Muscle mitochondrial DNA in encephalomyopathy and ragged red fibres: a Southern blot analysis and literature review

1991 ◽  
Vol 238 (3) ◽  
pp. 171-176 ◽  
Author(s):  
C. Geny ◽  
V. Cormier ◽  
C. Meyrignac ◽  
P. Cesaro ◽  
J. D. Degos ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Literature Review ◽  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis

scholarly journals Quantification of Mitochondrial DNA Deletion, Depletion, and Overreplication: Application to Diagnosis

2003 ◽  
Vol 49 (8) ◽  
pp. 1309-1317 ◽  
Author(s):  
Béatrice Chabi ◽  
Bénédicte Mousson de Camaret ◽  
Hervé Duborjal ◽  
Jean-Paul Issartel ◽  
Georges Stepien
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Southern Blot ◽  
Quantitative Pcr ◽  
Blot Analysis ◽  
Copy Number ◽  
Southern Blot Analysis ◽  
Mtdna Copy Number ◽  
Mtdna Depletion ◽  
Real Time Quantitative Pcr

Abstract Background: Many mitochondrial pathologies are quantitative disorders related to tissue-specific deletion, depletion, or overreplication of mitochondrial DNA (mtDNA). We developed an assay for the determination of mtDNA copy number by real-time quantitative PCR for the molecular diagnosis of such alterations. Methods: To determine altered mtDNA copy number in muscle from nine patients with single or multiple mtDNA deletions, we generated calibration curves from serial dilutions of cloned mtDNA probes specific to four different mitochondrial genes encoding either ribosomal (16S) or messenger (ND2, ND5, and ATPase6) RNAs, localized in different regions of the mtDNA sequence. This method was compared with quantification of radioactive signals from Southern-blot analysis. We also determined the mitochondrial-to-nuclear DNA ratio in muscle, liver, and cultured fibroblasts from a patient with mtDNA depletion and in liver from two patients with mtDNA overreplication. Results: Both methods quantified 5–76% of deleted mtDNA in muscle, 59–97% of mtDNA depletion in the tissues, and 1.7- to 4.1-fold mtDNA overreplication in liver. The data obtained were concordant, with a linear correlation coefficient (r2) between the two methods of 0.94, and indicated that quantitative PCR has a higher sensitivity than Southern-blot analysis. Conclusions: Real-time quantitative PCR can determine the copy number of either deleted or full-length mtDNA in patients with mitochondrial diseases and has advantages over classic Southern-blot analysis.

scholarly journals Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments]

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2566-2571 ◽  
Author(s):  
FM Stewart ◽  
RB Crittenden ◽  
PA Lowry ◽  
S Pearson-White ◽  
PJ Quesenberry
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Normal Male ◽  
Normal Donor ◽  
Long Term Stability ◽  
Donor Bone Marrow ◽  
Male Donor ◽  
Marrow Cells

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that “niches” need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.

scholarly journals Mutational analysis of the CDKN2 (MTS1/p16ink4A) gene in primary B-cell lymphomas

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2724-2731 ◽  
Author(s):  
T Uchida ◽  
T Watanabe ◽  
T Kinoshita ◽  
T Murate ◽  
H Saito ◽  
...  
Keyword(s):  
B Cell ◽  
Missense Mutation ◽  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Mutational Analysis ◽  
Direct Sequencing ◽  
Lymphocytic Leukemia ◽  
Sscp Analysis ◽  
Putative Tumor Suppressor Gene

Abstract The CDKN2 gene located on chromosome 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16. This gene is a putative tumor-suppressor gene because of its frequent alterations in many kinds of tumor cell lines. We analyzed the CDKN2 gene to evaluate its alterations in 52 primary specimens of non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL) of B-cell origin by Southern blot analysis, polymerase chain reaction-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, and direct sequencing. By Southern blot analysis, we showed homozygous deletion of the CDKN2 gene in 3 of 42 patients with B-NHL (7.1%). After screening by PCR-SSCP analysis, direct sequencing identified one missense mutation at codon 72 (nucleotide 233) and two frameshifts due to a 35-bp deletion arising at codon 49 (nucleotides 163 to 175) in patients with B-NHL (3 of 42, 7.1%). In the patient carrying the missense mutation, hemizygous deletion of the CDKN2 gene was also suspected. In this study, we detected alterations in CDKN2 in 6 of 42 patients (14.3%) with B-NHL and in none of 10 patients with B-CLL. Our results suggest that the CDKN2 alterations contribute in tumorigenesis in some patients with B-NHL.

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