Cross-validation of Peak Oxygen Consumption Prediction Models From OMNI Perceived Exertion.

This study cross-validated statistical models for prediction of peak oxygen consumption using ratings of perceived exertion from the Adult OMNI Cycle Scale of Perceived Exertion. 74 participants (men: n=36; women: n=38) completed a graded cycle exercise test. Ratings of perceived exertion for the overall body, legs, and chest/breathing were recorded each test stage and entered into previously developed 3-stage peak oxygen consumption prediction models. There were no significant differences (p>0.05) between measured and predicted peak oxygen consumption from ratings of perceived exertion for the overall body, legs, and chest/breathing within men (mean±standard deviation: 3.16±0.52 vs. 2.92±0.33 vs. 2.90±0.29 vs. 2.90±0.26 L·min(-1)) and women (2.17±0.29 vs. 2.02±0.22 vs. 2.03±0.19 vs. 2.01±0.19 L·min(-1)) participants. Previously developed statistical models for prediction of peak oxygen consumption based on subpeak OMNI ratings of perceived exertion responses were similar to measured peak oxygen consumption in a separate group of participants. These findings provide practical implications for the use of the original statistical models in standard health-fitness settings.

Differential investment in pre- vs. post-copulatory sexual selection reinforces a cross-continental reversal of sexual size dimorphism in Sepsis punctum (Diptera: Sepsidae).

Theory predicts that males have a limited amount of resources to invest in reproduction, suggesting a trade-off between traits that enhance mate acquisition and those that enhance fertilization success. Here, we investigate the relationship between pre- and post-copulatory investment by comparing the mating behaviour and reproductive morphology of four European and five North American populations of the dung fly Sepsis punctum (Diptera) that display a reversal of sexual size dimorphism (SSD). We show that the geographic reversal in SSD between the continents (male biased in Europe, female biased in North America) is accompanied by differential investment in pre- vs. post-copulatory traits. We find higher remating rates in European populations, where larger males acquire more matings and consequently have evolved relatively larger testes and steeper hyper-allometry with body size. American populations, in sharp contrast, display much reduced, if any, effect of body size on those traits. Instead, North American males demonstrate an increased investment in mate acquisition prior to copulation, with more mounting attempts and a distinctive abdominal courtship display that is completely absent in Europe. When controlling for body size, relative female spermathecal size is similar on both continents, so we find no direct evidence for the co-evolution of male and female internal reproductive morphology. By comparing allopatric populations of the same species that apparently have evolved different mating systems and consequently SSD, we thus indirectly demonstrate differential investment in pre- vs. post-copulatory mechanisms increasing reproductive success.

Two distinct genomic regions, harbouring the period and fruitless genes, affect male courtship song in Drosophila montana.

Acoustic signals often have a significant role in pair formation and in species recognition. Determining the genetic basis of signal divergence will help to understand signal evolution by sexual selection and its role in the speciation process. An earlier study investigated quantitative trait locus for male courtship song carrier frequency (FRE) in Drosophila montana using microsatellite markers. We refined this study by adding to the linkage map markers for 10 candidate genes known to affect song production in Drosophila melanogaster. We also extended the analyses to additional song characters (pulse train length (PTL), pulse number (PN), interpulse interval, pulse length (PL) and cycle number (CN)). Our results indicate that loci in two different regions of the genome control distinct features of the courtship song. Pulse train traits (PTL and PN) mapped to the X chromosome, showing significant linkage with the period gene. In contrast, characters related to song pulse properties (PL, CN and carrier FRE) mapped to the region of chromosome 2 near the candidate gene fruitless, identifying these genes as suitable loci for further investigations. In previous studies, the pulse train traits have been found to vary substantially between Drosophila species, and so are potential species recognition signals, while the pulse traits may be more important in intra-specific mate choice.

Multiple quantitative trait loci influence intra-specific variation in genital morphology between phylogenetically distinct lines of Drosophila montana.

The evolution of animal genitalia has gained renewed interest because of their potential roles during sexual selection and early stages of species formation. Although central to understanding the evolutionary process, knowledge of the genetic basis of natural variation in genital morphology is limited to a very few species. Using an outbred cross between phylogenetically distinct lines of Drosophila montana, we characterized quantitative trait loci (QTLs) affecting the size and shape of the distiphallus, a prominent part of the male intromittent organ. Our microsatellite-based linkage analysis shows that intra-specific variation in the distiphallus involves several QTLs of largely additive effect and that a highly significant QTL co-localizes with the same inversion where we have earlier localized a large QTL for a sexually selected courtship song trait. The latter indicates that inversions can play an important role in shaping the evolution of rapidly evolving traits with a potential influence on speciation.

A microsatellite linkage map for Drosophila montana shows large variation in recombination rates, and a courtship song trait maps to an area of low recombination.

Current advances in genetic analysis are opening up our knowledge of the genetics of species differences, but challenges remain, particularly for out-bred natural populations. We constructed a microsatellite-based linkage map for two out-bred lines of Drosophila montana derived from divergent populations by taking advantage of the Drosophila virilis genome and available cytological maps of both species. Although the placement of markers was quite consistent with cytological predictions, the map indicated large heterogeneity in recombination rates along chromosomes. We also performed a quantitative trait locus (QTL) analysis on a courtship song character (carrier frequency), which differs between populations and is subject to strong sexual selection. Linkage mapping yielded two significant QTLs, which explained 3% and 14% of the variation in carrier frequency, respectively. Interestingly, as in other recent studies of traits which can influence speciation, the strongest QTL mapped to a genomic region partly covered by an inversion polymorphism.

Phylogeographic patterns in Drosophila montana.

The Drosophila virilis species group offers valuable opportunities for studying the roles of chromosomal re-arrangements and mating signals in speciation. The 13 species are divided into two subgroups, the montana and virilis 'phylads'. There is greater differentiation among species within the montana phylad in both karyotype and acoustic signals than exists among members of the virilis phylad. Drosophila montana is a divergent species which is included in the montana phylad. Here, we analyse the phylogeography of D. montana to provide a framework for understanding divergence of acoustic signals among populations. We analysed mitochondrial sequences corresponding to the cytochrome oxidase I and cytochrome oxidase II genes, as well as 16 microsatellite loci, from 108 lines of D. montana covering most of the species' range. The species shows a clear genetic differentiation between North American and Scandinavian populations. Microsatellite allele frequencies and mitochondrial DNA haplotypes gave significant FST values between populations from Canada, USA and Finland. A Bayesian analysis of population structure based on the microsatellite frequencies showed four genetically distinct groups, corresponding to these three populations plus a small sample from Japan. A network based on mitochondrial haplotypes showed two Finnish clades of very different shape and variability, and another clade with all sequences from North America and Japan. All D. montana populations showed evidence of demographic expansion but the patterns inferred by coalescent analysis differed between populations. The divergence times between Scandinavian and North American clades were estimated to range from 450,000 to 900,000 years with populations in Canada and the USA possibly representing descendants of different refugial populations. Long-term separation of D. montana populations could have provided the opportunity for differentiation observed in male signal traits, especially carrier frequency of the song, but relaxation of sexual selection during population expansion may have been necessary.

Patterns of microsatellite variation through a transition zone of a chromosomal cline in Drosophila americana.

Chromosomal rearrangements have been considered as important barriers to gene flow and were often used in the delineation of species. The original taxonomic designation of Drosophila americana americana and Drosophila americana texana is based on the presence/absence of a centric fusion between the X- and fourth chromosomes. D. a. americana presents the derived fused state, whereas Drosophila a. texana presents the freely segregating ancestral state. The degree of genetic separation between the two chromosomal forms is still controversial, with different genetic markers yielding contrasting results even when the same populations were analyzed. Using 27 polymorphic microsatellites, we re-evaluated patterns of genetic differentiation between six D. americana populations sampled through a transition zone of both chromosomal forms in the central United States. Our results clearly reject a scenario of two differentiated species forming a hybrid zone in a region of parapatry and indicate that gene flow minimizes genome-wide differentiation associated with the two chromosomal arrangements.

The essential Drosophila melanogaster gene wds (will die slowly) codes for a WD-repeat protein with seven repeats.

We have isolated and characterized the will die slowly (wds) gene of Drosophila melanogaster, formerly known as l(1)zw8 or l(1)3Ad. The gene codes for a 2.0-kb RNA that is transcribed at all stages of development. The RNA has been localized by in situ hybridization to imaginal discs, larval brain, to nurse cells in the ovary, and to spermatogonia and spermatocytes in the testis. The putative translation product contains seven WD-repeats and is, therefore, a new member of the family of WD-proteins. Clear homologues of the Drosophila WDS protein exist in three other fully sequenced higher eukaryotes - human, Caenorhabditis elegans and Arabidopsis. A genomic fragment containing the wds transcription unit is able to rescue two different lethal wds alleles, thus proving that we have indeed isolated the wds gene.

Geographical patterns of genetic subdivision in the cellar spider Pholcus phalangioides (Araneae).

Geographical patterns of gene flow and drift were analysed in the commensal cellar spider Pholcus phalangioides to get insight into the causes affecting genetic variation in this species strictly associated with man. Our sampling consisted of 23 subpopulations collected over five urban regions in central Europe (distances ranged from 920 km to sites within the same building complex). Five variable allozyme loci showed significant interpopulation subdivision (theta=0.146) and isolation by distance over the area studied. On a regional scale (up to 70 km) significant differentiation was found, but the genetic pattern did not correlate with distance. Moreover, significant two-locus disequilibria were detected and a recent reduction in the effective population size was indicated within six sites. These results suggest that in P. phalangioides a high potential of dispersal and strong effects of drift within small, demographically unstable mating units seem to cause significant, but unpredictable genetic differentiation patterns at lower geographical scales. Our study documents strong effects of drift in a strictly commensal species outside the murine rodents.

Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer.

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3'-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Alterations in titer and distribution of high mobility group proteins during embryonic development of Drosophila melanogaster.

High mobility group proteins are thought to have an architectural function in chromatin. Here we describe changes in titers, extent of phosphorylation, and cellular distribution of the three abundant HMG proteins during embryonic development of Drosophila. The titers of the HMG proteins HMGD, HMGZ, and D1 are highest in ovaries and at the beginning of embryonic development. They decrease continuously until cellularization of the embryo. Relative to the histone H1 titer, the levels of HMGD and D1 remain almost constant during gastrulation and organogenesis, whereas the titer of HMGZ increases during late organogenesis. Up to gastrulation, the development is accompanied by dephosphorylation of D1. In contrast, HMGD and HMGZ appear to be constitutively phosphorylated. As the high extent of phosphorylation of D1 is also characteristic in ovaries, it is likely that the posttranslational modifications of this protein observed in early embryonic stages are of maternal origin. Using site specific antibodies against helices I and III of HMGD and HMGZ and against the AT-hook motif of D1, protein-specific staining patterns have been observed during embryonic development. Despite high levels of HMG proteins at the beginning of embryonic development, we were unable to detect any of these proteins in nuclei of stage 2 embryos. The accumulation of the HMG proteins correlates with the onset of transcription in stage 3. Our results argue against a proposal of a shared role of HMGD and histone H1 in Drosophila chromatin.

Protection patterns of tRNAs do not change during ribosomal translocation.

The translocation reaction of two tRNAs on the ribosome during elongation of the nascent peptide chain is one of the most puzzling reactions of protein biosynthesis. We show here that the ribosomal contact patterns of the two tRNAs at A and P sites, although strikingly different from each other, hardly change during the translocation reaction to the P and E sites, respectively. The results imply that the ribosomal micro-environment of the tRNAs remains the same before and after translocation and thus suggest that a movable ribosomal domain exists that tightly binds two tRNAs and carries them together with the mRNA during the translocation reaction from the A-P region to the P-E region. These findings lead to a new explanation for the translocation reaction.

The egghead gene product influences oocyte differentiation by follicle cell-germ cell interactions in Drosophila melanogaster.

Oogenesis in Drosophila is a useful model for studying cell differentiation. We have analyzed the role of the egh gene in these processes with the aid of a newly isolated viable but female sterile allele. This mutation results in diverse variable defects in oogenesis. The most frequent defect being follicles that have either more or less than the normal number of 16 germ cells. This is caused by erroneous splitting and/or fusion of correct clusters of 16 cystocytes. The entire follicle has a rather flexible structure in this allele, most obvious by a highly variable position of the oocyte within the follicle. Moreover, a second oocyte can also develop in egh clusters. This is exclusively observed in aberrant follicles that are generated by the aforementioned splitting/fusion process. Surprisingly, even a germ cell which is distinct from the two pro-oocytes can differentiate into an oocyte under these circumstances. Hence, determination of the oocyte is definitely not fixed when germ cell clusters are enveloped by prefollicular cells, and interactions between follicle cells and germ cells must play an important role in oocyte specification. Molecular analysis proves that the oocyte-specific transcript of the egh gene is drastically reduced in this viable allele.

Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila.

The aly, can, mia and sa genes of Drosophila are essential in males both for the G2-meiosis I transition and for onset of spermatid differentiation. Function of all four genes is required for transcription in primary spermatocytes of a suite of spermatid differentiation genes. aly is also required for transcription of the cell cycle control genes cyclin B and twine in primary spermatocytes. In contrast can, mia and sa are required for accumulation of twine protein but not twine transcript. We propose that the can, mia and sa gene products act together or in a pathway to turn on transcription of spermatid differentiation genes, and that aly acts upstream of can, mia and sa to regulate spermatid differentiation. We also propose that control of translation or protein stability regulates entry into the first meiotic division. We suggest that a gene or genes transcribed under the control of can, mia and sa allow(s) accumulation of twine protein, thus coordinating meiotic division with onset of spermatid differentiation.

Organisation of regulatory elements in two closely spaced Drosophila genes with common expression characteristics.

Sperm tail proteins that are components of a specific structure formed late during spermatid elongation have been found to be encoded by the Mst(3)CGP gene family. These genes have been demonstrated to be regulated both at the transcriptional as well as at the translational level. We report here on the dissection of the regulatory regions for two members of the gene family, Mst84Da and Mst84Db. While high level transcription and negative translational control of Mst84Da is mediated by a short gene segment of 205 nt (-152/+53), Mst84Db expression is controlled by a number of distinct regulatory elements with different effects that all reside within the gene itself. We identify a transcriptional control element between +154 and +216, a translational repression element around +216 to +275 and an RNA stability element within the 3'UTR. Irrespective of the final common expression characteristics, correct regulation for any individual member of the gene family seems to be achieved by very different means. This confirms earlier observations that did not detect any other sequence elements in common apart from the TCE (translational control element).

Mutational analysis of two highly conserved UGG sequences of 23 S rRNA from Escherichia coli.

The 23 S-type rRNA contains two phylogenetically conserved UGG sequences, which have the potential to bind the universal CCA-3'-ends of tRNAs at the ribosomal peptidyltransferase center by base pairing. The first two positions, UG, of these sequences at the helix-loop 80 (U2249G2250) and helix-loop 90 (Psi2580G2581) and some related nucleotides were tested by site-directed mutagenesis for their involvement in ribosomal function, i.e. peptidyltransferase. The plasmid-derived mutated 23 S rRNA comprised about 50% of the total 23 S rRNA. None of the single mutations caused an assembly defect, and all 50 S subunits carrying an altered 23 S rRNA could freely exchange with the pools of 70S ribosomes and polysomes. The mutations at the helix-loop 80 region hardly affected bacterial growth. However, mutations at the helix 90 caused severe growth effects and severely impaired the in vitro protein synthesis, showing that this 23 S rRNA region is of high importance for ribosomal function.

Conserved nucleotides of 23 S rRNA located at the ribosomal peptidyltransferase center.

Two nucleotides of the 23 S rRNA gene were mutated; the nucleotides correspond to the first two positions of the universally conserved sequence PsiGG2582 at the peptidyltransferase ring of 23 S rRNA. The ribosomes containing the altered 23 S rRNA were analyzed. Previously, it was shown that ribosomal assembly was indistinguishable from that in wild-type cells, that the flow of the corresponding 50 S subunit into the polysome fraction was not restricted, but that the ribosomes were strongly impaired in poly(Phe) synthesis (C. M. T. Spahn, J. Remme, M. A. Schäfer, and K. H. Nierhaus (1996) J. Biol. Chem. 271, 32849-32856). Here we apply assay systems exclusively testing the puromycin reaction of ribosomes carrying plasmid-born rRNA, a dipeptide assay using the minimal P site donor pA(fMet) and a translocation system not depending on the puromycin reaction. The mutations in helix 90 exclusively abolish or severely impair the ribosome capability to catalyze AcPhe-puromycin formation. A possible explanation of these observations is that G2581 and Psi2580 (and possibly also G2582) are part of the binding site of C75 of peptidyl-tRNA in the P site. The results suggest that in this case, however, such an interaction would disobey canonical base pairing.

[Phosphate sites of RNA-ligands interacting with ribosome at different stages of translation. Thiophosphate method of analysis]. / Fosfatnye uchastki RNK-ligandov, vzaimodeistvuiushchie s ribosomoi na razlichnykh stadiiakh transliatsii. Izuchenie s pomoshch'iu tiofosfatnogo metoda.

A novel footprinting method was recently developed which identifies phosphate groups of RNA involved in strong RNA-RNA and RNA-protein interactions. The method is based on iodine-dependent RNA cleavage at phosphothioate groups as long as these groups are not protected from iodine. Our recent studies of mRNA and tRNA regions protected in active ribosomes are summarized; initiation state of ribosomes as well as two elongation states in pre- and post-translocational states were analyzed. Only one phosphate group of mRNA, which was two positions upstream of the decoding codons, was weakly protected in longation complexes, whereas this group and the phosphate groups in the Shine-Dalgarno sequence were protected in the initiation complex. No protection was observed downstream of the decoding codons. On the contrary, numerous phosphate residues of tRNA were protected by the ribosome. The tRNA protection patterns significantly varied between two tRNAs simultaneously bound to the ribosome. The protection pattern of an individual tRNA was not significantly affected by translocation. The data indicate that both tRNA molecules are tightly bound to the ribosome, whereas mRNA is fixed predominantly by two tRNAs via codon-anticodon interaction. A possible translocation mechanism is suggested.

The elongating ribosome: structural and functional aspects.

We determined the positions and arrangements of RNA ligands within the ribosome with a new neutron-scattering technique, the proton-spin contrast-variation. Two tRNAs were bound to the ribosome in the pre-translocational and the post-translocational state. The mass centre of gravity of both tRNAs resides at the subunit interface of the body of the 30S subunit. Both tRNAs are separated by an angle of 50-55 degrees, and their mutual arrangement does not change during translocation. The mass centre of gravity moves by 13 +/- 3 A (1A = 0.1 nm) during translocation, corresponding well with the length of one codon. Using an RNase-digestion technique, the length of the mRNA sequence covered by the ribosome was determined to be 39 +/- 3 nucleotides before and after translocation. The ribosome moves like a rigid frame along the mRNA during translocation. In contrast, both tRNAs seem to be located on a movable ribosomal domain, which carries the tRNAs before, during, and after translocation, leaving the microtopography of the tRNAs with the ribosome unaltered. This conclusion was derived from an analysis of the contract patterns of thioated tRNAs on the ribosome. The results have led to a new model of the elongation cycle, which reinterprets the features of the previous "allosteric three-sites model" in a surprisingly simple fashion. Finally, a mutational analysis has identified a single nucleotide of the 23S rRNA essential for the peptidyltransferase activity.

Spectral and electron transfer properties of Sepharose 6MB-immobilized cytochrome c.

Cytochrome c has been covalently attached to Sepharose 6MB to study the effects of immobilization on the molecule. A detailed study of the resulting product was conducted based on its three characteristic properties: spectra, oxidation-reduction potential, and biological activity. The spectral properties demonstrated that cytochrome c was essentially the same after attachment. No major conformational changes were indicated. The redox potentials for most samples of immobilized cytochrome c loaded with different amounts of protein were generally 20-25 mV lower than native cytochrome c (270 mV). Heavily loaded samples, however, showed no difference in potential. The Km values for immobilized cytochrome c with cytochrome oxidase and reductase from submitochondrial particles were comparable to the soluble protein. Vmax values are more strongly affected by immobilization, especially for the reductase. It has been demonstrated that the submitochondrial particles cannot penetrate the pores of the support material and therefore only the cytochrome c molecules on the surface are available for reaction. As a support material, Sepharose 6MB, which is CNBr activated, hydrolyzes at a significant rate at the pH of the coupling reaction, and this must be considered in establishing coupling conditions for protein immobilization.