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PubMed2002

Comparative studies of the Acinetobacter genus and the species identification method based on the recA sequences.

The recA gene is indispensable for a maintaining and diversification of the bacterial genetic material. Given its important role in ensuring cell viability, it is not surprising that the RecA protein is both ubiquitous and well conserved among a range of prokaryotes. Previously, we reported Acinetobacter genomic species identification method based on PCR amplification of an internal fragment of the recA gene with subsequent restriction analysis (RFLP) with HinfI and MboI enzymes. In present study, the PCR products containing the internal fragment of the recA gene, for 25 Acinetobacter strains belonging to all genomic species, were sequenced. Based on the nucleotide sequences the restriction maps and phylogenetic tree were prepared. The restriction maps revealed that Tsp509I restriction enzyme is the most discriminating for RFLP. To verify the computer analysis, the amplified DNAs from all reference genomic species available (43 strains) and 34 clinical strains were digested with each of the three restriction endonucleases mentioned. The results of digestion confirmed the computer analysis. The reconstructed phylogenetic tree showed linkages between genomic species 1 (Acinetobacter calcoaceticus), 2 (Acinetobacter baumannii), 3, 'between 1 and 3', TU13 and 'close to TU13'; genomic species 4, 6, BJ13, BJ14, BJ15, BJ16 and BJ17; genomic species 7 (Acinetobacter johnsonii) and TU14; genomic species 10 and 11; genomic species 8 (Acinetobacter Iwoffii), 9, 12 (Acinetobacter radioresistens) and TU15; and genomic species 5 (Acinetobacter junii). It is interesting that one branch in the phylogenetic tree contains haemolytic species-genomic species 4 (A. haemolyticus), BJ13, BJ14, BJ15, BJ16 and BJ17. The proposed genotypic method clearly revealed that the RFLP profiles obtained with Tsp509I enzyme might be useful for species identification of Acinetobacter strains. In this context, recA/RFLP genotypic method should be seen as an ideal preliminary screening method for large numbers of isolates, with the ultimate confirmatory role reserved for DNA hybridization analysis.

Acinetobacter↗
PubMed1985

An electron microscopic heteroduplex study of the sequence relations between the bacteriophages LP52 and theta.

The genomes of the phylogenetically related but morphologically distinct bacteriophages LP52 and theta (theta) were compared by electron microscopic heteroduplex analysis. The heteroduplex maps were aligned with known restriction maps. In the heteroduplices of LP52 DNA (63.8 kb) with the DNA of the lytic phage theta c (65.9 kb) the tracts of homologous DNA cover about 50% of the genome length and are interspaced by four large and ten smaller non-base-paired regions. The largest block of non-homologous DNA (18.9 kb), represents the right-hand end and there is an unmatched piece of DNA at the left-hand end as well. Most of the heterology is due to substitution resulting in the conservation of the total length of DNA; the three insertions/deletions amount to less than 3.2% of the genome length. Heteroduplices between the DNAs of phage LP52 and the temperate phage theta 1 (65.0 kb) resembled those of LP52:theta c except for the absence of minor loops. Heteroduplex theta c:theta 1 displayed about 9% heterology in seven separate loops which coincided with sections of diversity on the restriction maps; 4.8% of theta 1 DNA did not hybridize with either theta c or LP52 DNA.

Bacillus↗
PubMed1990

Escherichia coli molecular genetic map (1000 kbp): update I.

The sequenced genes from Escherichia coli that are available in the EMBL library (release 21) have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al. (1987). One thousand kbp of sequenced DNA are incorporated in this update; this is equivalent to 23% of the total genome. The accuracy of the map is assessed, and it is corrected and updated where appropriate. A significant number of sites were missing from the original map, mainly involving two of the eight enzymes used by Kohara et al. (1987), ie. PvuII and EcoRV. The nucleotide environment of such missing sites was examined and, using an Artificial Intelligence approach, it appears that the site for these enzymes is sensitive to context effects. Several genes of known position on the E. coli chromosome could not be placed on the restriction map; this suggests that additional gaps are likely to exist on the restriction map, in addition to the original seven identified by Kohara et al. We have also obtained information about the probable direction of transcription of chromosomal genes with respect to the map. Most genes are transcribed in the same direction as the replication forks, particularly around oriC at 84 min.

Artificial Intelligence↗
PubMed1998

Gene synteny in species of Plasmodium.

We have attempted to establish the degree of linkage conservation between different species of the malaria parasite Plasmodium. Initially, the chromosome locations of 42 homologous genes were established in parasites from a rodent malaria species and the human malaria parasite P. falciparum. Of these genes, 26 appeared to be conserved within ten synteny groups between the two genomes. Several synteny groups were analysed further by long-range restriction mapping of digested chromosomes. Finally, a fine restriction map of one of the linkage groups was made from the rodent malaria parasites P. berghei and from P. falciparum and from the simian malaria parasite P. knowlesi. The fine-scale organisation of this linkage group appears to have remained intact among the three species, despite the evolutionary distance between them. This provides the first example of linkage conservation between the rodent, simian and human malaria species, which represent three different branches of the inferred phylogenetic tree of the genus Plasmodium.

Animals↗
PubMed1985

Characterization of cloned human alphoid satellite with an unusual monomeric construction: evidence for enrichment in HeLa small polydisperse circular DNA.

A recombinant DNA plasmid library was constructed from HeLa cell extrachromosomal circular DNA and the sequence organization of one family of clones, which contain sequences enriched in HeLa small polydisperse circular (spc) DNA, was studied by restriction mapping and base sequence analysis. Restriction mapping revealed each clone to be composed solely of imperfect tandem repeats of ca. 170 bp. The entire DNA sequence of one clone was determined and found to be alphoid satellite with a variant monomeric construction.

Base Sequence↗
PubMedDate not supplied

[Early transcription of FAV-1 (CELO) avian adenovirus genome].

The patterns of RNA from cells infected with avian adenovirus chicken embryo lethal orphan (CELO) virus were analyzed by Northern hybridization method. Early RNAs are specifically hybridized with several fragments of the Eco-RI restriction map: A (49-81%), B (0.2-20.3%), D (81-92.5%), and E fragments of the Bam-HI CELO DNA restriction map. Early RNAs were not hybridized with C (37.8-49%), G (31-37.8%), and E (20.3-31%) fragments of the Eco-RI DNA CELO restriction map. At least 19 types of virus-specific RNA molecules homologous to 4 different regions of the virus genome are produced in CELO-infected permissive cells. Several classes of virus-specific RNAs were identified in CELO virus-transformed cell strains.

Animals↗
PubMed1993

Subcloning and restriction enzyme mapping of bovine adenovirus type 2.

Bovine adenoviruses (BAVs), causing both respiratory and/or enteral diseases in cattle, have been isolated in many countries all over the world. In this study we report on the molecular cloning of the internal EcoRI fragments spanning 20.6-90.5%, and the internal SalI fragments spanning 3.1-65.2% of the BAV type 2 (BAV2; strain No. 19) genome into the plasmid vector pUC19. Moreover, the subcloning of the BAV2 genome facilitated the construction of a detailed physical restriction endonuclease map for BamHI, ClaI, EcoRI, HindIII, KpnI, NotI, NspV, PstI, PvuI, SalI, XbaI and XhoI.

Animals↗
PubMed1990

Extensive linkage disequilibrium in the achaete-scute complex of Drosophila melanogaster.

We have analyzed the level of gametic association between restriction map variants in a sample of 44 X chromosomes from a natural population of Drosophila melanogaster. Of 21 pairwise tests involving 7 restriction map polymorphisms in the yellow-achaete-scute complex, 17 were found to be significant, including some between restriction sites over 80 kb apart. Three-way linkage disequilibria and their variances were also estimated for all 35 three-way comparisons between these loci. Twelve such tests were found to be significant, again spanning distances of up to 80 kb on the restriction map. Only 9 of a possible 128 haplotypes were represented in the sample and 8 of these could be linked together by changes at a single site. The strength of these associations at y-ac-sc is unusual by comparison with studies on other regions of the genome of D. melanogaster, and is consistent with the very low level of recombination which has been reported for the complex. However, our estimate of nucleotide diversity in the region is not significantly different from those made for some other loci in this species.

Animals↗
PubMed1992

Restriction enzyme maps for equine adenovirus 1 genome.

Physical maps were constructed for the genome of equine adenovirus 1 (EAV1) using the restriction enzymes; DraI, EcoRV, NotI and SfiI. The total size of the EAV1 genome was 34.4 kb estimated by comparison with known DNA standards and the polarity of the fragment order, with respect to the left and right molecular ends, was determined by hybridization with known regions of the human adenovirus 2 (HAV2) genome.

Adenoviridae↗
PubMed1991

The derivation of a restriction endonuclease map for Alcelaphine herpesvirus 1 DNA.

The gammaherpesvirus Alcelaphine herpesvirus 1 (AHV-1), indigenous to the wildebeest (Connochaetes species), is a causative agent of the fatal lymphoproliferative disease malignant catarrhal fever in cattle and deer. The genome of the attenuated WC11 isolate of AHV-1 has previously been shown to possess a region of unique DNA together with multiple direct repeat sequences. Approximately 70% of the genome of WC11 has now been cloned into plasmid or bacteriophage vectors and these clones have been used in hybridisation experiments to construct a restriction endonuclease map of the WC11 unique DNA with respect to BamHI. EcoRI, SalI, SmaI and XhoI. The map allows the size of the unique region of the WC11 genome to be estimated as 130 kbp and thus the entire genome as 155-160 kbp. The results confirm a terminal location for the repeat sequences.

Bacteriophage lambda↗
PubMed1981

An improved method for estimating sequence divergence of DNA using restriction endonuclease mappings.

The method proposed by Kaplan and Langley for estimating the extent of sequence divergence between related DNA's using restriction endonuclease maps is modified so that the estimates are easier to compute. In the two-species case, these modifications lead via a maximum likelihood approach to an estimate which is closely related to one recently suggested by Nei and Li (1979) and Gotoh et al. (1979). Simulation studies show that the modified estimates are comparable to those of Kaplan and Langley, providing that there is sufficient homology in the DNA segments of the related species. The M-species case, M greater than or equal to 3, is also discussed.

Animals↗
PubMed1989

Molecular epidemiology and restriction site mapping of adenovirus type 3 genome types.

One hundred thirty-eight strains of adenovirus type 3 isolated from patients in the United States, West Germany, and other regions between 1961 and 1984 were analyzed with six restriction endonucleases; 18 genome types were found. BglII was the most discriminative enzyme. Mapping of altered restriction sites was also performed for all six enzymes. The genome types D1 (like the prototype) and D3 prevailed among 45 and 47 strains, respectively. All genome types could be divided into two groups related to D1 or D3. Several clusters of infections by strains with the same genome type were observed. Only hints of differences were found in the pathogenicities of individual genome types. D1 strains were present in the United States and in Europe; group D3 prevailed almost exclusively in the United States.

Adenoviridae Infections↗
PubMed1988

Anti-liver kidney microsome antibody recognizes a cytochrome P450 from the IID subfamily.

Children with autoimmune hepatitis have high serum titers of antibodies directed against a 50-kD protein of rat liver endoplasmic reticulum. Affinity-purified anti-50-kD antibodies were used to screen a rat liver cDNA library in lambda GT-11 expression vector. 12 immunopositive clones were obtained. Crossreactivities between fusion proteins of these clones and the 50-kD protein was demonstrated, and four clones were analyzed by restriction mapping, one of them by nucleotide sequencing. Complete identity was found between the restriction maps of two clones (LKMC1 and LKMC2) and that of the 5' end of the rat cytochrome P450 db2. Sequence of a 608-bp fragment of LKMC1 showed complete homology with the rat P450 db2 form. The restriction map of the other two clones (LKMC3 and LKMC4) was identical to that of rat P450 db1. These results suggest that the antigen recognized by LKMA is a P450 of the IID subfamily.

Animals↗
PubMed1987

Restriction enzyme map of herpesvirus of turkey DNA and its collinear relationship with Marek's disease virus DNA.

The genome of herpesvirus of turkey (HVT) was shown to consist of long and short unique regions flanked by inverted repeats (J. Cebrian, Kaschka-Dietrich, C., Berthelot, N., and Sheldrick, P., 1982, Proc. Natl. Acad. Sci. USA 79, 555-558). In this paper we report the construction of the linkage map of HVT DNA for BamHI, HindIII, and PstI restriction endonucleases. The maps were constructed by hybridization of 19 cloned BamHI fragments of HVT DNA to electrophoretically separated digests of genomic DNA. Our results indicate that the terminal and internal inverted repeats (TRL and IRL) flanking the long unique sequences (UL) are spanned by BamHI-F fragment and a -F-related terminal fragment, respectively, whereas the terminal and internal inverted repeats (TRS and IRS) flanking the short unique sequences (US) are mostly contained in BamHI-A fragment. Both BamHI-A and -F showed a heterogeneity in size, suggesting the presence of amplification of certain sequences in the inverted repeats. We also report that the HVT genome is collinear with the genetically related Marek's disease virus (MDV) genome, as determined by hybridization of labeled cloned HVT DNA fragments with electrophoretically separated MDV DNA fragments.

Animals↗
PubMed1988

'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.

DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds.

Amino Acid Sequence↗
PubMed1986

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta.

Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.

Aryldialkylphosphatase↗
PubMed2000

Restriction site mapping of the mitochondrial DNA of the four-wing flyingfish, Hirundichthys affinis.

Mitochondrial DNA was isolated from samples of the four-wing flyingfish, Hirundichthys affinis, collected in Barbados in January 1996 and subjected to restriction enzyme analysis, using 13 restriction endonucleases which recognise hexanucleotide sequences, in single and double digests. The resulting restricted DNA fragments were used to map the 14 enzyme recognition sites of 6 endonucleases (7 had no sites) on the flyingfish mtDNA molecule for the first time. In addition, the mtDNA D-loop region was positioned on the restriction site map, for the first time, by selective restriction digestion of the mtDNA molecule followed by polymerase chain reaction (PCR) amplification of the resulting fragments using specific mtDNA D-loop primers. The size of the flyingfish mtDNA molecule (18 kb) was also determined.

Animals↗
PubMed2002

Probing protein-DNA interactions by unzipping a single DNA double helix.

We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes (BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of approximately 25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (K(A)) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K(A) for EcoRI binding. The measured values are in good agreement with previous measurements of K(A) over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.

Base Sequence↗