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PubMed1995

Genome analysis of Clostridium botulinum type A by pulsed-field gel electrophoresis.

Genomic DNA from type A Clostridium botulinum was digested with restriction endonucleases that cut at rare sites, and the large fragments were separated by pulsed-field gel electrophoresis. Of 15 restriction enzymes tested, MluI, RsrII, SmaI, NruI, KspI, NaeI, and XhoI generated satisfactory digestion patterns of genomic DNA of various C. botulinum strains, enabling the use of the method for genomic fingerprinting. The genomes of four group I (type A) C. botulinum strains examined had similar restriction patterns. However, each strain had unique digestion patterns, reflecting genotypic differences. The genome size of C. botulinum strain 62A was estimated to be 4,039 +/- 40 kbp from the summation of restriction fragments from MluI, RsrII, and SmaI digestions. Genes encoding proteins involved in the toxinogenicity of C. botulinum, including neurotoxin, hemagglutinin A, and genes for a temperate phage, as well as various transposon Tn916 insertion sites in C. botulinum 62A, were mapped by pulsed-field gel electrophoresis. The genes encoding neurotoxin and hemagglutinin A-1, were located on the same fragment in several cases, indicating their probable physical linkage. The macrorestriction analysis established here should be useful for genetic and epidemiological studies of C. botulinum.

Base Sequence↗

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↗

PubMed1997

Identification and characterization of pbpA encoding Bacillus subtilis penicillin-binding protein 2A.

Amino acid sequence analysis of tryptic peptides derived from purified penicillin-binding protein PBP2a of Bacillus subtilis identified the coding gene (now termed pbpA) as yqgF, which had been sequenced as part of the B. subtilis genome project; pbpA encodes a 716-residue protein with sequence similarity to class B high-molecular-weight PBPs. Use of a pbpA-lacZ fusion showed that pbpA was expressed predominantly during vegetative growth, and the transcription start site was mapped using primer extension analysis. Insertional mutagenesis of pbpA resulted in no changes in the growth rate or morphology of vegetative cells, in the ability to produce heat-resistant spores, or in the ability to trigger spore germination when compared to the wild type. However, pbpA spores were unable to efficiently elongate into cylindrical cells and were delayed significantly in spore outgrowth. This provides evidence that PBP2a is involved in the synthesis of peptidoglycan associated with cell wall elongation in B. subtilis.

Amino Acid Sequence↗

PubMed1990

[Genetic analysis of the host range of the plasmid RSF1010].

32 host range mutants of pMO58, which contains a 8.0 kb fragment of RSF1010, were screened, using the transposon Tn5 insertion mutagenesis. To judge the genes which affect the host range of plasmid, the Tn5 insertion sites were located with restriction endonuclease mapping. The ability of the plasmid replication in different recipients was checked with DNA transformation. The results further confirmed the barrier to the host range of plasmid was their inability to replicate and maintain themselves in genetically different host. The repA repB and repC genes were necessary for the plasmid replication, but the repC gene may be a positive regulator and its function may be complemented by host gene products.

DNA Replication↗

PubMed2004

A mechanism for immediate reduction in mitral regurgitation after cardiac resynchronization therapy: insights from mechanical activation strain mapping.

OBJECTIVES: We tested the hypothesis that an immediate reduction in mitral regurgitation (MR) after cardiac resynchronization therapy (CRT) results from improved coordinated timing of the papillary muscle insertion sites, using the novel approach of mechanical activation strain mapping. BACKGROUND: Heart failure patients with left bundle branch block often benefit acutely from CRT; however, the role and mechanism of reduction of MR are unclear. METHODS: Twenty-six consecutive patients undergoing CRT with at least mild MR were studied (ejection fraction 24 +/- 6%; QRS duration 168 +/- 30 ms). Echocardiographic Doppler and strain imaging was performed immediately before and the day after CRT, as well as in 10 normal control subjects. Mechanical activation sequence maps were constructed using longitudinal strain from 12 basal and mid-LV sites, with color coding of time-to-peak strain. RESULTS: Mitral regurgitation by the volumetric method consistently decreased after CRT: regurgitant volume from 40 +/- 20 ml to 24 +/- 17 ml and regurgitant fraction from 40 +/- 12% to 25 +/- 14% (both: p < 0.001 vs. baseline). Normal controls had uniform segmental time-to-peak strain, with a difference of only 12 +/- 8 ms between all segments. In contrast, CRT patients at baseline had a 106 +/- 74 ms time delay between papillary muscle insertion sites (p < 0.001 vs. normal). This interpapillary muscle time delay shortened after CRT to 39 +/- 43 ms (p < 0.001 vs. baseline) and was significantly correlated with reductions in mitral regurgitant fraction (r = 0.77, p < 0.001). CONCLUSIONS: Cardiac resynchronization therapy significantly and immediately reduced MR. Improved coordinated timing of mechanical activation of papillary muscle insertion sites appears to be a mechanistic contributor to immediate MR reduction by CRT.

Aged↗

PubMed2003

Mapping multiple co-sequenced T-DNA integration sites within the Arabidopsis genome.

MOTIVATION: Insertion mutagenesis, using transgenes or endogenous transposons, is a popular method for generating null mutations (knockouts) in model organisms. Insertions are mapped to specific genes by amplifying (via TAIL-PCR) and sequencing genomic regions flanking the inserted DNA. The presence of multiple TAIL-PCR templates in one sequencing reaction results in chimeric sequence of intermittently low quality. Standard processing of this sequence by applying Phred quality requirements results in loss of informative sequence, whereas not trimming low-quality sequence causes inclusion of low-complexity homopolymers from the ends of sequence runs. Accurate mapping of the flanking sequences is complicated by the presence of gene families. RESULTS: Methods for extracting informative regions from sequence traces obtained by sequencing multiple TAIL-PCR fragments in a single reaction are described. The completely sequenced Arabidopsis genome was used to identify informative TAIL-PCR sequence regions. Methods were devised to define and select high quality matches and precisely map each insert to the correct genome location. These methods were used to analyze sequence of TAIL-PCR-amplified flanking regions of the inserts from individual plants in a T-DNA-mutagenized population of Arabidopsis thaliana, and are applicable to similar situations where a reference genome can be used to extract information from poor-quality sequence.

Arabidopsis↗

PubMedDate not supplied

[Simple method of cloning eukaryote DNA: production of certain new ribosomal genes of Drosophila].

We propose a simple method which allows to receive a collection of clones containing recombinant plasmids. It is based on the ligation of the longer fragment of pBR332 formed by EcoRI and BamH1 with eukaryotic DNA (from Drosophila melanogaster embryo in this case) partially cleaved with EcoRI and BamHI. This approach gave us 10(4) colonies from 1 microgram of Drosophila DNA and 0.1 microgram of the BamHI--EcoRI "vector". About 0.5% of all clones carried the fragments of ribosomal genes with insertions in the 26S gene. Ribosomal genes lacking insertions did not enter the collection due to some peculiarities in their restriction map. The sites of cleavage are mapped in eight recombinant plasmide for HindIII, BamHI and EcoRI. These maps show that some insertions within 26S gene have not been cloned earlier. The mean length of cloned fragments is 11.8 kilobases, the mean number of EcoRI and BamHI restriction sites are 1.2 and 1.0, respectively. The electrophoretical screening of plasmids using cetyl trimethyl ammonium bromide was developed.

Animals↗

PubMed1990

The human class I alcohol dehydrogenase gene cluster: three genes are tandemly organized in an 80-kb-long segment of the genome.

The class I alcohol dehydrogenases (ADH; EC 1.1.1.1) play a key role in hepatic alcohol catabolism. Three human class I ADH genes, ADH1, ADH2, and ADH3, which encode the alpha, beta, and gamma subunits respectively, have been isolated and mapped on chromosome 4q21-q23. Genomic cloning using a cosmid vector allowed us to obtain an 88-kb-long genomic segment, which was found to include an entire 80 kb of the class I ADH gene cluster. All three genes lie in the same transcriptional orientation and the order of genes is 5'-ADH3-ADH2-ADH1-3'. It may be of some significance that the order of transcriptional activation in the hepatic development, alpha----beta----gamma, is opposite to the order of gene arrangement. Several members of the AluI family and the KpnI (L1) family of interspersed repetitive sequences were mapped in this region. The divergence of insertional sites suggested that gene multiplication of the class I ADH genes had taken place in the earlier stages of human (or primate) evolution.

Alcohol Dehydrogenase↗

PubMed1988

Mapping of Escherichia coli chromosomal Tn5 and F insertions by pulsed field gel electrophoresis.

A low resolution Not I physical map of Escherichia coli was recently constructed. In this report we demonstrated that this map can be used to map Tn5 and F insertions physically. The transposon, Tn5, contains Not I recognition sequences in its IS50 sequences. F plasmid contains an unmapped Not I site. Hence, the location of Tn5 and F in the chromosome can be mapped by identifying the location of the introduced Not I sites using pulsed field gel electrophoresis. The physical mapping of genetically mapped Tn5 insertions confirm the previously constructed Not I map and helps align the E. coli physical and genetic maps. The use of Tn5 can assist the construction of both physical and genetic maps for microorganisms lacking such maps. Variations on this approach will facilitate physical mapping with a wide variety of organisms, enzymes, and genetic elements.

Alleles↗

PubMed2004

Large retrotransposon derivatives: abundant, conserved but nonautonomous retroelements of barley and related genomes.

Retroviruses and LTR retrotransposons comprise two long-terminal repeats (LTRs) bounding a central domain that encodes the products needed for reverse transcription, packaging, and integration into the genome. We describe a group of retrotransposons in 13 species and four genera of the grass tribe Triticeae, including barley, with long, approximately 4.4-kb LTRs formerly called Sukkula elements. The approximately 3.5-kb central domains include reverse transcriptase priming sites and are conserved in sequence but contain no open reading frames encoding typical retrotransposon proteins. However, they specify well-conserved RNA secondary structures. These features describe a novel group of elements, called LARDs or large retrotransposon derivatives (LARDs). These appear to be members of the gypsy class of LTR retrotransposons. Although apparently nonautonomous, LARDs appear to be transcribed and can be recombinationally mapped due to the polymorphism of their insertion sites. They are dispersed throughout the genome in an estimated 1.3 x 10(3) full-length copies and 1.16 x 10(4) solo LTRs, indicating frequent recombinational loss of internal domains as demonstrated also for the BARE-1 barley retrotransposon.

3' Untranslated Regions↗

PubMed1993

Identification of an Alu retrotransposition event in close proximity to a strong candidate gene for Huntington's disease.

Huntington's disease (HD) is a late-onset autosomal dominant neuropsychiatric disorder presenting in mid-adult life with personality disturbance and involuntary movements, cognitive and affective disturbance, and inexorable progression to death. The underlying genetic defect has been mapped to chromosomal band 4p16.3 (refs 2, 3). Analysis of specific recombination events in some families with HD has further refined the location of the HD defect to a 2.2 megabase DNA interval. Using a direct complementary DNA selection strategy we have identified at least seven transcriptional units within the minimal region believed to contain the HD gene. Screening with one of the cDNA clones identified an Alu insertion in genomic DNA from two persons with HD which showed complete cosegregation with the disease in these families but was not found in 1,000 control chromosomes. Two genes including the previously identified alpha-adducin gene and another that encodes for a 12-kilobase transcript, map in close proximity to the Alu insertion site. The 12-kilobase transcript should be regarded as a strong candidate for the HD gene.

Base Sequence↗

PubMed1982

"Frizzy" mutants: a new class of aggregation-defective developmental mutants of Myxococcus xanthus.

During fruiting-body formation in Myxococcus xanthus, cells aggregate into raised mounds, where they sporulate. A new class of aggregation-defective developmental mutants was identified within a collection of nonfruiting mutants of M. xanthus. The mutants failed to aggregate into discrete mounds, but rather aggregated into "frizzy" filaments. Many cells within the filaments sporulated normally. Pairwise mixtures of representative frizzy mutants were unable to stimulate each other to aggregate normally. Two strains of M. xanthus were isolated which contained transposon Tn5 insertions mapping near one frizzy mutation. A search through 36 mutants exhibiting the frizzy phenotype showed that all were linked to the same Tn5 insertion sites. Three-factor cross-analysis of 22 of these mutants allowed the mapping of these mutations into many loci. The localization of Tn5 inserts adjacent to this region make possible further manipulation of these genes.

DNA Transposable Elements↗

PubMed1982

Template-directed pausing in in vitro DNA synthesis by DNA polymerase a from Drosophila melanogaster embryos.

The activity of Drosophila melanogaster DNA polymerase alpha on DNA-primed single-stranded DNA templates has been examined. The DNA templates contain a 1471-nucleotide sequence from the heavy-strand origin region of mouse mtDNA inserted into the single-stranded bacteriophage vector M13Gori1. Preferred sites for pausing of in vitro DNA synthesis have been mapped within the cloned mtDNA insert and in the G4 cDNA strand origin which is contained within the vector DNA. Analysis of nascent DNA strands from replicative intermediates has revealed that pause sites are discrete and lie both at the positions of predicted stable dyads and in regions lacking the potential for formation of such structures. The patterns of kinetic pause sites observed for Escherichia coli DNA polymerase III holoenzyme is qualitatively similar to that found for DNA polymerase alpha. A subset of the observed kinetic pause signals are recognized by E. coli DNA polymerase I under similar conditions.

Animals↗

PubMed1998

Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor.

The kinase insert domain-containing receptor (KDR) for vascular endothelial growth factor (VEGF) has been shown to be involved in vasculogenesis and angiogenesis. This receptor is characterized by seven immunoglobulin (Ig)-like domains within its extracellular region. To identify the domains involved in VEGF binding, we constructed various deletion mutants of the extracellular region fused with the crystallizable fragment portion of an IgG and then examined the binding affinity with VEGF by means of the BIAcore biosensor assay. Deletion of the COOH-terminal two or three Ig-like domains out of a total of seven affected ligand dissociation rather than association. Further deletion of the fourth domain caused a drastic decrease in the association rate. Binding ability was abolished completely with removal of the third domain. The mutant KDR proteins lacking the NH2-terminal Ig-like domain exhibited a slightly higher association rate compared with those of the mutants having this domain. Deletion of the first two NH2-terminal Ig-like domains caused a drastic reduction in the association rate, but affinity to VEGF was retained. These results suggest that the third Ig-like domain is critical for ligand binding, the second and fourth domains are important for ligand association, and the fifth and sixth domains are required for retention of the ligand bound to the receptor molecule. The first Ig-like domain may regulate the ligand binding.

Binding Sites↗

PubMed1985

Molecular cloning and partial sequencing of hepatitis A viral cDNA.

Hepatitis A virus was purified from infected monkey kidney cell cultures, and the viral RNA was used to synthesize double-stranded cDNA. This cDNA was cloned either after insertion into a plasmid-primed synthesis system or after insertion into the PstI site of pBR322. The resulting clones were mapped by restriction endonuclease analysis and by cross hybridization of the viral inserts to generate a composite map which represented at least 97% of the viral genome, lacking ca. 220 bases from the 5' end of the genome. The clones were verified to be hepatitis A virus specific based on their positive hybridization to viral RNA and to total hepatitis A virus-infected cellular RNA from a heterologous marmoset host system. The nucleotide sequence of 3,054 base pairs of cDNA homologous to the 5' half of the viral genome was determined, and an open reading frame of 854 consecutive coding triplets was identified. In addition, sequences which encode the VP-1 and VP-3 viral structural proteins were located in the nucleotide sequence.

Amino Acid Sequence↗

PubMed1987

Characterization of vaccinia virus early promoters and evaluation of their informational content.

We have reported the isolation of cis-acting regulatory DNA sequences promoting expression of the herpes virus thymidine kinase gene in vaccinia virus recombinants. In this work we show that each of the inserts from recombinants VpT25, 28, 36 and 56 contains a vaccinia virus early promoter. The position of each of the early RNA start sites in the nucleotide sequence of these four vaccinia virus inserts was precisely mapped by an S1 nuclease mapping procedure. Among the four recombinants analysed only VpT56-infected cells also contained a substantial amount of a transcript with the same 5' end at late period. The insert present in VpT25 contained a new late RNA start site 50 nucleotides upstream from that of the early RNA. The four inserts were mapped on the vaccinia virus genome. We also localized the 5' end of the mRNA of a vaccinia virus host-range gene, whose DNA nucleotide sequence has recently been established. The 45 nucleotides preceding the RNA start site from most of 19 known vaccinia virus early promoters were found to be A + T-rich (at least 80%) and contained shorter A-rich (at least 60%) regions, beginning approximately 25 nucleotides upstream from the RNA start site. The information content, as expressed by the parameter Rsequence, of early vaccinia virus promoters revealed ten bits of information in the sequence of 28 nucleotides upstream from the early RNA start sites. Most of the information needed to locate an early promoter is contained within the nucleotide sequence upstream from an RNA start site. A consensus sequence consists of two blocks: the sequence AA(A/T)N(T/A)N(A/G)AAAANAANA starting at position -27 and the sequence (T/A)(C/T)N(A/T)T(A/G) starting at position -5. It was concluded that vaccinia virus early promoters may be characterized by an A + T-rich region of approximately 45 nucleotides preceding the RNA start site and include a specific 3'-terminal sequence of 28 nucleotides containing at least ten bits of information. A procedure for localizing putative early RNA start sites in nucleotide sequences is proposed.

Base Sequence↗

PubMed1998

Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes.

A series of modular mini-transposon derivatives which permit the rapid cloning and mapping of the DNA flanking the minitransposon's site of insertion has been developed. The basic plasposon, named TnMod, consists of the Tn5 inverted repeats, a conditional origin of replication, rare restriction endonuclease multiple cloning sites, and exchangeable antibiotic resistance cassettes. The broad host range and low target DNA sequence specificity of the Tn5 transposase, in combination with the flexibility afforded by the modular arrangement of TnMod, result in a versatile tool for the mapping of insertional mutations and the rapid recovery of clones from gram-negative bacteria.

DNA Transposable Elements↗

PubMed1994

Cosmid cloning and restriction endonuclease mapping of the herpesvirus of turkeys (HVT) genome.

The genomic libraries of the herpesvirus of turkeys (HVT) presented to date do not include the entire genome. To construct a complete genomic library, HVT DNA was partially digested with Sau3A and inserted into the double cos site vector pcos2EMBL. The PstI maps derived from the partial Sau3A library demonstrate that the left region of the genome compared to the right area is overrepresented. Similar to the libraries of the HVT genome established earlier, a defined portion of the middle genomic region, however, is not contained in the partial Sau3A library. Cloning of HVT DNA in pcos2EMBL, employing BamHI for the partial digestion step, enabled to find the recombinant cosmid cBL267 which carries the BamHI-C and -D fragment as DNA insertion. As shown by Southern blot hybridization, the BamHI-C fragment ranges in a size to close the gap in the partial Sau3A library and thus guarantees the completeness of the genomic library of HVT which consists of seven overlapping cosmid clones (cBL1, cBL328, cBL11, cBL267, cBL27, cBL33, and cBL34).

Animals↗