PvuII Fragment Research Articles

A Mutant RNA Polymerase Reveals a Kinetic Mechanism for the Switch between Nonproductive Stuttering Synthesis and Productive Initiation during Promoter Clearance

A Mutant RNA Polymerase Reveals a Kinetic Mechanism for the Switch between Nonproductive Stuttering Synthesis and Productive Initiation during Promoter Clearance

Characterization and phylogeny of the pfp gene of Amycolatopsis methanolica encoding PPi-dependent phosphofructokinase.

The actinomycete Amycolatopsis methanolica employs a PPi-dependent phosphofructokinase (PPi-PFK) (EC 2.7.1.90) with biochemical characteristics similar to those of both ATP- and PPi-dependent enzymes during growth on glucose. A 2.3-kb PvuII fragment hybridizing to two oligonucleotides based on the amino-terminal amino acid sequence of PPi-PFK was isolated from a genomic library of A. methanolica. Nucleotide sequence analysis of this fragment revealed the presence of an open reading frame encoding a protein of 340 amino acids with a high degree of similarity to PFK proteins. Heterologous expression of this open reading frame in Escherichia coli gave rise to a unique 45-kDa protein displaying a high level of PPi-PFK activity. The open reading frame was therefore designated pfp, encoding the PPi-PFK of A. methanolica. Upstream and transcribed divergently from pfp, a partial open reading frame (aroA) similar to 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase-encoding genes was identified. The partial open reading frame (chiA) downstream from pfp was similar to chitinase genes from Streptomyces species. A phylogenetic analysis of the ATP- and PPi-dependent proteins showed that PPi-PFK enzymes are monophyletic, suggesting that the two types of PFK evolved from a common ancestor.

Limited variation of DNA fingerprints (IS 6110 and IS 1081) in Korean strains of Mycobacterium tuberculosis

To establish the usefulness of DNA fingerprinting for the epidemiology of Mycobacterium tuberculosis isolated from Korean tuberculosis patients. Comparison of restriction fragment length polymorphism (RFLP) patterns produced by southern hybridization of PvuII-digested chromosomal DNA. IS6110-associated banding patterns of 41 isolates varied considerably, containing 1-13 copies. The RFLP pattern of the epidemiologically related M. tuberculosis isolates was identical in 8 of 10 groups of close contact patients. No noticeable differences in RFLP were observed between drug-sensitive and drug-resistant isolates. IS1081-containing restriction fragment analysis of 52 isolates showed 6 different banding patterns, and the C type was found dominant in Korea. Identification of G type M. tuberculosis, which has a 8.0 kb IS1081-containing PvuII fragment, is unusual because it has been observed only in M. bovis BCG so far. IS6110 was a very useful tool for tracing the transmission route of tuberculosis; IS1081 was also useful for subdividing M. tuberculosis into several groups.

Identification of cellular proteins involved in nikkomycin production in Steptomyces tendae Tü901

Expression of genes involved in nikkomycin production in Streptomyces tendae was investigated by two-dimensional gel electrophoresis of cellular proteins. Ten gene products (P1-P10) were identified that were synthesized when nikkomycin was produced; these proteins were not detected in non-producing mutants. N-terminal sequences of six of the 10 proteins were obtained by microsequencing of protein spots excised from preparative two-dimensional gels. Protein P8 was identified as L-histidine amino-transferase (HisAT), which has been previously correlated with nikkomycin production. By using oligonucleotide probes deduced from the N-terminal sequences of protein P2 and P6, we isolated an 8 kb BamHI fragment and a 6.5 kb PvuII fragment, respectively, from the genome of Streptomyces tendae Tü901. Restriction analyses revealed that both fragments overlapped within a region of 1.5 kb. Mapping of the oligonucleotide probe hybridizing sites indicated that the genes encoding protein P2 and P6 are closely spaced on the 8 kb BamHI fragment, and the latter is located on the overlapping region. DNA sequence analysis revealed that proteins P1 and P2 are encoded by a single gene, orfP1, that is translated at two initiation codons. The orfP1 gene was interrupted by homologous recombination using the integrating vector pWHM3. The gene-disrupted transformants did not produce nikkomycin, indicating that proteins P1 and P2 are essential for nikkomycin production. The data presented show that reverse genetics was successfully used to isolate genes involved in nikkomycin production.

A region of Haemophilus influenzae Rd hybridizes with repetitive human DNA.

A 17.7 kbp EcoR1 fragment of Haemophilus influenzae Rd (wild-type strain) DNA containing repeats has been cloned together with the DNA repair gene uvr3. The region containing repetitive DNA was subcloned on a 13 kbp EcoR1 fragment (pHi13). The insert hybridizes with restriction enzyme digests of H. influenzae Rd and H. parainfluenzae and exhibits a repetitive pattern. Discrete bands superimposed upon a polydisperse background were observed when pHi13 hybridizes with restriction digests of human DNA. The hybridization patterns indicate that H. parainfluenzae and human DNA contain cross-hybridizing sequences. The repeats of pHi13 are present on a 2.6 kbp PvuII fragment.

Streptococcus pyogenes type M12 protein shows selective binding to some human immunoglobulin G3 myeloma proteins

Purified, recombinant M12 protein from Streptococcus pyogenes CS24 has recently been demonstrated to bind human immunoglobulin G3 (IgG3). The binding site for IgG has been localized to an internal peptide encoded by a PvuII fragment of the gene emm12. We have investigated the ability of an isolated recombinant M12 protein consisting of the peptide encoded by the PvuII fragment to bind various monoclonal human IgG3 myeloma proteins representing a number of both Caucasian and Oriental IgG3 Gm(allotypic) phenotypes. Of nine Caucasian IgG3 myeloma proteins, only two bound strongly to the recombinant M12 protein in enzyme-linked immunosorbent assays. The allotypic phenotypes of the reactive proteins were IgG3m(b+)(g-) and IgG3m(b-)(g+). No binding was seen for seven IgG3 myeloma proteins of Oriental origin with G3m(st+)(u-)(b+)(g-), G3m(st-)(u+)(b+)(g-), G3m(st-)(u+)(b-)(g+), and G3m(st-)(u-)(b-)(g+) phenotypes. The binding of human IgG3 to M12 protein seems to be related to features other than its Gm allotypic markers. Selective reactivity of IgG3 myeloma proteins with M12 protein may provide another way to subclassify human IgG3 molecules. The biological significance of the selective reactivity is not known.

Genomic cloning and localization by FISH and linkage analysis of the human gene encoding the primary subunit NMDAR1 (GRIN1) of the NMDA receptor channel.

A cDNA clone of the NMDAR1 (isoform E) has been used to screen both lambda and cosmid genomic libraries. A genomic phage clone was identified and sequenced and was found to contain some of the 3' coding regions of the GRIN1 gene. This clone was used to localize the gene using fluorescent in situ hybridization (FISH) to normal chromosomes, and also to a lymphoblastoid cell line containing a translocation involving chromosomes 9 and 15. FISH localized the gene to chromosome 9q34.3. The clone was used to screen a panel of genomic DNAs cut with 20 restriction enzymes. A VNTR sequence 5' to the gene, which was polymorphic for a number of restriction enzymes, was detected. A PvuII fragment of the genomic clone was found to detect the VNTR on Southern hybridization. The polymorphic VNTR marker was mapped against chromosome 9q34 markers using linkage analysis in the CEPH families. The GRIN1 gene was linked to D9S7 with a maximum lod score of 20.09 at zero recombination fraction in males and 0.03% recombination in females.

Isolation and characterization of a gene encoding a Chlamydia pneumoniae 76-kilodalton protein containing a species-specific epitope.

Chlamydia pneumoniae is a human respiratory pathogen. Unlike the other two Chlamydia species, no species-specific antigen has been defined for C. pneumoniae. An immunoreactive clone containing a 0.8-kb fragment was isolated from a C. pneumoniae (AR-39) genomic library by using anti-C. pneumoniae rabbit immune serum. By Southern hybridization analysis of chromosomal digests of the different Chlamydia spp., the 0.8-kb fragment was shown to react specifically with C. pneumoniae. Subcloning of this fragment into the pGEX-1 lambda T expression vector resulted in the expression of a 62-kDa fusion protein. This fusion protein as well as the cleaved C. pneumoniae peptide were recognized by anti-C. pneumoniae rabbit immune serum, while the glutathione S-transferase moiety was not recognized. The fusion protein was used to produce monospecific rabbit antiserum. This antiserum was shown to react with a 76-kDa protein in all C. pneumoniae isolates tested, specifically recognize C. pneumoniae inclusions in tissue culture, and neutralize infectivity of C. pneumoniae in cell culture. No reactivity was observed with Chlamydia trachomatis or Chlamydia psittaci. To isolate the entire coding sequence of the 76-kDa protein, two partially overlapping fragments of C. pneumoniae DNA, a 3.2-kb HindIII fragment and a 1.2-kb PvuII fragment, were isolated, cloned, and sequenced. No significant sequence similarity was found with any previously reported nucleotide or amino acid sequence of the other Chlamydia species. This C. pneumoniae protein containing a species-specific epitope could play a role in pathogenesis and may be useful as a diagnostic tool.

Analysis of the self-defense gene ( fmrO) of a fortimicin A (astromicin) producer, Micromonospora olivasterospora: comparison with other aminoglycoside-resistance-encoding genes

The nucleotide (nt) sequence of a 3409-bp PvuII fragment carrying the self-defense gene ( fmrO) of a fortimicin A (FmA; astromicin) producer, Micromonospora olivasterospora, was determined; four open reading frames (ORFs) exist in this region. Expression analysis of fmrO using the lac promoter in Escherichia coli revealed that ORF-3 encodes fmrO. ORF-1 was considered to encode a FmKH epimerase ( fms11). ORF-2, ORF-3 and ORF-4 seemed to form an operon. No homology was detected between fmrO and the resistance-encoding gene fmrT of Streptomyces tenjimariensis, a producer of the Fm-group antibiotic, istamycin. Gene organization around the fmr genes differed considerably between M. olivasterospora and S. tenjimariensis. The deduced amino acid sequence of fmrO showed an identity of 30.8% to grmA of M. purpurea and 35.8% to grmB of M. rosea, respectively, suggesting that fmrO encodes a 16S rRNA methyltransferase. We found in M. purpurea a novel resistance gene distinct from grmA and highly similar to fmrO which conferred a resistance phenotype similar to that of fmrO. These results suggest that fmrO of M. olivasterospora and the two resistance-encoding genes of M. purpurea were derived from a common ancestral gene.

Type M12 protein from Streptococcus pyogenes is a receptor for IgG3.

Streptococcus pyogenes, serotype M12, was discovered to express a human IgG3 binding protein. Western blot analysis of partially purified M12 protein, exposed to IgG3 myeloma protein, showed that both M12 Ag and the receptor protein were the same apparent size. A lambda clone (lambda 4.1) containing the emm12 open reading frame expressed both the M12 Ag and the IgG3 binding protein. The emm12 open reading frame was amplified by the polymerase chain reaction and subcloned into the expression vector pJLA602. Based on Western blot analysis, one recombinant Escherichia coli (pD3) expressed M12 protein with IgG3 binding activity. This result confirmed that the M12 protein from strain CS24 is also an IgG3 receptor. Deletion analyses showed that a truncated M12 protein encoded by an internal PvuII fragment was sufficient for IgG3 binding activity. Further deletion studies suggested that the IgG3 binding domain was located in a 200 amino acid internal fragment containing two directly repeated sequences. Other experiments suggest that the receptor did not bind to the same IgG3 domain as that recognized by protein G. The M12 protein did not bind human IgG1, IgG2, IgG4, or Ig from several other animal species.

Cloning and sequencing of a novel endo-1,4-β-glucanase gene from Ruminococcus albus

The gene encoding an endo-1,4-β-glucanase from Ruminococcus albus F-40 was cloned in Escherichia coli JM109 using pBR322. The nucleotide sequence of the 1,798 bp PstI- PvuII fragment which includes a cellulase gene was determined. There was a single open reading frame (ORF) consisting of 936 bp encoding a peptide of 312 amino acid residues with a molecular weight of 35,766. The N-terminal amino acid sequence determined for the enzyme expressed in E. coli was identical to that deduced from the beginning of the ORF. A putative ribosome-binding site and a promoter were located upstream of the ORF. Activity was expressed from this fragment when it was subcloned in both orientations in pUC118 and pUC119, indicating that its own promoter functioned in E. coli. The amino acid sequence of the endoglucanase deduced from the nucleotide sequence showed 44% homology with CelA from Butyrivibrio fibrisolvens A46, suggesting that this enzyme was a member of family A2. The enzyme purified from E. coli exhibited the highest activity against carboxymethyl cellulose (CMC) at 40°C and pH around 7.0.

The use of a DNA probe and PCR to examine the distribution of the aac(6')-Ic gene in Serratia marcescens and other gram-negative bacteria.

A nucleotide probe for the chromosomal aminoglycoside 6' acetyltransferase gene (aac(6')-Ic) of Serratia marcescens was used in non-radioactive dot-blot hybridization experiments with 186 strains belonging to ten different species of Serratia. The gene was only detected in strains of S. marcescens (all strains tested), and positive hybridization was seen irrespective of whether or not strains were kanamycin-resistant. An additional 180 strains belonging to 28 Gram-negative bacterial species other than in the genus Serratia did not hybridize with the probe. A rapid PCR test for the aac(6')-Ic gene was developed and used to confirm that the aac(6')-Ic gene is only found in S. marcescens. Southern hybridization analysis of S. marcescens chromosomal DNA demonstrated that the gene was usually located on a PvuII fragment with a putative isoleucine tRNA-2 gene, but polymorphisms with respect to the size of this fragment were observed.

Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG.

Recently, the insertion element IS1081 from Mycobacterium bovis was identified. In this study, the usefulness of IS1081 in the epidemiology of tuberculosis was investigated. The host range of this insertion sequence was found to be restricted exclusively to the group of Mycobacterium tuberculosis complex bacteria, whereas none of the 10 mycobacterial species which do not belong to the M. tuberculosis complex contained IS1081-homologous DNA. All 99 M. tuberculosis complex strains investigated carried five or six copies of IS1081, and very limited IS1081-associated restriction fragment length polymorphisms were observed among the strains. Seven different IS1081-containing bands were distinguished in each strain, and the patterns differed only in one or two insertion sequence-containing bands. The banding pattern of M. bovis BCG differed in the presence of a 8.0-kb IS1081-containing PvuII fragment which was absent from all other M. tuberculosis complex strains.

Genetic and molecular analysis of pIP417 and pIP419: Bacteroides plasmids encoding 5-nitroimidazole resistance

This report describes a genetic and molecular analysis of two transferable Bacteroides plasmids, pIP417 and pIP419, which carry genetic determinants conferring low-level resistance to 5-nitroimidazoles. The restriction endonuclease cleavage sites for each plasmid were localized. The Ni R genetic determinants of pIP417 and pIP419 plasmids have been cloned into the Bacteroides cloning vector pBI191 (C.J. Smith, J. Bacteriol. 164, 294–301, 1985) as PvuII and Sau3A fragments, respectively. Both inserts had different restriction sites and did not cross-hybridize by Southern blot analysis. Genetic data obtained by cloning into pBI191 clearly show that the PvuII-generated fragments A (Rep) and B (Mob) of pIP417 are involved in plasmid replication and transfer, respectively. Although encoding resistance to the same antibiotic, both plasmids appeared different with regard to the 5-nitroimidazole resistance and replication genetic determinants. However, they share a homology in a region involved, at least in one case, in plasmid transfer. Considering the spontaneous high level of resistance to 5-nitroimidazole in Escherichia coli, this work, based on direct gene cloning into Bacteroides, demonstrates the value of such an approach.

Cloning and identification of the Escherichia coli murB DNA sequence, which encodes UDP-N-acetylenolpyruvoylglucosamine reductase

The murB gene, which complemented the UDP-N-acetylenolpyruvoylglucosamine reductase (EC 1.1.1.158) mutation in Escherichia coli ST5, was cloned from an E. coli chromosomal library. murB was subcloned on a 2.8-kb PvuII fragment into pUC19 and sequenced. A 1,029-bp open reading frame encoded a 342-amino-acid polypeptide of 37,859 Da. A DNA sequence homology search revealed that murB had almost 100% homology with a previously reported unidentified open reading frame, ORFII, at 89.9 min. Physical and genetic mapping results were consistent with this map position, and minicell analyses of murB subclones showed a plasmid-encoded protein of approximately 37,000 Da, which closely matched the calculated size of the murB protein.

Linkage of regression and malignant conversion of rabbit viral papillomas to MHC class II genes.

Human papillomaviruses associated with cutaneous and anogenital cancers induce intraepithelial precursor lesions which may regress spontaneously or progress into invasive carcinomas. Cell-mediated immune responses are probably involved in regression of precancerous lesions and the polymorphism of the genes responsible may thus have a key role in the variability of the host response. Skin warts and cancers induced in rabbits by Shope papillomavirus provide a model to test this hypothesis. We analysed a restriction-fragment-length polymorphism of major histocompatibility complex class I and class II genes and T-cell receptor beta-chain genes in infected domestic rabbits. We found a strong linkage between wart regression and a DR alpha EcoRI fragment, and an increased relative risk of malignant transformation associated with a DQ alpha PvuII fragment. This indicates a genetic control of wart evolution, involving genes in the class II region of the major histocompatibility complex.

Non-association between 9.2 KB PvuII RFLP and seronegative spondyloarthropathies in Spain.

Several studies of DNA restriction fragment length polymorphism (RFLP) in ankylosing spondylitis (AS) have been carried out. The association between a recently identified class I HLA 9.2 kb PvuII RFLP and AS remain controversial. In order to evaluate this possible association in an Euro-Caucasian population, the genomic DNA of 42 AS patients and 18 patients with Reiter's syndrome (RS) and 42 healthy controls was analysed. Non-association between 9.2 kb PvuII RFLP and AS or RS was observed. As described previously, a strong association between this fragment and HLA-A3 and/or HLA-A9 antigens was demonstrated. A study of two families showed that this RFLP segregates with HLA-A3 and/or HLA-A9 and independently of the HLA-B27. Our findings support the view that the 9.2 kb PvuII fragment is not universally associated with AS.

Replication and temperature-sensitive maintenance functions of lactose plasmid pSK11L from Lactococcus lactis subsp. cremoris

The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. cremoris) SK11, was isolated on a 14.8-kbp PvuII fragment by shotgun cloning into an Escherichia coli vector encoding erythromycin resistance and selection for erythromycin-resistant transformants of L. lactis subsp. lactis (L. lactis) LM0230. Deletion analysis and Tn5 mutagenesis of the resulting plasmid (pKMP1) further localized the replication region to a 2.3-kbp ScaI-SpeI fragment. DNA sequence analysis of this 2.3-kbp fragment revealed a 1,155-bp open reading frame encoding the putative replication protein, Rep. The replication origin was located upstream of rep and consisted of an 11-bp imperfect direct repeat and a 22-bp sequence tandemly repeated three and one-half times. The overall organization of the pSK11L replicon was remarkably similar to that of pCI305, suggesting that pSK11L does not replicate by the rolling-circle mechanism. Like pSK11L, pKMP1 was unstable in L. lactis LM0230. Deletion analysis allowed identification of several regions which appeared to contribute to the maintenance of pKMP1 in L. lactis LM0230. pKMP1 was significantly more stable in L. cremoris EB5 than in L. lactis LM0230 at all of the temperatures compared. This stability was lost by deletion of a 3.1-kbp PvuII-XbaI fragment which had no effect on stability in L. lactis LM0230. Other regions affecting stability in L. cremoris EB5 but not in L. lactis LM0230 were also identified. Stability assays conducted at various temperatures showed that pKMP1 maintenance was temperature sensitive in both L. lactis LM0230 and L. cremoris EB5, although the plasmid was more unstable in L. lactis LM0230. The region responsible for the temperature sensitivity phenotype in L. lactis LM0230 was tentatively localized to a 1.2-kbp ClaI-HindIII fragment which was distinct from the replication region of pSK11L. Our results suggest that the closely related L. lactis and L. cremoris subspecies behave differently regarding maintenance of plasmids.

Comparative anatomical distribution of 5-HT 1A receptor mRNA and 5-HT 1A binding in rat brain — a combined in situ hybridisation/in vitro receptor autoradiographic study

The present study examined the comparative distribution of 5-HT 1A receptor mRNA and 5-HT 1A receptors in rat brain using a combination of in situ hybridisation histochemistry and in vitro receptor autoradiography. 5-HT 1A mRNA was visualised using a 910 bp cRNA probe synthesised from a BalI- PvuII fragment of the rat 5-HT 1A reetor gene, while 5-HT 1A receptors were labelled with the 5-HT 1A-selective ligand 8-OH-DPAT. In general terms, there was a complementary distribution of cells expressing 5-HT 1A receptor mRNA and 5-HT 1A receptor sites. High levels of both 5-HT 1A mRNA and 5-HT 1A receptors were evident in the hippocampal formation (CA1, CA3, dentate gyrus), entorhinal cortex, and raphe nuclei and lower levels in neocortex and thalamus. Although 5-HT 1A mRNA was not expressed in any regions which did not also exhibit 5-HT 1A receptors, within both the diagonal band and the medial septal nucleus mRNA levels were proportionately higher than 5-HT 1A receptor levels, possibly reflecting receptor transport or a heterogeneity in 5-HT 1A receptor turnover mechanisms. 5-HT 1A receptor mRNA and 5-HT 1A binding sites were undetectable in caudate/putamen and cerebellar regions. The present data indicate the synthesis of 5-HT 1A receptors both in raphe serotonergic cells and anatomically specific serotonergic projection areas, further supporting both a presynaptic autoregulatory and postsynaptic modulatory role for this receptor in serotonergic transmission.

Isolation of a mouse genomic clone containing four tRNA Cys-encoding genes

A cluster of four tRNA Cys-encoding genes with the anticodon GCA was found on a murine genomic clone containing an 18-kb DNA insert. Three of the four genes encode the identical tRNA, whereas the fourth gene has an altered nucleotide (nt) sequence. Two of the genes within a 2-kb PvuII fragment have the same polarity and are separated by only 921 bp. These two tRNAs have a different primary sequence. The changes in the nt sequences occur within three stems of the tRNA cloverleaf structure and weaken the strength of the H-bonds within the stems. All four genes (designated i-iv) have the 3′ structural element that has been proposed as the transcription termination signal [Bogenhagen and Brown, Cell 24 (1981) 261–270]. The remainder of the flanking regions of the three identical tRNAs are very similar to each other, whereas the flanking regions of the fourth tRNA are distinctly different.