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Induction of endonucleolytic DNA cleavage is an essential event that links the initiating stimuli to the final effects of cells. The cleavage efficiency and thus the final yield could be affected by many factors, including structures of DNA substrates, composite structures of enzymes-substrates or enzymes-nucleic analogs and so on. However, it is not clear whether a nucleotide derivative-substituted in DNA substrates can influence the efficiency of enzymatic cleavage. To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2'-O-methyl modified nucleotides (OMeN) to modify DNA substrates of isocaudemers BamHI and BglII in this study, and used FRET assay as an efficient method for analysis of enzyme cleavage. Experimental results demonstrated that OMeN-substituted recognition sequences influenced the cleavage rates significantly in a position-dependent manner. OMeN substitutions can reduce the cleavage as expected. Surprisingly, OMeN substitutions can also enhance the cleavage rates. The kinetics parameters of Vmax and Km have been obtained by fitting the Michaelis-Menten kinetic equation. These 2'- OMe nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property could enrich our understanding about the endonuclease cleavage mechanism and enhance our ability to regulate the enzymatic cleavage efficiency for applications in synthetic biology.

Hypoxanthine (Hyp), a deaminated base of adenine (Ade), can be employed as a good probe molecule to reveal the significance of the minor groove of guanine (Gua) in biomolecular interactions because Hyp possesses a similar structure to Gua lacking its 2-amino group. In this study, we examined cleavage efficiencies of restriction endonuclease enzymes on DNA substrates with Hyp in their recognition sequences. As a substrate for BglII, EcoRI and BamHI, 24-mer DNA oligomer with Hyp (in place of Gua) was prepared together with its complementary sequences with cytosine (Cyt) or thymine (Thy) as the counter base. At 37 degrees C incubation for 1 h, BglII and EcoRI showed higher DNA cleavage reactivity on Hyp-containing DNA substrates than on normal ones, whereas BamHI showed lower values on Hyp-containing substrates. Such high cleavage performance of BglII and EcoRI on Hyp-containing DNA substrates is in contrast to the results obtained 20 years ago, in which short DNA substrates (8- or 10-mer) and low reaction temperatures (15-20 degrees C) were employed. These new results suggest that the lack of the exocyclic 2-amino group of Gua could contribute to enhanced recognition access of BglII and EcoRI to DNA substrates.

Objectives: To type mef genes in a nationwide collection of clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes as well as pharyngeal carrier strains of viridans streptococci in Norway., Methods: Erythromycin-resistant mef-positive multilocus sequence-typed (MLST) clinical isolates of S. pneumoniae (n = 36) and S. pyogenes (n = 12) from the National Surveillance Program for Antimicrobial Resistance (NORM) as well as viridans streptococci (n = 20) from healthy adults were included. PCR-amplified mef genes were initially discriminated by BamHI digestion. Selected mef genes from representatives of different sequence types (STs) of S. pneumoniae (n = 11) and S. pyogenes (n = 4), and viridans group streptococcal species (n = 8) were typed by sequencing and their strains examined for co-resistances. Hydropathy plots of different mef-encoded proteins were performed., Results: A predominance of mef(A) was detected in S. pneumoniae (23/36) and S. pyogenes (9/12) due to the clonal spread of ST9 and ST39, respectively. mef(E) was the most widely distributed mef determinant occurring in nine different STs of S. pneumoniae and in four different viridans species. A new mef allele was identified in two STs of S. pyogenes., Conclusions: mef(E) is the most widely distributed mef determinant in Norwegian clinical strains of S. pneumoniae and pharyngeal carrier strains of various viridans streptococci. However, mef(A) is more prevalent in S. pneumoniae and S. pyogenes due to clonal spread. A new mef allele was found in two different STs of S. pyogenes.

We report here the structure of BstYI, an "intermediate" type II restriction endonuclease with overlapping sequence specificities to BamHI and BglII. BstYI, a thermophilic endonuclease, recognizes and cleaves the degenerate hexanucleotide sequence 5'-RGATCY-3' (where R=A or G and Y=C or T), cleaving DNA after the 5'-R on each strand to produce four-base (5') staggered ends. The crystal structure of free BstYI was solved at 1.85A resolution by multi-wavelength anomalous dispersion (MAD) phasing. Comparison with BamHI and BglII reveals a strong structural consensus between all three enzymes mapping to the alpha/beta core domain and residues involved in catalysis. Unexpectedly, BstYI also contains an additional "arm" substructure outside of the core protein, which enables the enzyme to adopt a more compact, intertwined dimer structure compared with BamHI and BglII. This arm substructure may underlie the thermostability of BstYI. We identify putative DNA recognition residues and speculate as to how this enzyme achieves a "relaxed" DNA specificity.

Objective: To clone the prtH gene from Porphyromonas gingivalis (P.g) ATCC 33277 and analyze the polymorphism of prtH gene from 5 strains of P.g in order to explore the relationship between P.g and periodontitis., Methods: Using PCR, the prtH was amplified and cloned into pGEM-T vector. To illustrate the prtH polymorphism among P.g strains, the genomic DNAs were extracted and screened by PCR with three pairs of specific primers, dot blot and Southern blot hybridization using the biotin-labeled prtH sequence as probe., Results: Recombinant DNA pGEM-T- prtH was verified by restriction endonuclease and sequence assay. Strain W 381 and ATCC 33277 showed the identical results in PCR and hybridization assays, whereas strain ATCC 49417 and 14-3-2 revealed individual hybridization patterns. Strain 47A-1 seemed even not to contain prtH gene., Conclusions: Different prtH gene sequences exist in different P.g strains. This polymorphism may indicate various potential virulent effects during the infection and pathogenesis. Established PCR protocol is sensitive for identification of prtH gene.

A 1.6 Kb mobilization (Mob) fragment originating from broad host range IncP plasmid RP4 is effectively cloned into two different Col EI-origin based cloning vectors, pBluescript II SK+ and pT-Adv, to generate pPAR-I and pPAR-II, respectively. The vectors have different genetic markers and were demonstrated to get mobilized at significant frequency into a laboratory and an enteroroxigenic strain of Escherichia coli with all the genetic markers of the recombinant clones expressing efficiently in the recipient host cells. Important restriction endonuclease recognition sequences in the multiple cloning sites of the conjugal vector DNA molecules remained unique. Significance and relevance of the current study with regard to other gene delivery system in gram negative bacteria are discussed.

Objective: To establish a method for cloning thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus (Vp)., Methods: After enrichment by PCR, the trh gene, along with the plasmid DNA of pGEX-3X, was digested with two enzymes followed by linkage of the gene and the plasmid, the product of which was transferred into E.coli DH5alpha. Identification of the recombinant plasmid was performed by means of PCR, digestion and sequence analysis., Results: A fragment about 600 bp was identified in the PCR product of Vp14-91, which was also seen in the product of PCR and enzyme digestion of the recombinant fragment. Sequence analysis demonstrated a homology of 99.1% between the trh gene and the reference gene available in the GenBank., Conclusion: trh gene has been successfully cloned with its sequence analyzed, which prepares the ground for developing gene probe and protective vaccine against Vp.

A 2.6-kb BamHI fragment from the genome of the wild-type, nikkomycin-producing strain of Streptomyces tendae ATCC 31160 was cloned and sequenced. This 2.6-kb BamHI fragment corresponds to the DNA site where transposon Tn4560 had inserted to create a nikkomycin-nonproducing mutant. A possible ORF of 660 nucleotides was found in this 2.6-kb BamHI fragment, in which the third base of each codon was either G or C in 92% of the codons. The deduced amino acid sequence coded by this ORF (TarA, tendae autoregulator receptor) shows strong homology with several Gamma-butyrolactone-binding proteins that negatively regulate antibiotic production in other streptomycetes and have a helix-turn-helix DNA-binding motif. A portion (179 nucleotides) of tarA that encodes the helix-turn-helix motif was replaced with ermE, and wild-type S. tendae was transformed with this construct borne in pDH5, a gene-disruption vector. Southern hybridization indicated that ermE had inserted in the 2.6-kb BamHI region in one isolate that is erythromycin resistant. Northern hybridization indicated that tarA disruption significantly increased the amount of disrupted-tarA mRNA. This suggests that TarA negatively regulates its own synthesis. Nikkomycin production by the tarA disruptant was delayed but reached the wild-type level after longer incubation in production medium.

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1. 5 A resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar alpha/beta core, but in BglII, the core is augmented by a beta-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein-DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases.

Adult domestic cats (Felis catus) appear to have the most polymorphic beta-globin system of any species. Reversed-phase high-performance liquid chromatography (RP-HPLC) revealed one alpha-chain and six different beta-globin chains. Each cat may have one to four different beta-globins, and a total of 17 different beta-globin patterns were identified. Based on family studies, a beta-globin gene region with two linked beta-globin gene loci and two to five alleles was proposed. In order to further define the molecular basis of this polymorphism we performed Southern blot analysis of the beta-globin gene region from 25 cats with 13 different HPLC patterns. Genomic DNA was digested with five restriction endonucleases (BamHI, BglII, EcoRI, HindIII, and PstI) and blots were hybridized with a human beta-globin probe. Restriction fragment length polymorphisms (RFLPs) with one to five fragments of 2.2-23 kb in size were found with BglII, EcoRI, HindIII, and PstI. Cats with specific HPLC beta-globin patterns had a unique DNA restriction pattern. The similarly sized BamHI and HindIII fragments of 4.6 kb suggest the presence of two closely linked genes. Furthermore, family studies suggest an autosomal codominant mode of inheritance of the beta-globin chains with seven haplotypes. Thus the RFLP data analyzed in the context of the HPLC haplotypes provide evidence at the DNA level for a feline beta-globin gene region with two closely linked gene loci and two to five alleles.

The far upstream region of the rat Ha-ras gene has been characterized to determine whether possible repressor sequences may control the low level of Ha-ras gene transcription from its TATA-less, GC-rich strong promoter. The chloramphenicol acetyl transferase (CAT) gene under the control of the 3.8-kb Ha-ras upstream promoter was minimally expressed in HeLa cells. Surprisingly, CAT gene expression was increased by the deletion of a 0.7-kb BglII fragment containing non-coding exon minus 2 and TATA box promoter elements located 1.7 kb upstream of the GC-rich strong promoter. Far upstream (CA)25 repeats also appeared to repress Ha-ras gene activity. Sequences within the 0.7-kb BglII fragment suppressed CAT gene expression when placed upstream of a heterologous thymidine kinase (tk) gene promoter. Repressor activity was further localized to a 160-bp AvrII-BglII sub-fragment. Gel shift assays identified two sequence-specific DNA binding proteins. The results demonstrated for the first time that far upstream repressor sequences control normal transcription of the Ha-ras proto-oncogene., (Copyright 1998 Academic Press.)

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific alpha subunits and a common signal-transducing beta subunit (hbetac). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of hbetac. We report here a comprehensive screen of the entire hbetac molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of hbetac that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hbetac mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant beta subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hbetac activation, dissociate hbetac tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hbetac., (Copyright 1998 by The American Society of Hematology.)

In Xer site-specific recombination, two related recombinases, XerC and XerD, mediate the formation of recombinant products using Holliday junction-containing DNA molecules as reaction intermediates. Each recombinase catalyses the exchange of one pair of specific strands. By using synthetic Holliday junction-containing recombination substrates in which two of the four arms are tethered in an antiparallel configuration by a nine thymine oligonucleotide, we show that XerD catalyses efficient strand exchange only when its substrate strands are 'crossed'. XerC also catalyses very efficient strand exchange when its substrate strands are 'crossed', though it also appears to be able to mediate strand exchange when its substrate strands are 'continuous'. By using chemical probes of Holliday junction structure in the presence and absence of bound recombinases, we show that recombinase binding induces unstacking of the bases in the centre of the recombination site, indicating that the junction branch point is positioned there and is distorted as a consequence of recombinase binding.

Epizootics of a natural varicella-like disease occur in populations of nonhuman primates. Several primate herpesviruses have been isolated from these epizootics, but the relatedness of these isolates to each other is not well-defined. In this study, we demonstrated that the restriction endonuclease (REn) profiles of four epidemiologically distinct isolates were similar, although not identical, indicating that simian varicella epizootics are caused by various strains of simian varicella virus (SVV). The genetic variation among the isolates did not map to a specific region of the SVV genome and REn differences were detected within the SVV DNA long component and the inverted repeat region. Southern blot hybridization demonstrated that SVV is more closely related to varicella-zoster virus than to other primate herpesviruses. The study indicates that the current herpesvirus classification scheme should be changed to include SVV as a single taxonomic group within the Varicellovirus genus of alphaherpesviruses. In addition, REn profiles of SVV isolates, derived from primary and secondary episodes of simian varicella in the same monkey, were identical, providing evidence for SVV reactivation in a latently infected monkey.

A P1-based cloning system has been tested which depends upon (i) in vitro selection for vectors containing inserts mediated by methyltransferase (MTase) protection, and (ii) in vivo vector arm Cre-mediated recombination following electroporation. Specifically, chromosomal DNA was digested with BglII, dephosphorylated, methylated with BamHI MTase and ligated into the BamHI site of the vector, thereby destroying that site. Subsequent BamHI digestion acted as the in vitro selection, eliminating vector religation products prior to electroporation into cells expressing the Cre recombinase. Electroporation with linearized vector gave approx. 10(6) transformants per microgram vector, depending on vector concentration. Cloning of BglII fragments of gamma DNA using the in vitro selection system led to 1.3-4-fold fewer transformants per microgram vector. Plasmids recovered from these clones were all found to contain a gamma BglII fragment and the representation of fragments in the clones was independent of the length of the fragments. Both in vitro selection and electroporation are applicable to library construction using size-selected human DNA, with size selection either before or after ligation and BamHi digestion.

The HIS4LEU2 meiotic recombination hot spot specifies two double-strand break (DSB) sites, I and II. Results presented demonstrate that DSBs at site I occur at many positions throughout a region of approximately 150 bp; we infer that breaks occur in a sequence non-specific fashion. Single-strand nicks at sites I and II are not detectable. Analysis of the effects of a 36 bp linker insertion at site I reveals the existence of communication along and between homologs prior to DSB formation. In cis, the insertion allele causes an increase in DSBs at site I but a decrease in DSBs at site II. In trans, two effects are observed. One effect likely reflects very early pre-DSB interhomolog interactions; the second is suggestive of a later, more intimate interaction in which sites I and II on the two homologs all compete for DSBs. The existence of interhomolog interactions in early meiotic prophase can explain how the sites of crossovers come to lie between the homolog axes at pachytene.

This study was undertaken to assess the discriminatory value of restriction endonuclease (RE) digestion patterns of Streptococcus suis chromosomal DNA using polyacrylamide gel electrophoresis (SDS-PAGE) and DNA-rDNA hybridization. For the RE digestion patterns, DNAs were digested separately with the enzymes BamHI and BglII and the resultant fragments were separated by SDS-PAGE. An Escherichia coli rDNA probe derived from pKK3535 was used for the hybridization. Twenty-three S. suis capsular type 2 isolates recovered from diseased and clinically healthy pigs, from a human case, and from a cow were compared in this study. The majority of isolates associated with septicaemia belonged to one restriction endonuclease analysis (REA) profile group. Isolates associated with pneumonia belonged either to the REA profile group of isolates associated with septicaemia or to a second REA profile group. The REA profiles of isolates from clinically healthy animals were more heterogeneous. The REA profile of the type 2 reference strain, S735, which was originally isolated from a pig, was very different from those of the porcine and bovine isolates but similar to the profile of the human isolate. The profiles obtained after rDNA hybridization were more homogeneous. Although different patterns were detected in the 23 isolates, there was no correlation between the source of the isolate and the patterns observed with this technique.

The configuration of immunoglobulin (Ig) and T-cell receptor (TCR) genes was investigated in a case of atypical chronic myeloid leukemia (aCML) lacking a cytogenetically detectable Philadelphia chromosome and molecular evidence of breakpoint cluster region (bcr) rearrangement as determined by 5' and 3' bcr gene probes. Dual clonal rearrangement of Ig-heavy (H) and TCR-delta chain genes was identified. The genes for TCR-gamma and beta chain as well as Ig-kappa (k) chain were in germline configuration. These findings indicate transformation to have occurred in a hemopoietic stem cell with the capacity for antigen receptor gene rearrangement. The demonstration of cross-lineage rearrangement of both Ig and TCR genes lends support to evidence from G6PD alloenzyme studies that the target of transformation in aCML is an early stem cell not yet irreversibly committed to myeloid differentiation. Further studies are indicated to ascertain whether antigen receptor gene rearrangement constitutes a molecular marker useful in the diagnosis of aCML.

Site-specific DNA methylation is known to block cleavage by a number of restriction endonucleases. We show that methylation at 'non-canonical' DNA modification sites can also block methylation by five of 13 DNA methyltransferases (MTases) tested. Furthermore, MTases and endonucleases that recognize the same nucleotide sequence can differ in their sensitivity to non-canonical methylation. In particular, BamHI endonuclease can cut 5'-GGATCm5C efficiently, whereas M.BamHI cannot methylate this modified sequence. Methyltransferase/endonuclease pairs which differ in their sensitivity to non-canonical methylation can be exploited to generate rare DNA cleavage sites. For example, we show that M.HpaII, M.BamHI, and BamHI can be used sequentially in a three-step procedure to specifically cleave DNA at the 10-bp sequence 5'-CCGGATCCGG. Several highly selective DNA cutting strategies are made possible by these sequential double methylation-blocking reactions.