Showing papers on "Ribosomal DNA published in 1991"

16S ribosomal DNA amplification for phylogenetic study.

Abstract: A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.

Ribosomal DNA: molecular evolution and phylogenetic inference.

Abstract: Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organisms, and this approach has provided a wealth of information about phylogenetic relationships. Studies of rDNA sequences have been used to infer phylogenetic history across a very broad spectrum, from studies among the basal lineages of life to relationships among closely related species and populations. The reasons for the systematic versatility of rDNA include the numerous rates of evolution among different regions of rDNA (both among and within genes), the presence of many copies of most rDNA sequences per genome, and the pattern of concerted evolution that occurs among repeated copies. These features facilitate the analysis of rDNA by direct RNA sequencing, DNA sequencing (either by cloning or amplification), and restriction enzyme methodologies. Constraints imposed by secondary structure of rRNA and concerted evolution need to be considered in phylogenetic analyses, but these constraints do not appear to impede seriously the usefulness of rDNA. An analysis of aligned sequences of the four nuclear and two mitochondrial rRNA genes identified regions of these genes that are likely to be useful to address phylogenetic problems over a wide range of levels of divergence. In general, the small subunit nuclear sequences appear to be best for elucidating Precambrian divergences, the large subunit nuclear sequences for Paleozoic and Mesozoic divergences, and the organellar sequences of both subunits for Cenozoic divergences. Primer sequences were designed for use in amplifying the entire nuclear rDNA array in 15 sections by use of the polymerase chain reaction; these "universal" primers complement previously described primers for the mitochondrial rRNA genes. Pairs of primers can be selected in conjunction with the analysis of divergence of the rRNA genes to address systematic problems throughout the hierarchy of life.

Ehrlichia chaffeensis, a new species associated with human ehrlichiosis.

Abstract: The bacterial 16S rRNA genes from blood samples of two patients with human ehrlichiosis and from an isolate recovered from one of the patients were amplified by using the polymerase chain reaction. The amplimers were then cloned and sequenced. The 16S rRNA gene sequence was also determined for Ehrlichia canis (two strains), E. equi, E. phagocytophila (two strains), and E. sennetsu (two strains). These sequences, along with a previously published 16S rRNA gene sequence of E. risticii, were compared. The 16S rRNA gene sequences were identical for all three sources of the human ehrlichiosis agent. The sequence comparisons indicate that the human ehrlichiosis agent is a new species most closely related to E. canis (98.2%) and more distantly related to other Ehrlichia spp. We propose that this species be named Ehrlichia chaffeensis sp. nov., with the Arkansas strain as the type strain.

Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA

Abstract: We used the polymerase chain reaction (PCR) to amplify specific regions of the nuclear and mitochondrial genomes of fungi using DNA extracted from pure cultures as well as that directly from ectomy...

Evidence for Biased Gene Conversion in Concerted Evolution of Ribosomal DNA

Abstract: Concerted evolution is the production and maintenance of homogeneity within repeated families of DNA. Two mechanisms--unequal crossing over and biased gene conversion--have been the principal explanations of concerted evolution. Concerted evolution of ribosomal DNA (rDNA) arrays is thought to be largely the result of unequal crossing over. However, concerted evolution of rDNA in parthenogenetic lizards of hybrid origin is strongly biased toward one of two parental sequences, which is consistent with biased gene conversion as the operative mechanism. The apparent gene conversions are independent of initial genome dosage and result in homogenization of rDNA arrays across all nucleolar organizer regions.

Interspecific gene flow in sympatric oaks.

Abstract: Variation of chloroplast DNA and nuclear ribosomal DNA (DNA encoding ribosomal RNA) was studied for five species of white oak native to the eastern United States. Although these species differ in many morphological characters and have different (though overlapping) geographical ranges and ecological tolerances, they are interfertile and often grow in mixed stands, and hybrids are occasionally found in nature. All individuals studied were morphologically typical members of their respective species-i.e., showed no evidence of recent hybrid ancestry. Restriction site markers in the chloroplast DNA reveal several clear cases of localized gene exchange between species, showing that there is appreciable gene flow between sympatric species in this group. One length variant of the nuclear ribosomal DNA, however, is species specific. The sharp morphological and ecological differences between the species, together with the one ribosomal DNA variant, suggest that nuclear genes may be exchanged less freely between species than are chloroplast genotypes.

Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria.

Abstract: The 16S rDNA genes of an apparently pure culture of a psychrophilic and strict barophilic bacterium (WHB 46) were studied by PCR-mediated amplification and cloning into phage M13 mp18. Sequence analysis of five individual clones revealed the presence of two different 16S rDNA types. The homology value of 90% indicates that culture WHB 46 is actually composed of two closely related species (WHB 46-1 and 46-2). Both strains are members of the γ-subdivision of proteobacteria. Analysis of a sixth clone (WHB 46-1/2) leads to the conclusion that it represents a 16S rDNA hybrid molecule assembled during the PCR reaction. This hypothesis was confirmed by secondary structure analysis of the chimeric rDNA. The appearance of such hybrid molecules point to a potential risk in studies on the diversity of bacterial populations by analysis of rDNA pattern via PCR-mediated amplification because they suggest the existence of organisms that do not actually exist in the sample investigated.

Homoploid reticulate evolution in Helianthus (Asteraceae): evidence from ribosomal genes

Abstract: Phylogenetic relationships among the 21 taxa comprising Helianthus sect. Helianthus and three outgroup species were assessed by restriction site mapping ofthe 18S-25S nuclear ribosomal RNA gene family. Wagner parsimony analysis of the 41 restriction site or length mutations observed produced a single 59-step most parsimonious tree. This tree was then compared to a cytoplasmic-based plastid phylogeny for this group. Several major discrepancies were observed between the two phylogenies suggesting both recent and ancient introgression. Furthermore, three cases of diploid hybrid speciation are unambiguously documented and a fourth case is suggested. These data are interpreted to suggest that evolution in Heliathus is reticulate rather than exclusively dichotomous and branching.

Species-diagnostic differences in a ribosomal DNA internal transcribed spacer from the sibling species Anopheles freeborni and Anopheles hermsi (Diptera:Culicidae).

Abstract: Approximately 460 base pairs (bp) of DNA sequence that included the second internal transcribed spacer (ITS2) and some flanking 5.8S and 28S ribosomal RNA coding regions were compared between the two closely related and morphologically indistinguishable mosquito species Anopheles freeborni and A. hermsi and a third related species, A. occidentalis. Sequences were determined from 14 clones of polymerase chain reaction (PCR)-amplified DNA obtained from four colonies of A. freeborni, two colonies of A. hermsi, and one individual A. occidentalis. Four clones showed independent single bp differences from the consensus for the relevant species. Eleven sites differed between the consensus sequences of A. hermsi and A. freeborni; 28 sites differed between A. hermsi and A. occidentalis. With the exception of a single bp mismatch in the 5.8S and two single bp mismatches near the undetermined junction of the ITS2 and 28S regions, all differences were confined to the ITS2 region. A PCR-based species-diagnostic assay for the cryptic species A. hermsi and A. freeborni was developed; it uses four synthetic oligonucleotides, two derived from areas of interspecies sequence difference in the ITS2, and two derived from highly conserved regions in the flanking coding sequences. Small amounts of mosquito DNA amplified in the presence of these four primers produce fragments of diagnostic size for each species: 900 bp for A. freeborni, 350 bp for A. hermsi, and approximately 1.2-1.4 kb for various other Anopheles species tested. We believe that this general approach to the development of species-diagnostic assays can be extended easily to other complexes of closely related, morphologically indistinguishable species.

Phylogenetic analysis of a natural marine bacterioplankton population by rRNA gene cloning and sequencing.

Abstract: The identification of the prokaryotic species which constitute marine bacterioplankton communities has been a long-standing problem in marine microbiology. To address this question, we used the polymerase chain reaction to construct and analyze a library of 51 small-subunit (16S) rRNA genes cloned from Sargasso Sea bacterioplankton genomic DNA. Oligonucleotides complementary to conserved regions in the 16S rDNAs of eubacteria were used to direct the synthesis of polymerase chain reaction products, which were then cloned by blunt-end ligation into the phagemid vector pBluescript. Restriction fragment length polymorphisms and hybridizations to oligonucleotide probes for the SAR11 and marine Synechococcus phylogenetic groups indicated the presence of at least seven classes of genes. The sequences of five unique rDNAs were determined completely. In addition to 16S rRNA genes from the marine Synechococcus cluster and the previously identified but uncultivated microbial group, the SAR11 cluster [S. J. Giovannoni, T. B. Britschgi, C. L. Moyer, and K. G. Field. Nature (London) 345:60-63], two new gene classes were observed. Phylogenetic comparisons indicated that these belonged to unknown species of alpha- and gamma-proteobacteria. The data confirm the earlier conclusion that a majority of planktonic bacteria are new species previously unrecognized by bacteriologists.

Comparison of mitochondrial DNA sequences of seven morphospecies of black flies (Diptera: Simuliidae).

Abstract: Universal primers constructed from the 16S ribosomal RNA gene in the Drosophila yakuba mitochondrial genome were successfully used to amplify, via the polymerase chain reaction, the homologous regi...

Physical mapping of the 18S.26S rRNA multigene family in common wheat: Identification of a new locus

Abstract: In situ hybridization in conjunction with deletion mapping was used to map physically the 18S.26S multigene rRNA family in Triticum aestivum L. cv. Chinese Spring. Using in situ hybridization, we report a new locus in the 7DL arm of Chinese Spring and Aegilops squarrosa, and also confirm the nucleolus organizing region (Nor) locus in the short arm of chromosome 1A at the telomeric end in Chinese Spring. Based on in situ hybridization labeling patterns, we show that rDNA exists as condensed rDNA (heterochromatic) at each end and diffused rDNA within the secondary constriction region of the Nor-B1 (1B), Nor-B2 (6B) and Nor-D3 (5D) loci. In Nor-B1, 80% of the condensed rDNA domain lies in the proximal end and 20% in the distal end joined by diffuse rDNA threads. In Nor-B2, condensed rDNA is distributed evenly at each end joined by diffuse rDNA in the middle. In Nor-D3, the base of the satellite contains a greater concentration of condensed rDNA than the tip of the short arm. On the basis of these observations, we support the model that the usual state of rDNA is inactive (facultatively heterochromatic; Hilliker and Appels 1989). A small fraction of rDNA at a specific location (usually in the middle in wheat) exists as a diffuse region (active) in condensed chromosomes.

Characterization of anastomosis groups of binucleate Rhizoctonia species using restriction analysis of an amplified ribosomal RNA gene.

Abstract: Seven U.S. and 16 Japanese binucleate Rhizoctonia anastomosis tester isolates, representing 21 different anastomosis groups, were characterized by restriction analysis of a ribosomal RNA (rRNA) gene. Genomic DNA was extracted from each isolate and a region of DNA coding for a portion of the 25S rRNA (rDNA) was amplified using the polymerase chain reaction. Five tester isolates (CAG1, AGF, AGI, AGJ, and AGK) produced either two or three bands ranging from 1.4 to 1.8 kilobases (kb), whereas five other tester isolates (CAG5, AGBa, AGC, AGD, and AGH) produced a single, 1.8-kb fragment (...)

Human autoantibody to RNA polymerase I transcription factor hUBF. Molecular identity of nucleolus organizer region autoantigen NOR-90 and ribosomal RNA transcription upstream binding factor.

Abstract: In dividing eukaryotic cells, nucleoli disperse before mitosis and reform in daughter cells at sites of ribosomal RNA (rRNA) gene clusters that are at the secondary constrictions of chromosomes, called nucleolus organizer regions (NORs). In this study, cDNA clones for a NOR autoantigen (NOR-90) were selected using a specific human autoantibody probe and were subsequently identified to encode an alternative form of the reported human upstream binding factor (hUBF). Results from immunoprecipitation showed that anti-NOR-90 antibodies recognized both forms of hUBF/NOR-90. Our data therefore showed that UBF, a critical factor in the regulation of rRNA transcription, was tightly bound to NOR during mitosis even when rRNA synthesis was thought to be minimal. Furthermore, we identified a nucleolar transcription factor as a novel target for human autoimmune response.

Phylogenetic relationships among Frankia genomic species determined by use of amplified 16S rDNA sequences.

Abstract: Actinomycetes of the genus Frankia establish a nitrogen-fixing symbiosis with a large number of woody dicotyledonous plants. Hundreds of strains isolated from various actinorhizal plants growing in different geographical areas have recently been classified into at least nine genomic species by use of the DNA-DNA hybridization technique (M.P. Fernandez, H. Meugnier, P.A.D. Grimont, and R. Bardin, Int. J. Syst. Bacteriol. 39:424-429, 1989). A protocol based on the amplification and sequencing of 16S ribosomal DNA segments was used to classify and estimate the phylogenetic relationships among eight different genomic species. A good correlation was established between the grouping of strains according to their 16S ribosomal DNA sequence homology and that based on total DNA homology, since most genomic species could be characterized by a specific sequence. The phylogenetic tree showed that strains belonging to the Alnus infectivity group are closely related to strains belonging to the Casuarina infectivity group and that strains of these two infectivity groups are well separated from strains of the Elaeagnus infectivity group, which also includes atypical strains isolated from the Casuarina group. This phylogenetic analysis was also very efficient for classifying previously unclassified pure cultures or unisolatable strains by using total DNA extracted directly from nodules.

Nucleolar organization of HeLa cells as studied by in situ hybridization.

Abstract: The distribution of the ribosomal genes and their ribosomal RNA (rRNA) products in the different compartments of the nucleolus of HeLa cells was examined on thin sections of Lowicryl embedded material. The ribosomal nucleic acids were visualized after hybridization with a set of biotinylated double-stranded ribosomal DNA (rDNA) probes from different locations along the gene, followed by immunogold labelling of biotin. Ribosomal genes were detected over both the entire fibrillar centres (FCs) and some masses of intranucleolar condensed chromatin. As for the rRNA components, comparison of the signal levels obtained with the different probes provides some information about the compartmentalization of distinct stages of ribosome biogenesis. Thus a probe specific for the 5' external transcribed spacer (5'ETS) portion of pre-rRNA labels almost exclusively the dense fibrillar component (DFC) and the border of the FCs, while the interior of the FCs appears devoid of any kind of rRNA species. By contrast, probes recognizing either 18S or 28S mature rRNA sequences label both the DFC and the granular component (GC). Moreover, mature 18S rRNA sequences are markedly under-represented relative to mature 28S rRNA sequences in the GC, as compared with the other nucleolar compartments. Our observations are consistent with the view that DFCs contain elongating and 47S-45S precursor rRNA molecules whereas the subsequent various rRNA processing intermediates are mainly located within the GC. Since the border of FCs is the only site where both rDNA and newly synthesized pre-rRNA coexist, the transcription of ribosomal genes seems to take place at the periphery of the FCs, and not in the DFC, suggesting that elongating and newly completed transcripts are immediately transferred into the surrounding DFC where they transiently accumulate before undergoing processing reactions and transfer to the GC.

The evolutionary position of the rhodophyte Porphyra umbilicalis and the basidiomycete Leucosporidium scottii among other eukaryotes as deduced from complete sequences of small ribosomal subunit RNA

Abstract: The complete small ribosomal subunit RNA (srRNA) sequence was determined for the red algaPorphyra umbilicalis and the basidiomyceteLeucosporidium scottii, representing two taxa for which no srRNA sequences were hitherto known. These sequences were aligned with other published complete srRNA sequences of 58 eukaryotes. Evolutionary trees were reconstructed by a matrix optimization method from a dissimilarity matrix based on sections of the alignment that correspond to structurally conservative areas of the molecule that can be aligned unambiguously. The overall topology of the eukaryotic tree thus constructed is as follows: first there is a succession of early diverging branches, leading to a diplomonad, a microsporidian, a euglenoid plus kinetoplastids, an amoeba, and slime molds. Later, a nearly simultaneous radiation seems to occur into a number of taxa comprising the metazoa, the red alga, the sporozoa, the higher fungi, the ciliates, the green plants, plus some other less numerous groups. Because the red alga diverges late in the evolutionary tree, it does not seem to represent a very primitive organism as proposed on the basis of morphological and 5S rRNA sequence data.

Differentiation of Streptococcus uberis from Streptococcus parauberis by polymerase chain reaction and restriction fragment length polymorphism analysis of 16S ribosomal DNA.

Abstract: Streptococcus uberis type II has been proposed recently as a separate species designated Streptococcus parauberis (A. M. Williams and M. D. Collins, J. Appl. Bacteriol. 68:485-490, 1990). Differentiation of S. parauberis from S. uberis has been possible only by DNA-DNA hybridization or 16S rRNA sequencing, since the biochemical and serological characteristics of the two species are indistinguishable. A simple and reliable technique was developed for differentiating S. parauberis (S. uberis type II [ATCC 13386]) from S. uberis (S. uberis type I [ATCC 9927, ATCC 13387, and ATCC 27958]) by restriction fragment length polymorphism (RFLP) analysis of 1.4-kb 16S ribosomal DNA (rDNA). Oligonucleotide primers complementary to 16S rRNA genes were used to amplify 16S ribosomal gene fragments from genomic DNA by polymerase chain reaction. The 1.4-kb 16S rDNA fragment was digested with ScaI, NspI, DdeI, and AvaII restriction endonucleases. Restriction fragments produced by all four restriction endonucleases were characteristic for each species. RFLP analysis of 16S rDNA from 24 "S. uberis" isolates obtained from mammary secretions of dairy cows indicated that all 24 isolates were indeed S. uberis.

Restriction fragment length polymorphisms in the ribosomal genes for species identification and subtyping of aerotolerant Campylobacter species.

Abstract: Whole-cell chromosomal digests of 84 strains of aerotolerant Campylobacter (AC) were examined by using PvuII restriction fragment length polymorphisms of rRNA genes followed by hybridization with Escherichia coli 16S and 23S rRNA (ribotyping). The AC strains belonged to Campylobacter cryaerophila (n = 13) and a newly defined species, "C. butzleri" (n = 64). Strains of C. cryaerophila belonged to two hybridization groups: DNA group 1A (including the type strain of C. cryaerophila) and DNA group 1B (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). Six AC strains not classified as C. cryaerophila or "C. butzleri" were also included. All 35 sporadic human and animal isolates of "C. butzleri" sent to the Centers for Disease Control for identification showed different ribotype patterns. However, most "C. butzleri" strains contained common bands at approximately 3.0, 6.2, 12.0, and 15.0 kb; the 3.0-kb band was present in all but four strains. An additional 23 strains of "C. butzleri," isolated as part of special studies, contained the 3.0-kb band. Thus, on the basis of visual identification of the 3.0-kb band, 94% of available strains were correctly identified as "C. butzleri." Ribotyping demonstrated that C. cryaerophila strains (DNA groups 1A and 1B) were different from C. butzleri strains. All C. cryaerophila strains demonstrated a common ribosomal DNA restriction fragment of 3.2 kb; DNA group 1B strains contained an additional common band at 2.6 kb. Ribotyping patterns of AC species were easily distinguished from patterns of other Campylobacter, Helicobacter, and Wolinella species.(ABSTRACT TRUNCATED AT 250 WORDS)

Ribosomal DNA Variation in Daphnia pulex

Abstract: Variation in the ribosomal DNA (rDNA) gene family was surveyed in five cyclically parthenogenetic populations of Daphnia pulex from central Illinois and in obligately parthenogenetic clones from Illinois and Ontario. A total of 37 distinct rDNA repeat types were identified on the basis of restriction-site and repeat-length polymorphism in a sample of 90 isolates. Repeat-type diversity was high within cyclic populations; however, individuals possessed only a small subset of the repeat-type variation present in each population. The distribution of repeat types within and among individuals suggested that new variants spread within rDNA arrays much faster than arrays carrying the variants spread within populations. This observation is contrary to a model developed by Ohta and Dover for strictly sexual organisms. Previous surveys of isozyme and mitochondrial DNA variation in these populations showed that gene flow among them is limited. Hierarchical analysis of rDNA restriction-site variation was consistent with this observation and showed that genetic divergence accumulates between populations for rDNA almost as rapidly as it does for single-copy nuclear genes (isozymes). Analysis of rDNA variation in obligately parthenogenetic clones provided evidence that both intraand interchromosomal exchanges occur between rDNA arrays in the absence of meiosis. Moreover, individuals reproducing by obligate parthenogenesis possessed fewer rDNA repeat types, on average, than did individuals from cyclic populations, suggesting that there is a net loss of rDNA repeat-type variability within obligately clonal lineages over time. Preliminary analyses of two additional species, D. pulicaria and D. obtusa, revealed several restriction-site polymorphisms that were found in more than one of the three species. The existence of such shared polymorphisms advises caution in the use of multigene-family variation to infer phylogenies among species when levels of intraspecific variation have not been assessed.

Molecular evolution in hypotrichous ciliates: sequence of the small subunit ribosomal RNA genes from Onychodromus quadricornutus and Oxytricha granulifera (Oxytrichidae, Hypotrichida, Ciliophora)

Abstract: The small subunit ribosomal RNA (16S-like rRNA) coding regions of the hypotrichous ciliates Onychodromus quadricornutus and Oxytricha granulifera were amplified using polymerase chain reaction techniques. Complete sequences were determined for the amplified genes and compared to those of other ciliated protozoa. In phylogenetic trees inferred using distance matrix methods oxytrichids are not seen as a cohesive phylogenetic group. Oxytricha nova is most closely related to Stylonychia pustulata in a lineage that also includes O. quadricornutus. This phylogeny contradicts phylogenetic schemes in which Onychodromus is considered to be a primitive hypotrichous ciliate and suggests that O. nova was misidentified as members of the genus Oxytricha.

Assessing the Reliability of 5S rRNA Sequence Data for Phylogenetic Analysis in Green Plants

Abstract: Elimination of compensating substitutions in portions of 5S rRNA sequences that exhibit secondary structure and scoring of common gaps as homologous characters affects both the topology of phylogenetic estimates within green plants and measures of their reliability. Allowing for a transition / transversion bias, on the other hand, has little affect. Detailed analysis of these data results in phylogenetic estimates largely congruent with widely accepted hypotheses of relationships among green plants, but bootstrapping provides little statistical support for most of these groupings. The lack of statistical support is a direct result of both the small number of phylogenetically informative sites in the SS rRNA molecule and the high rate of change at those positions that are free to vary. When considered in conjunction with earlier distance-based analyses of phylogeny that use 5s rRNA, these results demonstrate that phylogenetic estimates based only on analyses of 5S rRNA sequences must be viewed with considerable caution.

Evolution of ribosomal RNA gene copy number on the sex chromosomes of Drosophila melanogaster.

Abstract: A diverse array of cellular and evolutionary forces-including unequal crossingover, magnification, compensation, and natural selection-is at play modulating the number of copies of ribosomal RNA (rRNA) genes on the X and Y chromosomes of Drosophila. Accurate estimates of naturally occurring distributions of copy numbers on both the X and Y chromosomes are needed in order to explore the evolutionary end result of these forces. Estimates of relative copy numbers of the ribosomal DNA repeat, as well as of the type I and type II inserts, were obtained for a series of 96 X chromosomes and 144 Y chromosomes by using densitometric measurements of slot blots of genomic DNA from adult D. melanogaster bearing appropriate deficiencies that reveal chromosome-specific copy numbers. Estimates of copy number were put on an absolute scale with slot blots having serial dilutions both of the repeat and of genomic DNA from nonpolytene larval brain and imaginal discs. The distributions of rRNA copy number are decidedly skewed, with a long tail toward higher copy numbers. These distributions were fitted by a population genetic model that posits three different types of exchange events-sister-chromatid exchange, intrachromatid exchange, and interchromosomal crossing-over. In addition, the model incorporates natural selection, because experimental evidence shows that there is a minimum number of fimctional elements necessary for survival. Adequate fits of the model were found, indicating that either natural selection also eliminates chromosomes with high copy number or that the rate of intrachromatid exchange exceeds the rate of interchromosomal exchange.

Molecular phylogenetic analysis of actin genic regions from Achlya bisexualis (Oomycota) and Costaria costata (Chromophyta).

Abstract: Actin genic regions were isolated and characterized from the heterokont-flagellated protists, Achlya bisexualis (Oomycota) and Costaria costata (Chromophyta). Restriction enzyme and cloning experiments suggested that the genes are present in a single copy and sequence determinations revealed the existence of two introns in the C. costata actin genic region. Phylogenetic analyses of actin genic regions using distance matrix and maximum parsimony methods confirmed the close evolutionary relationship of A. bisexualis and C. costata suggested by ribosomal DNA (rDNA) sequence comparisons and reproductive cell ultrastructure. The higher fungi, green plants, and animals were seen as monophyletic groups; however, a precise order of branching for these assemblages could not be determined. Phylogentic frameworks inferred from comparisons of rRNAs were used to assess rates of evolution in actin genic regions of diverse eukaryotes. Actin genic regions had nonuniform rates of nucleotide substitution in different lineages. Comparison of rates of actin and rDNA sequence divergence indicated that actin genic regions evolve 2.0 and 5.3 times faster in higher fungi and flowering plants, respectively, than their rDNA sequences. Conversely, animal actins evolve at approximately one-fifth the rate of their rDNA sequences.

Novel tRNA gene organization in the 16S-23S intergenic spacer of the Streptococcus pneumoniae rRNA gene cluster

Abstract: Isoleucine and alanine tRNAs are encoded tandemly within the 16S-23S intergenic spacer of some eubacterial rRNA gene clusters. Southern hybridization analysis and DNA sequence analysis demonstrated a novel gene organization for an rRNA gene cluster on the Streptococcus pneumoniae chromosome. A sequence specifying an alanine tRNA was found within the intergenic spacer, but no sequence specifying an isoleucine tRNA was found there. Southern hybridization analysis indicated that the location of the isoleucine tRNA gene was near the 5S rRNA gene in two of four rRNA gene clusters. Images