The tap‐tpg gene pair on the linear plasmid functions to maintain a linear topology of the chromosome in Streptomyces rochei

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.     

Terminal fragments of herpes simplex virus DNA produced by restriction endonuclease.

Digestion of HSV-1 DNA with λ 5′-exonuclease prior to digesting the DNA with the $\text{Eco R}$ 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 $\text{Eco R}$ I fragments and the partial refractoriness of HSV-1 DNA to λ 5′-exonuclease digestion is discussed.     

Selective resistance of bacterial polyadenylate-containing RNA to hydrolysis by guanosine 3'-5'-monophosphate-sensitive nuclease of Bacillus brevis

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.     

Linear plasmid DNAs of the plant pathogenic fungus Rhizoctonia solani with unique terminal structures

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.     

A linear DNA plasmid from Streptomyces rochei with an inverted terminal repetition of 614 base pairs.

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.     

Purification and properties of two ribonuclease H enzymes from yeast

Two ribonuclease H activities have been purified from Saccharomyces cerevisiae. The major protein, RNase H_A is an acidic protein with a molecular weight of 65,000. RNase H_B 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 H_A has an absolute requirement for Mg²⁺, while Mn²⁺ can replace Mg²⁺ for RNase H_B. RNase H_A is inhibited by low concentrations of N-ethylmaleimide, whereas RNase H_B activity is unaffected under similar conditions. Substrate specificity studies using various polyribonucleotide · poly-deoxynucleotide hybrids showed that RNase H_A preferentially degrades polycytidylate, while RNase H_B 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 H_A is an endonuclease producing 5′-phosphorylated oligonucleotides while yeast RNase H_B is a 5′-exonuclease producing 5′-AMP.     

Synthesis and Biological Properties of 2′-5′ Oligodeoxyribonucleotide,an Isomer of Biologic DNA

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.     

RecA independent,site-specific recombination between ColE1 or ColK and a miniplasmid they complement for mobilization and relaxation: Implications for the mechanism of DNA transfer during mobilization

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.     

Small cryptic plasmids of Streptococcus pneumoniae belong to the pC194/pUB110 family of rolling circle plasmids

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.     

Studeis on the biochemical basis of mutation. IV. Effect of amino acid substitution on the enzymatic and biological properties of bacteriophage T4 DNA polymerase

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.     

Endonuclease V-mediated deoxyinosine excision repair in vitro

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.     

Highly Specific Culture-Independent Detection of YGNGV Motif-Containing Pediocin-Producing Strains

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 Lactobacillus plantarum NCDC20) producing YGNGV motif-containing pediocins.     

Histone genes of the sea urchin (S. purpuratus) cloned in E. coli: Order,polarity, and strandedness of the five histone-coding and spacer regions

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:

     

Escherichia coli K-12 F′ plasmids carrying insertion sequences IS1 and IS5

A simple method is described for the detection of the insertion elements IS1 and IS5 in Escherichia coli F′ plasmids. Several of these insertion elements are normal constituents of the E. coli chromosome and are located on chromosomal regions carried by the F′ plasmids, while several others were probably acquired during the isolation or propagation of the F′ plasmids. The F′ plasmids carrying copies of IS1 or IS5 have been transferred into Salmonella (a host lacking chromosomal copies of IS1 and IS5) where individual copies can be examined for a variety of properties, including structural similarities and ability to transpose to new sites.