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Southern Blotting (southern + blotting)
Selected AbstractsForensic aspects of DNA-based human identity testingJOURNAL OF FORENSIC NURSING, Issue 4 2008Stephen M. Roper MS Abstract The forensic applications of DNA-based human identity laboratory testing are often underappreciated. Molecular biology has seen an exponential improvement in the accuracy and statistical power provided by identity testing in the past decade. This technology, dependent upon an individual's unique DNA sequence, has cemented the use of DNA technology in the forensic laboratory. This paper will discuss the state of modern DNA-based identity testing, describe the technology used to perform this testing, and describe its use as it relates to forensic applications. We will also compare individual technologies, including polymerase chain reaction (PCR) and Southern Blotting, that are used to detect the molecular differences that make all individuals unique. An increasing reliance on DNA-based identity testing dictates that healthcare providers develop an understanding of the background, techniques, and guiding principles of this important forensic tool. [source] Coexistence of Unverricht-Lundborg disease and congenital deafness: Molecular resolution of a complex comorbidityEPILEPSIA, Issue 6 2009Miljana Kecmanovi Summary Purpose:, We report on genetic analysis of a complex condition in a Serbian family of four siblings, wherein two had progressive myoclonic epilepsy (PME) and congenital deafness (CD), one had isolated congenital deafness (ICD), and one was healthy. Methods and Results:, Molecular diagnosis performed by Southern blotting confirmed Unverricht-Lundborg disease in the available sibling with PME/CD. In the sibling with ICD (heterozygote for expansion mutation in CSTB) we demonstrated recombination event between the D21S2040 marker and the CSTB gene and identified c.207delC (p.T70Xfs) mutation in the fourth exon of the transmembrane protease, serine-3 (TMPRSS3) gene (maps in close proximity to CSTB), responsible for nonsyndromic deafness in the sibling with PME/CD as well. Discussion:, To the best of our knowledge this is the first genetic confirmation of the coexistence of these two mutations. [source] ALK-positive anaplastic large-cell lymphoma: strong T and B anti-tumour responses may cause hypocellular aspects of lymph nodes mimicking inflammatory lesionsEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2003B. Borisch Abstract: The anaplastic large cell lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) is a rare type of non-Hodgkin lymphoma which occurs in children mostly. The ALK protein is highly immunogenic and elicits both humoral and cellular immune responses. A 15-yr-old child presented with fever and adenopathy and did not respond to antibiotics. Biopsy of the enlarged lymph node contained almost no lymphoid element except for a few CD8-positive T cells, plasma cells and isolated CD30-positive blasts. The patient's condition improved following lymphadenectomy but relapse occurred 3 months later with multiple nodes, high fever and an abdominal mass. This time an ALK-positive ALCL was diagnosed and the retrospective analysis of the initial biopsy revealed rare, isolated ALK+ cells. Molecular analysis showed T-cell clones and oligoclonal B cells in both biopsies and peripheral blood of the patient. The tumour cells harbour a t(2;5) translocation, revealing a null phenotype by immunohistochemistry and no evidence for T-cell clonality by Southern blotting. The patient's serum contained anti-ALK antibodies. Our findings suggest that the T-cell clones and anti-ALK antibodies in this patient constitute an anti-tumour response that caused the hypocellularity of the initial lymph node. Hypocellular and oedematous lymph nodes occurring in a child with evocative symptoms should be tested for the presence of ALK. [source] Comparative analysis of MLL partial tandem duplication and FLT3 internal tandem duplication mutations in 956 adult patients with acute myeloid leukemiaGENES, CHROMOSOMES AND CANCER, Issue 3 2003Christine Steudel Partial tandem duplication (PTD) of the MLL gene and internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor tyrosine kinase gene have been described in acute myeloid leukemia (AML) patients, preferentially in those with normal cytogenetics. These alterations have been associated with a poor prognosis. In our study, we analyzed the prevalence and the potential prognostic impact of these aberrations in a large unselected and well-defined cohort of 956 patients with AML. Results were correlated with cytogenetic data and clinical outcome. MLL PTD was detected by RT-PCR, subsequent nucleotide sequencing, and Southern blotting. The overall incidence was found to be 5.0% (48/956), whereas FLT3 ITD was detected in 19.2% (184/956). Sixteen cases were positive for both alterations. The rate of MLL PTD in FLT3 ITD positive patients was significantly higher than that in FLT3 ITD negative patients [16/184 (8.7%); 32/772 (4.1%); P = 0.025]. However, both aberrations were highly increased in patients with normal karyotype (MLL PTD 35/431, P = 0.004; FLT3 ITD 132/334, P < 0.001). When restricted to this subgroup, the rate of MLL PTD in patients with FLT3 mutations was not significantly increased. No statistically significant differences were detected between patients positive for MLL PTD and patients negative for MLL PTD in the rate of complete remissions or the overall survival, although we did see a significantly shorter disease-free survival in patients age 60 or younger. In conclusion, although there is an overlap in the mutational spectrum in AML with FLT3 ITD and MLL PTD mutations, our data do not support a common mechanistic basis. Although associated with inferior disease-free survival, the results of this study do not unequivocally support the notion that MLL PTD mutations represent an independent prognostic factor. © 2003 Wiley-Liss, Inc. [source] Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting,HUMAN MUTATION, Issue 10 2008Tomasz K. Wojdacz Abstract Beckwith Wiedemann syndrome (BWS) and Russell Silver syndrome (RS) are growth disorders with opposing epimutations affecting the H19/IGF2 imprinting center at 11p15.5. Overgrowth and tumor risk in BWS is caused by aberrant expression of the paternally expressed, imprinted IGF2 gene, occurring as a consequence of mosaic hypermethylation within the imprinting center, or to mosaic paternal uniparental disomy (UPD). RS is characterized by severe intrauterine growth retardation (IUGR). A subset of RS cases were recently shown to have mosaic hypomethylation within the H19/IGF2 imprinting center, predicted to silence paternally expressed IGF2 in early development. Molecular diagnosis for BWS and RS involves methylation analysis of the H19 locus, enabling discrimination of allelic methylation patterns. In this study, methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyze methylation within the intergenic region of the H19 locus. A total of 36 samples comprising normal control (11), BWS (19), and RS (six) DNA were analyzed in a blinded study and scored as hypermethylated, normal, or hypomethylated. Results were compared with those derived by methylation-sensitive Southern blotting using the restriction enzymes Rsa I and Hpa II. A total of 100% concordance was obtained for the Southern blotting and MS-HRM scores. A total of three samples with paternal duplication affecting the H19/IGF2 region were scored as equivocal by both methods; however, 33 out of 36 (92%) the samples were unambiguously scored as being hypermethylated, hypomethylated, or normally methylated using MS-HRM. We conclude that MS-HRM is a rapid, cost-effective, and sensitive method for screening mosaic methylation changes at the H19 locus in BWS and RS. Hum Mutat 0,1,6, 2008. © 2008 Wiley-Liss, Inc. [source] Hereditary angioedema: The mutation spectrum of SERPING1/C1NH in a large Spanish cohort,HUMAN MUTATION, Issue 2 2005Olga Roche Abstract Hereditary angioedema (HAE) is a disease caused by defects in the C1 inhibitor gene (SERPING1/C1NH). We screened the entire C1NH gene for mutations in a large series of 87 Spanish families (77 with type I, and 10 with type II HAE) by SSCP, sequencing, Southern blotting, and quantitative multiplex PCR of short fluorescent fragments (QMPSF), and we characterized several defects at the mRNA level. We found large rearrangements in 13 families, and point mutations or microdeletions/insertions in 74 families. The 13 large rearrangements included nine exon deletions, of which at least eight were distinct, two were distinct exon duplications, and two were rearrangements whose precise nature could not be determined. We confirmed that exon 4 is particularly prone to rearrangements. Thirty-six mutations were unreported, and included 10 microdeletions/insertions, 10 missense, five nonsense, eight splicing, and three splicing or missense mutations. Moreover, we detected six novel uncharacterized sequence variants (USV). RT-PCR studies showed that in addition to several intronic splice site mutations tested, the exonic mutations c.882C>G and c.884T>G, located near the 3, end of exon 5, also produced exon skipping. This is the first evidence of SERPING1/C1NH mutations in coding regions that differ from the canonical splice sites that affect splicing, which suggests the presence of an exonic splicing enhancer (ESE) in exon 5. Hum Mutat 26(2), 1,10, 2005. © 2005 Wiley-Liss, Inc. [source] Extranodal NK/T-cell lymphoma, nasal type, presenting after 5 years of remissionINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 3 2008Tomonobu Ito MD A 76-year-old woman with multiple edematous erythemas, erosions, and ulcers on the breast and abdomen was admitted to our hospital in June 2005. She had developed granulomatous bleeding lesions in the right nostril 6 years prior to her visit to our dermatology unit. She had been observed at the otorhinolaryngology department of our hospital, and a biopsy was taken from the nasal lesion. Computerized tomography and gallium scintigraphy (67Ga single-photon emission computed tomography) did not reveal any lesions corresponding to the diagnosis of malignant lymphoma. The histologic examination of the nasal specimen rendered a diagnosis of natural killer (NK)/T-cell lymphoma, nasal. Because imaging analysis indicated a small-sized tumor without metastases, oral prednisolone at 20 mg/day was administered for 1 month. The tumor decreased in size and disappeared after 19 months of low-dose steroid therapy. ,Five years after the initial treatment, the patient developed a fever of 38 °C with infiltrated erythemas and erosions on her breast. Erysipelas was initially suspected, but the antimicrobial agent did not show any effect and the multiple infiltrated erythemas and ulcers spread throughout her chest and abdomen (Fig. 1). The lymph nodes were not palpable. The right nasal cavity showed no granulomatous lesions or other signs of abnormality. The peripheral white blood cell count (3000/µL), red blood cell count (3.54 × 106/µL), and platelet count (112 × 103/µL) were reduced. Atypical lymphocytes were not observed. The serum lactic dehydrogenase (LDH; 1770 U/L; normal, 224,454 U/L), aspartate aminotransferase (AST; 140 U/L; normal, 10,30 U/L), and alanine aminotransferase (ALT; 57 U/L; normal, 3,29 U/L) levels were elevated. The soluble interleukin-2 (IL-2) receptor level was high (25,300 U/mL; normal, 167,497 U/mL). Epstein,Barr virus (EBV) serologic examination showed the immunoglobulin G (IgG) viral capsid antigen (VCA) at 1 : 320 and the EBV nuclear antigen (EBNA) at 1 : 40. IgM VCA and EBV early antigen-diffuse restricted antibody (EA) IgA and IgG were not detectable. Histologic findings from the left chest skin showed a distribution of atypical lymphocytes from the upper dermis to the subcutaneous tissue, and many foamy cells which had phagocytosed the hemocytes (Fig. 2a,b). Immunohistochemical analysis showed that the atypical lymphocytes were sCD3,, CD4,, CD8,, CD20,, CD56+, granzyme B+, and T-cell intracellular antigen (TIA-1) positive. Furthermore, EBV-encoded small RNAs (EBER), detected by in situ hybridization, exhibited a strong signal. The nasal lesions biopsied 6 years previously showed an identical staining pattern with the skin lesions immunohistochemically. Analysis of the T-cell receptor-, (TCR-,), TCR-,, and TCR-, gene did not reveal any clonal rearrangements, but the EBV gene was detected from the skin specimens by Southern blotting. Our patient's condition was diagnosed as a case of extranodal NK/T-cell lymphoma, nasal type, but the patient had concomitantly developed hemophagocytic syndrome (HPS). She was treated with a combination of steroid pulse therapy and chemotherapy (pirarubicin hydrochloride 30 mg/m2, cyclophosphamide 500 mg/m2, vincristine 1 mg/m2, prednisolone 30 mg/m2, etoposide 80 mg/m2). After the first session of chemotherapy, the lesions on the chest and abdomen diminished, but, 2 weeks later, the skin lesions recurred, and disseminated intravascular coagulation (DIC) induced by HPS supervened. The patient died as a result of multiple organ failure induced by HPS. Figure 1. Multiple infiltrated erythemas, erosions, and ulcers on the breast and abdomen Figure 2. Histologic findings of a skin biopsy specimen from the left chest (hematoxylin and eosin staining). (a) Dense infiltration of atypical lymphocytes from the upper dermis to the subcutaneous tissue (×40). (b) Many foamy cells had phagocytosed the hemocytes (×400) [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] Oxalate-degrading Providencia rettgeri isolated from human stoolsINTERNATIONAL JOURNAL OF UROLOGY, Issue 6 2005SANEHIRO HOKAMA Abstract Background:, Oxalate-degrading bacteria are thought to metabolize intestinal oxalate and thus decrease the urinary excretion of oxalate by reducing its intestinal absorption. Methods:, We have isolated several novel oxalate-degrading bacteria from human stools. Oxalate degrading bacteria were investigated to characterize their protein profiles with antibodies against oxalyl-coenzyme A decarboxylase (65 kDa) and formyl-coenzyme A transferase (48 kDa) purified from Oxalobacter formigenes. Results:, One of these isolates was identified as Providencia rettgeri, which showed two proteins (65 kDa and 48 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) that were not found in non-oxalate-degrading P. rettgeri. Antibodies reacted with the 65 and 48 kDa proteins from the P. rettgeri strain on Western blotting. An Oxalobacter formigenes formyl-coenzyme A transferase gene probe reacted with chromosomal DNA from P. rettgeri on Southern blotting under high stringency conditions, while an Oxalobacter formigenes oxalyl-coenzyme A decarboxylase gene probe did not react under the same conditions. Conclusions:, The mechamism of oxalate degradation by P. rettgeri appears to be similar to that of Oxalobacter formigenes. This is the first report of a facultative oxalate-degrading organism that is one of the Enterobacteriaceae. [source] TLRs antiviral effect on hepatitis B virus in HepG2 cellsJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2008C. Xia Abstract Aims:, A hepatoma cell line, HepG2, was used as a model system to detect Toll-like receptor (TLR) expression in hepatocytes and examine the antiviral effect on hepatitis B virus (HBV). Methods and Results:, Toll-like receptor expression was detected in HepG2 cells by RT-PCR. The TLRs, which were strongly expressed in HepG2 cells, were stimulated with specific ligands. Interferon (IFN) response was evaluated poststimulation with Western blotting for signal transduction and activators of transcription-1. Furthermore, HepG2 cells were transiently transfected with wild-type HBV 1·3-fold over-length plasmid and treated with specific ligands at indicated times. Replication of HBV DNA, transcription of HBV RNA intermediate and expression of HBV antigens were respectively detected by Southern blotting, real time PCR, ELISA and Western blotting. Activation of different TLRs induced antiviral effects on HBV to varying degrees. Conclusions:, The TLRs, which were strongly expressed in HepG2 cells, could be stimulated with specific ligands. Activation of TLRs induced apparent production of antiviral cytokines such as IFN-,/, and inhibited HBV lifecycle in the hepatocyte cell model. Significance and Impact of the Study:, Expression of TLRs in hepatocytes may be related to local immunity of liver and participate in the outcome of viral hepatitis. [source] Gene expression profile of transgenic mouse kidney reveals pathogenesis of hepatitis B virus associated nephropathy,JOURNAL OF MEDICAL VIROLOGY, Issue 5 2006J. Ren Abstract Hepatitis B virus (HBV)-associated nephritis has been reported worldwide. Immune complex deposition has been accepted as its pathogenesis, although the association between the presence of local HBV DNA and viral antigen and the development of nephritis remains controversial. To understand better the roles played by HBV protein expression in the kidney, the global gene expression profile was studied in the kidney tissue of a lineage of HBV transgenic mouse (#59). The mice expressed HBsAg in serum, and HBsAg and HBcAg in liver and kidney, but without virus replication. Full-length HBV genome (adr subtype, C genotype) isolated from a chronic HBV carrier was used to establish the transgenic mice #59. Similarly manipulated mice that did not express HBV viral antigens served as controls. Southern blotting, hybridization with HBV probe, and immuno-histochemical staining were used to study HBV gene expression. mRNA extracted from the kidney tissue was analyzed using Affymetrix microarrays. HBsAg and HBcAg were located mainly in the cytoplasm of tubular epithelium. Altogether 520 genes were "up-regulated" more than twofold and 76 genes "down-regulated" more than twofold in the kidney. The complement activation, blood coagulation, and acute-phase response genes were markedly "up-regulated". Compared to the controls, the level of serum C3 protein was decreased in #59 mice, while the level of C3 protein from kidney extract was increased. Results indicate that expression of HBsAg and HBcAg in tubular epithelial cells of the kidney per se can up-regulate complement-mediated inflammatory gene pathways, in addition to immune complex formation. J. Med. Virol. 78:551,560, 2006. © 2006 Wiley-Liss, Inc. [source] Identification of a unique BK virus variant in the CNS of a patient with AIDS,JOURNAL OF MEDICAL VIROLOGY, Issue 1 2003Gunn Eli Kimo Jřrgensen Abstract Human polyomavirus BK (BKV; GenBank or EMBL or DDBJ accession no. NC001538) is often reactivated in immunosuppressed patients. Reactivation has been associated primarily with excretion of the virus in the urine, and there have been few reports of renal and/or neurological disease caused by BKV in patients with acquired immunodeficiency syndrome (AIDS). Polymerase chain reaction, Southern blotting, and sequencing were used to detect and identify the noncoding control region (NCCR) of BKV in different tissues in an AIDS patient with meningoencephalitis, retinitis, and nephritis. An undescribed reorganized NCCR variant of the virus, completely different from the variants detected in peripheral blood leukocytes (PBLs) and urine, was identified in the cerebrospinal fluid (CSF) and CNS tissues. These results suggest that rearrangements in the NCCR of the virus have resulted in a BKV variant, which is better adapted to the host cell machinery of the cells in CNS tissue. The rearranged variant (BKV CNS) might have been involved in the initiation and/or development of the pathological lesions observed in the CNS-related tissues of this patient. J. Med. Virol. 70: 14,19, 2003. © 2003 Wiley-Liss, Inc. [source] Richter syndrome in B-cell chronic lymphocytic leukemiaPATHOLOGY INTERNATIONAL, Issue 4 2003Naoya Nakamura Richter syndrome (RS) is well known as a secondary high-grade lymphoma, mostly diffuse large B-cell lymphoma (DLBCL) developed in patients with B-cell chronic lymphocytic leukemia (B-CLL). In this review, we describe clinicopathological, histological, immunophenotypical and genetic findings of RS. The patients with RS, regardless of transformation of pre-existing clone or de novo malignant clone, were resistant to conventional combined chemotherapy and died within months of diagnosis. Molecular techniques can provide convincing results for the clonal relationship of RS to pre-existing B-CLL. When RS carries a same rearrangement band or a same sequence as B-CLL by Southern blotting or nucleotide sequence analyses of immunoglobulin heavy and/or light chain genes, it is suggested to that RS transforms from original B-CLL. These analyses have showed that approximately two-thirds of RS cases evolved from a B-CLL clone. How and where does the B-CLL clone evolve to RS? The genetic alteration of transforming B-CLL clone into RS has been addressed. Abnormalities of chromosomes 11 and 14 were most frequently involved in RS, but non-specific. In addition, RS does not include chromosomal translocation between Ig locus and oncogenes or rearrangements of bcl-6 gene, both of which were found in some de novo DLBCL. Several candidates, such as mutation of p53 gene and abnormalities of cyclin dependent kinase inhibitor, have been proposed to play an important role in the transformation of a part of B-CLL. However, there is still uncertainly as to how B-CLL progresses or develops into RS. [source] CD19 negative precursor B acute lymphoblastic leukemia presenting with hypercalcemiaPEDIATRIC BLOOD & CANCER, Issue 1 2004Iyad Sultan MD Abstract A 9-month-old infant presented with hypercalcemia and lytic bone lesions. Suspicion for malignancy led to a bone marrow examination, which showed replacement of the marrow by a small round blue cell infiltrate. Flow cytometric analysis of these cells showed an unusual immunophenotype in that these cells were dim CD45, HLA-DR, and CD10 positive, but CD19, CD20, CD79a, and CD34 negative. Southern blotting showed clonal rearrangement of immunoglobulin heavy chain (IgH) which confirmed a diagnosis of precursor B acute lymphoblastic leukemia (ALL). He received supportive treatment with hydration and pamidronate, but had recurrent episodes of hypercalcemia. Once the correct diagnosis of ALL was established, the patient was treated with an infantile ALL chemotherapeutic regimen and the hypercalcemia resolved. This case highlights the usefulness of immunoglobulin gene rearrangement studies in atypical cases of ALL. © 2004 Wiley-Liss, Inc. [source] ,, T-cell large granular lymphocyte (LGL) leukemia with spontaneous remissionAMERICAN JOURNAL OF HEMATOLOGY, Issue 3 2004Tsutomu Shichishima Abstract T-cell large granular lymphocyte (LGL) leukemia is a clonal disorder with an indolent clinical course. In July 1995, a 46-year-old Japanese man was admitted to our hospital because his anemia had progressed. He had a white blood cell count of 3.9 × 109/L with 75% lymphocytes, which were intermediate to large and had almost round nuclei and azurophilic granules, and anemia with a red blood cell count (RBC) of 2.69 × 1012/L, hemoglobin (Hb) of 9.5 g/dL, and hematocrit (Hct) of 28.3%. Electron microscopic examination showed that most of the lymphocytes had a parallel tubular array and dense core granules in their cytoplasm. Flow cytometry and Southern blotting of the T-cell antigen receptor (TCR) genes using the peripheral blood species showed monoclonal proliferation of LGLs with a CD3+, TCR,,+, CD4,, CD8,, CD16+, CD56,, CD57,, HLA-DR+ phenotype, and a TCR , gene rearrangement, respectively, suggesting that the patient was diagnosed as having ,, T-cell LGL leukemia. He had no symptoms, organomegaly, or skin lesions. About 1.5 years after diagnosis, the anemia gradually improved with disappearance and appearance of a rearranged band in the TCR-, gene and TCR-, gene, respectively. About 7 years after diagnosis, the anemia improved completely with a RBC of 5.01 × 1012/L, Hb of 14.8 g/dL, and Hct of 44.3%, and he was in complete remission without TCR-, and -, gene rearrangements. He had received no therapy. This is the first report of spontaneous remission of ,, T-cell LGL leukemia. Am. J. Hematol. 75:168,172, 2004. © 2004 Wiley-Liss, Inc. [source] A Cre::FLP fusion protein recombines FRT or loxP sites in transgenic maize plants,PLANT BIOTECHNOLOGY JOURNAL, Issue 8 2008Vesna Djukanovic Summary The coding sequences of Cre (site-specific recombinase from bacteriophage P1) and FLP (yeast 2-µm plasmid site-specific recombinase) were fused in frame to produce a novel, dual-function, site-specific recombinase gene. Transgenic maize plants containing the Cre::FLP fusion expression vector were crossed to transgenic plants containing either the loxP or FRT excision substrate. Complete and precise excisions of chromosomal fragments flanked by the respective target sites were observed in the F1 and F2 progeny plants. The episomal DNA recombination products were frequently lost. Non-recombined FRT substrates found in the F1 plants were recovered in the F2 generation after the Cre::FLP gene segregated out. They produced the recombination products in the F3 generation when crossed back to the FLP-expressing plants. These observations may indicate that the efficiency of site-specific recombination is affected by the plant developmental stage, with site-specific recombination being more prevalent in developing embryos. The Cre::FLP fusion protein was also tested for excisions catalysed by Cre. Excisions were identified in the F1 plants and verified in the F2 plants by polymerase chain reaction and Southern blotting. Both components of the fusion protein (FLP and Cre) were functional and acted with similar efficiency. The crossing strategy proved to be suitable for the genetic engineering of maize using the FLP or Cre site-specific recombination system. [source] Evidence of pAgK84 transfer from Agrobacterium rhizogenes K84 to natural pathogenic Agrobacterium spp. in an Italian peach nurseryPLANT PATHOLOGY, Issue 4 2009A. Raio Nine Italian peach nurseries, which use Agrobacterium rhizogenes strain K84 to protect plants from crown gall, were monitored for three years with the aim of determining whether transconjugant populations may arise following plasmid exchanges between K84 and autochtonous soil agrobacteria. Six hundred and seventy-eight Agrobacterium isolates were obtained from 120 tumours developed on apricot and peach rootstocks that had been treated in pre-planting with the antagonist. Agrobacteria were characterized for pathogenicity, biovar, opine catabolism and agrocin 84 sensitivity. Colony hybridization was used for screening the isolates harbouring plasmids pTi and/or pAgK84. Analysis of plasmid content and Southern blotting were performed on putative transconjugant agrobacteria found in tumours collected from one nursery where a biological control breakdown was observed. The RFLP analysis of 16S + IGS regions showed that pAgK84 was transferred from the antagonist to virulent and avirulent soil agrobacteria belonging to different ribotypes. Pathogenic transconjugants, inoculated on GF677 rootstocks, were not controlled in vivo by K84 and stably maintained pTi and pAgK84 in the bacterial cells for at least one year. At the end of a biocontrol trial, new transconjugant tumorigenic agrobacteria originated by the transfer of pAgK84 to the pathogen. Virulent and avirulent transconjugants may represent a real threat for biological control by K84 strain since all of them produced agrocin and were insensitive to it. Survival in soil of these populations could make the future application of K84 ineffective. [source] Comparison of PERV genomic locations between Asian and European pigsANIMAL GENETICS, Issue 1 2010W. Y. Jung Summary Xenotransplantation from pigs provides a possible solution to the shortage of human organs for allotransplantation. Porcine endogenous retroviruses (PERVs) are a possible obstacle to using porcine organs in addition to the immunological barriers. Three main types of PERVs (A, B and C) have been previously investigated in diverse pig breeds. To examine the copy numbers of PERVs and their genomic locations in the Korean native pig genome, we screened a BAC (Bacterial Artificial Chromosome) library with PERV-specific protease primers for initial recognition of PERV-positive clones and three sets of envelope-specific primers for the identification of PERV types. A total of 45 PERV-positive clones, nine PERV-A and 36 PERV-B, have been identified from the library screening and the BAC contigs were constructed using the primers designed from BAC end sequences (BESs). These primers were also used for SCH (Somatic Cell Hybrid) and RH (Radiation Hybrid) mapping of the PERV-positive clones. The results indicate that 45 PERV-positive BAC clones belong to nine contigs and a singleton. SCH and IMpRH (INRA-Minnesota Porcine Radiation Hybrid) mapping results indicated that there are at least eight separate PERV genomic locations, consisting of three PERV-A and five PERV-B. One contig could not be mapped, and two contigs are closely located on SSC7. Southern blotting indicates there may be up to 15 additional sites. Further investigation of these clones will contribute to a general strategy to generate PERV-free lines of pigs suitable for xenotransplantation. [source] Problem-solving test: Telomere replicationBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2010József Szeberényi Terms to be familiar with before you start to solve the test: DNA replication, template, primer, linear DNA, antiparallel orientation, telomere, origin of replication, chromatid, replicon, short tandem repeats, Okazaki fragments, leading strand, lagging strand, ribozyme, promoter, enhancer, terminal transferase, DNA polymerases, reverse transcriptase, RNA polymerase, topoisomerase, retroviral vector, Southern blotting, restriction endonuclease. [source] Vector fragmentation: Characterizing vector integrity in transfected clones by Southern blottingBIOTECHNOLOGY PROGRESS, Issue 1 2010Say Kong Ng Abstract The Chinese Hamster Ovary production cell line development process using methotrexate (MTX) amplification is well studied and commonly used for biopharmaceutical processes. However, successful MTX amplification varies from clone to clone and suggested reasons include vector fragmentation during the transfection process and genomic rearrangement of the Chinese Hamster Ovary chromosomes. Here, we elucidated the vector integration patterns of 40 transfected single-cell clones by Southern blotting and showed that vector fragmentation occurs at a significant level in our experiment. This concurs with MTX amplification studies implying that single-cell cloning is necessary to ensure a successful amplification process. Truncations at the ends of the integrated vectors were also observed, whereas gross DNA insertions were not detected in our data. This suggests that end deletions are common, whereas insertion events are rare in animal cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] | |||||||||||||