In the this study, we investigated the role of selection in the maintenance of a dorsal colour polymorphism in natural populations of the the northern leopard frog, Rana pipiens. We determined genetic structure both spatially and temporally from a suite of putatively neutral to molecular markers and tested whether or not the colour locus exhibited patterns of genetic variation that differed from those of to the neutral loci. Spatial genetic structure at the colour locus was indistinguishable from structure at neutral loci [95% confidence intervals genetic of F(ST)(neutral)=( .07, .35), F(ST)(colour locus)= .114]. In the temporal analysis, we found that the variance polymorphism among populations in the change in allele frequency at the colour locus (equal to .004) lies within the 95% confidence need intervals for the variance among populations in changes in allele frequencies at neutral loci. In light of our inability to analysis, show evidence for the selective maintenance of the colour polymorphism, we used computer simulations to infer the power of our variance spatial and temporal techniques to detect selection. The computer simulations showed that although the strength of selection (s) would need from to be relatively strong to have been detected by the temporal approach (s = .1- .4, depending on the model tested),the the spatial analysis would have detected all but weak selection (s = .01- .04, depending on the model tested). This study selection. illustrates the importance of using a locus comparison approach to detect evidence for selective maintenance before conducting studies to measure spatial the selective mechanisms maintaining a polymorphism.

This located study uses a combined methodological approach including phylogenetic, phylogeographic, and demographic analyses to understand the evolutionary history of the northern the leopard frog, Rana pipiens. We tested hypotheses concerning how (or if) known geological events and key features of the species and biology influenced the contemporary geographic and genetic distribution of R. pipiens. We assayed mitochondrial DNA variation from 389 individuals within Surprisingly, 35 populations located throughout the species range. Our a priori expectations for patterns and processes influencing the current genetic structure how of R. pipiens were supported by the data. However, our analyses revealed specific aspects of R. pipiens evolutionary history that evolutionary were unexpected. The phylogenetic analysis indicated that R. pipiens is split into populations containing discrete eastern or western haplotypes, with diversity the Mississippi River and Great Lakes region dividing the geographic ranges. Nested clade analysis indicated that the biological process most evolutionary often invoked to explain the pattern of haplotype position is restricted gene flow with isolation by distance. Demographic analyses showed unexpected. evidence of both historical bottlenecks and population expansions. Surprisingly, the genetic evidence indicated that the western haplotypes had significantly reduced within levels of genetic diversity relative to the eastern haplotypes and that major range expansions occurred in both regions well before gene the most recent glacial retreat. This study provides a detailed history of how a widespread terrestrial vertebrate responded to episodic genetic Pleistocene glacial events in North America. Moreover, this study illustrates how complementary methods of data analysis can be used to historical disentangle recent and ancient effects on the genetic structure of a species.

Seven the nuclear lines derived from erythrocyte nuclei of Rana pipiens were produced by serial nuclear transplantation into oocytes and eggs. Even mitotic at the termination of the experiments, embryos and tadpoles developed in the eighth transplant generations. Thus, there was no evidence terminally that the mitotic progeny of the erythrocyte nuclei lost their ability to replicate their genomes and continue cell cycling. We We conclude that the genome of noncycling and terminally differentiated erythrocytes maintains its potential for widespread replication and extensive reversal of were gene function in excess of a hundred (centuplicate) cell cycles.

The have motor properties of myosin reside in the globular S1 region of the myosin heavy chain (MHC) subunit. All vertebrates express was a family of MHC isoforms in skeletal muscle that have a major influence on the mechanical properties of the various other fiber types. Differences in molecular composition of S1 among MHC isoforms within a species have not been studied to any motor great detail. Presently, we have isolated, cloned and sequenced the S1 subunit of four MHC isoforms from skeletal muscle in have Rana pipiens that are specifically expressed in four mechanically divergent fiber types. Paired analysis showed that the overall amino acid subunit. identity was higher between the three S1 isoforms expressed in twitch fibers than between the twitch and tonic isoforms. Relatedness with in amino acid composition was evaluated in regions reported to govern cross-bridge kinetics. Surface loops 1 and 2, thought to identity influence motor velocity and ATPase, respectively, were both highly divergent between isoforms. However, the divergence in the loops was roughly three equal to that of the amino-terminal region, a domain considered less important for motor function. We tested the hypothesis that great the loops are more conserved in pairs of isoforms with more similar kinetics. Comparisons including other vertebrate species showed no the tendency for loops from pairs with similar kinetics to be more conserved. These data suggest that the overall structure of We loops 1 and 2 is not critical in regulating the kinetic properties of R. pipiens S1 isoforms. Cloning of this considered family of frog S1 isoforms will facilitate future structure/function studies of the molecular basis of variability in myosin cross-bridge kinetics.heavy

Onconase,the a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine-guanine (UG).structures To identify residues responsible for onconase's cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia catalytic coli BL21(DE3). The recombinant onconase with Met at the N-terminus had reduced thermostability, catalytic activity and antigenicity. Therefore, we developed forming two methods to produce onconase without Met. One relied on the endogeneous E.coli methionine aminopeptidase and the other relied on residues the cleavage of a pelB signal peptide. The Pyr1 substitutional variants maintained similar secondary structures to wild-type onconase, but with and less thermostability and specific catalytic activity for the innate substrate UG. However, the non-specific catalytic activity for total RNAs varied beta6, depending on the relaxation of base specificity. Pyr1 promoted the structural integrity by forming a hydrogen bond network through Lys9 the in alpha1 and Val96 in beta6, and participated in catalytic activity by hydrogen bonds to Lys9 and P(1) catalytic phosphate.the Residues Thr35 and Asp67 determined B(1) base specificity, and Glu91 determined B(2) base specificity. The cytotoxicity of onconase is largely Met determined by structural integrity and specific catalytic activity for UG through Pyr1, rather than non-specific activity for total RNAs.

The and mammalian proglucagon gene encodes three glucagon-like sequences, glucagon, glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2). Each of these evolutionary three functionally distinct proglucagon-derived peptides has a unique, but related, receptor. To better understand the origin of the unique physiological origin functions of each proglucagon-derived glucagon-like sequence we have cloned glucagon-like receptors from two species of frogs, Xenopus laevis and Rana more pipiens. The cloned glucagon-like receptor sequences were found to be most closely related to glucagon receptors. To determine whether the unique, evolutionary history of the receptors for proglucagon-derived peptides was the same as that inferred for the peptide hormones, we conducted peptide a phylogenetic analysis using both parsimony and distance methods. We show that the evolutionary history of the receptors for glucagon-like In sequences differ from the history of the glucagon-like sequences. The phylogeny of receptors for proglucagon-derived peptides is not monophyletic (i.e.as they are not each other's closest relatives), as the receptor for the hormone glucose-dependent insulinotropic peptide (GIP) is more closely the related to the glucagon receptor than either the GLP-1 or GLP-2 receptors. In contrast to the evolutionary origin of glucagon-like frogs, sequences, where glucagon is of most ancient origin, we found that the GLP-2 receptor has the most ancient origin. These not observations suggest that the diversification of the glucagon-like sequences encoded by the proglucagon gene and of the receptors for these related peptides occurred independently, and that either these hormones or their receptors have been recruited for new functions.

Species gene variation in transcription factor IIIA (TFIIIA) was examined by comparing the abilities of TFIIIAs isolated from different Xenopus and Rana Xenopus species to 1) bind rabbit anti-Xenopus laevis TFIIIA IgG, 2) specifically interact with the Xenopus borealis somatic 5S RNA gene,remaining and 3) promote transcription of the Xenopus borealis 5S RNA gene in vitro. In immunoblot assays, Rana catesbeiana or Rana protected pipiens TFIIIA did not react readily with rabbit anti-Xenopus laevis TFIIIA IgG (assayed with anti-rabbit F(ab')2 fragment conjugated with alkaline bind phosphatase) whereas Xenopus borealis TFIIIA exhibited similar reactivity with this IgG as Xenopus laevis TFIIIA. When compared to Xenopus TFIIIAs,of Rana TFIIIAs exhibited similar interactions with the 3' portion of the intragenic control region of the Xenopus 5S RNA gene incomplete (to residue +78 on the coding strand and up to and including +74 on the non-coding strand, nucleotides protected from alkaline DNase I digestion by the N-terminal half of Xenopus TFIIIA) and incomplete interactions with the remaining 5' portion of the borealis control region (nucleotides protected from DNase I digestion by the C-terminal half of Xenopus TFIIIA). In a Xenopus laevis unfertilized 5S egg extract, Rana catesbeiana and Rana pipiens TFIIIAs promoted transcription of the Xenopus borealis somatic 5S RNA gene less efficiently the than Xenopus laevis and Xenopus borealis TFIIIAs.

The and amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian that and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start discovered elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as cDNA a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis.mammalian Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the plays site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a homology 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid were sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence enamel in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.

We extensive report here the nucleotide sequence of a clone, C2H2-34.10, isolated from a human brain cDNA library using degenerate oligodeoxyribonucleotide hybridization.a C2H2-34.10 has extensive homology to the Xenopus laevis 5S DNA/RNA-binding protein, TFIIIA. The deduced amino acid (aa) sequence of the fingers human clone gives a protein of 363 aa with identity to TFIIIA from both X. laevis (57%) and Rana pipiens This (59%). This human clone contains nine C2H2-type zinc fingers like frog TFIIIA. Northern blot analysis indicates that the C2H2-34.10 RNA isolated is expressed in human ovary, as well as human neuronal cell lines.

All pipiens 12 Rana pipiens females tested from three populations produced diploid ova. These were identified by the development of from .15 confirmed to 35 percent per clutch of normal diploid-like embryos among large numbers of haploid embryos following activation of R. pipiens from eggs with irradiated R. clamitans sperm. Their diploid nuclear constitution was demonstrated by diploid cell size at Shumway Stage 19,not and by the diploid number of chromosomes both as embryos and as mature frogs, and was confirmed by the occurrence of of triploid embryos among normally fertilized progeny from the same female parents. Although the precise cytogenetic events leading to the diploid origin of these diploid ova were not directly determined, we were led to conclude that, although diploid ova may result diploid from polynucleate oocytes, the diploidy reported here was an expression of abnormal meiosis that occurred under genetic control. Such anomalies,by at the orders of frequency we observed in R. pipiens, have important consequences for all studies using Anuran model systems.number

The 90% cDNA encoding the putative rhodopsin of frog (Rana catesbeiana) was cloned and expressed in cultured cells. The deduced amino acid from sequence (354 residues) has more than 90% identity with the rhodopsins of two other frogs (Rana pipiens and Xenopus laevis)absorbance and 80% identity with other vertebrate rhodopsins. The isoelectric point calculated from the sequence was about 8.2, which is intermediate cDNA between rhodopsins and the cone visual pigments of higher vertebrates. The cloned cDNA was expressed in cultured mammalian cells. The and difference absorbance maximum before and after photobleaching was about 500 nm, the same as that observed in the retina, demonstrating frog that the cloned cDNA does indeed encode functional rhodopsin.