Showing papers on "XhoI published in 1986"

Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes.

Abstract: A gene cluster encoding biphenyl- and chlorobiphenyl-degrading enzymes was cloned from a soil pseudomonad into Pseudomonas aeruginosa PAO1161. Chromosomal DNA from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 was digested with restriction endonuclease XhoI and cloned into the unique XhoI site of broad-host-range plasmid pKF330. Of 8,000 transformants tested, only 1, containing the chimeric plasmid pMFB1, rendered the host cell able to convert biphenyls and chlorobiphenyls to ring meta cleavage compounds via dihydrodiols and dihydroxy compounds. The chimeric plasmid contained a 7.9-kilobase XhoI insert. Subcloning experiments revealed that the genes bphA (encoding biphenyl dioxygenase), bphB (encoding dihydrodiol dehydrogenase), and bphC (encoding 2,3-dihydroxybiphenyl dioxygenase) were coded for by the 7.9-kilobase fragment. The gene order was bphA-bphB-bphC. The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acids, and was encoded by a putative bphD gene, was missing from the cloned 7.9-kilobase fragment.

Analysis of 15 different genome types of adenovirus type 7 isolated on five continents.

Abstract: A total of 15 different genome types of adenovirus type 7 (Ad7), i.e., Ad7p, Ad7p1, Ad7a, Ad7a1 to Ad7a5, Ad7b, Ad7c, Ad7d, Ad7d1, Ad7e, Ad7f, and Ad7g, were identified among 40 selected strains isolated in Europe, Asia, North America, South America, and Australia by using restriction endonucleases BamHI, BclI, BglI, BglII, BstEII, EcoRI, HindIII, HpaI, SalI, SmaI, XbaI, and XhoI. Eight of them, Ad7p1, Ad7a1 to Ad7a5, Ad7d1, and Ad7g, are newly discovered. All 15 genome types could be distinguished by the four restriction endonucleases BamHI, BclI, BglI, and XbaI. At least four restriction sites differed between Ad7d and Ad7g. Pairwise analyses of comigrating DNA restriction fragments of all 15 Ad7 genome types were performed and presented in a schematic fashion. According to the degree of comigration of DNA restriction fragments, the 15 genome types could be divided into three clusters. Ad7b was the dominant genome type in different parts of the world and may have evolved in China into Ad7d and further to Ad7d1.

Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii.

Abstract: Multiple genomic regions homologous to nifH were found in the diazotroph Azotobacter vinelandii. The nifHDK gene cluster, located on a 12.8-kilobase (kb) XhoI fragment and two additional XhoI fragments (7.4 and 8.4 kb) hybridized to a nifH-specific DNA template but the 7.4- and 8.4-kb fragments did not hybridize to nifD- or nifK-specific DNA probes. In vivo transcription of the nifHDK gene cluster was ammonia-repressible and required the presence of at least 50 nM molybdenum in the derepression medium. Three mRNA species were transcribed from the nifHDK gene cluster, a 4.2-kb transcript homologous to nifH-, nifD-, and nifK-specific DNA templates, a 2.6-kb transcript homologous to nifH- and nifD-specific DNA templates, and a 1.2-kb transcript homologous only to the nifH-specific DNA template. In strain CA11, a nifHDK deletion mutant, the nifHDK-specific transcripts were not produced and the strain was unable to grow in N-free medium in the presence of Na2MoO4 at concentrations of 50 nM or higher. However, at concentrations of 25 nM Mo or less, growth occurred in N-free medium. Under these conditions two nifH-homologous (but not nifD- or nifK-homologous) transcripts were observed (1.2 and 1.8 kb). Presumably these were transcribed from the additional nifH-homologous sequences present in the genome. These results are consistent with the existence of two N2 fixation systems in A. vinelandii which are regulated by molybdenum at the level of transcription. Images

Cloning of a gene involved in regulation of exotoxin A expression in Pseudomonas aeruginosa.

Abstract: We have cloned a gene from Pseudomonas aeruginosa that stimulates the expression of exotoxin A. A recombinant library of genomic DNA from strain PA103 constructed with a broad-host-range plasmid vector containing chromosomal insert fragments generated by Sau3A was used to transform the hypotoxigenic mutant strain PA103-29. A recombinant plasmid, pFHK6, was isolated from a PA103-29 transformant which displayed increased toxin production. From pFHK6, which contained a 20-kilobase-pair chromosomal insert, a 3-kilobase-pair XhoI fragment was isolated and subcloned into the plasmid cloning vector pVK101 to give pFHK10. In toxigenic P. aeruginosa strains containing pFHK10, toxin expression was increased 10-fold and high levels of iron in the culture medium only partially inhibited the overproduction. Expression studies suggested that pFHK10 did not contain the toxin structural gene. In addition, Southern analysis with the 3-kilobase-pair XhoI fragment suggested that the putative toxin regulatory gene is common among different strains of P. aeruginosa including previously reported nontoxigenic strains.

Terminal structure and heterogeneity in human cytomegalovirus strain AD169.

Abstract: We have characterized the heterogeneity occurring at the junction of the long (L) and short (S) segments and at the termini of the strain AD169 human cytomegalovirus (HCMV) genome by restriction endonuclease mapping and nucleotide sequence analyses. The HCMV a sequence was identified by its position at both termini and inverted orientation at the L-S junction. Heterogeneity at both termini and the L-S junction was generated by the presence of fused and tandem a sequences. Some S termini lacked an a sequence. In addition, near the L terminus and at the L-S junction there were a variable number of 217-base-pair (bp) XhoI fragments arranged in tandem. The 217-bp fragments consisted of a portion of the a and adjacent b sequences (in the L-segment repeat) bounded by the same direct repeats (DR1) found at the boundaries of the a sequence. A model for the generation of these heterogeneous fragments is presented. We also determined the sequence of seven cloned terminal fragments, five from the L terminus and two from the S terminus. All L termini contained identical terminal sequences ending with base 32 of a 33-bp DR1. The S termini differed from each other and from the L-segment termini. One S terminus lacked an a sequence and terminated within S-segment repeat (c) sequences. The second S terminus contained an a sequence and terminated with bases 20 to 33 of a 33-bp DR1. A comparison of the cloned L and S terminal sequences with cloned L-S junction sequences suggested that the termini contained 3' single base extensions which were removed during the cloning. We also show that the herpesvirus conserved sequence is in a similar position relative to the termini of HCMV and several other herpesviruses, thus adding further support for the role of the sequence in the maturation of viral DNA.

A restriction map of naphthalene catabolic plasmid pWW60-1 and the location of some of its catabolic genes

Abstract: SUMMARY: A restriction map for the 87 kbp IncP9 naphthalene catabolic plasmid pWW60-1 is presented. Transposon mutants were obtained using direct Tn5 insertion or using an indirect method involving the intermediate formation of unstable cointegrates between RP4 and pWW60-1. Insertions which affected expression of the early enzymes of the pathway (naphthalene to salicylate) were separated from inserts which affected expression of salicylate hydroxylase (nahG) or catechol 2,3-oxygenase (nahH). nahABC were cloned on a contiguous region of the plasmid on either a 6.9 kbp HindIII fragment (HE) or a 5.7 kbp XhoI fragment (XD). nahGH were cloned on a region situated about 30 kbp from nahABC on an XhoI fragment (XC), but nahH was only expressed on the corresponding but smaller XhoI fragments from two derivative plasmids of pWW60-1 with a deletion in that region. The detailed restriction map of nahH shows no similarities with the restriction maps of the genes for catechol 2,3-oxygenases from TOL plasmids, although the cloned gene did hybridize with genes for catechol 2,3-oxygenase from two different TOL plasmids. The organization of the catabolic genes on pWW60-1 suggests a separation into two operons as also described for the naphthalene catabolic plasmid NAH7 (alternatively called pIG7), but the relative directions of their transcription differs from that in NAH7.

Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes.

Abstract: Structural genes for catechol 2,3-oxygenase (C23O) were cloned from the TOL plasmids pWW5, pWW14, pWW74, pWW84, and pWW88 isolated from Pseudomonas strains of diverse geographical origins. Each pKT230-based C23O+ recombinant plasmid carried a 2.05-kilobase XhoI insert which showed strong homology in Southern hybridizations with the xylE gene from the archetype TOL plasmid pWW0. Fragments were mapped for restriction endonuclease sites and were classified into two closely related groups on the basis of restriction maps. C23O structural genes were located on cloned fragments by a combination of subcloning and site-specific mutagenesis. All five TOL plasmids examined yielded clones whose maps differed from that of xylE of pWW0 by only a single XbaI site, but in addition plasmids pWW5, pWW74, and pWW88 carried a second, homologous C23O gene with seven further restriction site differences. The remaining plasmids, pWW14 and pWW84, carried a second nonhomologous C23O gene related to the second C23O gene (C23OII) of TOL plasmid pWW15 described previously (H. Keil, M. R. Lebens, and P. A. Williams, J. Bacteriol. 163:248-255, 1985). Thus, each naturally occurring TOL plasmid in this study appears to carry genes for two meta cleavage dioxygenases.

Structural organization of the Amy locus In seven strains of Drosophila melanogaster

Abstract: Restriction maps were made by Southern blot analysis of the Amy (alpha-amylase) region in 7 strains of D. melanogaster using endonucleases SalI, XhoI and EcoRI. These were compared to the map of lambda Dm65 which contains the cloned Amy region. Strains used produce either two amylase variants, a single variant, or no amylase, yet all 7 strains carry two Amy genes as inverted repeats at the Amy locus. This and the orientation of the repeats resembles the situation in lambda Dm65. Most restriction sites mapped are conserved but two strains contain a large insertion which differs in size and position between strains. A complex anomaly, probably an inversion, exists at the Amy locus in a null strain. Maps for our Amy1,3 strain and the lambda Dm65 clone are identical, the DNA of each having been derived from a Canton-S wild stock. Restriction and genetic maps of the Amy region were aligned and alleles assigned to the proximal and distal genes, Amy-p and Amy-d.

Chloroplast genome organisation in sugar beet and maize.

Abstract: The XhoI and SmaI restriction map of the chloroplast genome from the fertile cytoplasm of sugar beet has been constructed from overlapping cosmid clones. The genome was found to be typical of that of a dicotyledonous species, being 147.3 kb in size and having an inverted repeat. RbcL for the large subunit of ribulose-1,5-bisphosphate carboxylase, psbA for the 32 kD protein of the photosystem II reaction centre, and the 16S ribosomal RNA were located using heterologous probes. In both sugar beet and maize the inverted repeats recombine giving two isomeric forms of the genome.

Structural alterations in a transcribed region of the T type cytoplasmic male sterile maize mitochondrial genome.

Abstract: H. maydis toxin resistance, and cytoplasmic reversion from sterility to fertility in Zea mays T type cytoplasm are correlated with sequence changes in a 6.6 kb Xhol fragment of T mitochondrial DNA. Comparative Northern blot analysis of N (normal male fertile), and cmsT (cytoplasmic male sterile) mitochondrial RNAs using subclones of the 6.6 kb Xho I region of cmsT as probes, reveals different sized transcripts. In cmsT these RNA coding sequences have been mapped onto a 1.5 kb AvaI fragment completely internal to the 6.6 kb XhoI region. Comparative Southern analysis of AvaI digested mitochondrial DNA from cmsT, N and a T-type cytoplasm which has reverted to fertility (designated V3) (Brettel et al. 1979), positions the RNA coding sequences on a 1.5 kb, 2.1 kb and 2.1 kb fragment respectively. These sequence rearrangements in the fertile T revertants create a novel mtRNA transcript. Southern hybridization experiments of other higher plant mitochondrial DNAs using the 1.5 kb Ava I fragment from cmsT mtDNA as a probe, indicate the presence of homologous sequences.

Isolation of a deoxycytidylate methyl transferase capable of protecting DNA uniquely against cleavage by endonuclease R.Aqu I (isoschizomer of Ava I)

Abstract: A sequence-specific modification methylase (M.AquI) was isolated and purified from Agmenellum quadruplicatum (Synechococcus PCC 7002). This enzyme uniquely methylates the deoxycytidylate residue in the sequence *CYCGRG indicated by the asterisk. It was shown to protect DNA against cleavage by restriction endonucleases AvaI, SmaI and XhoI, which recognize the sequences CYCGRG, CCCGGG, and CTCGAG, respectively.

The repeated sequences (incB) preceding the protein E gene of plasmid mini-F are essential for replication.

Abstract: At the XhoI site (45.08F) of plasmid mini-F a deletion of 649 bp was generated employing exonuclease Bal31. By this deletion nucleotide sequences functioning as origin II and the four 19 bp direct repeats constituting the incB region in front of the E protein gene were removed from the plasmid. Analysis of proteins radioactively labelled in Escherichia coli mini-cells indicated that all mini-F encoded proteins are expressed. However, the plasmid carrying the deletion was not capable of replicating from the primary origin (origin I, 42.6F). Recently a smaller deletion at the XhoI site (45.08F) of about 300 bp, removing only the region functioning as origin II and replicating from origin I, was described by Tanimoto and Iino (1984, 1985). The data presented suggest that the incB repeats are essential for the initiation of replication from origin I, and possibly also from origin II, and seem not to be engaged in the autoregulation of E protein expression.

Methylation of spore DNA in Bacillus coagulans strain 26

Abstract: SUMMARY: The modification status of DNA throughout the life cycle of Bacillus coagulans strain 26 was analysed by restriction analysis with methylation-sensitive enzymes. A significant fraction of the GATC sequences (dam target) in spore DNA contain N 6-methyladenine, a modification that is lacking during the vegetative phase. From the modulation of the modification pattern of GATC sites, the existence of a de novo methylase may be inferred. Spore DNA was more sensitive than vegetative cell DNA to BamHI, HpaI, SalI and XhoI, indicating that the sites for these enzymes are modified during the vegetative growth phase.

[Studies on new adenovirus types of subgenus D causing conjunctivitis--antigenic and restriction endonuclease analysis of adenovirus types 19 and 37].

Abstract: Neutralization test and DNA restriction endonuclease analysis were performed in strains of adenovirus types 19 (Ad-19) and 37 (Ad-37) isolated from patients with acute conjunctivitis in Sapporo, Kao-Hsiung (Taiwan) and Pusan (Korea) in the period of 1979 and 1984. Although Ad-19 and Ad-37 were differentiated, they were related to each other in neutralization test. There was no difference between prototype strain and isolates in Ad-19 and Ad-37 strains. By DNA restriction endonuclease analysis Ad-19 isolates were clearly distinct from Ad-19 prototype strain (AV-587) and Ad-37 strains. And it was suggested that these Ad-19 isolates from East Asia were as same as isolates from Europe named Ad-19a. Ad-37 isolated strains were as same as Ad-37 prototype strain (GW) by SmaI, SacI, SalI, BamHI, EcoRI, XhoI, HindIII restriction analysis. But some strains were distinct from prototype strain by only HindIII restriction analysis, and they were new subtype of Ad-37 which had never been reported.

Physical map of Pseudomonas aeruginosa phage phi kF77 genome. Localization of sites sensitive to restriction endonucleases

Abstract: A physical map of the P. aeruginosa bacteriophage phi kF77 has been constructed using the restriction endonucleases SalI, HindIII, EcoRI, EcoRV, MuI, XbaI, ClaI. The phi kF77 DNA is resistant to cleavage by the restriction endonucleases BamHI, BglII, HpaI, PstI, PvuII, SmaI, XhoI.