High Stringency Conditions Research Articles

A DA/DAPI positive human 14p heteromorphism defined by fluorescence in-situ hybridisation using chromosome 15-specific probes D15Z1 (satellite III) and p-TRA-25 (alphoid).

We have investigated the use of the satellite III probe, D15Z1, as an alternative to DA/DAPI staining in the identification of chromosome 15-derived markers. The probe hybridises to the short arm of chromosome 15 under high stringency conditions. We have screened 100 randomly selected patients, by fluorescence in-situ hybridisation (FISH), using co-hybridisation experiments with D15Z1 and a whole chromosome library, pBS-15. 88 individuals showed the expected pattern of two D15Z1 signals on the p-arm of both homologues of chromosome 15, whereas 12 individuals showed an additional signal on a third acrocentric D-group chromosome. Sequential GTC banding and D15Z1 hybridisation revealed that in each case it was one homologue of chromosome 14 that was D15Z1 positive. This pattern always correlated with positive DA/DAPI staining. In contrast, the 15 centromere-specific alphoid probe, pTRA-25, gave the expected two signals on both homologues of chromosome 15 in every case. Thus, D15Z1 and DA/DAPI signals co-localize while pTRA-25 is 15 centromere-specific and should be applied for unequivocal identification of chromosome 15-derived markers in clinical studies. The chromosome 14 heteromorphism, as identified by D15Z1, and defined by pTRA-25, may have arisen by intrachromosomal amplification or interchromosomal exchange.

Organization of the KpnI family of chromosomal distal-end satellite DNAs in Silene latifolia.

The major satellite DNAs of the dioecious plant Silene latifolia are represented by the repetitive sequences X43.1, RMY1 and members of the SacI family, which are located at the distal ends of chromosomes. To characterize the satellite DNAs at the distal ends of the chromosomes in S. latifolia ( Sl-distal-satDNA), we isolated a bacterial artificial chromosome clone (number 15B12) that contained multiple repeat sequences with KpnI restriction sites, and subcloned a portion of this sequence into a plasmid vector. Sequencing analysis confirmed that recognition or degenerate sites for KpnI were repeated 26 times at intervals of 310-324 bp in the inserted DNA. The phylogenetic tree that was constructed with the 26 KpnI repeat units contained clustered branches that were independent of the SacI family. It is clear that the KpnI repeat belongs to an Sl-distal-satDNA family that is distinct from the SacI family. We designated this family as " KpnI" after the restriction enzyme that does not have a site in the SacI family. Multi-colored fluorescent in situ hybridization was performed with the KpnI family and RMY1 probes under high stringency conditions. The results suggest that chromosome 7 is unique and that it carries the KpnI family at only one end.

Construction of a deep coverage BAC library from Capsicum annuum, 'CM334'.

A bacterial artificial chromosome (BAC) library consisting of 235,000 clones with an average insert size of 130 kb was constructed from Capsicum annuum, 'CM334'. Based on a pepper haploid genome size of 2,702 Mbp/C, the BAC library is estimated to contain approximately 12 genome equivalents and represents at least 99% of the pepper genome. Screening of the library with mitochondrial DNA probes (coxII, coxIII, atp6 and atp9) and chloroplast DNA probes (atpB, rbcL) indicated that contamination with cytoplasmic DNA was less than 0.5%. To estimate the possibility of isolating a specific clone, the library was screened with single or low-copy gene-specific probes and RFLP probes. Screening of high density BAC filters with RFLP markers linked to L (TMV resistance), y (fruit color), C2 (fruit color) and C (pungency) loci under high stringency conditions revealed that at least three positive BAC clones were found per each probe. This fact indicates that the library is highly reliable and represents a resource for map-based cloning, physical mapping, and characterization of upstream and downstream regulations of the chili pepper genes.

Cross-protection experiments with parasitoids in the genus Microplitis (Hymenoptera: Braconidae) suggest a high level of specificity in their associated bracoviruses

The immunological and developmental effects of bracoviruses (BVs) from three parasitoids in the genus Microplitis (Braconidae: Microgastrinae) were compared in the hosts Pseudoplusia includens and Heliothis virescens (Lepidoptera: Noctuidae). Southern blotting experiments indicated that viral DNAs from Microplitis demolitor bracovirus (MdBV) cross-hybridized with viral DNAs from Microplitis croceipes bracovirus (McBV) and Microplitis mediator bracovirus (MmBV) under conditions of high stringency. Injection of calyx fluid plus venom from each parasitoid species dose-dependently delayed development of P. includens and H. virescens. Each virus also inhibited pupation of P. includens but not H. virescens. In situ hybridization experiments indicated that MdBV and McBV persistently infect hemocytes in both hosts while MmBV persistently infects hemocytes in P. includens but not H. virescens. While MdBV infection induced a loss of adhesion by most plasmatocytes, McBV and MmBV infection induced a loss of adhesion in less than 50% of cells. Cross-protection experiments indicated that calyx fluid plus venom from one species usually protected progeny of another species from encapsulation but did not always promote successful development.

Molecular characterization of genes encoding cytosolic Hsp70s in the zygomycete fungus Rhizopus nigricans.

Previous studies have shown that some stressors, including steroid hormones 21-OH progesterone and testosterone, stimulate the accumulation of heat shock protein 70 (hsp70) messenger ribonucleic acid (mRNA) population in the zygomycete filamentous fungus Rhizopus nigricans. In this study we report the cloning of 3 R nigricans hsp70 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding cytosolic Hsp70s. With a Southern blot experiment under high stringency conditions we did not detect any additional highly homologous copies of the cytosolic hsp70 genes in the R nigricans genome. Sequence analyses showed that all 3 genes contain introns within the open reading frame. The dynamics of the R nigricans molecular response to progesterone, 21-OH progesterone, and testosterone, as well as to heat shock, copper ions, hydrogen peroxide, and ethanol was studied by temporal analysis of Rnhsp70-1 and Rnhsp70-2 mRNA accumulation. Northern blot experiments revealed that the Rnhsp70-2 transcript level is not affected by testosterone, whereas mRNA levels of both genes are rapidly increased with all the other stressors studied. Moreover, the decrease of transcript levels is notably delayed in ethanol stress, and a difference is observed between the profiles of Rnhsp70-1 and Rnhsp70-2 transcripts during heat stress.

Cloning and characterization of genes encoding homologues of the B subunit of cholera toxin and the Escherichia coli heat-labile enterotoxin from clinical isolates of Citrobacter freundii and E. coli.

We identified and characterized a gene encoding a homologue of the B subunits of cholera toxin (CTB) and heat-labile enterotoxin (LTB) of Escherichia coli from a clinical isolate of Citrobacter freundii that was found to produce a factor in the culture supernatant that cross-reacted with antibodies to CTB and LTB when assayed by enzyme-linked immunosorbent assay (ELISA). The gene encoding the ELISA-positive factor, cfxB, consisted of 375 nucleotides and was located downstream of an 852-nucleotide open reading frame, cfxA, with a 56-nucleotide intergenic space. The cfxB gene was predicted to encode a 125-amino-acid polypeptide, which had 73.8 and 72.8% identities with the amino acid sequences of LTB and CTB, respectively. However, the amino acid sequence of the deduced polypeptide CFXA had no homologies to those of the A subunits of CT or LT. DNA probes developed from the sequences of cfxA and cfxB were used to screen 67 C. freundii isolates and 152 E. coli isolates from diarrheal patients by colony blot hybridization. Two strains, C. freundii 48 and E. coli 176, reacted with both DNA probes under conditions of high stringency. We cloned homologues of the cfxA and cfxB genes from E. coli 176 and designated them ecxA and ecxB, respectively. The ecxA gene and the ecxB gene comprise 855 and 375 nucleotides, respectively, with a 50-nucleotide intergenic space, and encode a 285- and a 125-amino-acid residue polypeptides, respectively. The results of the present study may provide important clues to the origin and evolution of immunologically related factors sharing a common enterotoxin-like A and B subunit structures.

A Multiplex-PCR approach to identification of the Brazilian intermediate hosts of Schistosoma mansoni.

Due to difficulties concerning morphological identification of planorbid snails of the genus Biomphalaria, and given a high variation of characters and in the organs with muscular tissue, we designed specific polymerase chain reaction (PCR) primers for Brazilian snail hosts of Schistosoma mansoni from available sequences of internal transcribed spacer 2 (ITS2) of the ribosomal RNA gene. From the previous sequencing of the ITS2 region, one primer was designed to anchor in the 5.8S conserved region and three other species-specific primers in the 28S region, flanking the ITS2 region. These four primers were simultaneously used in the same reaction (Multiplex-PCR), under high stringency conditions. Amplification of the ITS2 region of Biomphalaria snails produced distinct profiles (between 280 and 350 bp) for B. glabrata, B. tenagophila and B. straminea. The present study demonstrates that Multiplex-PCR of ITS2-DNAr showed to be a promising auxiliary tool for the morphological identification of Biomphalaria snails, the intermediate hosts of S. mansoni.

Cosmid library of the marine macroalga Bryopsis maxima (Bryopsidales, Ulvophyceae)

SUMMARY The purpose of this study was to construct a cosmid library from chromosomal DNA of a marine macroalga, Bryopsis maxima Okamura ex Segawa (Bryopsidales, Ulvophyceae), in a rapid, simple and inexpensive manner. In the DNA purification, polysaccharides were removed by covalently binding them to resin particles containing free boric acid groups. The DNA yield was 20 μg g−1 of B. maxima fresh weight. This DNA was 100–200 kb in length, and its A260/A280 and A230/A260 ratios were 1.8 and 0.4, respectively. It was of sufficient quality for molecular research. The cloning procedures were carried out in the following steps: controlled partial shearing of purified DNA through a microsyringe, optimal size separation of the DNA by biased sinusoidal field gel electrophoresis, ligation of the DNA to the cosmid vector in the gel, and in vitro packaging into the lambda phage. The library consisted of 2.0 × 103 independent clones with an average insert size of 40 kb. The fragment amplified by polymerase chain reaction in the library was hybridized with a DNA fragment (328 bp) encoding B. maxima glutamate dehydrogenase under high-stringency conditions by Southern blot analysis, thus demonstrating that the library contained B. maxima chromosomal DNA. This cosmid library is the first to be constructed for any species of marine macroalgae.

Cloning of a gene encoding a unique haemolysin from Klebsiella pneumoniae and its potential use as a species-specific gene probe

A gene, designated khe, that encodes a haemolysin of Klebsiella pneumoniae CMC-1 has been cloned and sequenced. When expressed in Escherichia coli, a unique peptide of approximately 20kDa was identified. Nucleotide sequence analysis predicted a single open reading frame (ORF) of 486bp encoding a 162 amino acid polypeptide with an estimated pI of 6·77. No extensive sequence homology could be identified between khe and any reported sequence at either the nucleotide or amino acid level. Furthermore, DNA hybridizations under high stringency conditions failed to show any cross hybridizations to several bacteria including K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. These data indicate that we have cloned a unique gene, which is highly conserved among tested K. pneumoniae isolates.

Unique Molecular Architecture of Silk Fibroin in the Waxmoth,Galleria mellonella

Proteins of silk fibers are characterized by reiterations of amino acid repeats. Physical properties of the fiber are determined by the amino acid composition, the complexity of repetitive units, and arrangement of these units into higher order arrays. Except for very short motifs of 6-10 residues, the length of repetitive units and the number of these units concatenated in higher order assemblies vary in all spider and lepidopteran silks analyzed so far. This paper describes an exceptional silk protein represented by the 500-kDa heavy chain fibroin (H-fibroin) of the waxmoth, Galleria mellonella. Its non-repetitive N-terminal (175 residues) and C-terminal (60 residues) parts, the overall gene organization, and the nucleotide sequence around the TATA box show that it is homologous to the H-fibroins of other Lepidoptera. However, over 95% of the protein consists of highly ordered repetitive structures that are unmatched in other species. The repetitive region includes 11 assemblies AB(1)AB(1)AB(1)AB(2)(AB(2))AB(2) of remarkably conserved polypeptide repeats A (63 amino acid residues), B(1) (43 residues), and B(2) (18 residues). The repeats contain a high proportion of Gly (31.6%), Ala (23.8%), Ser (18.1%), and of residues with long hydrophobic side chains (16% for Leu, Ile, and Val combined). The presence of the GLGGLG and SSAASAA(AA) motifs suggests formation of pleated beta-sheets and their stacking into crystallites. Conspicuous conservation of the apolar sequence VIVI followed by DD or ED is interpreted as indicating the importance of hydrophobicity and electrostatic charge in H-fibroin cross-linking. The environment of G. mellonella larvae within bee cultures requires continuous production of silk that must be both strong and elastic. The spectacular arrangement of the repetitive H-fibroin region apparently evolved to meet these requirements.

An Enterococcus faecalis ABC homologue (Lsa) is required for the resistance of this species to clindamycin and quinupristin-dalfopristin.

Enterococcus faecalis isolates are resistant to clindamycin (CLI) and quinupristin-dalfopristin (Q-D), and this is thought to be a species characteristic. Disruption of a gene (abc-23, now designated lsa, for "lincosamide and streptogramin A resistance") of E. faecalis was associated with a > or =40-fold decrease in MICs of Q-D (to 0.75 microg/ml), CLI (to 0.12 to 0.5 microg/ml), and dalfopristin (DAL) (to 4 to 8 microg/ml) for the wild-type E. faecalis parental strain (Q-D MIC, 32 microg/ml; CLI MIC, 32 to 48 microg/ml; DAL MIC, 512 microg/ml). Complementation of the disruption mutant with lsa on a shuttle plasmid resulted in restoration of the MICs of CLI, Q-D, and DAL to wild-type levels. Under high-stringency conditions, lsa was found in 180 of 180 isolates of E. faecalis but in none of 189 other enterococci. Among 19 erm(B)-lacking Enterococcus faecium strains, 9 (47%) were highly susceptible to CLI (MIC, 0.06 to 0.25 microg/ml) and had DAL MICs of 4 to 16 microg/ml; for the remaining erm(B)-lacking E. faecium strains, the CLI and DAL MICs were 4 to > 256 and 2 to > 128 microg/ml, respectively. In contrast, none of 32 erm(B)-lacking E. faecalis strains were susceptible (CLI MIC range, 16 to 32 microg/ml; DAL MIC range, > or =32 microg/ml). When lsa was introduced into an E. faecium strain initially susceptible to CLI, the MICs of CLI and DAL increased > or =60-fold and that of Q-D increased 6-fold (to 3 to 6 microg/ml). Introduction of lsa into two DAL-resistant (MICs, > 128 microg/ml), Q-D-susceptible (MICs, 0.5 and 1.5 microg/ml) E. faecium strains (CLI MICs, 12 and >256 microg/ml) resulted in an increase in the Q-D MICs from 3- to 10-fold (to 8 and >32 microg/ml), respectively. Although efflux was not studied, the similarity (41 to 64%) of the predicted Lsa protein to ABC proteins such as Vga(A), Vga(B), and Msr(A) of Staphylococcus aureus and YjcA of Lactococcus lactis and the presence of Walker A and B ATP-binding motifs suggest that this resistance may be related to efflux of these antibiotics. In conclusion, lsa appears to be an intrinsic gene of E. faecalis that explains the characteristic resistance of this species to CLI and Q-D.

The natural occurrence of a begomovirus in sunn hemp (Crotalaria juncea) in India

Sunn hemp (Crotalaria juncea) is cultivated in India and other tropical countries as a fibre or green manure crop. During July/August 2001, a severe virus-like disease affected 80–90% of sunn hemp plants in experimental plots at the National Botanical Research Institute, Lucknow. The symptoms of the disease consisted of interveinal chlorosis, yellowing, curling and shortening of leaves, and stunting of plants. Whiteflies (Bemisia tabaci) transmitted the associated virus from diseased to healthy sunn hemp plants which developed symptoms similar to those occurring in naturally infected plants. Total DNA was isolated from the infected-leaf tissues and subjected to polymerase chain reaction (PCR) using begomovirus specific universal primers (AV494/AC1048) which were designed from the conserved sequences of the core region of the coat protein (CP) genes of several begomoviruses (Wyatt & Brown, 1996). As expected, a fragment of c. 500 bp was amplified from the DNA extracted from diseased, but not healthy, leaves. The authenticity of the PCR-amplified DNA fragment was confirmed by Southern hybridization in which DNA probes, prepared from the CP gene of Indian tomato leaf curl virus (IToLCV; Srivastava et al., 1995), cross hybridized with the PCR fragment under high stringency conditions. Although Sunn-hemp mosaic virus and some strains of Tobacco mosaic virus infect sunn hemp (Brunt et al., 1996), a begomovirus has not hitherto been reported to naturally infect this important crop species. Transmission of the sunn hemp virus by whiteflies, PCR amplification of its DNA with begomovirus specific primers and its strong hybridization with a DNA-A probe of IToLCV strongly indicate that it is a begomovirus. The extent of its relationship to IToLCV (Family Geminiviridae; Genus Begomovirus) and other begomoviruses is now being further investigated.

Evidence that the enterococcal polysaccharide antigen gene (epa) cluster is widespread in Enterococcus faecalis and influences resistance to phagocytic killing of E. faecalis.

In previous studies, we cloned a cluster of genes involved in polysaccharide biosynthesis (epa) from Enterococcus faecalis strain OG1RF and showed that this gene cluster mediated synthesis of a polysaccharide in Escherichia coli. Disruption of two open reading frames in the epa gene cluster of OG1RF generated two mutants, TX5179 and TX5180, which were attenuated in a mouse peritonitis model. In the current study, Western blotting was performed with serum from a patient with E. faecalis endocarditis and polysaccharide extracts from OG1RF and the mutants TX5179 and TX5180. OG1RF showed a smear in the high-molecular-weight region and discrete bands in the low-molecular-weight region, which were missing from the mutants; periodate treatment and carbohydrate staining confirmed the polysaccharide nature of this material. In a neutrophil killing assay using OG1RF-absorbed normal human serum, the mutants TX5179 and TX5180, respectively, were 50 and 2.4 times more susceptible to killing than wild-type OG1RF (P < or = 0.01). With a fluorescence phagocytosis assay, 2.5 to 3 times more of the mutants were taken up by neutrophils than OG1RF (P < or = 0.001). Finally, with restriction digestion and hybridization under high-stringency conditions, the epa gene cluster of OG1RF (which is also present in the sequenced E. faecalis strain V583) was detected in 12 of 12 other clonally distinct E. faecalis strains tested: a similar polysaccharide pattern was detected for the 12 strains on Western blots using an E. faecalis endocarditis patient serum, and sera from four other patients with E. faecalis endocarditis all reacted with polysaccharide extracts of OG1RF. These results indicate that the epa gene cluster is widespread among E. faecalis and confers some protection against human host defenses.

The Association of Begomovirus with Bitter Melon in India.

Bitter melon, Momordica charantia (Cucurbitaceae), is a vegetable of nutritive and medicinal value that is cultivated throughout India and other tropical countries. In September 2001, a severe disease of bitter melon with virus-like symptoms was observed at Lucknow, India. Symptoms consisted of upward curling, shortening, and distortion of leaves. Diseased melon fruits were stunted and deformed. Disease incidence was as high as 100%. Whitefly (Bemicia tabaci) can transmit the associated virus from diseased bitter melon to Nicotiana tabacum cv. White burley. The development of leaf curl symptoms in N. tabacum indicated the pathogen could be a begomovirus. Total nucleic acids were extracted from diseased bitter melon leaves, and polymerase chain reaction (PCR) tests were performed. Three pairs of primers, AV494 and AC1048 (1), CL-CR/F2 and CL-CR/R2, CL/11F and CL10/R (2), specific to DNA-A of begomoviruses were used in PCR. Virus-specific DNA-A fragments of expected sizes were identified (≈0.5, 0.7 and 1.2 kb, respectively). The presence of a begomovirus in all PCR-amplified DNA fragments was confirmed by Southern hybridization. Cloned DNA-A fragments of Tomato leaf curl virus and Cotton leaf curl virus (both begomoviruses) cross-hybridized with the PCR products gave strong signals under high stringency conditions. These data suggest that a begomovirus is associated with this bitter melon disease. Watermelon mosaic 1 virus is the only virus previously reported to naturally infect bitter melon; however, this virus has not been identified in India. Bitter melon is also an experimental host of Ribgrass mosaic virus (genus Tobamovirus) and Trichosanthes mottle virus (genus Potyvirus). To our knowledge, this is the first report of the occurrence of begomovirus infecting bitter melon.

Changes in gene expression following induction of ischemic tolerance in rat brain: detection and verification.

Tolerance against ischemic insults can be elicited in the CA1 region of rat hippocampus by inducing a short ischemic period 2-3 days prior to the ischemic insult. To detect genes whose expression changes following induction of ischemic tolerance (IT), we applied a differential display technique called restriction fragment differential display-PCR (RFDD-PCR). RFDD-PCR displays the coding region of mRNA and allows detection of differentially expressed mRNA. Double-stranded cDNA generated using a T25V primer is digested by the endonuclease TaqI, and adapters are ligated onto the cDNA fragments. When amplifying the adapter-containing cDNA fragments under high-stringency conditions, reproducible PCR profiles are obtained. By comparing these profiles from naïve and ischemia-tolerant rat brains statistically, significant expression changes of 20 fragments were identified. To verify the observed changes, quantitative PCR and in situ hybridization were performed for three fragments representing proteins with quite different functions (GluR2-flop, SC1, and p68 RNA helicase). Quantitative PCR displayed the same degree of regulation as RFDD-PCR, but in situ hybridization did not display any regulation. As the applied PCR-based techniques detect only polyadenylated mRNA, whereas in situ hybridization detects both nonadenylated and adenylated mRNA, changes in the polyadenylation state of the mRNA, rather than inconsistent changes in the total amount of mRNA, probably explain this discrepancy. Thus, our results show that the expression of genes hitherto not related to IT changes with the induction of IT and that the degree of regulation displayed by RFDD-PCR can be verified by quantitative PCR.

Human papillomavirus infection in cyclosporin-induced gingival overgrowth in renal allograft recipients.

Host immunity plays an important role in the development of human papillomavirus (HPV)-associated disease. The HPV infection in oral cyclosporin-induced gingival overgrowth in renal transplant recipients has not been investigated previously. The aim of this study was to establish the HPV infection of cyclosporin-induced gingival hyperplasia in renal transplant recipients through morphological changes and use of the in situ hybridization technique. We examined 13 renal transplant recipient biopsies with gingival overgrowth lesions and 4 healthy mucosa samples of these patients. The histopathological diagnoses were established on the basis of widely accepted criteria, and the pathologist was not aware of the HPV result. An in situ molecular hybridization was carried out under low stringent conditions to detect HPV species with mixed biotin-labeled probes of HPV 6 and HPV 11, and under high stringent conditions with HPV 6, HPV 11, HPV 16, and HPV 18 probes for HPV typing. The HPV prevalence among the 13 samples studied was 92.31% (12/13), of which 4 tested positive for HPV 6-11 and 1 for HPV 16. The 4 biopsies of normal mucosa from gingival overgrowth patients were also reactive for HPV DNA. In 11/12 (91.7%) HPV-positive cases, koilocytotic atypia was found. The suppression of T-cell function by cyclosporin therapy can result in an increase of HPV infection, adding to the proliferative activity of cyclosporin in the oral mucosa.

GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP.

The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of approximately 4000 genes in five developmental stages produced by cDNA-AFLP.

Transcription Factor BACH1 Is Recruited to the Nucleus by Its Novel Alternative Spliced Isoform

The transcription factor Bach1 is a member of a novel family of broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) basic region leucine zipper factors. Bach1 forms a heterodimer with MafK, a member of the small Maf protein family (MafF, MafG, and MafK), which recognizes the NF-E2/Maf recognition element, a cis-regulatory motif containing a 12-O-tetradecanoylphorbol-13-acetate-responsive element. Here we describe the gene structure of human BACH1, including a newly identified promoter and an alternatively RNA-spliced truncated form of BACH1, designated BACH1t, abundantly transcribed in human testis. The alternate splicing originated from the usage of a novel exon located 5.6 kilobase pairs downstream of the exon encoding the leucine zipper domain, and produced a protein that contained the conserved BTB/POZ, Cap'n collar, and basic region domains, but lacked the leucine zipper domain essential for NF-E2/Maf recognition element binding. Subcellular localization studies using green fluorescent protein as a reporter showed that full-length BACH1 localized to the cytoplasm, whereas BACH1t accumulated in the nucleus. Interestingly, coexpression of BACH1 and BACH1t demonstrated interaction between the molecules and the induction of nuclear import of BACH1. These results suggested that BACH1t recruits BACH1 to the nucleus through BTB domain-mediated interaction.

DNase-resistant DNA in the extracellular and cell wall-associated fractions of Frankia strains R43 and CcI3.

DNases were shown to be present in the extracellular fraction of Frankia strains R43 and CcI3. In spite of this, DNA was found in both the extracellular and cell wall fractions of these strains, and it was shown that extracellular DNA was resistant to the DNases secreted into the culture medium of both Frankia strains. Furthermore, Southern blot analysis under high stringency conditions revealed the chromosomal origin of the cell wall-adsorbed DNA (CW-DNA). Mobility gel band shift assays suggested that the extracellular DNA and the CW-DNA are engaged in complexes with other molecules, most likely proteins, which are probably responsible for the enzymatic resistance observed against extracellular DNase activities. In addition, it was shown that lysis of a small proportion of the cells in the exponential growth phase may account for the DNA being released into the supernatant and adsorbed to the cell wall.

Cloning of a beta-hemolysin gene of Brachyspira (Serpulina) hyodysenteriae and its expression in Escherichia coli.

Brachyspira (Serpulina) hyodysenteriae induces a mucohemorrhagic diarrheal disease in pigs. The production of a beta-hemolysin has been considered a major virulence attribute of this organism. Previous reports have failed to correlate a specific cloned gene sequence with a purified beta-hemolytic protein sequence. Thus, questions still remain concerning the structural gene sequence of the hemolysin. To answer this question unequivocally, the beta-hemolytic toxin was purified from extracts of log-phase spirochetes, and the N-terminal amino acid sequence was determined (K-D-V-V-A-N-Q-L-N-I-S-D-K) and compared with the translated sequences of previously cloned genes, tlyA to tlyC. The lack of homology between tlyA to tlyC translated sequences and the purified beta-hemolytic toxin sequence resulted in the study that is reported here. A degenerate probe was designed based on the N-terminal amino acid sequence of the purified beta-hemolysin and used to screen a B. hyodysenteriae genomic library. Three overlapping clones were identified, and one was sequenced to reveal an open reading frame coding for a putative 8.93-kDa polypeptide containing the N-terminal sequence of the purified beta-hemolysin. To distinguish this gene from the tlyA to tlyC genes, it has been designated hlyA. A hemolysis-negative Escherichia coli strains containing hlyA was beta-hemolytic on blood agar media. Also, the hemolytic activity of the recombinant protein had identical protease and lipase sensitivities and electrophoretic mobility to those of native B. hyodysenteriae beta-hemolysin. Based on sequence analysis, the translated protein had a pI of 4.3, an alpha-helical structure, and a phosphopantetheine binding motif. Hybridization analysis of genomic DNA indicated that the hlyA gene was present in B. hyodysenteriae and B. intermedia but was not detected in B. innocens, B. pilosicoli, or B. murdochii under high-stringency conditions. The location of hlyA on the chromosomal map was distinct from the locations of tlyA, tlyB, and tlyC.