Characterization of inverted repeats from plasmid-like DNAs and the maize mitochondrial genome

Summary Integrated inverted repeat (IR) sequences similar to those of the S plasmids have been isolated from the genomes of the normal and S type male-sterile cytoplasms of maize mitochondria. The nucleotide sequences of both the IRs and their flanking regions have distinguished and characterized several different types of repeats. The repeats may be involved in the recombinational process that occurs continuously in the mitochondrial genome. One cloned fragment, derived from a fertile revertant and containing sequences similar to S-2, does not appear to act as atypical transposable element during reversion. Several of the flanking regions examined contain a small repeat of 34 base pairs, in which a nonanucleotide segment is found with similarity to the yeast mitochondrial promoter.     

Estimation of the size of the chromosome 17p11.2 duplication in Charcot-Marie-Tooth neuropathy type 1a (CMT1a). HMSN Collaborative Research Group.

We have previously shown a duplication in 17p11.2 with probe pVAW409R3 (D17S122) in 12 families with hereditary motor and sensory neuropathy type I (HMSN I) or Charcot-Marie-Tooth disease type 1 (CMT1). In this study we aimed to estimate the size of the duplication using additional polymorphic DNA markers located in 17p11.2-p12. Two other 17p11.2 markers, pVAW412R3 (D17S125) and pEW401 (D17S61), were found to be duplicated in all HMSN I patients tested. Furthermore, all HMSN I patients showed the same duplication junction fragment with probe pVAW409R3. On the genetic map the duplicated markers span a minimal distance of 10 cM while on the physical map they are present in the same NotI restriction fragment of 1150 kb. The discrepancy between the genetic and physical map distances suggests that the 17p11.2 region is extremely prone to recombinational events. The high recombination rate may be a contributing factor to the genetic instability of this chromosomal region.     

Inversions and recombinations in mitochondrial DNA of the (SG-1) cytoplasmic mutant in two Neurospora species

Summary The mitochondrial DNAs of [SG-1] cytoplasmically-mutant and wild-type strains of Neurospora crassa and Neurospora sitophila were examined by comparative restriction endonuclease analyses. The mtDNA of N. sitophila wild type of Whitehouse differs from type II mtDNA of N. crassa by insertions of 3.3 kb in EcoRI-9, and 1.2 kb in EcoRI-3, and a deletion of 1.1 kb in EcoRI-5. These DNA heteromorphisms provided convenient markers for tracing N. crassa [SG-1] mtDNA during and after its transfer into N. sitophila. The [SG-1] cytoplasmic mutant in both N. crassa and N. sitophila has a distinctive inversion that connects the fragment EcoRI-4 with HindIII-10a. The [SG-1] mtDNA from N. crassa remained essentially intact after it was transferred by crosses into N. sitophila. In each species, a unique second inversion occured in the [SG-1] mtDNA after the transfer was made. In N. sitophila, polar recombination in heteroplasmons between [SG-1] and wild-type preferentially yields strains with mtDNAs that contain the maximum possible number of insertions in the cob and co-1 loci of the EcoRI-3 region of the mitochondrial chromosome.     

Role of sequence amplification in the generation of nematode mitochondrial DNA polymorphism

Summary Romanomennis culicivorax, an obligate parasitic nematode of mosquitos, possesses an unusually large mitochondrial genome. Individuals are monomorphic for one of several mitochondrial DNA (mtDNA) size variants ranging from 26–32 kb. In this report, we demonstrate that the mitochondrial genome size differential in three isofemale lineages is due to the presence of mtDNA sequences amplified to different copy numbers within each mtDNA molecule. Restriction enzyme analysis and DNA sequencing studies reveal that each mitochondrial genome contains one of two 3.0 kb repeat types that differ by approximately 30 bp. This difference is primarily due to a short (23 bp) imperfect tandem duplication present within the larger of two polymorphic repeating units. The 3.0 kb reiterated DNA sequences are present as direct, tandem repeats and as inverted portions of the same sequence located elsewhere in the genome. Based on mtDNA analysis of an independently reared R. culicivorax culture, we conclude that events resulting in mitochondrial genome rearrangement occurred in natural field populations prior to propagation within the laboratory.     

Nasal natural killer/T-cell lymphoma and its association with type "i"/XhoI loss strain Epstein-Barr virus in Chile

BACKGROUND: Nasal T/natural killer (NK)-cell lymphoma is an aggressive type of non-Hodking's lymphoma associated with Epstein-Barr virus (EBV) and striking geographical variations worldwide. AIM: To characterise nasal NK/T-cell lymphoma associated with genotypes of EBV in Chile, a Latin American country, where multiple strains of EBV, including two new recombinant strains, in healthy individuals were recently found. METHODS: Cases with diagnosis of primary nasal lymphoma were selected for histological and immunohistochemical analysis (CD3, CD3e, CD4, CD8, CD79a, CD56, CD57 and TIA-1) and in-situ hybridisation, serology and genotyping analysis for EBV. RESULTS: Out of 22 cases, 9 (41%) cases fulfilled the World Health Organization criteria for nasal NK/T-cell lymphoma; of these 7 (78%) cases were positive for EBV. Genotyping analysis revealed 6 cases of type 1 EBV and wildtype F at the BamHI-F region, 4 cases type "i" EBV at the BamHI-W1/I1 region; XhoI wild type was found in 2 and XhoI loss in 4 cases, respectively. Cosegregation analysis of the BamHI-W1/I1 region and XhoI restriction site showed the new recombinant strain type "i"/XhoI loss in 3 cases and type "i"/XhoI wild-type strain in 1 case. Most patients were treated with combined anthracycline-containing regimens. Half of the cases attained complete remission. CONCLUSION: Although nasal NK/T-cell lymphomas from Chile share similar clinicopathological features, high association with EBV and unfavourable prognosis with those described elsewhere, genotype analysis shows that the new recombinant type "i"/XhoI loss strain might contribute to explain the intermediate incidence of nasal NK/T-cell lymphomas in Latin America.     

慢性进行性眼外肌瘫痪和Kearns-Sayre综合征的线粒体DNA突变分析

目的 探讨慢性进行性眼外肌瘫痪(chronic progressive external ophthalmoplegia，CPEO)和Kearns-Sayre综合征(Kearns—Sayre syndrome，KSS)的线粒体DINA(mitochondrial DNA，mtDNA)突变特点。方法 用Southern印迹方法检测7例CPEO和4例KSS患者的肌肉组织mtDNA，并进一步用聚合酶链反应产物直接测序来明确缺失的具体范围；用聚合酶链反应-限制性内切酶分析法检测有无mtDNA A3243G点突变。结果 发现5例患者(2例CPEO和3例KSS)存在mtDNA的大片段缺失；1例KSS患者存在A3243G点突变。5例大片段缺失的大小及缺失范围各不相同，从3．0～8．0kb不等，缺失型mtDNA占总mtDNA的比例为37．6％～87．0％。聚合酶链反应产物测序表明这5例缺失类型均未见文献报道。结论 与CPEO和KSS患者相关的最常见的mtDNA突变为大片段缺失，A3243G点突变也可在少数患者中检测到。     

Reaching for the ring: the study of mitochondrial genome structure

The linear molecules that comprise most of the mitochondrial DNA (mtDNA) isolated from most organisms result from the artifactual degradation of circular genomes that exist within mitochondria. This view has been adopted by most investigators and is based on DNA fragment mapping data as well as analogy to the genomesized circular mtDNA molecules obtained in high yield from animals. The alternative view that linear molecules actually represent the major form of DNA within mitochondria is supported by two observations: (1) over a 1000-fold range of genome size among fungi and plants we find the same size distribution of linear mtDNA molecules, and (2) linear mtDNA molecules much larger than genome size can be found for some fungi and plants. The circles that represent only a small fraction of the mtDNA obtained from most eukaryotes could be optional sequence forms unimportant for mitochondrial function; they may also participate in mtDNA replication. The circles might result from incidental recombination events between directly repeated sequences within or between tandemly arrayed genome units on linear mtDNA molecules.     

Detection of restriction fragment length polymorphisms in clinical isolates and serially passaged Pseudomonas aeruginosa strains.

An 800-base-pair HindIII-PstI fragment that flanks a hot spot for Tn7 insertion was isolated from the chromosome of Pseudomonas aeruginosa and cloned into pUC12. The fragment was used to probe XhoI digests of genomic DNA from 18 P. aeruginosa isolates collected from sputum samples of seven cystic fibrosis patients. Only two XhoI restriction fragment length polymorphisms (RFLPs), of 3.7 and 7.7 kilobases (kb), were detected. Isolate WSU3531-1 (3.7-kb XhoI fragment) and WSU3860 (7.7-kb XhoI fragment), while isolated from the same patient, showed different RFLPs. Serial passages of isolate WSU3531-1 demonstrated that this strain was phenotypically stable. In contrast, colony and pigment variants were readily isolated at a frequency of 1% from serial passages of isolate WSU3860. When XhoI-digested genomic DNA from phenotypic variants of serially passaged WSU3860 were probed with the 800-base-pair HindIII-PstI fragment, the probe hybridized to a 10.4-kb XhoI fragment from three isolates. Restriction analysis of the genomic DNA digested with a variety of restriction enzymes showed that a 2.7-kb insertion occurred in the same region for all three isolates. There appeared to be no correlation between changes in the RFLP and changes in colony morphology.     

Moving pictures and pulsed-field gel electrophoresis show only linear mitochondrial DNA molecules from yeasts with linear-mapping and circular-mapping mitochondrial genomes

  The mobility of mitochondrial DNA (mtDNA) in pulsed-field gel electrophoresis (PFGE) and its appearance in moving pictures from fluorescence microscopy were used to investigate the mitochondrial genome structure for five Pichia and Williopsis strains of yeast. An apocytochrome b-gene hybridization probe identified only linear mtDNA molecules for each strain when total cellular DNA was fractionated by PFGE. Most of the mass of DNA isolated from mitochondria for one linear-mapping and one circular-mapping mitochondrial genome was found in linear molecules much larger than the genome size of 50 kb; some molecules were as long as 1500 kb, but only a trace amount of apparently circular mtDNA was found for the strain with the circular-mapping genome. Probes for both the apocytochrome-b and mitochondrial small rRNA subunit genes hybridized strongly to mtDNA of approximately 50–100 kb, but weakly to the larger DNA from mitochondria of these two strains. For the four linear-mapping strains, PFGE revealed two or three distinct bands of linear mtDNA, larger than the genome size, within a smear of approximately 50–100 kb, but a smear without bands was found for the circular-mapping strain. Received: 28 June 1995 / 15 January 1996     

2型糖尿病患者mtDNA变异筛查研究

目的探讨线粒体基因突变与2型糖尿病的关系。方法随机筛查222例散发2型糖尿病患者和191名正常对照，以聚合酶链反应、限制性内切酶片段长度多态性及T-A克隆测序和变性高效液相色谱分析技术验证等方法检测线粒体基因突变。结果糖尿病组线粒体基因（3153—3551nt）突变总的发生率（24．32％）明显高于正常组（7．33％）（P〈0．05）；发现3个尚未见报道的新突变位点：A3209T、T3253G和A3467C，而C3497T则在糖尿病中是首次报道；起病年龄、体重指数、空腹血糖、糖化血红蛋白、高密度脂蛋白和糖尿病肾病等指标是线粒体基因突变的相关因素（P〈0．05）。结论温州地区糖尿病患者存在多种线粒体基因点突变，其在糖尿病的发生发展中起重要作用。     

Mitochondrial DNA D-loop in pancreatic cancer: somatic mutations are epiphenomena while the germline 16519 T variant worsens metabolism and outcome

We ascertained the frequency of mitochondrial DNA (mtDNA) D-loop region somatic mutations in pancreatic cancer (PC) and verified whether polymorphisms were linked to diagnosis, prognosis, and PC-associated diabetes mellitus (DM) in 99 PC cases, 42 chronic pancreatitis (CP) cases, 18 pancreatobiliary tract tumors, and 87 healthy control subjects (CSs). Tissue samples were obtained from 19 patients with PC and 5 with CP. The D-loop region was sequenced from all tissue samples and from blood DNA of the same patients and 12 CSs. D-loop somatic mutations were found in 3 PC tissue samples (16%). Four single nucleotide polymorphisms (SNPs; T152C, T16189C, T16519C, A73G), more frequently found in PC than in CS, were analyzed by denaturing high-performance liquid chromatography-restriction fragment length polymorphism using blood DNA as the starting template in all cases. The T allele of 16519 SNP correlated with DM. The survival of patients with PC correlated with tumor stage and grade and with DM at diagnosis. When survival analysis was performed considering only patients with locally advanced disease, the T allele of mtDNA 16519 SNP correlated with shorter life expectancy. mtDNA D-loop somatic mutations, rarely found in PC, cannot be considered causative events for this tumor type and probably are epiphenomena; the mtDNA D-loop 16519 variant, which worsens PC prognosis, seems to be a predisposing genetic factor for DM.     

Partial mitochondrial DNA sequence of the crustaceanDaphnia pulex

A 3667-base pair (bp) fragment of the mitochondrial genome of the crustaceanDaphnia pulex has been sequenced and found to contain the complete genes for the small subunit ribosomal RNA, ND2, seven tRNAs and the control region. This organization is identical to that found inDrosophila yakuba mtDNA yetD. pulex mtDNA exhibits several unique features when compared to other mitochondrial sequences. The sequenced fragment is only 62.6% A+T which is much lower than that of any other arthropod mtDNA sequenced to date.D. pulex mtDNA also exhibits length conservation having shorter coding and non-coding regions. The putative control region is 689 bp in length and includes a sequence that has the potential to fold into a hairpin structure with a perfect 20-bp pair stem and a 22-base loop.     

Involvement of a large inverted repeated sequence

Summary Southern hybridization of the total DNA of Agrocybe aegerita with cloned mitochondrial (mt) probes revealed a sequence homology between two distant mitochondrial restriction fragments. From the mtDNA restriction map and the distribution of restriction sites on the cross-hybridizing mitochondrial fragments, two copies of a large inverted repeated sequence (IR) of 3 kbp were located on the mitochondrial genome. These IR sequences divided the 80 kbp mtDNA into two singlecopy regions of 24 kbp (SSC) and 50 kbp (LSC). For the first time in higher fungi, this IR sequence has been shown to be involved in an intramolecular homologous recombinational event. Such a rearrangement led to an inversion of the orientation of the two unique-copy regions, without any change in mtDNA complexity. The location of the recombinational event was compared with previously reported plant and fungal mitochondrial rearrangements and the potential role of the IR sequence was discussed.