Dot-blot Hybridization Studies Research Articles

Expression patterns of cotton chloroplast genes during development: implications for development of plastid transformation vectors

Although most plastid transformation studies have focused on chloroplast expression, plastid transformation can also be used to express genes in plastids of a wide variety of plant tissues by using appropriate plastid promoters. Based on the sequence of the Gossypium hirsutum chloroplast genome, we developed primers and amplified segments of 20 different plastid genes. The PCR products were labeled and used in filter dot blot hybridization studies to characterize their expression levels and patterns in total RNA isolated from light- and dark-grown cotton tissues at different developmental stages. A subset of 6 genes among these was further characterized by real time PCR. Highest expression levels were observed for rrn16 and psbA. Four genes were expressed in all samples at relatively constant levels: accD, atpA, matK and rrn16. Expression in root tissue was generally low. The results of our study can be used to predict which operons and promoters are most likely to be preferentially expressed in the plastids of tissues of interest at levels that would result in the desired phenotype, facilitating the development of plastid transformation vectors.

Expression patterns of cotton chloroplast genes during development: implications for development of plastid transformation vectors

Although most plastid transformation studies have focused on chloroplast expression, plastid transformation can also be used to express genes in plastids of a wide variety of plant tissues by using appropriate plastid promoters. Based on the sequence of the Gossypium hirsutum chloroplast genome, we developed primers and amplified segments of 20 different plastid genes. The PCR products were labeled and used in filter dot blot hybridization studies to characterize their expression levels and patterns in total RNA isolated from light- and dark-grown cotton tissues at different developmental stages. A subset of 6 genes among these was further characterized by real time PCR. Highest expression levels were observed for rrn16 and psbA. Four genes were expressed in all samples at relatively constant levels: accD, atpA, matK and rrn16. Expression in root tissue was generally low. The results of our study can be used to predict which operons and promoters are most likely to be preferentially expressed in the plastids of tissues of interest at levels that would result in the desired phenotype, facilitating the development of plastid transformation vectors.

RT-PCR based diagnosis revealed importance of human group B rotavirus infection in childhood diarrhoea

Background Human group B rotavirus was first identified as causative agent of a large outbreak of severe gastroenteritis affecting more than 1 million people, predominantly adults in China in 1982–1983. In spite of serological evidences for the presence of group B rotavirus in many countries of the world, the virus has been detected only from China, India and Bangladesh, where most of the cases were from adults. Objectives To ascertain the role of group B rotavirus as an aetiological agent of diarrhoea among children in Kolkata, India. Study design An active surveillance was conducted for rotavirus infection in children in a leading referral paediatric hospital and a few samples were also collected from adults of another hospital in Kolkata, India over a period of 3 years (2002–2004). After primary screening of rotaviruses by RNA electrophoresis in polyacrylamide gel, 200 of 412 samples negative by PAGE were screened by reverse transcription polymerase chain reaction for group B rotaviruses. The group B rotavirus positives samples were also confirmed by dot-blot hybridization. Result During the study period, we detected 37 (18.5%) sporadic cases of human group B rotavirus infection in children below 3 years of age of which 15 (7.5%) showed mixed infection with group A rotaviruses by RT-PCR. In dot-blot hybridization studies the RNA of all rotavirus positive samples hybridized with the nonisotopic psoralen-biotin labeled total RNA probe generated from a human group B rotavirus CAL-1 strain confirming the samples as group B rotaviruses. Conclusion The shift in age preference of group B rotavirus infection from adult to children and mixed infection of group B and group A rotaviruses reveals the importance of group B rotavirus as an etiological agent of childhood diarrhoea. Therefore, future vaccination strategy should include both group A and B rotaviruses to control rotavirus diarrhoea.

Rapid, species-specific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridization and an electrochemical sensor array

Development of rapid molecular approaches for pathogen detection is key to improving treatment of infectious diseases. For this study, the kinetics and temperature-dependence of DNA probe hybridization to uropathogen species-specific sequences were examined. A set of oligonucleotide probes were designed based on variable regions of the 16S gene of the Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, and Pseudomonas aeruginosa. A universal bacterial probe and probes-specific for gram-positive and gram-negative organisms were also included. The oligonucleotide probes discriminated among 16S genes derived from 11 different species of uropathogenic bacteria applied to nylon membranes in a dot-blot format. Significant binding of oligonucleotide probes to target DNA and removal of nonspecific binding by membrane washing could both be achieved rapidly, requiring as little as 10 min. An oligonucleotide probe from the same species-specific region of the E. coli 16S gene was used as a capture probe in a novel electrochemical 16-sensor array based on microfabrication technology. Sequence-specific hybridization of target uropathogen 16S rDNA was detected through horseradish peroxidase acting as an electrochemical transducer via a second, detector probe. The sensor array demonstrated rapid, species-specific hybridization in a time course consistent with the rapid kinetics of the dot-blot hybridization studies. As in the dot-blot hybridization studies, species-specific detection of bacterial nucleic acids using the sensor array approach was demonstrated both at 65 °C and at room temperature. These results demonstrate that molecular hybridization approaches can be adapted to rapid, room temperature conditions ideal for an electrochemical sensor array platform.

Helicobacter sp. flexispira 16S rDNA taxa 1, 4 and 5 and Finnish porcine Helicobacter isolates are members of the species Helicobacter trogontum (taxon 6).

The term 'flexispira' refers to micro-organisms with a particular morphology: fusiform-shaped with helical periplasmic fibrils and bipolar tufts of sheathed flagella. Two flexispira taxa have been formally named, Helicobacter bilis and Helicobacter trogontum, a third named species is Helicobacter aurati and eight additional 16S rRNA sequence-based flexispira taxa have been described by Dewhirst et al. (Int J Syst Evol Microbiol 50, 1781-1787, 2000) and given the provisional designation Helicobacter sp. flexispira taxa 1-5, 7, 8 and 10. In the present study, seven gastric or intestinal flexispira isolates from seven Finnish pigs originating from different farms were characterized. Morphologically, all these porcine isolates had typical flexispira morphology. Analysis of the 16S rDNA sequences of five isolates showed that they were most closely related to the sequences of flexispira taxa 4 and 5 and H. trogontum (taxon 6), but less closely related to taxa 1-3 and 8, H. bilis and H. aurati. Phenotypic characterization, analysis of RFLPs of 16S and 23S rDNAs and SDS-PAGE profiles revealed that all of the porcine isolates, reference strains of flexispira taxa 1, 4 and 5 and the type strain of H. trogontum (ATCC 700114T) had highly related characteristics that differed from those of the reference strains of taxa 2, 3 and 8 and H. bilis. Furthermore, a high DNA-DNA binding rate was found, in dot-blot hybridization studies, between the Finnish porcine strains, taxa 1, 4 and 5 reference strains and H. trogontum ATCC 700114T. In conclusion, polyphasic characterization of novel porcine flexispira isolates and previously described taxa 1, 4 and 5 reference strains showed that they all belong to a validly described species, H. trogontum, and that the taxonomy of known flexispiras is less complicated than proposed on the basis of 16S rDNA sequence analysis.

Specific Detection of Cherry Mottle Leaf Virus Using Digoxigenin-Labeled cDNA Probes and RT-PCR.

Cherry mottle leaf virus (CMLV)-associated double-stranded RNA (dsRNA) was isolated from the propagation host Chenopodium quinoa. The dsRNA band, with a molecular weight estimated at 7.0 × 106 Da, was used to produce cDNA. Two recombinant plasmids from the cloned cDNA library were identified that specifically bound with CMLV-associated RNA in dot blot hybridization studies. The cDNA inserts were sequenced, and oligonucleotide primers were designed that specifically amplify an 848-bp fragment of the CMLV genome by reverse-transcription polymerase chain reaction. Also, a poly(T) primer was reliably used for reverse transcription, with specific amplification using the CMLV primers, suggesting polyadenylation of the virus genome. Search of the database revealed some sequence homology of the partially characterized genome of CMLV with that of apple chlorotic leafspot virus. Additional sequence data are required, however, to establish the taxonomic position of the filamentous CMLV.

Localization of nod, nif, and acidic exopolysaccharide determinants on a large plasmid in slow-growing cowpea Rhizobium sp. S2.

Slow-growing cowpea Rhizobium sp. S2, a pigeon pea isolate, showed excessive synthesis of exopolysaccharide (EPS) and nod factor(s) only when grown in the presence of corresponding host root exudate (RE) or naringenin (a flavonoid present in RE of pigeon pea). muc- strain, a plasmid-cured derivative of the parent strain, showed negligible EPS production and failed to synthesize the nod factor(s) under similar growth conditions. The nod factor(s) was extracted and partially purified from the total EPS of the culture supernatant of S2 grown in the presence of either RE or naringenin by the butanol-soluble, ethyl acetate-insoluble method. The parent strain also showed ex-planta nitrogenase activity, whereas in muc- it was not detectable. In contrast to observations of the location of nod, nif- and EPS-synthesizing genes on the chromosome in commonly studied cowpea Rhizobia and Bradyrhizobium sp. colony blot and dot blot hybridization studies revealed the presence of nod (common nod genes), nif (nif KDH); and EPS (pss) determinants to be on the large plasmid in the parent strain and therefore absent in muc-, which is a plasmid-cured derivative.

Molecular cloning, sequence analysis and differential expression of an intron-containing gene encoding tomato histone H1.

A tomato genomic clone has been identified which encodes histone H1. The deduced polypeptide is 287 amino acids in length, and exhibits the tripartite organization typical of histones H1. The central globular domain is highly similar to those regions from other H1 molecules, and the carboxyl-terminal domain contains a repeating hexapeptide motif, variants of which are conserved among H1 molecules. RNA gel blotting revealed that histone H1 mRNA is expressed at higher levels in organs which contain meristematic tissue and/or which have a high proportion of actively cycling cells. DNA gel blotting and dot-blot hybridization studies revealed that histone H1 in tomato is encoded by a small gene family. By employing the polymerase chain reaction on genomic DNA and on cDNA, it was determined that the gene is interrupted by an intron. The location and approximate length of the intron are conserved in both the tomato and Arabidopsis genes, with the intron separating the 'nose' region (encoded by exon 1) from the central globular domain (exon 2). The promoter region was found to contain several conserved sequence motifs which likely participate in the regulation of the gene.

Proposals for a new classification of iridescent viruses

The need for comparative studies of iridoviruses to elucidate the relationships between them has been well appreciated. Sixteen iridoviruses, including type species from each of the four recognized genera of the Iridoviridae, were compared by restriction endonuclease characterization, hybridization to the major structural protein (MSP) gene of an invertebrate iridescent virus (IV) isolate at various stringencies, PCR amplification of the MSP gene region and by dot-blot hybridization studies. The results broadly supported previous serological studies. The vertebrate iridoviruses, frog virus 3 (genus Ranavirus) and flounder lymphocystivirus (genus Lymphocystivirus), appeared distinct from one another and from the invertebrate isolates. Naming and numbering invertebrate IV isolates according to history and host is no longer useful since IVs infect a number of species. A revised system, involving names based on the geographical origin of the isolate is proposed, in line with other virus families. The large IVs of invertebrates represented by Vero Beach IV (previously IV3 or mosquito IV; genus Chloriridovirus) showed little similarity to any other IVs. Members of the genus Iridovirus, the small invertebrate IVs, fell into three distinct groups of interrelated isolates. The largest group, containing the Plowden (IV1), Tia (IV2), Nelson (IV9, IV10 and IV18), Aberystwyth (IV22), Srinagar (IV24), Fort Collins (IV29) and Stoneville (IV30) iridoviruses, is named the Polyiridovirus complex. The Plowden iridovirus (IV1) is suggested as type species for this complex given the data available on its molecular biology. Based on previously published data, Timaru (IV16 and IV19) and Uitenhage (IV23) iridoviruses are also assigned to this complex. The second but smaller group is named the Oligoiridovirus complex, which includes Dazaifu (IV6) as the type species and contains Ntondwe (IV21 and IV28) on a tentative basis. Riverside IV (IV31) was distinct from both of the other groups, and is proposed as a third complex, Crustaceoiridovirus.

A second form (beta isoform) of nucleoside diphosphate kinase from rat. Isolation and characterization of complementary and genomic DNA and expression.

Complementary and genomic clones for a second form (the beta isoform) of rat nucleoside diphosphate kinase were isolated. Structural studies revealed that nucleotide and deduced amino acid sequences of the beta isoform were quite similar to those of the alpha isoform (identities were 82 and 89%, respectively), which were delineated in our previous study (Kimura, N., Shimada, N., Nomura, K., and Watanabe, K. (1990) J. Biol. Chem. 265, 15744-15749). The gene encoding the beta isoform, covering 10 kilobases and comprising five exons, was located in tandem in the immediate vicinity (at a 3-kilobase distance) of the 5' upstream of the alpha isoform gene which was recently reported from this laboratory (Ishikawa, N., Shimada, N., Munakata, Y., Watanabe, K., and Kimura, N. (1992) J. Biol. Chem. 267, 14366-14372), suggesting their generation by gene duplication. The exon-intron junctions were exactly conserved between the two genes. Southern blot analyses showed that unidentified fragments cross-reacted with the beta isoform cDNA probe besides those containing the genuine gene, and at least two of them were identified as possible processed pseudogenes. Northern and dot blot hybridization studies demonstrated that the alpha isoform was more expressed than the beta isoform in rat tissues examined except brain, from which the isoform designation was derived. These results suggest independent expression and specific roles of these isoforms in the cell. Comparative studies between rat and human isoforms indicate that the isoforms could have differentiated before the two species evolutionally diverged.

Differential levels of thyroid peroxidase and thyroglobulin messenger ribonucleic acids in congenital goiter with defective thyroglobulin synthesis

The biosynthesis of thyroid hormones requires iodide, thyroid peroxidase (TPO), thyroglobulin (Tg) and H2O2. We have studied two sisters with congenital large goiters and hypothyroidism. Perchlorate tests were negative. Serum T3 and T4 were decreased, TSH was increased and Tg was within the lower limit of normal. Biochemical and molecular studies were performed on goiter samples obtained after surgery. Tg content in both tissues was negligible. Paper chromatography of labeled iodocompounds showed a decrease in T4, and the presence of a pronase/pancreatin-resistant iodoprotein. TPO activity was normal in the tissues. Sephacryl S-300 gel filtration demonstrated labeled iodoalbumin-like protein and the absence of a Tg peak. Salting out studies of soluble protein fraction gave an abnormal pattern. Agarose gel electrophoresis showed the presence of an iodoalbumin-like protein and the absence of Tg in the tissues. This last finding was confirmed by immunoelectrophoresis. The Tg and TPO mRNAs levels were also analyzed. Dot-blot hybridization studies with pM5 (TPO cDNA) and phTgM2 (Tg cDNA) probes showed increased and decreased signals, respectively. The increase in TPO mRNA can be explained as a compensatory mechanism vis a vis an increase in serum TSH caused by decreased serum T3 and T4 due to the impairment in Tg mRNA. The Tg mRNA of both patients was further studied with four different probes covering 5' and 3' regions (phTgM1, phTgB1, phTgB2 and phTgB3). Hybridization was observed with all four probes, thus excluding a dramatic deletion defect. Northern transfer showed a clear signal of hybridization with the phTgB1 probe in the 8-9 Kb range. We may conclude that the biochemical and molecular abnormality of these patients is characterized by a decrease of Tg mRNA and of Tg translation.

Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis

A tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1.1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10(-7) per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10(-8) per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9.5 and 11.0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.

Selective inhibition of formation of suppressor glutamine tRNA in moloney murine leukemia virus-infected NIH-3T3 cells by Avarol

Avarol is a sesquiterpenoid hydroquinone, which displays no inhibitory potencies on mammalian DNA polymerases α, β, and γ, on mammalian RNA polymerases I, II, and III, or on reverse transcriptases from Moloney murine leukemia virus (Mo-MuLV) and from HIV. For a further elucidation of the antiviral effect of Avarol, we used NIH-3T3 cells infected with Mo-MuLV as a model system. The results show that in uninfected NIH-3T3 cells Avarol (i) causes a 50% reduction of the growth rate only at the high concentration of 29.6 μ M and (ii) is accumulated in the cytoplasm close to the nucleus. At the much lower concentrations of 1–3 μ M Avarol causes an almost complete inhibition of viral progeny release. Moreover, it is shown that at 3 μM Avarol, the increase of the Mo-MuLV-induced UAG suppressor glutamine tRNA (tRNA UmUG Gln) was reduced to the normal level. Dot blot hybridization studies revealed that Avarol displays no inhibitory activity on cellular and viral m RNA synthesis. Taking the processing pathway of viral polyprotein Pr180 gag, pol to p80 (reverse transcriptase) as an example, our Western blotting experiments showed that the final maturation process, conversion of pl 10 to p80, is inhibited in Avarol-treated cells. From these data we conclude that Avarol prevents the suppression of the UAG termination codon at the gag-pol junction of the retroviral genome. The functional consequence of this event is very likely an inhibition of the readthrough of the retroviral protease gene which overlaps the pol and gag genes, resulting in the reduction of the protease synthesis which is necessary for the viral proliferation.

Differential effects of gamma-interferon on collagen and fibronectin gene expression.

The interferons are a group of endogenous proteins that exhibit a variety of biological functions in addition to their ability to induce resistance to viruses. In order to evaluate the anti-fibrogenic actions of interferon, we have delineated the level of regulation responsible for gamma-interferon-induced changes in collagen and fibronectin gene expression in cultured fibroblasts. Confluent human skin fibroblasts were exposed to 500 anti-viral units/ml of gamma-interferon. RNA was then extracted from the cells, and steady-state mRNA levels were determined by Northern and dot blot hybridization studies. Cells exposed to interferon had type I procollagen mRNA levels that were 23% of control and type III procollagen mRNA levels only 7% of control. The interferon-treated cells also had beta-actin mRNA levels that were decreased to 51% that of untreated cells but had fibronectin steady-state mRNA levels that were 560% of control levels. Nuclear run-on assays revealed that interferon did not affect the transcriptional rates of types I and III collagen or beta-actin, but it did increase the transcriptional rate of fibronectin to 670% of control levels. These findings demonstrate that gamma-interferon causes a marked decrease in types I and III procollagen mRNA levels in vitro by a post-transcriptional mechanism while inducing fibronectin expression at a transcriptional level.

Distribution of Vitamin D-Dependent Calcium-Binding Protein Messenger Ribonucleic Acid in Rat Placenta and Duodenum*

The vitamin D-dependent calcium-binding protein (CaBP), cholecalcin or calbindin, is one of the best documented molecular expressions of 1,25-dihydroxyvitamin D, the hormonal metabolite of vitamin D. In this report, DNA/RNA hybridization assays have been used to examine cholecalcin (CaBP) mRNA production in the placenta and duodenum of 21-day pregnant rats. A cloned CaBP cDNA which codes for the rat intestinal 9000 mol wt cholecalcin (9KCaBP) was radiolabeled and used in hybridization assays to explore 1) the size and relative quantities of CaBP mRNA extractable from placenta and duodenum by molecular hybridization, and 2) the localization and quantification, by in situ hybridization histochemistry, of CaBP mRNA in specific cells in rat placenta and duodenum. Northern hybridization studies show that the [32P]cDNA sequence hybridizes to a single 500- to 600-nucleotide species in the placenta as in the duodenum and, therefore, demonstrate identical 9KCaBP mRNA processing in both tissues. Dot blot hybridization studies show that the concentration of 9KCaBP mRNA was greatest in the duodenum, while that of the inner (fetal) placenta was about 50% the duodenal level. Considerably less CaBP mRNA was found in the outer (maternal) placenta. The observed differences in 9KCaBP mRNA levels correlate well with the in vivo variations in 9KCaBP concentrations. In situ hybridization histochemistry using [3H]cDNA reveals that 9KCaBP mRNA visualized by silver grains was concentrated in the inner placenta over the cytoplasm of syncytial cells in the trophoblastic epithelium of the labyrinth and much less frequently in the cells of the outer placenta. In the duodenum, 9KCaBP mRNA was found only in the absorptive epithelial cells from the crypt region to the upper part of the villi. The silver grains were distributed throughout the cytoplasm of the columnar cells; they were densest in the perinuclear region and rarest in the nuclear region. The concentration was greater in the cells at the villous tips than in those of the crypts. This difference in 9KCaBP mRNA levels correlates well with the distribution of the protein itself along the villi. CaBP mRNA quantities detected by hybridization histochemistry showed greater labeling in the syncytial cells of the trophoblastic epithelium of the labyrinth than in the absorptive epithelial cells of the upper part of the villi (200% less), indicating accumulation of CaBP mRNA at a greater rate in the trophoblastic epithelium than in the absorptive epithelial cells. These results indicate that in the rat, 9KCaBP is synthesized in both the absorptive cells of the duodenum and the cells of the trophoblastic epithelium of the placenta.

A study of the messenger RNA encoding pyruvate kinase of Neurospora crassa.

In Neurospora crassa, there is a single pyruvate kinase (PK) consisting of four identical subunits of approximately 60k daltons. Northern and dot blot hybridization studies, using most of the yeast pyruvate kinase gene as a probe, suggest the presence of two distinct mRNA species for pyruvate kinase, separable on the basis of the length of their polyadenylated tails, by oligo(dT)cellulose chromatography. These messages are present in polysomes, immuno-precipitated by anti-PK antibodies, indicating probable translation in vivo. Fractions containing both messages were translated in vitro in the heterologous systems as well as in a homologous N. crassa lysate, the newly-synthesized PK being detected by immunoadsorption. Protection studies using S1-nuclease suggest no major structural differences in the 5'-untranslated and most of the coding regions of the two messages.