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PubMed1988

Identification by a random linker insertion method of a region which suppresses the transforming activity of the raf oncogene.

Activation mechanism of raf oncogene was studied by applying in vitro mutagenesis to its cDNA. Previous studies suggested the presence of an activation suppressing sequence in the amino-terminal half of c-raf product. Loss of the sequence by genetic rearrangement was presumed to convert c-raf to possess transforming activity. To identify such sequence, we prepared cDNA mutants by random linker insertion. Synthetic oligonucleotide linker was inserted into the plasmid containing cDNA at a single and random site. Coupling two different mutants, in-frame deletion mutants were constructed systematically. Analysis of these deletion mutants revealed a region, the loss of which made c-raf activated.

Animals↗
PubMed1987

Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene. Identification of an element, upstream from the promoter, required for efficient expression of phoE protein.

The phoE gene of Escherichia coli codes for an outer membrane pore protein whose expression is induced under phosphate limitation. The promoter of this gene contains a 17 base-pair fragment, designated a pho box, which is present also in other phosphate-controlled promoters. The mRNA start site was determined and found to be located downstream from the pho box, such that this element is located in the -35 region of the phoE promoter. A set of promoter deletions was generated in vitro and analysis of these deletions revealed that sequences upstream from the pho box are required for the efficient expression of phoE. The required upstream region is located (in part) between positions -106 and -121 relative to the mRNA start site, and contains sequences homologous to a pho box and a correctly spaced Pribnow box, but in the reversed orientation relative to the regular -35 and -10 regions. A proper spacing between this upstream region and the -35 region appears to be important, since an oligonucleotide insertion in the intervening region interferes with phoE expression. By cloning the upstream region in a lacZ operon fusion vector, a weak phosphate limitation-inducible promoter activity could be detected.

Bacterial Outer Membrane Proteins↗
PubMedDate not supplied

Identification of Drosophila mutant with memory defects after acquisition of conditioned reflex suppression of courtship.

Four lines were selected from a collection of 33 lines prepared by P insertion mutagenesis using a single-copy P-element system; the males of these four lines showed memory defects after acquisition of conditioned reflex suppression of courting. In two lines (P171 and P95), the dynamics of retention of the conditioned reflex in the repeated impregnated-female courting test were similar to those of known short-term memory mutants dnc and rut. In line P153, the dynamics were more reminiscent of the memory dynamics in a known medium-term memory mutant, amn. In line P124, the learning index was insignificant immediately after training was completed, which may indicate that this line was unable to acquire conditioned reflex suppression of courting. Determination of the positions of the P elements (P171: 48A-B; P153: 49B-C; P124: 67B-68A; P95: 77C-D) showed no correspondence with previously known mutations producing memory lesions.

Animals↗
PubMed1985

Identification of a Tn5 determinant conferring resistance to phleomycins, bleomycins, and tallysomycins.

Tn5 conferred resistance to the related antibiotics, phleomycins, bleomycins, and tallysomycins in Escherichia coli and Salmonella typhimurium. For pure phleomycins the level of resistance was influenced by the structure of the terminal basic group. Deletion derivatives of a pBR322::Tn5 plasmid were used to show that the phleomycin resistance determinant is located between the previously identified neomycin and streptomycin resistance determinants. The pattern of expression of phleomycin and neomycin resistance in the deletion derivatives suggests that the phleomycin resistance gene is transcribed from the same promoter, PL, which is essential for expression of neomycin and streptomycin resistance. The location of the phleomycin resistance determinant correlates with the location of an open reading frame in the Tn5 sequence, which codes for a polypeptide of 126 amino acids.

Bleomycin↗
PubMed1983

Identification and amplification of the E. coli phr gene product.

We have constructed a series of multicopy plasmids that complement mutations in the phr gene of Escherichia coli. By subcloning into a tac plasmid vector we obtained a phr plasmid that upon induction overproduces two proteins of Mr's 49,000 and 20,000. Tn1000 insertions into the phr gene caused the disappearance of the 49,000 dalton protein, thus demonstrating this protein to be the phr gene product, DNA photolyase. The photolyase encoded by the phr gene makes up about 15% of total cellular proteins after induction of cells carrying a tac-phr plasmid. This protein binds specifically to UV (254 nm) irradiated DNA and upon exposure to near UV (300-500 nm) illumination repairs the UV damage and dissociates from DNA.

DNA Repair↗
PubMed1992

Cloning and identification of the Escherichia coli murB DNA sequence, which encodes UDP-N-acetylenolpyruvoylglucosamine reductase.

The murB gene, which complemented the UDP-N-acetylenolpyruvoylglucosamine reductase (EC 1.1.1.158) mutation in Escherichia coli ST5, was cloned from an E. coli chromosomal library. murB was subcloned on a 2.8-kb PvuII fragment into pUC19 and sequenced. A 1,029-bp open reading frame encoded a 342-amino-acid polypeptide of 37,859 Da. A DNA sequence homology search revealed that murB had almost 100% homology with a previously reported unidentified open reading frame, ORFII, at 89.9 min. Physical and genetic mapping results were consistent with this map position, and minicell analyses of murB subclones showed a plasmid-encoded protein of approximately 37,000 Da, which closely matched the calculated size of the murB protein.

Carbohydrate Dehydrogenases↗
PubMed2005

Isolation and characterization of FecA- and FeoB-mediated iron acquisition systems of the spirochete Leptospira biflexa by random insertional mutagenesis.

The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.

Amino Acid Sequence↗
PubMed1980

Identification of the dnaA and dnaN gene products of Escherichia coli.

A specialized transducing lambda phage carrying the dnaN genes of Escherichia coli specifies two proteins of about 41 and 48 kilodaltons (kd). The temperature-sensitive mutations, dnaN59 and dnaA167, were found to result in altered isoelectric points of the 41 and 48 kd proteins, respectively. Thus the dnaN gene product was identified as a weakly acidic 41 and 48 kd protein. The synthesis of the dnaN gene product is greatly reduced by insertion of a transposon Tn3 in the dnaA gene and by deletion in the gene at the distal end to the dnaN gene. Temperature-sensitive dnaA mutations, on the dnaN gene product. These results indicate that the synthesis of the dnaN gene product is dependent on the structural integrity of the dnaA gene.

Bacterial Proteins↗
PubMed1989

Identification and sequence analysis of the Rhizobium meliloti dctA gene encoding the C4-dicarboxylate carrier.

Transposon Tn5-induced C4-dicarboxylate transport mutants of Rhizobium meliloti 2011 which could be complemented by cosmid pRmSC121 were subdivided into two classes. Class I mutants (RMS37 and RMS938) were defective in symbiotic C4-dicarboxylate transport and in nitrogen fixation. They were mutated in the structural gene dctA, which codes for the C4-dicarboxylate carrier. Class II mutants (RMS11, RMS16, RMS17, RMS24, and RMS31) expressed reduced activity in symbiotic C4-dicarboxylate transport and in nitrogen fixation. These mutants were mutated in regulatory dct genes which do not play an essential role in the symbiotic state. Thin sections of alfalfa nodules induced by the wild type and class I and class II mutants were analyzed by light microscopy. Class mutants induced typical Fix- nodules, showing a large senescent zone, whereas nodules induced by class II mutants only differed in an enhanced content of starch granules compared with wild-type nodules. Class I mutants could be complemented by a 2.1-kilobase SalI-HindIII subfragment of cosmid pRmSC121. DNA sequencing of this fragment resulted in the identification of an open reading frame, which was designated dctA because Tn5 insertion sites of the class I mutants mapped within this coding region. The dctA gene was preceded by a nif consensus promoter and an upstream NifA-binding element. Upstream of the dctA promoter, the 5' end of the R. meliloti dctB gene could be localized. The amino acid sequence of the N-terminal part of the R. meliloti DctB protein shared 49% homology with the corresponding part of the R. leguminosarum DctB protein. The DctA protein consisted of 441 or 453 amino acids due to two possible ATG start codons, with calculated molecular masses of 46.1 and 47.6 kilodaltons, respectively. The hydrophobicity plot suggests that DctA is a membrane protein with several membrane passages. The amino acid sequences of the R. meliloti and the R. leguminosarum DctA proteins were highly conserved (82%).

Amino Acid Sequence↗
PubMed2005

Flavobacterium johnsoniae gliding motility genes identified by mariner mutagenesis.

Cells of Flavobacterium johnsoniae glide rapidly over surfaces. The mechanism of F. johnsoniae gliding motility is not known. Eight gld genes required for gliding motility have been described. Disruption of any of these genes results in complete loss of gliding motility, deficiency in chitin utilization, and resistance to bacteriophages that infect wild-type cells. Two modified mariner transposons, HimarEm1 and HimarEm2, were constructed to allow the identification of additional motility genes. HimarEm1 and HimarEm2 each transposed in F. johnsoniae, and nonmotile mutants were identified and analyzed. Four novel motility genes, gldK, gldL, gldM, and gldN, were identified. GldK is similar in sequence to the lipoprotein GldJ, which is required for gliding. GldL, GldM, and GldN are not similar in sequence to proteins of known function. Cells with mutations in gldK, gldL, gldM, and gldN were defective in motility and chitin utilization and were resistant to bacteriophages that infect wild-type cells. Introduction of gldA, gldB, gldD, gldFG, gldH, gldI, and gldJ and the region spanning gldK, gldL, gldM, and gldN individually into 50 spontaneous and chemically induced nonmotile mutants restored motility to each of them, suggesting that few additional F. johnsoniae gld genes remain to be identified.

Bacteriophages↗
PubMed2004

Early intermediates of mariner transposition: catalysis without synapsis of the transposon ends suggests a novel architecture of the synaptic complex.

The mariner family is probably the most widely distributed family of transposons in nature. Although these transposons are related to the well-studied bacterial insertion elements, there is evidence for major differences in their reaction mechanisms. We report the identification and characterization of complexes that contain the Himar1 transposase bound to a single transposon end. Titrations and mixing experiments with the native transposase and transposase fusions suggested that they contain different numbers of transposase monomers. However, the DNA protection footprints of the two most abundant single-end complexes are identical. This indicates that some transposase monomers may be bound to the transposon end solely by protein-protein interactions. This would mean that the Himar1 transposase can dimerize independently of the second transposon end and that the architecture of the synaptic complex has more in common with V(D)J recombination than with bacterial insertion elements. Like V(D)J recombination and in contrast to the case for bacterial elements, Himar1 catalysis does not appear to depend on synapsis of the transposon ends, and the single-end complexes are active for nicking and probably for cleavage. We discuss the role of this single-end activity in generating the mutations that inactivate the vast majority of mariner elements in eukaryotes.

Animals↗
PubMed1998

Identification and characterization of IS1302, a novel insertion element from Wolinella succinogenes belonging to the IS3 family.

A new insertion sequence (IS) designated IS1302 was identified in Wolinella succinogenes. IS1302 is 1,306 bp in size with 36-bp imperfect terminal inverted repeats. It contains only one open reading frame (tnpA), which encodes a putative transposase whose sequence is similar to that of transposases of various IS elements of the IS3 family. IS1302 was identified in the genome of a W. succinogenes fumarate reductase deletion mutant in which the frd operon had been replaced by the kan gene. The insertion of IS1302 occurred when the mutant was propagated in the presence of a high concentration of kanamycin. Two different target sites of IS1302 were found immediately upstream of the kan gene, where the insertion of IS1302 resulted in a duplication of 3 bp of the target DNA. Upon insertion of IS1302, new possible promoter structures of the kan gene were created, which might lead to a stimulated transcription of the kan gene and result in a selective advantage of cells containing IS1302 at one of the two target sites. Southern blot analysis suggested the presence of at least 13 copies of IS1302 in the genome of W. succinogenes. This is the first IS element discovered in W. succinogenes.

Amino Acid Sequence↗
PubMed1985

Expression of a Rhizobium phaseoli Sym plasmid in R. trifolii and Agrobacterium tumefaciens: incompatibility with a R. trifolii Sym plasmid.

Identification of the Sym plasmid in Rhizobium phaseoli strain RCC3622 is described. Introduction of this plasmid into R. trifolii or Agrobacterium tumefaciens strains resulted in bacteria capable of forming characteristic spherical root nodules on beans. This Sym plasmid, designated pSym9, was characterized as 275 MDa and nonconjugative. pSym9 was incompatible with the R. trifolii Sym plasmid pSym5, and carries genes determining a melanin-like black pigment. A second plasmid of 135 MDa, pRph3622a, was also transferred from R. phaseoli to R. trifolii and A. tumefaciens. Transconjugants carrying this plasmid did not form root nodules on beans. In contrast to other Rhizobium plasmids, pRph3622a was unstable in A. tumefaciens.

Conjugation, Genetic↗
PubMed2007

Identification of a novel mutation in a Korean patient with oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disorder characterized by progressive dysphagia and bilateral ptosis. Mutations in the polyadenylate binding protein nuclear 1 (PABPN1) gene have been found to cause OPMD. The typical mutation is a stable trinucleotide repeat expansion in the first exon of the PABPN1 gene, in which (GCG)(6) is the normal repeat length. We investigated a Korean patient with OPMD and identified a novel mutation: a heterozygous insertion of a 9-bp sequence [(GCG)(GCA)(GCA); c.27_28insGCGGCAGCA] instead of the (GCG) repeat expansion, resulting in an in-frame insertion of three alanines (p.A10insAAA). To the best of our knowledge, this is the first report of a genetically confirmed case of OPMD in Korea.

Blepharoptosis↗
PubMed2006

Comparative genomics of Neisseria meningitidis: core genome, islands of horizontal transfer and pathogen-specific genes.

To better understand Neisseria meningitidis genomes and virulence, microarray comparative genome hybridization (mCGH) data were collected from one Neisseria cinerea, two Neisseria lactamica, two Neisseria gonorrhoeae and 48 Neisseria meningitidis isolates. For N. meningitidis, these isolates are from diverse clonal complexes, invasive and carriage strains, and all major serogroups. The microarray platform represented N. meningitidis strains MC58, Z2491 and FAM18, and N. gonorrhoeae FA1090. By comparing hybridization data to genome sequences, the core N. meningitidis genome and insertions/deletions (e.g. capsule locus, type I secretion system) related to pathogenicity were identified, including further characterization of the capsule locus, bioinformatics analysis of a type I secretion system, and identification of some metabolic pathways associated with intracellular survival in pathogens. Hybridization data clustered meningococcal isolates from similar clonal complexes that were distinguished by the differential presence of six distinct islands of horizontal transfer. Several of these islands contained prophage or other mobile elements, including a novel prophage and a transposon carrying portions of a type I secretion system. Acquisition of some genetic islands appears to have occurred in multiple lineages, including transfer between N. lactamica and N. meningitidis. However, island acquisition occurs infrequently, such that the genomic-level relationship is not obscured within clonal complexes. The N. meningitidis genome is characterized by the horizontal acquisition of multiple genetic islands; the study of these islands reveals important sets of genes varying between isolates and likely to be related to pathogenicity.

Bacterial Capsules↗
PubMed2006

Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants.

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.

Animals↗
PubMed1998

Systematic identification of essential genes by in vitro mariner mutagenesis.

Although the complete DNA sequences of several microbial genomes are now available, nearly 40% of the putative genes lack identifiable functions. Comprehensive screens and selections for identifying functional classes of genes are needed to convert sequence data into meaningful biological information. One particularly significant group of bacterial genes consists of those that are essential for growth or viability. Here, we describe a simple system for performing transposon mutagenesis on naturally transformable organisms along with a technique to rapidly identify essential or conditionally essential DNA segments. We show the general utility of this approach by applying it to two human pathogens, Haemophilus influenzae and Streptococcus pneumoniae, in which we detected known essential genes and assigned essentiality to several ORFs of unknown function.

Base Sequence↗
PubMed2000

TnAraOut, a transposon-based approach to identify and characterize essential bacterial genes.

Identification of genes that encode essential products provides a promising approach to validation of new antibacterial drug targets. We have developed a mariner-based transposon, TnAraOut, that allows efficient identification and characterization of essential genes by transcriptionally fusing them to an outward-facing, arabinose-inducible promoter, PBAD, located at one end of the transposon. In the absence of arabinose, such TnAraOut fusion strains display pronounced growth defects. Of a total of 16 arabinose-dependent TnAraOut mutants characterized in Vibrio cholerae, four were found to carry insertions upstream of known essential genes (gyrB, proRS, ileRS, and aspRS) whereas the other strains carried insertions upstream of known and hypothetical genes not previously shown to encode essential gene products. One of the essential genes identified by this analysis appears to be unique to V. cholerae and thus may represent an example of a species-specific drug target.

Arabinose↗