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PubMed1986

A 'Southern Cross' method for the analysis of genome organization and the localization of transcription units.

A 'Southern Cross' hybridization method is described which permits the rapid restriction mapping of DNA molecules, up to 40 kb in size, for at least ten enzymes in a single operation. The procedure allows the full set of 32P-end-labelled fragments derived from one restriction enzyme digest to intersect and attempt to hybridize to the gel-separated fragments of as many as ten unlabelled digests immobilized on parallel sheets of filter paper. A two-dimensional array of hybridization spots is revealed on each recipient paper, indicating which radioactive and non-radioactive DNA fragments have sequences in common. A restriction map can then be directly and simply deduced from the matrix of hybridization spots in each cross-blot. The method affords advantages over other procedures for obtaining restriction maps in terms of the time required, the number of restriction enzymes that can be mapped, and the potential for eliminating ambiguity. It is also sufficiently sensitive to detect DNA rearrangements and restriction-site polymorphisms in moderately complex genomes. Furthermore, the procedure is applicable to other aspects of the study of genome organization: for example, the exon and intron areas of a segment of cloned genomic DNA can be identified by cross-hybridizing a set of radioactive restriction fragments from the genomic clone against immobilized RNA from a cell type of interest.

DNA Restriction Enzymes↗
PubMed2002

[Cloning and identification of human tissue-type plasminogen activator gene].

OBJECTIVE: To acquire the gene fragments coding for human tissue-type plasminogen activator (ht-PA). METHOD: Total RNA was extracted from Bowes Melanoma cell line and reverse transcriptase-"touchdown" PCR was employed to amplify ht-PA cDNA that underwent digestion with the restriction enzymes and was subsequently cloned into vector pSP72 for transforming E.coli TG1. The cloned fragment was subjected to restriction mapping and sequence analysis for confirmation. RESULT: The restriction maps of the selected clones were consistent with those generated by computer-based analysis and was identical with that of reported ht-PA cDNA. CONCLUSION: The gene fragment encoding ht-PA was successfully cloned, which lays a foundation for further study and artificial modification of this gene.

Base Sequence↗
PubMed1993

Physical linkage of the murine Hox-b cluster and nerve growth factor receptor on yeast artificial chromosomes.

Evidence derived from sequence comparisons and the genomic organization of the murine Antennapedia-class homeobox gene clusters suggest that they arose from a primordial cluster through a process of gene duplication and divergence followed by cluster duplication. A large chromosomal domain surrounding the ancestral homeobox cluster also appears to have been duplicated and has remained relatively stable since the divergence of humans and rodents. To test the extent of the duplicated chromosomal domain, we have initiated physical mapping studies of the regions surrounding the four murine homeobox clusters using pulsed-field gel electrophoresis and yeast artificial chromosome cloning. In this study, we present a long-range restriction map of mouse chromosome 11 spanning 1500 kb in the region surrounding the Hox-b cluster. We have determined that the gene for the nerve growth factor receptor is tightly linked to the Hox-b complex and is located within 50 kb of the Hox-b 1 gene at the 3' end of the cluster. Four yeast artificial chromosomes have been isolated and characterized by the polymerase chain reaction, long-range restriction mapping, and Southern blotting. Two clones of 150 and 300 kb contain the entire Hox-b cluster and the nerve growth factor receptor gene. A 440-kb clone contains the 3' end of the Hox-b cluster, the nerve growth factor receptor gene, and extends downstream. A 210-kb clone contains the 5' end of the Hox-b cluster and extends upstream. These clones confirm the pulsed-field restriction map of uncloned mouse DNA and represent a contig of approximately 600 kb of cloned material from mouse chromosomes 11.

Animals↗
PubMed1980

Physical map of Caulobacter crescentus bacteriophage phi Cd1 DNA.

A restriction map of the Caulobacter crescentus bacteriophage phi Cd1 genome was constructed by using the restriction endonucleases HindIII and HpaI. A total of 12 fragments, ranging in molecular weight from 7.7 X 10(6) to 0.25 X 10(6), were produced by HindIII, and 7 fragments, ranging in molecular weight from 9.0 X 10(6) to 0.24 X 10(6), were generated by HpaI. The molecular weight of the genome was estimated to be approximately 28.8 X 10(6) on the basis of the relative electrophoretic mobilities of the restriction fragments. The relative order of the cleavage fragments was determined by specific cleavage of isolated restriction fragments, terminal labeling of both the whole genome and isolated fragments, and hybridization of isolated fragments to restriction fragments generated by other restriction enzymes. The genome of phi Cd1 was found to be terminally repetitive, and analysis of previously determined in vivo and in vitro RNA transcripts showed that the restriction map could be oriented such that transcription began on the left and proceeded to the right end of the genome.

Bacteriophages↗
PubMed1988

MIC2: a human pseudoautosomal gene.

MIC2 and XGR are the only known pseudoautosomal genes in man. MIC2 encodes the 12E7 antigen, a human cell-surface molecule of unknown function. XGR regulates, in cis, the expression of the XG and MIC2 genes. DNA probes derived from the MIC2 locus have been used in the construction of a meiotic map of the pseudoautosomal region and a long range restriction map into the X- and Y-specific chromosome domains. MIC2 is the most proximal marker in the pseudoautosomal region and recombination between the sex chromsomes only rarely includes the MIC2 locus. Our long-range restriction maps and chromosome walking experiments have localized the pseudoautosomal boundary within 40 kilobases adjacent to the 3' end of the MIC2 gene. The same maps have been used to predict the chromosomal location of TDF.

Amino Acid Sequence↗
PubMed1987

Cosmid mapping of the human chorionic gonadotropin beta subunit genes by field-inversion gel electrophoresis.

A cosmid clone containing the entire hCG beta gene cluster has been isolated. The restriction map of this clone has been determined by an indirect-end-label FIGE (field inversion gel electrophoresis) method. Analysis of this cosmid clone shows that there are 6 hCG beta genes in human genomic DNA. A previously uncloned portion of the hCG beta cluster, termed the "gap" region, has been shown not to contain any sequences homologous to the hCG beta cDNA. The restriction mapping method employed in this study takes advantage of the superior resolution of FIGE for high molecular weight DNA fragments in the size range 15-50 kb. This method is broadly applicable and permits rapid and accurate restriction mapping for extended regions of genomic DNA that have been cloned into cosmid or lambda vectors.

Chorionic Gonadotropin↗
PubMed1979

Restriction endonuclease mapping of the human gamma globin gene loci.

The restriction endonuclease sites in and around the human gamma globin gene loci have been mapped using the gel blotting technique of Southern, in both normal DNA and DNA from an individual with hereditary persistence of fetal hemoglobin (HPFH). In normal DNA, the gamma genes are linked to the delta (and beta) globin genes, and the orientation of these genes with respect to transcription is (5') G gamma leads to A gamma leads to delta leads to beta (3'). The distance between the G gamma and A gamma genes is 3.5 kb and that between the A gamma and delta genes is 16 kb. In both normal DNA and HPFH DNA, the gamma genes are interrupted by an intervening sequence, approximately 1 kb in length that is situated between codon positions 99 and 121 of the coding sequence. In different DNA samples, there is polymorphism for the presence or absence of a Hind III site in the intervening sequence of either gamma golbin gene. In HPFH DNA, a deletion of at least 16 kb of DNA has been detected. This deletion starts at a point approximately 12.5 kb from the 3'-end of A gamma gene and extends through the delta and beta globin genes to a point at least 3 kb beyond the 3'-end of beta globin gene.

Base Sequence↗
PubMed1982

Restriction endonuclease mapping of the proviral DNA of the exogenous RIII murine mammary tumor virus.

Cellular DNA containing integrated murine mammary tumor virus (MuMTV) was isolated from FeI/C6 feline kidney cells and CCL64 mink lung cells infected with milkborne RIII MuMTV. By using restriction enzyme HpaI, intact RIII MuMTV provirus (length, 8.7 kilobases [kb]) was excised from the cellular DNA. Subsequent restriction endonuclease analysis of this HpaI fragment with KpnI, HindIII, EcoRI, BamHI, BglII, PstI, SstI, SalI, and XhoI enabled us to construct a map of the RIII virus genome. A comparison of this map with the maps of the GR and C3H MuMTV's revealed that there are greater sequence differences between the RIII virus and the GR and C3H MuMTV proviruses than there are between the GR and C3H proviruses. The following are features of the restriction map unique to the RIII provirus: the presence of three BamHI and two EcoRI cleavage sites, a HpaI cleavage site in the terminal 3'-5' repeat unit of the provirus, and the absence of an XhoI cleavage site. Another distinguishing feature of the RIII provirus is that the sizes of some of the restriction fragments produced by cleavage of the RIII provirus with PstI are different from the sizes of the fragments obtained by PstI cleavage of the GR and C3H proviruses. Like the GR proviral DNA, the RIII proviral DNA has three SstI (SacI) cleavage sites, whereas the C3H provirus has only two SstI sites. HpaI digestion of MuMTV-infected mink lung cell DNA revealed only one class of provirus (an 8.7-kb fragment); however, we observed several minor classes of RIII proviral DNA in addition to the major class of provirus DNA in infected cat kidney cells. PstI digestion of the HpaI 8.7-kb fragments from both feline and mink cells generated a 3.7-kb DNA fragment identical in size to a PstI-generated fragment that has been found in GR and C3H milkborne virus-infected cells. Although a fragment similar in size to the milkborne 3.7-kb PstI fragment has been found as an endogenous component in many C3H and GR mouse tissues, we did not observe such an endogenous fragment in the RIII mouse strain. Therefore, the 3.7-kb fragment may be useful as a marker for the milkborne RIII MuMTV provirus in RIII mice.

Animals↗
PubMed1979

Restriction endonuclease cleavage map and location of unique features of the DNA of hepatitis B virus, subtype adw2.

DNA of hepatitis B virus (HBV) of hepatitis B surface antigen (HBsAg) subtype adw2 made fully double stranded by the virion DNA polymerase and radiolabeled either by the virion DNA polymerase reaction or by nick-translation with 32P-labeled deoxynucleoside triphosphates was used to establish a map of restriction endonuclease cleavage sites by the method double and triple enzyme digestion and to determine the relative positions of several unique physical features of this DNA. The five restriction sites for enzyme HincII, the two sites each for BamHI, Ava I, and Bgl II, and the single sites for EcoRI, Pst I, Hpa I, and Taq I were positioned relative to each other. Within this map, the single-stranded region in HBV DNA has been localized and the locations of nicks in each strand (a and b) have been determined with respect to restriction sites on the circular map. Comparison of restriction endonuclease cleavage patterns of DNAs of HBV of HBsAg subtypes adw2, ayw3, and adrq+ revealed consistent differences among subtypes and occasional differences within subtypes.

Chromosome Mapping↗
PubMed1985

Evolutionary conservation of genes coding for meta pathway enzymes within TOL plasmids pWW0 and pWW53.

Pseudomonas putida MT53 contains a TOL plasmid, pWW53, that encodes toluene-xylene catabolism. pWW53 is nonconjugative, is about 105 to 110 kilobase pairs (kbp) in size, and differs significantly in its restriction endonuclease digestion pattern and incompatibility group from the archetypal TOL plasmid pWW0. An RP4::pWW53 cointegrate plasmid, pWW53-4, containing about 35 kbp of pWW53 DNA, including the entire catabolic pathway genes, was formed, and a restriction map for KpnI, HindIII, and BamHI was derived. The entire regulated meta pathway genes for the catabolism of m-toluate were cloned into pKT230 from pWW53 on a 17.5-kbp HindIII fragment. The recombinant plasmid supported growth on m-toluate when mobilized into plasmid-free P. putida PaW130. A restriction map of the insert for 10 restriction enzymes was derived, and the locations of xylD, xylL, xylE, xylG, and xylF were determined by subcloning and assaying for their gene products in both Escherichia coli and P. putida hosts. Good induction of the enzymes by m-toluate and m-methylbenzyl alcohol but not by m-xylene was measured in P. putida, but little or no regulation was found in E. coli. The restriction map and the gene order showed strong similarities with published maps of the DNA encoding both the entire meta pathway operon (xylDLEGFJIH) and the regulatory genes xylS and xylR on the archetype TOL plasmid pWW0, suggesting a high degree of conservation in DNA structure for the catabolic operon on the two different plasmids.

Alcohol Oxidoreductases↗
PubMed1993

Characterization and physical mapping of the genome of bacteriophage phi Aa from Actinobacillus actinomycetemcomitans.

The size, configuration and restriction map of Actinobacillus actinomycetemcomitans bacteriophage phi Aa DNA was determined by means of restriction endonuclease analysis. Digestion of the phi Aa DNA with restriction enzymes Hind III, Eco RI and Sal I produced 6, 5, and 4 fragments, respectively. Based upon the sum of the sizes of the restriction fragments of these enzymes, the DNA was estimated to be 47.2 kilobase pairs in length. A restriction map was constructed using Hind III and Sal I. Incubation with exonuclease Bal 31 for increasing lengths of time resulted in progressive hydrolysis of the DNA, as expected for a linear molecule. No sub-molar fragments or diffuse bands were observed in the agarose gels of the restriction endonuclease digests of the phi Aa DNA. Attempts at ligating the ends of the DNA were consistently unsuccessful. Therefore, we found no evidence for cohesive ends, a circular permutation of the genome or for headful packaging mechanism from a concatameric DNA precursor.

Aggregatibacter actinomycetemcomitans↗
PubMed1984

Identification of beta variant hemoglobins by DNA restriction endonuclease mapping.

An alternative method for identifying beta variant hemoglobins is described. Computer analysis of restriction sites was used to predict which beta variants could be detected by DNA mapping. 61 of 217 variants were shown to have changes in restriction fragment patterns which were useful markers for the abnormal hemoglobin. A further 25 could be identified by polyacrylamide electrophoresis. Implications of DNA analysis in diagnosis of variant hemoglobins are discussed.

Base Sequence↗
PubMed1982

Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2.

The promoter-distal region of the tra operon of the F sex factor Escherichia coli K-12 was analyzed, using the chimeric plasmid pRS31, which contains the F EcoRI restriction fragments f17, f19, and f2 cloned into the EcoRI site of pSC101. A series of deletion plasmids of pRS31, extending increasing distances from a site in f17 through f19 and ending in f2, were isolated. These plasmids were examined by heteroduplex analysis with the parent DNA, and a restriction map of this region of DNA was constructed. A series of Tn5 insertion derivatives of pRS31 were also isolated and mapped, using both heteroduplex analysis and restriction mapping. Both the insertion and deletion mutants were tested in minicells for the synthesis of radioactively labeled proteins. This allowed the identification of the individual gene products and mapping of the genes. The result is a saturated physical map of this region of DNA from fragment f17 through to the IS3 insertion sequence near the promoter-distal end of f2.

Bacterial Proteins↗
PubMed1982

Restriction endonuclease mapping of unintegrated proviral DNA of Kirsten murine sarcoma virus.

Linear unintegrated Kirsten murine sarcoma virus DNA synthesized after acute infection of NIH/3T3 cells or by detergent-disrupted virions, was studied by restriction enzyme cleavage and agarose gel electrophoresis. Labelled DNA (cDNA) synthesized by reverse transcription of a virion RNA template was used to detect viral DNA sequences by filter hybridization. The sites of cleavage for eleven enzymes were located on the genome. Hybridization studies using a cDNA probe specific for the 3' end of the genome defined the orientation of the physical map with respect to virion RNA and identified terminally redundant sequences on the genome. Further evidence for terminal redundancy was obtained by restriction mapping of linear and circular viral DNA, where the duplication was shown to be 0.34 x 10(6) (500 base pairs) in length and in a tandem orientation. Circular viral DNA was found predominantly in the nucleus of acutely infected cells and it existed in two major size classes. The larger size class represented an exact circularization of linear DNA (with no other sequence permutation) whilst in the smaller DNA species one of the terminally redundant sequences was absent.

Animals↗
PubMed1995

Construction of a pulsed-field map around D3S3 using partially demethylated DNA.

Heavy methylation of restriction sites in the relevant chromosomal region can be a major problem in the construction of a long-range restriction map. In the region around the locus D3S3 there appeared to be few accessible restriction sites. Therefore, we cultured cells in the presence of 5-azacytidine and used the partially demethylated DNA to construct a relatively detailed restriction map spanning approximately 1 Mb. Using partially demethylated DNA is a recommendable approach when a genome region appears inaccessible for pulsed-field analysis because excessively long restriction fragments are obtained.

Azacitidine↗
PubMed1995

Cloning and restriction endonuclease mapping of herpes simplex virus type-1 strains H129 and +GC.

EcoRI fragments of herpes simplex virus I (HSV-1) strains H129 and +GC were cloned and the EcoRI and BglII restriction enzyme sites were mapped. Comparison of these enzyme sites with the sequence of HSV-1 strain 17syn+ demonstrated that all EcoRI sites were identical. For H129, the BglII sites were also found to match strain 17syn+ BglII sites. With one exception, the BglII sites in strain +GC also aligned with the strain 17syn+ sequence. The one exception was a missing BglII site from strain +GC located between bases 25,149 and 25,154 in the EcoRI D fragment within the viral deoxyribonuclease gene (UL12). The BglII site represents the first difference to be mapped within HSV-1 strains H129 and +GC which have unique pathobiological properties in animal models of acute and reactivated infections.

Cloning, Molecular↗