False-positive diagnosis of a single, large-scale mitochondrial DNA deletion by Southern blot analysis: the role of neutral polymorphisms.

Single, large-scale deletions of mitochondrial DNA (mtDNA) are a common finding in the molecular investigation of patients with suspected mitochondrial disorders and are typically detected by Southern blot analysis of muscle DNA that has been linearized by a single cutter enzyme (BamHI or PvuII). We describe our investigations of a 47-year-old woman with exercise intolerance, myalgia, and ptosis who underwent a muscle biopsy for a suspected mitochondrial genetic abnormality. Southern blot analysis after digestion of muscle DNA with BamHI revealed the apparent presence of two mtDNA species, indicative of a heteroplasmic deletion of 2.0-2.5 kb in length involving approximately 50% of all molecules. Contrary to this observation, longrange polymerase chain reaction (PCR) amplified only wild-type mtDNA. Sequence analysis revealed that the patient harbored two previously recognized control region polymorphisms, a homoplasmic 16390G>A variant that introduces a new BamHI site and a heteroplasmic 16390G>A change that abolishes this site, thus explaining the initial false-positive testing for a heteroplasmic mtDNA deletion. Our findings highlight the potential problems associated with the diagnosis of mitochondrial genetic disease and emphasize the need to confirm positive cases of mtDNA deletions using more than one enzyme or an independent method such as long-range PCR amplification.

Validity of SmaI-defined genotypes of Campylobacter jejuni examined by SalI, KpnI, and BamHI polymorphisms: evidence of identical clones infecting humans, poultry, and cattle.

We describe here an examination of the validity of molecular types of Campylobacter jejuni as defined by separation of SmaI-digested DNA using pulsed-field gel electrophoresis (PFGE), recently suggested as part of a molecular subtyping scheme. Thirty-four Danish strains from humans, water, poultry and cattle were assigned to one of six SmaI 'profile groups' (PGs), with two additional strains included as genotypically distinct controls. The interstrain relationships were reexamined by PFGE of SalI, KpnI and BamHI-digested DNA, and also by serotyping with heat-stable antigens. All outbreak-related strains were indistinguishable by all criteria, as were two sets of two randomly-isolated human strains. Two groups of indistinguishable isolates contained randomly isolated strains from more than one source (poultry, humans and/or cattle), a finding with significant epidemiological connotations. All 'genetically identical' strains belonged to the same serotype, whereas genetic differences were detected between strains assigned to the same SmaI PG but differing in serotype. We conclude that PFGE-based genetic fingerprinting can yield invaluable data for epidemiological studies of sporadic C. jejuni infection, but that results based on one restriction site polymorphism must be checked with another enzyme.

Enhancement of frequencies of restriction endonuclease-induced chromatid breaks by arabinoside adenine in normal human and ataxia telangiectasia cells.

The effect of ara A (9-beta-D-arabino furanosyladenine) a potent inhibitor of DNA synthesis on the frequencies of chromatid breaks induced by restriction endonucleases (RE) has been investigated in normal human and ataxia telangiectasia (AT) lymphoblastoid cells. PvuII, PstI and BamHI which cause blunt-ended, 3'-overhang and 5'-overhang cohesive-ended DNA double-strand breaks (dsb) respectively, were introduced into two AT cell lines (AT-KM and AT-PA) and a normal human (N-SW) cell line by the use of streptolysin-O poration. Controls were exposed to gamma-irradiation and similarly treated with or without ara A. Both AT cell lines were found to exhibit higher frequencies of chromatid breaks when treated with RE alone as compared with the normal cell line. The pattern of chromatid response to the three RE was shown to be similar in all three cell lines i.e. PvuII was most clastogenic while PstI and BamHI were both less effective at inducing chromosomal aberrations. Incubation of cells with ara A resulted in an increase in frequencies of chromatid breaks in PvuII and PstI treated cells but no increase was observed in BamHI treated cells. Normal cells showed most response to ara A following treatment with PvuII and PstI (enhancement ratios 4.63 and 3.75 respectively) while AT cells were affected by ara A to a lesser extent indicating a reduced expression of damage by ara A in these lines. Since ara A is a potent inhibitor of DNA synthesis, it was concluded from the elevated frequency of chromosomal aberrations in the presence of ara A that rejoining of RE-induced dsb in genomic DNA of human cells involves nucleotide insertion at dsb termini prior to ligation.

Restriction analysis of the MD infectious bursal disease virus strain.

The variant MD strain of infectious bursal disease virus (IBDV) was examined using restriction fragment length polymorphism (RFLP) and the molecular variation was compared with published sequences of both variant and classic IBDV strains. Viral RNA was transcribed into cDNA using reverse transcriptase and then amplified by the polymerase chain reaction (PCR). Three separate but overlapping fragments of 1603 bp, 1496 bp, and 1066 bp containing the entire coding region for VP2, VP4, and VP3, respectively, were amplified. These amplified products were initially cloned using the TA Cloning kit and further subcloned into the pGEM-3Zf and pUC19. Eight restriction enzymes, PstI, BamHI, AvaI, EcoRI, HindII, XmaI, SmaI, and XbaI, were tested for their ability to digest the MD PCR products. The resulting RFLP was analyzed and compared with the published genome segment A sequences of the classic IBDV strain STC and the variant IBDV strain GLS. Differences in restriction fragment profiles were observed after digestion with BamHI, EcoRI, XmaI, and SmaI, with the absence of the EcoRI site in both variant strains, GLS and MD. The MD PCR products lacked both XmaI and SmaI sites, which were present in both STC and GLS. The MD PCR products contained a BamHI site within the hypervariable region of VP2, which is present in STC but not in the variant GLS. An additional BamHI site was identified in the VP4 gene of MD and GLS but not in STC.

Intrachromosomal localization of breakpoints induced by the restriction endonucleases AluI and BamHI in Chinese hamster ovary cells treated in S phase of the cell cycle.

The restriction endonucleases (REs) AluI and BamHI were electroporated into Chinese hamster ovary (CHO) cells during S phase of the cell cycle and breakpoints in G-banded metaphases were mapped to Giemsa-light or -dark bands or to band junctions. The majority of AluI- and BamHI-induced breakpoints were located in Giemsa-light bands. Both REs induced similar distributions of breakpoint clusters. The localization pattern of S phase-induced breakpoints in CHO cells is similar to the pattern of G1-induced breakpoints reported earlier. These data show that breakpoint localization for both REs is independent of the cell cycle stage (G1 or S) in which aberrations are induced and give further support to the hypothesis that nuclease hypersensitive regions (NHRs) associated with active genes play an important role in the distribution of breakpoints.

Localization of chromosome breakpoints induced by AluI and BamHI in Chinese hamster ovary cells treated in the G1 phase of the cell cycle.

Intact Chinese hamster ovary cells were exposed to the restriction endonucleases (REs) AluI or BamHI. In metaphase spreads from these cells, 300 breakpoints per RE were localized in G-banded chromosome type aberrations (dicentrics, translocations, rings, terminal and interstitial deletions). The majority of breakpoints induced by both REs were localized in G-light bands and showed a similar distribution of breakpoint clusters. RE digestion of metaphase spreads with AluI induced C-banding, and with BamHI G-banding. The data indicate that nuclease sensitive sites associated with active genes are mainly responsible for the distribution of breakpoints.

Response of ataxia telangiectasia cells to restriction endonuclease induced DNA double-strand breaks: I. Cytogenetic characterization.

Ataxia telangiectasia (AT) and normal human lymphoblastoid cell lines have been treated with either X-rays or the restriction endonucleases PvuII and BamHI using streptolysin-O poration, and the frequencies of micronuclei or chromosomal aberrations measured. We report that AT cells (AT-PA) are hypersensitive to the restriction endonucleases PvuII and BamHI, inducing DNA double-strand breaks (dsb) with either blunt or cohesive termini, respectively. Our data indicates that AT-PA cells have a dsb processing defect that leads to a higher rate of conversion of dsb into chromosomal aberrations than in normal cells. AT-PA cells showed up to a 5-fold enhanced sensitivity to PvuII over the normal (N-SW) line, a result of an increase in frequencies of chromatid aberrations. Chromosome-type aberrations appeared not to be increased in AT-PA cells over those induced in the normal N-SW line. Particularly striking was the appearance in AT-PA of high frequencies of chromatid aberrations at the 24 h sampling time. BamHI also caused enhanced aberration frequencies in AT-PA cells although the cohesive-ended dsb caused by BamHI still appeared to be less effective in causing chromosomal aberrations than the blunt-ended dsb caused by PvuII in both AT-PA and N-SW, as we have previously reported for Chinese hamster cells. The enhanced effectiveness of cohesive-ended dsb in AT-PA cells over normal cells may be a result of altered processing of dsb by AT-PA cells or may be caused by conversion of some cohesive-ended dsb into blunt-ended dsb by exonuclease digestion before ligation can take place.

[Study of growth hormone gene in non-familial complete somatotropin deficiency]. / Etude du gène de l'hormone de croissance dans le déficit somatotrope complet non familial.

Familial growth hormone deficiency has been often associated to homozygous gene deletions. In this work we have looked for the possible absence of this gene in patients with isolated GH deficiency. The patient genomic DNAs have been digested with two restriction enzymes and hybridized with a 32P labelled growth hormone cDNA. The presence of the growth hormone gene has been proved in the patients. This situation, in which the gene is present but not expressed, might be due to changes in gene regulation or to punctual gene deletions or mutations.

Induction of ferritin synthesis in vitro: problems of specificity inherent in the use of iron compounds.

The potential importance of heme as a translational regulator has been recognized for nearly 30 years. However, the ability to distinguish the specific regulatory effects of heme from the nonspecific effects has been hampered by its high reactivity and its tendency to generate reactive by-products in most systems. In this article we discuss some of the technical difficulties in studying the effects of iron salts and compounds, notably heme, in biochemical systems in vitro. Data are presented which show that the nonspecific inhibitory effects of heme on two restriction endonucleases can be eliminated by (a) including a redox buffer in the reaction mixture, and (b) maintaining a sufficiently high total protein concentration. Under these conditions, the specific effects of hemin on the ferritin repressor protein are still observed. A possible relationship between these observations and the status of 'free' heme in vivo is considered.