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PubMed1990

Definition of the limits of the Wilms tumor locus on human chromosome 11p13.

In a previous report, we described a contiguous restriction map of chromosome band 11p13 that localized the Wilms tumor locus to a small group of NotI fragments. In an effort to identify and isolate the 11p13-associated sporadic Wilms tumor locus, we developed a panel of NotI fragment-specific DNA probes. These probes were selected from genomic libraries constructed using the Chinese hamster ovary-human somatic cell hybrid carrying only human 11p. The libraries were prepared from NotI-digested DNA after size selection by pulsed-field gel electrophoresis. The selected NotI fragments had been previously targeted on the basis of deletion mapping as having a high probability of containing the Wilms tumor locus. We used these newly identified 11p13-specific probes to improve the resolution of the restriction map spanning the Wilms tumor locus. The locus has been defined by a homozygous deletion in a sporadic Wilms tumor. Using these probes, the region of homozygous deletion in this tumor and presumably all or part of the Wilms tumor gene have been confined to two small SfiI fragments spanning less than 350 kb.

Animals↗
PubMed1999

Optical mapping and its potential for large-scale sequencing projects.

Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.

Base Sequence↗
PubMed1994

Non-cloning amplification of specific DNA fragments from whole genomic DNA digests using DNA 'indexers'.

A highly systematic, non-cloning method of distinguishing and isolating every fragment in a class-IIS or interrupted palindrome restriction digest has been developed in our laboratory. These enzymes produce informative, non-identical cohesive ends which can be selectively modified by ligation to individual synthetic oligodeoxyribonucleotides with the corresponding complementary ends. In this way, polymerase chain reaction and sequencing primer sites and labels can be introduced specifically into a single fragment in a total genomic digest. Known and unknown fragments from genomes of the complexity of Escherichia coli can be isolated directly in sequencable form without the necessity of synthesizing unique primers. Human DNA has also been assessed in this way. Problems intrinsic to cloning (selective fragment loss, mutation and sequence rearrangement) are avoided. Systematic characterization of DNA fragments by their cohesive ends and length provides tremendous power and flexibility for analysis of any DNA molecule without specific clones, probes or libraries. We report proof of principle of this remarkable system and indicate potential applications in DNA sequence tagged site and restriction mapping, sequencing, restriction-fragment-length polymorphism analysis and DNA diagnostics.

Base Sequence↗
PubMed1985

Restriction endonuclease mapping of R27 (TP117), an incompatibility group HI subgroup 1 plasmid from Salmonella typhimurium.

A circular map of the IncHI plasmid R27 corresponding to a genome size of 182 kb was established using the restriction endonucleases ApaI, XbaI, and PstI. The map was derived from the results obtained by hybridizing individual ApaI and XbaI fragments to blotted digests of the plasmid, as well as from complete and partial digests. Analysis of a deletion mutant derived by in vitro digestion with PstI and of transfer-defective and tetracycline-sensitive deletion mutants of R27 derived by Tn5 insertion were instrumental in determining the positions of some fragments.

DNA Restriction Enzymes↗
PubMed1994

High-resolution mapping of the N-myc amplicon core domain in neuroblastomas.

The N-myc proto-oncogene is amplified in 25% of neuroblastomas. Amplification is strongly correlated with advanced disease stage and rapid tumor progression. We have constructed a detailed restriction map of the amplified core region in neuroblastomas which will allow the identification of structural features such as joint fragments, rearrangements and CpG islands. Using probes that had been obtained previously, twenty YACs were isolated from a library constructed from a double-minute-containing neuroblastoma cell line with 150-fold amplification of N-myc. Twenty-one YACs also were isolated from two normal human libraries. Normal and neuroblastoma YAC contiguous arrays (contigs), each spanning over 1 Mb of DNA, have been assembled (Molec Cell Biol 12:5563, 1992). A high-resolution restriction map of over 200 kb of contiguous DNA containing N-myc has been generated by subcloning YACs into cosmids. Using cosmids from this region plus additional amplified probes, we have determined that the amplicons from 33 neuroblastomas range in size from 350 kb to over 1 Mb. Rearrangements and deletions were identified in both tumors and cell lines. However joint fragments were not always amplified to the same level as the major amplicon, and may therefore represent a subset of amplicons. We have defined a 130 kb region which was amplified in 32 of the 33 tumors, and one additional tumor had deleted 65 kb of this region from its amplicon. The only CpG island found in this region was within the N-myc gene. Cosmids were screened with radiolabeled total cDNA probes and no highly expressed genes other than N-myc were found in the amplicon.

DNA, Neoplasm↗
PubMed1990

Genetic tagging, cloning, and DNA sequence of the Synechococcus sp. strain PCC 7942 gene (gnd) encoding 6-phosphogluconate dehydrogenase.

A genetic approach was used for the cloning of the Synechococcus sp. strain PCC 7942 (Synechococcus strain R2) gnd gene which encodes 6-phosphogluconate dehydrogenase (6PGD). A restriction map of the gnd locus was prepared by Southern analysis using the Escherichia coli gene as a heterologous probe. The Synechococcus strain R2 gene was genetically tagged by restriction site-specific insertion of the nptII gene of Tn903 into a pUC19 plasmid library of Synechococcus strain R2 chromosomal DNA. Synechococcus strain R2 was transformed with this insertion mutation library, and isolates carrying the gnd::nptII gene were identified as mutants hypersensitive to incubation in the dark. The interrupted gene was cloned from one of the mutants. A plasmid carrying the gnd::nptII gene was reintroduced into Synechococcus strain R2, and kanamycin-resistant transformants were selected. Transformants arising by gene replacement were dark sensitive and missing 6PGD activity. Transformants arising by plasmid insertion were dark resistant and had 6PGD activity. The wild-type gene was then cloned from a transformant containing a plasmid insertion, making use of the restriction map derived from the interrupted gene. Synechococcus strain R2 6PGD was expressed in E. coli when the cloned gnd gene was transcribed from the lacZ promoter resident on the vector. The boundaries of the gene and the direction of transcription were determined from the phenotypes conferred by plasmids carrying deletions entering gnd from either end. The nucleotide sequence was determined. The deduced amino acid sequence of Synechococcus strain R2 6PGD has 56% homology to that of the E. coli K-12 enzyme.

Amino Acid Sequence↗
PubMed2001

Physical mapping of restriction fragment length polymorphism (RFLP) markers in homoeologous groups 1 and 3 chromosomes of wheat by in situ hybridization.

Using wheat ditelosomic lines and in situ hybridization of biotin-labelled DNA probes, 18 restriction fragment length polymorphism (RFLP) markers were physically located on homoeologous groups 1 and 3 chromosomes of wheat. Most of the markers hybridized to chromosome arms in a physical order concordant with the genetic maps. A majority of the markers studied were clustered in non-C-banded, distal euchromatic areas, indicating the presence of recombination hot spots and cold spots in those regions. However, on IBS the markers were well dispersed, which could be due to the abundance of heterochromatin throughout the arm. An inversion between Xpsr653 and Xpsr953 was observed on 1AL. One new Xpsr688 locus, approximately 20-26% from the centromere, was found on 1AS and 1BS. The physical location of Xpsr170 on group 3 chromosomes probably represents an alternative to the loci on the genetic map. Finally, Xpsr313 was mapped to two physical loci on IDL. Five markers were located to bins consistent with the deletion-based physical maps.

Chromosomes↗
PubMed1997

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.

DNA↗
PubMed1989

Molecular mapping of the oncogene MYB and rearrangements in malignant melanoma.

The human cellular oncogene MYB has been mapped to 6q22-q23. Deletions and translocations involving this region of the long arm of chromosome 6 occur frequently in human malignant melanoma, and there are anecdotal reports of MYB gene rearrangements in this cancer. In the current study, Southern blotting and pulsed field gel electrophoresis (PFGE) have been performed to determine whether MYB or its flanking regions are commonly altered in malignant melanoma. Southern blotting failed to document obvious rearrangement of the MYB gene in 15 cases studied. To extend analysis of the MYB region, a long-range restriction map was established by PFGE. This map was then linked to the known restriction map of frequent cutting enzymes. Based on the mapping data and analysis of the MYB region in melanomas, ClaI tissue-specific variation due to methylation was demonstrated. Also, two melanomas (containing alterations in band 6q13) also demonstrated by PFGE a unique restriction fragment for the MYB gene. These results extend significantly the physical map surrounding the MYB locus and provide further evidence for the rearrangement of chromosome 6 in malignant melanoma.

Blotting, Southern↗
PubMed1985

Statistical analysis of molecular genetic data.

Recent advances in molecular biology have made it possible for geneticists to collect data at the DNA sequence level, and so make observations on genes directly, instead of on characters affected by genes. This new nature of genetic data is posing new problems of statistical analyses, as is shown in this paper. After an explanation of the methodology of recombinant DNA studies, separate consideration is given to the statistical analyses of restriction-map and complete-sequence data. The initial observations from restriction-enzyme studies are of the number and sizes of the fragments produced by digesting a DNA region with the enzymes, and it is necessary to infer the location of the breakage points. Model-based analyses are then necessary to infer characteristics of the DNA sequence from the frequency of these break points. An important application of such restriction maps is the detection of human disease genes, with the most notable success to date being the chromosomal allocation of the gene for Huntington's chorea. Complete sequence data present conflicting problems of scale. The amount of information collected per individual is so large that analyses must always be done on a computer, but the number of individuals studied in a population is too low to allow the magnitudes of between- and within-population variation to be assessed. Statistical problems also include the detection and testing of sequence patterns and the comparison of sequences. Any analyses are going to have to incorporate the observed high levels of association between adjacent sequence members. While national data bases now contain about three million sequence elements, very little is known about the error rate in the generation or recording of these sequences.

DNA↗
PubMed1991

Molecular evolution of inversions in Drosophila pseudoobscura: the amylase gene region.

The amylase region of the third chromosome of Drosophila pseudoobscura has been cloned and localized to cytological band 73A. It is contained within a series of highly polymorphic inversions and serves as a convenient tool for a molecular evolutionary analysis of the inverted gene arrangements. Amylase in D. pseudoobscura is a family of three genes, and some chromosomes have deletions for one or two of them. Two overlapping clones covering 26 kilobases were isolated and used as probes to survey DNA restriction map polymorphism among 28 lines, representing five of the major inversion types found in natural populations, as well as single chromosomes from the closely related species Drosophila persimilis and Drosophila miranda. Restriction-site differences are considerably greater among the various gene arrangements than among chromosomes with the same gene arrangement. Clustering the restriction map haplotypes yielded a dendrogram concordant with the phylogeny generated independently from cytogenetic considerations. The inversion polymorphism is estimated to be about 2 million years old.

Amylases↗
PubMed1993

Physical mapping and YAC contig analysis of the region surrounding Xist on the mouse X chromosome.

The Xist sequence has been proposed as a potential candidate for the X-inactivation center based both on its localization within the candidate region for the X-inactivation center in man and mouse and on its unique pattern of expression from the inactive X chromosome. We have cloned 550 kb of DNA surrounding the mouse Xist sequence in contiguously overlapping YAC clones and have developed a long-range restriction map that spans almost 1 Mb of this region and includes this YAC contig. The detailed restriction map we have established provides a framework for the identification of expressed sequences other than Xist that may equally exhibit unusual expression characteristics associated with X inactivation. The presence of possible structural or methylation differences within this region between the active and inactive X chromosomes has been investigated through comparative analysis of male and female genomic DNA, and we report here the identification of certain CpG-containing restriction sites around Xist that have an interesting differential methylation status on the inactive and active X chromosomes.

Animals↗
PubMed1988

Physical mapping of the human pseudo-autosomal region; comparison with genetic linkage map.

A long-range restriction map of the pseudo-autosomal or exchange pairing region (corresponding to the terminal parts of the short arms of the human sex chromosomes) has been established using pulsed field gel electrophoresis. A total of seven loci have been located on this physical map based essentially on the analysis of 45,X Turner genomes. The region spans a total of 2600 kb. The 5' end of the MIC2 gene maps at less than 80 kb from the proximal pseudo-autosomal boundary. Since the total pseudo-autosomal linkage interval represents approximately 50% of recombination at male meiosis, 1 cM corresponds to 50-60 kb. This is consistent with the almost 20-fold increase in recombination frequency observed in male versus female meiosis in this region. The present data show no distortion between both physical and linkage maps. The distribution of the CpG-rich restriction sites is notably disequilibrated. A large subset of these sites is concentrated within the 500 kb closest to the telomere whereas others appear in clusters (probably HTF islands) scattered in the rest of the pseudo-autosomal region.

Blotting, Southern↗
PubMed1982

Characterization of an infective molecular clone of the B-tropic, ecotropic BL/Ka(B) murine retrovirus genome.

Using molecular cloning techniques, we amplified the unintegrated, linear proviral DNA of the BL/Ka(B) virus, a non-leukemogenic retrovirus of mouse strain C57BL/Ka. Two independent clones in lambda phage vector 607 and one subclone in pBR322 were infective when transfected into mouse fibroblasts. Analysis of the progeny virus revealed biological properties and a restriction map identical to those of the parental viral shock. Comparison of the restriction map with the maps of other ecotropic murine viruses reveals many similarities. Particularly interesting is the comparison of the N-tropic Akv virus and the B-tropic BL/Ka(B) virus. The long terminal repeats of the two viruses are virtually identical, as are 22 of 23 restriction sites located outside of the region which spans from 1.8 to 3.8 kilobases from the left end of the genome. Within this region, however, only three of nine sites examined are shared. This suggests that the BL/Ka(B) virus was derived from an endogenous N-tropic virus closely related to Akv by recombinational events which altered the sequence in the last half of the gag gene and the first third of the pol gene. This change is probably responsible for the observed difference in the Fv-1 tropism of the two viruses.

Animals↗
PubMed1994

Physical map of mouse chromosome 17 in the region relevant for positional cloning of the Hybrid sterility 1 gene.

Hybrid sterility 1 (Hst1) is the major gene responsible for sterility of male hybrids between Mus musculus and certain laboratory strains. Thus, Hst1 is of importance in studying both postreproductive isolation of closely related species and male fertility. It has been mapped to mouse chromosome 17 in the region corresponding to the third inversion of the t haplotypes. The aim of the present study was to construct a physical map of the Hst1 region as the first step in an effort to clone the gene. Three yeast artificial chromosome (YAC) libraries (Princeton, Whitehead, and ICRF) were screened with polymerase chain reaction (PCR) oligonucleotide primers and DNA probes specific for loci previously mapped into the region of the third inversion. The isolated YAC clones were restriction mapped and arranged into contigs. Sixteen YAC clones were arranged into a single contig encompassing a region approximately 2000 kb long based on restriction mapping of highly overlapping but independently derived YAC clones. Five new loci in the region of the third inversion were mapped and the order and approximate physical distances of 12 loci established in this contig. The Hst1 gene maps approximately 0.2 cM proximal to the D17Ph1 locus encompassed by the YAC contig. Since the contig extends at least 1200 kb proximal to D17Ph1, it should contain the Hst1 gene.

Animals↗
PubMed1981

Characterization of candidate adenovirus 37 by SDS-polyacrylamide gel electrophoresis of virion polypeptides and DNA restriction site mapping.

A new human adenovirus has been isolated from patients with keratoconjunctivitis and/or genital infection since 1976. This adenovirus, designed candidate adenovirus 37 (Ad 37) is serologically distinct but related to Ad 10, 13, 19, and 30 (see the accompanying paper by de Jong et al). SDS-Polyacrylamide gel electrophoresis of Ad 37 virion polypeptides showed that this adenovirus is a member of subgroup D. DNA restriction endonuclease analysis of DNA from Ad 37 and related serotypes belonging to subgroup D showed that Ad 37 is a new genome type belonging to subgroup D but clearly distinct from the 20 serotypes classified into this subgroup.

Adenoviruses, Human↗
PubMed1991

The Adh in Drosophila: chromosomal location and restriction analysis in species with different phylogenetic relationships.

Restriction analysis of the genomic region containing the Adh gene and in situ hybridization assays were performed in six Drosophila species belonging to three different subgenera: D. ambigua, D. subobscura, D. madeirensis and D. guanche (sg. Sophophora); D. immigrans (sg. Drosophila); and D. lebanonensis (sg. Pholadoris). In agreement with previous observations, comparison of restriction maps of the Adh region shows that D. subobscura and D. madeirensis are very closely related. Partial homology is also observed with the rest of the obscura group species. Nevertheless, no resemblance at the restriction map level is detected when more distantly related species are compared. In D. ambigua, D. immigrans and D. lebanonensis in situ hybridization assays reveal a single chromosomal location for Adh, which in D. lebanonensis appears to be sex linked. In contrast, in D. subobscura, D. madeirensis and D. guanche multiple sites of hybridization with homologous and heterologous probes are observed. For example, in D. subobscura and D. madeirensis the functional Adh gene is located on the U chromosome and additional homologous retrosequences are found on the E chromosome.

Alcohol Dehydrogenase↗