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At least 253 records · Page 14Linked to original sources

PubMed1991

Mapping catalytically important regions of an enzyme using two-codon insertion mutagenesis: a case study correlating beta-lactamase mutants with the three-dimensional structure.

Two-codon insertion mutants throughout the beta-lactamase (Bla)-encoding gene were characterized. Second site revertants of various mutants were isolated, mapped and sequenced. The activity of the mutants and the ability to get revertants showed a positive correlation with increasing distance from the active site, based on the three-dimensional structure of Bla. This observation is discussed as it may pertain to the generalized use of two-codon insertion mutagenesis in mapping important catalytic regions in enzymes.

Amino Acid Sequence↗

PubMed1983

Amelioration of the ultraviolet sensitivity of an Escherichia coli recA mutant in the dark by photoreactivating enzyme.

When a recA strain of Escherichia coli is transformed with a multicopy plasmid, pKY1, carrying the phr gene of E. coli, its extreme ultraviolet sensitivity is decreased. Derivatives of pKY1 were prepared in which the phr gene was inactivated by inserting transposon Tn1000. None of the 20 phr- derivatives decreased the UV sensitivity of the recA strain. In an analogous experiment, we obtained 11 derivatives which failed to decrease the UV sensitivity of the recA strain. None of them complemented phr strain. Furthermore, TN1000 insertion sites in both types of derivatives were mapped in the same region of the plasmid. From these observations, we propose that the E. coli phr gene product has repair activity in the dark.

Bacterial Proteins↗

PubMed2001

Identification of nonessential Helicobacter pylori genes using random mutagenesis and loop amplification.

Analysis of the published genome sequences of Helicobacter pylori revealed that approximately 40% of the predicted open reading frames (ORFs) were of unknown function. We have developed the random mutagenesis and loop amplification (RMLA) strategy, and used this approach both to characterize individual virulence factors and to collectively screen comparatively large numbers of H. pylori mutants to identify genes that are not essential for viability in vitro. The mini-Tn3-Km transposon was used to generate a random mutant library in H. pylori strain G27. By screening the library of mutants we were able to demonstrate that the transposon integrated randomly into the chromosome of H. pylori and that RMLA was able to identify mutants in known virulence genes (urease and catalase). To test whether this strategy could be used as a high-throughput approach for the simultaneous identification of a series of nonessential genes of H. pylori, the transposon-chromosomal junctions of a pool of mutants were amplified by inverse PCR using circular fragments of genomic DNA obtained after chromosomal DNA extracted from the pool of mutants had been digested with HindIII and self-ligated. The amplification products were radioactively labelled and hybridized to a high density macroarray membrane containing a duplicated target sequence for every gene of H. pylori strain 26695. For the positive ORFs the precise site of transposon insertion was confirmed by PCR mapping. In total 78 H. pylori genes were unambiguously identified as nonessential for viability in vitro, including 20 with orthologues of unknown function in other species and 21 which were H. pylori-specific.

DNA Transposable Elements↗

PubMed1984

Identification of a rhizosphere protein encoded by the symbiotic plasmid of Rhizobium leguminosarum.

A protein was identified which was made by wild-type strains of Rhizobium leguminosarum but not by nodulation-deficient derivatives which had deletions of their symbiotic plasmids. The protein, which had a subunit molecular weight of ca. 24,000 ( 24K ), was found to be present in large amounts within bacteria that had been reisolated from the surface of inoculated pea roots but was not detected in bacteroids isolated from nodules. The protein could also be induced during growth of R. leguminosarum on nutrient medium and was purified from the cytoplasmic fraction of broken cells. Antiserum raised against the purified protein was used to screen transposon-induced mutants of R. leguminosarum, and four independent mutants were isolated which lacked the protein. The sites of the Tn5 insertions were found to map between the nitrogenase and nodulation genes on symbiotic plasmid pRL1JI , ca. 5 kilobases from the nitrogenase genes and 13 kilobases from the nodulation genes. Genetic determinants for the 24K protein were found to be closely linked to plasmid-borne nodulation genes for all strains of R. leguminosarum tested. However, the mutants which lacked the 24K protein still formed normal nitrogen-fixing nodules on peas, and the function of the protein is unknown.

Bacterial Proteins↗

PubMed1980

Unusual features in the nucleotide sequence of a cDNA clone derived from the common region of avian sarcoma virus messenger RNA.

We have constructed a recombinant plasmid containing a 700-base pair (bp) cDNA copy of the common region present at the 3' end of Schmidt-Ruppin avian sarcoma virus (ASV) 21S mRNA. The cDNA was inserted into plasmid pBR322 at the Pst I site by the G-C tailing method. A restriction map of the cloned insert from a recombinant plasmid pSRI indicates that it corresponds to the 3' end of the ASV genome. R-loop analysis with ASV genomic RNA indicates that the insert is colinear with the ASV genome over most of its length. The sequence of 331 bp at the 3' end of the DNA insert was determined and shows that the insert contains extra sequences not found at the 3' end of ASV genomic RNA. Following the terminally redundant sequence of 20 bp that has been found at the extreme 3' end of genomic RNA is a sequence of 79 bp that is almost identical to that located immediately next to the 20-bp repeat at the 5' end of ASV genomic RNA. This is followed by 18 bp of unique sequence, possibly of host origin. The structure of the clone suggests that ASV mRNA may differ from genomic RNA at its 3' end and that 21S mRNA is transcribed from integrated ASV DNA and contains at its 3' end sequences derived both from the 5' end of the ASV genome and from host DNA adjacent to the site of integration. The presence of termination codons in all three reading frames suggests that the common region probably does not contain coding sequences. However, the presence of sequences that resemble probable promoter sites supports the possibility that this region may be involved in the regulation of transcription.

Alpharetrovirus↗

PubMed1998

Fowl adenovirus recombinant expressing VP2 of infectious bursal disease virus induces protective immunity against bursal disease.

The right hand end Nde I fragment 3 (90.8-100 map units) of the fowl adenovirus serotype 10 (FAV-10) was characterised so as to allow the location of an insertion site for recombinant vector construction. Infectious bursal disease virus (IBDV) VP2 gene from the Australian classical strain 002/73, under the control of the FAV-10 major late promoter/leader sequence (MLP/LS) was inserted into a unique Not I site that was generated at 99.5 map units. This recombinant virus was produced without deletion of any portion of the FAV-10 genome. When administered to specific pathogen free (SPF) chickens intravenously, intraperitoneally, subcutaneously or intramuscularly, it was shown that the FAV-10/VP2 recombinant induced a serum VP2 antibody response and protected chickens against challenge with IBDV V877, an intermediate virulent classical strain. Birds were not protected when the recombinant was delivered via the conjunctival sac.

Animals↗

PubMed2005

Genetic linkage maps of the red flour beetle, Tribolium castaneum, based on bacterial artificial chromosomes and expressed sequence tags.

A genetic linkage map was constructed in a backcross family of the red flour beetle, Tribolium castaneum, based largely on sequences from bacterial artificial chromosome (BAC) ends and untranslated regions from random cDNA's. In most cases, dimorphisms were detected using heteroduplex or single-strand conformational polymorphism analysis after specific PCR amplification. The map incorporates a total of 424 markers, including 190 BACs and 165 cDNA's, as well as 69 genes, transposon insertion sites, sequence-tagged sites, microsatellites, and amplified fragment-length polymorphisms. Mapped loci are distributed along 571 cM, spanning all 10 linkage groups at an average marker separation of 1.3 cM. This genetic map provides a framework for positional cloning and a scaffold for integration of the emerging physical map and genome sequence assembly. The map and corresponding sequences can be accessed through BeetleBase (http://www.bioinformatics.ksu.edu/BeetleBase/).

Animals↗

PubMed2002

Transgene insertion induced dominant male sterility and rescue of male fertility using round spermatid injection.

Transgene insertions in the mouse often cause mutations at chromosomal loci. Analysis of insertion mutations that cause male sterility may lead to the identification of novel molecular mechanisms implicated in male fertility. Here we show a line of transgenic mice with dominant inheritance of male sterility (DMS) that was found amid several lines that were normally fertile. Transgene-positive males from this line invariably were sterile, whereas transgenic females and transgene-negative male littermates were fertile. Histologic analysis and TUNEL staining for apoptotic cells in DMS testis showed spermatogenesis arrest at metaphase of meiosis I (M-I), accompanied by massive apoptosis of spermatocytes. Meiosis I arrest was incomplete, however, as small numbers of spermatids and spermatozoa were found. Both round spermatids and spermatozoa were evaluated for their permissiveness in the assisted reproductive technologies intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI). Surprisingly, ROSI but not ICSI gave live offspring, suggesting that mature sperm had deteriorated by the time of recovery from the epididymis. Mapping the transgene insertion by fluorescence in situ hybridization revealed a site on chromosome 14 D3-E1. Two candidate genes, GFR alpha 2 and GnRH, that were previously mapped to that region and the functions of which in spermatogenesis are well established were not altered in DMS. As a consequence, positional cloning of the DMS locus will be essential to identify new molecules potentially involved in arrest at M-I. Furthermore, mice carrying this genetic trait might be useful for studies of assisted reproductive technologies and male contraceptives.

Animals↗

PubMed2001

Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence.

PlcR is a pleiotropic regulator of virulence factors in the insect pathogen Bacillus thuringiensis and in the opportunistic human pathogen Bacillus cereus. It activates the transcription of at least 15 genes encoding extracellular proteins, including phospholipases C, proteases and enterotoxins. Expression of the plcR gene is autoregulated and activated at the onset of stationary phase. Here, we used mini-Tn10 transposition to generate a library of B. thuringiensis mutants, with the goal of characterizing genes involved in the expression of the plcR gene. Three mutant strains were identified carrying distinct mini-Tn10 insertions. The mutations impaired plcR expression and caused a deficient haemolytic phenotype, similar to the phenotype of a B. thuringiensis strain in which the plcR gene had been disrupted. The insertion sites of the three mini-Tn10 transposons mapped in a five-gene operon encoding polypeptides homologous to the components of the oligopeptide permease (Opp) system of Bacillus subtilis, and with a similar structural organization. By analogy, the five B. thuringiensis genes were designated oppA, B, C, D and F. In vitro disruption of the B. thuringiensis oppB gene reproduced the effect of the mini-Tn10 insertions (i.e. the loss of haemolytic activity) and reduced the virulence of the strain against insects. These phenotypes are similar to those of a DeltaplcR mutant. Opp is required for the import of small peptides into the cell. Therefore, plcR expression might be activated at the onset of stationary phase by the uptake of a signalling peptide acting as a quorum-sensing effector. The opp mutations impaired the sporulation efficiency of B. thuringiensis when the cells were cultured in LB medium. Thus, Opp is on the pathway that ultimately regulates Spo0A phosphorylation, as is the case in B. subtilis. However, analysis of plcR expression in DeltaoppB, Deltaspo0A and DeltaoppB Deltaspo0A mutants indicates that Opp is required for plcR expression via a Spo0A-independent mechanism.

Animals↗

PubMed2005

Localization of a disease-associated mutation site in the three-dimensional structure of the cardiac muscle ryanodine receptor.

The cardiac muscle ryanodine receptor (RyR2) functions as a calcium release channel in the heart. Up to 40 mutations in RyR2 have been linked to genetic forms of sudden cardiac death. These mutations are largely clustered in three regions of the sequence of the polypeptide: one near the N terminus, one in the central region, and the third in the C-terminal region. The central region includes 11 mutations, and an isoleucine-proline motif (positions 2427 and 2428) in the same region is predicted to contribute to the binding of FKBP12.6 protein. We have mapped the central mutation site in the three-dimensional structure of RyR2 by green fluorescent protein insertion, cryoelectron microscopy, and single-particle image processing. The central mutation site was mapped to a "bridge" of density that connects cytoplasmic domains 5 and 6, which have been suggested to undergo conformational changes during channel gating. Moreover, the location of this central mutation site is not close to that of the FKBP12.6-binding site mapped previously by cryoelectron microscopy.

Animals↗

PubMed1986

Structural and regulatory divergence among site-specific recombination genes of lambdoid phage.

The lambdoid bacteriophage phi 80 and P22 have site-specific recombination systems similar to that of lambda. Each of the three phage has a different insertion specificity, but structural analysis of their attachment sites suggests that the three recombination pathways share similar features. In this study, we have identified and sequenced the int and xis genes of phi 80 and P22. phi 80 int and xis were identified using a plasmid recombination assay in vivo, and the P22 genes were mapped using Tn1 insertion mutations. In all three phage, the site-specific recombination genes are located directly adjacent to the phage attachment site. Interestingly, the transcriptional orientation of the phi 80 int gene is opposite to that of lambda and P22 int, resulting in convergent transcription of phi 80 int and xis. Because of its transcriptional orientation, phi 80 int cannot be expressed by the major leftward promoter, PL, and the regulatory strategy of phi 80 integration and excision must differ significantly from that of lambda. The deduced amino acid sequences of the recombination proteins of the three systems show surprisingly little homology. Sequences homologous to the lambda PI promoter are more conserved than the protein-coding sequences. Nevertheless, the Int proteins are locally related in the C-terminal sequences, particularly for a stretch of some 25 amino acid residues that lie approximately 50 residues from the C terminus. The Xis proteins can be aligned at their N termini.

Amino Acid Sequence↗

PubMed1996

Genomic structure and precise mapping of a thymic regulatory region on mouse chromosome 17 revealed by a c-myc transgene insertion.

In one transgenic strain harboring a human c-myc proto-oncogene construct, the transgene was actively and exclusively expressed in the thymus, where it contributed to the development of lymphoma that corresponded to CD4(+)CD8(+) cells. Here, we have pursued the analysis of transgene expression in healthy transgenic mice and show that transgene activation occurs in the thymus 3 days before birth, at a time when CD4(+)CD8(+) lymphocytes emerge. In the adult, its expression is restricted to the CD4(+)CD8(+) cells. The region flanking the transgene insertion site was isolated and made it possible to map the preintegration locus, hereafter called Tsil (for thymus-specific integration locus) on chromosome 17 between D17Rp11e and Ras12-3. A YAC that contains both Tsil and the Pim2 locus, previously shown to be involved in progression of T-cell lymphoma, was isolated. Analysis of Tsil offers a unique opportunity to identify a regulatory region or a gene that might play an important role in T-cell maturation.

Animals↗

PubMed1990

Probe mapping to facilitate transposon-based DNA sequencing.

A promising strategy for DNA sequencing exploits transposons to provide mobile sites for the binding of sequencing primers. For such a strategy to be maximally efficient, the location and orientation of the transposon must be readily determined and the insertion sites should be randomly distributed. We demonstrate an efficient probe-based method for the localization and orientation of transposon-borne primer sites, which is adaptable to large-scale sequencing strategies. This approach requires no prior restriction enzyme mapping or knowledge of the cloned sequence and eliminates the inefficiency inherent in totally random sequencing methods. To test the efficiency of probe mapping, 49 insertions of the transposon gamma delta (Tn1000) in a cloned fragment of Drosophila melanogaster DNA were mapped and oriented. In addition, oligonucleotide primers specific for unique subterminal gamma delta segments were used to prime dideoxynucleotide double-stranded sequencing. These data provided an opportunity to rigorously examine gamma delta insertion sites. The insertions were quite randomly distributed, even though the target DNA fragment had both A + T-rich and G + C-rich regions; in G + C-rich DNA, the insertions were found in A + T-rich "valleys." These data demonstrate that gamma delta is an excellent choice for supplying mobile primer binding sites to cloned DNA and that transposon-based probe mapping permits the sequences of large cloned segments to be determined without any subcloning.

Animals↗

PubMed1985

Delineation of two distinct regulatory domains in the 5' region of the nar operon of Escherichia coli.

A detailed restriction site map was determined for an 8.4-kilobase DNA fragment containing the 5' regulatory and promoter region of the nar operon of Escherichia coli. The 5' end of the nar operon was subcloned as a 2.5-kilobase fragment, and an intact nar operon was constructed from this subcloned fragment and an EcoRI fragment containing the remainder of the nar operon. A set of Bal 31 deletions extending into the 5' region of the intact operon was selected, mapped, and characterized. Based on the synthesis of the alpha and beta subunits of nitrate reductase in a nar::Tn5 mutant, three categories of deletions were found: (i) those which permitted normal expression, (ii) those which completely prevented expression, and (iii) those which permitted anaerobic expression of the operon but prevented any additional induction by nitrate. The nucleotide sequence was determined for a segment of the nar promoter region starting at one of the latter deletion end points and extending into the first structural gene of the operon. The position of the deletion end point relative to the translation start site for the first structural gene, narG, was defined by identifying the nucleotide sequence for the first 20 N-terminal amino acid residues of the alpha subunit of nitrate reductase. Deletions terminating 161 base pairs (bp) and approximately 200 bp upstream from the narG translation start site permitted anaerobic formation of nitrate reductase but interfered with the stimulation of nar operon expression by nitrate. A maximum size for the regulatory region was defined by two Tn5 insertions, which mapped approximately 550 bp 5' from the translation start site and did not interfere with the normal expression of nitrate reductase under anaerobic conditions with or without nitrate. We conclude that the nar operon 5' regulatory region is divided into two distinct regions: the 100 to 150 bp immediately 5' to the narG gene include a transcriptional start site and the signals necessary for anaerobic expression of the operon, and an adjacent region of 50 to 400 bp is required for the stimulation of operon expression by nitrate.

Base Sequence↗

PubMed1994

A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes.

A shuttle cosmid vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi. This vector contains two cos sequences separated by a single XbaI restriction site. pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15-20 micrograms of genomic DNA. Due to the presence of a functional hygromycin B phosphotransferase gene (hph) transformation of fungal protoplasts with pAN-sCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants. The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts.

Cloning, Molecular↗

PubMed1994

Transgenic mouse model of hemifacial microsomia: cloning and characterization of insertional mutation region on chromosome 10.

The 643 transgenic mouse line carries an autosomal dominant insertional mutation that results in hemifacial microsomia (HFM), including microtia and/or abnormal biting. In this paper, we characterize the transgene integration site in transgenic mice and preintegration site of wildtype mice. The locus, designated Hfm (hemifacial microsomia-associated locus), was mapped to chromosome 10, B1-3, by chromosome in situ hybridization. We cloned the transgene insertion site from the transgenic DNA library. By using the 5' and 3' flanking sequences, the preintegration region was isolated. The analysis of these regions showed that a deletion of at least 23 kb DNA occurred in association with the transgene integration. Evolutionarily conserved regions were detected within and beside the deleted region. The result of mating between hemizygotes suggests that the phenotype of the homozygote is lethality in the prenatal period. These results suggest that the Hfm locus is necessary for prenatal development and that this strain is a useful animal model for investigating the genetic predisposition to HFM in humans.

Animals↗

PubMed1982

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

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

Alcaligenes↗

PubMed1999

A YAC-based physical map of the mouse genome.

A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.

Animals↗