HindIII Fragment Research Articles

Konstruksi Vektor Biner Mengandung Gen Hibrid cryIB-cryIAa untuk Transformasi Agrobacterium Tanaman Padi (Oryza sativa L)

This study aimed to obtain binary vectors carrying cryIB-cryIAa hybrid gene suitable for Agrobacterium-mediated transformation of two Indonesian rice cultivars, Rajalele (Javanica) and Cisadane (Indica). Introduction of hybrid Bt toxin, encoded by cryIB-cryIAa hybrid gene with different binding receptor in the insect gut driven by maize ubiquitin promoter, was intended to reduce the possibility of the appearance of stem borer resistant to Bt toxin. A HindIII fragment containing the ubi cryIB-cryIAa hybrid gene was excised from pBKS ubi cryIB-cryIAa and cloned into the HindIII site of binary vector pCAMBIA 1301, 1303, or 1304. Restriction analysis of recombinant plasmids confirmed the insertion of the gene and demonstrated that the gene inserted in two orientations. The recombinant plasmids were transformed into electrocompetent Agrobacterium tumefaciens EHA105 by electroporation. Embryogenic calli of Rajalele and Cisadane were co-cultivated with A. tumefaciens EHA 105 harbouring binary vector pCAMBIA 1301, 1303, or 1304 containing ubi cryIB-cryIAa. GUS histochemical assay revealed that high transformation efficiency (75-100%) on both cultivars were obtained from embryogenic calli transformed with pCAMBIA 1303 or 1304.

Characterization of a nucleopolyhedrovirus variant of the gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae) in Turkey

ABSTRACTThe gypsy moth (Lymantria dispar L., Lepidoptera: Lymantriidae) is one of the most serious pest of various forestal, food and industrial crops worldwide. We have characterized a new Lymantria dispar nucleopolyhedrovirus (LdMNPV-T4) variant, which was isolated from dead L. dispar larvae in Turkey. Scanning electron microscope observations showed that the polyhedral occlusion bodies (OBs) of the LdMNPV-T4 were irregularly shaped. Transmission electron microscopy revealed that OBs of LdMNPV-T4 were occupied with several virions in which multiple nucleocapsids packaged by viral envelope. Restriction analysis of the LdMNPV-T4 DNA purified from the viral inclusion bodies yielded BamHI, BglII, EcoRI and HindIII fragments. The mean size estimated for the complete LdMNPV-T4 genome was calculated to be 163.3 kb. Phylogenetic analysis of amplified polh, lef-8 and lef-9 sequences showed its relation to the other NPVs from Lymantria species. Mortality values of the LdMNPV-T4 at four different concentrations against third instar larvae of L. dispar ranged from 45% to 88%. These results suggest that LdMNPV-T4 isolated from Turkey is a promising microbial control agent to be utilized for the biological control of L. dispar.

Identification of an antibacterial protein by functional screening of a human oral metagenomic library.

Screening of a bacterial artificial chromosome (BAC) library containing metagenomic DNA from human plaque and saliva allowed the isolation of four clones producing antimicrobial activity. Three of these were pigmented and encoded homologues of glutamyl-tRNA reductase (GluTR), an enzyme involved in the C5 pathway leading to tetrapyrole synthesis, and one clone had antibacterial activity with no pigmentation. The latter contained a BAC with an insert of 15.6 kb. Initial attempts to localize the gene(s) responsible for antimicrobial activity by subcloning into pUC-based vectors failed. A new plasmid for toxic gene expression (pTGEX) was designed enabling localization of the antibacterial activity to a 4.7-kb HindIII fragment. Transposon mutagenesis localized the gene to an open reading frame of 483 bp designated antibacterial protein1 (abp1). Abp1 was 94% identical to a hypothetical protein of Neisseria subflava (accession number WP_004519448.1). An Escherichia coli clone expressing Abp1 exhibited antibacterial activity against Bacillus subtilis BS78H, Staphylococcus epidermidis NCTC 11964 and B4268, and S. aureus NCTC 12493,ATCC 35696 and NCTC 11561. However, no antibacterial activity was observed against Pseudomonas aeruginosa ATCC 9027, N. subflava ATCC A1078, E. coli K12 JM109 and BL21(DE3) Fusobacterium nucleatum ATCC 25586 and NCTC 11326, Prevotella intermedia ATCC 25611, Veillonella parvula ATCC 10790 or Lactobacillus casei NCTC 6375.

Detection of monoclonal integration of bovine leukemia virus proviral DNA as a malignant marker in two enzootic bovine leukosis cases with difficult clinical diagnosis.

Monoclonal integration of bovine leukemia virus (BLV) proviral DNA into bovine genomes was detected in peripheral blood from two clinical cases of enzootic bovine leukosis (EBL) without enlargement of superficial lymph nodes. A BLV-specific probe hybridized with 1 to 3 EcoRI and HindIII fragments in these 2 atypical EBL cattle by Southern blotting and hybridization, as well as in 3 typical EBL cattle. The probe also hybridized to a large number of EcoRI and HindIII fragments in 5 cattle with persistent leukosis. These results suggest that the detection of monoclonal integration of BLV provirus into the host genome may serve as a marker of monoclonal proliferation and malignancy in difficult to diagnose EBL cattle.

A possible breakage of linkage disequilibrium between mitochondrial and chloroplast genomes during Emmer and Dinkel wheat evolution

In wheat (Triticum) and Aegilops, chloroplast and mitochondrial genomes have been studied for over three decades to clarify the phylogenetic relationships among species, and most of the maternal lineages of polyploid species have been clarified. Mitochondrial genomes of Emmer (tetraploid with nuclear genome AABB) and Dinkel (hexaploid with AABBDD) wheat are classified into two different types, VIIa and VIIb, by the presence-absence of the third largest HindIII fragment (named H3) in the mitochondrial DNA. Although the mitochondrial genome in the genera often provides useful information to clarify the phylogenetic relationship among closely related species, the phylogenetic significance of this dimorphism has yet not been clarified. In this study, to facilitate analysis using a large number of accessions, a sequence characterized amplified region (SCAR) marker that distinguishes the type VIIb mitochondrial genome from type VIIa was first developed. Mitochondrial genome type was determined for each of 30 accessions of wild and cultivated Emmer wheat and 25 accessions of Dinkel wheat. The mitochondrial genome type for each accession was compared with the plastogroup that had been determined using chloroplast microsatellite markers. Unexpectedly, the distribution of mitochondrial genome type was not in accordance with that of the plastogroups, suggesting occasional paternal leakage of either the mitochondrial or chloroplast genome during speciation and differentiation of Emmer and Dinkel wheat. An alternative possibility that substoichiometric shifting is involved in the observed dimorphism of the mitochondrial genome is also discussed.

Homologous Elements hs3a and hs3b in the 3′ Regulatory Region of the Murine Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

SCREENING AND CLONING OF BACTERIAL β-GLUCOSIDASE GENE THAT CAN DEGRADE SALICIN FROM SOME NIF AND VIRULENT BACTERIA

β-Glucosidase activity of the most virulent strains of Agrobacterium tumefaciens was found to be much higher than that of the weakly virulent strains. It appears that conversion of coniferin to coniferyl alcohol via bacterially encoded β-glucosidase affects genes induction and thus virulence. If indeed β-glucosidase is a necessary component of the pathway, this will be the first reported evidence of plant signal compound processing by Agrobacterium tumefaciens. The isolation, characterization of β-glucosidase gene from Agrobacterium tumefaciens B3/73 and sequencing of cbgl were done. The encoded enzyme catalyzes controlled hydrolysis of coniferin but not cellobiose (Morris and Morris, 1990). The Agrobacterium tumefaciens has two β-glucosidase genes cbgl and Cbgl, Cbg1 activity hydrolyzes coniferin but not cellobiose. Agrobacterium tumefaciens B3/73 beta-glucosidase genes Cbgl was cloned and sequenced, cbg1 expressed in Escherichia coli. The 88-kDa predicted product of cbg1 β-Glucosidase is highly similar to another bacterial β-Glucosidase and several fungal β-glucosidases (Morris and Morris, 1990). To confirm that Agrobacterium tumefaciens has strongly β-glucosidase activity and slight reaction, clones screened showed that two categories were indeed present DNA from individual clones was digested with HindIII and restriction fragment patterns were compared. (Linda et al., 1992). Two type of pattern were obtained, the relative ability of each gene to cleave coniferin was assessed. Clones containing Agrobacterium tumefaciens B3/73 DNA rapidly and completely hydrolyzed coniferin to coniferyl alcohol. Over the same period, type 2 clones were completely inactive. The different substrate specificities of clones were also evident from their ability to grow on cellobiose. Agrobacterium tumefaciens B3/73 was able to use cellobiose as the sole carbon source. Escherichia coli DH5α and type 1 clones were not able to grow on cellobiose. Other clones were able to utilize cellobiose but grew very slowly. (Linda et al., 1992). The 5.7 kb HindIlI fragment common to all type 1 clones was purified and ligated into pBR322. Clones with inserts in either orientation were able to cleave X-glucose, indicating that the entire β-glucosidase gene was probably located within this insert. An EcoRI, BamHI, BglII, and PstI restriction map of the insert showed that a 3.5 kb BamHI-PstI fragment with an internal PstI site was found to have the activitty to cleave X-glucose when cloned into pUC19. The sequence surrounding the EcoRI site in the pUC19:3.5 kb BamHI-PstI clone and the sequence were done. (Linda et al., 1992). Woodward and Wiseman (1982) reported that there are two constitutive, β-glucosidase genes in Agrobacterium tumefaciens B3/73 were appeared. Those represented by clones able to hydrolyze X-glucose and coniferin but not cellobiose. On the other hand, those represented by other clones having lower activity on X-glucose, non activity on coniferin and able to utilize cellobiose for growth. β -glucosidase, α-glucosidase, and β-galactosidase activities have been reported to be associated with 45 strains of rhizobia, and some cellulolytic and pectinolytic activities have been detected in Rhizobium leguminosarum. (Singh and Singh, 1985). Several glycosidases have also been detected in Bradyrhizobium lupini A β-glucosidase that is particularly active with cellobiose has been purified from Agrobacterium faecalis. In addition, a β-galactosidase and a β-glucosidase have been purified from the periplasmic space of Rhizobium trifolii, and an endoglucanase gene from Azorhizobium caulinodans has been cloned. (Geelen et al., 1995). Listeria spp. transcription of bvrB gene was induced by cellobiose and salicin but not by arbutin. Disruption of the bvr operon by replacing part of bvrAB with an interposon abolished the repression by cellobiose and salicin but not that by arbutin indicating that the bvr locus encodes a β-glucoside-specific sensor that mediates virulence gene repression upon detection of cellobiose and salicin. Bvr is the first sensory system found in Listeria. monocytogenes that is involved in environmental regulation of virulence genes (Klaus et al., 1999). Caulobacter crescentus ability to utilize lactose was examined to obtain an additional genetic tool for study this model organism, Identified a gene lacA that required for growth on lactose as the sole carbon source involved in the catabolism of two glucosides salicin and trehalose., this enzymatic activity is inducible and increased lac expression in the presence of lactose and salicin. (Benjamin et al., 2010).

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction

We designed a novel DNA probe and novel PCR primer sets for detecting the genes coding for Staphylococcus hyicus (S. hyicus) exfoliative toxin (ET). In dot blot hybridization, the novel DNA probe hybridized with chromosomal DNA of ExhA-, ExhB-, ExhC-, ExhD-, and SHETA-producing strains. This probe also hybridized with the plasmid DNA of a SHETB-producing strain. In Southern blot hybridization, the probe hybridized with a 1.5 kb HindIII fragment of chromosomal DNA from a SHETA-producing strain. The above fragment was cloned into E. coli and the nucleotide sequence of the SHETA gene determined, this gene proved to have almost the same homology (99.6%) as the ExhB gene. It was therefore thought that SHETA is a subtype of ExhB. In multiplex PCR using five primer sets, each gene gave a band distinguishable from the others. This multiplex PCR system has high specificity among the well-known S. hyicus ET genes. Of the 69 known ET-producing S. hyicus strains, 38, 19, 10, 2 and 1 strains have exhB, exhD exhA, shetb and exhC genes, respectively.

ZFP260 Is an Inducer of Cardiac Hypertrophy and a Nuclear Mediator of Endothelin-1 Signaling

Pressure and volume overload induce hypertrophic growth of postnatal cardiomyocytes and genetic reprogramming characterized by reactivation of a subset of fetal genes. Despite intense efforts, the nuclear effectors of cardiomyocyte hypertrophy remain incompletely defined. Endothelin-1 (ET-1) plays an important role in cardiomyocyte growth and is involved in mediating the neurohormonal effects of mechanical stress. Here, we show that the phenylephrine-induced complex-1 (PEX1), also known as zinc finger transcription factor ZFP260, is essential for cardiomyocyte response to ET-1 as evidenced in cardiomyocytes with PEX1 knockdown. We found that ET-1 enhances PEX1 transcriptional activity via a PKC-dependent pathway which phosphorylates the protein and further potentiates its synergy with GATA4. Consistent with a role for PEX1 in cardiomyocyte hypertrophy, overexpression of PEX1 is sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. Importantly, transgenic mice with inducible PEX1 expression in the adult heart develop cardiac hypertrophy with preserved heart function. Together, the results identify a novel nuclear effector of ET-1 signaling and suggest that PEX1 may be a regulator of the early stages of cardiac hypertrophy.

Methylation-independent DNA Binding Modulates Specificity of Repressor of Silencing 1 (ROS1) and Facilitates Demethylation in Long Substrates

DNA cytosine methylation is an epigenetic mark that promotes gene silencing and performs critical roles during reproduction and development in both plants and animals. The genomic distribution of DNA methylation is the dynamic outcome of opposing methylation and demethylation processes. In plants, active demethylation occurs through a base excision repair pathway initiated by 5-methycytosine (5-meC) DNA glycosylases of the REPRESSOR OF SILENCING 1 (ROS1)/DEMETER (DME) family. To gain insight into the mechanism by which Arabidopsis ROS1 recognizes and excises 5-meC, we have identified those protein regions that are required for efficient DNA binding and catalysis. We have found that a short N-terminal lysine-rich domain conserved in members of the ROS1/DME family mediates strong methylation-independent binding of ROS1 to DNA and is required for efficient activity on 5-meC.G, but not for T.G processing. Removal of this domain does not significantly affect 5-meC excision from short molecules, but strongly decreases ROS1 activity on long DNA substrates. This region is not required for product binding and is not involved in the distributive behavior of the enzyme on substrates containing multiple 5-meC residues. Altogether, our results suggest that methylation-independent DNA binding allows ROS1 to perform a highly redundant search for efficient excision of a nondamaged, correctly paired base such as 5-meC in long stretches of DNA. These findings may have implications for understanding the evolution of structure and target specificity in DNA glycosylases.

One time intranasal vaccination with a modified vaccinia Tiantan strain MVTT ZCI protects animals against pathogenic viral challenge

To combat variola virus in bioterrorist attacks, it is desirable to develop a noninvasive vaccine. Based on the vaccinia Tiantan (VTT) strain, which was historically used to eradicate the smallpox in China, we generated a modified VTT (MVTT ZCI) by removing the hemagglutinin gene and an 11,944 bp genomic region from HindIII fragment C2L to F3L. MVTT ZCI was characterized for its host cell range in vitro and preclinical safety and efficacy profiles in mice. Despite replication-competency in some cell lines, unlike VTT, MVTT ZCI did not cause death after intracranial injection or body weight loss after intranasal inoculation. MVTT ZCI did not replicate in mouse brain and was safe in immunodeficient mice. MVTT ZCI induced neutralizing antibodies via the intranasal route of immunization. One time intranasal immunization protected animals from the challenge of the pathogenic vaccinia WR strain. This study established proof-of-concept that the attenuated replicating MVTT ZCI may serve as a safe noninvasive smallpox vaccine candidate.

Phosphorylation of Histone H3 by Protein Kinase C Signaling Plays a Critical Role in the Regulation of the Developmentally Important TBX2 Gene

The mechanism(s) regulating the expression of the TBX2 gene, a key regulator of development, is poorly understood and thus limits an understanding of its function(s). Here we demonstrate that 12-O-tetradecanoylphorbol-13-acetate (TPA) induces TBX2 expression in normal human fibroblasts in a protein kinase C (PKC)-dependent and MAPK-independent manner. Our data further reveal that TPA activates transcription of TBX2 through activating MSK1, which leads to an increase in phosphorylated histone H3 and the recruitment of Sp1 to the TBX2 gene. In addition, TPA was shown to activate MSK1 in a PKC-dependent and MAPK-independent manner. This study is the first to provide evidence that phosphorylation of histone H3 leads to the transcriptional activation of the TBX2 gene and to link MSK1 to PKC.

Glucocorticoid Receptor Activation of the Ciz1-Lcn2 Locus by Long Range Interactions

The cellular response to glucocorticoid receptor (GR) activation involves a highly orchestrated series of regulatory actions influenced at multiple levels by a variety of mechanisms including the action of transcription factors and chromatin modifiers. Because the majority of GR binding sites (glucocorticoid-responsive elements (GREs)) are distant from promoters, it is likely that interactions at a distance play an important role in GR action. To determine whether long range chromosomal associations play a role in transcription regulation by GR, we utilized a chromosome conformation capture-based technique (associated chromosome trap) to identify unknown, remote sequences that interact with the GR-induced Lipocalin2 (Lcn2) gene. Our screen revealed that the Lcn2 GRE interacts with the Ciz1 gene, nearly 30 kb upstream. Ciz1 was subsequently found to be a novel GR-responsive gene. The GRE proximal to the Lcn2 promoter apparently functions to regulate both the Lcn2 gene and the distal Ciz1 gene. Using quantitative chromosome conformation capture, we find that a loop structure is organized between these two genes. This structure is hormone-independent and present only in cell types where the genes are active. The strong correlation between gene expression and loop structure in different cell lines suggests that high order interactions play a role in determining tissue-specific gene regulation.

Molecular structure and chromosome distribution of three repetitive DNA families in Anemone hortensis L. (Ranunculaceae)

The structure, abundance and location of repetitive DNA sequences on chromosomes can characterize the nature of higher plant genomes. Here we report on three new repeat DNA families isolated from Anemone hortensis L.; (i) AhTR1, a family of satellite DNA (stDNA) composed of a 554-561 bp long EcoRV monomer; (ii) AhTR2, a stDNA family composed of a 743 bp long HindIII monomer and; (iii) AhDR, a repeat family composed of a 945 bp long HindIII fragment that exhibits some sequence similarity to Ty3/gypsy-like retroelements. Fluorescence in-situ hybridization (FISH) to metaphase chromosomes of A. hortensis (2n = 16) revealed that both AhTR1 and AhTR2 sequences co-localized with DAPI-positive AT-rich heterochromatic regions. AhTR1 sequences occur at intercalary DAPI bands while AhTR2 sequences occur at 8-10 terminally located heterochromatic blocks. In contrast AhDR sequences are dispersed over all chromosomes as expected of a Ty3/gypsy-like element. AhTR2 and AhTR1 repeat families include polyA- and polyT-tracks, AT/TA-motifs and a pentanucleotide sequence (CAAAA) that may have consequences for chromatin packing and sequence homogeneity. AhTR2 repeats also contain TTTAGGG motifs and degenerate variants. We suggest that they arose by interspersion of telomeric repeats with subtelomeric repeats, before hybrid unit(s) amplified through the heterochromatic domain. The three repetitive DNA families together occupy approximately 10% of the A. hortensis genome. Comparative analyses of eight Anemone species revealed that the divergence of the A. hortensis genome was accompanied by considerable modification and/or amplification of repeats.

Identification and Characterization of a Novel Melanoma Tumor Suppressor Gene on Human Chromosome 6q21

By characterizing a complex chromosome rearrangement involving 6q and 17p in melanoma cell line UACC-930, we isolated a candidate tumor suppressor gene at 6q21, named prenyl diphosphate synthase subunit 2 (PDSS2), which was interrupted by an inversion breakpoint. The purpose of this study was to determine the tumor-suppressive potential of PDSS2 in the development of melanoma. To isolate the rearranged 6q in UACC-930 cells, a bacterial artificial chromosome clone (RP1-67A8) covering the breakpoint at 6q21 was digested with HindIII and each DNA fragment was used as the probe for the breakpoint in Southern blotting. The HindIII fragment probe covering the breakpoint was then used to screen an EcoRI-digested DNA library generated from UACC-930. To characterize the tumor-suppressive potential of PDSS2, PDSS2 was stably transfected into a highly tumorigenic melanoma cell line, UACC-903. The tumor-suppressive function of PDSS2 was shown by both in vitro and in vivo assays. The differential expression of PDSS2 in benign nevi and primary melanoma samples was also studied. Down-regulation of PDSS2 was observed in 59 of 87 (67.8%) primary melanomas, which was significantly higher than that in benign nevi (7 of 66, 10.6%; P < 0.001). In addition, an overexpression of the PDSS2 in UACC-903 cells could inhibit tumor cell growth, decrease the colony-forming ability in soft agar, and totally abrogate the tumorigenicity of UACC-903 in nude mice. Our results support the proposal that PDSS2 is a novel tumor suppressor gene that plays an important role in the development of malignant melanoma.

The Cytochrome c Maturation Components CcmF, CcmH, and CcmI Form a Membrane-integral Multisubunit Heme Ligation Complex

Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus.

Four different integrative recombination events involved in the mobilization of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 in Escherichia coli

We identified and characterized four different recombination mechanisms involved in the cointegrative transfer of the Neisseria gonorrhoeae beta-lactamase plasmid pSJ5.2 by the gonococcal 41 kb tet( M) and the Gram negative self-transmissible plasmids N3 and R64 drd-33 using an Escherichia coli recA-background. Mobilization of pSJ5.2 by the tet( M) plasmid occurred by cointegration through a replicative transposition of two IS1 elements inserted upstream from the beta-lactamase gene of pSJ5.2 and creating a IS1::beta-lactamase hybrid promoter. Two types of recombinational events occurred within the 1.8 kb BamH1–HindIII fragment of pSJ5.2 with the N3 and R64 plasmids. A non-homologous recombination was found at coordinates 1817 and 2849 of pSJ5.2 with sequences from R64. A non-homologous recombination combined with an IS26-mediated one-ended transposition was found at coordinates 1817 and 3010 of pSJ5.2 with N3. In both recombinational events, a deletion of over 1 kb of pSJ5.2 occurred. The fourth recombination event was detected in the 1.0 kb BamH1–HindIII fragment of pSJ5.2 by homologous recombination between DNA from the truncated Tn3 resolvase gene of pSJ5.2 and the resolvase sequences from R64 and N3.

Complete nucleotide sequencing and analysis of the 65-kb highly conjugativeEnterococcus faeciumplasmid pMG1: identification of the transfer-related region and the minimum region required for replication

pMG1 (65.1 kb) is a highly conjugative, pheromone-independent Enterococcus faecium plasmid that carries a Tn4001-like transposon encoding gentamicin resistance. The complete nucleotide sequence (65 029 bp) of the pMG1 plasmid was determined and 73 ORFs lying in the same transcription orientation were identified. Sixty-one of the 73 ORFs showed a high degree of similarity (90-100% identity at the amino acid level) to the ORFs of the pHTbeta plasmids. Like the pHTbeta plasmid, 22 of the pMG1 plasmid ORFs showed homology with ORFs present on the pXO2 plasmid (96.2 kb), which is the virulence plasmid essential for capsular formation by Bacillus anthracis. Analysis of tra mutants created by Tn917 insertion and Northern analysis of transcripts indicated that ORFs 15-49, lying in the 31.7 kb region between 13.6 and 45.3 kb on the plasmid map, were related to transfer. This region was designated as the Tra I region of pMG1. A 5.9-kb HindIII fragment that replicates autonomously in Enterococcus faecalis was cloned and analysis of this fragment by deletion and in vitro insertion mutations showed that ORF10 (rep) and the inverted repeat sequence in the noncoding region between ORF8 and ORF9 were necessary for pMG1 replication.

Molecular Characterization of a Poxvirus Isolated from an American Flamingo (Phoeniconais ruber rubber)

An avian poxvirus from the beak scab of an American flamingo (Phoeniconais ruber rubber) was isolated by inoculation on the chorioallantoic membrane (CAM) of specific-pathogen-free (SPF) chicken embryos. The virus produced multifocal areas of epithelial hyperplasia along with foci of inflammation in the CAM, and rare cells contained small eosinophilic intracytoplasmic bodies. Chickens inoculated with the isolated virus in the feather follicle of the leg did not develop significant lesions. Nucleotide sequence comparison of a PCR-amplified 4.5 kb HindIII fragment of the genome of flamingo poxvirus (FlPV) revealed very high homology (99.7%) with condor poxvirus (CPV), followed by approximately 92% similarity with canary poxvirus (CNPV) and Hawaiian goose poxvirus (HGPV), but less similarity (approximately 69%) to fowl poxvirus (FPV), the type species of the genus Avipoxvirus of family Poxviridae. As in the cases with CPV, CNPV, and HGPV, genetic analysis of FlPV revealed an absence of three corresponding FPV open reading frames (ORF199, 200, and 202) and an absence of any reticuloendotheliosis virus (REV) sequences in this region. There are only nine nucleotide substitutions observed between FlPV and CPV in the 4.5 kb fragment; those were clustered in the ORF201 region, which in FPV genome is a site for integration of REV sequences. Phylogenetic analysis of the predicted amino acid sequences of the ORF201-coded hypothetical protein demonstrated FlPV to be more closely related to CPV, as well as to CNPV and HGPV, than to FPV.

Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

Dentin sialophosphoprotein (DSPP), an important odontoblast differentiation marker, is necessary for tooth development and mineralization. Bone morphogenetic protein 2 (BMP2) plays a vital role in odontoblast function via diverse signal transduction systems. We hypothesize that BMP2 regulates DSPP gene transcription and thus odontoblast differentiation. Here we report that expression of BMP2 and DSPP is detected during mouse odontogenesis by in situ hybridization assay, and BMP2 up-regulates DSPP mRNA and protein expression as well as DSPP-luciferase promoter activity in mouse preodontoblasts. By sequentially deleting fragments of the mouse DSPP promoter, we show that a BMP2-response element is located between nucleotides -97 and -72. By using antibody and oligonucleotide competition assays in electrophoretic mobility shift analysis and chromatin immunoprecipitation experiments, we show that the heterotrimeric transcription factor Y (NF-Y) complex physically interacts with the inverted CCAAT box within the BMP2-response element. BMP2 induces NF-Y accumulation into the nucleus increasing its recruitment to the mouse DSPP promoter in vivo. Furthermore, forced overexpression of NF-Y enhances promoter activity and increases endogenous DSPP protein levels. In contrast, mutations in the NF-Y-binding motif reduce BMP2-induced DSPP transcription. Moreover, inhibiting BMP2 signaling by Noggin, a BMP2 antagonist, results in significant inhibition of DSPP gene expression in preodontoblasts. Taken together, these results indicate that BMP2 mediates DSPP gene expression and odontoblast differentiation via NF-Y signaling during tooth development.