Dot Hybridization Analysis Research Articles

Kinetic analyses of trichloroethylene cometabolism by toluene-degrading bacteria harboring a tod homologous gene

We have previously constructed a bacterial library consisting of toluene-degrading bacteria isolated from soil, activated sludge, and trichloroethylene (TCE) waste. In the present study, the library was subjected to colony hybridization with todC1C2-encoding terminal oxygenase of a multicomponent enzyme that is responsible for the first oxidation of toluene, toluene dioxygenase (TDO). Three strains, A1071, IB5, and B6122, which showed positive signals with todC1C2 by the colony hybridization, were selected on the basis of results of a preliminary degradation assay for detail analyses of degradation kinetics of toluene and TCE. Their possession of the gene homologous to todC1, a large subunit of TDO of Pseudomonas putida F1, was confirmed by dot hybridization analysis using their chromosomal DNA extracts as templates. Results of degradation experiments using resting cells revealed the variety in TCE degradation ability among these strains; A1071 demonstrated degradation kinetics similar to the strain F1 while B6122 could not degrade TCE at all. The interaction between toluene and TCE on their degradation could be simply expressed by competitive inhibition.

Expression of tumor related genes NGX6, NAG-7, BRD7 in gastric and colorectal cancer.

NGX6, NAG-7 and BRD7 genes are tumor related genes, which have been newly cloned by positional candidate cloning strategy. This study was designed to investigate the expression levels of NGX6, NAG-7 and BRD7 genes in human gastric and colorectal cancer tissues, and their corresponding normal tissues, and to investigate whether these genes play a role in the pathogenesis of gastric and colorectal cancers. Reverse transcription-polymerase chain reaction (RT-PCR), dot hybridization and Northern blot analysis were used to compare the expression levels of NGX6, NAG-7 and BRD7 genes in 34 gastric cancer tissues and 34 colorectal cancer tissues with their corresponding normal tissues of the same patients, respectively. Among the 34 colorectal cancer specimens and the 34 gastric cancer specimens, the expression of NGX6 in 25 colorectal cancer tissues was absent or very weak (73.5 %) by RT-PCR analysis. The down-regulation rate of NGX6 in colorectal cancer tissues was significantly higher than that in corresponding normal tissues (26.5 %,9/34) (P<0.005). Moreover, the down-regulation of NGX6 was significantly correlated with lymph node and/or distance metastases. Patients with lymph node and/or distance metastasis had much higher down-regulation rate of NGX6 than patients without metastases (93.8 % vs 55.6 %, P<0.05). However no correlation was found between the expression of NGX6 and pathologic type of colorectal cancer in this study, and also the expression of NGX6 did not display any difference between gastric cancer and corresponding normal tissues (58.8 % vs 70.6 %, P>0.25). Dot hybridization and Northern blot analysis confirmed the results of RT-PCR. Furthermore, NAG-7 and BRD7 mRNA was not up- or down-regulated in gastric and colorectal cancers compared with their corresponding normal tissues in our study. The down-regulation of NGX6 may be closely associated with tumorigenesis and metastasis of colorectal carcinoma. However, it may not contribute to the development and progression of gastric carcinoma. In addition, the expression levels of NAG-7, and BRD7 did not alter in gastric and colorectal cancers. This seems to suggest that NAG-7 and BRD7 genes may not play a role in gastric and colorectal carcinogenesis.

S100A6 protein as a marker for differential diagnosis of cholangiocarcinoma from hepatocellular carcinoma

A new marker useful for differential diagnosis of cholangiocarcinoma (CC) from hepatocellular carcinoma (HCC) has been identified and investigated. cDNA clone for S100A6 (calcyclin) was identified as a upregulated gene in intrahepatic tumors by differential dot hybridization and Northern blot analysis using normal and tumor intrahepatic tissues. Significant increase in the expression level of S100A6 mRNA was recognized in intrahepatic tissues in three of three CC cases but none of six primary HCC. Immunohistochemical examination of the tumor tissues using monoclonal antibody specific to S100A6 showed that 14 of 18 (77.8%) CC tissue samples were positively stained but only two of 20 (10%) specimens from the patients with primary HCC were weakly stained. These results suggest that the levels of S100A6 mRNA expression and protein in the tissue specimens examined by Northern blot and immunohistochemical staining may be additional useful marker for the differentiation of CC from HCC.

Specific genomic DNA fragment analysis of different geographical clinical samples of shrimp white spot syndrome virus

White spot syndrome (WSS) has been found in many species of shrimp and crabs, not just In Asia but globally. The causative agent is known as white spot syndrome virus (WSSV). In order to clarify the relatedness of WSSV from various geographic regions, we compared the viral DNA of a number of clinical samples of WSSV. (1) China96-116A from Penaeus chlnens~s, (2) India95-314 from Penaeus monodon, (3) grocery store95-204 and grocery store96-l15 from P. rnonodon possibly originating from Thailand, (4) crayfish97-25 from Orconectespunctimanuscollected from the U.S. National Zoo, (5) Thailand95-46 from experimentally infected Penaeus vannamei. (6) South Carolina97-64 from P. vannamei, and (7) Texas95-242 and Texas96-7 from P. vannarnei. These specimens were first examined by dot hybridization analysis with nucleic acid probes derived from a WSSV Taiwan isolate. Although the intensity of the hybridization signals varied, and although some specimens of India95314, crayfish97-25, Texas95-242 and Texas96-7 failed to give a detectable hybriduation signal with certain probes, the broad consistency of dot hybridization data suggests that these WSSV clinical samples from different geographical locations are closely related. Following this analysis, all the specimens were examined using 10 virus-specific polymerase chain reactions (PCR). The amplification products were subsequently digested with Cfo I, Hae 111, Hpa I1 and Rsa I restriction endonucleases to determine if there were any DNA fragment polymorphisms in the WSSV clinical samples. The results highlighted the genetic relatedness of all the WSSV clinical samples with the possible exception of a series of Texas viral samples which could be distinguished from the other geographic samples in some of the PCR-based tests.

The radiobiologic characteristics of DNA polymerase β in hepatomas

DNA polymerase beta activity, its content and gene transcription levels in SMMC-LTNM hepatoma were investigated, using 3H-TTP incorporation, immunocytochemistry and cytoplasmic dot hybridization, respectively. The relations between the biological properties of the enzyme and DNA repair synthesis induced by gamma-ray irradiation were also studied. It was found that DNA polymerase beta activity, its content and the amount of its mRNA were much higher in hepatoma than those in normal hepatocytes (P < 0.01). Following whole-body irradiation of the nude mouse bearing SMMC-LTNM with 2 Gy of gamma ray, the polymerase beta activity in hepatoma increased temporarily and the gene transcription of the enzyme seemed to be more active. DNA polymerase beta participated in DNA repair synthesis and this effect was different between hepatoma and hepatocyte because of the biological differences of DNA polymerase beta. The results presented here indicated that DNA polymerase beta could affect radiation damage and radiotherapy of cancer.

Basic and acidic fibroblast growth factor increase prolactin mRNA in a dose-dependent and specific manner in GH3 cells

In the present report we have studied the effects of acidic and basic molecular forms of the fibroblast growth factor (aFGF, bFGF) on prolactin (PRL) mRNA production and PRL secretion in GH3 cells, a rat pituitary cell line, and their interactions with 17β-estradiol (βE 2). To meet this purpose we measured mRNA levels in the cells by both Northern blot and dot blot hybridization analysis, and rPRL immunoreactivity in the culture medium by specific RIA. We observed a marked increase in PRL mRNA levels following 24 h incubation with both basic and acidic FGF. This effect was dose-dependent, with maximal responses ranging between 300 and 600% above the control values. bFGF appeared to be much more potent than aFGF (10–50 times), considering the ED 50 of the dose-response curves. Prior incubation with βE 2 (10 −8 M) produced an enhancement in the responses to low doses of bFGF and aFGF, but not to high doses, as revealed by dot-hybridization analysis. Norther blot analysis showed also that both aFGF and bFGF, may have a partially additive effect with βE 2, upon the mature form (1 kb) of rPRL mRNA in GH3 cells. Considering that bFGF is present at high levels in the pituitary, our results suggest that FGF could be a physiological regulatory factor for prolactin production and secretion.

A Salmonella-Specific DNA Probe and Its Use in Southern Hybridization for Differentiation of Salmonella enteritidis

Chromosomal DNA from phage type 13a and Phage type 8 of Salmonella enteritidis (SE) was cloned in Escherichia coli using the plasmid pUC8. A 2.1-kilobase-pair (kbp) DNA fragment specific for Salmonella spp. was identified by colony, dot, and Southern hybridization analyses. When labeled and used as a probe (C7), this recombinant clone hybridized with the DNA of 18 Salmonella species but did not hybridize with the DNA of seven other enteric and non-enteric bacterial species. In Southern blot hybridization, this probe hybridized specifically to a 6.0 kbp BglI-digested DNA fragment of 17 SE isolates from human as well as avian sources, thus differentiating this fragment from DNA of 17 other Salmonella species. The C7 probe also hybridized to a 5-kbp fragment of one SE DNA derived from human and two SE DNA from poultry sources. This may indicate variation among SE isolates.

Stored forms of vitellogenin mRNA in fat body cells of Locusta migratoria

Vitellogenin (Vg), a major yolk protein precursor for locust ( Locusta migratoria) eggs, is synthesized by female fat body. At the time of oviposition Vg production decreases greatly and Vg mRNA is stored for reinitiation of Vg synthesis in the next reproductive cycle. Sedimentation analysis of fat body extracts and dot-hybridization analysis of RNA have revealed at least three stored forms of Vg mRNA. Two of these sediment in the preribosome zone and one in the postribosome zone. CsCl density gradient analysis has revealed that all of them have buoyant density in the range 1.51–1.57 g/cm 3. In contrast to Vg, synthesis of apolipophorin (apoLp-III) is relatively constant in locust fat body. Unlike Vg mRNA, the stored form of ApoLp-III mRNA has density and sedimentation characteristics close to those of stored mRNA forms from other systems. This supports the conclusion that the unusual stored forms of Vg mRNA found are not an experimental artifact but have a function in the locust's reproductive cycle.

Enzymatic amplification of mini-exon-derived RNA gene spacers of Leishmania donovani: primers and probes for DNA diagnosis

The multicopy mini-exon-derived RNA (med RNA) locus of Leishmania donovani was enzymatically amplified by the polymerase chain reaction (PCR). The major 180 bp PCR product contained conserved med RNA gene sequences flanking the variable intergenic spacer from the med RNA gene tandem repeat. The oligonucleotide primers cross-reacted with other Leishmania species. In serial dilution experiments, positivity in the PCR assay was observed down to the genomic DNA equivalent of less than a single Leishmania cell. When the major PCR products from Indian L. donovani isolates were cloned and used as probes in dot hybridization analyses, they discriminated between L. donovani and L. amazonensis, L. major and L. infantum under high stringency conditions. DNA from spleen biopsies and blood samples of confirmed kala azar patients was positive, as were two skin biopsies from patients with post-kala azar dermal leishmaniasis (PKDL). These observations demonstrate that PCR amplification of med RNA intergenic spacers is sufficiently sensitive for clinical diagnosis of kala azar and PKDL, and furthermore, that cloned intergenic spacer probes may be useful for identification and classification of L. donovani.

Identification and analysis of a genomic strain cluster of mycoplasmalike organisms associated with Canadian peach (eastern) X disease, western X disease, and clover yellow edge.

Genetic interrelatedness among 13 strains of mycoplasmalike organisms (MLOs) from various sources was evaluated by dot hybridization and restriction fragment length polymorphism analyses using cloned DNA probes derived from Canadian peach X (CX) and western X (WX) MLOs. Dot hybridization analysis indicated that CX, WX, and clover yellow edge MLOs are closely related and form a distinct strain cluster that is only distantly related to the 10 other MLOs. Similarity coefficients derived from restriction fragment length polymorphism analysis revealed that CX, WX, and clover yellow edge MLOs represent three distinct genomic types.

Genetic Interrelatedness among Clover Proliferation Mycoplasmalike Organisms (MLOs) and Other MLOs Investigated by Nucleic Acid Hybridization and Restriction Fragment Length Polymorphism Analyses

DNA was isolated from clover proliferation (CP) mycoplasmalike organism (MLO)-diseased periwinkle plants (Catharanthus roseus (L.) G. Don.) and cloned into pSP6 plasmid vectors. CP MLO-specific recombinant DNA clones were biotin labeled and used as probes in dot hybridization and restriction fragment length polymorphism analyses to study the genetic interrelatedness among CP MLO and other MLOs, including potato witches'-broom (PWB) MLO. Results from dot hybridization analyses indicated that both a Maryland strain of aster yellows and a California strain of aster yellows are distantly related to CP MLO. Elm yellows, paulownia witches'-broom, peanut witches'-broom, loofah witches'-broom, and sweet potato witches'-broom may be very distantly related, if at all, to CP MLO. A new Jersey strain of aster yellows MLO, tomato big bud MLO, clover phyllody MLO, beet leafhopper-transmitted virescence MLO, and ash yellows MLO are related to CP MLO, but PWB MLO is the most closely related. Similarity coefficients derived from restriction fragment length polymorphism analyses revealed that PWB and CP MLOs are closely related strains and thus provided direct evidence of their relatedness in contrast to reliance solely on biological characterization.

Expression of the Collagenolytic and Ras-Induced Cysteine Proteinase Cathepsin L and Proliferation-Associated Oncogenes in Synovial Cells of MRL/I Mice and Patients with Rheumatoid Arthritis

Based on the observation that rheumatoid joint destruction is related to the presence of transformed-appearing proliferating synovial lining cells attached to cartilage and bone at the site of early destruction, we searched for the expression of proliferation- and transformation-associated oncoproteins in synovial tissues from patients with early destructive rheumatoid arthritis (RA). Immunolocalization of Ras and Myc proteins was found in about 70% of the RA cases and was restricted to the proliferating synovial lining cells. The cysteine proteinase, cathepsin L, which has been shown to be the major ras-induced protein in ras-transformed murine NIH 3T3 cells, was detected in 50% of the RA cases, predominantly in synovial cells attached to cartilage and bone at the site of joint destruction. Moreover, utilizing cytoplastic dot hybridization analysis, we demonstrated the presence of RNA sequences complementary to human cathepsin L in primary cultures of human synovial cells from RA joints and complementary to murine cathepsin L in synovial lining cells derived from MRL/l mice developing spontaneously a RA-like disease. Significant levels of ras oncogene transcripts and products in human RA synovial cells associated with an increased expression of the cathepsin L gene indicate that this collagen-degrading enzyme may contribute to the destruction of cartilage and bone in RA.

Nonradioactive Screening Method for Isolation of Disease-Specific Probes To Diagnose Plant Diseases Caused by Mycoplasmalike Organisms

DNA fragments of tomato big bud (BB) mycoplasmalike organism (MLO) in diseased periwinkle plants (Catharanthus roseus L.) were cloned to pSP6 plasmid vectors and amplified in Escherichia coli JM83. A nonradioactive method was developed and used to screen for MLO-specific recombinants. Cloned DNA probes were prepared by nick translation of the MLO recombinant plasmids by using biotinylated nucleotides. The probes all hybridized with nucleic acid from BB MLO-infected, but not healthy, plants. Results from dot hybridization analyses indicated that several MLOs, e.g., those of Italian tomato big bud, periwinkle little leaf, and clover phyllody, are closely related to BB MLO. The Maryland strain of aster yellows and maize bushy stunt MLOs are also related to BB MLO. Among the remaining MLOs used in this study, Vinca virescence and elm yellows MLOs may be very distantly related, if at all, to BB MLO. Potato witches' broom, clover proliferation, ash yellows, western X, and Canada X MLOs are distantly related to BB MLO. Southern hybridization analyses revealed that BB MLO contains extrachromosomal DNA that shares sequence homologies with extrachromosomal DNAs from aster yellows and periwinkle little leaf MLOs.

Preferential association of a transcriptionally active gene with the nuclear matrix of rat fibroblasts transformed by a simian-virus-40–pBR322 recombinant plasmid

To study the relationship between the structural organization and function of the eukaryotic genome, DNA associated with nuclear matrix was analysed by using a transformed rat fibroblast cell line. The nuclear matrices were prepared from the isolated nuclei of pSV1-FR, a rat fibroblast cell line transformed by a pBR322-based recombinant plasmid containing an early gene region, which codes for large T-antigen, of simian virus 40. This transformed cell contained a single copy of the plasmid sequence integrated into the chromosomal DNA of the host cell. The early gene of this plasmid was constitutively expressed, as demonstrated by positive immunofluorescence staining of the cell for large T-antigen and by RNA-blot analysis for its specific mRNA. DNAs were extracted from whole isolated nuclei and nuclear-matrix preparations of the cells, and the relative amounts of the sequence similar to that of the plasmid were compared between these DNA preparations. By employing dot hybridization and Southern-blot analyses we found that the plasmid sequence was more enriched in the DNA extracted from the nuclear matrices than in the DNA extracted from the whole nuclei. When an albumin gene sequence that was not transcribed in this cell line was compared similarly as a control, we found no significant enrichment of this sequence in the DNA associated with the nuclear matrix. Our results strongly support the concept that a transcriptionally active gene is preferentially associated with the nuclear matrix.

Detection of Chrysanthemum Yellows Mycoplasmalike Organism by Dot Hybridization and Southern Blot Analysis

(...) Of five probes used in Southern blot analyses, one probe containing extrachromosomal DNA from O-1 MLO hybridized with extrachromosomal but not chromosomal DNA of CY MLO. Three probes containing chromosomal DNA of AY MLO and one containing chromosomal DNA of O-1 MLO hybridized with chromosomal DNA of CY MLO. The nucleotide sequence homologies between the probes and nucleic acid of CY MLO are evidence for genetic relatedness of a European MLO and American MLOs and provide diagnostic tools for CY MLO detection

Heat shock of cultured GC cells enhances the level of triiodothyronine induced growth hormone (GH) and GH messenger ribonucleic acid.

We have previously proposed that the effects of heat shock on thyroid hormone-responsive rat pituitary tumor (GC) cells may be a model relevant to the in vivo effects of nonthyroidal disease on thyroid hormone action. To determine the effects of heat shock on thyroid hormone responses, GC cells (normally cultured at 37 C) were studied after incubation at 41 C. After 18 h at 41 C there was enhanced synthesis of proteins (mol wt, 70,000 and 90,000) considered to be universal markers of the cellular response to heat shock. Incubation at 41 C also resulted in a significant decrease in GC cell viability and (after 24 h) arrest of GC cell growth. However, the induction of GH synthesis by T3 was significantly enhanced in GC cells stressed by incubation at 41 C. The addition of 5 nM T3 to thyroid hormone-depeleted GC cells resulted in a significantly greater (P less than 0.001) accumulation of GH (2642 +/- 280 ng/18 h) during 41 C incubation than during 37 C incubation (1223 +/- 175 ng/18 h). The enhanced T3-induced production of GH was coincident with a proportional increase (P less than 0.05) in cellular GH mRNA determined by dot hybridization analysis. Thus, the stress of 41 C incubation elicits a heat shock response in GC cells characterized by decreased viability and growth arrest, but enhanced accumulation of GH mRNA in response to T3. Our recent report on the identical effects due to the stress of implantation of the Walker 256 carcinoma on T3-induced rat pituitary GH mRNA in vivo suggests that heat shock of cultured GC cells is a valid in vitro model of nonthyroidal disease.

Developmental patterns of gene expression of secreted proteins in brain and choroid plexus

The proteins secreted by the choroid plexus throughout rat brain development were analyzed by two-dimensional polyacrylamide gel electrophoresis following biosynthetic labeling of choroid plexus pieces with [ 14C]leucine in vitro. Approximately 20 major protein species were resolved which, with the exception of transferrin, transthyretin, and α 2-macroglobulin, appear to be unrelated to proteins found in high concentrations in plasma. Several patterns of developmental regulation were observed. At least two of the proteins were synthesized and secreted at high levels only by fetal choroid plexus, whereas the secretion of several other proteins including transferrin and proteins comigrating with cystatin C and α 2-macroglobulin increased only after birth. The levels of mRNA coding for transferrin, ceruloplasmin, cystatin C, α 2-macroglobulin, β 2-microglobulin, and transthyretin were measured in the brain during development by dot hybridization and northern gel analysis. No mRNA was detected coding for the proteins α-fetoprotein, α 1-antitrypsin, haptoglobin, and thiostatin in the brain at any stage. For those proteins, which are produced in other parts of the brain as well as by the choroid plexus, the changes in their corresponding mRNA levels measured in whole brain paralleled the changes in their secretion by the choroid plexus. The results presented in this paper show that the choroid plexus is active in protein secretion at all stages studied. The changing pattern of protein secretion by the choroid plexus, combined with its early development compared with other tissues in the brain, suggests that it is active in providing the appropriate extracellular environment for the growth and differentiation of the brain.

Induction of cellulase by cello-oligosaccharides in Trichoderma koningii G-39

A fraction of cello-oligosaccharides with a high degree of polymerization (Gx, degree of polymerization greater than 10) was found to be a potent cellulase inducer of Trichoderma koningii G-39. It was prepared by partial sulfuric acid hydrolysis of cellulose. A comparison of the induction potential and efficiency of Gx and sophorose indicated that the latent period of induction was longer and the maximal enzyme production lower for Gx than for sophorose. The optimal concentrations are 1% with Gx and 0.1% (w/v) with sophorose with a corresponding latent period and a maximum carboxymethylcellulase (CMCase) activity, which accumulated in the culture filtrate, of about 8 h and 5.4 units for Gx and 4 h and 7.2 units (in mg glucose equivalent per ml culture filtrate) for sophorose. The apparent differences between the two compounds are attributable to the interplay of catabolite repression in the inducing effect of Gx which is subjected to cellulase degradation with glucose as the major end products. HPLC sugar analysis of the culture filtrate incubated with Gx suggests that the inducing potential and efficiency of cello-oligosaccharides depend on its degree of polymerization. The protein patterns as revealed by SDS-polyacrylamide gel analysis of the culture filtrates from incubation with Avicel, phosphoric acid-swollen Avicel, Gx or sophorose showed no inducerspecific protein bands except variation in intensity and induction time. A dot hybridization analysis of poly(A) RNA from washed mycelium incubated with Gx or sophorose using a 650 basepair cDNA fragment of cellobiohydrolase I gene as the probe indicates that the inductive formation of cellulase by both inducers in this strain is operative at the transcriptional level.

Multiple mechanisms by which glutamine synthetase levels are controlled in murine tissue culture cells

We report the isolation of a complimentary DNA (cDNA) clone encoding glutamine synthetase, derived from a population of methionine sulfoxime-resistant mouse GF1 fibroblasts. When GF1 cells are incubated for 48 h in the presence of the glucocorticoid hormone dexamethasone, the specific activity of glutamine synthetase (GS), assayed as glutamyltransferase activity, increases by threefold. Based on dot hybridization analysis, hormonal treatment also produces a similar increase in the level of GS mRNA. When GF1 cells or mouse Neuro 2A neuroblastoma cells are transferred from medium containing 4 m m glutamine to glutamine-free medium, glutamyltransferase activity increases by at least fivefold. However, the presence or absence or glutamine in the medium does not affect the relative level of glutamine synthetase mRNA in either cell line. With both GF1 and Neuro 2A cells, the half-time for the decline in glutamine synthetase enzyme activity on addition of glutamine to the medium is approximately 1.5 h. This rapid decline, coupled with the lack of effect of glutamine on the level of GS messenger RNA in Neuro 2A cells, renders it unlikely that neural cells alter glutamine synthetase levels in response to glutamine by a biosynthetic mechanism, as suggested by previous authors [ L. Lacoste, K. D. Chaudhary, and J. Lapointe (1982) J. Neurochem. 39, 78–85 ].

Biological and biochemical comparison of nuclear polyhedrosis virus isolates pathogenic for the oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae)

Nuclear polyhedrosis virus (NPV) isolated from Mythimna separata populations in China (MsN-PV C), India (MsNPV I), and Japan (MsNPV J), and NPV from a closely related host insect Pseudaletia unipuncta (PuNPV) were compared. Bioassays in M. separata showed that the homologous NPVs were all significantly more virulent than the heterologous NPV, with MsNPV C and MsNPV I having the highest virulence and MsNPV J having a slightly lower but faster acting virulence. In restriction endonuclease and dot hybridization analyses the genomes of MsNPV C and MsNPV I were found to be closely related, reflecting their similar biological properties, while the genomes of MsNPV J and PuNPV were found to be different and quite distinct. The importance of comparing the characteristics of homologous and heterologous NPVs in the selection of a suitable candidate for biological control is discussed.