Rapid characterization of Mycobacterium fortuitum-chelonei complex by restriction fragment length polymorphism of ribosomal RNA genes

Using labelled, gamma-32P rRNA of mycobacteria as a probe restriction fragment length polymorphism (RFLP) of rRNA genes of strains belonging to the Mycobacterium fortuitum-chelonei complex was analysed. Each DNA sample was cleaved with EcoRI restriction endonuclease, the fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose membrane. Fragments of DNA containing rRNA genes were identified by hybridization with gamma-32P-labelled rRNA. Patterns were found to be species specific and both the species were distinguishable from each other. Results indicate that this approach can be used for rapid genomic characterization of the Mycobacterium fortuitum-chelonei complex.     

Restriction endonuclease recognition and Southern hybridization of bromodeoxyuridine-substituted genomic DNA

Abstract. Human glioma cell lines exposed to various concentrations of bromode-oxyuridine (BrdUrd) were studied to determine the effect of BrdUrd substitution on restriction endonuclease recognition and Southern hybridization of genomic DNA. BrdUrd substitution had no effect on the recognition of restriction endonucleases. When the exposure to BrdUrd was 2 h or less and the BrdUrd substitution rate was less than 40%, there was no difference in the density of hybridized bands after Southern hybridization using human non-recombinant complementary DNA as a probe. Hybridization was suppressed significantly by exposures longer than 24 h or BrdUrd substitution rates greater than 40%. These results suggest that the BrdUrd substitution rate and the exposure time to BrdUrd influence the hybridization reaction by a DNA probe. Brief exposure (up to 2 h) to BrdUrd does not influence restriction endonuclease recognition or Southern hybridization of genomic DNA.     

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.     

Physical analysis and mapping of the Mycoplasma pneumoniae chromosome.

Field inversion gel electrophoresis was used for analysis of the chromosome of Mycoplasma pneumoniae. The restriction endonuclease SfiI (5'-GGCCNNNNNGGCC-3') generated 2 M. pneumoniae DNA fragments of approximately 437 and 357.5 kilobase pairs (kbp), whereas 13 restriction fragments ranging in size from 2.4 to 252.0 kbp resulted from digestion with ApaI (5'-GGGCCC-3'). Totaling the sizes of the individual restriction fragments from digestion with SfiI or ApaI yielded a genome size of 794.5 or 775.4 kbp, respectively. A physical map of the M. pneumoniae chromosome was constructed by using a combination of techniques that included analysis by sequential or partial restriction endonuclease digestions and use as hybridization probes of cloned M. pneumoniae DNA containing ApaI sites and hence overlapping adjacent ApaI fragments. Genetic loci for deoC, rrn, hmw3, and the P1 gene were identified by using cloned DNA to probe ApaI restriction fragment profiles.     

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization.

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.     

The isolation and partial sequencing of human bone alkaline phosphatase gene

1. A charon 4A human fetal liver genomic library was screened for human alkaline phosphatase sequences using the cloned human bone cDNA as a hybridization probe. 2. A positive clone was obtained and then characterized by restriction endonuclease cleavage analysis, hybridization experiments and partial DNA sequencing.     

A nonradioactive DNA methyltransferase assay adaptable to high-throughput screening

We have developed a nonradioactive assay method for DNA methyltransferases based on the ability to protect substrate DNA from restriction. DNA immobilized to a microplate well was treated sequentially with methyltransferase and an appropriate endonuclease. The amount of methylated DNA product is reflected by a proportional decrease in endonuclease cleavage, which is in turn reflected by increased retention of the end-labeled affinity probe. A single universal substrate was designed to assay multiple methyltransferases including those that do not have a cognate endonuclease. The methodology developed is suited to screen a large number of compounds for inhibitors of various methyltransferases.     

Common physical map of four Brucella bacteriophage genomes

Abstract DNAs isolated from four strains of Brucella bacteriophages were studied by restriction endonuclease mapping and Southern blot analysis. In all strains the genome was composed of a 38 kb (25.1 × 10⁶ dalton) double-stranded circular DNA. The physical map was the same for the four genomes and Southern blot hybridization of restriction endonuclease fragments with the Tbilissi strain DNA as a probe showed complete homology between the four DNAs. Thus, the four phage strains appear to be identical, the specific host range of each originating from minor changes in phage or Brucella receptors or both.     

TWO NEUTRALITY TESTS OF Y-LINKED RDNA VARIATION IN DROSOPHILA MELANOGASTER

The evolutionary significance of Y-chromosomal ribosomal DNA sequence variation was tested by two different means. A single sample of males and females was collected from a peach orchard in central Pennsylvania. Wild-caught males and sons of wild-caught, wild-inseminated females were crossed to virgin females having an X-linked rDNA deficiency. Genomic DNA from male progeny of these crosses was extracted and digested with the single restriction endonuclease, DraI. Southern blots of these digestions, when probed with the complete rDNA probe, revealed 10 distinct patterns of restriction fragments. A chisquare test for the homogeneity of the frequency distributions of the sample of wild males and sons of wild females failed to reject a neutral null hypothesis. The allele frequency configuration was tested with the Ewens-Watterson test, and the departure from the infinite alleles neutral model was not significant. Simulations were performed to test the sensitivity of the tests to misclassification and to quantify the power of the two tests.     

ApoA-I related DNA polymorphism in humans with coronary heart disease

Summary A genetic analysis of atherosclerotic patients as well as healthy subjects using an apoA-I gene specific probe confirmed that an EcoRI restriction fragment length polymorphism is related to the development of atherosclerosis. Three subjects with severe coronary heart disease were found to be homozygous for a 6.5 kb fragment hybridizing to the apoA-I probe. In the atherosclerotic patient group 44% were heterozygous for this fragment, compared to 9.5% in the control group. The distribution of genotypes in the atherosclerotic and control groups was signficantly different. Among the heterozygous subjects, specific differences were found after digestion of their DNA with Bam HI restriction endonuclease.     

Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA)

We have developed a new method for identifying specific single- or double-stranded DNA sequences called nicking endonuclease signal amplification (NESA). A probe and target DNA anneal to create a restriction site that is recognized by a strand-specific endonuclease that cleaves the probe into two pieces leaving the target DNA intact. The target DNA can then act as a template for fresh probe and the process of hybridization, cleavage and dissociation repeats. Laser-induced fluorescence coupled with capillary electrophoresis was used to measure the probe cleavage products. The reaction is rapid; full cleavage of probe occurs within one minute under ideal conditions. The reaction is specific since it requires complete complementarity between the oligonucleotide and the template at the restriction site and sufficient complementarity overall to allow hybridization. We show that both Bacillus subtilis and B. anthracis genomic DNA can be detected and specifically differentiated from DNA of other Bacillus species. When combined with multiple displacement amplification, detection of a single copy target from less than 30 cfu is possible. This method should be applicable whenever there is a requirement to detect a specific DNA sequence. Other applications include SNP analysis and genotyping. The reaction is inherently simple to multiplex and is amenable to automation.     

Isolation and molecular identification of Leishmania chagasi from a bat (Carollia perspicillata) in northeastern Venezuela

This report describes the isolation of a Leishmania chagasi strain from a bat (Carollia perspicillata), and its identification using biological methods and molecular characterization. The parasites were isolated in an artificial culture medium from a blood sample extracted from a bat heart. The isolate was then inoculated into the footpads of Balb/c mice, which subsequently developed a typical nodular leishmanial lesion; the parasites were confirmed as Leishmania by smear and histopathology. Molecular characterization of the parasites was performed by polymerase chain reaction with species-specific primers, kDNA restriction pattern following Hae III endonuclease digestion and dot blot hybridization using a kDNA probe. This report demonstrates that bats can be hosts for L. chagasi species and suggests the need for studies to determine whether they may be involved in foci of visceral leishmaniasis.     

High capacity gel preparative electrophoresis for purification of fragments of genomic DNA

We have designed a high-capacity gel electrophoretic device for the purification of large amounts of restriction endonuclease fragments of genomic DNA. This device exploits the high resolution of gel electrophoresis in conjunction with an electronic system permitting discontinuous sample elution over a large gel surface area. This feature preserves resolution and greatly increases capacity and yield. The resulting DNA fractions may be used in restriction endonuclease, ligation, transfection, and transformation reactions without further extensive manipulation. Furthermore, DNA fragments of a broad size range are recovered with high efficiency from the gel.     

Identification of Atlantic salmon × brown trout hybrids with a nuclear marker useful for evolutionary studies

A new nuclear DNA marker is described to identify Atlantic salmon, brown trout, and their hybrids. Species-specific banding patterns are obtained by digestion of genomic DNA with the restriction endonuclease Sac I and hybridization with a histone DNA probe. FISH analyses in hybrid metaphases illustrate the potential of interspecific hybrids for evolutionary studies.     

Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin.

Cryptosporidium parvum oocysts isolated from different hosts and geographical areas were compared by restriction endonuclease analysis of repetitive DNA: Iowa (bovine), Florida (bovine), New York (bovine), Peru (human), Brazil (human), and Mexico (human). Southern blot hybridization analysis was performed using the restriction endonuclease enzyme Eco RI and the DNA probe pV47-2. The probe hybridized with 18 bands present in all the isolates. The Brazilian, Mexican, and Peruvian human isolates had an additional common band of 4.3 kbp that was absent in the bovine isolates. Two extra bands of 14 and 12 kbp were present in the Brazilian isolate whereas the Mexican isolate had an extra band of 14 kbp. When the Iowa and Peru C. parvum isolates were passed twice through calves, oocysts recovered from both passages showed identical banding patterns, suggesting that recombination of the repetitive sequences was not altered during sexual reproduction. The DNA digested with other restriction endonucleases were tested confirming differences between isolates. A genomic DNA library is currently being produced to better define isolate variation in C. parvum.     

Genomic arrangement of repeated PS700 elements in the nematode Panagrellus silusiae

When genomic DNA from the free-living nematode Panagrellus silusiae is digested with the restriction endonuclease BamHI and separated by electrophoresis, a band in the 700 base pair size range is evident after ethidium bromide staining. One of the 0.7-kilobase fragments (PS700-1) was characterized and found to be a member of a moderately repetitive DNA family (T. Warren and J.J. Pasternak. 1988. Nucleic Acids Res. 16: 10,833-10,847). In the current study, DNA sequence analyses of three independently isolated copies of the PS700 DNA family showed the same nucleotide sequence and greater than 98% similarity to PS700-1. Four EMBL-4 bacteriophage clones were isolated from a Panagrellus genomic DNA library with PS700-1 as the probe and were analyzed by restriction endonuclease site mapping and Southern blot DNA hybridization. These clones contain 31 copies of the PS700 DNA family. In each case, the units are arranged in head-to-tail arrays. One of the EMBL-4 clones contains copies of a novel variant of the PS700 elements. The maintenance of both nucleotide sequence and restriction endonuclease restriction site homogeneity among members of the dispersed PS700 DNA family may denote a functional role for these sequences.     

Differential organization of a LINE-1 family in Indian pygmy field mice

Southern blot hybridization analysis of genomic DNAs digested with restriction endonuclease EcoR I and Ava II from Mus musculus domesticus, Mus booduga and Mus terricolor with a cloned repetitive DNA fragment of Mus booduga as a probe showed difference in restriction pattern of this DNA in these three species. Further Southern analysis of the BamH I digested genomic DNAs from these species hybridized with cloned DNA fragment as a probe and sequencing of the cloned DNA revealed that this 252 bp cloned DNA fragment is a part of BamHI repeat element of genus Mus and is 87% homologous to the contiguous portion of the Mus musculus domesticus LINE-1 element. The species specific fragment pattern generated by different restriction endonucleases using this DNA as a probe revealed difference in the organization of LINE-1 repetitive element in the three species of genus Mus.     

新扬州鸡DNA指纹J带与生产性能的相关性研究

以EAV(禽内原性反转录病毒片段)为探针,EcoRI为限制性内切酶,对新扬州鸡DNA指纹图谱进行了的研究。结果表明：DNA指纹J带在新扬州鸡上存在,并对新扬州鸡早期增重具有减效效应,可作为新扬州鸡早期增重的遗传标记进行标记辅助选择。     

Existence of<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> genomic fragment hybridizing with the<Emphasis Type="Italic">nifM</Emphasis> gene of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A novel finding that genomic restriction fragments of symbiotic nitrogen fixer S. meliloti hybridized with nifM gene probe of the free-living diazotroph Klebsiella pneumoniae is reported. When SmaI endonuclease was used to digest S. meliloti DNA, a unique hybridizing band was obtained.     

New procedure using a psoralen derivative for analysis of nucleosome associated DNA sequences in chromatin of living cells.

Sites for restriction endonuclease cleavage in double helical DNA are blocked from cleavage when the photoaffinity drug trimethylpsoralen is photobound at or near the site. In general, Hind III sites are about 15 fold more sensitive to inactivation than the other restriction sites which were tested, although sensitivity of different Hind III sites seems to vary somewhat depending on base sequences adjacent to the site. Hind III sites can be inactivated in two ways; one which completely blocks action of the specific restriction endonuclease and one permitting the introduction of a swivel which relaxes DNA supercoiling without producing a double strand break. Nucleosomes and perhaps other protein-DNA complexes can protect the underlying DNA sequence from trimethylpsoralen photobinding and thus protect restriction sites from inactivation. This property can be exploited to determine if specific sites are accessible to the psoralon probe in vivo and thus to establish if specific nucleotide sequences are nucleosome associated. Using this procedure evidence is obtained that nucleosomes on SV40 DNA in living infected cells are either distributed randomly or at many discrete alternate sites that approach a random distribution.