly homogeneous in size and physical map are detected in all strains tested. Both in D. melanogaster and in D. simulans a relatively high level of intraspecific insertion site polymorphism is detectable, suggesting that in both species Bari-1 is or has been actively transposing. The main difference between the two sibling species is the presence of a large tandem array of the element in a well-defined heterochromatic location of the D. melanogaster genome, whereas such a cluster is absent in D. simulans. The presence of Bari-1 elements with apparently identical physical maps in all D. melanogaster and D. simulans strains examined suggests that Bari-1 is not a recent introduction in the genome of the melanogaster complex. Structural analysis reveals unusual features that distinguish it from other inverted repeat transposons, whereas many aspects are similar to the widely distributed Tc1 element of C. elegans.

Animals↗
PubMed1991

Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons.

Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA- background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5' CTAAAACAAAGTTA 3' (hs2). The double-site-specific recombination occurred with a frequency of 10(-5)-10(-6). The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a transacting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.

Base Sequence↗