Restriction-fragment map of the temperate Bacillus subtilis bacteriophage SPO2.

The endonucleases BglI, BglII, EcoRI, SalI, SmaI, and XbaI were used to fragment the phage SPO2 DNA. Electrophoretic analysis using ethidiumbromide agarose gels showed the phage to have nine BglI sites, one BglII site, four EcoRI sites, one SalI site, one SmaI site, and six XbaI sites. Using partial digestions, multiple endonuclease digestion, and autoradiography the fragments were sized and ordered into a circular map of 23 Md. Such an analysis locates the endonuclease sites, indicates which endonucleases are potentially useful in cloning with SPO2, and allows insertions and/or deletions in the SPO2 DNA to be characterized.     

Pulsed-field gel analysis of alpha-satellite DNA at the human X chromosome centromere: high-frequency polymorphisms and array size estimate

Using pulsed-field gel analysis (PFGE), we have characterized the large array of alpha-satellite DNA located in the centromeric region of the human X chromosome. The tandem repetitive nature of this DNA family lends itself to examination by PFGE using restriction enzymes that cleave frequently in unique sequence DNA but which cut only rarely within the repetitive alpha-satellite array. Several such restriction enzymes (BglI, BglII, KpnI, ScaI) have proven highly informative in sizing the alpha-satellite array and in following the segregation of individual X-chromosome centromeres using PFGE polymorphisms. Among 29 different X chromosomes, alpha-satellite array length varied between 1380 and 3730 kb (mean = 2895 kb; SD = 537). In three large CEPH families comprising 24 meioses, inheritance of these PFGE polymorphisms was strictly Mendelian, with no indication of intraarray recombination. Such DXZ1 alpha-satellite polymorphisms, therefore, may prove useful in the study of pericentromeric X-linked disorders.     

Restriction enzyme cleavage map of Tn10, a transposon which encodes tetracycline resistance.

A cleavage map of a recombinant plasmid carrying Tn10 was constructed for 13 different restriction enzymes. The Tn10 region of this plasmid contains cleavage sites for BamHI, AvaI, BglI, BglII, EcoRI, XbaI, HincII, HindIII, and HpaI. Restriction enzymes PstI, SmaI, KpnI, XhoI, SalI, and PvuI do not cleave within the Tn10 element. This map confirms the previously reported structure of this transposon; it is composed of a unique sequence (approximately6,400 base pairs long), which in part codes for the tetracycline resistance functions and is bounded by inverted repeats (approximately 1,450 base pairs long).     

Construction of new cloning vehicles with genes of the tryptophan operon of Escherichia coli as genetic markers

In vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes AosI, AvaI, BglI, BglII, HindIII, HpaI, PvuII, SalI, SstI and XhoI. The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trpE and trpA genes and contains single BglII and SstI sites in trpE, a single HindIII site located between trpE and trpA, and single EcoRI, SalI and XhoI sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trpB and trpA genes and single BglII, HindIII, EcoRI, SalI and XhoI sites. Plasmids pHP39 (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one EcoRI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.     

Polymorphism and gene arrangement among plastomes of ten Epilobium species

Summary Plastid DNAs of ten different Epilobium species from four continents have been analysed using the restriction endonucleases BamHI, BglI, BglII, EcoRI, PstI, PvuII and SalI. With respect to the position of cleavage sites of those enzymes, each species has a specific plastome. Fragment patterns of different species from the same continent show a higher degree of similarity than those from different continents. Physical maps of the circular plastid DNA molecule have been constructed for each of the ten species by localising the cleavage sites of the enzymes BglI, PvuII and SalI. As in most other higher plants, the plastid DNA of Epilobium is segmentally organized into two inverted repeats separated by a large and a small single copy region. In heterologous hybridization experiments using radioactively labelled gene probes, the positions of structural genes coding for the rRNAs and for seven polypeptides have been determined. In contrast to its closest relative, Oenothera, the gene arrangement of Epilobium plastomes has the same order as in spinach. This indicates that changes in gene arrangement may be genus-specific and not the result of one or several events affecting all members of a plant family.Abbreviations kbp kilobase pairs - ptDNA plastid DNA - rDNA ribosomal DNA - rRNA ribosomal RNA - SDS sodium dodecyl sulfate     

Physical mapping of the BglI,BglII, PstI and EcoRI restriction fragments of staphylococcal phage ϕ11 DNA

Molecular Genetics and Genomics - A cleavage map of the generalized transducing staphylococcal phage ϕ11 DNA has been constructed by reciprocal double digestion. All three BglI, the six BglII,...     

Isolation and characterization of viable deletion mutants of Bacillus subtilis bacteriophage SPO2.

Spontaneous deletion mutants of the bacteriophage SPO2, which are viable and retain their temperate character, were isolated using a heat-EDTA enrichment step. They were identified by endonuclease digestion and agarose-gel electrophoresis of phage DNA. Two of the nine mutants were characterized in detail. Both mutants have a 2.3 Md deletion removing the single BglII site and two of the XbaI fragments. The deletion extends 1.0 Md to one side of the former BglII site and 1.3 Md on the other side. This region of the SPO2 genome is non-essential for either lysogeny or viable phage production and thus is a suitable region for the insertion of exogenous DNA fragments.     

Comparative restriction analysis of HindIII-F-fragments of DNA from 4 strains of vaccinia virus

The localization of KpnI, SacI, XhoI, AvaI, PstI, BglI, BamHI, EcoRI, PmiI, SalI, BglII, restriction endonuclease cleavage sites in HindIII-F-fragments of DNA from vaccinia strains WR, Copenhagen, LIVP and neurovaccine has been detected. The fragments have been shown to differ in the number of AvaI, EcoRI and BamHI sites. The fragments also differ from the analogue of Tian Tan vaccinia strain in the pattern of restriction by AvaI, XhoI, PstI, EcoRI and BamHI endonucleases.     

Physical mapping of the immunity region of phage phi 80

Mapping of the sites of cleavage of five restriction endonucleases (BglI, BglII, EcoRV, KpnI and PvuII) in the immunity region of bacteriophage phi 80 DNA is described. The order of the 27 restriction sites was established. This helped to localize the phi 80 cI gene within the 640 bp fragment of the immunity region. Recombinant plasmids carrying phi 80 "kil" function were constructed. The function is suppressed by the cI-coded repressor. The deletion AB43 which is present in the phi 80 vir mutant is located within the phi 80 DNA fragment carrying the cI gene.     

The human "interferon-beta 2/hepatocyte stimulating factor/interleukin-6" gene: DNA polymorphism studies and localization to chromosome 7p21

The human interferon-beta 2 gene (IFNB2) product is identical to that for the B-cell stimulation factor-2 (BSF-2), the hybridoma growth factor (HGF) ("interleukin-6"), and the hepatocyte stimulating factor (HSF). Proteins derived from this gene mediate the plasma protein response to tissue injury (acute-phase response) and regulate the growth and differentiation of both B and T cells. By using the enzymes MspI, BstNI, and BglI, three polymorphic systems were detected with probes for the IFNB2 gene. The MspI and BglI polymorphisms are likely to be due to base pair substitutions; the BstNI polymorphism was revealed by nine other enzymes and is likely to be due to DNA insertions within 1 kb of the 3' flanking region of the gene. This region is rich in AT dinucleotides, and slippage at DNA replication may generate the insertions of between 0.07 and 0.23 kb that were observed. The polymorphic MspI site also lies within the vicinity of the fifth exon. The BglI polymorphic site is likely to lie in 5' flanking DNA. The three polymorphisms are separate, and a variety of haplotypes was observed. A low level of linkage disequilibrium exists between the MspI and the BglI alleles. MspI and BstNI polymorphisms were observed in Caucasoids, CAR Pygmies, Zaire Pygmies, Melanesians, and Chinese but at differing frequencies, and not all alleles were present in all populations. The BglI polymorphism was observed in Caucasoids and Africans only. Linkage studies involving the IFNB2 gene and 27 other chromosome 7 markers have localized it to between D7S135 and D7S370 at 7p22-p21.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulating restriction endonuclease activities and specificities using neutral detergents

It is well known that type II restriction enzyme activities and specificities can be modulated by altering solution conditions. The addition of co-solvents such as dimethyl sulfoxide (DMSO), alcohols and polyols can promote star activity, which is the cleavage of non-cognate sequences. While neutral detergents are often used to control protein aggregation, little is known about the effect of neutral detergents on restriction enzyme activities and specificities. We report here that BamHI, BglI, BglII, EcoRI, EcoRV, HindIII, MluI, PvuII, SalI and XhoI restriction endonucleases are remarkably tolerant of high concentrations of neutral detergents Triton X-100, CHAPS and octyl glucoside. In most cases, lambda DNA cleavage rates were comparable to those observed in the absence of detergent. Indeed, the specific activities of SalI and XhoI were appreciably increased in the presence of Triton X-100. For all enzymes active in the presence of detergents, sequence specificity toward lambda DNA was not compromised. Assays of star cleavage of pUC18 by EcoRI, PvuII and BamHI endonucleases in equimolar concentrations of Triton X-100 and sucrose revealed reduced star activity in the detergent relative to the sucrose co-solvent. Interestingly, under star activity-promoting conditions, PvuII endonuclease displayed greater fidelity in Triton X-100 than in conventional buffer. Taken altogether, these results suggest that in some cases, neutral detergents can be used to manipulate restriction endonuclease reaction rates and specificities.     

A restriction map of plasmid pDC1 from the filamentous cyanobacterium Nostoc sp. MAC PCC 8009

A plasmid designated pDC1 from the cyanobacterium Nostoc sp. MAC PCC 8009 was incubated with 16 different restriction enzymes, of which 8 cleaved pDC1. Plasmid pDC1 has a single site for ClaI, two sites for each of BglI, EcoRI, EcoRV, and MluI, three sites for HpaI, and four for HindIII. A restriction map of pDC1 for these 7 enzymes was constructed.     

Reactions of BglI and other type II restriction endonucleases with discontinuous recognition sites

Type II restriction enzymes generally recognize continuous sequences of 4-8 consecutive base pairs on DNA, but some recognize discontinuous sites where the specified sequence is interrupted by a defined length of nonspecific DNA. To date, a mechanism has been established for only one type II endonuclease with a discontinuous site, SfiI at GGCCNNNNNGGCC (where N is any base). In contrast to orthodox enzymes such as EcoRV, dimeric proteins that act at a single site, SfiI is a tetramer that interacts with two sites before cleaving DNA. BglI has a similar recognition sequence (GCCNNNNNGGC) to SfiI but a crystal structure like EcoRV. BglI and several other endonucleases with discontinuous sites were examined to see if they need two sites for their DNA cleavage reactions. The enzymes included some with sites containing lengthy segments of nonspecific DNA, such as XcmI (CCANNNNNNNNNTGG). In all cases, they acted at individual sites. Elongated recognition sites do not necessitate unusual reaction mechanisms. Other experiments on BglI showed that it bound to and cleaved DNA in the same manner as EcoRV, thus further delineating a distinct group of restriction enzymes with similar structures and a common reaction mechanism.     

Identification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage

Fenton chemistry [Fenton (1894) J. Chem. Soc. 65, 899-910] techniques were employed to identify the residues involved in metal binding located at the active sites of restriction endonucleases. This process uses transition metals to catalytically oxidize the peptide linkage that is in close proximity to the amino acid residues involved in metal ligation. Fe2+ was used as the redox-active transition metal. It was expected that Fe2+ would bind to the endonucleases at the Mg2+-binding site [Liaw et al. (1993) Biochemistry 32, 7999-4003; Ermácora et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387; Soundar and Colman (1993) J. Biol. Chem. 268, 5264-5271; Wei et al. (1994) Biochemistry 33, 7931-7936; Ettner et al. (1995) Biochemistry 34, 22-31; Hlavaty and Nowak (1997) Biochemistry 36, 15515-15525). Fe2+-mediated oxidation was successfully performed on TaqI endonulease, suggesting that this approach could be applied to a wide array of endonucleases [Cao and Barany (1998) J. Biol. Chem. 273, 33002-33010]. The restriction endonucleases BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, BsoBI, EcoRI, EcoRV, MspI, and HinP1I were subjected to oxidizing conditions in the presence of Fe2+ and ascorbate. All proteins were inactivated upon treatment with Fe2+ and ascorbate. BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, and BsoBI were specifically cleaved upon treatment with Fe2+/ascorbate. The site of Fe2+/ascorbate-induced protein cleavage for each enzyme was determined. The Fe2+-mediated oxidative cleavage of BamHI occurs between residues Glu77 and Lys78. Glu77 has been shown by structural and mutational studies to be involved in both metal ligation and catalysis [Newman et al. (1995) Science 269, 656-663; Viadiu and Aggarwal (1998) Nat. Struct. Biol. 5, 910-916; Xu and Schildkraut (1991) J. Biol. Chem. 266, 4425-4429]. The sites of Fe2+/ascorbate-induced cleavage for PvuII, FokI, BglI, and BsoBI agree with the metal-binding sites identified in their corresponding three-dimensional structures or from mutational studies [Cheng et al. (1994) EMBO J. 13, 3297-3935; Wah et al. (1997) Nature 388, 97-100; Newman et al. (1998) EMBO J. 17, 5466-5476; Ruan et al. (1997) Gene 188, 35-39]. The metal-binding residues of BglII, SfiI, and BssSI are proposed based on amino acid sequencing of their Fe2+/ascorbate-generated cleavage fragments. These results suggest that Fenton chemistry may be a useful methodology in identifying amino acids involved in metal binding in endonucleases.     

Analysis of 15 different genome types of adenovirus type 7 isolated on five continents.

A total of 15 different genome types of adenovirus type 7 (Ad7), i.e., Ad7p, Ad7p1, Ad7a, Ad7a1 to Ad7a5, Ad7b, Ad7c, Ad7d, Ad7d1, Ad7e, Ad7f, and Ad7g, were identified among 40 selected strains isolated in Europe, Asia, North America, South America, and Australia by using restriction endonucleases BamHI, BclI, BglI, BglII, BstEII, EcoRI, HindIII, HpaI, SalI, SmaI, XbaI, and XhoI. Eight of them, Ad7p1, Ad7a1 to Ad7a5, Ad7d1, and Ad7g, are newly discovered. All 15 genome types could be distinguished by the four restriction endonucleases BamHI, BclI, BglI, and XbaI. At least four restriction sites differed between Ad7d and Ad7g. Pairwise analyses of comigrating DNA restriction fragments of all 15 Ad7 genome types were performed and presented in a schematic fashion. According to the degree of comigration of DNA restriction fragments, the 15 genome types could be divided into three clusters. Ad7b was the dominant genome type in different parts of the world and may have evolved in China into Ad7d and further to Ad7d1.     

Mapping of functions in the R-plasmid R388 by examination of deletion mutants generated in vitro.

Mutant plasmids in which large segments of R388 DNA are deleted were constructed in vitro from two R388::TnA (Tn801) plasmids, using the BamHI site of TnA and the BamHI and BglII sites of R388. These deletion mutants permitted mapping of genetic functions into the restriction map of R388.     

A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA. The structures reveal two different binding states of SfiI: one with both DNA-binding sites fully occupied and the other with fully and partially occupied sites. These two states provide details on how SfiI recognizes and cleaves its target DNA sites, and gives insight into sequential binding events. The SfiI recognition sequence (GGCCNNNN[downward arrow]NGGCC) is a subset of the recognition sequence of BglI (GCCNNNN[downward arrow]NGGC), and both enzymes cleave their target DNAs to leave 3-base 3' overhangs. We show that even though SfiI is a tetramer and BglI is a dimer, and there is little sequence similarity between the two enzymes, their modes of DNA recognition are unusually similar.     

The recognition site of type II restriction enzyme BglI is interrupted

The Type II restriction endonuclease BglI recognizes the interrupted DNA sequence 5'-G-C-C-N-N-N-N-N-G-G-C-. This sequence occurs at all locations in over 33 000 base pairs of DNA sequence where the enzyme was found to cut DNA and nowhere else. All six of the specified bases are essential parts of the site since all groups of five of the six bases occur in the DNA sequences tested and none of them are cut by BglI. The length of the block of intervening unspecified positions must be exactly five since all other sizes between zero and 15 occur in the DNA sequences searched and none are cut by BglI. The 5'-terminal nucleotides of BglI cleaved phage G4 replicative form DNA and plasmid pER18 DNA were compared with the DNA sequences near the BglI sites on these DNAs. These results indicated that BglI cuts within the intervening unspecified region and produces single-stranded 3' termini that are three bases long. The BglI recognition site and cleavage points can thus be represented as follows: (Formula: see text). This study of the BglI recognition site was facilitated by the use of inexpensive microcomputers. A system of programs was developed that allowed analysis of over 33 kb of DNA sequences stored on flexible magnetic disks or audio cassettes. While these programs were generally written in the higher level language BASIC, some assembly language subroutines were utilized to reduce execution time.     

MutH complexed with hemi- and unmethylated DNAs: coupling base recognition and DNA cleavage

MutH initiates mismatch repair by nicking the transiently unmethylated daughter strand 5' to a GATC sequence. Here, we report crystal structures of MutH complexed with hemimethylated and unmethylated GATC substrates. Both structures contain two Ca2+ ions jointly coordinated by a conserved aspartate and the scissile phosphate, as observed in the restriction endonucleases BamHI and BglI. In the hemimethylated complexes, the active site is more compact and DNA cleavage is more efficient. The Lys residue in the conserved DEK motif coordinates the nucleophilic water in conjunction with the phosphate 3' to the scissile bond; the same Lys is also hydrogen bonded with a carbonyl oxygen in the DNA binding module. We propose that this Lys, which is conserved in many restriction endonucleases and is replaced by Glu or Gln in BamHI and BglII, is a sensor for DNA binding and the linchpin that couples base recognition and DNA cleavage.     

Involvement of E. coli dcm methylase in Tn3 transposition

The effects of DNA methyltransferases on Tn3 transposition were investigated. The E. coli dam (deoxyadenosine methylase) gene was found to have no effect on Tn3 transposition. In contrast, Tn3 was found to transpose more frequently in dcm+ (deoxycytosine methylase) cells than in dcm- mutants. When the EcoRII methylase gene was introduced into dcm- cells (E. coli strain GM208), the frequency of Tn3 transposition in GM208 was dramatically increased. The EcoRII methylase recognizes and methylates the same sequence as does the dcm methylase. These results suggest that deoxycytosine methylase modified DNA may be a preferred target for Tn3 transposition. Experiments were also performed to determine whether the Tn3 transposase was involved in DNA modification. Plasmid DNA isolated from dcm- E. coli containing the Tn3 transposase gene was susceptible to ApyI digestion but resistant to EcoRI digestion, suggesting that Tn3 transposase modified the dcm recognition sequence. In addition, restriction enzymes TaqI, AvaII, BglI and HpaII did not digest this DNA completely, suggesting that the recognition sequences of TaqI, AvaII, BglI and HpaII were modified by Tn3 transposase to a certain degree. The type(s), the extent and mechanism(s) of this modification remain to be investigated.