On the relationship between the frequency of two types of lethal-mutation and x-ray doses in Drosophila

The dose-frequency relationship for each of 2 types of lethal mutations, fractional- and whole-lethal, was obtained using X-rays on Drosophila melanogaster. The results show that fractional-lethal mutations are induced by X-rays, and also that the proportion of fractional-lethal mutations in the total of mutations tends to decrease with increasing doses, namely, 61% at o R, 47% at 500 R, 37% at 1000 R and 20% at 2000 R. The same tendency is observed with visible mutations. In order to consider the problems related to the above results, the relationship between the true frequency and the observed frequency of the induced lethal mutations is discussed, taking into consideration the existence of the ostensible whole-lethal and the ostensible normal.     

Molecular diversity and relationship within Lactococcus lactis, as revealed by randomly amplified polymorphic DNA (RAPD)

Lactococcus lactis strains are widely used in industrial dairy fermentations. Conventional phenotypic tests have been used for years to classify members of this species into two subspecies, lactis and cremoris, and play a key role in the choice of strains to be used in particular cheese fermentations. DNA hybridisation techniques have also been used for strain classification, giving rise to two genome homology groups. However, results showed discrepancies between the two methods of classification. We applied the randomly amplified polymorphic DNA fingerprinting (RAPD) technique to resolve previous contradictions in lactococcal classifications. Unlike usual RAPD methods, we use three primers to classify 113 strains and integrate the resulting information by a digitised programme used for this purpose. Our analysis revealed three major RAPD groups, designated G1, G2 and G3. G1 and G3 contain strains of the lactis subspecies, and G2 contains strains of the cremoris subspecies, as previously defined by phenotypic characteristics. Moreover, group G1 corresponds to one genome homology group, and groups G2 and G3 correspond to the second one. The taxonomic structure within L. lactis is therefore unusual: two distinct genetic groups of strains show indistinguishable phenotypes, while conversely, two phenotypically distinct groups are genetically homologous. We hypothesize that a subfamily of the subsp. lactis group gave rise to the cremoris subspecies.     

Comparisons of two polymorphic species of Ostertagia and phylogenetic relationships within the Ostertagiinae (Nematoda: Trichostrongyloidea) inferred from ribosomal DNA repeat and mitochondrial DNA sequences

The first internal transcribed spacer DNA (ITS-1) (rDNA) and the mitochondrial (mt) DNA-derived cytochrome oxidase I gene (COX-1) were enzymatically amplified, cloned and sequenced from 6 nominal species of Ostertagiinae as well as Haemonchus contortus and Haemonchus placei. The portion of the COX-1 gene analyzed was 393 base pairs (bp) in length and contained 33 within species polymorphic base changes at 28 synonymous sites. The ITS-1 rDNA consensus sequences ranged from 392 bp (Ostertagia ostertagi/Ostertagia lyrata, Teladorsagia circumcincta) to 404 bp (H. contortus, H. placei). These data were used both in a distance analysis to assess the concept of polymorphic species within the genus Ostertagia and in parsimony analysis to assess phylogenetic relationships within a limited group of Ostertagiinae. Pairwise similarity scores of both ITS-1 and COX-1 data showed the highest number of conserved sites between the proposed dimorphic species of Ostertagia. The level of similarity was lower in the COX-1 data due to the high number of synonymous base changes. Analysis by maximum parsimony of the same data did not refute O. ostertagi/O. lyrata and Ostertagia mossil/Ostertagia dikmansi as dimorphic species and supported monophyly of these ostertagiines relative to representatives of the haemonchine outgroup. In the single most parsimonious tree from ITS-1 rDNA data, a subclade of Ostertagia spp. included forms possessing parallel synlophes and long esophageal valves that typically occur in cervid hosts.     

A satellite DNA of the Sparidae family (pisces, perciformes) associated with telomeric sequences

This paper gives an overview of a lecture scheduled for the opening of the 10th European Bioenergetics Congress. In this lecture I plan to first reflect on the accomplishments of some of the individuals who were involved in research on the ATP synthase during the past 50 years. Then I will give a brief view of the present information about rotational catalysis by the ATP synthase. This will be followed by a discussion of some results from my laboratory that call for additional experimentation. Finally I will direct attention to other questions about the ATP synthase that should be addressed in future studies.     

Comparison between the complete mtDNA sequences of the blue and the fin whale, two species that can hybridize in nature

The sequence of the mitochondrial DNA (mtDNA) molecule of the blue whale (Balaenoptera musculus) was determined. The molecule is 16,402 bp long and its organization conforms with that of other eutherian mammals. The molecule was compared with the mtDNA of the congeneric fin whale (B. physalus). It was recently documented that the two species can hybridize and that male offspring are infertile whereas female offspring may be fertile. The present comparison made it possible to determine the degree of mtDNA difference that occurs between two species that are not completely separated by hybridization incompatibility. The difference between the complete mtDNA sequences was 7.4%. Lengths of peptide coding genes were the same in both species. Except for a small portion of the control region, disruption in alignment was usually limited to insertion/deletion of a single nucleotide. Nucleotide differences between peptide coding genes ranged from 7.1 to 10.5%, and difference at the inferred amino acid level was 0.0-7.9%. In the rRNA genes the mean transition difference was 3.8%. This figure is similar in degree to the difference (3.4%) between the 12S rRNA gene of humans and the chimpanzee. The mtDNA differences between the two whale species, involving both peptide coding and rRNA genes, suggest an evolutionary separation of > or = 5 million years. Although hybridization between more distantly related mammalian species may not be excluded, it is probable that the blue and fin whales are nearly as different in their mtDNA sequences as hybridizing mammal species may be.     

The genetic relationship between the Finns and the Finnish Saami (Lapps): analysis of nuclear DNA and mtDNA

The genetic relationships between two Finno-Ugric-speaking populations, the Finns and the Finnish Saami (Lapps), were studied by using PCR for six nuclear-DNA marker loci, mitochondrial restriction-site polymorphism, and sequence variation of a 360-bp segment of the mitochondrial control region. The allele frequencies of each of the nuclear-DNA marker loci and the frequencies of mtDNA restriction haplotypes were significantly different between the populations. The Saami showed exceptionally low variation in their mtDNA restriction sites. The 9-bp deletion common in East Asian populations was not observed, nor did the haplotype data fit into the haplogroup categorization of Torroni et al. The average number of nucleotide substitutions from the mtDNA haplotype data indicated that the Finnish Saami may be closer to the Finns than to the other reference populations, whereas nuclear DNA suggested that the Finns are more closely related to the European reference populations than to the Finnish Saami. The similarity of the Finns to the other Europeans was even more pronounced according to the sequence data. We were unable to distinguish between the Finns and either the Swiss or Sardinian reference populations, whereas the Finnish Saami clearly stood apart. The Finnish Saami are distinct from other Circumarctic populations, although two of the lineages found among the Saami showed closer relationship to the Circumarctic than to the European lineages. The sequence data indicated an exceptionally high divergence for the Saami mtDNA control lineages. The distribution of the pairwise nucleotide differences in the Saami suggested that this population has not experienced an expansion similar to what was indicated for the Finns and the reference populations.     

Cytobiological studies on hemocyanin metabolism in the branchial heart complex of the common cuttlefish Sepia officinalis (Cephalopoda, Dibranchiata)

The present study confirms previous investigations that demonstrated a high copper content in the branchial heart and its appendage, and that gave the first indication that this organ complex might be involved in hemocyanin metabolism in Sepia officinalis L. Immunocytochemical localization of hemocyanin molecules within the endocytotic lysosomal system of the ovoid cells and tracer experiments with 125I-labeled Sepia hemocyanin suggest its endocytotic uptake. Energy-dispersive X-ray microanalysis and histochemical methods reveal a high copper content within the ovoid cells of the branchial heart. In view of the turnover of the respiratory pigment in the branchial heart of Sepia officinalis L., we believe that the ovoid cells are a site of hemocyanin catabolism.     

Extensive direct-tandem organization of a long repeat DNA sequence on the Y chromosome of chinook salmon (Oncorhynchus tshawytscha)

A long repetitive DNA sequence (OtY8) has been cloned from male chinook salmon and its genomic organization has been characterized. The repeat has a unit length of 8 kb and is present approximately 300 times per diploid male nucleus. All internal fragments within the 8-kb repeat segregate from father to son, suggesting that the entire repeat unit is located on the Y chromosome. The organization of this sequence into an 8-kb repeat unit is restricted to the Y chromosome, as are several male-specific repeat subtypes identified on the basis of restriction-site variation. The repeat possesses only weak internal sequence similarities, suggesting that OtY8 has not arisen by duplication of a smaller repeat unit, as is the case for other long tandem arrays found in eukaryotes. Based on a laddered pattern arising from partial digestion of genomic DNA with a restriction enzyme which cuts only once per repeat unit, this sequence is not dispersed on the Y chromosome but is organized as a head-to-tail tandem array. Pulse-gel electrophoresis reveals that the direct-tandem repeats are organized into at least six separate clusters containing approximately 12 to 250 copies, comprising some 2.4 Mb of Y-chromosomal DNA in total. Related sequences with nucleotide substitutions and DNA insertions relative to the Y-chromosomal fragment are found elsewhere in the genome but at much lower copy number and, although similar sequences are also found in other salmonid species, the amplification of the repeat into a Y-chromosome-linked tandem array is only observed in chinook salmon. The OtY8 repetitive sequence is genetically tightly associated with the sex-determination locus and provides an opportunity to examine the evolution of the Y chromosome and sex determination process in a lower vertebrate.     

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.     

Molecular basis of hereditary methaemoglobinaemia, types I and II: two novel mutations in the NADH-cytochrome b5 reductase gene

Hereditary methaemoglobinaemia, caused by deficiency of NADH-cytochrome b5 reductase (b5R), has been classified into two types, an erythrocyte (type I) and a generalized (type II). We analysed the b5R gene of two Thai patients and found two novel mutations. The patient with type II was homozygous for a C-to-T substitution in codon 8 3 that changes Arg (CGA) to a stop codon (TGA), resulting in a truncated b5R without the catalytic portion. The patient with type I was homozygous for a C-to-T substitution in codon 178 causing replacement of Ala (GCG) with Val (GTG). To characterize effects of this missense mutation, we investigated enzymatic properties of mutant b5R (Ala 178 Val). Although the mutant enzyme showed normal catalytic activity, less stability and different spectra were observed. These results suggest that this substitution influenced enzyme stability due to the slight change of structure. In conclusion, the nonsense mutation led to type II because of malfunction of the truncated protein. On the other hand, the missense mutation caused type I, due to degradation of the unstable mutant enzyme with normal activities in patient's erythrocytes, because of the lack of compensation by new protein synthesis during the long life-span of erythrocytes.     

Dielectric study of the interaction between DNA and an oligopeptide (lysine-tyrosine-lysine)

Interaction between Na-DNA and the oligopeptide lysine-tyrosine-lysine (LTL) is studied by a dielectric method. The comparison between conductivities (at the frequence of 5MHz) of LTL alone and of the complex LTL-DNA allows us to show up an electrostatic interaction between LTL and phosphates sites of DNA. During the formation of the complex LTL-DNA, a certain fraction of Na+ counter-ions is ejected from the phosphates sites.     

Molecular effects of 2',2'-difluorodeoxycytidine (Gemcitabine) on DNA replication in intact HL-60 cells

The ability of pH-step alkaline elution to isolate different size species of nascent DNA (nDNA) from intact cells was utilized to study the effects of 2',2'-difluorodeoxycytidine (dFdC) on DNA replication in HL-60 cells. Preincubation with dFdC caused a concentration-dependent decrease in overall [3H]thymidine incorporation into DNA, accompanied by an increase in the proportion of radiolabel accumulated in small nDNA fragments. Twenty-four hours following removal of dFdC, radiolabel progressed from smaller to larger fragments and into genomic-length DNA. At initial concentrations of exposures to dFdC or cytosine arabinoside (ara-C) that caused 50% lethality (LC50) to HL-60 cells (40 and 50 nM, respectively), slower and less complete transit of nDNA from small subreplicon-length fragments through larger intermediates to genomic-length DNA was observed for nDNA fragments containing incorporated [3H]dFdC than for fragments containing [3H]ara-C. This was accomplished with less [3H]dFdC incorporated into DNA than [3H]ara-C at these extracellular concentrations of drug. Pulse-chase studies, using higher concentrations of radiolabeled drug, similarly revealed that nDNA fragments containing incorporated dFdC, like those containing ara-C, progressed with respect to time into larger nDNA intermediates and ultimately into genomic-length DNA; however, such progression for nDNA fragments containing dFdC was less complete than for fragments containing ara-C. The radioactivity incorporated into DNA represented authentic dFdC, as determined by DNA degradation studies, and was stable in DNA for at least 48 hr after removal of extracellular [3H]dFdC. Some of the effects of dFdC on ribonucleotide reduction in HL-60 cells were assessed by measurement of the intracellular pools of dCTP and dGTP. The drug had a greater effect on pools of dGTP than of dCTP, with transient reductions in dGTP observed at concentrations that encompass the LC50 for dFdC. These studies suggest that the interaction with DNA synthesis is an important component of the cytotoxicity of dFdC in HL-60 cells. Because it is incorporated progressively through nDNA compartments and ultimately into genomic-length DNA, dFdC should be categorized as an agent that slows DNA elongation in the intact cell, and not as a chain terminator in the absolute sense.     

Vasopressin in the forebrain of common marmosets (Callithrix jacchus): studies with in situ hybridization, immunocytochemistry and receptor autoradiography

The distribution of vasopressin (AVP) producing cells, their projections and AVP receptors was examined in the brain of common marmosets (Callithrix jacchus) using in situ hybridization, immunocytochemistry and receptor autoradiography. Clusters of cells labeled for AVP mRNA or stained for AVP immunoreactivity (AVP-ir) were found in the paraventricular (PVN), supraoptic (SON) and suprachiasmatic nuclei (SCN) of the hypothalamus. Scattered AVP producing cells were also found in the lateral hypothalamus and the bed nucleus of the stria terminalis (BST). Neither AVP mRNA-labeled nor AVP-ir cells were detected in the amygdala. Although AVP-ir fibers were evident outside of the hypothalamic-neurohypophyseal tract, a plexus of fibers in the lateral septum, as observed in the rat brain, was not detected. Receptor autoradiography using 125I-linear-AVP revealed specific binding for AVP receptors in the nucleus accumbens, diagonal band, lateral septum, the BST, SCN, PVN, amygdala, anterodorsal and ventromedial nucleus of the hypothalamus, indicating sites for central AVP action in the marmoset brain. Together, these data provide a comprehensive picture of AVP pathways in the marmoset brain, demonstrating differences from rodents in the distribution of cell bodies, fibers and receptors.     

Molecular phylogeny of forest and field mice of the genus Apodemus (Muridae, Rodentia) based on the data on restriction analysis of total nuclear DNA

Based on restriction-fragment length polymorphism (RFLP) of total nuclear DNA (nDNA), analyses of phylogenetic relations and genetic similarity were performed in nine species of forest and field mice of the genus Apodemus. Genetic distances calculated for different species pairs ranged from 0.24 to 12.53%; i.e., the differences were 50-fold. The estimated evolutionary age of the genus Apodemus is approximately 12 million years. In general, the obtained data on genetic similarity and phylogenetic relationship allow us to differentiate at least three groups of species: (1) southern Paleoarctic (A. argenteus), (2) eastern (A. peninsulae, A. speciosus, and A. agrarius), and (3) western (A. sylvaticus, A. flavicollis, A. ponticus, A. uralensis, and A. fulvipectus) ones. The latter two groups are related to the northern Paleoarctic. Such a division into groups corresponds to characteristic features of karyotype organization and segmentation of satellite DNA (satDNA) of these species, as well as the nature of variation in isozymes and in a fragment of the enzyme-encoding sequence of cytochrome b gene isolated from the mitochondrial genome. Species groups (1) and (3) exhibited a high probability of a monophyletic origin (70 and 99%, respectively). Group (2) is unlikely to be monophyletic, and the genetic distances in it are significantly greater than those in group 3. A. argenteus is the most diverged, both phenogenetically and phylogenetically. The data are consistent with a new zoological classification, which assumes the division of the unified genus Apodemus into two taxa of generic rank and suggest that the southern Paleoarctic forest mouse should be regarded as a separate taxon of at least subgeneric rank.     

The relationship between capillary and venous concentrations of the antimalarial drug lumefantrine (benflumetol)

The study aimed to determine the antibacterial therapy effective in the cure of endocarditis caused by Enterococcus faecalis resistant to clinically achievable levels of vancomycin. Isolation of the causative enterococcus had been achieved by direct inoculation of the resected valve into the culture medium in theatre. The patient was known to have had an aortic valve defect since childhood and had recently undergone splenectomy following trauma. Blood cultures were negative prior to valve replacement. A perivalvular abscess was noted at operation. In vitro minimal bactericidal results and serum activity were the basis of the postoperative choice of drugs. The minimal bactericidal level of teicoplanin was 250 micrograms/ml and that of amoxycillin 64 micrograms/ml. Neither is achievable with the advocated dosage. A combination of these two cell-wall-active agents successfully eliminated the infection. Acting at two different sites in the synthesis of the bacterial cell wall, teicoplanin and amoxycillin were found to be bactericidal in vitro at the trough levels of the antibiotics in the serum. The patient recovered fully.     

Molecular evolution of two lineages of L1 (LINE-1) retrotransposons in the california mouse, Peromyscus californicus

The large number of L1 [long interspersed elements (LINE)-1] sequences found in the genome is due to the insertion of copies of the retrotransposon over evolutionary time. The majority of copies appear to be replicates of a few active, or "master" templates. A continual replacement of master templates over time gives rise to lineages distinguishable by their own unique set of shared-sequence variants. A previous analysis of L1 sequences in deer mice, Peromyscus maniculatus and P. leucopus, revealed two active L1 lineages, marked by different rates of evolution, whose most recent common ancestor predates the expansion of the Peromyscus species. Here we exploit lineage-specific, shared-sequence variants to reveal a paucity of Lineage 2 sequences in at least one species, P. californicus. The dearth of Lineage 2 copies in P. californicus suggests that Lineage 2 may have been unproductive until after the most recent common ancestor of P. californicus and P. maniculatus. We also show that Lineage 1 appears to have a higher rate of evolution in P. maniculatus relative to either P. californicus or P. leucopus. As a phylogenetic tool, L1 lineage-specific variants support a close affinity between P. californicus and P. eremicus relative to the other species examined.     

Molecular phylogeny of the New World monkeys (Platyrrhini, primates) based on two unlinked nuclear genes: IRBP intron 1 and epsilon-globin sequences

Nuclear sequences of the 1.8 kilobase (kb) long intron 1 of the interstitial retinol-binding protein gene (IRBP), previously determined for 11 of the 16 extant genera of New World monkeys (superfamily Ceboidea, infraorder Platyrrhini), have now been determined for the remaining 5 genera. The maximum parsimony trees found, first with IRBP sequences alone and then with tandemly combined IRBP and epsilon-globin gene sequences from the same species, supported a provisional cladistic classification with the following clusters. Subtribes Callitrichina (Callithrix, Cebuella), Callimiconina (Callimico), Leontopithecina (Leontopithecus) and Saguina (Saguinus) constitute subfamily Callitrichinae, and subfamilies Callitrichinae, Aotinae (Aotus), and Cebinae (Cebus, Saimiri) constitute family Cebidae. Subtribes Chiropotina (Chiropotes, Cacajao) and Pitheciina (Pithecia) constitute tribe Pitheciini; and tribes Pitheciini and Callicebini (Callicebus) constitute subfamily Pitheciinae. Subtribes Brachytelina (Brachyteles, Lagothrix) and Atelina (Ateles) constitute tribe Atelini, and tribes Atelini and Alouattini (Alouatta) constitute subfamily Atelinae. The parsimony results were equivocal as to whether Pitheciinae should be grouped with Atelinae in family Atelidae or have its own family Pitheciidae. The cladistic groupings of extant ceboids were also examined by different stochastic evolutionary models that employed the same stochastic process of nucleotide substitutions but alternative putative phylogenetic trees on which the nucleotide substitutions occurred. Each model, i.e., each different tree, predicted a different multinomial distribution of nucleotide character patterns for the contemporary sequences. The predicted distributions that were closest to the actual observed distributions identified the best fitting trees. The cladistic relationships depicted in these best fitting trees agreed in almost all cases with those depicted in the maximum parsimony trees.     

The relationship of the -5, -8, and -24 variant alleles in African Americans to triosephosphate isomerase (TPI) enzyme activity and to TPI deficiency

In 424 African-American and 75 white subjects, we found that the -5 (TPI 592 A-->G), -8 (TPI 589 G-->A), and -24 (TPI 573 T-->G) variants in the triosephosphate isomerase (TPI) gene occurred frequently (41.0%) in the African-American subjects but did not occur in the whites. These data suggest that this set of polymorphisms may turn out to be one of the higher-incidence molecular markers of African lineage, a surprising finding because others had reported that these nucleotide substitutions were restricted to a small subset of African Americans who had been characterized as TPI-deficiency heterozygotes. Additionally, we investigated the relationship of these variants to TPI-enzyme activity. Although the variant substitutions (occurring in three haplotypes: -5 alone, -5 -8, and -5 -8 -24) were associated with moderate reduction in enzyme activity, severe-deficiency heterozygotes could not be identified with certainty, and none of the haplotypes were restricted to subjects with marked reduction of enzyme activity. Three subjects were homozygous for the -5 -8 haplotype, a finding inconsistent with the putative role of this haplotype as the cause of a null variant incompatible with life in homozygotes. Despite these findings, the possibility remains that the -5 -8 or -5 -8 -24 haplotypes may in some instances contribute to compound heterozygosity and clinical TPI deficiency.