Primary structure of an EcoRI fragment of lambda imm434 DNA containing regions cI-cro of phage 434 and cII-o of phage lambda.

Digestion of phage lambda imm434 DNA with restriction endonuclease EcoRI yields 7 fragments. The shortest among them (1287 bp) contains the right part of the phage 434 immunity region and the phage DNA portion proximal to it. The complete primary structure of this fragment has been determined using the chemical method of DNA sequencing. Hypothetical amino-acid sequences of proteins coded by the cro gene of phage 434 and the cII gene of phage lambda, as well as NH2-terminal amino-acid sequences of the cI protein of phage 434 and the O protein of phage lambda, have been deduced solely on the basis of the DNA sequence. The fragment studied contains also the pR and probably prm promoters and the oR operator of phage 434. The sequence coding for them differs from the respective DNA sequence of phage lambda.     

Fluorescence study of secondary structure of DNA within bacteriophage lambda

Bromoacetaldehyde (BAA) was used to study the secondary structure of DNA in lambda-phage particles. It was determined that about 1% of the adenines in the intraphage lambda-DNA reacts readily with BAA, thus, they are placed in DNA sites with disturbed complementary interactions. These adenines are close to the tryptophan residues of the phage protein. Fluorescence emission of epsilon A in the intraphage DNA is dramatically quenched. This, apparently, indicates the interaction between epsilon A and Trp- and/or Tyr- and/or Met-residues of phage protein.     

The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations

Summary Restriction endonucleases and agarose gel electrophoresis have been used to demonstrate extensive nucleotide sequence diversity in mitochondrial DNA (mtDNA) within and between conspecific populations of rodents and other mammals. Cleavage of mtDNA samples with a relatively small number of endonucleases provides information concerning the phylogenetic relatedness of individual organisms which cannot now be readily obtained by any other type of molecular analysis. This information is qualitatively different from that available from the study of nuclear genes or gene products because the mitochondrial genome is inherited intact from the female parent and is not altered by recombination or meiotic segregation.The requirements for large tissue samples and laborious DNA purification procedures have imposed severe limitations on the kinds of population surveys in which this technique could be utilized. Here, we show that these difficulties can be overcome by using DNA-DNA hybridization to detect minute amounts of mtDNA in crude tissue fractions which can be more easily and rapidly prepared from very small amounts of tissue without the use of expensive and immobile laboratory equipment. The techniques are described in detail in an effort to make restriction analysis of mtDNA available to biologists who may be unfamiliar with current DNA technology.     

DNA cleavage of lambda and phi 80 transducing phages carrying the argA, argECBH, argF and argI operons of Escherichia coli K-12 with the restriction endonucleases EcoRI, SmaI and HindIII.

DNA isolated from each of the seven arginine transducing phages lambdaargA2cI857susS7, phi80ppc argECBH, phi80argF, phi80argF ilambdacI857, lambdaargF2, lambdaargF23 and lambdaargI valScI857susS7 has been specifically cleaved by the restriction endonucleases EcoRI, SmaI and HindIII. The DNA fragments resulting from single, and in some cases, double endonuclease digests were separated by electrophoresis in agarose and also in polyacrylamide gel. The electrophoretic patterns thus obtained were compared with those produced by digestion of DNA isolated from the corresponding lambda and phi80 parental phages. The majority of cleavage sites produced by the action of these restriction enzymes on arginine transducing DNA have been physically mapped.     

斜纹刺蛾核型多角体病毒及其核酸的限制性内切酶酶解分析

本文描述了从自然罹死的斜纹刺蛾(Oxyplax orhracea(moore))幼虫中分离出一种核型多角体病毒。用快速简便方法提取核酸,经限制性内切酶EcoRⅠ、HindⅢ酶解,获得该病毒核酸的酶解带谱。以λDNA的EcoRⅠ酸解片段在凝胶中的迁移率与相应DNA片段分子量的对数值做标准曲线求得其平均分子量为102.09×10~6道尔顿,即为147.77kb。     

Cloning of restriction and modification genes in E. coli: The HhaII system from Haemophilus haemolyticus

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R·PstI site, (5′)C-T-G-C-A^↓-G (3′), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3′-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3′-OH termini, thus regenating the PstI cleavage site sequence. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r^?m^?recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage λcI·O). A single phage-resistant clone was found. The recombinant plasmid, pDI10, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.     

A simplified method for the characterization of nuclear polyhedrosis virus genomes

Abstract A simplified restriction endonuclease analysis procedure is described which allows the characterization of baculovirus DNA obtained directly from a single larvae without purification of virus. This rapid method was used to demonstrate the genomic stability of nuclear polyhedrosis viruses (NPVs) from Agrotis segetum, Euxoa messoria and Mamestra brassicae after several passages in Euxoa scandens     

HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli genome, and location of the tonB gene.

The HindII and HindIII restriction maps of the attphi80-tonB-trp region of the Escherichia coli chromosome are presented. Analysis of phage DNAs carrying tonB mutations has allowed identification of a 1,730-base pair HindII fragment containing at least part of the tonB gene. This fragment is 4,020 base pairs from the end of trpA, with the total distance from attphi80 to trpA being 6,550 +/- 800 base pairs. Properties of hybrid plasmids containing insertions of various tonB+ restriction fragments suggest that tonB lies completely within the 1,730-base pair fragment. In addition, apparent fusions of beta-galactoside to proteins within the tonB region suggest that the entire region codes for more than one polypeptide.     

The N protein of bacteriophage lambda, defined by its DNA sequence, is highly basic.

Nucleotide sequence has been determined for the restriction fragments and cloned DNA from the pL-N-tL1 region of bacteriophage lambda. A unique reading frame for the N gene is defined by the absence of natural nonsense codons and by the presence of seven nonsense codons generated by mutations in N. This reading frame is initiated at two alternative ATG codons, the second of which is probably the in vivo translation start. Reading is stopped at a single TAG codon. The protein coded is therefore 133 or, more probably, 107 amino acids long, rich in lysine, arginine and proline.     

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .     

Cloning of bacteriophage T5 DNA fragments in plasmid pBR322 and bacteriophage lambda gtWES

Bacteriophage T5 was digested with the restriction endonucleases HindIII and EcoRI and the resulting fragments were inserted into the plasmid pBR322 and the bacteriophage lambda gtWES as vectors. Approx. 15% of the phage genome was recovered in recombinant clones. The recombinants were characterized by restriction analysis, DNA/DNA hybridization employing Southern blots, and ability to complement or recombine with amber mutants of T5. The results obtained allow revisions of the physical map of the T5 genome and partial correlation of the physical map with the genetic map.     

Physical map of the dispensable region of the genome of E. coli bacteriophage BF23

Using 13 deletion mutants of bacteriophage BF23, physical as well as genetic structures of that portion of the genome which is dispensable for phage growth were investigated. The dispensable region covers at least 15% of the genome of wild type BF23, extending from about 0.2 to 0.35 map unit. Restriction endonuclease (EcoRI and HindIII) cleavage sites and the sites of single-strand interruptions in this dispensable region were localized. It was found that the dispensable region contains an interruption site, which is missing in the mutant BF23st(0) used by Okada and Shimura (1980). Wild-type phage DNA is heterogeneous in the presence or absence of specific single-strand interruptions in this or in a neighboring region of the genome.     

The biochemical and cytological characterization of Vicia faba DNA by means of MboI,AluI and Bam HI restriction endonucleases

Summary Restriction endonucleases were employed to characterize both cytologically and electrophoretically the DNA of Vicia faba. The electrophoretic pattern of total DNA digested with AluI and MboI shows a continuous smear. Bam HI also shows a continuous smear for the bigger polynucleotide fragments and several bands in the lower part of the lane. Digestion of fixed chromosomal DNA produces metaphase longitudinal differentiation when MboI and AluI are used, while no appreciable banding pattern is present when Bam HI is employed. These results are discussed in relation to the organization of chromosomal DNA, to other data in the literature on chromosome banding and on the digestion of total DNA of other species.     

HindII, HindIII,and HpaI restriction fragment maps of the left arm of bacteriophage λ DNA

The sites on the left arm of bacteriophage λ DNA cleaved by the restriction endonucleases isolated from Hemophilus influenzae strain Rc (HincII) and Rd (HindII+III), and Hemophilus parainfluenzae (HpaI) were localized on the λ physical map, and the fragments resulting from these cleavages were identified by gel electrophoresis. The restriction sites within the b2 region of λ were mapped by analysis of the digestion profiles of deletion and substitution derivatives of λ, as well as by digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease. The restriction sites on the λ genome between the left vegetative end and the b2 region were mapped entirely by successive digestion experiments. The restriction fragment map for the right arm of λ may be found in the accompanying paper (Robinson and Landy, 1977).     

The solution structure of the bacteriophage lambda head-tail joining protein,gpFII

The bacteriophage lambda FII protein (gpFII) is a 117 residue structural protein found in the phage particle that is required for the joining of phage heads and tails at the last step of morphogenesis. We have performed biophysical experiments to show that gpFII is stable, monomeric, and reversibly folded. We have also determined the atomic resolution structure of gpFII using NMR spectroscopy. gpFII is shown to possess a novel fold consisting of seven beta-strands and a short alpha-helix. It also displays two large unstructured regions at the N terminus (residues 1-24) and in a large loop near the middle of the protein (residues 46-62). We speculate that these unstructured regions become structured when gpFII assembles into the phage particle, and that these conformational changes play an important role in regulating the assembly pathway. Alignment of the gpFII sequence with those of homologues from other lambdoid phages has allowed us to putatively identify distinct surfaces on the gpFII structure that mediate binding to the phage head and tail.     

Megaplasmids in Thermus oshimai isolates from two widely separated geographical areas: restriction fragment profiling and DNA homology

Megaplasmid DNA was detected in ten isolates belonging to the recently described thermophilic eubacterial species Thermus oshimai and isolated from hot springs in Portugal (eight isolates) and Iceland (two isolates). The estimated size of the large plasmids purified from T. oshimai SPS-18 from S. Pedro do Sul, Portugal, and from isolate JK-91 from Hveragerdhi-Hengill, Iceland, was 214 and 275 kb, respectively. No sequence homologous to isolate SPS-18 megaplasmid is present in chromosomal DNA as indicated by Southern hybridization analysis. Overall examination of the HindIII fragment profiles of megaplasmid DNAs purified from isolates from the same geographical area gave similar but not always identical restriction profiles on agarose gels. Restriction fragment length polymorphism (RFLP) was higher for megaplasmids present in isolates purified from the Portuguese and Icelandic isolates than for megaplasmids from the same hot spring. Megaplasmid RFLP correlated with previous results obtained on the polymorphism of macrorestriction patterns of whole genomic DNA and with the RFLP of co-resident small plasmid DNA that was found in one half of the isolates examined. The 16-kb HindIII–HindIII fragment from isolate SPS-18 megaplasmid showed DNA–DNA homology with restriction fragments of similar size generated by the large plasmids present in all the other isolates, even in those from hot springs of widely separated geographical areas. This suggests a high degree of sequence conservation in T. oshimai megaplasmids. Received: 12 May 1997 / Accepted: 17 July 1997     

Physical mapping of bacteriophage lambda DNA with restriction endonuclease HpaI

The restriction endonuclease from Haemophilus parainfluenzae, endoR·HpaI cleaves λcI857s7 DNA into 14 fragments. The sizes of these fragments were determined and a physical map was constructed. The ordering of the fragments was carried out using different deletion and substitution mutants of λ phage, double cleavages with another restriction enzyme, endoR·BamHI, and partial protection of individual HpaI recognition sites by the antibiotics distamycin A and actinomycin D. HpaI produces fragments from the left arm of the λ DNA genome, which may help in investigating the structure and function of this part of the phage.     

An agarose gel resolving a wide range of DNA fragment lengths

To resolve DNA fragments ranging from several kilobases to some tens of base pairs in length, an agarose slab gel of steadily increasing thickness has been designed. During electrophoresis a gradient of decreasing electric-field strength is generated throughout the gel from the cathode end to the anode end. Shorter fragments which migrate further are decelerated, resulting in an increased linearity of the relationship between mobility and molecular weight.     

Cleavage maps for human cytomegalovirus DNA strain AD169 for restriction endonucleases EcoRI, BglII, and HindIII.

We have used cloned EcoRI fragments of the human CMV (HCMV) genome, strain AD169, to prepare restriction endonuclease maps of the DNA. Individual 32P-labeled cloned fragments were hybridized to Southern blots of HCMV DNA cleaved to completion with the restriction endonucleases BglII and HindIII and cleaved partially with EcoRI. By determining which EcoRI fragments hybridized to the same band on a Southern blot, we were able to establish linkage groups. This information coupled with the data derived from digestion of the cloned fragments with the enzymes BglII and HindIII (Tamashiro et al., J. Virol. 42:547-557, 1982) provided the basis for the construction of detailed maps for the enzymes EcoRI, BglII, and HindIII. We also identified the EcoRI fragments derived from the termini of this genome and mapped them with respect to the BglII and HindIII terminal fragments. From our mapping data, we conclude that the genome of HCMV is approximately 240 kilobases in length and is divided into long (198 kilobases) and short (42 kilobases) regions. Both regions consist of a unique sequence bounded by inverted repeats (11 to 12 kilobases for the long region and 2 to 3 kilobases for the short region). Furthermore, the long and short regions can invert relative to each other.