Streptomyces rochei 7434AN4 carries three linear plasmids, pSLA2‐L (211 kb), pSLA2‐M (113 kb) and pSLA2‐S (18 kb), their complete nucleotide sequences having been determined. Restriction and sequencing analysis revealed that the telomere sequences at both ends of the linear chromosome are identical to each other, are 98.5% identical to the right end sequences of pSLA2‐L and pSLA2‐M up to 3.1 kb from the ends and have homology to those of typical Streptomyces species. Mutant 2‐39, which lost all the three linear plasmids, was found to carry a circularized chromosome. Sequence comparison of the fusion junction and both deletion ends revealed that chromosomal circularization occurred by terminal deletions followed by nonhomologous recombination. Curing of pSLA2‐L from strain 51252, which carries only pSLA2‐L, also resulted in terminal deletions in newly obtained mutants. The tap‐tpg gene pair, which encodes a telomere‐associated protein and a terminal protein for end patching, is located on pSLA2‐L and pSLA2–M but has not hitherto been found on the chromosome. These results led us to the idea that the tap‐tpg of pSLA2‐L or pSLA2‐M functions to maintain a linear chromosome in strain 7434AN4. This hypothesis was finally confirmed by complementation and curing experiments of the tap‐tpg of pSLA2‐M. 相似文献
Digestion of HSV-1 DNA with λ 5′-exonuclease prior to digesting the DNA with the I restriction endonuclease specifically affects two of the fragments normally obtained after restriction endonuclease digestion. Therefore these two fragments contain the sequences which occur at the termini of HSV-1 DNA. One of the fragments affected is a “minor” fragment which is always present in less than molar yield. The possible relationship between the occurrence of minor I fragments and the partial refractoriness of HSV-1 DNA to λ 5′-exonuclease digestion is discussed. 相似文献
Earlier experiments had shown that the degradation of newly synthesized RNA in permeable cells of Bacillus brevis is mediated primarily by a guanosine 3′,5′-monophosphate-sensitive 3′-exonuclease [N. Sarkar and H. Paulus (1975) J. Biol. Chem. 250, 684–690]. More recently, we found that a substantial fraction of pulse-labeled RNA in B. brevis is polyadenylylated [N. Sarkar, D. Langley, and H. Paulus (1978) Biochemistry 17, 3468–3474], and it was thus of interest to examine the effect of polyadenylylation on the susceptibility of RNA to degradation by the 3′-exonuclease. Purified 3′-exonuclease from B. brevis hydrolyzed the unadenylylated fraction of pulse-labeled RNA from B. brevis much more rapidly than poly(A)-containing RNA. Similar results were obtained with the pulse-labeled unadenylylated and polyadenylylated RNA fractions from Bacillus subtilis. Control experiments showed that the differential hydrolysis of the labeled RNA preparations by 3′-exonuclease was not due to the presence of inhibitors or activators. These results suggest that the stability of mRNA in Bacillus species may be regulated by polyadenylylation. 相似文献
Summary Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization analysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3-exonuclease and 5-exonuclease even after treatment with proteinase K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of the plasmids is discussed. 相似文献
The terminal structure of a linear plasmid pSLA2 , which was isolated from Streptomyces rochei , was analysed. The 5' ends of pSLA2 DNA were blocked by the association of a protein probably covalently bonded with the DNA. This block is removed by alkali treatment and blunt ends with 5'-phosphate and 3'-hydroxy termini were released. The two terminal fragments of pSLA2 were cloned and the nucleotide sequence was determined. An inverted terminal repetition of 614 bp was found along with the presence of further interrupted homologous sequences beyond this area up to 800 bp. These are the first inverted terminal repeat sequences found in microbial linear plasmids. 相似文献
Expression of human recombinant plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli has led to crystallization of ‘latent’ PAI-1. Cleavage with restriction endonucleases of a cDNA clone encoding PAI-1 yielded an 1127 base pair fragment encoding residues 2–376 of the 379 amino acid serpin. Synthetic DNA linkers were ligated to the 5′ and 3′ ends of the subclone to add an initiation codon and restore the full coding sequence, and the resulting semisynthetic gene was incorporated into an expression plasmid, pPAIST-7, under the control of the E. coli trp promoter. Transformation of E. coli GE81 with pPAIST-7 led to expression of unglycosylated PAI-1. Lysates of expression cultures contained PAI-1 activity and PAI-1 protein with the predicted Mr. Unglycosylated PAI-1 from E. coli exhibited characteristic properties of authentic PAI-1: (1) it was recovered in both active and inactive (latent) forms; (2) its activity declined during incubation at 37°C; (3) latent PAI-1 was activated by treatment with 4 M guanidine hydrochloride; (4) reactivated PAI-1 formed a detergent-stable complex with tissue plasminogen activator. Latent PAI-1 accounted for more than 85% of PAI-1 in cell lysates and was purified by ammonium sulfate fractionation, anion-exchange chromatography and hydrophobic interaction chromatography. The purified latent PAI-1 was crystallized. 相似文献
Two ribonuclease H activities have been purified from Saccharomyces cerevisiae. The major protein, RNase HA is an acidic protein with a molecular weight of 65,000. RNase HB is a basic protein with molecular weight of 54,000. Both RNases are active at alkaline pH range and require divalent cations for activity. RNase HA has an absolute requirement for Mg2+, while Mn2+ can replace Mg2+ for RNase HB. RNase HA is inhibited by low concentrations of N-ethylmaleimide, whereas RNase HB activity is unaffected under similar conditions. Substrate specificity studies using various polyribonucleotide · poly-deoxynucleotide hybrids showed that RNase HA preferentially degrades polycytidylate, while RNase HB is specific for polyadenylate. Kinetic analysis of the degradation of specifically end-labeled polymers and analysis of the products of the two yeast RNase H enzymes showed that yeast RNase HA is an endonuclease producing 5′-phosphorylated oligonucleotides while yeast RNase HB is a 5′-exonuclease producing 5′-AMP. 相似文献
Abstract Oligonucleotide having 2′-5′ phosphodiester linkage has been synthesised on solid support using indigenously prepared 3′-deoxy-2′-phosphoramidites. The 2′-5′ oligonucleotide showed higher half-life when subjected to 3′-exonuclease, SVPD, digestion. This oligonucleotide formed a stable duplex with complementary RNA but not with DNA. Similarly, it did not form triplex as well either with DNA or RNA duplex. 相似文献
The DNA sequences of two related plasmids pPR1 and pPR3 described previously in Streptococcus pneumoniae isolates from Germany and Spain were now determined. Both plasmids belong to a family of rolling circle (RC) plasmids found in a variety of bacteria. Their GC content with 32% is lower than that of the S. pneumoniae chromosomal DNA. The plasmid pPR3 has a molecular size of 3160 bp with four putative open reading frames, whereas pPR1 contained a deletion of 313 bp that included the 5′-part of ORF2 and upstream regions and differed by three bp from pPR3. The predicted protein of ORF1 showed high similarity to replication proteins of RC plasmids with 74% identical amino acids to RepA of Streptococcus thermophilus plasmids. Sequences similar to the plus origin of replication of ssDNA plasmids were present in both plasmids. They also contained a 152-bp region with over 83% identity to the minus origin of replication of the Streptococcus agalacticae plasmid pMV158. 相似文献
A novel method based on (1) initial microbiological screening and (2) a highly specific PCR is described for selection of strains expressing YGNGV motif-containing pediocin. Initial screening is carried out using spot on the lawn assay for selection of acid-free, hydrogen peroxide (H2O2)-free and secreted heat-stable inhibitory activity producing strains. This is followed by highly specific PCR for amplification of 406-bp fragment using forward primer: 5′-tggccaatatcattggtggt-3′ targeting signal peptide sequence of pediocin structural gene and reverse primer: 5′-ctactaacgcttggctggca-3′ encoding N-terminus of immunity gene. The assay was validated with Pediococcus pentosaceus NCDC273 and Pediococcus acidilactici NCDC252 using (1) digestion of amplified 406-bp fragment with HindIII restriction enzyme-producing two restriction fragments of expected sizes (227 and 179 bp), (2) nucleotide sequencing of 406-bp fragment from both strains found these pediocins identical to pediocin PA-1/AcH and (3) identification of both pediocins as pediocin PA-1 at protein level using RP-HPLC. The assay was used for screening six strains (3 pediococci, 2 lactobacilli and an Enterococcus faecium) producing acid-free, hydrogen peroxide (H2O2)-free and secreted heat-stable inhibitory activity. This resulted in the detection of three new strains (P. pentosaceus NCDC35, E.faecium NCDC124 and Lactobacillusplantarum NCDC20) producing YGNGV motif-containing pediocins. 相似文献
We report a novel type of recA independent recombination between plasmids ColE1 or ColK and a naturally occurring miniplasmid (pLG500). This miniplasmid can be complemented for mobilization and relaxation in the presence of ColE1 or ColK. Recombination between ColE1 and pLG500, or ColK and pLG500, was site-specific, and was only detected following the mobilization of these plasmids. The composite plasmids thus formed were stable, but recombination (resulting in dissociation of their component replicons) was again detected following mobilization. For ColE1, the site at which cointegration with pLG500 occurred was mapped to within 47 base pairs of the relaxation nicking site; for ColK, the recombination site was localized to the same region as its genetically defined transfer origin. The generation of these cointegrate plasmids is consistent with the hypothesis that mobilization entails relaxation nicking, transfer of the nicked single strand of DNA, and recircularization of the transferred single strand by ligation of 3′ and 5′ termini by the relaxation protein bound to the 5′ nick terminus. Since both plasmids are mobilized by the same proteins, their cointegration can be explained as a consequence of the ligation of the 5′ end of one plasmid to the 3′ end of the other, and vice versa. 相似文献
The effects of substituting specific amino acids at specified loci in the bacterio-phage T4 DNA polymerase molecule have been studied. Gene 43 (DNA polymerase) amber mutants grown on suppressor strains which substitute serine, glutamine, or tyrosine at specific sites in the polymerase molecule, produce enzymes with substantially different physical, enzymatic and biological properties when compared to wild type. When amB22, a gene 43 mutant which makes a DNA polymerase fragment with only 3′-exonuclease activity, was grown in Escherichia coli B40(sup+1), -(sup+ 2) or -(sup+3), enzymes with different temperature sensitivities and nuclease to polymerase ratios were produced. Measurements of spontaneous mutation rates in these suppressed strains indicated that the two with higher than normal exonuclease activity were antimutators, and the one with a slightly lower exonuclease activity was a mutator. The substituted amino acids at the amB22 site perturbed the 3′-exonuclease activity creating either antimutator or mutator phenotypes. Thus, the B22 enzymes provide additional biochemical evidence to support the hypothesis that the exonuclease to polymerase ratio may influence the spontaneous mutation rate in phage T4. 相似文献
Vectors were constructed that allow the expression of large quantities of fused proteins in Escherichia coli. The plasmids carry the E. coli lac UV5 promotor and different portions of the coding sequence of the E. coli lacZ gene. The truncated lacZ genes are flanked by a polylinker region either at their 5′ end or their 3′ end, which has several unique restriction sites. Thus, coding sequences of any prokaryotic and eukaryotic gene can be ligated in frame to the truncated lacZ genes. These vectors were used for high-level production of herpes simplex virus type 1 envelope glycoprotein D antigens. 相似文献
Deoxyinosine (dI) in DNA can arise from hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the nfi gene product, in Escherichia coli. Repair was studied in vitro using M13mp18 derived heteroduplexes containing a site-specific deoxyinosine. Unpaired dI/G mismatch resides within the recognition site for XhoI restriction endonucleases, permitting evaluation of repair occurring on deoxyinosine-containing DNA strand. Our results show that dI lesions were efficiently repaired in nfi+E. coli extracts but the repair level was much reduced in nfi mutant extracts. We subjected the deoxyinosine-containing heteroduplex to a purified system consisting of soluble endonuclease V fusion protein, DNA polymerase I, and DNA ligase, along with the four deoxynucleoside triphosphates. Interestingly we found these three proteins alone are sufficient to process the dI lesion efficiently. We also found that the 3′-exonuclease activity of DNA polymerase I is sufficient to remove the dI lesion in this minimum reconstituted assay. 相似文献
Sea urchin (S. purpuratus) histone DNA of constructed plasmid chimeras cloned in E. coli was cleaved with the restriction endonucleases Eco RI, Hind III, Sal I, Bam I, and Hha I. The resulting fragments were ordered and isolated directly from agarose gels or cloned into other plasmids. Each fragment hybridized to one or another of the five histone mRNAs and elucidated the order of the histone genes in each of the cloned fragments. Some DNA did not hybridize to histone mRNAs and was identified as spacer DNA located between coding regions.Total sea urchin DNA was cleaved with restriction endonucleases, fractionated on agarose gels, and hybridized to histone mRNAs or histone DNA. The results revealed the order of the five histone genes in the histone gene repeat unit and demonstrate that the histone spacer DNAs have little sequence homology to other genes. Exonuclease III digestion of specific linear chimeric histone DNA plasmids followed by hybridization with mRNAs demonstrated the existence of all five histone genes on one strand of DNA and the 5′-3′ polarity of that strand. These results, in conjunction with the data of Wu et al. (1976), allow us to construct a map of coding and spacer sequences in the transcribed strand of the S. purpuratus histone gene repeat unit: 相似文献