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Southern Hybridization (southern + hybridization)
Terms modified by Southern Hybridization Selected AbstractsAmplification and overexpression of prosaposin in prostate cancerGENES, CHROMOSOMES AND CANCER, Issue 4 2005Shahriar Koochekpour We identified prosaposin (PSAP) as a secreted protein expressed in androgen-independent (AI) prostate cancer cells by cloning/sequencing, after probing a PC-3 cDNA library expressed in the ,TriplEx phagemid expression vector with a polyclonal rabbit antibody generated against pooled human seminal plasma. PSAP is a neurotrophic molecule; its deficiency or inactivation has proved to be lethal in man and mice, and in mice, it leads to abnormal development and atrophy of the prostate gland, despite normal testosterone levels. We used Southern hybridization, quantitative real-time polymerase chain reaction, and/or single nucleotide polymorphism (SNP) array analysis, and we now report the genomic amplification of PSAP in the metastatic AI prostate cancer cell lines, PC-3, DU-145, MDA-PCa 2b, M-12, and NCI-H660. In addition, by using SNP arrays and a set of 25 punch biopsy samples of human prostate cancer xenografts (LAPC3, LuCaP 23.1, 35, 49, 58, 73, 77, 81, 86.2, 92.1, 93, 96, 105, and 115), lymph nodes, and visceral-organ metastases, we detected amplification of the PSAP locus (10q22.1) in LuCaP 58 and 96 xenografts and two lymph node metastases. In addition, AI metastatic prostate cancer cell lines C4-2B and IA8-ARCaP over-expressed PSAP mRNA without evidence of genomic amplification. Taken together with prior data that demonstrated the growth-, migration-, and invasion-promoting activities, the activation of multiple signal transduction pathways, and the antiapoptotic effect of PSAP (or one of its active domains, saposin C) in prostate cancer cells, our current observation of PSAP amplification or overexpression in prostate cancer suggests, for the first time, a role for this molecule in the process of carcinogenesis or cancer progression in the prostate. © 2005 Wiley-Liss, Inc. [source] A deletion/insertion mutation in the BRCA2 gene in a breast cancer family: A possible role of the Alu -polyA tail in the evolution of the deletionGENES, CHROMOSOMES AND CANCER, Issue 1 2001Tieling Wang Patients with breast and/or ovarian cancer were screened for gross rearrangements in the BRCA2 gene by Southern hybridization, with exon 10 and a fragment of exon 11 used as probes. One breast cancer patient with a positive family history had a 6.2-kb deletion including exons 12 and 13. The deletion breakpoint in intron 11 was in the 3, polyA tail of an Alu element, where a track of ,60 adenine nucleotide residues was inserted. Expansion of the Alu -polyA tail may have resulted from polymerase slippage during replication, representing a novel mechanism in which Alu elements mediate deletion/insertion mutations. © 2001 Wiley-Liss, Inc. [source] Cloning and characterization of a 70 kDa heat shock cognate gene (HSC70) from two species of ChironomusINSECT MOLECULAR BIOLOGY, Issue 1 2003N. K. Karouna-Renier Abstract In the present study we carried out the isolation and characterization of an HSC70 gene from two midges, Chironomus tentans and C. yoshimatsui. The HSC70 cDNAs are approximately 2424 (C. tentans) and 2464 bp (C. yoshimatsui) long, and contain 1950 and 1956 bp open reading frames, respectively. Analysis of genomic DNA revealed the presence of two introns in these genes. The 5, untranslated regions of the HSC70 genes are adenosine-rich, a feature found in inducible HSP70 genes. The nucleotide and amino acid sequences exhibit high identity with cytosolic HSC70s from other Dipterans. Northern hybridization indicated that HSC70 is expressed at all developmental stages, from embryo to adult, and Southern hybridization confirmed the presence of multiple HSP70 genes in Chironomus. [source] Germ-line transformation of the South American malaria vector, Anopheles albimanus, with a piggyBac/EGFP transposon vector is routine and highly efficientINSECT MOLECULAR BIOLOGY, Issue 4 2002O. P. Perera Abstract Stable and efficient germ-line transformation was achieved in the South American malaria vector, Anopheles albimanus, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Transgenic mosquitoes were identified from four independent experiments at frequencies ranging from 20 to 43% per fertile G0. Fluorescence was observable throughout the body of larvae and pupae, and abdominal segments of adults. Transgenic lines analysed by Southern hybridization had one to six germ-line integrations, with most lines having three or more integrations. Hybridized transposon vector fragments and insertion site sequences were consistent with precise piggyBac -mediated integrations, although this was not verified for all lines. The piggyBac/PUbnlsEGFP vector appears to be a robust transformation system for this anopheline species, in contrast to the use of a piggyBac vector in An. gambiae. Further tests are needed to determine if differences in anopheline transformation efficiency are due to the marker systems or to organismal or cellular factors specific to the species. [source] A case of mucosal leishmaniasis: beneficial usage of polymerase chain reaction for diagnosisINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 12 2001Hironori Onuma MD A 36-year-old woman, who had emigrated from Japan to Paraguay as a 4-year-old child before returning to Japan in 1991, visited our clinic on November 10, 1997. She had suffered from a persistent ulcer on her forearm as a 6-year-old child and received intravenous injections for a few months, although she did not remember the details of therapy. Since May 1997, she had been aware of redness and swelling on her nose and had been treated with topical corticosteroid, but no improvement had been noted. Physical examination revealed erythematous plaque with crust from the left internal naris to nasolabial region (Fig. 1a). The atrophic plaque that had resulted from prolonged ulceration was found on the right forearm (Fig. 1b). In a biopsy specimen from the erythematous plaque on the nasolabial region, mononuclear dermal infiltrate, consisting of lymphocytes and histiocytes, was seen (Fig. 2a). The histiocytes were filled with Leishman-Donovan (L-D) bodies on a Giemsa staining sample (Fig. 2b). Fiberscopic examination revealed white plaque in the pharynx. The biopsy from the affected mucosa showed the same histopathological finding as with the skin. Figure 1. (a) Erythematous plaque with crust from the left internal naris to nasolabial region. (b) Atrophic plaque on the right forearm Figure 2. (a) In the biopsy specimen from the erythematous plaque on the nasolabial region, a mononuclear dermal infiltrate consisting of lymphocytes and histiocytes was seen. (Hematoxylin-Eosin stain, × 100) (b) The histiocytes were filled with Leishman-Donovan bodies. (Giemsa staining, × 400) Total DNA was purified from the skin biopsy specimen for polymerase chain reaction (PCR) analysis using a specific primer for L (V) braziliensis.1,2 A 70-bp product was amplified (Fig. 3a); furthermore, the specificity of the PCR product was confirmed by Southern hybridization with the probe for L (V) braziliensis (Fig. 3b) and DNA sequence analysis (data not shown). From December 2, 1997, the patient received 20 mg/kg/day sodium stibogluconate (PentostamTM) intravenously for 20 days. After 5 days of treatment, the redness and swelling of the skin lesion was improved, and faint erythema remained at the end of 20 days' treatment. After a 2-week interval, since the erythema remained, another 20-day treatment was performed. All of the skin lesion became scar tissue and L-D bodies could not be found in a skin biopsy specimen. However, L-D bodies were still found in a biopsy from the pharyngeal mucosa that had a normal appearance. Though another additional treatment was planned, the patient refused it. Figure 3. (a) The results of PCR. 70-bps bands appear in lanes 2 and 6. Lane 1, a size marker (pUC19/HapII); lane 2, DNA extracted from the formalin-fixed patient's sample; lane 3, DNA extracted from a formalin-fixed control sample; lane 4, DNA (,); lane 5, DNA extracted from L (V) tropica; lane 6, DNA extracted from L (V) braziliensis. (b) Results of Southern blotting using the PCR products. The PCR products were transferred from agarose gel as shown in Fig. 3 (a). Specific probes were hybridized with 70-bps bands on lanes 2 and 6 [source] Molecular characterization of polyphosphate (PolyP) operon from Serratia marcescensJOURNAL OF BASIC MICROBIOLOGY, Issue 2 2006Seung-Jin Lee The polyphosphate (polyP) operon was cloned from a genomic library of Serratia marcescens KCTC 2172 by Southern hybridization using E. coli ppk gene as a probe. The polyP operon was composed of a polyphosphate promoter, polyphosphate kinase (ppk ) and exopolyphosphatase (ppx ). A potential CRP binding site and pho box sequence were found in the region upstream of the putative promoter in the regulatory region. The ppk gene comprises 2,063 nucleotides and encodes 686 amino acids yielding a protein with a molecular mass of 70 kDa. The ppx gene contains 1611 nucleotides and encodes 536 amino acids with a molecular 58 kDa. An E. coli strain transformed with the ppk gene had a 16-fold increased in polyphosphate kinase activity, while introduction of the ppx gene produced a 25-fold increase in polyphosphatase activity. E. coli strains transformed with ppk and ppx genes also displayed increased accumulation of polyphosphate. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Association of viral factors with non-familial breast cancer in Taiwan by comparison with non-cancerous, fibroadenoma, and thyroid tumor tissues,JOURNAL OF MEDICAL VIROLOGY, Issue 2 2005Ju-Hsin Tsai Abstract To study the etiologic factors of non-familial breast cancer, the polymerase chain reaction (PCR) and Southern hybridization were used to detect six viruses including human papillomavirus (HPV), cytomegalovirus (CMV), Epstein,Barr virus (EBV), herpes simplex virus (HSV)-1, HSV-2, and human herpesvirus (HHV)-8 DNA in 69 patients with breast cancer and 60 specimens from non-cancerous or other individuals with thyroid tumors or fibroadenoma (non-breast cancer controls). Two specimens from patients with a familial history of breast cancer and five breast cancer specimens with negative results for ,-globin, which was used as internal control, were excluded from this study. Eight (12.9%) HSV-1, 28 (45.2%) EBV, 47 (75.8%) CMV, 8 (12.9%) HPV, and 28 (45.2%) HHV-8 positive samples out of the 62 breast cancer specimens were detected; no HSV-2 DNA was detected in any group. Among the viral gene-positive breast cancer samples, 12 (23.1%) were positive for 1 virus, 16 (30.8%) were positive for 2 viruses, 21 (40.4%) were positive for 3 viruses, and 3 (5.8%) were positive for 4 viruses. Among the viral gene-positive specimens of the control groups, only one virus, CMV, was found in the non-cancerous and thyroid tumor specimens, while multiple viruses were found in the fibroadenoma specimens. The viruses associated with breast cancer were HHV-8,>,EBV (P,<,0.01). The viruses associated with fibroadenoma were HSV-1 and HHV-8,>,EBV (P,<,0.01). The presence of more than one virus was found predominantly in breast cancer and exclusively found in fibroadenoma. CMV was the only virus associated with thyroid tumors. J. Med. Virol. 75:276,281, 2005. © 2004 Wiley-Liss, Inc. [source] Lack of susceptibility of Chacma baboons (Papio ursinus orientalis) to hepatitis C virus infectionJOURNAL OF MEDICAL VIROLOGY, Issue 4 2002N.P. Sithebe Abstract The main reason to ascertain whether baboons are susceptible to infection with hepatitis C virus (HCV) is the need to replace chimpanzees, which are endangered, as an animal model for undertaking research into the biology and host,virus interactions of HCV, and for developing a vaccine against this virus. A second reason is that baboons are a possible source of xenografts for human liver transplantation. We inoculated serum containing HCV into four Chacma baboons and monitored them for 52 weeks for evidence of infection. Serum was tested for antibody to HCV, HCV RNA, and aminotransferase concentrations at 2-week intervals for 26 weeks and thereafter at 4-week intervals. Liver tissue was examined at 28 and 52 weeks for histopathological changes and viral RNA, and at 52 weeks for viral particles using electron microscopy. Reverse transcription-polymerase chain reaction assay was used to detect HCV RNA, and the results were confirmed by Southern hybridization. Serum aminotransferase concentrations remained within the normal range and liver histology was normal during the follow-up period. Passive transmission of anti-HCV to the baboons was observed during the first 4 weeks. HCV RNA was not detectable in any serum or liver sample and electron microscopy failed to reveal viral particles in liver tissue. In conclusion, we did not find Chacma baboons to be susceptible to infection with HCV, although we cannot deny that in an immunosuppressed liver transplant recipient, infection of a baboon xenograft might occur. Another animal model for HCV infection must be sought. J. Med. Virol. 66:468,471, 2002. © 2002 Wiley-Liss, Inc. [source] The Yersinia HPI is present in Serratia liquefaciens isolated from meatLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2003C. Olsson Abstract Aims: The aim of the study was to screen the Enterobacteriaceae flora of meat for the presence of bacteria harbouring the Yersinia high-pathogenicity island (HPI). Methods and Results: Bacteria from 29 meat and 29 liver samples were isolated on violet,red bile glucose agar. A total of 197 isolates were screened for the presence of the irp2 gene, encoded within the HPI, by PCR. One isolate that was positive for irp2 gene was also positive for the fyuA, irp1, ybtP/ybtQ, ybtX/ybtS and int/asn tRNA genes by PCR. The presence of fyuA, irp1 and irp2 genes was confirmed by Southern hybridization. Conclusions: The isolate was identified as Serratia liquefaciens by sequencing of the 16S rRNA gene and by ribotyping. Significance and Impact of the Study: This is the first report of a Serratia harbouring the Yersinia HPI. Serratia is a frequently occurring Enterobacteriaceae genus in chill-stored meat. [source] Rapid detection of Haemophilus influenzae by hel gene polymerase chain reactionLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2003M.C. Yadav Abstract Aims: To evaluate the efficiency of hel gene polymerase chain reaction (PCR) to detect Haemophilus influenzae in various clinical/non-clinical samples. Methods and Results: Seventy-four clinical samples (cerebrospinal fluid, blood, sputum, throat and nasal swabs) and throat swabs of 17 asymptomatic carriers were collected. Primers were used to amplify the hel gene of H. influenzae encoding P4 outer membrane protein directly from the processed samples. The samples were also examined by conventional culture methods and the results were compared with those of PCR. The culture methods showed positive results in 60 (65·9%) of 91 samples in contrast to 62 (68·12%) samples tested positive by PCR. None of the culture-positive samples were PCR-negative while two of the culture-negative samples were PCR-positive. The specificity of the products was confirmed by Southern hybridization and failure of various other organisms to amplify the hel gene product. The sensitivity of the PCR assay was found to be 50 pg of DNA. Conclusions: These findings suggest that the hel gene PCR is a rapid, sensitive and a specific new method for direct identification of H. influenzae. Significance and Impact of the study: Thus, this PCR test can improve the detection rate of H. influenzae in suspected clinical samples as compared with that of conventional culture methods. [source] Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticityMOLECULAR MICROBIOLOGY, Issue 6 2000R. M. Warren Southern hybridization was used in combination with IS6110 insertion-locus-specific probes in a comparative study to determine the structure of chromosomal domains flanking IS6110 elements in clinical isolates of Mycobacterium tuberculosis. The resulting restriction fragment length polymorphism (RFLP) data demonstrated three mutational mechanisms responsible for the polymorphisms observed: IS6110 insertion, chromosomal mutation and deletion. The frequency of IS6110 insertion within many of the chromosomal regions demonstrates that preferential integration regions are common in M. tuberculosis. Mapping the IS6110 insertion positions and chromosomal deletions in relation to the M. tuberculosis H37Rv and M. bovis BCG genome sequences reveals numerous disruptions of predicted open reading frames (ORFs). A phylogenetic tree, based on the mutational data, showed a number of independently evolving lineages of M. tuberculosis, while analysis of the mutational events occurring at each branch point suggests both divergent and convergent evolution. A significant positive correlation was demonstrated between the mutation rate and the frequency of occurrence of different isolates in families of strains, suggesting that evolution may impact on strain ,fitness' or that strain proliferation may increase the chance of mutation. We conclude that the genome of clinical isolates of M. tuberculosis continues to evolve. [source] cDNA characterization and expression analysis of two arylphorin-like hexameric protein genes from the diamondback moth, Plutella xylostella (L.)ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2007Muhammad Ashfaq Abstract We cloned and characterized two hexameric storage protein genes, PxAry1 and PxAry2, from Plutella xylostella and investigated the expression pattern in different developmental stages and in response to treatment by a juvenile hormone (JH) analog. The complete coding sequences of PxAry1 and PxAry2 are comprised of 2,097 and 2,094 bp with 699 and 698 amino acid residues, respectively. Signal peptides of 16 amino acids are predicted at the N-termini. According to both the phylogenetic analysis and amino acid composition (>16% aromatic amino acids), PxAry1 and PxAry2 belong to the arylphorin-like protein genes. Analysis using Northern hybridization and RT-PCR showed varying levels of genes expression in the developmental stages with a small difference between sexes. Expression of both genes in fourth instar larvae was suppressed after treatment with a JH-analog. Southern hybridization revealed the presence of multiple arylphorin genes in the genome. Arch. Insect Biochem. Physiol. 64:175,185, 2007. © 2007 Wiley-Liss, Inc. [source] Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentationBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2005Ying Zhu Abstract Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta -deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose. © 2005 Wiley Periodicals, Inc. [source] Germ line transformation of the yellow fever mosquito, Aedes aegypti, mediated by transpositional insertion of a piggyBac vectorINSECT MOLECULAR BIOLOGY, Issue 2 2002N. F. Lobo Mosquito-vectored diseases such as yellow fever and dengue fever continue to have a substantial impact on human populations world-wide. Novel strategies for control of these mosquito vectored diseases can arise through the development of reliable systems for genetic manipulation of the insect vector. A piggyBac vector marked with the Drosophila melanogaster cinnabar (cn) gene was used to transform the white-eyed khw strain of Aedes aegypti. Microinjection of preblastoderm embryos resulted in four families of cinnabar transformed insects. An overall transformation frequency of 4%, with a range of 0% to as high as 13% for individual experiments, was achieved when using a heat-shock induced transposase providing helper plasmid. Southern hybridizations indicated multiple insertion events in three of four transgenic lines, while the presence of duplicated target TTAA sites at either ends of individual insertions confirmed characteristic piggyBac transposition events in these three transgenic lines. The transgenic phenotype has remained stable for more than twenty generations. The transformations effected using the piggyBac element establish the potential of this element as a germ-line transformation vector for Aedine mosquitoes. [source] Rapid assessment of the sex of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) eggs and larvaeJOURNAL OF APPLIED ENTOMOLOGY, Issue 4 2009I. Fuková Abstract Two different methods were tested to identify the sex of the early developmental stages of the codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) with a WZ/ZZ (female/male) sex chromosome system. First, it was shown that the sex of all larval stages can be easily determined by the presence or absence of sex chromatin, which is formed by the female-specific W chromosome in interphase nuclei. This trait can also be used to identify the sex of newly hatched larvae but it does require care and accuracy. Secondly, a new sexing technique was developed based on a molecular marker of the codling moth W chromosome. Flanking regions of an earlier described W-specific sequence (CpW2) were isolated and sequenced and a 2.74 kb sequence (CpW2- EcoRI), specific for the W chromosome, was obtained. Several PCR tests were conducted, which confirmed that the CpW2- EcoRI sequence is a reliable marker for the sex identification in codling moth samples of different geographical origin. In addition, a fragment of a codling moth gene, period (Cpper) was isolated and sequenced. Results of southern hybridization of the Cpper probe with female and male genomic DNA suggested that the Cpper gene is located on the Z chromosome. Then a multiplex PCR assay was developed, which co-amplified the CpW2- EcoRI sequence to identify the W chromosome and the Z-linked Cpper sequence, which served as a positive control of accurate processing of tested samples. The multiplex PCR provides an easy and rapid identification of the sex of embryos and early larval instars of the codling moth. [source] | |||||||||||||