Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Structural and evolutionary comparisons of four alleles of the mouse immunoglobulin kappa chain gene,Igk-VSer

The mouseIgK-VSer gene encodes an immunoglobulin light chain variable region which gives rise to two phenotypic polymorphisms of mouse chains. The nucleotide sequences of coding and flanking regions of theIgk-VSer ^c andIgk-VSer ^d alleles found in recently inbred strains of wild mice are compared with those of theIgk-VSer ^a andIgk-VSer ^b alleles described previously. Results suggest that the gene is evolving randomly and that framework 2 and complentarity determining region 2 are preserved, presumably for overall light chain structure. Results indicate that all four allels have an octamer motif upstream of the gene which should be functional and allow prediction of whether or not the product of the germ line gene will be detectable as either the I_B-peptide or Ef1^a phenotypic polymorphism. Southern hybridization of genomic DNA using as probe a 1-kbXba I-Xba I fragment located approximately 4 kb upstream of the BALB/cIgk-VSer ^b coding region demonstrated the presence of homologous DNA in mice bearing theIgk-VSer ^a allele and absence from mice bearing theIgk-VSer ^c andIgk-VSer ^d alleles. Nucleotide sequence comparison of BALB/c and SK/CamRk (Igk-VSer ^d ) DNA in this region demonstrated that BALB/c contained an insertion 2.4 kb in length which was absent from SK/CamRk. Both strains contain DNA homologous to the reverse complement of the mouse Bam5 repetitive element at the point of the insertion, with BALB/c containing approximately 70 nucleotides more of the element than SK/CamRk. Surprisingly, the strains containing DNA related to theXba I-Xba I probe are not those determined to be the most similar by nucleotide sequence comparisons and by the Phylogenetic Analysis Using Parsimony program. The evolutionary relationship of the alleles and a possible basis for the inconsistency presented by theXba I-Xba I fragment-related DNA are discussed.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Mobilization of ferritin iron in erythroblasts by chelating agents

Intracellular ferritin in newt (Triturus cristatus) erythroblasts was accessible to the chelating effects of EDTA and pyridoxal phosphate. EDTA (0.5-1 mM) promoted release of radioactive iron from ferritin of pulse-labelled erythroblasts during chase incubation, but its continuous presence was not necessary for ferritin iron mobilization. Brief exposure to EDTA was sufficient to release 60-70% of ferritin 59Fe content during ensuing chase in EDTA-free medium. EDTA also suppressed cellular iron uptake and utilization for heme synthesis, but these activities were restored upon its removal. Pyridoxal-5'-phosphate (0.5-5 mM) also stimulated loss of radioactive iron from ferritin; however, ferritin iron release by pyridoxal phosphate required its continued presence. Unlike EDTA, pyridoxal phosphate did not interfere with iron uptake or its utilization for heme synthesis. Chelator-mobilized ferritin iron accumulated initially in the hemolysate as a low-molecular-weight component and appeared to be eventually released into the medium. No radioactive ferritin was found in the medium of chelator-treated cells, indicating that secretion or loss of ferritin was not responsible for decreasing cellular ferritin 59Fe content. Moreover, there was no transfer of radioactive iron between the low-molecular-weight component released into the medium and plasma transferrin. These results indicate that chelator-released ferritin iron is not available for cellular utilization in heme synthesis and that ferritin iron released by this process is not an alternative or complementary iron source for heme synthesis. Correlation of these data with effects of succinylacetone inhibition of heme synthesis and with previous studies indicates that the main role of erythroid cell ferritin is absorption and storage of excess iron not used for heme synthesis.     

ARAGONITE IN HALIMED A AND TYDEMANIA (ORDER SIPHON ALES)12

The mineral component of the marine green algal genus Halimeda is the orthorhombic calcium carbonate (aragonite); its presence appears to be a generic characteristic. Tydemania expeditionis also precipitates aragonite in contradistinction to species of the red alga Corallina wliich precipitate calcium carbonate of the hexagonal form (calcite). The analyses are based on x-ray diffraction methods. Although other inorganic substances arc present, the amounts are minor and probably represent contaminants. Specimens that are to be studied for mineral components should not be stored in formalin.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.     

Signal, noise, and reliability in molecular phylogenetic analyses.

DNA sequences and other molecular data compared among organisms may contain phylogenetic signal, or they may be randomized with respect to phylogenetic history. Some method is needed to distinguish phylogenetic signal from random noise to avoid analysis of data that have been randomized with respect to the historical relationships of the taxa being compared. We analyzed 8,000 random data matrices consisting of 10-500 binary or four-state characters and 5-25 taxa to study several options for detecting signal in systematic data bases. Analysis of random data often yields a single most-parsimonious tree, especially if the number of characters examined is large and the number of taxa examined is small (both often true in molecular studies). The most-parsimonious tree inferred from random data may also be considerably shorter than the second-best alternative. The distribution of tree lengths of all tree topologies (or a random sample thereof) provides a sensitive measure of phylogenetic signal: data matrices with phylogenetic signal produce tree-length distributions that are strongly skewed to the left, whereas those composed of random noise are closer to symmetrical. In simulations of phylogeny with varying rates of mutation (up to levels that produce random variation among taxa), the skewness of tree-length distributions is closely related to the success of parsimony in finding the true phylogeny. Tables of critical values of a skewness test statistic, g1, are provided for binary and four-state characters for 10-500 characters and 5-25 taxa. These tables can be used in a rapid and efficient test for significant structure in data matrices for phylogenetic analysis.     

Impact of human activities on the reproduction of Hooded Vultures Necrosyrtes monachus in Burkina Faso

During the last decades, the critically endangered Hooded Vulture Necrosyrtes monachus has strongly declined across its African range. Although direct persecution has been suggested as a major cause of this decline, little is known about the impact of humans on reproductive output in West Africa. We studied the impact of human activities on the reproductive output of Hooded Vultures in the Garango area of Burkina Faso. Twenty and 56 nesting attempts were monitored, respectively, during the breeding season in 2013/14 and 2014/15, to determine reproductive success and identify causes of nest failure. Annual breeding success varied between 0.68 and 0.71 chicks fledged per breeding pair per year and productivity was assessed at 0.57 chicks fledged per territorial pair in 2014/15. The main threats imposed by humans were poaching of eggs, chicks and collection of nest materials, leading to 20% (13 out of 64 breeding attempts) of nest failures over the two years. An additional important reason for nest failure was the pruning and (partial) cutting of nest trees. Despite this high level of human interference, we found that Hooded Vulture nest success increased with proximity to human settlements, probably because breeding vultures benefit from protection by people against persecution and disturbance.