Restriction endonuclease mapping of ribosomal RNA genes: Sequence divergence and the origin of the tetraploid treefrog Hyla versicolor

Hyla chrysoscelis (2n=24) and H. versicolor (2n=48) are a diploid-tetraploid species pair of treefrogs. Restriction endonuclease mapping of ribosomal RNA (rRNA) gene repeat units of diploids collected from eastern and western populations reveals no differences within rRNA gene coding regions but distinctive differences within the nontranscribed spacers. A minimum of two physical maps is required to construct an rRNA gene map for the tetraploid, whose repeat units appear to be a composite, with about 50% of the elements resembling the western diploid population and about 50% resembling the eastern population. These results imply that this population of the tetraploid species may have arisen from a genetically hybrid diploid. Alternatively, the dual level of sequence heterogeneity in H. versicolor may reflect some type of gene flow between the two species. The coding region of the rRNA genes in the tetraploid differs from that in either diploid in about 20% of all repeat units, as exemplified by a BamHI site located near the 5 terminus of the 28 S rRNA gene. If the 20% variant class of 28 S rRNA gene coding sequences is expressed, then there must be two structural classes of ribosomes; if only the 80% sequence class is expressed, then a genetic control mechanism must be capable of distinguishing between the two different sequence variants. It is postulated that the 20% variant sequence class may be correlated with a partial functional diploidization of rRNA genes in the tetraploid species.This research was supported, in part, by NSF Grants CDP-8002341 and PRM-8106947 and by faculty research grants from Miami University to J.C.V.     

Quantitative determination of Coomassie R bound to proteins in polyacrylamide gel. A facilitated method for multi-spot analysis of electrograms

Dimethylsulfoxide (DMSO) was found to be an efficient solvent for extraction of Coomassie Blue R 250 (Coomassie R) from stained proteins on polyacrylamide gels. Kinetic measurements show that the extraction of the dye from a 2-D gel reached equilibrium in 48 h. Staining of E. coli ribosomal proteins by Coomassie R dissolved in trichloroacetic acid exhibited two types of dye-protein complexes, the majority of them yield a blue-purple colour, while the rest are stained with a light-blue tone and fluorescent appearance as well. The absorbance spectra of the complexes in the gel matrix differ significantly from each other. However, the DMSO-extracted Coomassie show identical absorbance profiles with lambda max at 602 nm, thus the amount of the bound dye can easily be measured spectrophotometrically.     

Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A)+RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S17 protein (pRS17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid (aa) sequence of S17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an Mr of 15 377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an Mr of 12 652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3'-noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].     

Systematic placement of the basidiomycetous yeastCystofilobasidium lari-marini comb. nov. as predicted by rRNA nucleotide sequence analysis

Based on similarities in basidial morphology and nucleotide sequences of the V3 variable region in the large sub-unit ribosomal RNA, the yeastLeucosporidium lari-marini is considered phylogenetically related to the genusCystofilobasidium. Therefore the new combinationCystofilobasidium lari-marini is proposed.     

Evolutionary relationships of eukaryotic kingdoms

The evolutionary relationships of four eukaryotic kingdoms—Animalia, Plantae, Fungi, and Protista—remain unclear. In particular, statistical support for the closeness of animals to fungi rather than to plants is lacking, and a preferred branching order of these and other eukaryotic lineages is still controversial even though molecular sequences from diverse eukaryotic taxa have been analyzed. We report a statistical analysis of 214 sequences of nuclear small-subunit ribosomal RNA (srRNA) gene undertaken to clarify these evolutionary relationships. We have considered the variability of substitution rates and the nonindependence of nucleotide substitution across sites in the srRNA gene in testing alternative hypotheses regarding the branching patterns of eukaryote phylogeny. We find that the rates of evolution among sites in the srRNA sequences vary substantially and are approximately gamma distributed with size and shape parameter equal to 0.76. Our results suggest that (1) the animals and true fungi are indeed closer to each other than to any other crown group in the eukaryote tree, (2) red algae are the closest relatives of animals, true fungi, and green plants, and (3) the heterokonts and alveolates probably evolved prior to the divergence of red algae and animal-fungus-green-plant lineages. Furthermore, our analyses indicate that the branching order of the eukaryotic lineages that diverged prior to the evolution of alveolates may be generally difficult to resolve with the srRNA sequence data.