Identifying and distinguishing sibling species in the Tetrahymena pyriformis complex (Ciliophora, Oligohymenophorea) using PCR/RFLP analysis of nuclear ribosomal DNA

We describe a riboprinting strategy for identifying and distinguishing among sibling species in the Tetrahymena pyriformis complex. It involves use of the polymerase chain reaction to amplify a large segment of the nuclear ribosomal DNA and internal transcribed spacers, and digestion of this DNA with restriction enzymes. Unique restriction fragment length patterns or haplotypes were then used to distinguish species into: (1) six taxa that were identifiable to the species level, (2) eight taxa that were separated into four pairs, and (3) a group of eight taxa that were identical to each other. The latter result indicates that a more variable molecule is needed to distinguish the most closely related species in the complex. There was no intraspecific variation between two strains from one species (Tetrahymena thermophila) nor among multiple isolates from another species (Tetrahymena empidokyrea). This approach provides an alternative to traditional techniques for identifying T. pyriformis species that require living reference specimens and/or that reveal high levels of intraspecific variation.     

Alternative splicing of Pax6 in bovine eye and evolutionary conservation of intron sequences

There are now six recognized neuropeptide Y (NPY) receptor subtypes (Y1-Y4 and two recently cloned distinct receptors labeled Y5), of which Y1 and one of the Y5's have been suggested could mediate the effect of NPY on feeding. The fragments NPY(2-36) and NPY(3-36), which bind Y1 only poorly, were injected intracerebroventricularly (icv) and found to have similar dose-response relationships to NPY in the stimulation of feeding. However NPY (13-36), which stimulates both Y2 and Y5, caused no increase in food intake, even at high doses. Maximal stimulation with the classical Y1 agonist [Pro34]-NPY produced only 50% of the maximum effect of NPY itself despite fully inhibiting adenylyl cyclase activity in vitro in a Y1 system. The novel fragment [Pro34]-NPY(3-36) is as effective at stimulating food intake as the classical Y1 analogue [Pro34]-NPY but bound to the Y1 receptor with only 1/20th of the affinity of NPY and failed to inhibit adenylyl cyclase through this receptor. [Pro34]-NPY(3-36) is therefore a relatively appetite-selective ligand. Coadministration of high dose NPY(13-36) and [Pro34]NPY did not enhance feeding compared with [Pro34]-NPY alone. In addition, the NPY Y1 receptor antagonist BIBP-3226, which does not bind Y2, Y4, or Y5 receptors, significantly reduced NPY induced feeding. These results indicate that the feeding effect of icv NPY involves a novel receptor and that it is functionally distinct from the recognized receptor subtypes.     

Amount of DNA produced during extra S phases in Tetrahymena

A protein mixture named LTx, was released from human tonsillar lymphocytes, when incubated at 37 degrees C in either "individual" or "mixed" cultures. LTx caused a decrease in the incorporation of 14-C- labelled amino acids of the target lymphocytes, while the release of 51- Cr from labelled target cells increased. Target cells were found to bind the "toxic" protein.     

Large differences in substitutional pattern and evolutionary rate of 12S ribosomal RNA genes

We demonstrate using Drosophila, periodical cicadas, and hominid primates, that the molecular clock based on animal mitochondrial small-subunit (12S) rRNA genes ticks at significantly different relative rates depending on which taxa and which region of the gene are examined. Drosophila, which are commonly used as model taxa, are evolving in a highly peculiar manner with the majority of sites in the 3' half of the 12S gene apparently invariant. The analogous 3' half of the mitochondrial large-subunit rRNA gene (16S) appears to be similarly constrained. It is surprising that these regions that are already highly constrained in all animals should be even more constrained in Drosophila, especially when the Drosophila mitochondrial genome as a whole does not display a similar rate slowdown. This extreme 12S rate slowdown is not apparent in periodical cicadas or hominid primates and appears to be related to strong structural and functional constraints rather than a depressed mutation rate. Finally, the slow average rate of evolution in the third domain of Drosophila does not imply that the few variable sites lack multiple hits.     

Identification, mapping and linkage analysis of randomly amplified DNA polymorphisms in Tetrahymena thermophila

Using the random amplified polymorphic DNA (RAPD) technique and exploiting the unique genetics of Tetrahymena thermophila, we have identified and characterized 40 DNA polymorphisms occurring between two inbred strains (B and C3) of this ciliated protozoan. These RAPD markers permit the PCR amplification of a DNA species using template DNA from SB1969 (B strain) but fail to do so using DNA from C3-368-5 (C3 strain). Polymorphisms were mapped to chromosomes using a panel of monosomic strains constructed by crossing B strain-derived nullisomic strains to inbred strain C3. They map to all five chromosomes and appear to be evenly distributed throughout the genome. Chromosomal groups were then analyzed for linkage using meiotic segregants; four linkage groups were identified in chromosomes 1R 2L, 3 and 5. The RAPD method appears useful for the construction of a genetic map of the Tetrahymena genome based on DNA polymorphisms.     

Mapping of the genes for ribosomal proteins S26, L19, and L32 on human chromosomes

A fragment of cDNA and an intron-containing fragment of the human L19 ribosomal protein (RPL19) gene, and introns of the human ribosomal proteins S26 (RPS26) and L32 (RPL32) genes were cloned and sequenced. The intron-containing genes of these ribosomal proteins were mapped to human chromosomes by means of polymerase chain reaction (PCR) using a human/rodent hybrid DNA panel.     

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.     

Characterization of Borrelia lusitaniae sp. nov. by 16S ribosomal DNA sequence analysis

We determined the complete sequence of the rrs gene from five strains of genomic species PotiB2. Both distance and parsimony methods were used to infer the evolutionary relationships of the rrs gene sequence of this genomic species in comparison with the rrs gene sequence of Borrelia valaisiana and the rrs gene sequences of Borrelia burgdorferi sensu lato species obtained from sequence databases. The phylogenetic analysis revealed that the genomic species PotiB2 strains clustered in a separate lineage, which was consistent with data from previous DNA-DNA hybridization experiments (D. Postic, M. V. Assous, P. A. D. Grimont, and G. Baranton, Int. J. Syst. Bacteriol. 44:743-752, 1994). A PCR-restriction fragment length polymorphism analysis was used to identify genomic species PotiB2 and to differentiate it from B. burgdorferi sensu lato species. Moreover, signature nucleotide positions were identified for each B. burgdorferi sensu lato species. In accordance with DNA relatedness values, our findings suggest that genomic species PotiB2 can be more clearly defined and identified, and we propose that it should be referred to as a new species, Borrelia lusitaniae. The type strain is PotiB2.     

Orthologous DNA sequence variation among 5S ribosomal RNA gene spacer sequences on homoeologous chromosomes 1B, 1D, and 1R of wheat and rye

5S ribosomal gene spacer sequences from the short-spacer arrays of wheat and rye were isolated by PCR. The 29 new DNA sequences displayed noticeable heterogeneity at scattered positions. Nevertheless, based on shared DNA sequence polymorphisms, sequence alignment clearly classified the sequences into three groups. Group-specific primer sets were designed to allow chromosomal assignment by PCR on nullitetrasomic wheat stocks, as well as on wheat-rye translocation and addition lines. The three groups were assigned to orthologous loci 5S-Rrna-B1, 5S-Rrna-D1, and 5S-Rrna-R1 on homoeologous chromosomes 1B, 1D, and 1R, respectively. Hence, group-specific DNA sequence variation could be related to fixed orthologous DNA sequence variation between 5S rRNA multigene families on the homoeologous group 1 chromosomes. In addition, members of the three groups showed fixed orthologous length polymorphism. Four sequenced 5S-Rrna-B1 units, however, had a duplication in the gene encoding region and are probably representatives of a nontranscribed subfamily of 5S rDNA repeating units. The observed chromosome-specific polymorphisms among sequences belonging to a multigene family with thousands of copies suggests that this type of polymorphism may exist in many genes and gene families in polyploid wheats. The implication of this finding in relation to the construction of molecular tools for wheat-genome analysis and manipulation is discussed.     

Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 40 S ribosomal subunit proteins Sa, Sc, S3a, S3b, S5', S9, S10, S11, S12, S14, S15, S15', S16, S17, S18, S19, S20, S21, S26, S27', and S29

The proteins of the small subunit of rat liver ribosomes were separated into five main groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Twenty-one proteins (Sa, Sc, S3a, S3b, S5', S9, S10, S11, S12, S14, S15, S15', S16, S17, S18, S19, S20, S21, S26, S27', and S29) were isolated from three groups (A40, C40, and D40) by ion exchange chromatography on DEAE-cellulose, carboxymethylcellulose, and phosphocellulose and by filtration through Sephadex. The amount of protein obtained varied from 0.1 to 11 mg. Six of the proteins (S5', S10, S11, S18, S19, and S27') had no detectable contamination; the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.     

Germination, growth, and sporulation of Bacillus thuringiensis subsp. israelensis in excreted food vacuoles of the protozoan Tetrahymena pyriformis

Spores of Bacillus thuringiensis subsp. israelensis and their toxic crystals are bioencapsulated in the protozoan Tetrahymena pyriformis, in which the toxin remains stable. Each T. pyriformis cell concentrates the spores and crystals in its food vacuoles, thus delivering them to mosquito larvae, which rapidly die. Vacuoles containing undigested material are later excreted from the cells. The fate of spores and toxin inside the food vacuoles was determined at various times after excretion by phase-contrast and electron microscopy as well as by viable-cell counting. Excreted food vacuoles gradually aggregated, and vegetative growth of B. thuringiensis subsp. israelensis was observed after 7 h as filaments that stemmed from the aggregates. The outgrown cells sporulated between 27 and 42 h. The spore multiplication values in this system are low compared to those obtained in carcasses of B. thuringiensis subsp. israelensis-killed larvae and pupae, but this bioencapsulation represents a new possible mode of B. thuringiensis subsp. israelensis recycling in nontarget organisms.     

Detection of human herpesvirus 8 DNA sequences in tissues and bodily fluids

Human herpesvirus 8 (HHV-8) has been proposed as a sexually transmitted etiologic agent of Kaposi's sarcoma (KS). In this study, by use of a sensitive polymerase chain reaction assay, HHV-8 DNA was detected in the skin lesions (92%), normal skin (23%), peripheral blood mononuclear cells (PBMC) (46%), plasma (7%), saliva (37%), and semen (12%) but not stool samples from KS patients. The average number of HHV-8 copies per microgram of positive target DNA was 64, 000, 9000, 40, 33,000, and 300 for skin, PBMC, plasma, saliva, and semen samples, respectively. Only 1 non-KS donor sample, of saliva, was positive for HHV-8. Sequencing showed 5% divergence among HHV-8 strains. The data suggest that saliva may be more important than semen or stool in the sexual transmission of HHV-8. The relatively high prevalence of HHV-8 in PBMC raises the question as to why there is no evidence for bloodborne virus transmission.     

The phylogenetic relationships of Rhodosporidium dacryoidum Fell, Hunter et Tallman based on the partial sequences of 18S and 26S ribosomal RNAs: the proposal of Sakaguchia gen. nov., a heterobasidiomycetous yeast genus

The partial base sequences of 18S and 26S rRNAs of Rhodosporidium fluviale, R. lusitaniae, and Erythrobasidium hasegawianum were analyzed. In the 26S rRNA partial base sequencings, R. fluviale CBS 6568 and R. lusitaniae IGC 4599 and IGC 4641 had 81-82 and 77 percent similarities compared with R. toruloides (type species of genus Rhodosporidium) IFO 0559 and IFO 0880. Erythrobasidium hasegawianum IFO 1058 showed 69-71, 59, 63, and 61 percent similarities with R. toruloides IFO 0559 and IFO 0880, L. scottii (type species of genus Leucosporidium) IFO 1923, R. dacryoidum IFO 1930 and IFO 1931, and Kondoa malvinella IFO 1936, respectively. In the 18S rRNA partial base sequencings, R. fluviale CBS 6568 and R. lusitaniae IGC 4599 and IGC 4641 had zero and two base differences with R. toruloides. Erythrobasidium hasegawianum IFO 1058 showed ten, sixteen, three, and twenty base differences with R. toruloides IFO 0559 and IFO 0880, L. scottii IFO 1923, R. dacryoidum IFO 1930 and IFO 1931, and K. malvinella IFO 1936, respectively. Based on the sequence data obtained, a new genus, Sakaguchia was proposed for R. dacryoidum with a new combination, Sakaguchia dacryoides.     

The isolation of eukaryotic ribosomal proteins. The purification and characterization of the 40 S ribosomal subunit proteins S2, S3, S4, S5, S6, S7, S8, S9, S13, S23/S24, S27, and S28

The proteins of the small subunit of rat liver ribosomes were separated into five groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5 (Collatz, E., Lin, A., St?ffler, G., Tsurugi, K., and Wool, I.G., (1976) J. Biol. Chem. 251, 1808-1816). From the several groups, 12 proteins (S2,S3, S4, S5, S6, S7, S8, S9, S13, S23/S24, S27, and S28) wereisolated by ion exchange chromatography on carboxymethylcellulose, by chromatography on sulfopropyl-Sephadex, and by gel filtration through Sephadex G-75. The amount of protein obtained varied from 1 to 9 mg depending on the number of steps required for the preparation; several proteins had no detectable contamination and the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyazrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.     

Studies on the amount and location of the tRNA and 5-S rRNA genes in Tetrahymena pyriformis GL

The occurrence of a deficiency of adenosine deaminase (ADA) activity in some patients with severe combined immunodeficiency suggests a possible relationship between the activity of ADA and the aberration of the immune system. To help delineate the function of ADA in the immune response we have examined its role in monocyte maturation. When incubated in vitro, peripheral blood monocytes transformed, within 3 days, to macrophagea as assessed by phase-contrast microscopy and an increase in the specific activity of the lysosomal enzyme acid phosphatase. The specific activity of ADA increased as much as ninefold, reaching a peak after the 1st day in culture, while the activities of other enzymes involved in the purine salvage pathway were not altered. Sucrose density ultracentrifugation of extracts prepared immediately after the isolation of monocytes revealed the presence of two forms of ADA with molecular weights of approximately 30,000 and 110,000. The increase in ADA specific activity during monocyte cultivation correlated with an increase in the activity of the smaller molecular species. A specific inhibitor ADA, erythro-9-(2-hydroxy-3-nonyl) adenine, prevented the increase in acid phosphatase activity, as well as the morphological changes associated with the monocyte maturation. These data suggest a role for ADA in monocyte to macrophage maturation. In view of the central role of macrophages in immune function, this observation may relate to the association of combined immunodeficiency and a deficiency of this enzyme.     

16S ribosomal DNA typing for identification of pathogens in patients with bacterial keratitis

The identification of pathogens in patients with bacterial keratitis remains problematic because standard diagnostic tests are negative for 40 to 60% of patients. A cross-sectional study was undertaken to determine if PCR and sequence analysis of 16S ribosomal DNA (rDNA) could be used to detect bacterial pathogens in patients with keratitis. Corneal specimens were collected for culture and rDNA typing. Variable segments of each rDNA specimen were amplified by PCR, sequenced, and aligned with the sequences in GenBank. Eleven patients had microbiologically documented bacterial keratitis, while 17 patients had keratitis due to other causes. Nine (82%) of 11 bacterial keratitis patients were PCR positive; each sequencing result matched the culture results. Seventeen (100%) patients with nonbacterial keratitis were PCR negative. Our data suggest that 16S rDNA typing holds promise as a rapid alternative to culture for identifying pathogens in patients with bacterial keratitis.     

Detection of false DNA aneuploidy and false DNA multiploidy in flow cytometric DNA analysis

Although false DNA aneuploid peaks have previously been described in normal tissue, criteria for distinguishing them from 'true' near-diploid peaks have not been established. Normal thyroid (n = 4) and kidney (n = 1) tissue were allowed to autolyze over a fixed period of time and DNA content was analyzed by flow cytometry (FCM). Autolysis was associated with the development of distinct separate G0/G1 peaks which had low DNA indices (1.09-1.18) and showed decreased forward light scatter (FSC) when compared to fresh tissue. Using DNA content and FSC measurements similar false DNA aneuploid peaks were identified in 29/94 surgical specimens. These cases included both benign and malignant lesions from thyroid (n = 63) with the remaining 31 neoplastic cases being from breast (16), lymphoma (8), sarcoma (4), lung (2) and uterine (1) tissue. In addition, false DNA multiploidy was identified. None of these cases showed histological evidence of necrosis. In a parallel comparison study using image cytometry (ICM) on the thyroid nodules, the presence of false DNA aneuploidy was supported. Investigators should routinely employ quality control criteria to identify possible cases of false DNA aneuploidy when measuring DNA content using FCM.