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PubMed1987

Serotype-converting bacteriophage D3 of Pseudomonas aeruginosa: vegetative and prophage restriction maps.

D3 is a temperate serotype-converting bacteriophage of Pseudomonas aeruginosa. A restriction map, based upon BamHI, PstI, PvuI, HindIII and SmaI sites, indicates that the phage genome is 56.4 kb long, and that it possesses cohesive ends. The prophage map suggests a unique insertion site in the strain AK1380 genome. Phage DNA integration occurs upon the circularization of D3 genome with the integration point approximately equidistant from the two ends.

Bacteriophages↗

PubMed1980

Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants.

Cleavage sites for the restriction endonucleases XhoI, BamHI, HindIII, and EcoRI were mapped on the pTN2 plasmid, a recombinant of TOL and RP4, which specifies the toluene-degrading enzymes in the same way as the wild-type TOL plasmid. The pTN2 plasmid, purified from a strain of Escherichia coli, contained the entire length of the RP4 plasmid (about 54 kilobase pairs [kb]) and the TOL segment (about 56 kb). The TOL segment is inserted at about 12 and 5 kb away from the EcoRI and BamHI cleavage sites of RP4, respectively. Cleavage sites for XhoI, BamHI, HindIII, and EcoRI were also mapped on an insertion mutant, pTN1, and two deletion mutants, pTN81 and pTN9. Analysis of pTN81 and pTN9 allowed estimation of the region of the gene cluster for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, as well as the region required for toluate oxygenase activity. Induction of TOL enzymes directed by pTN1 suggested the location and orientation of transcription of the gene cluster for catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase. Analysis of strains carrying both pTN9 and a xylR mutant of the TOL plasmid demonstrated that xylR+ is trans dominant over xylR.

Alcohol Oxidoreductases↗

PubMed2003

Distribution and organization of auxotrophic genes on the multichromosomal genome of Burkholderia multivorans ATCC 17616.

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To determine the distribution and organization of the amino acid biosynthetic genes on the genome of this beta-proteobacterium, various auxotrophic mutations were isolated using a Tn5 derivative that was convenient not only for the determination of its insertion site on the genome map but also for the structural analysis of the flanking regions. Analysis by pulsed-field gel electrophoresis revealed that 20 out of 23 insertion mutations were distributed on the 3.4-Mb chromosome. More detailed analysis of the his, trp, arg, and lys mutations and their flanking regions revealed the following properties of these auxotrophic genes: (i) all nine his genes were clustered on the 3.4-Mb chromosome; (ii) seven trp genes were organized within two distinct regions, i.e., a trpEGDC cluster on the 3.4-Mb chromosome and a trpFBA cluster on the 2.5-Mb chromosome; (iii) the leu gene cluster, leuCDB, was also located close to the trpFBA cluster; and (iv) lysA and argG genes were located on the 2.5-Mb chromosome, in contrast to the argH gene, which was located on the 3.4-Mb chromosome. Southern hybridization analysis, allelic exchange mutagenesis of ATCC 17616, and complementation tests demonstrated that all of the genes examined were functional and existed as a single copy within the genome. The present findings also indicated that the 2.5-Mb chromosome carried various auxotrophic genes with no structural or functional counterparts on the remaining two chromosomes.

Amino Acids↗

PubMed2006

Mapped Ds/T-DNA launch pads for functional genomics in barley.

A system for targeted gene tagging and local saturation mutagenesis based on maize transposable elements (Ac/Ds) was developed in barley (Hordeum vulgare L.). We generated large numbers of transgenic barley lines carrying a single copy of the non-autonomous maize Ds element at defined positions in the genome. Independent Ds lines were either generated by activating Ds elements in existing single-copy lines after crossing with AcTPase-expressing plants or by Agrobacterium-mediated transformation. Genomic DNA flanking Ds and T-DNA insertion sites from over 200 independent lines was isolated and sequenced, and was used for a sequence based mapping strategy in a barley reference population. More than 100 independent Ds insertion sites were mapped and can be used as launch pads for future targeted tagging of genes in the vicinity of the insertion sites. Sequence analysis of Ds and T-DNA flanking regions revealed a sevenfold preference of both mutagens for insertion into non-redundant, gene-containing regions of the barley genome. However, whilst transposed Ds elements preferentially inserted adjacent to regions with a high number of predicted and experimentally validated matrix attachment regions (nuclear MARs), this was not the case for T-DNA integration sites. These findings and an observed high transposition frequency from mapped launch pads demonstrate the future potential of gene tagging for functional genomics and gene discovery in barley.

Base Composition↗

PubMed1985

Genes aroA and serC of Salmonella typhimurium constitute an operon.

Genetic analysis of aroA554::Tn10 derivatives of two mouse-virulent Salmonella typhimurium strains, "FIRN" and "WRAY," and of a nonreverting derivative of each constructed for use as a live vaccine, showed the site of the insertion among mapped aroA point mutants. The WRAY live-vaccine strain gave no aro+ recombinants in crosses with aroA point mutations to one side of the insertion, indicating a deletion from Tn10 through the sites of these point mutations. The FIRN live-vaccine strain gave wild-type recombinants with all tested point mutants; it probably has a deletion or inversion extending from Tn10 into aroA but not as far as the nearest point mutation. Some tetracycline-sensitive mutants of aroA554::Tn10 strains required serine and pyridoxine, indicating loss of serC function, and some that were found to be SerC- did not produce gas from glucose, indicating a loss of pfl function. These results show the gene order pfl-serC-aroA, as in Escherichia coli. Ampicillin enrichment applied to pools of tetracycline-sensitive mutants of strains with Tn10 insertions near aroA (i.e., zbj::Tn10 strains) yielded Aro- SerC- Pfl-, Aro- SerC+ Pfl+, and Aro- SerC- Pfl+ mutants but none which were Aro+ SerC-. All of the mutants are explicable by deletions or inversions extending clockwise from zbj::Tn10 into or through an operon comprising serC (promoter-proximal) and aroA. Such an operon was also shown by the identification of two Tn10 insertions causing phenotype Aro- SerC-, each able to revert to Aro+ SerC+ by precise excision. serC corresponds to the open reading frame promoter-proximal to aroA that was identified elsewhere by base sequencing of a cloned aroA segment of S. typhimurium (Comai et al., Science 221:370-371, 1983). Both serine and chorismate are precursors of enterochelin; this may be why serC and aroA are in a single operon.

Bacterial Vaccines↗

PubMed1992

Genetic mapping of two DNA markers, D16Ros1 and D16Ros2, flanking the mutation site in the chakragati mouse, a transgenic insertional mutant.

We present here the genetic mapping of two novel loci, D16Ros1 and D16Ros2, to mouse Chromosome (Chr) 16. The probes for these loci were genomic fragments isolated from the chakragati mouse, a behavioral mutant resulting from insertional mutagenesis during the course of making transgenic mice. D16Ros1 and D16Ros2 were first mapped by recombinant inbred (RI) strain analysis and subsequently by the analysis of 145 progeny of two interspecific backcrosses between Mus domesticus and Mus spretus. These progeny had been typed for the centromere and this allowed mapping of D16Ros1 and D16Ros2 relative to the centromere. The other markers included in this study were Prm-1, Gap43 and Sod-1. The genetic map generated spanned 47.5 cM from the centromere to Sod-1, the most distal marker mapped here. The linkage data presented here should prove useful in mapping other loci relative to the centromere of Chr 16.

Animals↗

PubMed1994

Physical map of Mycoplasma gallisepticum.

Physical chromosomal maps of two Mycoplasma gallisepticum strains, R and ATCC 19610, were constructed by using field inversion gel electrophoresis. To assist in the ordering of chromosomal fragments and the construction of the chromosomal maps, the gram-positive transposon Tn4001 was modified to serve as a mobile restriction site. The total sizes of the M. gallisepticum R and ATCC 19610 genomes were estimated to be 1,037 and 998 kb, respectively. The restriction enzyme locations for EagI and SmaI were determined along with several transposon insertion sites. The two strain maps were similar except for three small deletions and one additional EagI site in strain ATCC 19610.

Base Sequence↗

PubMed1989

Location and nucleotide sequence of the 25K protein missing from baculovirus few polyhedra (FP) mutants.

Wild-type and few polyhedra (FP) mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) were studied to identify and sequence the gene encoding the 25-kDa (25K) protein normally present in AcMNPV-infected Spodoptera frugiperda cells but which is often missing from FP mutant-infected cells. Our previous study had mapped two overlapping late transcripts to the insertion site of host cell DNA within the HindIII-I fragment (33.8 to 37.7 map units) of wild-type AcMNPV. An FP mutant, AcFP875-2, had a 1.6-kbp insertion of S. frugiperda DNA near the 5' end of these transcripts which by S1 analysis were shown to initiate within the host cell sequence. Primer extension analysis revealed that the transcription start for this gene in wild-type virus occurred within a conserved 12-base sequence found near the transcription start sites of several baculovirus late and hyper-expressed genes. A similar 12-base sequence was found at the transcription start site within this 1.6-kbp pair host cell DNA sequence in AcFP875-2. mRNAs from wild-type virus-infected cells were hybridization-selected using a 542-bp SalI subfragment of the 3.2-kbp EcoRI-HindIII fragment (35.0 to 37.7 map units). These mRNAs directed the synthesis of a 25K protein which in size was identical to the 25K protein in wild-type virus-infected cells and the translation product of a 1.15-kb cRNA transcribed from a RsaI fragment (36.4 to 37.4 map units). Comparison of gel band patterns following partial proteolysis of the translation product of the 1.15 cRNA and the 25K protein from wild-type virus-infected cells revealed that the two proteins were closely related if not identical. Nucleotide sequence analysis within this EcoRI-HindIII fragment revealed an open reading frame which encodes a 25K protein. Insertion of the Escherichia coli lacZ gene encoding the beta-galactosidase enzyme into the transcribed portion of this EcoRI-HindIII fragment yielded a recombinant virus which lacked a 25K protein and exhibited an altered (FP) plaque phenotype.

Animals↗

PubMed1981

Analysis of mini-F plasmid replication by transposition mutagenesis.

Derivatives of a mini-F plasmid in which Tn3 is inserted in F deoxyribonucleic acid were obtained, and the sites of insertion for 40 of the derivatives were mapped. Tn3 was found to insert at many sites within mini-F, but most insertions were within the 43.0- to 43.7-kilobase (kb), 44.2- to 44.7-kb, and 45.9- to 46.3-kb segments. Hence, these segments are unnecessary for mini-F replication. Most of the Tn3 derivatives were similar to their parent miniplasmid with respect to copy number, stability, and incompatibility. Insertions at 45.15 kb and near 46.0 kb caused a moderate disruption of copy number control, and insertion at 47.6 kb resulted in unstable maintenance. Deletion derivatives lacking deoxyribonucleic acid between 40.3 and 44.76 kb and between 45.92 and 49.4 kb were obtained. This observation suggests either that mini-F contains a third origin, in addition to those already reported to be at 42.6 and 44.4 kb, or that the reported position of the secondary origin, 44.4 kb, is incorrect and that this origin is between 44.76 and 45.92 kb.

Base Sequence↗

PubMed1985

Mapping of DNase I-hypersensitive sites in the 5' and 3' long terminal repeats of integrated moloney murine leukemia virus proviral DNA.

The chromatin state of integrated Moloney murine leukemia virus (M-MuLV) proviral DNA was investigated. Nuclei from M-MuLV-infected mouse NIH 3T3 cells were digested with limited amounts of DNase I, and hypersensitive (HS) sites were mapped by the indirect end labeling technique. Particular emphasis was placed on the 5' long terminal repeat (LTR), since viral transcription initiates there. M-MuLV proviral DNA showed two strong DNase I-HS sites in the 5' LTR, one coincident with the transcription initiation (cap) site and the other with the transcriptional enhancers. Two weaker DNase I-HS sites were also detected in internal proviral DNA. The 3' LTR also showed a strong HS site in the region of the enhancers, but an HS site at the cap site of the 3' LTR was not detected. Thus, the chromatin configurations of the 5' and 3' LTRs of integrated M-MuLV proviruses appear to be different. The chromatin configuration of M-MuLV proviruses which contain LTR insertions of polyomavirus enhancer sequences was also studied. The 5' LTR of M-MuLV proviruses containing polyoma enhancer sequences substituted for the M-MuLV enhancers showed two strong HS sites, one in the polyoma sequences and one at the cap site. The 5' LTR of M-MuLV proviruses containing polyoma enhancer sequences inserted into the wild-type M-MuLV LTR between the cap site and the M-MuLV enhancers showed three HS sites. Two HS sites corresponded to those of the wild-type M-MuLV LTR, whereas the third mapped to the inserted polyoma sequences. The HS site associated with the inserted polyoma sequences was considerably stronger than the M-MuLV-associated HS sites.

Animals↗

PubMed1984

Location of a structural gene for xylose-H+ symport at 91 min on the linkage map of Escherichia coli K12.

Mutations in the xylose-H+ transport activity of Escherichia coli K12 were isolated using Mud(ApRlac). The initial selection was for simultaneous acquisition of ampicillin and xylose resistance in an fda background. Colonies were then screened for xylose-inducible beta-galactosidase and for growth on xylose of their fda+ derivatives. Two of the xylose-positive derivatives were shown to be impaired in xylose-H+ symport in whole cells and in xylose transport into subcellular vesicles. Their xylose transport in whole cells showed increased sensitivity to arsenate. The site of prophage insertion was mapped to 91.4 min on the E. coli genome between pgi and malB. It is proposed that the gene for the xylose-H+ symport system be called xylE.

Biological Transport↗

PubMed2003

Insertional mutation in the intron 1 of Unc5h3 gene induces ataxic, lean and hyperactive phenotype in mice.

Mice carrying a mutation in the first intron of Unc5h3 were accidentally produced by transgenic insertion and characterized for their homozygous mutant phenotypes. Morphological and histological analysis revealed cerebellar and midbrain abnormalities, which are similar to the previously reported phenotypes of the Unc5h3 mutant. Behavioral analysis showed higher ambulatory activity and circling, and defects in habituation in a novel environment. Their body weights were 10-30% less than wildtype mice from 2-3 weeks of age to 22 months possibly due to reduced accumulation of adipose tissues. The transgenic insertion site was identified and mapped to the intron 1 of Unc5h3 gene with approximately 50 kb deletion of the intron sequence. This intronic mutation interfered with the mRNA expression of the Unc5h3 gene not in testes, but in many tissues including the brain, implying that this intronic region may play a role in regulating tissue-specific transcription of Unc5h3.

Animals↗

PubMed2001

Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana.

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.

Alleles↗

PubMed1988

Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region.

Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon Tn5 mutagenesis. The Tn5 insertion sites of 30 Nif- mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of Tn5 insertions (21 mutants) map within nif region A, characterized by two ClaI fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::Tn5 insertions, and the three remaining mutations are located on a 32-kb ClaI fragment of nif region C. Hybridization experiments using all 17 Klebsiella pneumoniae nif genes individually as probes revealed homology to nifE, nifS, nifA, and nifB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nif region A, was identified.

Chromosome Mapping↗

PubMed1995

Integrated maps of the chromosomes in Dictyostelium discoideum.

Detailed maps of the six chromosomes that carry the genes of Dictyostelium discoideum were constructed by correlating physically mapped regions with parasexually determined linkage groups. Chromosomally assigned regions were ordered and positioned by the pattern of altered fragment sizes seen in a set of restriction enzyme mediated integration-restriction fragment length polymorphism (REMI-RFLP) strains each harboring an inserted plasmid that carries sites recognized by NotI, SstI, SmaI, BglI and ApaI. These restriction enzymes were used to digest high molecular weight DNA prepared from more than 100 REMI-RFLP strains and the resulting fragments were separated and sized by pulsed-field gels. More than 150 gene probes were hybridized to blots of these gels and used to map the insertion sites relative to flanking restriction sites. In this way, we have been able to restriction map the 35 mb genome as well as determine the map position of more than 150 genes to with approximately 40 kb resolution. These maps provide a framework for subsequent refinement.

Animals↗

PubMed2004

Analysis of a Ty1-less variant of Saccharomyces paradoxus: the gain and loss of Ty1 elements.

Because Ty elements transpose through an RNA intermediate, element accumulation through retrotransposition must be regulated or offset by element loss to avoid uncontrolled genome expansion. Here we examine the fate of Ty sequences in Saccharomyces strain 337, a strain that is reported to lack Ty1 and Ty2 elements, but contains remnant solo long terminal repeats (LTRs). Although strain 337 was initially classified as Saccharomyces cerevisiae, our work indicates that this strain is more closely related to S. paradoxus. Several degenerate Ty1 and Ty2 LTRs were mapped to the same insertion sites as full-length Ty1 and Ty2 elements in S. cerevisiae, suggesting that this strain lost Ty elements by LTR-LTR recombination. Southern analysis indicates that strain 337 also lacks Ty4 and Ty5 elements. We estimated the rates of element gain and loss in this strain by introducing a single transposition-competent Ty1 element. The results indicate that Ty1 retrotransposition occurs at a much higher rate than elimination, suggesting that copy-number-dependent co-factors or environmental conditions contribute to the loss of Ty elements in this genome.

Blotting, Southern↗

PubMed1987

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation.

Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod-) or nitrogen fixation (Fix-). Seven Nod- mutants were isolated from strain USDA110 and from strain 61 A101 C, 4 Nod- mutants and 14 Fix- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His- Nod- mutants of USDA110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three non-auxotrophic Nod- mutants and one His-Nod- mutant of USDA110. Homogenotization of the cloned fragments into wild-type strain USDA110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.

DNA Transposable Elements↗

PubMed2003

Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans.

The phaZ gene of Pseudomonas resinovorans codes for a poly(hydroxyalkanoates) (PHA) depolymerase. Two phaZ mutants of Pseudomonas resinovorans NRRL B-2649, FOAC001 and FOAC002, were constructed by an in vitro transposition procedure followed by chromosomal integration via homologous recombination. A detailed mapping of the transposon insertion sites and an analysis of the resultant sequences showed that putative fusion polypeptides PhaZ(FOAC001) (239 amino-acid residues) and PhaZ(FOAC002) (85 amino-acid residues) could result from the mutant phaZ genes of FOAC001 and FOAC002, respectively. In vivo PHA degradation data indicated that PhaZ(FOAC001) might still retain a partial PHA depolymerization activity, while PhaZ(FOAC002) is completely devoid of this function. The cell yields and PHA contents of B-2649, FOAC001, and FOAC002 were similar when the cells were grown either under a limiting nitrogen-source (low-N) condition for up to 5 days or in excess N-source (high-N) for 3 days. A dramatic decrease in PHA content was observed in the PhaZ-active B-2649 and FOAC001 cells during prolonged cell growth (5 days) in high-N medium or in response to a shift-up in nitrogen-source. The repeat-unit compositions of the PHAs from FOAC001 and FOAC002 contained slightly lower amounts of beta-hydroxyoctanoate and higher beta-hydroxytetradecenoate than that of the wild-type B-2649 when grown under a high-N condition. While the molecular masses of the PHAs from FOAC001 and FOAC002 did not vary under any conditions used in this study, those of the wild-type B-2649 were markedly increased in cells either grown for 5 days under a high-N condition or subjected to a nitrogen-source shift-up. These phaZ mutants thus provide a valuable system to study the influence of PHA depolymerase on the accumulation and properties of medium-chain-length PHA.

Carboxylic Ester Hydrolases↗