Phenotype of FAECB (Facility for Automated Experiments in Cell Biology) Chinese hamster ovary mutants with minimal UV-sensitivity

Mitochondrial DNA (mtDNA) haplogroup U, defined by the polymorphism 12308A>G, may constitute a risk factor for an occipital stroke in migraine. We therefore identified 14 patients with an occipital stroke and with 12308A>G. We determined complete mtDNA coding region sequence for the patients and for population controls by conformation sensitive gel electrophoresis (CSGE) and direct sequencing. Sequence information was used to construct a phylogenetic network of mtDNA haplogroups U and K, which was found to be composed of subclusters U2, U4, U5 and a new subcluster U7, as well as cluster K. Five patients with a migrainous stroke belonged to subcluster U5 (P=0.006; Fisher's exact test). Many unique mutations were found among the patients with an occipital stroke including two tRNA mutations that have previously been suggested to be pathogenic. Analysis of mtDNA sequences by CSGE and comparison of the sequences through phylogenetic analysis greatly enhances the identification of mtDNA clusters in population and detection of mtDNA mutations in patients.     

Uncoupling the roles of the SUV3 helicase in maintenance of mitochondrial genome stability and RNA degradation

Yeast SUV3 is a nuclear encoded mitochondrial RNA helicase that complexes with an exoribonuclease, DSS1, to function as an RNA degradosome. Inactivation of SUV3 leads to mitochondrial dysfunctions, such as respiratory deficiency; accumulation of aberrant RNA species, including excised group I introns; and loss of mitochondrial DNA (mtDNA). Although intron toxicity has long been speculated to be the major reason for the observed phenotypes, direct evidence to support or refute this theory is lacking. Moreover, it remains unknown whether SUV3 plays a direct role in mtDNA maintenance independently of its degradosome activity. In this paper, we address these questions by employing an inducible knockdown system in Saccharomyces cerevisiae with either normal or intronless mtDNA background. Expressing mutants defective in ATPase (K245A) or RNA binding activities (V272L or ΔCC, which carries an 8-amino acid deletion at the C-terminal conserved region) resulted in not only respiratory deficiencies but also loss of mtDNA under normal mtDNA background. Surprisingly, V272L, but not other mutants, can rescue the said deficiencies under intronless background. These results provide genetic evidence supporting the notion that the functional requirements of SUV3 for degradosome activity and maintenance of mtDNA stability are separable. Furthermore, V272L mutants and wild-type SUV3 associated with an active mtDNA replication origin and facilitated mtDNA replication, whereas K245A and ΔCC failed to support mtDNA replication. These results indicate a direct role of SUV3 in maintaining mitochondrial genome stability that is independent of intron turnover but requires the intact ATPase activity and the CC conserved region.     

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

Phylogenetic network of the mtDNA haplogroup U in Northern Finland based on sequence analysis of the complete coding region by conformation-sensitive gel electrophoresis

Mutations in mtDNA have accumulated sequentially, and maternal lineages have diverged to form population-specific genotypes. Classification of the genotypes has been made based on differences found in restriction fragment analysis of the coding region or in the sequence of the hypervariable segment I. Both methods have shortcomings, as the former may not detect all the important polymorphisms and the latter makes use of a segment containing hypervariable nucleotide positions. Here, we have used conformation-sensitive gel electrophoresis (CSGE) to detect polymorphisms within the coding region of mtDNA from 22 Finns belonging to haplogroup U. Sixty-three overlapping PCR fragments covering the entire coding region were analyzed by CSGE, and the fragments that differed in their migration pattern were sequenced. CSGE proved to be a sensitive and specific method for identifying mtDNA substitutions. The phylogenetic network of the 22 coding-region sequences constituted a perfect tree, free of homoplasy, and provided several previously unidentified common polymorphisms characterizing subgroups of U. After contrasting this data with that of hypervariable segment I, we concluded that position 16192 seems to be prone to recurrent mutations and that position 16270 has experienced a back mutation. Interestingly, all 22 samples were found to belong to subcluster U5, suggesting that this subcluster is more frequent in Finns than in other European populations. Complete sequence data of the mtDNA yield a more reliable phylogenetic network and a more accurate classification of the haplogroups than previous ones. In medical genetics, such networks may help to decide between a rare polymorphism and a pathogenic mutation; in population genetics, the networks may enable more detailed analyses of population history and mtDNA evolution.     

A 370-kb Cosmid Contig of the Serpin Gene Cluster on Human Chromosome 14q32.1: Molecular Linkage of the Genes Encoding α1-Antichymotrypsin, Protein C Inhibitor, Kallistatin, α1-Antitrypsin, and Corticosteroid-Binding Globulin

The human genes encoding α1-antitrypsin (α1AT, gene symbol PI), corticosteroid-binding globulin (CBG), α1-antichymotrypsin (AACT), and protein C inhibitor (PCI) are related by descent, and they all map to human chromosome 14q32.1. This serine protease inhibitor (serpin) gene cluster also contains an antitrypsin-related sequence (ATR, gene symbol PIL), but the precise molecular organization of this region has not been defined. In this report we describe the generation and characterization of an 370-kb cosmid contig that includes all five serpin genes. Moreover, a newly described serpin, kallistatin (KAL, gene symbol PI4), was also mapped within the region. Gene order within this interval is cen–CBG–ATR–α1AT–KAL–PCI–AACT–tel. The genes occupy 320 kb of genomic DNA, and they are organized into two discrete subclusters of three genes each that are separated by 170 kb. The distal subcluster includes KAL, PCI, and AACT; it occupies 63 kb of DNA, and all three genes are transcribed in a proximal-to-distal orientation. Within the subcluster, there is 12 kb of intergenic DNA between KAL and PCI and 19 kb between PCI and AACT. The proximal subcluster includes α1AT, ATR, and CBG; it occupies 90 kb of genomic DNA, with 12 kb of DNA between α1AT and ATR and 40 kb between ATR and CBG. These genes are all transcribed in a distal-to-proximal orientation. This represents the first detailed physical map of the serpin gene cluster on 14q32.1.     

Mitochondrial DNA variation, but not nuclear DNA, sharply divides morphologically identical chameleons along an ancient geographic barrier

The Levant is an important migration bridge, harboring border-zones between Afrotropical and palearctic species. Accordingly, Chameleo chameleon, a common species throughout the Mediterranean basin, is morphologically divided in the southern Levant (Israel) into two subspecies, Chamaeleo chamaeleon recticrista (CCR) and C. c. musae (CCM). CCR mostly inhabits the Mediterranean climate (northern Israel), while CCM inhabits the sands of the north-western Negev Desert (southern Israel). AFLP analysis of 94 geographically well dispersed specimens indicated moderate genetic differentiation (PhiPT = 0.097), consistent with the classical division into the two subspecies, CCR and CCM. In contrast, sequence analysis of a 637 bp coding mitochondrial DNA (mtDNA) fragment revealed two distinct phylogenetic clusters which were not consistent with the morphological division: one mtDNA cluster consisted of CCR specimens collected in regions northern of the Jezreel Valley and another mtDNA cluster harboring specimens pertaining to both the CCR and CCM subspecies but collected southern of the Jezreel Valley. AMOVA indicated clear mtDNA differentiation between specimens collected northern and southern to the Jezreel Valley (PhiPT = 0.79), which was further supported by a very low coalescent-based estimate of effective migration rates. Whole chameleon mtDNA sequencing (∼17,400 bp) generated from 11 well dispersed geographic locations revealed 325 mutations sharply differentiating the two mtDNA clusters, suggesting a long allopatric history further supported by BEAST. This separation correlated temporally with the existence of an at least 1 million year old marine barrier at the Jezreel Valley exactly where the mtDNA clusters meet. We discuss possible involvement of gender-dependent life history differences in maintaining such mtDNA genetic differentiation and suggest that it reflects (ancient) local adaptation to mitochondrial-related traits.     

Cryptic clonal lineages and genetic diversity in the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) inferred from nuclear and mitochondrial DNA analyses

In the loach Misgurnus anguillicaudatue, the asexual lineage, which produces unreduced clonal diploid eggs, has been identified. Among 833 specimens collected from 54 localities in Japan and two localities in China, 82 candidates of other lineage(s) of cryptic clones were screened by examining RFLP (restriction fragment length polymorphism)-PCR haplotypes in the control region of mtDNA. This analysis was performed because triploid loaches arise from the accidental incorporation of the sperm nucleus into unreduced diploid eggs of a clone. The categorization of members belonging to three newly identified lineages (clones 2–4) and the previously identified clonal lineage (clone 1) was verified by evaluating the genetic identity between two or more individuals from each clonal lineage based on RAPD (random amplified polymorphic DNA)-PCR and multilocus DNA fingerprints. We detected 75 haplotypes by observing the nucleotide status at variable sites from the control region of mtDNA. Phylogenic trees constructed from such sequences showed two highly diversified clades, A and B, that were beyond the level common for interspecific genetic differentiation. That result suggests that M. anguillicaudatus in Japan is not a single species entity. Two clone-specific mtDNA sequences were included in clade A, and the loaches with such sequences may be the maternal origin of the clones. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Circular mitochondrial DNA molecules from petite mutants of Saccharomyces cerevisiae: resolution by polyacrylamide gel electrophoresis

Mitochondrial DNA (mtDNA) from petite strain K45 ofSaccharomyces cerevisiae contains about 7% circular DNA molecules which comprise a simple oligomeric series based on a monomeric size of 1.7 kilobase pairs. Electrophoresis of K45 mtDNA on a polyacrylamide-agarose slab gel fractionates the mtDNA into a major band (containing linear DNA) and several faster running minor bands each containing particular size class of circular DNA molecules. From study of mtDNA from K45 and two other simple petites it was found that the mobility of circles is inversely proportional to the logarithm of the circle size. Polyacrylamide gel electrophoresis thus permits the separation of circular mtDNA from the linear mtDNA of simple petites, and physically resolves circles of different size from one another.     

Retrospective Estimation of Genetic Diversity of an Extinct Oriental White Stork (Ciconia boyciana) Population in Japan Using Mounted Specimens and Implications for Reintroduction Programs

The Japanese regional population of the Oriental white stork (Ciconia boyciana) became extinct in 1986. Mitochondrial DNA (mtDNA) D-loop region from 20 mounted specimens preserved at public facilities in Toyooka City, Hyogo Prefecture, Japan and its vicinity (n = 17), the area inhabited by the last of the Japanese population, and samples originating from China (n = 3) which were kept at a zoo was analyzed. After extracting DNA from small pieces of skin from mounted specimens, a 1210-bp region of the mtDNA D-loop region was analyzed. The haplotypes among 11 specimens of storks captured or found dead at Toyooka City just before the population became extinct were completely identical. Four haplotypes observed among the mounted specimens preserved in the vicinity of Toyooka City were differentiated from those of captive storks originating from China and Russia in a previous study. Therefore, the last Japanese population might have been a genetically unique group. However, phylogenetic analysis using the maximum likelihood method showed that haplotypes found in the Japanese regional population were closely related to the Chinese and Russian lineages (sequence difference = 2.1%). One mounted specimen collected in 1935 at Izushi village, in the vicinity of Toyooka City, showed the same haplotype as the captive storks from China, suggesting that genetic flow may have historically occurred between the populations of Japan and the continent. Recently, reintroduction for the Oriental white stork has been planned in Toyooka City. The planning for the recovery of extinct populations should not only involve translocation of species to the range from which it disappeared, but also reconstruction of regional populations while considering the genetic lineage between the extinct and introduced populations.     

Genomic structures and population histories of linguistically distinct tribal groups of India

There are various conflicting hypotheses regarding the origins of the tribal groups of India, who belong to three major language groups--Austro-Asiatic, Dravidian and Tibeto-Burman. To test some of the major hypotheses we designed a genetic study in which we sampled tribal populations belonging to all the three language groups. We used a set of autosomal DNA markers, mtDNA restriction-site polymorphisms (RSPs) and mtDNA hypervariable segment-1 (HVS-1) sequence polymorphisms in this study. Using the unlinked autosomal markers we found that there is a fair correspondence between linguistic and genomic affinities among the Indian tribal groups. We reconstructed mtDNA RSP haplotypes and found that there is extensive haplotype sharing among all tribal populations. However, there is very little sharing of mtDNA HVS-1 sequences across populations, and none across language groups. Haplogroup M is ubiquitous, and the subcluster U2i of haplogroup U occurs in a high frequency. Our analyses of haplogroup and HVS-1 sequence data provides evidence in support of the hypothesis that the Austro-Asiatic speakers are the most ancient inhabitants of India. Our data also support the earlier finding that some of the western Eurasian haplogroups found in India may have been present in India prior to the entry of Aryan speakers. However, we do not find compelling evidence to support the theory that haplogroup M was brought into India on an "out of Africa" wave of migration through a southern exit route from Ethiopia. On the contrary, our data raise the possibility that this haplogroup arose in India and was later carried to East Africa from India.     

An economical mtDNA SNP assay detecting different mitochondrial haplogroups in identical HVR 1 samples of Caucasian ancestry

BackgroundWe had sequenced 329 Caucasian samples in Hypervariable Region 1 (HVR 1) and found that they belong to eleven different mitochondrial DNA (mtDNA) haplotypes. The sample set was further analysed by an mtDNA assay examining 32 single nucleotide polymorphisms (SNPs) for haplogroup discrimination.In a validation study on 160 samples of different origin it was shown that these SNPs were able to discriminate between the evolved superhaplogroups worldwide (L, M and N) and between the nine most common Caucasian haplogroups (H, I, J, K, T, U, V, W and X).ResultsThe 32 mtDNA SNPs comprised 42 different SNP haplotypes instead of only eleven haplotypes after HVR 1 sequencing. The assay provided stable results in a range of 5 ng genomic DNA down to virtually no genomic DNA per reaction. It was possible to detect samples of African, Asian and Eurasian ancestry, respectively.DiscussionThe 32 mtDNA SNP assay is a helpful adjunct to further distinguish between identical HVR 1 sequences of Caucasian origin. Our results suggest that haplogroup prediction using HVR 1 sequencing provides instable results. The use of coding region SNPs for haplogroup assignment is more suited than using HVR 1 haplotypes.     

The Complete Mitochondrial Genome of the Hagfish Myxine glutinosa: Unique Features of the Control Region

The complete sequence of the mitochondrial DNA of the hagfish Myxine glutinosa has been determined. The hagfish mtDNA (18,909 bp) is the longest vertebrate mtDNA determined so far. The gene arrangement conforms to the consensus vertebrate type and differs from that of lampreys. The exceptionally long (3628-bp) control region of the hagfish contains the typical conserved elements found in other vertebrate mtDNAs but is characterized by a large number of putative hairpins, which can potentially fold into a highly compact secondary structure that appears to be unique to hagfish. The comparison of the mtDNAs of two M. glutinosa specimens, excluding the control region, shows a 0.6% divergence at the nucleotide level as a sample of intraspecies polymorphism. Received: 21 August 2000 / Accepted: 2 March 2001     

Somatic mutations in mitochondrial genome and their potential roles in the progression of human gastric cancer

Background

Somatic mutation in mitochondrial DNA (mtDNA) has been proposed to contribute to initiation and progression of human cancer. In our previous study, high frequency of somatic mutations was found in the D-loop region of mtDNA of gastric cancers. However, it is unclear whether somatic mutations occur in the coding region of mtDNA of gastric cancers.

Methods

Using DNA sequencing, we studied 31 gastric cancer specimens and corresponding non-cancerous stomach tissues. Moreover, a human gastric cancer SC-M1 cell line was treated with oligomycin to induce mitochondrial dysfunction. Cisplatin sensitivity and cell migration were analyzed.

Results

We identified eight somatic mutations in the coding region of mtDNAs of seven gastric cancer samples (7/31, 22.6%). Patients with somatic mutations in the entire mtDNA of gastric cancers did not show significant association with their clinicopathologic features. Among the eight somatic mutations, five point mutations (G3697A, G4996A, G9986A, C12405T and T13015C) are homoplasmic and three mutations (5895delC, 7472insC and 12418insA) are heteroplasmic. Four (4/8, 50%) of these somatic mutations result in amino acid substitutions in the highly conserved regions of mtDNA, which potentially lead to mitochondrial dysfunction. In addition, in vitro experiments in SC-M1 cells revealed that oligomycin-induced mitochondrial dysfunction promoted resistance to cisplatin and enhanced cell migration. N-acetyl cysteine was effective in the prevention of the oligomycin-enhanced migration, which suggests that reactive oxygen species generated by defective mitochondria may be involved in the enhanced migration of SC-M1 cells.

General Significance

Our results suggest that somatic mtDNA mutations and mitochondrial dysfunction may play an important role in the malignant progression of gastric cancer.     

Analysis of ancient DNA from a prehistoric Amerindian cemetery

The Norris Farms No. 36 cemetery in central Illinois has been the subject of considerable archaeological and genetic research. Both mitochondrial DNA (mtDNA) and nuclear DNA have been examined in this 700-year-old population. DNA preservation at the site was good, with about 70% of the samples producing mtDNA results and approximately 15% yielding nuclear DNA data. All four of the major Amerindian mtDNA haplogroups were found, in addition to a fifth haplogroup. Sequences of the first hypervariable region of the mtDNA control region revealed a high level of diversity in the Norris Farms population and confirmed that the fifth haplogroup associates with Mongolian sequences and hence is probably authentic. Other than a possible reduction in the number of rare mtDNA lineages in many populations, it does not appear as if European contact significantly altered patterns of Amerindian mtDNA variation, despite the large decrease in population size that occurred. For nuclear DNA analysis, a novel method for DNA-based sex identification that uses nucleotide differences between the X and Y copies of the amelogenin gene was developed and applied successfully in approximately 20 individuals. Despite the well-known problems of poor DNA preservation and the ever-present possibility of contamination with modern DNA, genetic analysis of the Norris Farms No. 36 population demonstrates that ancient DNA can be a fruitful source of new insights into prehistoric populations.     

Stock Structure and Homing Fidelity in Gulf of Mexico Sturgeon (Acipenser Oxyrinchus Desotoi) Based on Restriction Fragment Length Polymorphism and Sequence Analyses of Mitochondrial DNA

Efforts have been proposed worldwide to restore sturgeon populations through the use of hatcheries to supplement natural reproduction and to reintroduce sturgeon where they have become extinct. We examined the population structure and inferred the extent of homing in the anadromous Gulf of Mexico (Gulf) sturgeon (Acipenser oxyrinchus desotoi). Restriction fragment length polymorphism and control region sequence analyses of mitochondrial DNA (mtDNA) were used to identify haplotypes of Gulf sturgeon specimens obtained from eight drainages spanning the subspecies' entire distribution from Louisiana to Florida. Significant differences in haplotype frequencies indicated substantial geographic structuring of populations. A minimum of four regional or river-specific populations were identified (from west to east): (1) Pearl River, LA and Pascagoula River, MS, (2) Escambia and Yellow rivers, FL, (3) Choctawhatchee River, FL, and (4) Apalachicola, Ochlockonee, and Suwannee rivers, FL. Estimates of maternally mediated gene flow between any pair of the four regional or river-specific stocks ranged between 0.15 to 1.2. Tandem repeats in the mtDNA control region of Gulf sturgeon were not perfectly conserved. This result, together with an absence of heteroplasmy and length variation in Gulf sturgeon mtDNA, indicates that the molecular mechanisms of mtDNA control region sequence evolution differ among acipenserids.     

Damage to mitochondrial DNA induced by the quinolone Bay y 3118 in embryonic turkey liver

Quinolones are a class of antibiotics that induce damage to and loss of DNA from bacteria. The structural organization of bacterial DNA is more similar to eukaryotic mitochondrial DNA (mtDNA) than to eukaryotic chromosomal or nuclear DNA (nDNA). Antibiotics affecting the bacterial genome may therefore preferentially damage mtDNA rather than nDNA. We investigated the effect of a quinolone on mtDNA in avian embryonic hepatocytes in ovo. The quinolone Bay y 3118 (1-cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8-yl) 6-fluoro-8-chloro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride, chemical structure see Bremm et al. [K.D. Bremm, U. Petersen, K.G. Metzger, R. Endermann, In vitro evaluation of Bay-y 3118, a new full-spectrum fluoroquinolone, Chemotherapy 38 (1992) 376-387] was injected into fertilized turkey eggs 8 days before hatching at doses of 1, 3, 10 and 30 mg per egg. The embryos were removed from the eggs after 4 days and liver samples were shock frozen. Mitochondrial DNA was purified from samples of the embryonic liver. The integrity of mtDNA was investigated by electrophoresis on agarose gels with native mtDNA and with ribonuclease-treated mtDNA. Fluorescent staining of the electrophoresis gels allows the densitometric quantification of the mtDNA of the regular band at 16 kilobases (kb) and the amount of DNA fragments of irregular size (smear). The genotoxic nitrosamine nitrosodiethylamine (NDEA) has previously been shown to reduce the content of mtDNA of the regular size of 16 kb and to induce the occurrence of smaller fragments of mtDNA [H. Enzmann, C. Kühlem, E. L?ser, P. Bannasch, Damage to mitochondrial DNA induced by the hepatocarcinogen, diethylnitrosamine in ovo, Mutation Res. 329 (1995) 113-120]. After exposure to 10 and 30 mg Bay y 3118, a dose-dependent induction of damage to the mtDNA was found, whereas exposure to 3 and 1 mg showed no effect. NDEA (25 mg) was used as positive control. Testing chemical compounds in the in ovo model is a simple and rapid approach for investigations on chemically induced alterations of mtDNA.     

In situ photochemical crosslinking of HeLa cell mitochondrial DNA by a psoralen derivative reveals a protected region near the origin of replication.

The in vivo association with proteins of HeLa cell mitochondrial DNA (mtDNA) has been investigated by analyzing the pattern of in situ crosslinking of the DNA by 4'-hydroxymethyl-4, 5',8-trimethylpsoralen (HMT). Either isolated mitochondria or whole cells were irradiated with long wavelength UV light in the presence of ths psoralen derivative, and the mtDNA was then isolated and analyzed in the electron microscope under totally denaturing conditions. No evidence of nucleosomal structure was found. The great majority of the molecules (approximately 90%) had a double-stranded DNA appearance over most of their contour length, with one to several bubbles occupying the rest of the contour, while the remaining 10% of the molecules appeared to be double-stranded over their entire length. Analysis of restriction fragments indicated the presence, in approximately 80% of the molecules, of a protected segment (300 to 1500 bp long) in a region which was centered asymmetrically around the origin of replication so as to overlap extensively the D-loop. Control experiments showed that at most 30% of the bubbles found near the origin could represent D-loops or expanded D-loops: furthermore, it could be excluded that some sequence peculiarity would account for the preferential location of bubbles near the origin of replication. The data have been interpreted to indicate that, in at least 55% of HeLa cell mtDNA molecules, the region around the origin is protected from in situ psoralen crosslinking by proteins or protein complexes which are associated in vivo with the DNA.     

Incorporating historical museum specimens into molecular systematic and conservation genetics research

We successfully amplified mitochondrial DNA (mtDNA) and microsatellites from historical museum specimens of lizards and frogs dating from 1894 through to 1998. Ancient DNA techniques were used to extract whole genomic DNA from a number of different tissues, including liver, leg muscle and toe clips, followed by standard polymerase chain reaction techniques for amplification. We had a 78% success rate amplifying mtDNA from 14 museum specimens and a 57% success rate amplifying microsatellite markers for seven museum specimens. Our study demonstrates the feasibility of incorporating historical museum specimens into molecular systematic and conservation genetic studies.     

DNA sequence variation at the mitochondrial cytochrome oxidase I subunit among pheromotypes of the sibling taxa Diachrysia chrysitis and D. tutti (Lepidoptera: Noctuidae)

We surveyed variation in the mtDNA cytochrome oxidase subunit I (COI) gene in the noctuid sibling species Diachrysia chrysitis and D. tutti , whose taxonomic status has been queried. Taxonomically, these taxa are separated on the basis of wing pattern and time of flight period. Samples were field-collected from different geographical sites where pheromone traps were baited to attract males containing different mixtures of two blends of pheromone components: (Z)-5-decenyl acetate and (Z)-7-decenyl acetate. Most specimens were sequenced over a 709-bp segment of the COI gene. Single specimens each of D. chrysitis and D. tutti were sequenced over a region of 1.5 kilobases. mtDNA variation within and among D. chrysitis and D. tutti is most simply interpreted as DNA polymorphism within a complex of closely related, but well-differentiated pheromotypes. Maximal nucleotide difference per site among haplotypes was 0.28%, which is at the lower end of the range for interspecific mtDNA nucleotide diversity in Lepidoptera. Coefficient of differentiation G _st was c . 76.3% ± 11.7%, a typical value at the intraspecific level. Sequences revealed stable diagnostic differences between pheromotypes irrespective of geographical origin. Identification of pheromone-trapped males based on morphology remained vague and uncorrelated to mtDNA haplotypes. The survey illustrated the potential utility of direct DNA sequencing in assessing lineage structures or taxon limits among moths that have been previously found to be different using the pheromone mate recognition system, but which have not been subjected to DNA analysis. The results of mtDNA analyses presented here support recognition of chrysitis and tutti lineages as presented in previous allozyme studies.