Showing papers on "Restriction map published in 1994"

Computer analysis of sequence data.

Abstract: GCG: Fragment Assembly Programs. GCG: Drawing Linear Restriction Maps. GCG: Drawing Circular Restriction Maps. GCG: Displaying Restriction Sites and Possible Translations in a DNA Sequence. GCG: Assembly of Sequences into New Sequence Constructs. GCG: Comparison of Sequences. GCG: Production of Multiple-Sequence Alignment. GCG: Database Searching. GCG: Pattern Recognition. GCG: Translation of DNA Sequence. GCG: Analysis of Protein Sequences. GCG: The Analysis of RNA Secondary Structure. GCG: Preparing Sequence Data for Publication. MicroGenie: Introduction and Restriction Enzyme Analysis. MicroGenie: Shotgun DNA Sequencing. MicroGenie: Translation. MicroGenie: Protein Analysis. MicroGenie: Homology Searches. PC/GENE: Sequence Entry and Assembly. PC/GENE: Restriction Enzyme Analysis. PC/GENE: Translation and Searches for Protein Coding Regions. PC/GENE: Sequence Comparisons and Homologies. PC/GENE: Database Searches. PC/GENE: Searches for Functional Sites in Nucleic Acids and Proteins. Using the FASTA Program to Search Protein and DNA Sequence Databases. Converting Between Sequence Formats. Obtaining Software via INTERNET. Submission of Nucleotide Sequence Data to EMBL/GenBank/DDBJ. Index.

Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.

Abstract: Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes.

Complete Dna-Sequence Of Yeast Chromosome-Xi

Abstract: The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

A DNA-based approach to the identification of insect species used for postmortem interval estimation

Abstract: Insect larvae found on a corpse can be used for estimating postmortem intervals. Here, we describe a molecular method for rapid identification of these insects. Specific insect DNA fragments were amplified using the polymerase chain reaction (PCR), followed by direct DNA sequencing of the amplification products. We sequenced 2300 base pairs of mitochondrial DNA from each of three blowfly species: Phormia regina, Phaenicia sericata and Lucilia illustris. All three species are important in forensic entomology. We found 118 nucleotide differences between the L. illustris and P. sericata sequences, 186 between L. illustris and P. regina, and 192 between P. sericata and P. regina. Based on these abundant DNA sequence differences, we can unambiguously identify the immature larval stages of these insects. These DNA sequence differences were also used to predict species-specific, diagnostic restriction sites in the amplified DNA, and these predictions were verified by digestion with nine restriction enzymes. The DNA sequences reported here encode the mitochondrial COI, COII and tRNA-leucine genes.

Tagged mutations at the Tox1 locus of Cochliobolus heterostrophus by restriction enzyme-mediated integration.

Abstract: We have used the restriction enzyme-mediated integration insertional mutagenesis procedure to tag the Tox1 locus in the filamentous Ascomycete Cochliobolus heterostrophus. Mutations at other, unselected, loci were also identified and a high proportion (30-50%) of them were tagged. This procedure may be of general utility for simultaneously mutating and tagging genes in fungi and in other eukaryotes. The Tox1 locus of C. heterostrophus has been defined by Mendelian analysis as a single genetic element that controls production of T toxin, a linear polyketide involved in virulence of the fungus to its host plant, corn. To tag Tox1, protoplasts of a Tox1+ (T-toxin producing) strain were transformed with a linearized, nonhomologous plasmid along with an excess of the restriction enzyme used to linearize the plasmid. Of 1310 transformants recovered, two produced no detectable T toxin in culture or on corn plants. In each of these transformants, the Tox- mutation mapped at Tox1, was tagged with the selectable marker (hygB) on the transforming plasmid, and was tightly linked to the other tagged Tox- mutation. The two mutations, however, represent two different points of plasmid insertion at the Tox1 locus.

Identification of an imprinted U2af binding protein related sequence on mouse chromosome 11 using the RLGS method

Abstract: A new imprinted gene has been discovered in mice using the technique of restriction landmark genomic scanning (RLGS) with methylation sensitive enzymes. Eight out of 3,100 strain-specific NotI and BssHII spots were identified as imprinted in reciprocal F1 hybrids. Subsequently, we isolated a genomic clone for one locus on proximal chromosome 11 near the Glns locus, an imprinted region in uniparental disomic mice, and its corresponding cDNA clone. Expression of this transcript from the paternal allele was established using RT-PCR of reciprocal F1-hybrid mice. The amino-acid sequence deduced from the cDNA showed significant homology to the U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit.

Two Mhc class I and two Mhc class II genes map to the chicken Rfp-Y system outside the B complex

Abstract: Gene sequences highly similar to major histocompatibility complex (Mhc) class I and class II genes were recently recognized as mapping to a site in the genome of the chicken separate from the Mhc class I, class II, and B-G genes of the major histocompatibility (B) complex. The present study was undertaken to see whether this complex of Mhc-like genes designated as restriction fragment pattern Y (Rfp-Y) might reside in one of three clusters of cosmid clones contained within the molecular map of chicken Mhc genes, since only two of the three clusters can be assigned to the B system. To determine whether the third cluster (cluster II/IV) might contain Rfp-Y, a subclone (18.1) from within cluster II/IV near a polymorphic lectin gene was used to analyze the DNA of families in which Rfp-Y haplotypes are known to be segregating. The restriction fragment polymorphisms revealed by the 18.1 probe were found to segregate in parallel with the restriction fragment polymorphisms defining the Rfp-Y haplotypes, thus establishing the location of Rfp-Y within cosmid cluster II/IV. Two of six Mhc class I genes and two of five Mhc class II genes map to cosmid cluster II/IV, so a substantial fraction of chicken Mhc genes, including at least one that may be expressed, are located in a chromosomal region separate from the B system. In further linkage analyses, Rfp-Y was found to assort independently from more than 400 markers in the present linkage map of the chicken genome.

Extension of Autographa californica nuclear polyhedrosis virus host range by interspecific replacement of a short DNA sequence in the p143 helicase gene

Abstract: Recombinant baculoviruses obtained by coinfection of insect cells with Autographa californica and Bombyx mori nuclear polyhedrosis viruses (AcNPV and BmNPV, respectively) possess a wider in vitro host range than either parent virus. To localize the DNA sequences responsible for this species specificity, we used a two-step method of production and selection of recombinant viruses with altered specificity. Sf9 cells, which are permissive for AcNPV, were first cotransfected with genomic AcNPV DNA and a complete or incomplete set of BmNPV restriction fragments. AcNPV-BmNPV recombinants from the Sf9 supernatant were then selected on the basis of ability to replicate in B. mori Bm5 cells, which are not permissive for AcNPV. Cotransfection of AcNPV DNA with the 7.6-kbp BmNPV Sma I-C fragment was sufficient to produce recombinants able to infect both Sf9 and Bm5 cells. A series of cotransfections with subclones of this fragment defined a 79-nt sequence within the p143 helicase gene capable of extending AcNPV host range in vitro. In this 79-nt region, BmNPV and AcNPV differ at six positions, corresponding to four amino acid substitutions. The involvement of the 79-nt region in species specificity control was confirmed by cotransfecting AcNPV DNA and gel-purified polymerase chain reaction products derived from the BmNPV p143 gene. Replacement in the AcNPV genome of three AcNPV-specific amino acids by the three corresponding BmNPV-specific amino acids at positions 556, 564, and 577 of the p143 protein extends AcNPV host range to B. mori larvae.

Ordered cosmid library and high-resolution physical-genetic map of Helicobacter pylori strain NCTC11638.

Abstract: Helicobacter pylori is a Gram-negative bacterium that infects the human gastric mucosa, causes gastritis and contributes to the development of peptic ulcers and gastric cancer. To facilitate molecular genetic analysis of this pathogen, we constructed a approximately 20-fold redundant cosmid library and physical/genetic map of strain NCTC11638. Genomic DNA fragments were cloned into the cosmid vector Lorist6, and clones were ordered by hybridization with several types of probes: (i) ends of cloned DNAs; (ii) chromosomal Notl digest fragments; (iii) cosmids containing Notl sites; and (iv) specific genes. Seven hundred and fifty-one cosmids were mapped to one of three contigs covering > 90% of the chromosome, and are represented by a 68-cosmid miniset. The order of cosmids was confirmed and extents of overlap among them were estimated by restriction analysis. All currently known H. pylori genes were mapped, including those for a cytotoxin (vacA), cytotoxin-associated protein (cagA), urease and regulatory functions (ureAb, ureD and ureH), catalase (katA), major and minor flagellins (flaA and flaB), heat-shock (stress) and chaperone proteins (dnaK, htA, hspB (groEL)), prokaryotic ferritin (pfr), an adhesin subunit (hpaA), a surface protein (26 kDa), and 16S and 23S ribosomal RNAs (two genes each). The orientations of eight genes or clusters were determined, and two repetitive sequences were also found. The gene order and rRNA gene copy number determined here differed from that reported for an unrelated strain, which suggests considerable flexibility in H. pylori genome organization.

Multiple replicons constituting the genome of Pseudomonas cepacia 17616.

Abstract: Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasmid present in this strain gave an overall estimate of genome size of 7 Mb. Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence elements identified previously in this strain. The distribution of SwaI, PacI, and PmeI sites on the three replicons was determined. A derivative of Tn5-751 carrying a SwaI site was used to inactivate and map genes on the 2.5- and 3.4-Mb replicons. Mutants were isolated in which the 2.5- and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively. The loss of DNA from the 2.5-Mb replicon was associated with lysine auxotrophy, beta-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis. The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates.

Pausing of the restriction endonuclease EcoRI during linear diffusion on DNA.

Abstract: Linear diffusion is a mechanism to accelerate association rates beyond their three-dimensional diffusional limit It is employed by the restriction endonuclease EcoRI as well as many other proteins interacting with specific DNA sequences to locate their target sites on the macromolecular substrate In order to investigate biochemical and biophysical details of the linear diffusion process, we have developed a competitive cleavage assay which allows us to assess with great accuracy the influence of sequence, sequence context, and other structural features on the linear diffusion of EcoRI on DNA We show here that linear diffusion is not a hopping but a sliding movement in which EcoRI follows the helical pitch of the DNA, because it does not "overlook" any cleavage site Linear diffusion is slowed when EcoRI encounters sites on the DNA which resemble its recognition site ("star" sites) Pauses of up to 20 s are induced, depending on sequence and orientation of the star site These data suggest that EcoRI can bind to DNA in two binding modes: one tight, specific, and immobile, leading to DNA cleavage, and another one loose and nonspecific, allowing for linear diffusion Depending on the similarity between the recognition sequence and the DNA sequence being encountered by EcoRI, there will be a continuous transition between these binding modes Other proteins bound to the DNA and irregular DNA structures such as bent DNA or a triple helix constitute a barrier that cannot easily be passed by EcoRI

Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90.

Abstract: Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.

Presence of Two Sets of Ribosomal Genes in Phytopathogenic Mollicutes

Abstract: DNA from 28 strains of phytopathogenic mycoplasmalike organisms that represented five primary taxonomic clusters was digested with restriction endonucleases and hybridized with several ribosomal probes. The results indicate the presence of two sets of ribosomal genes in all strains examined. Restriction maps of the two ribosomal operons for a group of 12 aster yellows mycoplasmalike organisms were constructed. Images

Detection and species identification of Cryptosporidium oocysts using a system based on PCR and endonuclease restriction

Abstract: The polymerase chain reaction (PCR) was used to produce a 556 bp nucleotide stretch, employing primers based on the published sequence of the 18S rRNA genes in Cryptosporidium parvum and C. muris. This sequence was found to contain 3 Mae I endonuclease restriction sites, 1 of which was present only in C. parvum. Mae I restriction of PCR products from 2 C. parvum isolates (one of human origin and the other of bovine origin), 1 C. muris isolate, and 1 C. baileyi isolate, showed a specific and reproducible profile for C. parvum that was different from the one obtained for both C. muris and C. baileyi. From these data, new Mae I restriction maps were proposed for the three species. The system was then used to screen 6 C. parvum isolates (from human and bovine hosts), and the C. parvum-specific profile was obtained for all isolates examined. It should be possible to adapt this protocol to detect small numbers of C. parvum oocysts in environmental samples (e.g. in water supplies).

Polydnavirus of the parasitic wasp Chelonus inanitus (Braconidae): characterization, genome organization and time point of replication.

Abstract: Ultrastructural analysis of the polydnavirus of the braconid wasp Chelonus inanitus revealed that virions consist of one cylindrical nucleocapsid enveloped by a single unit membrane. Nucleocapsids have a constant diameter of 33.7 +/- 1.4 nm and a variable length of between 8 and 46 nm. Spreading of viral DNA showed that the genome consists of circular dsDNA molecules of variable sizes and measurement of the contour lengths indicated sizes of between 7 and 31 kbp. When virions were exposed to osmotic shock conditions to release the DNA, only one circular molecule was released per particle suggesting that the various DNA molecules are singly encapsidated in this bracovirus. The viral genome was seen to consist of at least 10 different segments and the aggregate genome size is in the order of 200 kbp. By partial digestion of viral DNA with HindIII or EcoRI in the presence of ethidium bromide and subsequent ligation with HindIII-cut pSP65 or EcoRI-cut pSP64 and transfection into Escherichia coli, libraries of 103 HindIII and 23 EcoRI clones were obtained. Southern blots revealed that complete and unrearranged segments were cloned with this approach, and restriction maps for five segments were obtained. Part of a 16.8 kbp segment was sequenced, found to be AT-rich (73%) and to contain six copies of a 17 bp repeated sequence. The development of the female reproductive tract in the course of pupal-adult development of the wasp was investigated and seen to be strictly correlated with the pigmentation pattern. By the use of a semiquantitative PCR, replication of viral DNA was observed to initiate at a specific stage of pupal-adult development.

Cloning and Sequencing of a Rhodothermus marinus Gene, bglA, Coding for a Thermostable β-Glucanase and its Expression in Escherichia coli

Abstract: A gene library of the thermophilic eubacterium, Rhodothermus marinus, strain 21, was prepared in pUC18 and used to transform Escherichia coli. Of 5400 transformants, two produced halos on lichenan plates after Congo-red staining. Restriction mapping showed that the two clones shared an overlapping 1200-bp DNA fragment, which was used for DNA sequencing. Five potential methionine (Met) translational-initiation codons were identified. A putative signal peptide of 30 amino acids was identified with a hydrophobic core of nine hydrophobic amino acids. The molecular mass of the mature enzyme was estimated to be 29.7 kDa. A comparison of the primary protein sequence of β-glucanase of Rhodothermus marinus with other glycosyl hydrolases showed 38.5% identity to the C-terminal part of the β-1,3-glucanase of Bacillus circulars and limited identity to bacterial endo-β-1,3–1,4-glucanases. The amino acid sequence showed high similarity to regions surrounding the catalytic Glu residue of bacterial β-glucanases. A gene fragment of 889 bp containing the catalytic domain was overexpressed in E. coli using the pET23, T7-phage RNA polymerase system. The enzyme showed activity on lichenan, β-glucan and laminarin but not on CMC cellulose or xylan. The expressed enzyme was purified by heat treatment of the host. The enzyme had a temperature and pH optima of 85°C and pH 7.0, respectively, and was shown to retain full activity after incubation for 16 h at 80°C and have a half life of 3 h at 85°C.

Physical mapping of the mitochondrial genome of Arabidopsis thaliana by cosmid and YAC clones

Abstract: As part of the worldwide efforts at molecular analysis of Arabidopsis thaliana as a model plant the complete structure of the mitochondrial genome has been determined. The mitochondrial DNA molecules were mapped by restriction fragment analysis of more than 300 cosmid clones and purified mitochondrial DNA. The entire genome of 372 kb is contained in three different configurations of circular molecules and is split into two additional subgenomic molecules of 234 kb and 138 kb, respectively. These arrangements result from recombinations of the two sets of repeats present in combinations of inverted and/or direct orientation. Alignment of YAC clones confirms the in vivo presence of continuous DNA molecules of more than 300 kb in A. thaliana mitochondria. The presence of this comparatively large mitochondrial genome in a plant with one of the smallest nuclear genomes shows that different size constraints act upon the different genomes in plant cells.

Infectious Bursal Disease Viruses: Molecular Differentiation of Antigenic Subtypes among Serotype 1 Viruses

Abstract: Published nucleotide sequence data for IBDV variant viruses (A and 1048E) and classic viruses (STC, 52-70, PBG98, Cu-1, and 002-73) were used to identify restriction enzyme sites that could potentially be used to differentiate these strains of IBDV. To test this hypothesis, the genomes of IBDV strains STC, MD, NC, and OH were converted to cDNA using reverse transcriptase (RT) and then amplified using the polymerase chain reaction (PCR). The PCR primers were selected from relatively conserved sequence regions in the VP2 gene, and they were used to amplify a 394-base-pair fragment. The restriction enzymes (RE) DraI, EcoRII, SacI, Sau3AI, StyI, and TaqI were tested for their ability to digest the RT/PCR products. The STC classic virus was SacI-, StyI-, and EcoRII-positive and DraI-, Sau3AI-, and TaqI-negative. The MD, NC, and OH viruses were DraI-, Sau3AI-, and TaqI-positive and SacI-, StyI-, and EcoRII-negative. The classic STC strain could be differentiated from the variant MD strain using the RT/PCR-RE assay. Based on these results and the presence or absence of other restriction sites that were predicted by published nucleotide sequence data, the RT/PCR-RE assay has the potential to differentiate IBDV isolates MD, A, and 1048E. Published nucleotide sequence data and the RT/PCR-RE results obtained using STC and MD indicated that variant viruses MD, A, and 1048E could be differentiated from classic viruses STC, 52-70, PBG98, Cu-1, 002-73, and NC.

Chloroplast DNA restriction site variation and phylogenetic relationships in the genus Thlaspi sensu lato (Brassicaceae).

Abstract: The classification of the genus Thlaspi s.l. is difficult and controversial. All hypotheses have been based on morphological and anatomical data and no cladistic analyses have been per- formed. In the current study restriction site variation of chloroplast DNA among 45 populations of 22 taxa was employed to assess phylogenetic relationships in the genus Thlaspi. Although only 22 taxa have been analyzed herein (ca. 30% worldwide) these species represent all sections. One length mutation and 114 restriction site mutations were detected. Cladistic analysis of the chloroplast DNA data supported four groups that are congruent with four segregate genera (Thlaspi s. str., Microthlaspi, Noccaea, and Raparia), previously recognized by Meyer on the basis of seed anatomy. Sequence divergence between these groups was higher than usually found in intrageneric analyses and comparable to levels of divergence between related genera of other angiosperm families. The chloroplast DNA phylogeny suggests that the perennial taxa are not ancestral to the annual taxa and that the annual habit originated more than once in Thlaspi s.l. A paucity of restriction site mutations precludes resolving relationships among species of Noccaea, whereas the two intraspecific taxa of the T. perfoliatum polyploid complex (Microthlaspi) were separated by at least 17 restriction site mutations. Consideration of chloroplast DNA divergence within the clades Noccaea and Mi- crothlaspi indicates that the rate of plastome evolution is uncoupled from rates of morphological/ anatomical diversification. Chloroplast DNA polymorphisms were detected among eight popula- tions of T. perfoliatum subsp. perfoliatum with different ploidy levels. There seems to be geographic partitioning of the two plastome types found, possibly warranting taxonomic recognition. The genus Thlaspi L. s.l. comprises approxi-

Fluorescent Pseudomonas species categorized by using polymerase chain reaction (PCR)/restriction fragment analysis of 16S rDNA.

Abstract: A rapid procedure for the identification of fluorescent pseudomonads, based on the polymerase chain reaction (PCR) and restriction fragment analysis of 16S rDNA genes is described. Thirty-one strains belonging to 10 different Pseudomonas species of the Pseudomonas fluorescens rRNA branch were characterized. Amplified rDNA was digested with 13 different restriction endonucleases. The combined data from restriction analysis enabled the definition of 17 different 16S rDNA genotypes. All type strains belonging to different species were differentiated. The good correlation between grouping obtained using restriction analysis with other molecular classification criteria demonstrates the value of the described method to characterize rapidly fluorescent Pseudomonas strains at the species level.