Filter Hybridization Technique Research Articles

Quantification of Escherichia coli Genomic DNA Contamination in Recombinant Protein Preparations by Polymerase Chain Reaction and Affinity-Based Collection

This study describes the development of a novel assay for the quantification of Escherichia coli genomic DNA contamination in recombinant protein samples. The technique is based on PCR amplification and digoxygenin labeling of the genes encoding 5S ribosomal RNA followed by affinity-based collection and detection. Samples containing 1 pg mL−1 of extracted E. coli genomic DNA (gDNA) could be measured using this method. Using extracted E. coli gDNA as standards, a 35-cycle PCR reaction exhibited a linear response versus template concentration between 1 pg mL−1 and1 ng mL−1 genomic DNA even when diluted in a variety of buffering conditions. Comparison of the novel assay with a traditional filter binding and hybridization technique using recombinant protein samples confirmed that the procedure was accurate and sensitive. The assay described in this report is a safer and less expensive alternative to radioactive techniques employed for DNA quantification, utilizing readily available reagents and apparatus.

Application of fluorescently labeled poly(dU) for gene expression profiling on cDNA microarrays.

The membrane filter hybridization technique has been widely used for gene expression profiling. The preparation of sensitive and reliable probes is critical for quantitative analysis in this technique. We report a method in which fluorescently labeled poly(dU) is used to detect poly(A)-containing mRNA that hybridizes to specific gene targets. The probe can be used commonly for every sample, alleviating problems encountered in preparing cDNA probes by reverse transcription, particularly when many samples are to be analyzed. Moreover, the sensitivity is at least comparable to cDNA probes prepared by conventional protocols, and intensities of signals after hybridization are independent of mRNA sizes and solely dependent on copy numbers. This method was also shown to be applicable to DNA chip technology.

Identity of the xerophilic species Aspergillus penicillioides: Integrated analysis of the genotypic and phenotypic characters.

We examined the identity of Aspergillus penicillioides, the typical xerophilic and strictly anamorphic species, using an integrated analysis of the genotypic and phenotypic characters. Our experimental methods on two genotypic characters, i.e., DNA base composition using the HPLC method and DNA relatedness using the nitrocellulose filter hybridization technique between A. flavus, A. oryzae, and their close relations revealed a good agreement with the values by buoyant density (for DNA base composition) and spectrophotometric determination (for DNA relatedness) reported by Kurtzman et al. in 1986. On the basis of these comparisons, we examined DNA base composition and DNA relatedness of six selected strains of A. penicillioides, including IFO 8155 (originally described as A. vitricola), one strain of A. restrictus, and the respective strains from Eurotium amstelodami, E. repens, and E. rubrum. As a result, five strains within A. penicillioides, including the neotype strain NRRL 4548, had G+C contents of 46 to 49 mol%, whereas IFO 8155 had 50 mol%. A. restrictus had 52 mol%, and three Eurotium species ranged from 46 to 49 mol%. The DNA relatedness between A. penicillioides (five strains), except for IFO 8155, exhibited values greater than 70%, but the DNA complementarity between four strains and IFO 8155 in A. penicillioides revealed values of less than 40%. DNA relatedness values between three species of Eurotium were 65 to 72%. We determined 18S, 5.8S, and ITS rDNA sequences as other genotypic characters from A. penicillioides (six strains), A. restrictus, and related teleomorphic species of Eurotium. In three phylogenetic trees inferred from these sequences, five strains of A. penicillioides, including the neotype strain, were closely related to each other, whereas IFO 8155 was distantly related and grouped with other xerophilic species. Our results have suggested that A. penicillioides typified by NRRL 4548 and A. penicillioides IFO 8155 (ex holotype of A. vitricola) are not conspecific. The enzyme patterns as a genotypic character and general morphology and conidial ornamentation types as phenotypic characters supported this conclusion. Therefore the name A. vitricola Ohtsuki, typified by the holotype strain IFO 8155, should be revived. Evolutionary affinities among Aspergillus species and related teleomorphs, including the xerophilic taxa, are discussed.

Quantification of cytochrome P450 gene expression by RNase protection analysis.

Many attempts to describe the regulation of mdivldual cytochrome P450 (CYP) gene products in animal tissues have been frustrated by the lack of sensitivity and specificity of the assays used. Most enzymatic assays are hampered by the overlapping substrate specificity of these enzymes, and the generation of high-affinity antibodies that are specific for individual isoforms is not a trivial task. Many CYP isoforms have close relatives, some of which can have as much as 98% identity at the RNA nucleotlde-sequence level, and filter-hybridization techniques using labeled cDNA probes are unable to dlscrlminate between such mRNAs. Although noncoding portions of the cDNAs may be used as probes, as they are more likely to offer regions of divergence within subfamllles, this is not often possible because

Baculovirus-directed high level expression of the hepatitis delta antigen in Spodoptera frugiperda cells

The hepatitis delta antigen (HDAg) is a multifunctional protein. It forms the core-like structure of the hepatitis delta virus (HDV) but also enhances replication of HDV in the nucleus of the hepatocyte. A cDNA fragment encoding HDAg was inserted adjacent to the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus present in the baculovirus transfer vector pVL941. After transfection of Spodoptera frugiperda (Sf9) cells a recombinant baculovirus Ac delta 1 was isolated and purified using filter hybridization techniques. Sf9 cells infected with Ac delta 1 express the HDAg as a non-fused, non-glycosylated protein with an abundance of up to 25% of the total cellular protein mass. Immunoblot analysis using a human polyclonal anti-HD conjugate identified a 22K and a 24K protein in the nucleus of Ac delta 1-infected Sf9 cells. Electron microscopic studies using immunogold labelling showed that the recombinant HDAg (recHDAg) was associated with the hetero-chromatin of the Sf9 cells. The recHDAg produced by Sf9 cells elicited anti-HD antibodies in chimpanzees when injected intramuscularly.

Mutation screenig of bleomycin-induced V79 Chinese hamster hprt mutants using multiplex polymerse chain reaction

We have shown by the filter hybridization technique that bleomycin (BLM) induces different types of mutations at the hprt gene locus of V79 Chinese hamster cells. DNA of mutants identified by Southern blots as partial deletions was subjected to further analysis using multiplex polymerase chain reaction (PCR) to localize the endpoints of the deletions over the hprt gene. The PCR analysis revealed that deletions occur in all parts of the hprt gene but are distributed non-randomly. Deletions occurred most frequently at the 3'-end of the hprt gene suggesting a possible existence of a hot spot for deletions in this region; exons 1, 2 and 3 appeared to be less affected by deletions. As PCR can detect microdeletions which are below the limit of resolution of Southern blot hybridization we analysed 25 HPRT- mutants with Southern wild-type pattern to distinguish between point mutations and small deletions. Of these HPRT- mutants, all except five showed PCR amplification products identical to that of V79 wild-type cells. These results are consistent with previous Southern analyses indicating that a large portion of BLM-induced HPRT- mutants are real point mutations. Five mutants, however, showed differences in fragment sizes of single PCR products or did not yield one single exon fragment and thus are probably the result of deletions which were not to be detected by Southern analyses.

Comparative Analysis of Human Papillomavirus Detection by Polymerase Chain Reaction and Virapap/Viratype Kits

Cervical samples from 270 women referred by area physicians were analyzed for the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 by the polymerase chain reaction (PCR) followed by Southern hybridization. Samples from 154 patients were concurrently analyzed by a commercial filter hybridization technique (Virapap and Viratype Kits, Life Technologies, Bethesda Research Labs, Gaithersburg, MD). The sensitivity of the Southern blot procedure combined with PCR was significantly higher than that of the Virapap and Viratype methods. HPV was detected in 67% of women who had positive results for dysplasia by PCR and in 47% by the Virapap method. HPV types 16/18 were found more commonly than types 6/11 in every diagnostic category. More than one HPV type was detected in 12% of HPV-positive patients. The prevalence of HPV in cytologically negative or indefinite patients as measured by PCR was 22% and 40%; in contrast, by the Virapap method, these values were 7% and 10%. These results demonstrate that PCR combined with Southern hybridization provides a higher level of sensitivity than methods that use hybridization without amplification of HPV DNA and also show that the prevalence of HPV is highest in cytologically positive smears.

Human papillomavirus DNA in CO 2 laser-generated plume of smoke and its consequences to the surgeon

Carbon dioxide laser energy is absorbed by intracellular water but not by proteins or nucleic acids. The possibility of dispersing viral DNA during laser therapy of human papillomavirus (HPV)-containing genital infections was explored using a filter hybridization technique. Samples were taken using dacron swabs from 110 patients in nine separate treatment sessions as well as from five pre-filter canisters, four fume vacuum tubes, and from the nasopharynx, eyelids, and ears of the laser surgeon before and after laser surgery. The viral RNA probes were specific for groups of HPV types 6/11, 16/18, and 31/33/35. Human papillomavirus DNA was identified in swabs from 65 of 110 (60%) of histologically unequivocal condylomata and cervical intraepithelial neoplasias. One of the five pre-filter canisters (20%) tested was HPV DNA-positive after laser treatment of 65 patients; it contained HPV DNA type 6. The four fume vacuum tubes tested in the remaining 45 patients were HPV DNA-negative, as were the nasopharynx, eyelids, and ears of the operator. Although HPV DNA may be released during laser vaporization of genital HPV infections, contamination of the operator is unlikely provided appropriate equipment for evacuating HPV DNA-positive smoke is used.

Differentiation of respiratory syncytial virus subgroups with cDNA probes in a nucleic acid hybridization assay

A new approach to respiratory syncytial (RS) virus subgroup determination was developed by using a simple nucleic acid filter hybridization technique. By this method, virus-infected cells are bound and fixed in a single step, and the viral RNA in the fixed-cell preparation is characterized directly by its ability to hybridize to cDNA probes specific for either the A or B subgroups of RS virus. The subgroup-specific probes were constructed from cDNA clones that corresponded to a portion of the extracellular domain of the RS virus G protein of either a subgroup B RS virus (8/60) or a subgroup A RS virus (A2). The cDNA probes were labeled with 32P and used to analyze RS virus isolates collected over a period of three decades. Replicate templates of infected cell preparations were hybridized with either the subgroup A or B probe. The subgroup assignments of 40 viruses tested by nucleic acid hybridization were in agreement with the results of subgroup determinations based on their reactivities with monoclonal antibodies, which previously has been the only method available for determining the subgroup classification of RS virus isolates. The nucleic acid hybridization assay has the advantage of providing broad-based discrimination of the two subgroups on the basis of nucleic acid homology, irrespective of minor antigenic differences that are detected in assays in which monoclonal antibodies are used. The nucleic acid hybridization technique provides a reliable method for RS virus subgroup characterization.

Long-term survival of and plasmid stability inPseudomonas andKlebsiella species and appearance of nonculturable cells in agricultural drainage water

One year after introduction into agricultural drainage waterPseudomonas fluorescens R2f (RP4),Pseudomonas putida CYM318 (pRK2501), andKlebsiella aerogenes NCTC418 (pBR322) could be recovered on agar media. Survival of the introduced strains depended on competition with the indigenous microflora, the presence of nutrients, and the availability of air.In contrast toK. aerogenes NCTC418 (pBR322), bothPseudomonas species lost their plasmids, as indicated by the consistently lower colony counts on selective medium compared with the counts on nonselective medium. The plasmid loss did not depend on nutrient status and oxygen supply. P. fluorescens R2f cells could be detected with the immunofluorescence (IF) technique. Total cell counts determined by IF were consistently higher than corresponding colony counts. Even in samples where no colonies were recovered, R2f cells could be detected by IF. This indicated the occurrence of nonculturable R2f cells in drainage water. Homology with(32)P-labelled plasmid RP4 DNA was found in several drainage water samples that originally receivedP. fluorescens R2f (RP4), by using the cell suspension filter hybridization technique. P. putida CYM318 andK. aerogenes NCTC418 cells could also be detected in sterile drainage water samples, after nonspecific staining with fluorescein isothiocyanate. Cell counts of both strains were consistently higher than corresponding plate counts.

Underwear: Contamination by human papillomaviruses

Genital human papillomavirus-related lesions occurring in 74 patients and cellular swab samples taken from their underwear were analyzed with a filter hybridization technique (ViraPap-ViraType, Life Technologies Inc., Gaithersburg, Md.) for human papillomavirus deoxyribonucleic acid. Human papillomavirus deoxyribonucleic acid was found in 54 of 74 (72%) lesional tissues and 13 of 74 (17%) swabs from the underwear. Recurrence rates in patients with and without positive underwear swabs were 61% and 29% (p less than 0.05), respectively.

A repeated sequence probe for the C genome in Avena (Oats)

The genus Avena consists of at least 23 species composed of three ploidy levels. Cytogenetic analysis has characterised four distinct karyotypes. These are the A, B, C and D genomes. We have isolated a repeated sequence clone that can be used for the detection of the C genome in Avena by filter hybridization techniques. This clone, termed RS-1, is a genomic DNA clone containing at least one highly repeated sequence that is abundant in Avena species containing the C genome. This sequence or a related sequence is also present, but at much reduced levels, in species that do not contain the C genome. Because of its abundance and the characteristic Southern blot pattern, we have termed this clone a C genome specific clone. We have also done similar analysis of the Avena genus using a rDNA clone from wheat. The results of these experiments demonstrate that clearly definable C genome-specific markers can be identified with both probes. These molecular probes can be useful in studying the genomic relationships of Avena and can provide some clues as to the origin of the cultivated Avena species. These results can, therefore, provide breeders with directions for the efficient transfer of desirable traits of wild Avena species into commencal varieties.

An assay for quantitative nucleic acid hybridization on membrane filters

A quantitative nucleic acid hybridization assay based on a 6-mm-diameter nitrocellulose membrane filter and only 20 μl of hybridization mixture per determination is described. As a consequence of the small ratio of hybridization volume to membrane surface, the hybridization rates reached in this system are higher than those obtained at volume to surface ratios of conventional protocols, allowing even RNA at very low concentrations to complete hybridization. This advantage, together with the high reproducibility and hybrid stability obtained with the assay, increases the ability of the filter hybridization technique to analyze quantitatively minute amounts of RNA.

Human papillomavirus (HPV) DNA in CO 2-laser-generated plume of smoke and its' consequences to the surgeon

Carbon dioxide laser energy is absorbed by intracellular water but not by proteins or nucleic acids. The possibility of dispersing viral DNA during laser therapy of human papillomavirus (HPV)-containing genital infections was explored using a filter hybridization technique. Samples were taken using dacron swabs from 110 patients in nine separate treatment sessions as well as from five pre-filter canisters, four fume vacuum tubes, and from the nasopharynx, eyelids, and ears of the laser surgeon before and after laser surgery. The viral RNA probes were specific for groups of HPV types 6/11, 16/18, and 31/33/35. Human papillomavirus DNA was identified in swabs from 65 of 110 (60%) of histologically unequivocal condylomata and cervical intraepithelial neoplasias. One of the five pre-filter canisters (20%) tested was HPV DNA-positive after laser treatment of 65 patients; it contained HPV DNA type 6. The four fume vacuum tubes tested in the remaining 45 patients were HPV DNA-negative, as were the nasopharynx, eyelids, and ears of the operator. Although HPV DNA may be released during laser vaporization of genital HPV infections, contamination of the operator is unlikely provided appropriate equipment for evacuating HPV DNA-positive smoke is used.

Isolation of mesoderm-specific genes expressed in the Drosophila embryo

Tissue differentiation during embryonic development involves activation of specific genes. To isolate genes selectively expressed in mesodern and nervous system in the Drosophila embryo, we have screened a cDNA library with molecular probes enriched in specific gene sequences from both tissues. In this way, we have isolated six mesoderm-specific genes, as demonstrated by in situ hybridization to embryo sections. Two of these genes, expressed during muscle differentiation, are described here for the first time. These genes have been localized in the 17A region of the first chromosome and in the 60A region of the second chromosome, respectively. No neural-specific genes were identified using this approach, most probably because of the low sensitivity of detection methods which combine filter hybridization techniques with the use of complex probes.

Involvement of protein kinase C in the transcriptional regulation of ornithine decarboxylase gene expression by 12- O-tetradecanoylphorbol-13-acetate in T24 human bladder carcinoma cells

We have analyzed the molecular mechanisms involved in ornithine decarboxylase (ODC) induction by the tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA) in T24 cells, an easily manipulable human epithelial cell line. The addition of as low as 10 −9 m TPA to T24 cells, cultured in a serum-free medium, resulted in ODC induction, with peak ODC activity occurring at about 6 h after TPA treatment. The induction of ODC activity correlates with the steady-state levels of ODC mRNA increased by TPA in T24 cells. TPA treatment did not elicit any change in the size (2.1 kb) of ODC mRNA as determined by Northern blot analysis by hybridization to nick-translated 32P-labeled cDNA prepared from either mouse or human ODC mRNA. Using the DNA-excess filter hybridization technique, we found that increased steady-state levels of ODC mRNA after TPA treatment may be the result of enhanced accumulation of newly synthesized ODC mRNA. The magnitude of the induction of ODC activity was proportional to the amount of ODC mRNA synthesis caused by TPA. In a pulse-chase experiment, we failed to detect any difference in the half-life of ODC mRNA in T24 cells after treatment with either TPA or the vehicle ethanol; the half-life of ODC mRNA was about 6 h in both cases. Furthermore, as determined by the “nuclear runoff transcription assay,” the rate of transcription of ODC-gene was increased by treatment of T24 cells with TPA. These results provide direct evidence of the role of transcription-initiation in ODC-gene expression. The examination of the role of protein kinase C in ODC-gene transcription revealed that TPA or diacylglycerol did not induce the synthesis of ODC mRNA in PKC-deficient T24 cells. Taken together these results indicate that the TPA-increased synthesis of steady-state levels of ODC mRNA in T24 cells may be mediated by protein kinase C and is regulated at the transcriptional level.

The use of filter hybridization techniques for the identification, differentiation and classification of plant viruses

In attempts to use dot-blot hybridization tests for the identification of viruses or for assigning them to a certain taxonomic group we found that hybridization signals may be given not only by the homologous virus, but also by heterologous viruses belonging to the same or different taxonomic groups. Possible reasons for this phenomenon, which was observed with uncloned as well as with cloned cDNAs, are discussed. Quantitative dot-blot hybridization tests with extracted viral RNAs proved to be very sensitive in differentiating closely related viruses which were barely distinguishable in serological tests. Estimates on the degree of homology between the RNAs of different viruses may be influenced by a number of experimental parameters, such as competition for the available cDNA between homologous and heterologous RNAs or homologous RNAs in different concentrations on the same sheet of nitrocellulose, saturation phenomena due to close packaging of highly concentrated RNA on the blot and, of course, stringency conditions during washing procedures. Taking these parameters into account we have reestimated the degree of homology between the RNAs of 5 tombusviruses. Our new data suggest that the order of sequence for the relationships among these 5 tombusviruses is similar to that proposed by Koenig and Gibbs (1986) on the basis of serological data.

Detection of specific types of human papillomavirus in cervical scrapes, anal scrapes, and anogenital biopsies by DNA hybridization.

Specific varieties of human papillomavirus (HPV) infecting the anogenital region were detected in clinical samples by use of a filter hybridization technique suitable for rapid screening of cervical and anal scrapes. In this way possibly benign types (HPV6 and HPV11) could be differentiated from types thought to be capable of malignant transformation (HPV 16 and HPV 18). Cervical or anal canal cells were applied directly to nylon filters and fixed by u.v. irradiation before hybridization with mixed viral DNA probes under both low- and high-stringency conditions. In addition, probe for the human Alu-repeated DNA sequence was used to assess the relative amount of total nucleic acids in each sample applied to the filter. HPV DNA was detected in 3 of 19 cervical scrapes from patients with no past or present history of wart virus infection or cervical dysplasia. Within a positive study group totalling 71 patients, HPV (6/11 or 16/18) was detected in cervical scrapes from 24% of 41 patients who did not have visible genital dysplasia, 30% of 27 patients with visible genital dysplasia or cervical intraepithelial neoplasia (CIN) I, and in 1 of 3 patients with past CIN II/III. In addition, HPV6/11 or 16/18 DNA was detected in anal scrapes from 3 of 6 male patients and in 85% of genital biopsies. A notably high proportion (4/6) of vaginal condylomata were positive with both the HPV6/11 and the HPV16/18 mixed viral DNA probes. Of the biopsies prepared for histopathology and positive for HPV DNA, the HPV group-specific antigen could be detected in only 60%.

A survey of human cancers for human papillomavirus DNA by filter hybridization.

We examined 217 tissue samples of various human malignancies for the presence of human papillomavirus (HPV) DNA using low-stringency filter hybridization techniques. These techniques were sufficiently sensitive for crosshybridization of the HPV DNA probes to all the known types of papillomavirus DNAs, both human and animal. Approximately 2% of the cancers analyzed contained HPV DNA. These included carcinomas of the lung, cecum, tongue, and neck. Three of four cancers contained HPV-16-related nucleotide sequences. Thus, in addition to previous data demonstrating the association of HPV DNA with certain cancers of the skin and genital tract, data is presented that indicates that several additional human cancers also contain HPV-related nucleotide sequences.

Cross hybridization between organelle RNAs and mitochondrial and chloroplast genomes in Brassica

Mitochondrial and chloroplast RNAs from male fertile and cytoplasmically male sterile Brassica napus cultivars were labelled by in organello uptake and incorporation of 32P-UTP. Filter hybridization techniques indicated substantial sequence homology between mitochondrial transcripts and chloroplast DNA restriction fragments. The converse was also found, i.e., hybridization of chloroplast transcripts to mitochondrial DNA restriction fragments, although to a lesser degree. The data suggest that the large number of homologous sequences present in mitochondrial and chloroplast genomes remain transcriptionally active after transfer and integration into the other organellar genome.