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1.
The DNA of the temperate bacteriophage Aaφ23 isolated from the oral bacterium Actinobacillus actinomycetemcomitans was examined structurally both in the phage head and in the prophage. The DNA in phage particles comprises 44?kb linear molecules with a terminal redundancy of 1.6?kb, which represent circular permutations. Thus, DNA is packaged into phage heads by the headful mechanism. The Aaφ23 prophage is integrated into the host chromosome.  相似文献   

2.
A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10 752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100 kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180 bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80 kb <85% <120 kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing. Received: 26 November 1997 / Accepted: 19 February 1998  相似文献   

3.
The φ29-like phage genus of Podoviridae family contains phages B103, BS32, GA-1, M2, Nf, φ15, φ29, and PZA that all infect Bacillus subtilis. They have very similar morphology and their genomes consist of linear double-stranded DNA of approximately 20 kb. The nucleotide sequences of individual genomes or their parts determined thus far show that these phages evolved from a common ancestor. A terminal protein (TP) that is covalently bound to the DNA 5′-end primes DNA replication of these phages. The same mechanism of DNA replication is used by the Cp-1 related phages (also members of the Podoviridae family) and by the phage PRD1 (member of the Tectoviridae family). Based on the complete or partial genomic sequence data of these phages it was possible to analyze the evolutionary relationship within the φ29-like phage genus as well as to other protein-primed replicating phages. Noncoding regions containing origins of replication were used in the analysis, as well as amino acid sequences of DNA polymerases, and with the φ29-like phages also amino acid sequences of the terminal proteins and of the gene 17 protein product, an accessory component of bacteriophage DNA replicating machinery. Included in the analysis are also results of a comparison of these phage DNAs with the prophages present in the Bacillus subtilis genome. Based on this complex analysis we define and describe in more detail the evolutionary branches of φ29-like phages, one branch consisting of phages BS32, φ15, φ29, and PZA, the second branch composed of phages B103, M2, and Nf, and the third branch having phage GA-1 as its sole member. In addition, amino acid sequences of holins, proteins involved in phage lysis were used to extend the evolutionary study to other phages infecting Gram-positive bacteria. The analysis based on the amino acid sequences of holins showed several weak points in present bacteriophage classification. Received: 14 April 1998 / Accepted: 31 July 1998  相似文献   

4.
Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400 kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100 kb, spanned by nine overlapping cosmid clones. Received: 13 March 1997 / Accepted: 11 may 1997  相似文献   

5.
AnE. coli-Xanthomonas shuttle vector, p2GP (3.3 kb), was constructed by fusing pOK12 with a DNA fragment carrying gene II andori from filamentous phage φLf. p2GP retains the advantages afforded by pOK12 and can replicate in differentX. campestris pathovars. Superinfection ofX. c. pv.campestris cells harboring p2GP or its derivatives with φLf causes the release of ssDNA-containing recombinant phage particles which are sources of single-stranded template for sequencing the inserts.  相似文献   

6.
A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C. Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997  相似文献   

7.
8.
This study describes the isolation of temperate B. anthracis phages, from 4 out of 20 B. anthracis strains screened, by use of the inducing agents mitomycin C and UV light. Phage φ20 isolated from B. anthracis Sterne 34F2 (pXO1+ pXO2) was shown to have double-stranded DNA of size 48756 bp and a restriction site map showing nine sites for enzymes BamHI, BglII, and SstI is included. The φ20 genome was found to exist as a plasmidial prophage and the phage itself to have a polyhedral head of diameter 65 nm and tail 217 nm long and 15 nm wide.  相似文献   

9.
We describe the cDNA sequence for ARG7, the gene that encodes argininosuccinate lyase – a selectable nuclear marker – in Chlamydomonas reinhardtii. The 5′ end of the cDNA contains one more exon and the organisation of the mRNA is different from that predicted from the genomic sequence. When expressed under the control of the endogenous RbcS2 promoter, the 2.22-kb cDNA complements the arg7 mutation as well as the genomic DNA. A linear cDNA fragment lacking promoter sequences is also able to complement, suggesting that it could be used in promoter-trapping experiments. Despite the presence of a sequence encoding a potential chloroplast transit peptide in the cDNA the protein is not targeted to the chloroplast, nor can it complement the arg7 mutation when expressed there. By inserting a T7 bacteriophage promoter into the plasmid, a version of the cDNA which is able to complement both the C. reinhardtii arg7 mutant and the Escherichia coli argH mutant has been created. This modified Arg7 cDNA provides two advantages over the genomic DNA currently in use for gene tagging: it is shorter (6.2 kb versus 11.9 kb for pARG7.8φ3), and the selectable marker used in C. reinhardtii is the same as that used in E. coli, making plasmid rescue of the tag much more likely to succeed. Received: 2 June 1998 / Accepted: 25 September 1998  相似文献   

10.
Lactobacillus delbrueckii subsp. lactis (Lb. lactis) CNRZ 326 is widely used in the propagation of Lb. delbrueckii bacteriophages. In this study, evidence is presented that this strain possesses a restriction-modification (R/M) system. The mitomycin C-induced temperate bacteriophage lb539 has a reduced efficiency of plaquing (EOP) on CNRZ 326 cells (EOP = 10−3), but after several passages on this strain, or on the indicator strain Lb. lactis LKT, the recovered phages (phages lb539.326 and lb539.LKT) have an EOP equal to 1. Restrictive development on CNRZ 326 was also observed after phage lb539.326 was propagated on the strain Lb. lactis CRL 934. The R/M system was also active against the virulent Lb. delbrueckii phage ll-h. Plasmid DNA was not detected in CNRZ 326, which suggests that the R/M system described is chromosomally encoded. Received: 11 September 1997 / Accepted: 21 October 1997  相似文献   

11.
In wheat mitochondria, the gene coding for subunit 2 of the NADH-ubiquinone oxidoreductase (nad2) is divided into five exons located in two distant genomic regions. The first two exons of the gene, a and b, lie 22 kb downstream of exons c, d, and e, on the same DNA strand. All introns of nad2 are group II introns. A trans-splicing event is required to join exons b and c. It involves base pairing of the two precursor RNAs in the stem of domain IV of the intron. A gene coding for tRNATyr is located upstream of exon c. In addition to splicing processes, mRNA editing is also required for the correct expression of nad2. The mature mRNA is edited at 36 positions, distributed over its five exons, resulting in 28 codon modifications. Editing increases protein hydrophobicity and conservation. Received: 11 August 1997 / Accepted: 2 February 1998  相似文献   

12.
It was previously demonstrated that while lysogenic development of bacteriophage λ in Escherichia coli proceeds normally at low temperature (20–25° C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the p E promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage λcIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37° C, while no phage progeny are observed at 20° C. Contrary to previous reports, it is possible to demonstrate that p E promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20° C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage λ is neither inhibited at 20° C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between λ phage and λ plasmid DNA at low temperature. Received: 30 December 1997 / Accepted: 25 February 1998  相似文献   

13.
The envelope properties of B. subtilis cultures grown in LB and LBN hyperosmotic media (LB + 1.5 M NaCl) were compared. Since hypertonic cultures showed a Spo-phenotype, a Spo-mutant grown in LB was also analyzed. LBN cultures showed extensive filamentation and presented different sensitivities toward phage infection (φ29 and φ105), or antibiotics whose targets are at wall (lysozyme, penicillin G) or membrane level (polymyxin B, phosphonomycin). Results of the biochemical composition revealed that during hyperosmotic growth, the cell wall increased in thickness, and among the membrane lipids, glycolipid and cardiolipin increased in parallel with a decrease in phosphatidylglycerol. The fatty acid composition was also modified, and an increase in saturated straight chain with a decrease of saturated iso-branched fatty acids was observed. The increase of monounsaturated 18-1 (ω-9) fatty acid was probably related to the absence of sporulation observed in hypertonic media, since its increase has been shown to inhibit the KinA sensor of sporulation. The significance of the other wall and membrane composition variations (and hydrophobic surface properties) in relation to the osmotic adaptation are discussed. Received: 16 June 1997 / Accepted: 22 July 1997  相似文献   

14.
During a study of the genetic diversity of Stenotrophomonas strains, we found an autonomous replicating DNA molecule in chromosomal DNA preparations of the clinical Stenotrophomonas maltophilia strain c5. The entire sequence of 6,907 bp of the isolated DNA molecule was determined, which was called φSMA9. Seven ORFs, which code for proteins with considerable similarity to proteins in databases, were identified in the DNA sequence. The largest ORF shows high sequence similarities to the pI protein of the filamentous phage φLf, which was later shown to be identical to toxin Zot of Vibrio cholerae. Beside the Zot-like protein, six other proteins with similarities to known phage proteins such as a phage replication protein RstA and phage absorption or coat protein are encoded on φSMA9, which indicate that this circular DNA molecule represents the replicative form of a linear phage genome. A PCR-based screening showed that only five from the totally investigated 47 Stenotrophomonas strains of clinical and environmental origin harbor these genes. Altogether, we describe the first genome of a phage for the nosocomial pathogen Stenotrophomonas, which contains a Zot toxin like gene and might be regarded as the first Stenotrophomonas virulence factor.  相似文献   

15.
A DNA primase was isolated from a nuclear fraction from leaves of tobacco (Nicotiana tabacum L. cv. Samsun) and from purified nuclei prepared from tobacco suspension culture cells. The DNA primase was purified to homogeneity (i) for preparations from leaves, by ammonium sulphate fractionation, followed by chromatography on columns of phosphocellulose, Q-Sepharose, heparin-Sepharose and single-stranded DNA cellulose, and sedimentation in a glycerol gradient, or (ii) for preparations from cells, by chromatography on single-stranded DNA cellulose, followed by ammonium sulphate precipitation and chromatography on columns of High Q, heparin-Sepharose and Mono Q. In glycerol gradients, the DNA primase sedimented at a rate corresponding to a molecular mass of about 120 kDa. In SDS-polyacrylamide gel electrophoresis, the primase was resolved into two polypeptide subunits of 63 kDa and 53 kDa, which are similar in size to the primase subunits of animal and yeast DNA polymerase α-primase complexes. On poly(dT) or phage M13 single-stranded DNA templates, the DNA primase catalysed the synthesis of oligoribonucleotides up to 20 nucleotides in length, which could serve as primers for DNA synthesis catalysed by Escherichia coli DNA polymerase. Primase activity was dependent on a template, magnesium ions and ATP; it was resistant to aphidicolin and rifampicin, but was strongly inhibited by N-ethylmaleimide. This is the first report of the purification to homogeneity of a plant DNA primase. Received: 8 May 1997 / Accepted: 5 June 1997  相似文献   

16.
A tandem DNA duplication carried on a ColE1-derived plasmid segregates at high frequency upon generalized transduction by phage P22 HT. Transductional segregation of the plasmid-borne duplication can be promoted either by RecA or by the Erf function of P22, indicating that transductional segregation is a consequence of the recombination events that re-circularize the plasmid in the recipient cell. RecA-mediated and Erf-mediated transduction give similar frequencies of duplication segregation and yield the same types of segregation products, indicating that two distinct recombination machineries (RecA + RecBCD and Erf + RecBCD) perform similar or identical recombination reactions on plasmid DNA substrates transduced by bacteriophage P22 HT. Received: 4 September 1997 / Accepted: 23 March 1998  相似文献   

17.
Bacillus subtilis strain 168 is known to possess two genes that encode thymidylate synthases, thyA and thyB. We have identified genes similar to the thyA and thyB genes in several Bacillus strains by Southern hybridization and by DNA amplification with sequence-specific primers. Analysis of thyA genes cloned from B. subtilis W23 strain 2A6, B. subtilis ATCC6633, B. amyloliquefaciens S18 and B. atrophaeus S223 reveals that they are very similar to the thyA genes from B. subtilis 168 and its phage φ3T, but differ considerably from the majority of known prokaryotic and eukaryotic thymidylate synthases. Received: 6 November 1997 / Accepted: 13 January 1998  相似文献   

18.
    
The mbrC17 mutation in Escherichia coli had been shown to cause conditional growth defects and an increase in the quantity of DNA per cell. The present work was aimed at identifying the mutation. Sequencing showed that the MbrC17 phenotype does not involve glr (murI), as previously suggested. P1 transduction data indicated that the mbrC17 mutation is closely linked to rpoB, and allele exchange showed it to lie within the secE-nusG operon. A single change relative to wild type was found in the secE-nusG region from the mbrC17 strain, a G → A mutation 23 bp upstream of the secE coding sequence. This mutation causes a two-fold increase in the concentration of secE-nusG mRNA. Received: 26 March 1997 / Accepted: 2 June 1997  相似文献   

19.
20.
During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (Mr = 37 008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5′ end of the hemB mRNA was determined and the −10 and −35 regions of a potential σ70-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg2+-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg2+-dependent activity was directly demonstrated. Received: 11 July 1997 / Accepted: 9 October 1997  相似文献   

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