Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule.

The structure of DNA from the temperate Bacillus subtilis phage phi105 was examined by using the restriction endonuclease EcoRI and by sedimentation analysis. The DNA contains six EcoRI cleavage sites. Although eight DNA fragments were identified in the EcoRI digests, the largest of these was shown to consist of the two fragments that carry the cohesive ends of the phage DNA. In neutral gradients, the majority of whole phi105 DNA sedimented as nicked circles and the remainder as oligomers. No unit-length linear structures were detected. The associated cohesive ends could be sealed by DNA ligase from Escherichia coli and could be cleaved by S1 nuclease. On the basis of these results and previously reported studies, it appears that, as isolated from phage particles, phi105 DNA is a circular molecule that is formed from the linear structure by the association of complementary single-stranded DNA.     

Characterization of bacteriophage N3 DNA.

The DNA from Haemophilus influenzae temperate phage N3 was characterized by centrifugation and by electrophoresis after nuclease digestion. The double-stranded DNA, with a mass of 25.8 X 10(6) daltons, had single-strand cohesive ends. Strand association through cohesion was reduced by heat and removed by S1 nuclease digestion. N3 DNA contained five EcoRI, one KpnI, two SacI, six XbaI, and four XhoI cleavage sites. The cohesive end segments were identified by heating the digests before electrophoresis. This was the first step in the construction of the physical maps of this DNA.     

Restriction endonuclease/nick translation of fixed mouse chromosomes: A study of factors affecting digestion of chromosomal DNA in situ

We used a restriction endonuclease/nick translation procedure to study the ability of certain enzymes, known to cleave mouse satellite DNA in solution, to attack satellite DNA in fixed mouse chromosomes. Although AvaII and Sau96I readily attack the mouse major satellite in fixed chromosomes, BstNI and EcoRII do not normally do so, although if the heterochromatin is uncondensed as a result of culture in the presence of 5-azacytidine, BstNI can attack it. No clear evidence was obtained for digestion in situ of the minor satellite of mouse chromosomes by MspI, the only enzyme reported to cleave this satellite. Our results show that the DNA of mouse heterochromatin is not merely not extracted by certain restriction enzymes, but is actually not cleaved by them. Chromatin conformation is therefore shown to be an important factor in determining patterns of digestion of chromosomes by restriction endonucleases.by D. Schweizer     

Specific hydrolysis of the cohesive ends of bacteriophage lambda DNA by three single strand-specific nucleases.

Procedures have been worked out for Aspergillus nuclease S1 and mung been nuclease to quantitatively cleave off both of the 12-nucleotide long, single-stranded cohesive ends of lambdaDNA. This cleavage is indicated by the almost complete elimination of the repair incorporation of radioactive nucleotides by DNA polymerase into the digested DNA. With S1 nuclease, cleavage was complete at 10 degrees as well as at 30 degrees. Under the conditions for quantitative cleavage of the single-stranded regions there was no digestion of the double-stranded lambdaDNA. The mung bean nuclease cleaved off the cohesive ends completely at 30 degrees but at 5 degrees, the cleavage was not complete even at high enzyme concentration. The nearest neighbor analysis of the repaired DNA indicates that at 5 degrees about four nucleotides remained undigested. The mung bean nuclease also introduced, under the conditions used, some nicks into double-stranded DNA as determined by the repair incorporation. The Escherichia coli exonuclease VII cleaved off part of the cohesive ends of lambdaDNA, leaving two nucleotides on each end as single-stranded tails.     

Cloning a defined region of DNA using a limited action of DNA polymerase: application to dissection of hepatitis B virus surface antigen gene

Using dodecadeoxynucleotides as primers for DNA synthesis and 3'-o-chlorophenyl-phosphorylated dodecadeoxynucleotides as "stoppers" for chain elongation, pre-defined regions of a gene previously cloned in M13 single-stranded (ss) DNA phage were converted into double-stranded (ds) DNA utilizing the action of the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). The resulting ds DNA was freed from the ss region by S1 nuclease treatment. This method can be used to obtain DNA fragments of any size with pre-defined 5' and 3' ends. About 15% of the input ss DNA template molecules are converted into ds DNA fragments. This technique was used to synthesize several DNA fragments from different portions of the hepatitis B virus surface antigen (HBsAg) gene. The products were then ligated into a yeast plasmid vector that carries the E. coli lacZ gene which is located downstream from the yeast acid-phosphatase promotor. Using this system, several fragments of HBsAg were produced in the form of beta-galactosidase fused protein.     

DNA sequence analysis. Terminal sequences of bacteriophage phi80.

Sequences of the cohesive ends and the 3'-terminal regions of phi80 DNA have been determined. Sequences of the cohesive ends were obtained through the use of two standard methods. The first method involved the incorporation of all four labeled deoxyribonucleotides into the phi80 cohesive ends using DNA polymerase I. The DNA was then partially digested with micrococcal nuclease or pancreatic DNase. The products were separated by two-dimensional electrophoresis and characterized by composition, 3'-terminal, and nearest neighbor analyses. The second method involved partial incorporation using one, two, or three labeled deoxyribonucleotides followed by similar analyses. Sequences of the double-stranded regions adjacent to the cohesive ends were determined by three new methods. These methods were: (a) the DNA was specifically labeled at the 3' terminus and then partially degraded. Labeled oligonucleotide products were sequenced by their mobilities on various separation systems. (b) The cohesive ends were enlarged by limited degradation with exonuclease III. After this treatment, the DNA was partially repaired with labeled nucleotides, digested, and the products were analyzed. (c) A synthetic ologonucleotide primer was bound to phi80 DNA which had been repaired with DNA polymerase I, and then partially digested with lambda-exonuclease. The primer was extended into the region of interest by partial repair with labeled nucleotides. The extended primer was isolated and analyzed.     

Specific cleavage of chromatin by restriction nucleases.

Digestion of mouse and rat liver nuclei with a restriction nuclease from Bacillus subtilis (Bsu) is examined in continuation of previous work from this laboratory (Pfeiffer et al., 1975, Nature 258, 450). The finding of more than 95% C in the 5'-termini of the DNA fragments generated during digestion with Bsu shows that the participation of endogenous nucleases in Bsu digestion is extremely small. The restriction nuclease Hae III, an isoschizomer of Bsu, yields identical degradation patterns. The patterns conform to what one expects from statistical calculations based on a nucleosome structure of chromatin with a region preferentially accessible to the nuclease of 40-50 nucleotide pairs per nucleosome. Integrity of the histones is maintained during digestion with restriction nucleases. Digestion of mouse liver nuclei with EcoRII shows that most if not all of the satellite DNA is organized in a nucleosome structure. Also in rat liver, much of the repetitive DNA appears to be present in nucleosomes.     

Mapping and cloning of Eco RI-fragments of bacteriophage T5+ DNA.

The Eco RI-fragments of bacteriophage T5 DNA were mapped using a technique which involves primarily length measurements of molecules observed in the electron microscope. Since Eco RI cleavage generates termini with 4-nucleotide long cohesive ends, fragments of complete and partial Eco RI digests were covalently circularized with DNA ligase at dilute DNA concentrations before measuring relative to internal length standards. This established the order of the internal Eco RI fragments. The two external Eco RI fragments, which had only one Eco RI terminus, were positioned relative to the internal fragments by identifying the location of some of the naturally-occurring nicks in partially denatured linear Eco RI fragments. An attempt was made to clone each of the internal Eco RI-fragments of T5 DNA via transformation into E. coli after ligation in vitro with the plasmid pMB 9. Only one fragment could be cloned and this fragment did not specify any new polypeptides in minicells of either the E. coli EK1 host, X1411, or the EK 2 host, X1776.     

Characterization of the genome of the Rhodococcus rhodochrous bacteriophage NJL.

Temperate bacteriophage NJL of Rhodococcus rhodochrous has a 49-kb linear double-stranded DNA with cohesive ends (cos). NJL DNA has unique target sites for HindIII and SspI, two target sites each for NheI and ScaI, and no cleavage site for AxyI, DraI, EcoRI, SacI, and SphI. The single-stranded regions of cos ends were ligated to each other with T4 DNA ligase, removed with mung bean nuclease, or blunted with the Klenow large fragment of DNA polymerase I; then the sequences of the cos ends were determined. Comparison of these sequences revealed that the single-stranded regions are complementary and 18 bases long and protrude at the 3' ends; they have the following sequences: 5'-TTGGCACCGTGGGAGGAG-3' and 3'-AACCGTGGCAC CCTCCTC-5'. A physical map of NJL was constructed by a cos mapping method based on information about the structure of the cohesive ends and multiple digestions with restriction endonucleases.     

Characterization of total deoxyribonucleic acid of Mycobacterium paratuberculosis (ATCC 19698) and of M. avium complex (ATCC 25291) using restriction enzymes

Total DNA was extracted from M. paratuberculosis (ATCC 19698) and from M. avium complex (ATCC 25291) cultivated on RVB-10 enriched liquid media. Restriction endonuclease analysis was conducted of Total DNA using 34 enzymes and DNA digestion profiles were compared. Fifteen enzymes revealed important differences between the two species. Two pairs of enzymes (EcoRII, BstNI) and (MboI, Sau3AI) provide evidence for the presence of dcmI and dam methylation in DNA of M. avium complex and M. paratuberculosis. The differences in DNA fragments of these two species could be of potential value in differentiating these clinically significant mycobacteria.     

Morphological and biochemical effects of endonucleases on isolated mammalian chromosomes in vitro

Endonuclease digestion of isolated and unfixed mammalian metaphase chromosomes in vitro was examined as a means to study the higher-order regional organization of chromosomes related to banding patterns and the mechanisms of endonuclease-induced banding. Isolated mouse LM cell chromosomes, digested with the restriction enzymes AluI, HaeIII, EcoRI, BstNI, AvaII, or Sau96I, demonstrated reproducible G- and/or C-banding at the cytological level depending on the enzyme and digestion conditions. At the molecular level, specific DNA alterations were induced that correlated with the banding patterns produced. The results indicate that: (1) chromatin extraction is intimately involved in the mechanism of endonuclease induced chromosome banding. (2) The extracted DNA fragments are variable in size, ranging from 200 bp to more than 4 kb in length. (3) For HaeIII, there appears to be variation in the rate of restriction site cleavage in G- and R-bands; HaeIII sites appear to be more rapidly cleaved in R-bands than in G-bands. (4) AluI and HaeIII ultimately produce banding patterns that reflect regional differences in the distribution of restriction sites along the chromosome. (5) BstNI restriction sites in the satellite DNA of constitutive heterochromatin are not cleaved intrachromosomally, probably reflecting an inaccessibility of the BstNI sites to enzyme due to the condensed nature of this chromatin or specific DNA-protein interactions. This implies that some enzymes may induce banding related to regional differences in the accessibility of restriction sites along the chromosome. (6) Several specific nonhistone protein differences were noted in the extracted and residual chromatin following an AluI digestion. Of these, some nonhistones were primarily detected in the extracted chromatin while others were apparently resistant to extraction and located principally in the residual chromatin. (7) The chromatin in constitutive heterochromatin is transiently resistant to cleavage by micrococcal nuclease.     

Analysis of highly repeated DNA sequences of rat with EcoR1 endonuclease

Cleavage of rat liver nuclear DNA with EcolR1 restriction endonuclease yields 14 discrete fragments ranging from 2300 to 93 base pairs in length, representing approx. 10.5% of the rat genome. Fragments of 1500, 180, and 93 base pairs are reiterated over 100 000 times; fragments of 2300, 880, 290, and 200 base pairs are reiterated over 20 000 times; the remaining fragments are present in over 1000 copies per genome. When compared to whole rate DNA, 11 were 1-5% richer in A . T base pairs and five were 1.5-2.5 times more methylated. From the criteria of the banding patterns in complete and incomplete digests, base composition and extent of methylation, none of these fragments appeared to be generated as oligomers of a basic shorter repeat. The reassociation of EcoR1 fragments was monitored on hydroxyapatite and by S1 nuclease treatment in order to assess band reiteration frequency and the possibility of interpersion or short internal repeats. The renaturation of the four smallest EcoR1 fragments gave no indication of short internal repeats from hyperpolymer formation nor interpersion with lower frequency sequences by size reduction after S1 nuclease treatment. Anomalous renaturation of several large fragments was observed, possibly due to internal repeats.     

cDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Escherichia coli exonuclease III.

cDNA encoding the human homologue of mouse APEX nuclease was isolated from a human bone-marrow cDNA library by screening with cDNA for mouse APEX nuclease. The mouse enzyme has been shown to possess four enzymatic activities, i.e., apurinic/apyrimidinic endonuclease, 3'-5' exonuclease, DNA 3'-phosphatase and DNA 3' repair diesterase activities. The cDNA for human APEX nuclease was 1420 nucleotides long, consisting of a 5' terminal untranslated region of 205 nucleotide long, a coding region of 954 nucleotide long encoding 318 amino acid residues, a 3' terminal untranslated region of 261 nucleotide long, and a poly(A) tail. Determination of the N-terminal amino acid sequence of APEX nuclease purified from HeLa cells showed that the mature enzyme lacks the N-terminal methionine. The amino acid sequence of human APEX nuclease has 94% sequence identity with that of mouse APEX nuclease, and shows significant homologies to those of Escherichia coli exonuclease III and Streptococcus pneumoniae ExoA protein. The coding sequence of human APEX nuclease was cloned into the pUC18 SmaI site in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain and BW9109 (delta xth) strain cells of E. coli. The transformed cells expressed a 36.4 kDa polypeptide (the 317 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide derived from the part of pUC18 sequence), and were less sensitive to methylmethanesulfonate and tert-butyl-hydroperoxide than the parent cells. The N-terminal regions of the constructed protein and APEX nuclease were cleaved frequently during the extraction and purification processes of protein to produce the 31, 33 and 35 kDa C-terminal fragments showing priming activities for DNA polymerase on acid-depurinated DNA and bleomycin-damaged DNA. Formation of such enzymatically active fragments of APEX nuclease may be a cause of heterogeneity of purified preparations of mammalian AP endonucleases. Based on analyses of the deduced amino acid sequence and the active fragments of APEX nuclease, it is suggested that the enzyme is organized into two domains, a 6 kDa N-terminal domain having nuclear location signals and 29 kDa C-terminal, catalytic domain.     

Classification of type-II restriction endonucleases and cloning of non-identical cohesive-end fragments without self-polymerization using nonpalindromic oligodeoxyribonucleotide adapters.

Enzymatic partial filling-in of recessed 3'-end sequences, left after digestion of DNA by the restriction endonucleases (ENases) Sau3A and SalI, with the Klenow fragment of E. coli DNA polymerase I allows the forced ligation of the resulting fragments; this technology is already used for subcloning and for genomic bank construction. To simplify and generalize its utilization, class-II ENases have been arranged into 16 different families according to the composition of the 5'-protruding sequences present after cleavage. Moreover, this system was extended to allow the joining of noncompatible ends by the use of nonpalindromic complementary oligodeoxyribonucleotides (NPCOs) containing two nucleotides protruding at each 5' end. The use of these synthetic adapters maintains all the advantages of the initial gap-filling cloning technique: only one insert can be cloned per vector molecule and no self-ligation or -polymerization can occur with any of the DNA molecules involved. Only 22 such oligodeoxyribonucleotides are needed to generate the 60 NPCO pairs necessary to ligate to each other any member of twelve ENase families when the regeneration of ENase recognition sites is not required.     

Isolation and characterization of adenovirus core nucleoprotein subunits.

Digestion of adenovirus type 2 (Ad2) or Ad5 cores with micrococcal nuclease generated four nucleoprotein species that could be resolved by electrophoresis in low-ionic-strength polyacrylamide gels: these nucleoproteins displayed mobilities equivalent to those of DNA fragments of 900 to 1,025, 775 to 850, 650 to 725, and 525 to 600 base pairs (bp) and thus were readily distinguishable from HeLa cell mononucleosomes. The DNA fragments associated with the core nucleoprotein species were more than 250 to 90 bp long. Nucleoproteins containing 150, 120, or 90 bp of DNA were the most stable. Polypeptide VII was associated with each of the nucleoprotein species liberated from Ad2 cores. These data suggest that polypeptide VII and viral DNA of 90 to 150 bp comprise the unit particle of the Ad2 or Ad5 core nucleoproteins.     

Specificity of restriction endonucleases and DNA modification methyltransferases a review (Edition 3)

The properties and sources of all known class-I, class-II and class-III restriction endonucleases (ENases) and DNA modification methyltransferases (MTases) are listed and newly subclassified according to their sequence specificity. In addition, the enzymes are distinguished in a novel manner according to sequence specificity, cleavage position and methylation sensitivity. Furthermore, new nomenclature rules are proposed for unambiguously defined enzyme names. In the various Tables, the enzymes are cross-indexed alphabetically according to their names (Table I), classified according to their recognition sequence homologies (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174, and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328, and the microorganisms from which they originate. Other tabulated properties of the ENases include relaxed specificities (integrated within Table II), the structure of the generated fragment ends (Table III), interconversion of restriction sites (Table IV) and the sensitivity to different kinds of DNA methylation (Table V). Table VI shows the influence of class-II MTases on the activity of class-II ENases with at least partially overlapping recognition sequences. Table VII lists all class-II restriction endonucleases and MTases which are commercially available. The information given in Table V focuses on the influence of methylation of the recognition sequences on the activity of ENases. This information might be useful for the design of cloning experiments especially in Escherichia coli containing M.EcodamI and M.EcodcmI [H16, M21, U3] or for studying the level and distribution of site-specific methylation in cellular DNA, e.g., 5'- (M)CpG-3' in mammals, 5'-(M)CpNpG-3' in plants or 5'-GpA(M)pTpC-3' in enterobacteria [B29, E4, M30, V4, V13, W24]. In Table IV a cross index for the interconversion of two- and four-nt 5'-protruding ends into new recognition sequences is complied. This was obtained by the fill-in reaction with the Klenow (large) fragment of the E. coli DNA polymerase I (PolIk), or additional nuclease S1 treatment followed by ligation of the modified fragment termini [P3]. Interconversion of restriction sites generates novel cloning sites without the need of linkers. This should improve the flexibility of genetic engineering experiments [K56, P3].(ABSTRACT TRUNCATED AT 400 WORDS)     

Oligo[d(C).(G)] runs exhibit a helical repeat of 11.1 bp in solution and cause slight DNA curvature when properly phased.

We have inserted d(C)10 in a set of DNA fragments with bent segments on both ends, which are rotated with respect to each other by base pair wise increasing insertions. The electrophoretic mobilities on polyacrylamide gels of these DNA fragments were used to identify insertion sizes with cis conformations of the bent ends. These experiments revealed a helical repeat in solution of d(C).d(G) tracts of 11.1 +/- 0.08 bp. The electrophoretic mobilities of ligation ladders with properly phased d(C)5 and d(C)16 runs demonstrate a small but clearly detectable curvature of these fragments.     

Presence of random single-strand gaps in mycobacteriophage I3 DNA

The genomic double-stranded DNA of mycobacteriophage I3, when denatured with alkali, heat, formamide or dimethylsulfoxide, breaks down to heterogeneous-sized single-strand (ss) fragments smaller than the expected intact unit genome length suggesting the presence of random ss interruptions on both the strands. The occurrence of the interruptions at random is also demonstrated by two-dimensional gel electrophoresis of the restriction fragments of I3 DNA. These interruptions have no adverse effect on the phage infectivity or DNA transfectivity. Studies with nuclease BAL 31 and end-labeling analysis confirm the presence of random interruptions. Detailed analysis using T4 DNA ligase, nuclease S1 and DNA polymerase I Klenow fragment revealed that the interruptions are in the form of small gaps rather than single phosphodiester bond breaks. The average length of the gap is about 10 nucleotides long and there are 13 to 14 such gaps per DNA molecule.     

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

Restriction fragments hybridizing to phage HP1c1 DNA were identified in digests of DNA from lysogenic strains of Haemophilus influenzae. The results showed that the cohesive ends of the mature phage DNA were joined in lysogens and that the phage genome was covalently linked to the host DNA, indicating that lysogeny involves recombination between specific sites on the phage and host chromosomes. The site on the phage chromosome at which this recombination occurred was between 110 and 750 base pairs of the left end on the mature phage genome.     

Structure of eukaryotic chromatin. Evaluation of periodicity using endogenous and exogenous nucleases.

DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endonuclease, (b) prostate chromatin digested in situ with micrococcal nuclease or pancreatic DNAase I, and (c) isolated liver chromatin digested with micrococcal nuclease or pancreatic DNAase I has been analyzed electrophoretically on polyacrylamide gels. The electrophoretic patterns of DNA prepared from chromatin digested in situ with either endogenous endonuclease (liver nuclei) or micrococcal nuclease (prostate nuclei) are virtually identical. Each pattern consists of a series of discrete bands representing multiples of the smallest fragment of DNA 200 +/- 20 base pairs in length. The smallest DNA fragment (monomer) accumulates during prolonged digestion of chromatin in situ until it accounts for nearly all of the DNA on the gel; approx. 20% of the DNA of chromatin is rendered acid soluble during this period. Digestion of liver chromatin in situ in the presence of micrococcal nuclease results initially in the reduction of the size of the monomer from 200 to 170 base pairs of DNA and subsequently results in its conversion to as many as eight smaller fragments. The electrophoretic pattern obtained with DNA prepared from micrococcal nuclease digests of isolated liver chromatin is similar, but not identical, to that obtained with liver chromatin in situ. These preparations are more heterogeneous and contain DNA fragments smaller than 200 base pairs in length. These results suggest that not all of the chromatin isolated from liver nuclei retains its native structure. In contrast to endogenous endonuclease and micrococcal nuclease digests of chromatin, pancreatic DNAase I digests of isolated chromatin and of chromatin in situ consist of an extremely heterogeneous population of DNA fragments which migrates as a continuum on gels. A similar electrophoretic pattern is obtained with purified DNA digested by micrococcal nuclease. The presence of spermine (0.15 mM) and spermidine (0.5 mM) in preparative and incubation buffers decreases the rate of digestion of chromatin by endogenous endonuclease in situ approx. 10-fold, without affecting the size of the resulting DNA fragments. The rates of production of the smallest DNA fragments, monomer, dimer, and trimer, are nearly identical when high molecular weight DNA is present in excess, indicating that all of the chromatin multimers are equally susceptible to endogenous endonuclease. These observations points out the effects of various experimental conditions on the digestion of chromatin by nucleases.