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PubMed1980

Internal organization of the major adult alpha- and beta-globin genes of X. laevis.

We describe the isolation of two recombinant lambda phages, each containing genomic DNA fragments encoding both the major adult alpha- and beta-globin mRNAs of X. laevis. The DNA fragment in the two clones have restriction maps which indicate that they are each derived from a different member of the pair of alleles present in the heterozygote used as the source of DNA for cloning. The characterization of these two clones by restriction mapping, R looping and DNA sequencing shows that the alpha 1- and beta 1-globin genes lie in the orientation separated by 7.7 kb of DNA. There are two introns in the alpha 1-globin gene and two in the beta 1-globin gene, and they interrupt the genes at exactly the same positions as the introns found in all known mammalian alpha- and beta-globin genes. The exon sequences proximal to the introns show a much higher degree of homology with mammalian sequences than the sequences distal to intron/exon junctions, and the introns in the beta 1-globin gene of X. laevis are very similar in length to the corresponding introns in the beta-globin genes of several mammals and the chicken.

Animals↗
PubMed1984

Restriction endonuclease map variation and gene activity in the Adh region in a population of Drosophila melanogaster.

Variation within a 12 kb region containing the Adh gene was studied in 16 highly inbred lines of independent extraction from a population of Drosophila melanogaster, "Texas". Out of a selection of 8 hexanucleotide specific restriction endonucleases, four restriction site and five DNA insertion/deletion variations were observed and the heterozygosity per nucleotide was 0.004. Linkage disequilibrium between two restriction sites, considerably removed from and on either side of the Adh transcriptional unit, identified two major classes of chromosome or haplotype in the population. These two haplotypes were strongly associated with the capacity of lines to respond to the environment (genotype-environment interaction) in ADH activity. It was concluded that a control or regulatory polymorphism was present in the population in association with the Adh transcriptional unit.

Alcohol Dehydrogenase↗
PubMed1988

Molecular structure and interrelationships of multiresistance beta-lactamase transposons.

Transposons coding for beta-lactamases OXA-3, OXA-4, OXA-5, LCR-1, and CARB-3 have been isolated and compared functionally and structurally with transposons for TEM-1, OXA-1, PSE-1, PSE-2, and PSE-4 enzymes. Each beta-lactamase gene type occurred in a unit together with resistance to other antibiotics, particularly streptomycin and sulfonamide but also chloramphenicol, mercuric ion, or gentamicin, kanamycin, and tobramycin. Restriction mapping, gene cloning, and DNA hybridization were used to compare the transposons and to localize their functional components. Although the multiresistance beta-lactamase transposons varied in size from 8 to 25 kb, the similarity of some of their restriction maps suggested a common derivation. Six of 12 transposons contained DNA segments homologous to the tnpR gene of transposon Tn21 and could complement a tnpR- Tn21 derivative. Consequently, these six transposons appear to have evolved from a common progenitor by acquisition of DNA coding for various beta-lactamases and other resistance genes.

DNA Restriction Enzymes↗
PubMed1981

Molecular cloning of the genome of poliovirus type 1.

Poliovirus cDNA.RNA hybrids were prepared from the Mahoney strain of poliovirus type 1 by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and cloned in the Escherichia coli plasmid pBR322. Bacteria colonies carrying recombinant plasmids were selected by in situ hybridization with virus-specific RNase T1-resistant oligonucleotides. Analysis of the cDNA inserts by restriction mapping and electron microscopy showed that the cloned cDNAs, the longest of which was 3.2 kilobase pairs, originated from various parts of the viral RNA, covering at least 99% of the genome length. Due to overlapping of the clones, the restriction map of the poliovirus genome could be reconstructed. The complete 5' end of the genome was successfully cloned in at least one of the recombinant plasmids, pPV1-366.

Cloning, Molecular↗
PubMed1978

Electron microscopy and restriction enzyme mapping reveal additional intervening sequences in the chicken ovalbumin split gene.

The Eco RI fragment "b" of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5' quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a "shotgun" approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5' quarter (approximately 500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Econ RI fragment "b" is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150-200 nucleotide long sequence at the 5' end of the ov mRNA similar to the "leader" sequences found at the 5' end of some adenovirus and SV40 mRNAs.

Animals↗
PubMed1988

Isolation of canine adenovirus-2 from the faeces of dogs with enteric disease and its unambiguous typing by restriction endonuclease mapping.

Fifty-four faecal specimens obtained from kennelled dogs with diarrhoeal disease were used to inoculate a range of cell types in tissue culture. Particles resembling adenovirus virions were seen in three specimens and 22 stools yielded an adenovirus upon culture. Viral DNA from each isolate was digested with the restriction endonucleases Bam H1 and Pst 1. Agarose gel electrophoresis revealed identical restriction patterns for all isolates. One isolate, 9228, was selected as a prototype and was compared with reference strains of canine adenovirus-2 (CAV-2) (Manhattan and Toronto A26/61) and CAV-1. Isolate 9228 was clearly distinct from CAV-1 but identical to both Manhattan and Toronto (A26/61) strains of CAV-2. However, restriction site polymorphism was observed in 9228 following digestion with Hpa II. Isolate 9228 was similarly compared with all commercially available vaccinal isolates of CAV-2 and was shown to be clearly distinct from these.

Adenoviridae↗
PubMed1984

Localization, cloning, and sequence determination of the conjugative plasmid ColB2 pilin gene.

ColB2 is a colicin-producing, 96-kilobase plasmid which encodes a conjugative system that is similar, but not identical, to F. A restriction map of this plasmid was generated, and DNA homology studies between F and ColB2 plasmids revealed homology only between their transfer operons. The locations of the ColB2 transfer operon and ColB2 pilin gene were localized on this restriction map. The gene encoding ColB2 pilin, traA, was cloned and sequenced. The pilin protein of ColB2 is identical to F, except at the amino terminus, where ala-gln of ColB2 pilin corresponds to Ala-Gly-Ser-Ser of F pilin. This is due to a 6-base-pair deletion in the ColB2 pilin gene. Biochemical studies on tryptic peptides derived from ColB2 pilin demonstrate the location of this gene to be correct. There is a putative signal peptidase cleavage site after the sequence Ala-Met-Ala, giving a signal peptide of 51 amino acids and a mature pilin protein of 68 amino acids (7,000 daltons). The amino terminus is blocked, probably with an acetyl group. A chimera containing the ColB2 pilin gene was able to complement an F traA mutant, demonstrating that the pilus assembly proteins of F can utilize the ColB2 pilin protein to form a pilus.

Amino Acid Sequence↗
PubMed1995

Linear mitochondrial genome organization in vivo in the genus Pythium.

Pulsed-field gel electrophoresis (PFGE) of isolates of Pythium oligandrum with linear mitochondrial genomes revealed a distinct band in ethidium bromide-stained gels similar in size to values estimated by restriction mapping of mitochondrial DNA (mtDNA). Southern analysis confirmed that these bands were mtDNA and indicated that linear genomes were present in unit-length size as well as multimers. Isolates of this species with circular mtDNA restriction maps also had low levels of linear mono- and multimers visualized by Southern analysis of PFGE gels. Examination of 17 additional species revealed similar results; three species had distinct linear mtDNA bands in ethidium bromide-stained gels while the remainder had linear mono- and multi-mers in lower amounts detected only by Southern analysis. Sequence analysis of an isolate of P. oligandrum with a primarily circular mitochondrial genomic map and a low amount of linear molecules revealed that the small unique region of the circular map (which corresponded to the terminal region of linear genomes) was flanked by palindromic intrastrand complementary sequences separated by a unique 194-bp sequence. Sequences with similarity to ATPase9 coding regions from other organisms were located adjacent to this region. Sequences with similarity to mitochondrial origins of replication and autonomously replicating sequences were also located in this region: their potential involvement in the generation of linear molecules is discussed.

Adenosine Triphosphatases↗
PubMed1988

Somatic cell mapping and restriction fragment analysis of bovine alpha and beta interferon gene families.

DNA from bovine x hamster hybrid cells preferentially segregating bovine chromosomes has been analyzed by blot hybridization with alpha and beta interferon probes. Retention or loss of bovine interferon genes was compared to segregation of bovine isozyme loci representing previously described syntenic groups. Families of bovine alpha (IFNA) and beta (IFNB) interferon genes were segregated in concordance with each other and with aconitase-1 (ACO1) on bovine syntenic group U18. This syntenic relationship is conserved on human chromosome 9p and on the portion of mouse chromosome 4 proximal to the centromere. In addition, cattle restriction fragment length polymorphisms were identified with both IFNA and IFNB probes. Of particular interest is a polymorphism apparently due to duplication of IFNB genes.

Animals↗
PubMed2006

First restriction and genetic mapping of the genomic DNA of urease-positive thermophilic campylobacters (UPTC), and small restriction fragment sequencing.

A restriction and genetic map of urease-positive thermophilic campylobacter (UPTC) CF89-12 genome DNA is constructed using a pulsed-field gel electrophoresis procedure after digestion with SalI and SmaI and Southern blot hybridisation. Each of the six gene fragments (flaA, glyA, lysS, recA, sodB and ureAB) selected are mapped in only a fragment on the restriction map. Three DNA fragments for rrn operon probes are mapped in multiple regions on the map. When two SmaI-digested neighbouring small fragments hybridised with rrn probes are cloned and sequenced, a total sequence length of 7487 bp is determined. In the sequence, part of the pnp gene (734 bp) bearing a p-independent transcriptional termination region, a cluster of five tRNA genes including the putative promoter region, a hypothetical Cj0171-like 507-bp sequence containing an internal termination codon, and a part of the rrn operon including the putative promoter region (4700 bp) are identified. The 507 bp sequence carried both putative transcriptional promoter sequences, including a ribosome binding site upstream of the ATG start codon and a characteristic G9 structure, and a possible p-independent transcriptional termination region. A hypothetical Cj0170-like 204-bp sequence containing an internal termination codon also occurred, overlapping partly with the Cj0171-like sequence. Based on nucleotide sequence alignment analysis between the UPTC rrn operon examined here and the previously reported one, two different 16S-23S ribosomal DNA (rDNA) internal spacer regions are shown to exist.

Base Sequence↗
PubMed1995

Optical mapping of lambda bacteriophage clones using restriction endonucleases.

Optical mapping is an emerging single molecule approach for the rapid generation of ordered restriction maps, using fluorescence microscopy. We have improved the size resolution of optical mapping by imaging individual DNA molecules elongated and fixed onto derivatized glass surfaces. Averaged fluorescence intensity and apparent length measurements accurately determined the mass of restriction fragments 800 basepairs long. We have used optical mapping to create ordered restriction maps for lambda clones derived from the mouse pygmy locus.

Animals↗
PubMed1992

Polymorphism and divergence in the Mst26A male accessory gland gene region in Drosophila.

Drosophila males, like males of most other insects, transfer a group of specific proteins to the females during mating. These proteins are produced primarily in the accessory gland and are likely to influence the female's reproduction. The results of studies of DNA sequence polymorphism and divergence in two genes coding for male accessory gland proteins of Drosophila are reported here. The Mst26Aa and Mst26Ab transcription units are tandemly arranged in a approximately 1.6-kb segment in Drosophila sechellia, Drosophila mauritiana and Drosophila simulans as they were reported to be in Drosophila melanogaster. The DNA sequences of 10 alleles from D. melanogaster and one allele each from the three sibling species reveals a high degree of amino acid replacement variation. A substantial part of the variation is due to insertion/deletion differences. Possible functional significance of these amino acid sequence changes is discussed. Statistical analyses based on the neutral theory of molecular evolution show that the distribution of polymorphism over the 1.6-kb region is inconsistent with the pattern of divergence between the species. The amount of 4-cutter restriction map polymorphism in a larger sample of 75 alleles from the same D. melanogaster population is similar to that obtained from the DNA sequence of the 10 alleles (a pairwise average of 0.007 difference per site). The 6-cutter restriction map survey of a 18-kb region containing the Mst26A genes indicates that polymorphism in the region flanking these genes maybe higher. The failure of polymorphisms and divergence in the Mst26A region to conform to the expectations of a simple mutation-drift-equilibrium model indicates that selection in or near this region has played a role in the history of these genes.

Amino Acid Sequence↗
PubMed1982

Mapping of a plasmid, coding for colonization, factor antigen I and heat-stable enterotoxin production, isolated from an enterotoxigenic strain of Escherichia coli.

The non-autotransferring plasmid NTP113 codes for production of colonization factor antigen I and heat-stable enterotoxin, NTP113, which has a molecular weight of 58 X 10(6), was digested with BamHI, EcoRI, and HindIII and combinations of these restriction endonucleases, and the products of these digestions were analyzed by agarose gel electrophoresis. The results were used to construct a partial restriction map of NTP113. Transposons coding for resistance to ampicillin, kanamycin, and tetracycline were inserted into NTP113, and we obtained a series of deletion mutants, as determined by the loss of tetracycline or kanamycin resistance from strains carrying the insertion mutants. A number of plasmid mutants obtained by insertion or deletion did not code for colonization factor antigen I, but most of these mutants still coded for heat-stable enterotoxin production. The position of the inserted transposons and of the deletions were determined on the restriction map. Two regions of NTP113 were required for the expression of colonization factor antigen I, and the two sites were separated by a length of DNA corresponding to a molecular weight of about 25 X10(6).

Antigens, Bacterial↗
PubMed1985

Analysis of cosmids using linearization by phage lambda terminase.

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Animals↗
PubMed1990

Isolation of a plasmid from "canine" Staphylococcus epidermidis mediating constitutive resistance to macrolides and lincosamides.

A small plasmid of 2.5 kB mediating constitutive resistance to macrolide-lincosamide-(ML)antibiotics could be detected in a "canine" Staphylococcus epidermidis-culture. This plasmid, designated as pSES 1, was identified by interspecies protoplast transformation into Staphylococcus aureus RN 4220. A detailed restriction map of pSES 1 could be constructed using the restriction endonucleases Acc I, Bcl I, Cfo I, Cla I, Hind III, Hinf I, Mbo I, Sst I and Taq I. This map allowed structural comparisons of pSES 1 with plasmids from "human" Staphylococcus- and Bacillus-species, also mediating macrolide-lincosamide resistance (MLR). On the basis of its restriction map, pSES 1 proved to be similar to the plasmids pNE 131 from "human" S. epidermidis, pE 194 from "human" S. aureus and pIM 13 from B. subtilis.

Animals↗
PubMed2005

[Construction of eukaryotic expression vector of siRNA specific to bcr/abl fusion gene].

OBJECTIVE: To construct eukaryotic expression vector of siRNA specific to bcr/abl and to initially investigate the effect of recombinant plasmid on bcr/abl and P210 protein expression in K562 cells. METHODS: siRNA (small interfering RNA) was designed according to the Tuschl's principle of RNAi-based medicine, and was converted into cDNA coding expression of shRNA (small hairpin RNAs)of siRNA for bcr/abl fusion gene. The cDNA was synthesized and inserted into plasmid pTER. The pTER117 and pTER363 of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymerase III, identified by the restriction map and the sequence analysis, and transfected into K562 cells by Lipofectamine. Expression of bcr/abl mRNA was assayed by RT-PCR; expression of P210 protein was detected by immunohistochemistry. RESULTS: The pTER117 and pTER363 of recombinant plasmid identified by the restriction map and the sequence analysis completely coincided with the designs. 24 hours after transfection in K562 cells, the recombinant plasmid could down regulate the expression of the bcr/abl mRNA and bcr/abl protein(P210) in K562 cells. CONCLUSION: The siRNA eukaryotic expression vector against bcr/abl mRNA has been successfully conctructed, and it effectively inhibits the expression of bcr/abl in K562 cells.

Base Sequence↗