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PubMed1992

Involvement of a large inverted repeated sequence in a recombinational rearrangement of the mitochondrial genome of the higher fungus Agrocybe aegerita.

Southern hybridization of the total DNA of Agrocybe aegerita with cloned mitochondrial (mt) probes revealed a sequence homology between two distant mitochondrial restriction fragments. From the mtDNA restriction map and the distribution of restriction sites on the cross-hybridizing mitochondrial fragments, two copies of a large inverted repeated sequence (IR) of 3 kbp were located on the mitochondrial genome. These IR sequences divided the 80 kbp mtDNA into two single-copy regions of 24 kbp (SSC) and 50 kbp (LSC). For the first time in higher fungi, this IR sequence has been shown to be involved in an intramolecular homologous recombinational event. Such a rearrangement led to an inversion of the orientation of the two unique-copy regions, without any change in mtDNA complexity. The location of the recombinational event was compared with previously reported plant and fungal mitochondrial rearrangements and the potential role of the IR sequence was discussed.

Basidiomycota↗

PubMed1997

Optical mapping approaches to molecular genomics.

A variety of physical mapping methods exist for the analysis of nucleic acids or genomes, including hybridization, sequence tagged site mapping, restriction enzyme fingerprinting, radiation hybrid mapping and optical mapping. Single-molecule approaches offer numerous advantages, including very high resolution, small sample size requirements, and parallel sample processing. The convergence of recent advances in new single molecule techniques, surface chemistry and machine vision technology has contributed to novel approaches to genome analysis.

Biotechnology↗

PubMed1982

Cloning and endonuclease restriction analysis of uidA and uidR genes in Escherichia coli K-12: determination of transcription direction for the uidA gene.

The two genes of the Escherichia coli K-12 uid region (the structural gene uidA and the regulatory gene uidR) were isolated on a ColE1-uid hybrid plasmid from the bank of Clarke and Carbon. We made a restriction map of this region and correlated it with the genetic map by subcloning the uid restriction fragments into plasmids pBR322, pBR325, and pACYC177. In these plasmids, amplification of the products of the uidA and uidR genes occurred. The enzyme coded for by uidA was identified by polyacrylamide gel electrophoresis of crude extracts from strains containing the uidA plasmid. A 1-megadalton EcoRI-BamHI segment contained the uidAo operator, and the direction of transcription of the uidA gene was determined. The restriction analysis also suggested that the order of the loci in this region is manA, uidA, uidAo, uidR.

Cloning, Molecular↗

PubMed1993

Mapping of three plasmids from Lactobacillus helveticus ATCC 15009.

Three plasmids isolated from Lactobacillus helveticus ATCC 15009 were analysed by restriction digestion. A restriction map of the 22.0, 6.0 and 3.5 kb plasmids is presented. The existence of genetic markers such as carbohydrate fermentation, lactose metabolism, antibiotics and heavy metal ions resistance was also investigated but the plasmids remain cryptic.

Lactobacillus↗

PubMed1996

Cloning, chromosomal localization, physical mapping, and genomic characterization of HKR3.

The Krüppel-type zinc finger proteins are members of a conserved family of transcription factors that are important in developmental regulation. Altered expression of several of these proteins has been implicated in human diseases, including cancer. We report the cloning, mapping, and characterization of the zinc finger gene Human Krüppel-Related 3 (HKR3). Genomic clones of HKR3 were isolated from a P1 library and localized to human chromosome subband 1p36.3 by human-rodent somatic cell hybrid mapping and fluorescence in situ hybridization. The gene was physically mapped to within 40 kb of D1S214 by YAC content and long-range restriction mapping. HKR3 spans 9.5 kb of genomic DNA and is contained in 11 exons. Sequencing defined each of the exon/intron splice site junctions and identified a CpG island in the 5' region of the gene. HKR3 is ubiquitously expressed in human tissues as at least two major transcripts, the shorter of which excludes a conserved finger-associated box and a putative acidic activation domain contained in the full-length transcript. HKR3 is a novel zinc finger gene that maps to a region of the genome commonly rearranged or deleted in human cancers.

Adult↗

PubMed1980

Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12.

The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322. Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA. The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment. Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated. To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4. A restriction map of pLC44-14 was obtained for nine restriction enzymes. The orientation of this map was determined relative to the E. coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.

Bacterial Proteins↗

PubMed1994

Physical map and set of overlapping cosmid clones representing the genome of the archaeon Halobacterium sp. GRB.

We have constructed a complete, five-enzyme restriction map of the genome of the archaeon Halobacterium sp. GRB, based on a set of 84 overlapping cosmid clones. Fewer than 30 kbp, in three gaps, remain uncloned. The genome consists of five replicons: a chromosome (2038 kbp) and four plasmids (305, 90, 37, and 1.8 kbp). The genome of Halobacterium sp. GRB is similar in style to other halobacterial genomes by being partitioned among multiple replicons and by being mosaic in terms of nucleotide composition. It is unlike other halobacterial genomes, however, in lacking multicopy families of insertion sequences.

Chromosome Mapping↗

PubMed1994

Molecular analysis of Gpdh null mutations that arose in mutation accumulation experiments in Drosophila melanogaster.

In order to clarify the cause of null mutations in enzyme loci, the molecular structure of six null mutations in the Gpdh locus (encoding alpha GPDH: alpha glycerol-3-phosphate dehydrogenase (NAD+), E.C. 1.1.1.8; map position at 2-17.8) that arose in mutation accumulation experiments was examined. A restriction map analysis showed that five of the mutations are insertional mutations whereas the sixth is a deletion. The Gpdh regions of these null mutations were then cloned and sequenced. The inserted DNA fragments are all internally deleted P elements measuring 1.1 kb in length. Two are a KP element and two others are a HP element. All the insertions occur in the region near the initiation signal of transcription. The deletion encompasses the seventh and eighth exons over a length of 1.1 kb. These results therefore indicate that the null mutation rate at the Gpdh locus is largely influenced by P elements.

Animals↗

PubMed1997

The structure of the human ALL-1/MLL/HRX gene.

The human ALL-1/MLL/HRX gene on chromosome 11q23 is the site of many locally clustered chromosomal alterations associated with several types of acute leukemias, including deletions. partial duplications and reciprocal translocations. Structurally variant proteins derived from an altered ALL-1 gene presumably make essential contributions to the malignant transformation of hematopoietic progenitor cells. The ALL-1 gene is spread over approximately 92 kb and consists of at least 37 exons. An exon/intron map including the position of the 3'-end of the gene and a detailed restriction map were produced and an updated map is presented. Data from other laboratories were incorporated where compatible. Exon/intron boundaries were sequenced and an intron-phase analysis was performed. The results are expected to contribute to a better understanding of those structural alterations of the gene that conserve the open reading frame and produce presumably oncogenic variants of the ALL-1 protein. They will also facilitate the rapid molecular diagnosis of structural alterations of this gene and the choice of therapeutic options. Mechanisms that may potentially account for the striking clustering of the translocation breakpoints in the breakpoint cluster region of the gene are discussed.

Base Sequence↗

PubMed1995

A physical map of the 85 kb virulence plasmid of Rhodococcus equi 103.

A physical map of the 85 kb virulence plasmid pOTS from Rhodococcus equi 103 was constructed. The restriction map contains 2 AsnI, 5 BglII, 9 EcoRI, 4 HindIII, and 3 XbaI sites. The positions of the EcoRI and HindIII of pOTS are identical to that of the 85 kb virulence plasmid of R. equi ATCC 33701 reported recently by others. EcoRI restriction fragment sizes were similar in the 85 kb plasmids isolated from 4 horse derived R. equi but, except apparently for the 28.3 and possibly 2.0 and 1.5 kb fragments, were different in an 80.1 kb plasmid isolated from a pig source R. equi.

Animals↗

PubMed1992

GeneScape: a relational database of Escherichia coli genomic map data for Macintosh computers.

We present a relational database program developed in FoxBase+/Mac for the viewing and manipulation of ordered restriction maps and associated features of the Escherichia coli genome including sequenced genes and the Kohara miniset of bacteriophage lambda clones. Use of this program allows easy access to the wealth of information being collected in a dataset of DNA sequences, maps and genetic data known as EcoSeq, EcoMap and EcoGene respectively.

Algorithms↗

PubMed1999

Novel plasmids from alkaliphilic halomonads.

Seventeen alkaliphilic halomonads were examined for the presence of plasmids. Of these, eight strains harbored one or more from 5.3 to 33 kb in size, the first plasmids to be identified from an alkaliphilic halomonad source. Restriction and hybridization analysis revealed three strains that maintained an identical 5.9-kb plasmid which we named pAH1, two that had an identical 33-kb plasmid, and three others, of which one carried two plasmids of 5.3 and 15 kb, the former being designated pAH2. The two final strains maintained plasmids of 15 and 20.5 kb. Restriction mapping of both pAH1 and pAH2 indicated that they have a number of unique restriction sites and are of a small enough size to make them suitable for vector construction.

DNA, Archaeal↗

PubMed1994

Isolation of two contigs of overlapping cosmids derived from human chromosomal band 3p21.1 and identification of 5 new 3p21.1 genes.

Consistent loss of DNA sequences from several regions on the short arm of human chromosome 3 has suggested that multiple tumor suppressor genes reside on chromosome 3p in various types of cancer cells. We have focused our efforts on an analysis of chromosomal band 3p21.1 since aminoacylase-1 (ACY1), which is localized to this band, has been shown to have lower levels of expression in several small cell and non-small cell lung cancer cell lines. Starting with two cosmids within 3p21.1, D3S92 and D3S93, we have isolated two separate contigs of overlapping cosmids within 3p21.1, by screening a library of 5700 chromosome 3-specific cosmid clones. Detailed restriction maps for these two contigs show that they contain multiple clusters of rare cutting restriction endonuclease sites. One contig extends for 100 kb and encompassed both ACY1 and D3S92, and the other extends about 80 kb around the D3S93 locus. Many different restriction fragments derived from these two contigs were found to be evolutionarily conserved and hybridized to distinct message transcripts. These fragments were used to identify homologous cDNAs from an adenogastric cDNA library, and several of these cDNAs were partially sequenced. We have identified five new genes from these two contigs and there is evidence to suggest that several additional genes reside within these cosmid contigs. The genes identified from 3p21.1 were then hybridized to DNA, isolated from a series of lung cancer cell lines and matched normal and tumor DNA from lung cancer patients. No alterations were detected with any of these probes, both at the DNA or RNA levels. A similar analysis with DNA fragments derived from these two genomic regions also failed to detect any alterations.

Animals↗

PubMed1997

Molecular delineation of Cylicocyclus nassatus and C. ashworthi (Nematoda:Strongylidae).

The nucleotide sequences of the first internal transcribed spacer (ITS-1), 5.8S gene and second internal transcribed spacer (ITS-2) of ribosomal DNA have been determined for Cylicocyclus nassatus, C. ashworthi and C. insignis. Pairwise comparisons revealed sequence differences between the taxa ranging from 3.8 to 6.2% for the ITS-2 and 2.2-2.7% for the ITS-1. For the ITS-1, the level of the sequence difference between C. ashworthi and C. nassatus (2.2%) was equivalent to that between C. nassatus and C. insignis (2.2%), indicating that C. ashworthi and C. nassatus represent separate species. Theoretical restriction maps were constructed from the sequence data, and a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-linked RFLP) technique was established to unequivocally distinguish C. ashworthi from C. nassatus.

Animals↗

PubMed1993

Chromosomal structure of Rhodobacter capsulatus strain SB1003: cosmid encyclopedia and high-resolution physical and genetic map.

A combination of cosmid genome walking and pulsed-field gel electrophoresis was used to construct a high-resolution physical and genetic map of the 3.8-megabase (Mb) genome of Rhodobacter capsulatus SB1003. The mapping was done by hybridization of pulsed-field gel blots and by grouping and further mapping of the cosmids and bacteriophages from genomic libraries. Cosmid clones formed two uninterrupted and ordered groups, one corresponding to the chromosome of R. capsulatus, the other to its 134-kb plasmid. Cos site end-labeling and partial EcoRV digestion of cosmids were used to construct a high-resolution EcoRV map of the genome. Overlapping of the cosmids was confirmed by the resemblance of the cosmid restriction maps and by direct end-to-end hybridization with SP6- and T7-specific transcripts. Twenty-three previously cloned genes and eight groups of repeated sequences, revealed in this work, were located in the ordered gene library and mapped with an accuracy of 1-10 kb. Blots of a minimal set of 192 cosmids, covering the chromosome and the plasmid with the known map position of each cosmid, give to R. capsulatus the same advantages that the Kohara phage panel gives to E. coli.

Chromosome Mapping↗

PubMed1998

[Recombinational origin of natural transposable phages of related species belonging to group B3, active in Pseudomonas aeruginosa species].

A heteroduplex analysis of four related transposable phages--B3, PM57, PM62, and Hw12--of the Pseudomonas aeruginosa B3 group was performed. Heteroduplex structures, restriction maps, and data on DNA-DNA hybridization obtained upon hybridization of phage DNA restriction fragments with labeled probes representing different regions of the phage genomes are in good agreement. The data obtained strongly confirmed the recombinational origin of the analyzed phages. Thus, all natural transposable phages of P. aeruginosa, including phages from both group B3 and species D3112, were shown to have a recombinational origin.

DNA Transposable Elements↗

PubMed1999

Interpretation of variability of mitochondrial genomes in the species Aspergillus carbonarius.

For interpretation of intraspecific polymorphism and the considerable differences in the size of mtDNAs among three groups of A. carbonarius, restriction maps were constructed from several enzymes. Functional maps were also developed to compare genome organisations and gene content. The appearance of various mtDNAs of A. carbonarius strains are different in size, but their gene content is almost identical. The 1.1 kb size difference between two closely related subgroups (1a, 1b) can be attributed to the presence or absence of an intron in cox2 gene. This phenomenon demonstrates that the migration of introns is possibly responsible for the development of variable mitochondrial genomes in nature. The striking differences in size and restriction patterns between two main mtDNA groups might derive from both the intronal variations and the altered intergenic organisation.

Aspergillus↗

PubMed1990

Mapping DNA by stochastic relaxation: a new approach to fragment sizes.

Instead of the traditional manipulations with given fixed fragment lengths in the restriction map construction a method of varying the lengths is proposed and realized under the simulated annealing algorithm scheme. The described approach has no upper limit on the number of fragments mapped with even ordinary hardware. A program has been derived from the algorithm combined with the last-squares refinement procedure for both linear and circular maps. The algorithm's ability to pick up missed maps is illustrated and the problem of reducing the number of solutions is discussed.

Algorithms↗