Showing papers on "XhoI published in 1995"

Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5).

Abstract: Previous work from our laboratory has shown that cosmid clone pFV100, containing a 26 kb insert, is able to restore O-antigen synthesis in serotype O5 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV100 into two P. aeruginosa semi-rough (SR) mutants, AK14O1 and rd7513, resulted in O-antigen expression, indicating that pFV100 may contain an O-polymerase (rfc) gene. pFV.TK6, a subclone of pFV100 that contains a 5.6 kb chromosomal insert, was able to complement O-antigen expression in these SR mutants. Mutagenesis of pFV.TK6 using Tn1000 exposed a 1.5 kb region that was essential for complementing O-antigen expression in AK14O1. A 2.0 kb XhoI-HindIII fragment, containing this region, was cloned into vector pUCP26 and the resulting plasmid called pFV.TK8. In Southern analysis of the 20 P. aeruginosa serotypes using a probe generated from the 1.5 kb XhoI fragment of pFV.TK8, the rfc probe hybridized to a common fragment of the cross-reactive O2-O5-O16-O18-O20 serogroup, suggesting that these serotypes may share a common O-polymerase gene. In functional studies of the rfc gene, the PAO1 (serotype O5) chromosomal rfc was mutated using a gene-replacement strategy. These knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype, which indicated that they were no longer producing a functional O-polymerase enzyme. Nucleotide sequence analysis of the insert DNA of pFV.TK8 revealed one open reading frame (ORF), designated ORF48.9, which could code for a 48.9 kDa protein. In comparisons of the P. aeruginosa rfc nucleotide and amino acid sequences with DNA and protein databases, no significant homology was found. However, the deduced structure of the P. aeruginosa Rfc protein indicated that it is very hydrophobic and contains 11 putative membrane-spanning domains. Therefore, the predicted structure is similar to that of other reported Rfc proteins. Furthermore, comparison of the amino acid composition and codon usage of the P. aeruginosa Rfc with other Rfc proteins revealed significant similarity between them.

Lymantria dispar nuclear polyhedrosis virus homologous regions: characterization of their ability to function as replication origins.

Abstract: Homologous regions (hrs) were identified in the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) genome. A 1.58-kb region surrounding hr4 was sequenced and found to have two distinct domains. Domain I (about 600 bp) is composed of seven repeats of about 80 bp including a series of palindromes containing MluI sites and overlapping XhoI and SacI sites. Domain II (about 700 bp) is composed of eight partially repeated sequences of 60 to 100 bp containing a 15- to 25-bp sequence that is 80 to 100% A+T in addition to a 6- to 10-bp palindrome containing an NruI site. Hybridization of a domain I sequence to cosmids containing the LdMNPV genome indicated its presence at eight positions (hr1 to -8) on the genome. In contrast, hybridization of domain II indicated that it was present only at the hr4 locus. A DpnI-based transient-replication assay was used to determine if subclones of hr4 transfected into LdMNPV-infected L. dispar cells functioned as replication origins. Subclones of hr4 containing either domain I or domain II replicated at very low or moderate levels, respectively. However, when domain I and domain II were linked on the same plasmid, high levels of replication were observed. A 1.4-kb region containing hr1 was also sequenced. It lies immediately upstream of the polyhedrin gene and contains six domain I-type repeats. Four-hundred-base-pair regions of domain I repeats from hr1 and hr4 showed 89% sequence identity. Plasmids containing the hr1 domain I replicated at low levels. However, hybrid plasmids in which the AT-rich hr4 domain II was inserted adjacent to hr1 domain I replicated to high levels, indicating that the AT-rich domain II greatly enhances replication. The orientation and position of domains I and II relative to each other did not have major effects on the levels of replication.

Genome analysis of Clostridium botulinum type A by pulsed-field gel electrophoresis.

Abstract: Genomic DNA from type A Clostridium botulinum was digested with restriction endonucleases that cut at rare sites, and the large fragments were separated by pulsed-field gel electrophoresis. Of 15 restriction enzymes tested, MluI, RsrII, SmaI, NruI, KspI, NaeI, and XhoI generated satisfactory digestion patterns of genomic DNA of various C. botulinum strains, enabling the use of the method for genomic fingerprinting. The genomes of four group I (type A) C. botulinum strains examined had similar restriction patterns. However, each strain had unique digestion patterns, reflecting genotypic differences. The genome size of C. botulinum strain 62A was estimated to be 4,039 +/- 40 kbp from the summation of restriction fragments from MluI, RsrII, and SmaI digestions. Genes encoding proteins involved in the toxinogenicity of C. botulinum, including neurotoxin, hemagglutinin A, and genes for a temperate phage, as well as various transposon Tn916 insertion sites in C. botulinum 62A, were mapped by pulsed-field gel electrophoresis. The genes encoding neurotoxin and hemagglutinin A-1, were located on the same fragment in several cases, indicating their probable physical linkage. The macrorestriction analysis established here should be useful for genetic and epidemiological studies of C. botulinum.

Genotyping of human deoxyribonuclease i polymorphism by the polymerase chain reaction

Abstract: We recently completely elucidated the molecular basis of genetic polymorphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE1*1, *2, *3 and *4. In this paper we describe a novel DNase I-genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the three nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitutions neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE1*1 (or *3) and the DNASE1*2 (or *4) alleles, and to detect the DNASE1*4 allele. An amplification refractory mutation system was employed to detect DNASE1*3. A 100% correlation was found between the results of this genotyping method and those obtained by phenotyping using conventional isoelectric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I-polymorphism in small DNA samples.

Rapid sex determination of chick embryos using the polymerase chain reaction 1

Abstract: The sex of early chicken embryos was determined by DNA dot‐blot hybridization and the polymerase chain reaction (PCR). An oligonucleotide probe, specific to the XhoI family of repetitive DNA of the W chromosome, was hybridized to crude and purified preparations of DNA obtained from blood or cells from embryos and detected using chemiluminescence. Complete agreement was observed in sexing 100 embryos using the dot‐blot assay and visual inspection of embryonic gonadal tissue. Although the dot‐blot system was useful for determining the sex of embryos prior to and throughout incubation, the time required for hybridization and detection did not allow for the immediate manipulation of embryos. Therefore, a PCR assay was developed using primers specific to the XhoI repetitive element and rapid thermocycling with an air thermocycler. A female‐specific amplification product was observed using DNA from as few as two cells in the PCR. Furthermore, female DNA was detected in mixtures of male and female DNA a...

Restriction fragment length polymorphisms of mitochondrial DNAs from seven Fusarium species causing fusarium head blight.

Abstract: Mitochondrial DNAs (mtDNAs) were purified by CsCl/bisbenzimide density-gradient ultracentrifugation from 21 strains of seven Fusarium species that cause fusarium head blight and mycotoxin contamination in wheat and other cereals. A partial PstI clone bank, from which one of twelve PstI fragments (14.7 kb) is missing, was constructed using mtDNA from strain KU-1615 of F. graminearum. Molecular sizes of mtDNAs of single representative strains from the seven species were determined after single-, double- and triple-digestion by four or five restriction enzymes (BamHI, MluI, PstI, PvuII and XhoI), while those of others were after single-digestion by BamHI and/or PstI. MtDNA size varied from the smallest 49 kb in one strain of F. avenaceum to the largest 116 kb in one strain of F. culmorum. Restriction fragment length polymorphism (RFLP) analysis revealed a large interspecific variation, thus all the species were identified by their restriction fragment patterns and assigned to individual clusters except for F. tricinctum in that a strain studied showed identical patterns to one of two strains of F. sporotrichioides. Considerable intraspecific variation including size variation was also detected. These results indicated a high incidence of insertions/deletions both between and within species. On the basis of results obtained by the cluster analysis, some aspects of taxonomy in these Fusarium species were discussed.

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences.

Abstract: Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5′end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.

Conversion of mRNA into Double-Stranded cDNA

Abstract: Enzymatic conversion of mRNA into double-stranded insert DNA can be accomplished by a number of different procedures. All of them involve the action of reverse transcriptase and oligonucleotide-primed synthesis of cDNA. After that, the procedures in common use diverge considerably. There are a number of methods for synthesizing the second strand and several procedures for producing suitable ends for making clonable DNA. The major goals of these procedures are to construct insert DNA that is as long as possible, with a high yield of conversion of mRNA into DNA that can ligate to vector DNA. The following protocols require only commercially available reagents and are usually successful in producing good cDNA libraries. The basic protocol describes a method for making blunt-ended cDNA that can then be ligated to linkers for subsequent cloning into a unique restriction site such as EcoRI. The Alternate Protocol is a variation that requires fewer enzymatic manipulations and allows construction of directional cDNA libraries, which are particularly desirable when the goal is to generate expression cDNA libraries. The Alternate Protocol takes advantage of a linker-primer consisting of (in order from 3' to 5') an oligo(dT) primer, a restriction site for the XhoI endonuclease, and a (GA)20 repeat to protect the restriction site during generation of the blunt-ended cDNA. The internal XhoI sites on the individual cDNA molecules are protected by incorporation of 5-methyl-dCTP in the first-strand nucleotide mix. The resulting cDNAs having unique ends can be cloned into EcoRI/XhoI-digested vectors after ligation of EcoRI adaptors to the 5' end and digestion by XhoI to release the 3' XhoI sites that were incorporated into the cDNA by the linker-primer. These changes result in a considerably streamlined procedure that is substantially faster and easier than the basic protocol.

A recombinatorial method useful for cloning dominant alleles in Saccharomyces cerevisiae.

Abstract: A major inconvenience in cloning a dominant mutation in Saccharomyces cerevisiae is the requirement for the construction of a genomic library from the mutant strain (1). To alleviate the need for library construction, we present an alternative that is based on the ability of yeast cells to carry out precise and efficient homologous recombination (2). In principle, a dominant mutation may be cloned by co-transforming (3) a genomic library of a wild-type strain with genomic DNA derived from the mutant strain and selecting for transformants with the mutant phenotype. These should represent the following events: (i) Recombination between the genomic DNA fragment from the mutant strain, carrying the mutant gene, and the respective locus in the genome of the wild-type recipient strain. (ii) Recombination between the same DNA and a library plasmid that contains the gene of interest, if they coexisted within the same cell. (iii) A spontaneous mutation resulting in the mutant phenotype. A fourth possibility where a few additional copies of a wild-type gene would result in the same mutant phenotype, is considered unlikely and would be a caveat even for the classical approach. Co-segregation of the mutant phenotype with the plasmid should facilitate the selection of the second class events that eventually result in the isolation of the mutant gene of interest. We initially exploited the possibility of correcting a defect in a known gene carried on a centromeric plasmid, by applying the above outlined scheme of recombination between a plasmid and a genomic DNA fragment. A gcn2 , ura3-52 strain (4), sensitive to l0 mM of the drug 3-aminotriazole (3-AT), was co-transformed with 20 g of a URA3 marked plasmid (YCp50) harbouring a defective GCN2 gene, along with 50 g of genomic DNA (5) from the wild-type strain S288C. The GCN2 gene on the plasmid had been inactivated by digesting, filling in and religating the unique XhoI site within the GCN2 ORF (Fig. 1). Out of 20 000 primary Ura+ transformants, seven were resistant to 3-AT after replica-plating on minimal plates containing l0 mM 3-AT. In six cases this resistance was shown to be plasmid borne following eviction of the plasmid by treating the transformed cells with 1.0 mg/ml 5-Fluoroorotic acid (5-FOA; ref. 2). The plasmids from these six transformants were recovered (6) and checked for the fidelity of the correction, i.e. restoration of the XhoI restriction site. The results are shown in Figure 1. In this experiment, a correction could not have resulted from exchange of information between the plasmid, Figure 1. Repair of a gcn2 allele following co-transformation with genomic DNA fragments. (Top) Diagrammatic representation of the GCN2 gene locus showing the positions of the unique XhoI and the flanking BamHI sites. (Bottom) Restriction analysis of GCN2 bearing plasmids. Lane 1, HindIII digest of phage DNA, used as size standard. Lane 2, the YCp50 plasmid carrying the gnc2 mutant gene generated by destroying the XhoI site, digested with BamHI and XhoI. Lane 3, BamHI/XhoI double digest of a plasmid recovered from a 3-AT sensitive transformant. No restoration of the XhoI is evident. Lanes 4–9, BamHI/XhoI double digests of the plasmids recovered from the 3-AT resistant transformants. In all cases the XhoI site has been restored. Lane 10, BamHI/XhoI control digest of YCp50 carrying the wild-type GCN2 gene.

Physical mapping of differences between the chloroplast DNAs of Beta vulgaris and B. webbiana

Abstract: A physical map of Beta webbiana (a wild species of the section Pro-cumbentes) chloroplast genome was constructed by localizing the cleavage sites of SmaI, PstI, PvuII, XhoI, and HindIII, and the map was then aligned with the map of sugarbeet (B. vulgaris) chloroplast DNA. This alignment shows 27 restriction-site changes and 11 insertions/deletions, most of which occur in the large single-copy region of the genome. A 0.7-kb long mutation, located within an unidentified open reading frame (ORF2280) in the inverted repeat, was also found.