Mitochondrial DNA as a genetic marker for brown trout, Salmo trutta L., populations

Mitochondrial DNA was examined in natural and hatchery-reared stocks of brown trout, using different methods of restriction analysis. The methods included the development of a brown trout mt DNA hybridization probe through cloning part of the brown trout mitochondrial genome. In addition, fragments were analysed by ethidium bromide staining and end-labelling. The relative merits of each of these methods in assessing levels of genetic relatedness between the natural and hatchery-reared brown trout stocks were evaluated. In addition, the study revealed a diagnostic mtDNA restriction pattern which could be used as a genetic marker for the discrimination of these two groups of brown trout.     

Intra- and inter-specific relationships in the six Italian species of the fairy shrimp genus Chirocephalus: combining allozyme and mtDNA data

This study analysed the levels of genetic differentiation within and among the six Italian species of the fairy shrimp genus Chirocephalus by analysing electrophoretic polymorphisms at 22 enzymatic loci and by sequencing a 665‐bp fragment of the mitochondrial gene encoding for subunit I of cytochrome oxidase. The allozyme data revealed different levels of intra‐specific differentiation; mean θ estimates were low in Chirocephalus salinus, higher and comparable in C. diaphanus and C. kerkyrensis, while C. ruffoi was the most genetically structured species. At the inter‐specific level, C. marchesonii was the most differentiated species, both for allozymes and mtDNA. Phylogenetic relationships deduced from allozymes and mtDNA were not always consistent with each other. This highlights the differences in performance of the two classes of molecular markers and the need of different independent strategies of data analysis to search for possible incongruence. Neither allozymes nor mtDNA supported monophyly of the diaphanus‐group, previously recognized on the basis of the morphology of appendages (antennae and penes). In contrast the molecular results were consistent with the great heterogeneity in resting egg morphology among representatives of the diaphanus‐group.     

Subcellular distribution of selenium during uptake and its influence on mitochondrial oxidations in germinatingVigna radiata L

The metabolic significance of Se in plants is not well documented, though the presence of many selenoenzymes in bacteria and the essentiality of Se in higher animals is established. Since germination is an active process in plant growth and metabolism, the effect of Se was investigated in germinatingVigna radiata L, a nonaccumulating Sedeficient legume. Growth and protein were enhanced in seedlings supplemented with selenium (Se) as sodium selenite in the medium up to 1 μg/mL. The pattern of uptake of⁷⁵Se in the differentiating tissues and the subcellular distribution were investigated. The percentage of incorporation of⁷⁵Se was greater in the mitochondria at the lowest level (0.5 μg/mL) of Se supplementation compared to higher levels of Se exposure. Proteins precipitated from the postmitochondrial supernatant fractions, when separated by means of polyacrylamide gel electrophoresis (PAGE), indicated a major selenoprotein in the seedlings germinated at 2.0 μg/mL Se. In seedlings grown with supplemented Se, enhanced respiratory control ratio and succinate dehydrogenase activity were observed in the mitochondria of tissues, indicative of a role for Se in mitochondrial membrane functions.     

Gene rearrangements in Chlamydomonas chloroplast DNAs are accounted for by inversions and by the expansion/contraction of the inverted repeat

To gain insight into the mutational events responsible for the extensive variation of chloroplast DNA (cpDNA) within the green algal genus Chlamydomonas, we have investigated the chloroplast gene organization of Chlamydomonas pitschmannii, a close relative of the interfertile species C. eugametos and C. moewusii whose cpDNAs have been well characterized. At 187 kb, the circular cpDNA of C. pitschmannii is the smallest Chlamydomonas cpDNA yet reported; it is 56 and 105 kb smaller than those of its C. eugametos and C. moewusii counterparts, respectively. Despite this substantial size difference, the arrangement of 77 genes on the C. pitschmannii cpDNA displays only three noticeable differences from the organization of the corresponding genes on the collinear C. eugametos and C. moewusii cpDNAs. These changes in gene order are accounted for by the expansion/contraction of the inverted repeat and one or two inversions in a single-copy region. In land plant cpDNAs, these kinds of events are also responsible for gene rearrangements. The large size difference between the C. pitschmannii and C. eugametos/C. moewusii cpDNAs is mainly attributed to multiple events of deletions/additions as opposed to the usually observed expansion/contraction of the inverted repeat in land plant cpDNAs. We also found that the mitochondrial genome of C. pitschmannii is a circular DNA molecule of 16.5 kb which is 5.5 and 7.5 kb smaller than its C. moewusii and C. eugametos counterparts, respectively.     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

An effective method for isolating DNA from historical specimens of baleen

DNA was isolated from an early twentieth century museum specimen of northern right whale baleen. A system of stringent controls and a novel set of cetacean specific primers eliminated contamination from external sources and ensured the authenticity of the results. Sequence analysis revealed that there were informative nucleotide positions between the museum specimen and extant members of the population and closely related species. The results indicate that museum specimens of baleen can be used to assess historical genetic population structure of the great whales.     

Cpn60 is exclusively localized into mitochondria of rat liver and embryonic Drosophila cells

Several reports have claimed that the mitochondrial chaperonin cpn60, or a close homolog, is also present in some other subcellular compartments of the eukaryotic cell. Immunoelectron microscopy studies, using a polyclonal serum against cpn60, revealed that the protein is exclusively localized within the mitochondria of rat liver and embryonic Drosophila cells (SL2). Furthermore, no cpn60 immunoreactive material could be found within the nucleus of SL2 cells subjected to a 1 h 37°C heat-shock treatment. In contrast to these findings, immunoelectron microscopy studies, using a cpn60 monoclonal antibody, revealed mitochondrial and extramitochondrial (plasma membrane, nucleus) immunoreactive material in rat liver cells. Surprisingly, the monoclonal antibody also reacted with fixed proteins of the mature red blood cell. The monoclonal antibody, as well as cpn60 polyclonal sera, only recognize mitochondrial cpn60 in Western blots of liver proteins. Furthermore, none of the cpn60 antibodies used in this study recognized blotted proteins from rat red blood cells. Therefore, we suggest that the reported extramitochondrial localization of cpn60 in metazoan cells may be due to cross-reactivity of some of cpn60 antibodies with conformational epitopes also present in distantly related cpn60 protein homologs that are preserved during fixation procedures of the cells. © 1995 Wiley-Liss, Inc.     

Outgroup heteroduplex analysis using temperature gradient gel electrophoresis: high resolution, large scale, screening of DNA variation in the mitochondrial control region

The ability of DNA screening techniques such as Temperature Gradient Gel Electrophoresis (TGGE) and Heteroduplex Analysis to provide resolution approaching that provided by DNA sequencing for a fraction of the time, effort and expense point to them as the logical successor to allozyme electrophoresis for population genetics. Here we present a novel alternative to the standard TGGE/Heteroduplex Analysis protocol - Outgroup Heteroduplex Analysis (OHA). We assess this technique's sensitivity in comparison to previous screening approaches using a known hierarchy of sequence differences. Our data show that Outgroup Heteroduplex Analysis has greatly increased sensitivity for screening DNA variants from that of TGGE used alone and is easily applicable to large numbers of samples. Using this technique we can consistently detect differences of as small as one base change in a 433-base-pair fragment of Control Region mitochondrial DNA from Melomys cerbinipes (an Australian rodent). The approach should easily be extendable to nuclear loci and is not necessarily dependent on the use of a denaturing gradient When combined with a targeted sequencing effort, OHA provides a sensitive and simple means of obtaining allele/haplotype frequencies and their phylogenies for population and phylogeographic studies in molecular ecology.