The complete mitochondrial genome of the hermaphroditic freshwater mussel Anodonta cygnea (Bivalvia: Unionidae): in silico analyses of sex-specific ORFs across order Unionoida.

BACKGROUND: Doubly uniparental inheritance (DUI) of mitochondrial DNA in bivalves is a fascinating exception to strictly maternal inheritance as practiced by all other animals. Recent work on DUI suggests that there may be unique regions of the mitochondrial genomes that play a role in sex determination and/or sexual development in freshwater mussels (order Unionoida). In this study, one complete mitochondrial genome of the hermaphroditic swan mussel, Anodonta cygnea, is sequenced and compared to the complete mitochondrial genome of the gonochoric duck mussel, Anodonta anatina. An in silico assessment of novel proteins found within freshwater bivalve species (known as F-, H-, and M-open reading frames or ORFs) is conducted, with special attention to putative transmembrane domains (TMs), signal peptides (SPs), signal cleavage sites (SCS), subcellular localization, and potential control regions. Characteristics of TMs are also examined across freshwater mussel lineages. RESULTS: In silico analyses suggests the presence of SPs and SCSs and provides some insight into possible function(s) of these novel ORFs. The assessed confidence in these structures and functions was highly variable, possibly due to the novelty of these proteins. The number and topology of putative TMs appear to be maintained among both F- and H-ORFs, however, this is not the case for M-ORFs. There does not appear to be a typical control region in H-type mitochondrial DNA, especially given the loss of tandem repeats in unassigned regions when compared to F-type mtDNA. CONCLUSION: In silico analyses provides a useful tool to discover patterns in DUI and to navigate further in situ analyses related to DUI in freshwater mussels. In situ analysis will be necessary to further explore the intracellular localizations and possible role of these open reading frames in the process of sex determination in freshwater mussel.

Sperm motility in Mytilus edulis in relation to mitochondrial DNA polymorphisms: implications for the evolution of doubly uniparental inheritance in bivalves.

Bivalves of the families Mytilidae, Unionidae, and Veneridae have an unusual mode of mitochondrial DNA (mtDNA) transmission called doubly uniparental inheritance (DUI). A characteristic feature of DUI is the presence of two gender-associated mtDNA genomes that are transmitted through males (M-type mtDNA) and females (F-type mtDNA), respectively. Female mussels are predominantly homoplasmic with only the F-type expressed in both somatic and gonadal tissue; males are heteroplasmic with the M-type expressed in the gonad and F-type in somatic tissue for the most part. An unusual evolutionary feature of this system is that an mt genome with F-coding sequences occasionally invades the male route of inheritance (i.e., a "role reversal" event), and is thereafter transmitted as a new M-type. Phylogenetic studies have demonstrated that the new or "recently masculinized" M-types may eventually replace the older or "standard" M-types over time. To investigate whether this replacement process could be due to an advantage in sperm swimming behavior, we measured differences in motility parameters and found that sperm with the recently masculinized M-type had significantly faster curvilinear velocity and average path velocity when compared to sperm with standard M-type. This increase in sperm swimming speed could explain the multiple evolutionary replacements of standard M-types by masculinized M-types that have been hypothesized for the mytilid lineage. However, our observations do not support the hypothesis that DUI originated because it permits the evolution of mitochondrial adaptations specific to sperm performance, otherwise, the evolutionarily older, standard M genome should perform better.

Reproductive function for a C-terminus extended, male-transmitted cytochrome c oxidase subunit II protein expressed in both spermatozoa and eggs.

Our previous study documented expression of a male-transmitted cytochrome c oxidase subunit II protein (MCOX2), with a C-terminus extension (MCOX2e), in unionoidean bivalve testes and sperm mitochondria. Here, we present evidence demonstrating that MCOX2 is seasonally expressed in testis, with a peak shortly before fertilization that is independent of sperm density. MCOX2 is localized to the inner and outer sperm mitochondrial membranes and the MCOX2 antibody's epitope is conserved across >65 million years of evolution. We also demonstrate the presence of male-transmitted mtDNA and season-specific MCOX2 spatial variation in ovaries. We hypothesize that MCOX2 plays a role in reproduction through gamete maturation, fertilization and/or embryogenesis.

Presence of a unique male-specific extension of C-terminus to the cytochrome c oxidase subunit II protein coded by the male-transmitted mitochondrial genome of Venustaconcha ellipsiformis (Bivalvia: Unionoidea).

Analyses of unionoidean bivalve male-transmitted (M) mtDNA genomes revealed an approximately 555 bp 3' coding extension to cox2. An antibody was generated against this predicted C-terminus extension to determine if the unique cox2 protein is expressed. Western blot and immunohistochemistry analyses demonstrated that the protein was predominantly expressed in testes. Weak expression was detected in other male tissues but the protein was not detected in female tissues. This is the first report documenting the expression of a cox2 protein with a long C-terminus in animals. Its universal presence in unionoidean bivalve testes suggests a functional significance for the protein.

Degree of selective constraint as an explanation of the different rates of evolution of gender-specific mitochondrial DNA lineages in the mussel mytilus.

Mussels of the genus Mytilus segregate for a maternally transmitted F lineage and a paternally transmitted M lineage of mitochondrial DNA. Previous studies demonstrated that these lineages are older than the species of the M. edulis complex and that the M lineage evolves faster than the F lineage. Here we show that the latter observation also applies to a region of the molecule with no assigned function. Sequence data for the mitochondrial COIII gene and the "unassigned" region of the F and M lineages of M. edulis and M. trossulus are used to evaluate various hypotheses that may account for the faster rate of evolution of the M lineage. Tests based on the proportion of synonymous and nonsynonymous substitutions suggest that the M lineage experiences relatively relaxed selection. Further support for this hypothesis comes from an examination of COIII amino acid substitutions at sites defined as either conserved or variable based on the pattern of variation in other mollusks and Drosophila. Most substitutions in the M lineage occur in regions that are also variable among non-Mytilus taxa. We suggest that these differences in selection pressure are a consequence of doubly uniparental mitochondrial DNA transmission in Mytilus.

Species-specific segregation of gender-associated mitochondrial DNA types in an area where two mussel species (Mytilus edulis and M. trossulus) hybridize.

In each of the mussel species Mytilus edulis and M. trossulus there exist two types of mtDNA, the F type transmitted through females and the M type transmitted through males. Because the two species produce fertile hybrids in nature, F and M types of one may introgress into the other. We present the results from a survey of a population in which extensive hybridization occurs between these two species. Among specimens classified as "pure" M. edulis or "pure" M. trossulus on the basis of allozyme analysis, we observed no animal that carried the F or the M mitotype of the other species. In most animals of mixed nuclear background, an individual's mtDNA came from the species that contributed the majority of the individual's nuclear genes. Most importantly, the two mtDNA types in post-F1 male hybrids were of the same species origin. We interpret this to mean that there are intrinsic barriers to the exchange of mtDNA between these two species. Because such barriers were not noted in other hybridizing species pairs (many being even less interfertile than M. edulis and M. trossulus), their presence in Mytilus could be another feature of the unusual mtDNA system in this genus.

Nerve conduction velocity in hypertensive patients.

Due to conflicting reports in the literature regarding nerve conduction velocities (NCVs) in hypertensives, peroneal and sural NCVs and facial nerve conduction latencies were studied in 30 hypertensives and in 30 controls. An improved technique of NCV measurement was used. Twenty-one of the hypertensives were retested after five weeks, and five of them were tested for motor and sensory NCVs of the median nerve during a short period of partial occlusion of blood flow in the arm. No changes were found that could be related to blood pressure, duration of hypertension, eyeground changes, or partial restriction of blood flow.

High fidelity of mitochondrial genome transmission under the doubly uniparental mode of inheritance in freshwater mussels (Bivalvia: Unionoidea).

Doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA) has been demonstrated in both mytilid and unionid bivalves. Under DUI, females pass on their mtDNA to both sons and daughters, whereas males pass on their mtDNA to only sons. In mytilids, the loss of an original male (or M) mitotype, with its subsequent replacement by that lineage's female (or F) mitotype, has been called a role-reversal or, more specifically, a masculinization event. Multiple masculinization events have been inferred during the evolutionary history of mytilids but not unionids. The perceived lack of role-reversal events in unionids may represent a significant difference in the evolutionary dynamics of DUI between the two bivalve taxa or simply a lack of sufficient taxon sampling in unionids. To evaluate these alternative hypotheses, six additional unionoidean bivalve genera were sampled for DUI including one genus from the sister taxon of the Unionidae, the Hyriidae. Phylogenetic analyses of 619 base pairs of cytochrome c oxidase I (COI) from eight genera (nine species) of unionoidean bivalves, plus the sister taxon to the Unionoida, Neotrigonia, revealed that the M and F unionoidean mitotypes were contained in gender-specific, topologically congruent clades. This supports the hypothesis that either role-reversal events do not occur in unionoideans or, if they do occur, their products are ephemeral in an evolutionary sense. Furthermore, the fact that the mantle-tissue-derived Neotrigonia mitotype is the sister mitotype to the unionoidean F mitotype clade suggests that DUI has been operating with high fidelity in unionoids for at least 200 million years. A relatively low incidence of interspecific hybridization in unionoideans and a possibly obligate role for the M mitotype in unionoidean gender determination are offered as potential explanations for the disparate evolutionary dynamics of DUI observed between mytilid and unionoidean bivalves.

Primary aldosteronism. A study in contrasts.

Two patient with primary aldosteronism, one with a solitary adrenal adenoma and the other with bilateral nodular hyperplasia, are described. Both patients showed the classic features of primary aldosteronism in electrolyte and hormone patterns, but there were important differences in the biochemistry of their excised adrenal tissue. In addition, the injection of plasma from the patient with bilateral adrenal hyperplasia into the sheep's transplanted adrenal gland elicited a definite aldosterone secretory response, but there was no aldosterone response to the injection of plasma from the patient with a solitary adrenal adenoma. The findings support the hypothesis that an extra-adrenal stimulus may contribute to the pathogenesis of bilateral adrenal hyperplasia.

Evolution of mtDNA D-loop sequences and their use in phylogenetic studies of shrews in the subgenus Otisorex (Sorex: Soricidae: Insectivora).

mtDNA D-loop sequences were examined in shrews of the genus Sorex. All specimens possessed an array of tandem repeats in which each repeat was 78 to 80 base pairs (bp) in length. Each specimen also possessed a 76-bp imperfect copy of the tandem repeats. Three observations are consistent with the tandem repeats being the product of concerted evolution: (1) the repeats are capable of forming secondary structures; (2) there was minimal sequence divergence between tandem repeats within individuals; and (3) although the tandem repeats and the imperfect repeat presumably arose due to a duplication event in an ancestor of the shrews, the imperfect repeat per se was not copied in any of the specimens observed. Interspecific homology can therefore be assumed for the imperfect repeat. Furthermore, given the apparent high rate of concerted evolution within a genome, tandem repeats in different individuals may be compared as though only a single copy were present. By including data from the imperfect repeat, the last tandem repeat, and the surrounding unique sequence cladistic and genetic distance approaches to phylogeny reconstruction indicated two sister groups within the subgenus Otisorex. One group was composed of Sorex fumeus and the S. cinereus species complex and the other group was composed of S. hoyi, S. monticolus, S. vagrans, and S. palustris. Resolution of relationships among recently evolved taxa demonstrated the usefulness of selected regions of the D-loop for molecular systematic studies.

Patterns of sequence variation in the mitochondrial D-loop region of shrews.

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D-loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.

Phylogenetic evidence for role-reversals of gender-associated mitochondrial DNA in Mytilus (Bivalvia: Mytilidae).

Distinct gender-associated mitochondrial DNA (mtDNA) lineages (i.e., lineages which are transmitted either through males or through females) have been demonstrated in two families of bivalves, the Mytilidae (marine mussels) and the Unionidae (freshwater mussels), which have been separated for more than 400 Myr. The mode of transmission of these M (for male-transmitted) and F (for female-transmitted) molecules has been referred to as doubly uniparental inheritance (DUI), in contrast to standard maternal inheritance (SMI), which is the norm in animals. A previous study suggested that at least three origins of DUI are required to explain the phylogenetic pattern of M and F lineages in freshwater and marine mussels. Here we present phylogenetic evidence based on partial sequences of the cytochrome c oxidase subunit I gene and the 16S RNA gene that indicates the DUI is a dynamic phenomenon. Specifically, we demonstrate that F lineages in three species of Mytilus mussels, M. edulis, M. trossulus, and M. californianus, have spawned separate lineages which are now associated only with males. This process is referred to as "masculinization" of F mtDNA. By extension, we propose that DUI may be a primitive bivalve character and that periodic masculinization events combined with extinction of previously existing M types effectively reset the time of divergence between conspecific gender-associated mtDNA lineages.

Molecular phylogeny and evolution of Sorex shrews (Soricidae: insectivora) inferred from mitochondrial DNA sequence data.

Shrews of the genus Sorex are characterized by a Holarctic distribution, and relationships among extant taxa have never been fully resolved. Phylogenies have been proposed based on morphological, karyological, and biochemical comparisons, but these analyses often produced controversial and contradictory results. Phylogenetic analyses of partial mitochondrial cytochrome b gene sequences (1011 bp) were used to examine the relationships among 27 Sorex species. The molecular data suggest that Sorex comprises two major monophyletic lineages, one restricted mostly to the New World and one with a primarily Palearctic distribution. Furthermore, several sister-species relationships are revealed by the analysis. Based on the split between the Soricinae and Crocidurinae subfamilies, we used a 95% confidence interval for both the calibration of a molecular clock and the subsequent calculation of major diversification events within the genus Sorex. Our analysis does not support an unambiguous acceleration of the molecular clock in shrews, the estimated rate being similar to other estimates of mammalian mitochondrial clocks. In addition, the data presented here indicate that estimates from the fossil record greatly underestimate divergence dates among Sorex taxa.

Male and female mitochondrial DNA lineages in the blue mussel (Mytilus edulis) species group.

In blue mussels of the Mytilus edulis species complex, mitochondrial DNA (mtDNA) inheritance is coupled with gender. Females receive their mother's mtDNA and pass it on to both their daughters and sons. In addition, males receive mtDNA from their father and transmit this male mtDNA to their sons. If this pattern of "doubly uniparental inheritance" is older than the M. edulis species complex, then all members of this group must have two distinct mtDNA lineages: a maternal lineage that is transmitted through females and a paternal lineage that is transmitted through males. To test this hypothesis, we scored mtDNA variation in two taxa in this complex, M. edulis and M. trossulus, by means of restriction fragment profiles of whole-mtDNA genomes and DNA sequence of a region of the cytochrome c oxidase subunit III gene (COIII). The various mitotypes present in these mussels were classified as "male" or "female" based on their gender association and as belonging to M. edulis or M. trossulus based on species-specific allozymes. Both maximum parsimony and neighbor-joining phylogenies based on the COIII sequences grouped female and male mtDNAs into two distinct lineages irrespective of specific origin in accordance with the hypothesis that the origin of these lineages predates the divergence of M. edulis and M. trossulus.

Prostate-specific antigen levels from completely sectioned, clinically benign, whole prostates.

Clinically benign whole, untrimmed prostates and pelvic lymph nodes were obtained from 105 patients at autopsy. All 105 patients had premortem serum from which prostate-specific antigen (PSA) levels were obtained. Sixty-eight did not have carcinoma of the prostate (CAP), 28 had CAP less than 1 ml and 9 had CAP larger than 1 ml. Eleven untrimmed prostates weighed 80 g or more and eight had elevated PSA levels (more than 4.0 ng/ml): five of eight without CAP, two of two with CAP less than 1 ml, and one of one with CAP larger than 1 ml. Ninety-four whole untrimmed prostates weighed less than 80 g and 20 had elevated PSA levels: ten of 60 without CAP, two of 26 with CAP less than 1 ml, and eight of eight with CAP larger than 1 ml. This study suggests that PSA levels from patients with untrimmed prostates weighing 80 g or more (equivalent to a 60-g trimmed prostate) are usually elevated regardless whether CAP is present. However, CAP less than 1 ml, in untrimmed prostates less than 80 g, usually does not elevate PSA levels whereas CAP larger than 1 ml usually does (P less than 0.0001). The likelihood that elevated PSA levels, from patients with untrimmed prostates less than 80 g, are due to CAP larger than 1 ml increases as the PSA level increases.