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To characterize the genotypes of Epstein-Barr virus (EBV) isolate present in North Africa, viruses were isolated from B-lymphoblastoid cell lines established from the saliva of both Algerian Nasopharyngeal Carcinoma (NPC) patients and EBV-positive normal individuals, Algerian Burkitt's lymphoma cell lines, and NPC biopsies. By nucleotide sequence analysis, we showed that there were two specific missense mutations in an 89 bp region of EBNA2 gene at position 49390-49479 of the EBV genome: a mutation at 49449 (C-->A) and another mutation at 49444 (T-->C), changing their amino acid sequence. The first mutation was found in all B cell lines established from the saliva and 50% of BL cell lines, as well as the W91 cell line, while the second mutation was found in EBV isolates from NPC biopsies, BL cell lines and the M-ABA isolate. A PCR-RFLP analysis on the BamHI DNA fragment H showed that the Hl-H2-polymorphism was specifically associated with M-ABA-like mutation, while H-polymorphism was linked with W91-like mutation. The latter was not identified in NPC biopsies, but was found rather in saliva from NPC patients, normal individuals and BL cell lines. The M-ABA-like mutation, on the other hand, was found in 100% of NPC biopsies and some BL cell lines. This suggests that EBV with H1-H2-polymorphism is tightly implicated in NPC development in North Africa rather than EBV with H-polymorphism.

The strategy for the design of photochemically controllable enzymes by manipulating the dimer interface is described. Employing a restriction endonuclease BamHI, the selective incorporation of amino acids having a photoremovable 6-nitroveratryl group into the specific position (Lys132) in the dimer interface of the BamHI mutant (H133A) was performed. The activity of the photofunctionalized BamHI mutant was significantly suppressed, and the following photoirradiation induced the recovery of the activity. In addition, uncaging of the 6-nitroveratryl group introduced to Lys132 did not seriously reduce the catalytic activity and affinity for the substrate. These results indicate that the activity of the enzyme can be effectively regulated by caging and uncaging of the specific amino acid in the dimer interface using the photoremovable group.

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.

Glutathione (GSH), an important antioxidant involved in stress response, is synthesized in two sequential reactions. Gamma-glutamylcysteine synthetase (GCS) catalyzes the first step in GSH biosynthesis, which is usually known to be rate-limiting. In this work, regulatory patterns of the GCS gene from the fission yeast Schizosaccharomyces pombe have been investigated. The 607 bp upstream region from the translational initiation point was amplified by the two synthetic primers. The amplified DNA was ligated into the BamHI/HindIII site of the shuttle vector YEp367R to generate the fusion plasmid pUGCS101. The GCS-lacZ fusion gene construct was confirmed by restriction mapping and nucleotide sequencing. The GCS-lacZ fusion gene was used to study effects of various agents on the transcription of the GCS gene. The synthesis of beta-galactosidase from the fusion plasmid pUGCS101 was enhanced by metals, oxidative and nitrosative stresses, and glutathione-depleting agents. The GCS mRNA level in the wildtype S. pombe cells was significantly elevated by the treatment with sodium nitroprusside or menadione, which was detected by RT-PCR. It was also induced by low concentrations of glucose and sucrose. These results suggest that the expression of S. pombe GCS gene is regulated by various stresses and carbon sources.

Efficient selection of the genetically modified cell population is a critical step to obtain the cells with desired properties. In this study, we propose an antigen-mediated genetically modified cell amplification (AMEGA) system employing an antibody/receptor chimera that triggers a growth signal in response to a non-toxic hapten dimer. An anti-fluorescein single-chain Fv fused to the extracellular D2 domain of erythropoietin receptor and transmembrane/intracellular domains of gp130 was expressed together with a model transgene, enhanced green fluorescent protein (EGFP) downstream of IRES sequence, by retroviral infection to IL-3-dependent Ba/F3 cells. Addition of fluorescein dimers connected by various oligo-DNA linkers induced selective growth of transfectants, thus leading to efficient expansion of EGFP-positive cell population. Also, digestion of the oligonucleotides by specific restriction endonuclease completely suppressed cell growth. Because these hapten dimers are not harmful for normal cells, the approach will be especially useful for reversible in vitro or in vivo expansion of genetically modified cell population employed for cell therapy and tissue engineering.

This report describes lytic infection with Epstein-Barr virus (EBV) in three cases of hemophagocytic syndrome (HPS) presumably associated with EBV-induced lymphoproliferative disorder. All cases were previously healthy females with ages ranging from 52 to 87 years who showed a fulminant clinical course with accompanying fever, liver dysfunction, and disseminated intravascular coagulation. Cases 1 and 2 showed proliferation of atypical T lymphocytes, and case 3 showed proliferation of atypical B lymphocytes. Hemophagocytic histiocytes were observed in the bone marrow, lymph nodes, spleen, and liver. Atypical lymphocytes in all cases showed a positive reaction for both EBV-encoded small RNA (EBER) indicating latent infection with EBV and immediate early mRNAs of the Bam HI fragment of lower stranded frame (BHLF), indicating lytic infection by in situ hybridization. Interestingly, BHLF-positive cells were predominant in all cases. It is possible that reactivation of EBV infection may be involved in triggering the induction of cytokines and abnormal activation of histiocytes.

There is still no clarity on whether the endonuclease incisions in apoptotic cells are induced randomly in the genome or induced in some preferable sites. In order to evaluate the intensity of DNA fragmentation in the chicken alpha-globin domain, AEV-virus transformed chicken erythroblasts (HD3) were incubated in a serum free medium, and their DNA was Southern blotted and hybridised with probes representing different fragments of the domain. Probes corresponding to the upstream areas of the domain mostly hybridised with high molecular weight DNA. Unlike these, the probe corresponding to the 2 Kb BamHI-BamHI fragment, containing the alphaA globin gene (B18), revealed a 5 Kb band on the hybridisation autoradiographs. The probe to the neighbouring upstream fragment did not reveal this band, but it was clearly seen on hybridisations with a downstream 1 Kb BamHI-BamHI fragment. The intensity of the band increased with overall apoptotic DNA degradation, hence its appearance should be coupled to apoptosis. Hybridisation of BamHI-digested DNA with B18 probe revealed a shortening of the 2 Kb band in preparations of DNA from apoptotic cells. The presumable positions of the cuts correspond to the formerly described DNase hypersensitive sites in the domain. Slot-blot and Northern hybridisation of RNA extracted from apoptotic HD3 cells revealed that the excision of the area of the B18 gene is coupled to a decrease in the intensity of alphaA globin gene transcription. Transcription of the non-erythroid NIK gene, transcribed in the upstream part of the domain, did not depend on the level of apoptotic DNA fragmentation.

Objective: To investigate the genomic variation of Epstein-Barr virus (EBV) and its significance in nasopharyngeal carcinogenesis., Methods: Forty nasopharyngeal carcinoma (NPC) biopsy tissues were used for detection of EBV BamHI f variant and LMP1 XhoI-loss by polymerase chain reaction (PCR), nested PCR, and RFLP (restriction fragment length polymorphism). Forty-eight samples of peripheral blood mononuclear cells (PBMC) taken from apparently healthy adult individuals were used for detection of LMP1 XhoI-loss. Three samples of amplified LMP1 exon 1 DNA from B95-8 cell line and 2 NPC tissues (one having XhoI-loss and the other having Wt-XhoI/XhoI-loss) were sequenced., Results: Thirty out of the 40 NPC cases (30/40, 75%) harbored EBV BamHI f variant and the remaining 10 (10/40, 25%) harbored BamHI F prototype. Thirty out of the 39 NPCs (30/39, 76.9%) showed single EBV LMP1 XhoI-loss, 7 (7/39, 18.0%) showed single LMP1 Wt-XhoI (presence of a XhoI site in exon 1 of LMP1 gene, as in B95-8 cell line), and 2 (2/39, 5.1%) showed both LMP1 Wt-XhoI and XhoI-loss. Thirty-eight of the 39 NPCs (97.4%) showed EBV LMP1 XhoI-loss or/and BamHI F variant. In the NPC tissue (1 case only) showing the prototype of Wt-XhoI/BamHI "f", there were several base substitutions, including 5 missense mutations and 2 silent mutations present in LMP1 exon 3, on DNA sequencing. On the other hand, 10 out of the 48 samples of PBMC taken from apparently healthy individuals could be amplified successfully by nested PCR for detection of LMP1 XhoI site. All of these 10 samples carried the prototype of EBV LMP1 Wt-XhoI., Conclusions: The majority of EBV present in neoplastic cells of NPC is of BamHI "f" variant and/or possesses LMP1 XhoI-loss, as compared with that in healthy individuals. This genomic variation of EBV may bear some roles in the development and progression of NPC.

Qbeta replicase (RNA-directed RNA polymerase of bacteriophage Qbeta) exponentially amplifies certain RNAs (RQ RNAs) in vitro. Here we characterize template properties of the 5' and 3' fragments obtained by cleaving one of such RNAs at an internal site. We unexpectedly found that, besides the 3' fragment, Qbeta replicase can copy the 5' fragment and a number of its variants, although they lack the initiator region of RQ RNA. This copying can occur as a 3'-terminal elongation or through de novo initiation. In contradistinction to RQ RNA and its 3' fragment, initiation on these templates occurs without regard to the 3'-terminal or internal oligo(C) clusters, is GTP-independent, and does not result in a stable replicative complex capable of elongation in the presence of aurintricarboxylic acid. The results suggest that, although Qbeta replicase can initiate and elongate on a variety of RNAs, only some of them are recognized as legitimate templates. GTP-dependent initiation on a legitimate template drives the enzyme to a "closed" conformation that may be important for keeping the template and the complementary nascent strand unannealed, without which the exponential replication is impossible. Triggering the GTP-dependent conformational transition at the initiation step could serve as a discriminative feature of legitimate templates providing for the high template specificity of Qbeta replicase.

The in vivo expression of the Epstein-Barr virus (EBV) BamHI-A rightward transcripts (BARTs) as well as the putative BART-encoded BARF0 and RK-BARF0 proteins in various EBV-associated malignancies was investigated. RT-PCRs specific for the different splice variants of the BARTs and both a nucleic acid sequence-based amplification assay and an RT-PCR specific for the BARF0 ORF were used. Abundant transcription of BARTs was found in EBV-associated Hodgkin's lymphomas, Burkitt's lymphomas (BL), T-cell non-Hodgkin's lymphomas, post-transplant lymphoproliferative disorders, AIDS-related lymphomas and gastric carcinomas. Using RNA in situ hybridization (RISH), BARTs were detected within the neoplastic cells of these malignancies. BARTs encoding RK-BARF0 were not detected. The BARTs detected were shown possibly to encode the RPMS1 and BARF0 proteins, based on their splicing. However, BARTs actually harbouring the BARF0 ORF were detected only in specimens containing a relatively large number of EBV-positive cells. New monoclonal antibodies against the BARF0 protein were generated that efficiently recognized prokaryotic and eukaryotic recombinant BARF0. However, the BARF0 protein was not detected in clinical samples, nor in EBV-positive cell lines, even though these were positive for BARTs by RISH and/or BARF0 RNA in vitro analysis. Using immunoblot analysis, no antibodies against baculovirus-expressed BARF0 protein were detected in the sera of nasopharyngeal carcinoma patients, BL patients and Hodgkin's disease patients, patients with chronic EBV infection, infectious mononucleosis patients or EBV-positive healthy donors. Thus, BARTs containing the BARF0 ORF are expressed in vivo but the BARF0 protein cannot be detected and may be expressed only marginally. It is concluded that the BARF0 protein is unlikely to play a role in vivo in EBV-positive malignancies.

We have used RT-PCR and low-stringency cDNA library screening to isolate the coding sequence of the sunflower auxin-binding protein (ABP1). All the clones analysed contained the same nucleotide sequence, suggesting that ABP1 is encoded by a single-copy gene in sunflower. The deduced amino acid sequence shows a high degree of similarity with ABP1 proteins from other plant species. Most remarkably, the sunflower protein lacks two cysteine residues present in all other plant ABPs known to date and shown to be involved in a disulfide bridge in the maize protein. Genomic Southern hybridization data support the existence of a single copy of the ABP1 gene in the sunflower genome. Northern hybridization corroborated earlier observations indicating that the steady-state level of ABP1 transcript is higher in actively dividing and growing organs than in the rest of the plant: it is more abundant in the shoot apex, floral buds and immature embryos than in mature leaves, stem, roots and ray flowers. To characterize the tissular ABP1 transcript distribution in sunflower, various organ sections were analysed upon in situ hybridization. Localized accumulation of the ABP1 transcript suggests that its spatial expression is highly regulated at the tissue level. In addition, the transcript preferentially accumulates in tissues having a high rate of cellular division, such as shoot and root apical meristems, leaf primordia and pro-vascular tissues. The ABP1 expression pattern was also studied at a temporal scale during lateral root formation. Real time PCR showed an elevation of the steady state level of the ABP1 transcript in root axes after 36 h of seed germination. In situ hybridization revealed that this global increase is the result of local accumulation of the ABP1 transcript in lateral root primordia, which are known to develop under auxin action. The possibility that a high ABP1 expression level correlates with a high cellular sensitivity to auxin is discussed.

Epstein-Barr virus (EBV) has been suggested to play a role in hepatocellular carcinoma (HCC). However, reports on detailed EBV transcript analyses in HCCs are limited. It was shown recently that expression of the transforming BARF1 (BamHI A rightward open reading frame 1) gene of EBV is restricted to latently EBV-infected epithelial malignancies, i.e. nasopharyngeal carcinoma and gastric carcinoma. The aim of this study was to test the presence of EBV in Dutch HCCs. A semiquantitative DNA PCR-enzyme immunoassay (PCR-EIA) for the BamHI W fragment of EBV was used to assess the presence of EBV in frozen and paraffin-embedded tissues of 16 HCCs. In addition, several RNA detection techniques, i.e. nucleic acid sequence-based amplification (NASBA), RT-PCR, RNA in situ hybridization (RISH) and immunohistochemistry (IHC), were applied. Five of 16 HCCs and two of four hepatitis C virus hepatitis samples were weakly positive for EBV DNA by PCR-EIA. Using sensitive RNA transcription techniques, no transcripts were found for BARF1, EBNA-1 and BARTs (BamHI A rightward transcripts) in any of the liver tissues tested. In addition, RISH for EBER1/2 and BARTs and IHC for EBNA-1, LMP-1 and ZEBRA, performed on the paraffin-embedded tissue of the PCR-EIA-positive cases and on adjacent non-neoplastic liver tissues, were negative. The absence of epithelial-specific BARF1 transcripts and other EBV transcripts and proteins in the EBV DNA PCR-positive cases argues strongly against a role for EBV in HCC.

We studied the kinetics of methyl group transfer by the BamHI DNA-(cytosine-N(4)-)-methyltransferase (MTase) from Bacillus amyloliquefaciens to a 20-mer oligodeoxynucleotide duplex containing the palindromic recognition site GGATCC. Under steady state conditions the BamHI MTase displayed a simple kinetic behavior toward the 20-mer duplex. There was no apparent substrate inhibition at concentrations much higher than the K(m) for either DNA (100-fold higher) or S-adenosyl-l-methionine (AdoMet) (20-fold higher); this indicates that dead-end complexes did not form in the course of the methylation reaction. The DNA methylation rate was analyzed as a function of both substrate and product concentrations. It was found to exhibit product inhibition patterns consistent with a steady state random bi-bi mechanism in which the dominant order of substrate binding and product release (methylated DNA, DNA(Me), and S-adenosyl-l-homocysteine, AdoHcy) was Ado-Met DNA DNA(Me) AdoHcy. The M.BamHI kinetic scheme was compared with that for the T4 Dam (adenine-N(6)-)-MTase. The two differed with respect to an effector action of substrates and in the rate-limiting step of the reaction (product inhibition patterns are the same for the both MTases). From this we conclude that the common chemical step in the methylation reaction, methyl transfer from AdoMet to a free exocyclic amino group, is not sufficient to dictate a common kinetic scheme even though both MTases follow the same reaction route.

Marek's disease (MD) in chickens is caused by MD herpesvirus (MDV), which induces T cell lymphomas. The early pathogenesis of MDV infection is characterized by a primary infection in B lymphocytes followed by infection of activated T lymphocytes. It has been speculated that a MDV-encoded homologue of interleukin-8 (vIL-8) may be important to attract activated T lymphocytes to infected B lymphocytes. Recently, more virulent strains of MDV have emerged, named very virulent plus (vv+)MDV, that cause earlier and more prolonged cytolytic infections compared to less virulent strains. In this report, it was found that vIL-8 mRNA expression in vivo was increased in very virulent (vv) and vv+MDV strains compared to mild (m) and virulent (v) strains, and could not be detected in two attenuated MDV strains examined using very sensitive real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. In order to identify potential mechanisms for the increased vIL-8 mRNA expression in more virulent strains, and lack thereof in attenuated strains, the vIL-8 gene and putative promoter sequences upstream of the vIL-8 gene were compared from 10 different MDV strains, including attenuated derivatives. Only the JM-16 strain (both non-attenuated and attenuated) and attenuated 584A (584Ap80C) encoded a predicted vIL-8 gene sequence different from all other strains examined. Within the putative vIL-8 gene promoter sequence, there was little difference among the non-attenuated strains; however significant deletions were identified in the attenuated JM-16/p71, Md11 (R2/23), and 584Ap80C strains. Additionally, these deletions were located within a previously hypothetical open reading frame (ORF) named LORF4. Rapid amplification of cDNA ends identified a full-length transcript of LORF4 in the MDV-transformed lymphoblastoid cell line MSB-1, and deletions within this ORF caused truncated predicted proteins in 4 out of 6 attenuated MDV strains examined.

Divalent metal ions are essential to many enzymatic reactions involving nucleic acids, but their critical and specific role still needs to be uncovered. Restriction endonucleases are a prominent group of such metal-requiring enzymes. Large scale accurate simulations of Mg- and Ca-BamHI elucidate the mechanism of the catalytic reaction leading to DNA cleavage and show that it involves the concerted action of two metal ions and water molecules. It is also established that what is decisive for the dramatically different behavior of magnesium (a cocatalyst) and calcium (an inhibitor) are kinetic factors and not the properties of the prereactive states of the enzymes. A new perspective is opened for the understanding of the functional role of metal ions in biological processes.

In this study the electrostatic and nonelectrostatic contributions to the binding free energy of a number of different protein-DNA recognition complexes are investigated. To determine the electrostatic effects in the protein-DNA association the Poisson-Boltzmann approach was applied. Overall the salt-dependent electrostatic free energy opposed binding in all protein-DNA complexes except one, and the salt-independent electrostatic contribution favored binding in more than half of the complexes. Further the salt-dependent electrostatic free energy increased with higher ionic concentrations and therefore complex association is stronger opposed at higher ionic concentrations. The hydrophobic effect in the protein-DNA complexes was determined from the buried accessible surface area and the surface tension. A majority of the complexes showed more polar than nonpolar buried accessible surface area. Interestingly the buried DNA-accessible surface area was preferentially hydrophilic, only in one complex a slightly more hydrophobic buried accessible surface area was observed. A quite sophisticated balance between several different free energy components seems to be responsible for determining the free energy of binding in protein-DNA systems.

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.

Bacterial artificial chromosome (BAC) library plays a pivotal role in genomics studies. A crucial step in BAC library construction is preparation of BAC vector DNA. Preparation of highly purified vector DNA is affected by a series of factors including digestion of restriction enzyme and dephosphorylation of linearized vector DNA. In our study, the BAC vector pECBAC1 was digested by the restriction enzyme of BamHI and dephosphorylated by HK phosphatase respectively. In order to improve the ligation capability of vector DNA, gel purification of linearized vector DNA was also conducted in our study. At the same time, we did a series of experiments to get high quality of vector DNA for construction of BAC library. They included the optimal concentration of restriction enzyme, optimal digestion time, the type of phosphatase and gel purification of linearized vector DNA.

Genomic signature tags (GSTs) are the products of a method we have developed for identifying and quantitatively analyzing genomic DNAs. The DNA is initially fragmented with a type II restriction enzyme. An oligonucleotide adaptor containing a recognition site for MmeI, a type IIS restriction enzyme, is then used to release 21-bp tags from fixed positions in the DNA relative to the sites recognized by the fragmenting enzyme. These tags are PCR-amplified, purified, concatenated, and then cloned and sequenced. The tag sequences and abundances are used to create a high-resolution GST sequence profile of the genomic DNA. GSTs are shown to be long enough for use as oligonucleotide primers to amplify adjacent segments of the DNA, which can then be sequenced to provide additional nucleotide information or used as probes to identify specific clones in metagenomic libraries. GST analysis of the 4.7-Mb Yersinia pestis EV766 genome using BamHI as the fragmenting enzyme and NlaIII as the tagging enzyme validated the precision of our approach. The GST profile predicts that this strain has several changes relative to the archetype CO92 strain, including deletion of a 57-kb region of the chromosome known to be an unstable pathogenicity island.

The ZEBRA protein encoded by the Epstein-Barr virus (EBV) genome activates a switch from the latent to the lytic gene expression programme of the virus. ZEBRA, a member of the basic leucine zipper family of DNA-binding proteins, is a transcriptional activator capable of inducing expression from several virus lytic cycle promoters by binding to activator protein 1 (AP-1)-like sites. The Epstein-Barr virus BamHI F promoter, Fp, was for some time believed to initiate EBNA1-specific transcription in EBV-transformed latent cells. More recent data, however, show that Fp is an early lytic promoter and that the dominant EBNA1 gene promoter in latent cells is Qp, located about 200 bp downstream of Fp. In the present investigation we confirm that Fp displays the characteristics of a lytic promoter. Fp is downregulated in latently EBV-infected cells, both in the endogenous virus genome and in reporter plasmids that carry Fp regulatory sequences upstream of position -136 and down to +10 relative to the Fp transcription start site (+1), and is activated on induction of the virus lytic cycle. We show that the repression of Fp in latent stages of infection can be abolished by ZEBRA, and demonstrate that ZEBRA activates Fp through a direct interaction with an AP-1-like site at position -52/-46 in the promoter-proximal Fp region.

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.

Vancomycin-resistant Enterococcus faecium (VREF) strains represent an important threat in hospital infections in the United States and are found at high frequencies in both the community and farm animals in Europe. We evaluated automated ribotyping for interlaboratory reproducibility by using the restriction enzymes EcoRI and BamHI and compared ribotyping to both amplification of fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) to assess its discriminatory power and capacity for the identification of epidemiologically important strains. Of 19 (EcoRI) and 16 (BamHI) isolates tested in duplicate in two laboratories, 18 (95%) and 16 (100%), respectively, showed reproducible ribotypes. These high reproducibility rates were obtained only after manual refinement of the automated fingerprint analysis. A group of 49 VREF strains initially selected to represent 32 distinct AFLP types were separated into 28 EcoRI ribotypes, 25 BamHI ribotypes, and 28 sequence types. Ribotyping with EcoRI and BamHI was able to discern the host-specific genogroups recently disclosed by AFLP typing and MLST and to distinguish most strains containing the esp gene, a marker specific for strains causing hospital outbreaks. An expandable ribotype identification library was created. We recommend EcoRI as the enzyme of choice for automated ribotyping of VREF strains. Given the high level of discrimination of VREF strains, the high rate of interlaboratory reproducibility, and the potential for the identification of epidemiologically important genotypes, automated ribotyping appears to be a very valuable approach for characterizing VREF strains.

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.

Monodisperse magnetic nanoparticles conjugated with complementary oligonucleotide sequences self-assemble into stable magnetic nanoassemblies resulting in a decrease of the spin-spin relaxation times (T2) of neighboring water protons. When these nanoassemblies are treated with a DNA cleaving agent, the nanoparticles become dispersed, switching the T2 of the solution back to original values. These qualities render the developed nanoparticles and their nanoassemblies as magnetic relaxation switches capable of screening for DNA-cleaving compounds by magnetic resonance methods such as MRI and NMR.

We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with approximately 46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of approximately 100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5-10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome.

The fundamental processes by which proteins recognize and bind to nucleic acids are critical to understanding cellular function. To explore the factors involved in protein-DNA recognition, we used hydrostatic pressure to perturb the binding of the BamHI endonuclease to cognate DNA, both in experiment and in molecular dynamic simulations. A new technique of high-pressure gel mobility shift analysis was used to test the effects of elevated hydrostatic pressure on the binding of BamHI to its cognate recognition sequence. Upon application of a pressure of 500 bar, the equilibrium dissociation constant of BamHI binding to the cognate site was found to increase nearly 10-fold. A challenge has been to link this type of pure thermodynamic measurement to functional events occurring at the molecular level. Thus, we used molecular dynamic simulations at both ambient and elevated pressures to reveal details of the direct and water-mediated interactions between BamHI and cognate DNA, which allow explanation of the effects of pressure on site-specific protein-DNA binding and complex stability.

The bacteriophage lambda gt10 DNA containing an insertion of 20 pairs of GC-bases capable of being arranged as Z-form was cloned. Two independent methodological approaches based on the main properties of Z-form were used to study the shape of the insertion: formation of transition bridges composed of unpaired nucleotides between left-rotating Z-forms and right-rotating B-forms of helix (j-domain) and high immunogenic activity of Z-form. O-beta-diethylaminoethylhydroxyamine (OHA), an analogue of hydroxylamine, is capable of reacting specifically with unpaired cytidines. In this work this modification was used to inhibit the process of restriction at BamH1-site adjacent to (gc)10 insertion, that N-Methyl-bis(2-chloethyl) amine (MBCA) is capable of fixing the Z-form of the insertion in situ. Fixed Z-form is conserved even after DNA has been isolated from bacteriophage, thereby providing an opportunity of its identification by anti-Z-antibodies. It was shown that from 4 to 6% of the total number of insertions are in the Z-form. The hypothesis of significant role of Z-form in the process of packing of DNA molecules in capsid is put forward.

BamHI is a type II restriction endonuclease that catalyzes the scission of the phoshodiester bond in the GAGTCC cognate sequence in the presence of two divalent metal ions. The first step of the reaction is the preparation of water for nucleophilic attack by Glu-113, which has been proposed to abstract the proton from the attacking water molecule. Alternatively, the 3'-phosphate group to the susceptible phosphodiester bond has been suggested to play a role as the general base. The two hypotheses have been tested by computer simulations using the semiempirical protein dipoles Langevin dipoles (PDLD/S) method. Deprotonation of water by Glu-113 has been found to be less favorable by 5.7 kcal/mol than metal-catalyzed deprotonation with a concomitant proton transfer to bulk solvent. The preparation of the nucleophile by the 3'-phosphate group is less favorable by 12.3 kcal/mol. These results suggest that both the general base and the substrate-assisted mechanisms in the first step of BamHI action are less likely than the metal-catalyzed reaction. The metal ions in the active site of BamHI make the largest contributions to the reduction of the free energy of hydroxide ion formation. On the basis of these findings we propose that the first step of endonuclease catalysis does not require a general base; rather, the essential attacking nucleophile in BamHI catalytic action is stabilized by the metal ions.

The type II restriction endonuclease, Bam HI, has been overexpressed in E. coli by cloning the Bam HI gene in frame with an E. coli Ribosome Binding Site (RBS) under the T7 promoter of an E. coli expression vector pRSET A. The expression level of Bam HI endonuclease using this construct was found to be higher than that reported of the overexpressing clone pAEK14. Our overexpressing clone, pAABRw in BL21 cells in presence of Bam HI methylase in pMAP6 following induction with IPTG yields about 9.2 x 10(6) units per gram wet cell paste. In vivo activity of the recombinant endonuclease could be confirmed by the SOS induction assay in JH139 cells even in the absence of T7 polymerase and cognate Bam HI methylase because of leaky expression in E. coli. This provides an alternate way to screen the active endonuclease and its variants.

The karyotypes of Eulemur species exhibit a high degree of variation, as a consequence of the Robertsonian fusion and/or centromere fission. Centromeric and pericentromeric heterochromatin of eulemurs is constituted by highly repeated DNA sequences (including some telomeric TTAGGG repeats) which have so far been investigated and used for the study of the systematic relationships of the different species of the genus Eulemur. In our study, we have cloned a set of repetitive pericentromeric sequences of five Eulemur species: E. fulvus fulvus (EFU), E. mongoz (EMO), E. macaco (EMA), E. rubriventer (ERU), and E. coronatus (ECO). We have characterized these clones by sequence comparison and by comparative fluorescence in situ hybridization analysis in EMA and EFU. Our results showed a high degree of sequence similarity among Eulemur species, indicating a strong conservation, within the five species, of these pericentromeric highly repeated DNA sequences.

Three specific mutants, C54I, C54W, and a double-mutant C54D:C64R of restriction endonuclease BamHI, were generated and studied to investigate the role, if any, of the 54th and 64th cysteine residues in the catalysis of BamHI. The mutation was achieved using the megaprimer approach for PCR. The mutant genes were cloned and characterized by sequencing. The mutant and the wild-type proteins were expressed and purified and their kinetic parameters were determined using short synthetic oligonucleotides as substrates. All mutants had higher K(m) values than that of the wild-type enzyme suggesting a decrease in the affinity of the enzyme for its substrate. The mutant protein C54W showed significant changes in the CD spectra vis-a-vis wild-type enzyme and had the lowest K(m)/K(cat) value among the mutants indicative of changes in the secondary structure of the protein. The melting curves of the mutant proteins overlapped that of the wild-type enzyme. Analysis of the K(cat) values in the context of cocrystal structure suggests that the effect of Cys54 mutation is probably through the perturbation of the local structure whereas reduced activity of the double mutant is consistent with the substrate-assisted catalysis mechanism., (Copyright 2001 Academic Press.)

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.

The human seminal vesicles secrete two proteins, semenogelin I and semenogelin II, at very high concentrations. It has previously been shown that the cotton-top tamarin (Sanguinus oedipus), a New World monkey, is lacking the semenogelin II gene. We have now determined the nucleotide sequence of DNA located 5--13 kilobases (kb) downstream of the tamarin semenogelin I gene---a region that in man is occupied by the semenogelin II gene. Two regions with homology to the human semenogelin II gene were identified in the tamarin DNA. The first region, of 3.5 kb, is homologous to DNA upstream of the human gene, and the second region, of 0.6 kb, is mainly derived from the second intron. Between these regions, equivalent to 594 base pairs (bp) upstream of the transcription initiation site to 12 bp downstream of the stop codon in the human semenogelin II gene, the cotton-top tamarin DNA carries a truncated LINE1 repeat. In another set of experiments, the tamarin DNA hybridizing to the mouse semenoclotin gene was investigated. It was concluded that hybridization is with the second intron of the semenoclotin gene, but very likely, the material does not represent a cotton-top tamarin semenoclotin gene. Thus, a mammalian ancestor probably carried a single gene that in the rodent lineage developed into the semenoclotin gene and in the primate lineage into a progenitor of the semenogelin genes.

Modifications of natural DNA by three anticancer heterocyclic ruthenium(III) compounds were studied by methods of molecular biophysics. These methods included DNA binding studies using atomic absorption spectrophotometry, inhibition of restriction endonucleases, mapping of DNA adducts by transcription assay, interstrand cross-linking employing gel electrophoresis under denaturing conditions, DNA unwinding studied by gel electrophoresis, circular dichroism analysis of the B-->Z transition in DNA, and DNA melting curves measured by absorption spectrophotometry. The results indicate that the complexes HIm[trans-Cl4Im2RuIII], HInd[trans-Cl4Ind2RuIII], and Na[trans-Cl4Im(Me2SO)RuIII] (Im and Ind stand for imidazole and indazole, respectively) coordinate irreversibly to DNA. Their DNA binding mode is, however, different from that of cisplatin. Interestingly, Na[trans-Cl4Im(Me2SO)RuIII] binds to DNA considerably faster than the other two ruthenium compounds and cisplatin. In addition, when Na[trans-Cl4Im(Me2SO)RuIII] binds to DNA it exhibits an enhanced base sequence specificity in comparison with the other two ruthenium complexes. Na[trans-Cl4Im(Me2SO)RuIII] also forms bifunctional intrastrand adducts on double-helical DNA which are capable of terminating RNA synthesis in vitro, while the capability of the other two ruthenium compounds to form such adducts is markedly lower. This observation has been interpreted to mean that the bifunctional adducts of HInd[trans-Cl4Ind2RuIII] and Na[trans-Cl4Im2RuIII] formed on rigid double-helical DNA are sterically more crowded by their octahedral geometry than those of Na[trans-Cl4Im(Me2SO)RuIII]. In addition, the adducts of all three ruthenium compounds affect the conformation of DNA, Na[trans-Cl4Im(Me2SO)RuIII] being most effective. It has been suggested that the altered DNA binding mode of ruthenium compounds in comparison with cisplatin might be an important factor responsible for the altered cytostatic activity of this class of ruthenium compounds in tumor cells.

The interaction of BamHI endonuclease with DNA has been studied crystallographically, but has not been characterized rigorously in solution. The enzyme binds in solution as a homodimer to its recognition site GGATCC. Only six base-pairs are directly recognized, but binding affinity (in the absence of the catalytic cofactor Mg(2+)) increases 5400-fold as oligonucleotide length increases from 10 to 14 bp. Binding is modulated by sequence context outside the recognition site, varying about 30-fold from the bes t (GTG or TAT) to the worst (CGG) flanking triplets. BamHI, EcoRI and EcoRV endonucleases all have different context preferences, suggesting that context affects binding by influencing the free energy levels of the complexes rather than that of the free DNA. Ethylation interference footprinting in the absence of divalent metal shows a localized and symmetrical pattern of phosphate contacts, with strong contacts at NpNpNpGGApTCC. In the presence of Mg(2+), first-order cleavage rate constants are identical in the two GGA half-sites, are the same for the two nicked intermediates and are unaffected by substrate length in the range 10-24 bp. DNA binding is strongly enhanced by mutations D94N, E111A or E113K, by binding of Ca(2+) at the active site, or by deletion of the scissile phosphate GpGATCC, indicating that a cluster of negative charges at the catalytic site contributes at least 3-4 kcal/mol of unfavorable binding free energy. This electrostatic repulsion destabilizes the enzyme-DNA complex and favors metal ion binding and progression to the transition state for cleavage., (Copyright 2001 Academic Press.)

The Epstein-Barr virus (EBV) BamHI-A rightward transcripts (BARTs) are expressed in all EBV-associated tumors as well as in latently infected B cells in vivo and cultured B-cell lines. One of the BART family transcripts contains an open reading frame, RPMS1, that encodes a nuclear protein termed RPMS. Reverse transcription-PCR analysis revealed that BART transcripts with the splicing pattern that generates the RPMS1 open reading frame are commonly expressed in EBV-positive lymphoblastoid cell lines and are also detected in Hodgkin's disease tissues. Experiments undertaken to determine the function of RPMS revealed that RPMS interacts with both CBF1 and components of the CBF1-associated corepressor complex. RPMS interaction with CBF1 was demonstrated in a glutathione S-transferase (GST) affinity assay and by the ability of RPMS to alter the intracellular localization of a mutant CBF1. A Gal4-RPMS fusion protein mediated transcriptional repression, suggesting an additional interaction between RPMS and corepressor proteins. GST affinity assays revealed interaction between RPMS and the corepressor Sin3A and CIR. The RPMS-CIR interaction was further substantiated in mammalian two-hybrid, coimmunoprecipitation, and colocalization experiments. RPMS has been shown to interfere with NotchIC and EBNA2 activation of CBF1-containing promoters in reporter assays. Consistent with this function, immunofluorescence assays performed on cotransfected cells showed that there was colocalization of RPMS with NotchIC and with EBNA2 in intranuclear punctate speckles. The effect of RPMS on NotchIC function was further examined in a muscle cell differentiation assay where RPMS was found to partially reverse NotchIC-mediated inhibition of differentiation. The mechanism of RPMS action was examined in cotransfection and mammalian two-hybrid assays. The results revealed that RPMS blocked relief of CBF1-mediated repression and interfered with SKIP-CIR interactions. We conclude that RPMS acts as a negative regulator of EBNA2 and Notch activity through its interactions with the CBF1-associated corepressor complex.

To investigate how protein interactions to DNA affects DNA-mediated hole transport, hole transport through the DNA bound to restriction endonuclease BamHI was examined. The DNA bound to BamHI retains a regular B-DNA type of conformation, but have a number of hydrogen bonding interaction. These hydrogen bondings in BamHI-DNA complex suppressed the guanine oxidation and the hole transport in sequence selective manner.

We have recently described the GS element, found in Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. silvaticum (MAS) and some isolates of Mycobacterium avium subsp. avium serotype 2 (MAAs2), which contains a set of genes of low GC% content, putatively associated with the biosynthesis, modification and transference of fucose to cell wall glycopeptidolipids. Here we describe a further gene of low GC% content (mpa), within the GS element in MAP. mpa is a putative acetyltransferase with homology to genes directly responsible for host specificity and virulence in Salmonella typhimurium and Shigella flexneri. Unlike other GS genes, strong homologues of mpa have not been found in related species, including Mycobacterium tuberculosis (MTB). In MAP, mpa encodes an ORF of 445aa, however, in MAS and MAAs2 mpa contains a single inserted copy of a novel insertion sequence. This element (IS1612) has two sets of inverted repeats at each terminus and encodes two ORFs with good homologies to transposase and helper proteins of IS21 (E. coli) and IS1415 (R. erythropolis). Sequence comparisons between mpa in MAP and MAS indicate the target site for IS1612 is duplicated on insertion to give a direct repeat at each end of the element. Immediately, downstream of the mpa gene in both MAP and MAS are a group of three genes with good homology to the daunorubicin resistance cluster. This cluster has a high GC% content which suggests a 'border' for the GS element. A short motif present at the beginning of this cluster matches with an inverted repeat of this motif at the beginning of the first gene in the GS element. This encapsulates the whole of this group of low GC% genes in MAP and further suggests its cassette-like nature. Homologues of the GS element in MTB show a marked similarity of organisation, suggesting a parallel role for these genes in both pathogens.

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 microg of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-microg preparations of total cellular DNA. Preparative gel electrophoresis of the PGK1 and p53 genes from 300 microg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 microM H2O2., (Copyright 2000 Academic Press.)

In this report, the effects of osmotic pressure on BamHI cognate binding and catalysis were investigated and compared with a previous study on EcoRI (Robinson, C. R. and Sligar, S. G. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2186-2191). Our observation of the dependence of binding and catalytic parameters on osmotic pressure has allowed for the comparison of hydration changes associated with site-specific DNA recognition for both endonucleases. Over a large range of osmotic pressures (pi), the dependence of BamHI on osmotic stress during cognate binding and catalysis was very different from that of the related endonuclease EcoRI. The binding of EcoRI to cognate DNA was dominated by a dehydration of the endonuclease-DNA complex, whereas binding by BamHI to its cognate sequence was accompanied by a solvent release corresponding to some 125 fewer waters. Catalytic analysis at elevated osmotic pressures indicated that both endonucleases had undergone a net hydration of the complex with BamHI displaying a much greater dependence on osmotic stress than EcoRI. Although the enzymes shared core structural motifs, comparisons of high resolution x-ray structures revealed many different secondary structural features of the complexed endonucleases. The large difference in hydration changes by both BamHI and EcoRI could be attributed to these dissimilar secondary structural features, as well as the functional differences of the two endonucleases during site-specific DNA recognition.

The results of a 3-ns molecular dynamics simulation of the dodecamer duplex d(TATGGATCCATA)(2) recognized by the BamHI endonuclease are presented here. The DNA has been simulated as a flexible molecule using an AMBER force field and the Ewald summation method, which eliminates the undesired effects of truncation and permits evaluation of the full effects of electrostatic forces. The starting B conformation evolves toward a configuration quite close to that observed through x-ray diffraction in its complex with BamHI. This configuration is fairly stable and the Watson-Crick hydrogen bonds are well maintained over the simulation trajectory. Hydration analysis indicates a preferential hydration for the phosphate rather than for the ester oxygens. Hydration shells in both the major and minor groove were observed. In both grooves the C-G pairs were found to be more hydrated than A-T pairs. The "spine of hydration" in the minor groove was clear. Water residence times are longer in the minor groove than in the major groove, although relatively short in both cases. No special long values are observed for sites where water molecules were observed by x-ray diffraction, indicating that water molecules having a high probability of being located in a specific site are also fast-exchanging.

The description and evaluation of a PCR-based assay for the detection and species identification of the eight known human herpesviruses are presented. Two primer pairs targeting well-conserved regions of the genome allowed the amplification of the DNAs of all known human herpesviruses at a high level of sensitivity (10 to 100 genome copies for most viruses). Identification of the virus species was achieved through restriction enzyme digestion with BamHI and BstUI, which yielded fragment sizes that were characteristic for each herpesvirus. Furthermore, it was demonstrated that this restriction enzyme panel allowed the discrimination between human herpesvirus 6 variant A and variant B. This assay format was validated over the course of 1 year in a clinical virology laboratory setting, where it was shown that it readily detected human herpesviruses, including occasional multiple infections, in a variety of clinical samples. The PCR assay was compared to isolation and electron microscopy for the detection of herpes simplex (HSV) and varicella-zoster virus (VZV) in clinical samples. All specimens positive by conventional methods were also positive by PCR. However, in a number of clinical specimens in which HSV or VZV could not be detected by conventional methods, PCR was able to demonstrate the presence of the virus.

The white gene within the transposon A(R)4-24P[white,rosy] inserted at cytological location 24D1-2 in the euchromatic portion of the Drosophila melanogaster genome exhibits a mosaic pattern of expression which is modified by temperature and Y-chromosome number, as in cases of classical position-effect variegation (PEV). The eye colour of the flies in this variegated stock remains mosaic in the presence of the PEV modifier Su(var)3-6, slightly less so with Su(var)3-9 and Su(var)2-5, and full suppression of variegation occurs in the presence of Su(var)3-7. We have induced further transposition of A(R)4-24 and isolated two mosaic stocks with this transgene at new cytological locations. In these stocks, the A(R)4-24 transposon was flanked by the same genomic DNA fragments as in the original location. Spontaneous loss of these fragments leads to reversion of the variegated eye colour to wild-type. We suggest that the flanking DNA fragments from 24D1-2 are capable of inducing position-effect variegation without any association with centromeric heterochromatin. In situ hybridisation and Southern analysis demonstrate that the 5' flanking genomic fragment contains repeated sequences which are abundantly present in heterochromatin.

Dna2 is a multifunctional enzyme in yeast that possesses endonuclease activity well suited to remove RNA-DNA primers of Okazaki fragments, raising the question of whether endonuclease activity is essential for in vivo Dna2 function. Systematic site-directed mutations of amino acid residues in Saccharomyces cerevisiae DNA2 conserved in the central region of many eukaryotic DNA2 homologs allowed us to identify mutant dna2 alleles that were divided into three groups based on the viability of the mutant cells: (i) viable; (ii) inviable only when expression was repressed; (iii) inviable. Biochemical analyses of recombinant mutant Dna2 proteins isolated from the latter two groups revealed that they possessed normal ATPase/helicase activity, but were impaired in their endonuclease activity. Cells expressing mutant Dna2 enzymes partially impaired in endonuclease activity were viable, but were unable to grow when expression of their mutant Dna2 enzymes was further reduced. Their growth was restored when the mutant Dna2 proteins decreased in nuclease activity were induced to overexpress. In contrast, mutant Dna2 proteins lacking endonuclease activity did not allow cells to grow under any conditions tested. These in vivo and in vitro results demonstrate that the endonuclease activity of Dna2 is essential for Okazaki fragment processing.

A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies.

The central problem faced by DNA binding proteins is how to select the correct DNA sequence from the sea of nonspecific sequences in a cell. The problem is particularly acute for bacterial restriction enzymes because cleavage at an incorrect DNA site could be lethal. To understand the basis of this selectivity, we report here the crystal structure of endonuclease BamHI bound to noncognate DNA. We show that, despite only a single base pair change in the recognition sequence, the enzyme adopts an open configuration that is on the pathway between free and specifically bound forms of the enzyme. Surprisingly, the DNA drops out of the binding cleft with a total loss of base-specific and backbone contacts. Taken together, the structure provides a remarkable snapshot of an enzyme poised for linear diffusion (rather than cleavage) along the DNA.

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.

We improved the integration replacement/disruption method (Shortle, D., Novic, P., and Botstein, D. Proc. Natl. Acad. Sci. USA 81: 4889-4893, 1984) for isolating mutants in any of essential genes of the yeast Saccharomyces cerevisiae by integrating mutagenized DNA into the wild type gene of interest. We adopted this method to isolate temperature-sensitive mutants of the MPC1 gene encoding the YLL031C ORF. To facilitate integration of the mutagenic plasmid at a site near the 5' end of the ORF, a BamHI site was created at 300 bp downstream of the 5' end of the truncated ORF to be mutagenized. The MPC1 gene was disrupted in the wild type haploid strain by integrating a 5'-truncated derivative of the gene with mutations induced by in vitro mutagenesis. Transformants thus obtained were subjected for diagnosis of conditional lethality by replica-plating onto an appropriate selection medium to detect mutants. A primary mutant isolated by this method reverted in a high frequency due to a tandem repeat created by mutagenic integration. We deviced a method to obtain a stable temperature-sensitive strain by disrupting the tandem duplication. Two stable temperature-sensitive mutants thus obtained were found to be remedial either with 1 M sorbitol or with 0.1 M Mg2+ and to be sensitive to local anestheticum, tetracaine, at 25 degrees C.