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PubMed1991

[New mutagen, glycidyl methacrylate (GMA) changed restriction enzyme map of plasmid].

Our present work showed that the mutant TcS ApR of plasmid pBR322 induced by GMA changed restriction enzyme map in the single and double enzyme digestions. Results indicated that the mutant plasmid has lots of mutated sites which are disappeared on some sites and appeared on others in resistance gene region, for example, sites of Bg I, EcoRI, HindIII and HincII, and occurred a new recognition site for HincII (252). The relationship between gene mutation and genotoxicity of GMA was discussed.

Animals↗

PubMed1978

Regulation of gene expression by site-specific inversion.

A site-specific inversion event is responsible for phase transition in Salmonella, as indicated by heteroduplex analysis of recombinant molecules carrying the gene coding for H2 flagellin in Salmonella. The inversion region corresponds to approximately 800 base pairs in length, and the inversion process does not appear to be dependent upon the E. coli RecA recombination pathway. Specific deletion derivatives of the cloned fragments no longer produce H2-specific flagella, effectively mapping the H2 gene within about 300 bp of the inversion region. Recombinant products of the hybrid molecules arose spontaneously, and they were used in the mapping of restriction sites within the inversion region. The restriction maps further demonstrate the extent and nature of the inversion.

Bacterial Proteins↗

PubMed1992

A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation.

We previously developed a cladistic approach to identify subsets of haplotypes defined by restriction endonuclease mapping or DNA sequencing that are associated with significant phenotypic deviations. Our approach was limited to segments of DNA in which little recombination occurs. In such cases, a cladogram can be constructed from the restriction site or sequence data that represents the evolutionary steps that interrelate the observed haplotypes. The cladogram is used to define a nested statistical design to identify mutational steps associated with significant phenotypic deviations. The central assumption behind this strategy is that any undetected mutation causing a phenotypic effect is embedded within the same evolutionary history that is represented by the cladogram. The power of this approach depends upon the confidence one has in the particular cladogram used to draw inferences. In this paper, we present a strategy for estimating the set of cladograms that are consistent with a particular sample of either restriction site or nucleotide sequence data and that includes the possibility of recombination. We first evaluate the limits of parsimony in constructing cladograms. Once these limits have been determined, we construct the set of parsimonious and nonparsimonious cladograms that is consistent with these limits. Our estimation procedure also identifies haplotypes that are candidates for being products of recombination. If recombination is extensive, our algorithm subdivides the DNA region into two or more subsections, each having little or no internal recombination. We apply this estimation procedure to three data sets to illustrate varying degrees of cladogram ambiguity and recombination.

Alcohol Dehydrogenase↗

PubMed1982

Promoter-distal region of the tra operon of F-like sex factor R100 in Escherichia coli K-12.

The distal region of the tra (transfer) operon of F-like plasmid R100 was investigated, using small plasmids derived from R100, primarily the plasmid pSM6. The transposon Tn5 (which confers kanamycin resistance) was inserted at different positions into pSM6, and the transposition derivatives were tested for ability to complement defined tra mutants of the F sex factor. Thus, the tra genes traH, G, T, and D were localized on the plasmid R100. A restriction map of pSM6 was constructed, and the locations of the insertions were mapped, using restriction endonuclease digestion of the plasmid DNA and exploiting the fact that several restriction sites are localized in the inverted repeat regions of the transposon. The gene products of the genes traG, S, T, and D were identified by radioactive labeling of proteins synthesized in minicells carrying the various insertion plasmids followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of another transfer gene, traI, was inferred from these data. Another protein, the r2-A protein, was also identified, and its gene was mapped. On the basis of the data, a best-fit physical map of this region of the tra operon of R100 was constructed. The results confirmed that the general order and size of the distal transfer genes is as in the F sex factor, but showed that differences exist with respect to all of the gene products. The significance of these differences are discussed in the light of the genetic and physical homology (Manning et al., J. Bacteriol. 150:76-88) of the transfer regions.

Bacterial Proteins↗

PubMed1995

The genetic and molecular organization of the Dopa decarboxylase gene cluster of Drosophila melanogaster.

We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis screens recovered 77 new Df(2L)TW130 recessive lethal mutations. These new alleles combined with 263 previously isolated mutations in the cluster to define 18 essential genes. In addition, seven new deficiencies were isolated and characterized. Deficiency mapping, restriction fragment length polymorphism (RFLP) analysis and P-element-mediated germline transformation experiments determined the gene order for all 18 loci. Genomic and cDNA restriction endonuclease mapping, Northern blot analysis and DNA sequencing provided information on exact gene location, mRNA size and transcriptional direction for most of these loci. In addition, this analysis identified two transcription units that had not previously been identified by extensive mutagenesis screening. Most of the loci are contained within two dense subclusters. We discuss the effectiveness of mutagens and strategies used in our screens, the variable mutability of loci within the genome of Drosophila melanogaster, the cytological and molecular organization of the Ddc gene cluster, the validity of the one band-one gene hypothesis and a possible purpose for the clustering of genes in the Ddc region.

Alleles↗

PubMed1983

Restriction endonuclease mapping of globin genes in beta-thalassaemia.

We have used the restriction endonuclease mapping technique to analyse the globin genes in local cases of beta-thalassaemia major. No gross deletions or rearrangements of the beta-globin gene locus were detected. In one individual the point mutation within the beta-globin gene which gives rise to a beta(0)-thalassaemia allele was detected by use of a restriction enzyme which cuts the gene at this site. Analysis of restriction fragment length polymorphisms at the beta-globin locus showed that antenatal diagnosis of the disorder could be accomplished in an indigenous family with thalassaemia. The application of the gene-mapping technique to the diagnosis of sickle cell anaemia and alpha-thalassaemia is also discussed.

Anemia, Sickle Cell↗

PubMed1979

Restriction cleavage map of SP01 DNA: general location of early, middle, and late genes.

A detailed restriction endonuclease map for the genome of Bacillus subtilis phage SP01 is presented. Sites of cleavage for the restriction enzymes BglII, EcoRI, HaeIII, and SalI were determined. This physical map showed that SP01 DNA was 140 kilobases in length and contained a repeated sequence of 12.4 kilobases at its termini. Combined with previously published information, we were also able to identify the general locations of genes expressed at early, middle, or late times in the phage lytic cycle. In particular, early genes were largely clustered in the terminal repeats, whereas a major cluster of late genes was located in the left-central portion of the genome.

Bacillus subtilis↗

PubMed1986

Charomids: cosmid vectors for efficient cloning and mapping of large or small restriction fragments.

Charomids are cosmid vectors up to 52 kilobases (kb) long, bearing 1-23 copies of a 2-kb spacer fragment linked in head-to-tail tandem arrays. Like cosmids and lambda phage, charomids can be packaged in vitro for efficient introduction into bacteria. Charomids contain a polylinker with nine unique restriction sites for cloning and can be used without preparing vector arms. Using a charomid of appropriate size, one can clone inserts of any size up to 45 kb. For example, charomid 9-36 (9 cloning sites, 36 kb long) is too small to be packaged efficiently without an insert and can be used to clone fragments of 2-16 kb. The structure of charomids facilitates restriction mapping of the insert DNA and, after cloning, all the spacer fragments can be removed easily. After enrichment by size fractionation in an agarose gel, a specific single-copy genomic sequence can be cloned rapidly from approximately 3 micrograms of DNA. Using charomid 9-36, we have cloned and mapped an amplified novel DNA fragment from a cell line resistant to N-(phosphonoacetyl)-L-aspartate and carrying about 100 copies of the CAD (carbamoyl-phosphate synthetase/aspartate carbamoyltransferase/dihydroorotase) gene. The fragment lies at the center of an inverted duplication of this gene.

Base Sequence↗

PubMed1985

Updating rDNA restriction enzyme maps of Tetrahymena reveals four new intron-containing species.

The extrachromosomal rDNA molecules from a number of Tetrahymena strains were characterized by restriction enzyme mapping using three different restriction enzymes combined with gel blotting and hybridization analysis. Strains from four out of six recently described species were found to contain an intron in the 26s rRNA coding region. The evolutionary relationship among the species of the T. pyriformis complex was examined on the basis of the rDNA maps with emphasis on similarities between two of the new species and the widely studied T. thermophila and T. pigmentosa. Examination of a large number of T. pigmentosa strains showed this species to exhibit an unusual polymorphism with respect to its rDNA. It is suggested that recombinational cross-over events play a role in the formation of new rDNA alleles in this species.

Alleles↗

PubMed1983

Dimethyl sulfoxide affects the amount of extrachromosomal spleen focus-forming virus DNA in murine erythroleukemia cells.

We used Southern blot hybridization to titrate and map restriction enzyme cleavage sites of a 6.3-kilobase-pair species of extrachromosomal viral DNA found in derivatives of the 745A line of murine erythroleukemia cells, which vary in their ability to be induced to differentiate by dimethyl sulfoxide (DMSO). Greater than an eightfold variation was observed in the amount of this DNA, with the largest amounts being found in cells that were resistant to the induction of differentiation by DMSO. This increase in the level of extrachromosomal viral DNA was found to be dependent upon the continued presence of DMSO in the culture medium. The increase was shown not to be due to an immediate stimulatory effect of this agent on the synthesis or maintenance of this DNA, since cell lines sensitive to the differentiation-inducing effects of DMSO were shown to undergo a transient reduction in the amount of extrachromosomal viral DNA after the addition of DMSO to the culture medium. In addition to the 6.3-kilobase-pair linear form found in the cytoplasm, in some preparations two hybridizing bands were observed that migrated in agarose gels in the position expected of covalently closed circular species of viral DNA. Restriction enzyme mapping of the cytoplasmic linear form indicated a close relationship of this DNA to two polycythemic strains of spleen focus-forming virus that have been molecularly cloned by other workers. No obvious change in the number or arrangement of chromosomal viral sequences could be detected after treating cells with DMSO. Thus, the exposure of murine erythroleukemia cells to DMSO caused an obvious change in the amount of extrachromosomal spleen focus-forming virus DNA but no obvious change in the integration of the provirus.

Animals↗

PubMed1996

1.8-megabases fine physical map encompassing IFNAR and AML1 loci on human chromosome 21q22.1.

A long-range restriction map of the 1.8-megabases (mb) region encompassing the area between the interferon-alpha receptor and the acute myelogenous leukemia loci on human chromosome 21q22.1 was constructed after analysis of both the contiguous yeast artificial chromosome (YAC) clones and genomic DNA. Analysis of pulsed-field gel electrophoresis of lymphoblastoid DNA digested with three rare-cutting enzymes, Not I, Mlu I, and Nru I, revealed the positions of 17 markers on each restriction map. The 1.8-mb YAC contig that covers this region was obtained through YAC walking mediated by sequence-tagged sites (STSs), with 29 STSs including 12 newly generated YAC end-specific STSs. The consensus restriction map from 15 overlapping YACs and the positioning of the STS markers on each clone allowed 24 markers including 4 Not I-linking STSs to be ordered and mapped physically. Comparison of the maps revealed that the proximal region contains more unmethylated CpG islands than the distal region, which suggests that many expressed genes are in the proximal region. This fine consensus physical map will be informative and useful for construction of contigs of cosmid, P1, or BAC clones for further large-scale sequencing in this gene-rich region.

Base Sequence↗

PubMed1986

Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functions.

The conjugative plasmid pCF-10 (58 kb) of Streptococcus faecalis has been mapped with restriction enzymes. By restriction mapping and Southern hybridization analysis, a 16-kb segment of the plasmid was shown to resemble closely the conjugative tetracycline resistance transposon, Tn916. Mutagenesis of the plasmid with the erythromycin resistance transposon Tn917 was used to localize a tetracycline resistance determinant and several regions involved in conjugal transfer. Fifty Tn917 insertions (outside the region of the plasmid homologous to Tn916) affecting mating behavior and the ability of donor cells to respond to the sex pheromone cCF-10 were mapped to nine distinct segments, or tra regions. Insertions into tra regions 1-3 and 7-9 led to an enhanced transfer ability of mutant plasmids relative to the transfer frequency obtained for the wild-type plasmid. Cells carrying these mutant plasmids differed in colony morphology or growth in broth culture from cells carrying pCF-10. Insertions into tra regions 4-6 resulted in reduced plasmid transfer, or completely eliminated the mating potential of donor cells. Insertions generating transfer-defective plasmids could be grouped further according to the ability of strains harboring the mutant plasmids to respond to cCF-10. HindIII fragments of pCF-10 coding for transfer functions have been cloned into Escherichia coli.

Conjugation, Genetic↗

PubMed1982

Restriction fragment maps of the genome of Bacillus subtilis bacteriophage SP beta.

Six different restriction endonucleases were used to generate restriction fragment maps of the genome of the temperate Bacillus subtilis phage SP beta. AvaI and SalI each had six target sites in the phage DNA, AvaII had three, BamHI had seven, PstI had twenty, and SacI had sixteen. Restriction analysis and heteroduplex analysis were used to locate a 10-kb region of DNA that is deleted in the clear-plaque mutant, SP beta cl. The deletion lay approx. 50 kb from the left end of the 126-kb phage genome.

Bacillus subtilis↗

PubMed1981