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At least 649 records · Page 36Linked to original sources
PubMed1993

Physical mapping and YAC-cloning connects four genetically distinct 4qter loci (D4S163, D4S139, D4F35S1 and D4F104S1) in the FSHD gene-region.

We have constructed a long-range restriction map of the region on chromosome 4q that contains the gene for facioscapulohumeral muscular dystrophy (FSHD). This region contains the linkage group cen ... D4S163-D4S139-D4F35S1-D4F104S1-FSHD ... 4qter, which spans a genetic distance of about 5 cM. Pulse field gel electrophoresis (PFGE) mapping indicated that these loci span a region not more than 1 Mb. STSs were developed for several of these loci, which served to isolate four overlapping yeast artificial chromosomes (YACs). These YACs confirmed the PFGE map and have allowed us to generate a more detailed restriction map using cosmid contig mapping. The physical distances were smaller than was expected on the basis of the genetic map. Two potential HTF islands have been detected within the cloned region. One HTF island maps about 100 kb centromeric from the tandem repeats involved in the FSHD mutation, whereas the other maps within these tandem repeats.

Base Sequence↗
PubMed1981

Constancy of somatic DNA organization in developmentally regulated regions of the Drosophila genome.

The purpose of this study was to test for the occurrence of changes in the organization, modification, or selective amplification of six developmentally regulated regions of genomic DNA during Drosophila development. Five of the regions contain structural genes, each of which maps at a single chromosomal site; the sixth region contains a dispersed segment that maps at about 30 different sites and appears to be transposable. The RNA transcripts and encoded proteins of the structural genes are major components of the fat body tissue in late third-instar larvae, in contrast to other larval tissues and young embryos in which the transcripts and proteins are hardly detectable. The dispersed segment shows the reverse developmental regulation: the amount of transcript is relatively high in young embryos and low in larval fat bodies. The test for changes in genomic DNA associated with the regulated expression of the six regions was based on comparative restriction mapping of the DNA from these sources. Genomic clones containing the transcribed and also flanking regions of DNA were used as probes to determine the positions of the complementary restriction fragments after electrophoresis in agarose gels. Each test, which involved digesting the genomic DNA samples with one of the endonucleases and probing with one of the clones, produced identical restriction maps for the different samples. The tests are capable of detecting changes in the organization of the cloned regions resulting from addition or removal either of endonuclease cleavage sites or of segments of DNA located between cleavage sites. Because the activities of several of the endonucleases, and probably all, are sensitive to methylation of one of the bases in the DNA recognition sequence, certain modifications of the cloned regions by methylation could also be detected. Although it is not certain that genomic reorganization or modification would have been detected by these tests, the number of endonucleases used was sufficient to provide persuasive evidence that such changes did not occur. Furthermore, the quantitative as well as qualitative similarities of the restriction maps in each test indicated that there was no significant selective amplification of the DNA within the cloned regions.

Adipose Tissue↗
PubMed1990

Characterization of expressed meiotic prophase repeat transcript clones of Lilium: meiosis-specific expression, relatedness, and affinities to small heat shock protein genes.

The inserts of plasmid cDNA clones for transcripts showing meiotic prophase specific expression show cross reassociation to varying degrees of intensity with one another. These clones were recovered from a cDNA library made from Lilium microsporocyte poly(A)+ RNA. RNA-dot and Northern-blot analyses indicate that these clones represent transcripts specific to the meiotic prophase interval in microsporocytes. The transcripts appear to constitute the most abundant class of meiosis-specific poly(A)+ RNAs. At least two subgroups can be distinguished by examining cloned transcripts from genes of this expressed meiotic prophase repeat (EMPR) sequence family. Members of each subgroup have similar although not identical restriction maps and show relatively high but varying fidelities of DNA cross reassociation between members. However, consensus restriction maps of the two subgroups are largely dissimilar and, except at low stringencies, cross reassociation is readily detected only at restriction fragments from a particular conserved internal segment. The DNA sequence of a representative EMPR clone has been determined, and the inferred peptide product has been found to show extensive sequence homology to that of a small heat-shock gene of Glycine max, particularly in the conserved region. Alignment of the sequences for the conserved regions of two EMPR subgroup representatives with the soybean sequence suggests that selection has acted to conserve similar blocks of amino acids in this area. These observations suggest that a major portion of the transcripts produced during the apparently unrelated processes of meiosis and heat shock in higher plants are derived from related gene sequences encoding similar products.

Amino Acid Sequence↗
PubMed1996

Restriction site mapping of a bovine adenovirus type 10 strain.

Physical maps of the genome of a bovine adenovirus (BAV) type 10 strain (isolate Belfast1) were constructed for eight restriction enzymes. The size of the viral genome was estimated to be around 29,800 base pairs (bp). The orientation of the maps was determined on the basis of partial DNA sequences of the cloned PstI/D fragment of Belfast1. This work is an introduction to DNA sequence and phylogenetic analysis of BAV-10 in order to clarify its taxonomic place.

Adenoviridae↗
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↗
PubMed1999

Optical PCR: genomic analysis by long-range PCR and optical mapping.

Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors.

DNA Restriction Enzymes↗
PubMed2000

Monitoring genome evolution ex vivo: reversible chromosomal integration of a 106 kb plasmid at two tRNA(Lys) gene loci in sequential Pseudomonas aeruginosa airway isolates.

The genome rearrangements in sequential Pseudomonas aeruginosa clone K isolates from the airways of a patient with cystic fibrosis were determined by an integrated approach of mapping, sequencing and bioinformatics. Restriction mapping uncovered an 8.9 kb deletion of PAO sequence between phnAB and oprL in clone K, and two 106 kb insertions either adjacent to this deletion or several hundred kilobases away, close to the pilA locus. These 106 kb blocks of extra DNA also co-existed as the circular plasmid pKLK106 in several clone K isolates and were found to be closely related to plasmid pKLC102 in P. aeruginosa clone C isolates. The breakpoints of the deletion in clone K and the attB-attP sequences for the reversible integration of the plasmid in clones C and K were located within the 3' end of the lysine tRNA structural genes (att site). pKLK106 sequentially recombined with either of the two tRNA(Lys) genes in clone K isolates. The att site of the pilA hypervariable region has been utilized by clone C to target its plasmid pKLC102 into the chromosome; the att site of the phnAB-oprL region has been employed by strain PAO to incorporate a DNA block encoding pyocin, transposases and IS elements. The use of typical phage attachment sites by conjugative genetic elements could be one of the major mechanisms used by P. aeruginosa to generate the mosaic genome structure of blocks of species-, clone- and strain-specific DNA. The example described here demonstrates the potential impact of systematic genome analysis of sequential isolates from the same habitat on our understanding of the evolution of microbial genomes.

Base Sequence↗
PubMed1990

Molecular analysis of the human MHC class I region using yeast artificial chromosome clones.

The cloning of large genomic fragments corresponding to the major histocompatibility complex (MHC) class I region provides the necessary framework for a better understanding of its organization and for the localization of new genes involved in MHC-associated disease. Two human genomic libraries constructed in yeast artificial chromosomes (YACs) have been prepared using complete Not I or Mlu I digestion of source DNA. From these libraries three YAC clones with inserts belonging to the MHC class I region have been isolated. They correspond to exact copies of three genomic fragments of 210, 145, and 50 kilobases (kb), respectively and have been precisely located in the restriction map of the region. Detailed rare-cutter restriction maps of the inserts have been generated. Within these clones we have demonstrated the presence of two class I genes, one of which is HLA-E, and of at least three Hpa II tiny fragment (HTF) islands, corresponding to three putative new transcribed sequences. End clones, which are of particular interest in the extension and refinement of the regional map, have been rescued by systematic subcloning of purified YACs.

Base Sequence↗
PubMed1995

Interchromosomal exchange of genetic information between gene arrangements on the third chromosome of Drosophila pseudoobscura.

During the last 60 years, the inversion polymorphism on the third chromosome of Drosophila pseudoobscura has become a case study of the evolution of linked blocks of genes, isolated from each other by the suppression of recombination in heterozygotes for different inversions. Due to its location within inverted regions in most gene arrangements, the amylase (Amy) gene region can be used to elucidate the molecular pattern of evolution in these inversions. We studied this region in the Tree Line phylad of gene arrangements, with regard to both restriction site polymorphisms (RSP) and nucleotide sequences. The analysis of restriction maps, encompassing 26 kb, corroborates the cytogenetic phylogeny established on the basis of inversion breakpoints. However, we found that the 2.7 kb of nucleotide sequences of the AmyI gene are identical in both Estes Park and Hidalgo arrangements, despite the fact that these inversions arose independently from Tree Line. These contrasting results suggest that a homogenizing force, most likely gene conversion, is able to bring about localized exchanges between otherwise isolated gene arrangements.

Amylases↗
PubMed2005

Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis.

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.

Bacterial Typing Techniques↗
PubMed1994

Large-scale structure conservation along the entire long arm of human chromosome 21.

Complete, contiguous NotI restriction maps were constructed for the long arm of human chromosome 21 contained in nine different cell lines. These maps span about 43 Mb and consist of about 60 distinct NotI fragments, about 80 markers, and 11 chromosomal breakpoints. Although some differences among the maps were noted, the overall large-scale structure of the chromosomes is remarkably similar, which strongly validates the use of cell lines for mapping studies. Remarkably few serious discrepancies were found between the order of markers on this NotI restriction map and various genetic and physical maps. However, when compared with the NotI physical map, both the genetic map and the radiation hybrid map of chromosome 21 appear to expanded near the centromere and especially near the telomere.

Animals↗
PubMed1992

Physical mapping of the Saccharomyces cerevisiae Ap4A phosphorylase I-encoding gene by the Achilles' cleavage method.

LacI-mediated Achilles' cleavage (AC) is a method for selective fragmentation of chromosomes at special lac operator sites introduced by gene targeting methods [Koob and Szybalski, Science 250 (1990) 271-273]. The Saccharomyces cerevisiae APA1 gene, coding for diadenosine 5', 5"'-P1, P4-tetraphosphate phosphorylase I, has previously been shown to be located on chromosome III [Kaushal et al., Gene 95 (1990) 79-84]. We have now used the AC method to map APA1 gene to a site 44 kb from the left terminus of the chromosome, between the HIS4 and HML genes. This location was confirmed by the comparison of restriction maps of the APA1 gene region to published restriction maps of chromosome III.

Electrophoresis, Gel, Pulsed-Field↗
PubMed1993

A direct demonstration of recombination between an injected virus and endogenous viral sequences, resulting in the generation of mink cell focus-inducing viruses in AKR mice.

We analyzed viral recombination events that occur during the preleukemic period in AKR mice. We tagged a molecular chimera between the nonleukemogenic virus Akv and the leukemogenic mink cell focus-inducing (MCF) virus MCF 247 with an amber suppressor tRNA gene, supF. We injected the supF-tagged chimeric virus that contains all of the genes of MCF 247 except the envelope gene, which in turn is derived from Akv, into newborn AKR mice to evaluate its pathogenic potential. Approximately the same percentage of animals developed leukemia with similar latent periods when injected with either the tagged or nontagged virus. DNA from tumors induced in AKR mice by the tagged chimeric virus was analyzed by Southern blotting with the supF gene as a probe. One set of tumors contained the injected supF-tagged virus. Two kinds of supF-tagged proviruses were found in a second set of tumors. One group of supF-tagged viruses had a restriction map consistent with that of the injected virus, while the other group of proviruses had restriction maps that suggested that the proviruses had acquired an MCF virus-like envelope gene by recombination with endogenous viral sequences. These results demonstrate that injected viruses recombine in vivo with endogenous viral sequences. Furthermore, the progression to leukemia was accelerated in mice that develop tumors containing proviruses with an MCF virus env gene, emphasizing the importance of the role of the MCF virus env gene product in transformation.

AKR murine leukemia virus↗
PubMed1978

Restriction endonuclease mapping of bacteriophage phi105 and closely related temperate Bacillus subtilis bacteriophages rho10 and rho14.

Cleavage maps of the three similar Bacillus subtilis temperate bacteriophages, phi105, rho10, and rho14, were constructed by partial digestion analysis utilizing the restriction endonuclease EcoRI. Comparison of the topography of these maps indicates that all phage DNAs posses cohesive ends and a number of EcoRI restriction sites; the fragments are conserved, and the estimated base substitution/nucleotide divergence between these phages is 0.03 to 0.07 based on conserved fragments or between 0.03 and 0.11 based on conserved cleavage sites. These lines of evidence indicate that phi105, rho10, and rho14 are closely related. Double-enzyme digestion analysis reveals that rho14 DNA has unique SalGI and BglII restriction sites and phi105 DNA has a unique SalGI restriction site, making these phages possible cloning vectors for B. subtilis.

Bacillus subtilis↗
PubMed1980

Restriction endonuclease mapping of ColE2-P9 and ColE3-CA38 plasmids.

Using single and double restriction-endonuclease digestions, 16 and 17 cleavage sites have been mapped for the ColE2-P9 and ColE3-CA38 plasmids, respectively. One or more sites for AvaI, BglI, EcoRI, HincII, PvuI, PvuII, SmaI and XhoI endonucleases were found in both plasmids, two BglII sites were found only in ColE2-P9, and one KpnI site was unique to ColE3-CA38. ColE2-P9 was found to be slightly smaller than ColE3-CA38, 4.4 Md compared to 4.6 Md. Eleven restriction sites are common to both plasmids in that they are identically placed relative to each other. These sites define a continuous DNA segment equal to over 60% of each plasmid. The remaining portions of the plasmids, which contain the non-homologous regions identified by Inselburg and Johns (1975) have no restriction sites in common, and differ in size by about 0.2 Md.

Bacteriocin Plasmids↗
PubMed1993

Mechanism of efficient elimination of protein D2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa.

Most imipenem-resistant Pseudomonas aeruginosa isolates produce an immunologically undetectable level of protein D2 (OprD2). To study the efficient elimination of the protein, we selected 23 independent imipenem-resistant mutants from a strain harboring the plasmid carrying cloned oprD and having a mutation in chromosomal oprD. All these oprD/oprD (plasmid/chromosomal) mutants expressed undetectable levels of OprD2, as shown from an assay by the immunoblotting method. Restriction maps of the DNAs from all 23 mutant plasmids could be divided into two groups. Restriction mapping and sequencing analysis of DNA from one representative plasmid from each group showed that both mutant oprD genes had a deletion. One had an 11-bp deletion in the coding region generating a frameshift mutation and a premature termination codon. Another had a large deletion encompassing the upstream site of its putative promoter region through the coding region. Northern blotting analysis showed that the gene with the 11-bp deletion was transcribed to about 1.5 kb of mRNA, but the gene with the large deletion produced undetectable RNA complementary to the oprD DNA probe. Since we analyzed only plasmid-borne oprD, we cannot exclude the possibility that the imipenem resistance caused by the chromosomal mutation is by a different mechanism(s). It is suggested, yet, that clear elimination of OprD2 from most imipenem-resistant P. aeruginosa isolates is due to efficient selection of the oprD deletion mutants.

Bacterial Proteins↗