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PubMed1997

Epitope mapping and tagging by recombination PCR mutagenesis.

We describe a rapid PCR method that directly inserts an epitope tag into an open reading frame (ORF) to facilitate protein detection. This project was performed within a varicella-zoster virus (VZV) system. In earlier work, we produced a monoclonal antibody (MAb 3B3) to one VZV ORF called gE. MAb 3B3 bound to its epitope under extreme denaturing conditions. To further characterize the epitope, we devised a technique that identified the epitope by its insertion into another protein of interest. The 3B3 epitope was mapped to 11 residues (residues 151-161; QRQYGDVFKGD) in the gE ectodomain by using the technique of recombination PCR. At the same time, the 3B3 epitope was inserted in-frame into another VZV protein for which no MAb was available. The end result, VZV gL3B3.11, was a unique construct possessing a 33-bp insertion that expresses gL-3B3 protein recognized by the MAb 3B3. The 3B3 epitope was verified to be both highly functional and stable. An important advantage of this recombination PCR method of epitope mapping and tagging is that the epitope sequence can be inserted anywhere along the nucleotide sequence of an ORF, regardless of existing restriction sites.

Amino Acid Sequence↗
PubMed1992

Rapid mapping of Escherichia coli::Tn5 insertion mutations by REP-Tn5 PCR.

We describe a novel method to map chromosomal Escherichia coli::Tn5 insertion mutations rapidly. This method utilizes the ends of Tn5 and the E. coli REP sequence as primer binding sites for the polymerase chain reaction (PCR). The unique E. coli chromosomal sequence located between these primer binding sites is amplified by PCR and used as a probe to identify the recombinant clones from the Kohara phage ordered E. coli miniset bank that contains the Tn5 mutated loci. We used this approach to map two Tn5 insertion mutations previously identified by their effect on glycerol metabolism. The insertion mutations mapped to glpD, the aerobic sn-glycerol-3-phosphate dehydrogenase gene. Phenotypic analysis of the mutant strains revealed one with partial GlpD activity, suggesting transposon-mediated alteration of promoter activity. This mapping method should be applicable to the rapid physical mapping of any insertion mutation in the E. coli chromosome.

Base Sequence↗
PubMed1998

[Mutation bursts of the yellow gene in a natural Drosophila melanogaster population is connected with insertion of the hobo transposon].

In 1981, a recurrent local burst of high mutability and high allele frequency of the yellow gene was recorded in the natural population of Drosophila melanogaster from Uman', Ukraine. A detailed genetic analysis showed that hypomorphic alleles y2 prevailed during the mutation burst. Mutations were strictly allele-specific and occurred only in two directions: from y2 to y+ and from y+ to y2. Alleles y2 isolated from the natural population differed in the degree of instability of mutant and wild-type derivatives, expression of the mutant phenotype, and complementation properties. In this work, the insertion nature of all unstable alleles derived from the natural population is confirmed by direct molecular methods. The mutation burst at the yellow locus is shown to result from insertion of a defective hobo copy. Six mutations y2 independently isolated from the natural population were caused by an insertion of hobo in the same site of the yellow regulatory region. The hobo copies were identical according to the restriction map. In three y2 alleles, the mutant phenotype was associated with inversion between hobo copies, one of which was located in the yellow locus and the other, in another unidentified region of the X chromosome. The remaining y2 alleles were associated with deletions that were located in the vicinity of the hobo insertion site and in the region of yellow enhancers.

Alleles↗
PubMed1978

Structure of recombinant plasmids containing synthetic human foetal globin gene sequences.

In vitro synthesized duplex DNA complementary to human foetal globin messenger RNA was integrated into bacterial plasmids and amplified by transformation of Escherichia coli. Recombinants carrying globin DNA were identified by hybridization of foetal globin messenger RNA to bacterial DNA in situ and by liquid hybridization of purified plasmids to specific globin complementary DNA probes. Heteroduplex mapping revealed either a simple insertion loop at the position of the EcoRI site of the parental plasmid DNA. We provide evidence to suggest that these deletions are the result of a site-specific nicking activity of the EcoRI preparations used in the formation of recombinant plasmids.

DNA Restriction Enzymes↗
PubMed1991

Design and synthesis of new enzymes based on the lactate dehydrogenase framework.

Analysis of the mechanism and structure of lactate dehydrogenases is summarized in a map of the catalytic pathway. Chemical probes, single tryptophan residues inserted at specific sites and a crystal structure reveal slow movements of the protein framework that discriminate between closely related small substrates. Only small and correctly charged substrates allow the protein to engulf the substrate in an internal vacuole that is isolated from solvent protons, in which water is frozen and hydride transfer is rapid. The closed vacuole is very sensitive to the size and charge of the substrate and provides discrimination between small substrates that otherwise have too few functional groups to be distinguished at a solvated protein surface. This model was tested against its ability to successfully predict the design and synthesis of new enzymes such as L-hydroxyisocaproate dehydrogenase and fully active malate dehydrogenase. Solvent friction limits the rate of forming the vacuole and thus the maximum rate of catalysis.

Amino Acid Sequence↗
PubMed1982

Cloning of genes determining the production of mannose-resistant fimbriae in a uropathogenic strain of Escherichia coli belonging to serogroup O6.

Chromosomal DNA from a uropathogenic strain of Escherichia coli was partially digested with the restriction enzyme EcoRI. The partial digests were ligated into a cosmid containing an ampicillin-resistant determinant and packaged into lambda phage particles. An ampicillin-resistant transductant of E. coli HB101 was found to possess mannose-resistant hemagglutinating activity associated with a 50-kilobase-pair plasmid. Subcloning of the mannose-resistant fimbrial genes revealed that the genetic determinants were encoded by a 6.9-kilobase-pair DNA fragment of a recombinant plasmid. Chimeric plasmids smaller in size were unable to transform E. coli to fimbrial production. Physical maps of the recombinant plasmids were prepared showing restriction endonuclease sites within the inserted DNA fragments. The hemagglutinating activities of the wild-type strain and of the recombinant derivative were compared. Both strains agglutinated human erythrocytes in the presence of D-mannose to the same degree and also failed to produce fimbriae after incubation at 18 degrees C. Also, both strains were agglutinated by antifimbrial serum at a high titer, whereas no such activity was observed when a strain of E. coli which did not possess a plasmid was used.

Cloning, Molecular↗
PubMed2003

Cytadherence-deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis.

Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gm(r) HA-negative (HA(-)) colonies displaying a stable HA(-) phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA(-) mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA(-) mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.

Adhesins, Bacterial↗
PubMed1983

Mapping of the pin locus coding for a site-specific recombinase that causes flagellar-phase variation in Escherichia coli K-12.

Although the vh2 mutation almost entirely prevents phase variation in Salmonella spp., an Escherichia coli strain that carried the Salmonella H1 and H2 region, including the vh2 mutation, showed phase variation. From this strain, EJ1076, a number of mutants defective in phase variation were isolated, and the symbol pin was assigned to their mutant gene. The pin locus was mapped between purB and trp near purB by interrupted matings using Tn10 sites inserted near pin. The locus was cotransduced with purB by P1 vir at a frequency of around 0.33. All the mutations tested were clustered at this locus. Three E. coli K-12 strains probably derived via different lines from the wild type have been tested for the presence of pin+ by introducing the two Salmonella H regions; two were pin+, and one was a pin mutant.

Chromosome Inversion↗
PubMed2001

Requirement of novel competence genes pilT and pilU of Pseudomonas stutzeri for natural transformation and suppression of pilT deficiency by a hexahistidine tag on the type IV pilus protein PilAI.

The ubiquitous species Pseudomonas stutzeri has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termed pilT. The deduced amino acid sequence has identity with PilT of Pseudomonas aeruginosa (94%), Neisseria gonorrhoeae (67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [(3)H]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of pilT a gene, termed pilU, coding for a putative protein with 88% amino acid identity with PilU of P. aeruginosa was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable P. aeruginosa. When the pilAI gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in pilAI::Gm(r) cells but allowed transformability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the pilT mutation. Considering the hypothesized role of pilT(+) in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the pilT-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.

Adenosine Triphosphatases↗
PubMed1983

Isolation of a nondefective recombinant between adenovirus type 5 and early region 1A of adenovirus type 12.

A nondefective recombinant between adenovirus type 5 (Ad5) and type 12 (Ad12), rc-1 (Ad5 dl312, carrying the Ad12 E1A gene), was isolated from hamster cell foci transformed by a defective recombinant, rcB-1 (dl312, carrying the Ad12 E1 gene). The recombinant rc-1 grew in human embryo kidney and KB cells in the absence of helper and synthesized Ad12 T antigen g, the product of the E1A gene. The genome of rc-1 has a deletion between 79.9 and 82.5 map units of Ad5 dl312 DNA with an insertion of 0.1 to 5.5 map units of Ad12 DNA at the deletion site. The mRNAs of Ad12 E1A were transcribed from the Ad12 E1A promoter, and unusual RNAs were abundantly transcribed from the Ad5 E3 promoter on the opposite strand. The frequency of cell transformation with rc-1 was lower than those with Ad5 and Ad12 wild types.

Adenoviruses, Human↗
PubMed1991

Pigmentation mutants produced by transposon mutagenesis in Antirrhinum majus.

New pigmentation mutants were generated by transposon mutagenesis in Antirrhinum majus, in three previously described loci, nivea, delila and incolorata, and two new loci, daphne and olive. The wild-type olive gene is required for the production of dark-green leaves, and the daphne gene for the synthesis of flavones. Five out of the six mutants were both germinally and somatically unstable, indicating that they resulted from transposon insertions. Molecular analysis of the mutant at nivea (niv-600) showed that it was caused by insertion of a new transposon, Tam4. The sequence of Tam4 suggests that it is unable to transpose autonomously and that it is related to Tam1 and Tam2. All three of these transposons have identical inverted repeats, produce 3 bp target duplications, leave similar excision footprints and share at one end a 600-700 bp region containing many palindromic copies of a motif sequence, possibly required in cis for transposition. The somatic excision of Tam4 in niv-600 is at a very low rate compared to germinal excision but it can be activated by crossing to lines carrying derivative alleles of a Tam1 insertion at niv. Molecular analysis of four different pigmentation mutants has shown that insertions of Tam1, Tam2, Tam3 and Tam4 have been obtained, illustrating the potential of general transposon mutagenesis for trapping and isolating new transposons as well as for tagging genes.

Base Sequence↗
PubMed1993

The P1 plasmid partition complex at parS. II. Analysis of ParB protein binding activity and specificity.

The P1 plasmid prophage is partitioned by a very high affinity protein complex at its partition site, parS, that contains the P1 ParB protein and Escherichia coli integration host factor (IHF). ParB binds to regions of parS that flank the IHF binding site. In this report, we have examined the sequences to which ParB binds, the spatial relationship between them, and the effect of IHF on ParB binding patterns. Methylation protection and interference experiments were performed on supercoiled plasmids. Mutations that interfered with the action of both proteins in vivo were identified following random mutagenesis of parS. These studies revealed that ParB binds to a complicated, nonsymmetrical region in the right side of parS. ParB recognizes a partial copy of this sequence, TCGCCA, in the left side of parS with much lower affinity. The presence of IHF greatly facilitates the interaction of ParB with parS such that both sides bind with an equal affinity that is much greater than to either side alone. The stimulation by IHF is strongly influenced by helical phasing. These observations support the proposal that ParB is directed, by the bend created by IHF, to bind simultaneously to properly placed sequences flanking the IHF site.

Bacterial Proteins↗
PubMed1995

DNA sequence of the deletion/insertion in early region 3 of Ad5 dl309.

dl309 is an adenovirus type 5 (Ad5) mutant that has been extensively utilized for construction of Ad5 mutants in early region 1 (E1), in developing vectors for use as viral vaccines, and in development of gene transfer vectors for gene therapy. Ad5 dl309 has been useful for vector construction because of its altered XbaI restriction pattern and lends itself to a variety of strategies for rescuing inserts or mutations into E1. It contains only one XbaI site at 3.7 map units (m.u.) as compared to wt Ad5 which contains 4(3.7, 29.5, 79.5, and 84.8 m.u.). The loss of the sites at 29.5 and 79.5 m.u. is due to deletions of a few bp but the loss of the site at 84.8 m.u. was the result of a deletion from approximately 83 to 85 m.u. and substitution with a fragment of foreign DNA. Because of the widespread use of dl309 and derivatives of this mutant in the construction of Ad5-based vectors and the need to have precise genetic information on the sequences present in vectors to be used as vaccines and in gene therapy, we have sequenced the alterations in dl309 which affect the XbaI sites at 79.5 and 84.8 m.u. and have determined which E3 proteins are expressed by this virus. The deletion that removes the XbaI site at 84.8 m.u. extends from Ad5 bp 30005-30750 and is substituted with 642-bp of heterologous DNA that shows homology to salmon DNA. This alteration deletes all or part of the coding sequences for the E3 14.7K, 14.5K and 10.4K proteins and these proteins are not detected in dl309 infected cells. The loss of the XbaI site at 79.5 m.u. is the result of a 6-bp deletion which removes two internal amino acids (18 and 19) from the E3 6.7K protein. The E3 6.7K protein and other E3 proteins whose coding sequences are unaffected by the alterations in dl309 (gp19K, 12.5K and 11.6K) were expressed in dl309 infected cells.

Adenoviridae↗
PubMed1994

Insertional mutation on mouse chromosome 18 with vestibular and craniofacial abnormalities.

A dominant mutation was generated in transgenic mice as a consequence of insertional mutation. Heterozygous mice from transgenic line 9257 (Tg9257) are hyperactive with bidirectional circling behavior and have a distinctive facial appearance due to hypoplasia of the nasal bone. Morphological analysis of the inner ear revealed asymmetric abnormalities of the horizontal canal and flattening or invagination of the crista ampullaris, which can account for the circling behavior. The sensory epithelium appeared to be normal. The transgene insertion site was localized by in situ hybridization to the B1 band of mouse chromosome 18. Genetic mapping in an interspecific backcross demonstrated the gene order centromere--Tg9257--8.8 +/- 3.4--Grl-1, Egr-1, Fgf-1, Apc--14.7 +/- 4.3--Pdgfr. The phenotype and the mapping data suggest that the transgene may be inserted at the Twirler locus. Homozygosity for the transgene results in prenatal lethality, but compound heterozygotes carrying the Tw allele and the transgene are viable. The function of the closely linked ataxia locus is not disrupted by the transgene insertion. This insertional mutant will provide molecular access to genes located in the Twirler region of mouse chromosome 18.

Amylases↗
PubMed1982

Structure and variation of human ribosomal DNA: the external transcribed spacer and adjacent regions.

A group of human ribosomal DNA (rDNA) recombinants that include the probable site for initiation of transcription have been examined for sequence polymorphism. A detailed restriction map of one rDNA insert was constructed using plasmid subclones and end-labeled segments. Comparison of 16 similar rDNA inserts by restriction and heteroduplex analysis demonstrated striking conservation of the external transcribed spacer and 18S gene regions, but defined a region where restriction sites for the enzymes Sma I, Hpa II, and Hha I become frequent or variable. This region extends for about 400--800 base pairs (bp) at the left end of the rDNA insert and is postulated to contain nontranscribed spacer sequences. The use of cloned rDNA segments as probes for the restriction analysis of genomic rDNA has demonstrated certain fixed sites in the nontranscribed spacer that do not vary significantly among different individuals or tumor cell lines. In contrast, restriction with the enzyme Sal I reveals several variable fragments, one of which has been found only in a retinoblastoma cell line.

Base Composition↗
PubMed2003

Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome.

In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modified Ds transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.

Cloning, Molecular↗
PubMed1991

Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis.

Linker insertion mutagenesis was employed to create structural disruptions of the lethal factor (LF) protein of anthrax toxin to map functional domains. A dodecameric linker was inserted at 17 blunt end restriction enzyme sites throughout the gene. Paired MluI restriction sites within the linker allowed the inserts to be reduced from four to two amino acids. Shuttle vectors containing the mutated genes were transformed into the avirulent Bacillus anthracis UM23C1-1 for expression and secretion of the gene products. Mutations at five sites in the central one-third of the sequence made the protein unstable, and purified protein could not be obtained. Mutated LF proteins with insertions at the other sites were purified and assessed for toxic activity in a macrophage lysis assay and for their ability to bind to the protective antigen (PA) component of anthrax toxin, the receptor binding moiety. Most insertions located in the NH2-terminal one-third of the LF protein eliminated both toxicity and binding to PA, while all four insertions in the COOH-terminal one-third of the protein eliminated toxicity without affecting binding to PA. These data support the hypothesis that the NH2-terminal domain contains the structures required for binding to PA and the COOH-terminal domain contains the putative catalytic domain of LF.

Antigens, Bacterial↗