Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.     

Monophyly of Endosymbiont Containing Trypanosomatids: Phylogeny versus Taxonomy

To obtain additional information on the phylogenetic relationships within the family Trypanosomatidae (order Kinetoplastida), we have sequenced the small subunit ribosomal RNA genes from the endosymbiont containing species Herpetomonas roitmani TCC080, Herpetomonas sp. TCC263, Crithidia oncopelti ATCC 12982 and a partial large subunit rRNA gene from H. roitmani. The small subunit sequences in the two isolates of Herpetomonas are very similar but not identical, and so are their restriction digest profiles of kinetoplast DNA. The size of minicircles in both isolates is 4.2 kilobases. The inferred ribosomal RNA phylogenetic trees shows the genera Herpetomonas and Crithidia as polyphyletic. Endosymbiont-bearing herpetomonads cluster with the endosymbiont-bearing crithidias and a blastocrithidia to form a monophyletic clade, whereas the endosymbiont-free members of these genera are found elsewhere in the tree. These data support the hypothesis of a monophyletic origin of endosymbiosis in trypanosomatid evolution and also suggest that a taxonomic revision is needed in order to better describe the natural affinities in this family.     

Kinetoplast DNA of Crithidia oncopelti. Recognition of the principles of structural organization of minicircular DNA molecules

The heterogeneity of the kinetoplast minicircular DNAs from Crithidia oncopelti was studied by electron microscopy and restriction analysis. The associate of kinetoplast DNA comprises five main classes of minicircles sizing 0.42, 0.51, 0.62, 0.78 and 0.83 microns. Examination of cleavage patterns revealed that each class of the minicircles is heterogeneous in base sequence, but there are restriction fragments of the same size common for all these classes. A model of structural arrangement of minicircles of Crithidia oncopelti was proposed. This model is based on the block structure of minicircular molecules. Each minicircle is composed of one variable block (V-block), 810 base pairs, and from 1 to 4 more conservative blocks (C-blocks) each containing 445 base pairs.     

The genes for cytoplasmic ribosomal ribonucleic Acid in higher plants

The genes for cytoplasmic ribosomal RNA are partially resolved from the bulk of the DNA by CsCl equilibrium centrifugation. Although in some plants the buoyant density of the ribosomal RNA genes is as expected from the base composition of ribosomal RNA, others show a large discrepancy which cannot be due to the presence of low G-C spacer-DNA. The cross-hybridization observed with 1.3 and 0.7 × 10⁶ molecular weight ribosomal RNAs and DNA, which varies greatly with different plant species, is not due to contamination of the ribosomal RNAs, and is specific for the ribosomal DNA of each species, probably largely restricted to those sequences coding for the two stable ribosomal RNAs. The double reciprocal plot may be used for the extrapolation of saturation values only with caution, because in these cases such plots are not linear over the whole of the hybridization reaction.     

Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana.

Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite- or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-type minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type- or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.     

Quantitation of in situ hybridization of ribosomal ribonucleic acids to human diploid cells

The hybridization of 5S and 28S ribosomal RNAs to human fibroblast and leukocyte cells was used as a model system to quantitate the technique of in situ hybridization for human diploid cell types. Quantitation consisted of counting (scoring) the number of grains formed over both interphase nuclei and metaphase chromosomes on slides after various hybridization procedures. The average number of grains/nucleus per slide was then used to determine hybridization percentages. As with nitrocellulose filter hybridizations the kinetics of in situ hybridizations can be fit with a single first-order rate constant. However, the in situ hybridization rate was approximately 10 times slower than the corresponding filter hybridization rate. The efficiency of in situ hybridization was found to range between 5 and 15% for both leukocyte and fibroblast cell types and for both metaphase and interphase nuclei. Determination of the parameters of the in situ hybridization reaction of ribosomal RNAs to diploid chromosomes define the experimental conditions needed for the localization of single copy genes to diploid chromosomes.     

Plastid-derived single gene minicircles of the dinoflagellate Ceratium horridum are localized in the nucleus

Recent reports show that numerous chloroplast-specific proteins of peridinin-containing dinoflagellates are encoded on minicircles-small plasmidlike molecules containing one or two polypeptide genes each. The genes for these polypeptides are chloroplast specific because their homologs from other photosynthetic eukaryotes are exclusively encoded in the chloroplast genome. Here, we report the isolation, sequencing, and subcellular localization of minicircles from the peridinin-containing dinoflagellate Ceratium horridum. The C. horridum minicircles are organized in the same manner as in other peridinin-containing dinoflagellates and encode the same kinds of plastid-specific proteins, as previous studies reported. However, intact plastids isolated from C. horridum do not contain minicircles, nor do they contain DNA that hybridizes to minicircle-specific probes. Rather, C. horridum minicircles are localized in the nucleus as shown by cell fractionation, Southern hybridization, and in situ hybridization with minicircle-specific probes. A high-molecular-weight DNA was detected in purified C. horridum plastids, but it is apparently not minicircular in organization, as hybridization with a cloned probe from the plastid-localized DNA suggests. The distinction between C. horridum and other peridinin-containing dinoflagellates at the level of their minicircle localization is paralleled by C. horridum thylakoid organization, which also differs from that of other peridinin-containing dinoflagellates, indicating that a hitherto underestimated diversity of minicircle DNA localization and thylakoid organization exists across various dinoflagellate groups.     

Mitochondrial genes are found on minicircle DNA molecules in the mesozoan animal Dicyema

Animal mitochondrial DNA genomes are generally single circular molecules, 14-20 kb in size, containing a number of functional RNAs and 13 protein-coding genes. Among these, the COI, COII and COIII genes encode three subunits of cytochrome c oxidase. We have isolated and characterized these three mitochondrial genes from the mesozoan Dicyema, a primitive multicellular animal. Surprisingly, the COI, COII and COIII genes are encoded on three small, separate circular DNA molecules (minicircles) of length 1700, 1599 and 1697 bp, respectively. We estimated the copy number of each minicircle at 100 to 1000 per cell, and have shown a mitochondrial localization of the minicircles by in situ hybridization. Furthermore, we could not detect a putative "maxicircle" DNA molecule containing any combination of the COI, COII and COIII genes using either PCR or genomic Southern hybridization. Thus, our results show a novel mitochondrial genome organization in the mesozoan animal Dicyema.     

Ribosomal RNA and ribosomal proteins in corynebacteria

Ribosomal RNAs (rRNAs) (16S, 23S, 5S) encoded by the rrn operons and ribosomal proteins play a very important role in the formation of ribosomes and in the control of translation. Five copies of the rrn operon were reported by hybridization studies in Brevibacterium (Corynebacterium) lactofermentum but the genome sequence of Corynebacterium glutamicum provided evidence for six rrn copies. All six copies of the C. glutamicum 16S rRNA have a size of 1523 bp and each of the six copies of the 5S contain 120 bp whereas size differences are found between the six copies of the 23S rRNA. The anti-Shine-Dalgarno sequence at the 3'-end of the 16S rRNA was 5'-CCUCCUUUC-3'. Each rrn operon is transcribed as a large precursor rRNA (pre-rRNA) that is processed by RNaseIII and other RNases at specific cleavage boxes that have been identified in the C. glutamicum pre-rRNA. A secondary structure of the C. glutamicum 16S rRNA is proposed. The 16S rRNA sequence has been used as a molecular evolution clock allowing the deduction of a phylogenetic tree of all Corynebacterium species. In C. glutamicum, there are 11 ribosomal protein gene clusters encoding 42 ribosomal proteins. The organization of some of the ribosomal protein gene cluster is identical to that of Escherichia coli whereas in other clusters the organization of the genes is rather different. Some specific ribosomal protein genes are located in a different cluster in C. glutamicum when compared with E. coli, indicating that the control of expression of these genes is different in E. coli and C. glutamicum.     

Fine structure of ribosomal RNA: I. Conservation of homologous regions within ribosomal RNA of eukaryotes

By molecular hybridization experiments the homologies between ribosomal RNAs from a unicellular organism (Gyrodinium cohnii), three invertebrates (Drosophila hydei, Chironomus thummi, Sciara coprophila), an amphibian (Xenopus laevis), and a mammal (mouse) were determined. Competition hybridization experiments demonstrated that portions of these homologous regions are the same in all the ribosomal RNAs tested, regardless of animal species. This conclusion based on hybridization data was confirmed by comparative fingerprint analysis. The ribosomal RNA sequences involved in heterologous hybridization have a higher A + T composition than the bulk ribosomal RNA. It appears from competition experiments of a heterologous hybridization that two thirds of the conserved similar regions are present in 18 S ribosomal RNA, and the remaining one third in 28 S ribosomal RNA. It is argued that these similar regions have been conserved during evolution due to their structural and/or functional role in ribosomal RNA.     

Characterization of Kinetoplast DNA from Phytomonas serpens

ABSTRACT. The restriction enzyme digestion of kinetoplast DNA from four Phytomonas serpens isolates shows an overall similar band pattern. One minicircle from isolate 30T was cloned and sequenced, showing low levels of homology but the same general features and organization as described for minicircles of other trypanosomatids. Extensive regions of the minicircle are composed by G and T on the H strand. These regions are very repetitive and similar to regions in a minicircle of Crithidia oncopelti and to telomeric sequences of Saccharomyces cerevisiae. Conserved Sequence Block 3, present in all trypanosomatids, is one nucleotide different from the consensus in P. serpens and provides a basis to differentiate P. serpens from other trypanosomatids. Electron microscopy of kinetoplast DNA evidenced a network with organization similar to other trypanosomatids and the measurement of minicircles confirmed the size of about 1.45 kb of the sequenced minicircle.     

Localization of the ribosomal genes in Caenorhabditis elegans chromosomes by in situ hybridization using biotin-labeled probes

The site of the ribosomal gene cluster on embryonic metaphase chromosomes of Caenorhabditis elegans has been mapped by in situ hybridization using probe DNAs that have been nick-translated to incorporate biotin-labeled UTP. The hybridized probe DNA was detected by a double-layer fluorescent antibody technique. Since chromosomes from wild-type C. elegans embryos are indistinguishable, in situ hybridization was carried out with chromosomes from C. elegans strains carrying cytologically distinct translocation or duplication chromosomes in order to identify the right end of linkage group I as the site of the ribosomal genes. Chromosomes carrying a lethal mutation, let-209 I displayed smaller hybridization signals than wild-type, suggesting that these chromosomes carried a partial deficiency of the ribosomal gene cluster. A duplication of the ribosomal genes, eDp20(I;II) rescued let-209 homozygotes. Chromosomes carrying the alterations in the ribosomal genes were combined with mnT12(IV;X) to facilitate the mapping of genes in C. elegans by in situ hybridization. Linkage groups I and II are then labeled by the distinctive hybridization signals from the ribosomal probes, linkage groups IV and X are together distinguishable morphologically and linkage group V is labeled by hybridization to a 5S gene probe.     

A computational screen for C/D box snoRNAs in the human genomic region associated with Prader-Willi and Angelman syndromes

Small nucleolar RNAs (snoRNAs) play a significant role in Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS), which are genomic disorders resulting from deletions in the human chromosomal region 15q11–q13. To identify snoRNAs in the region, our computational study employs key motif features of C/D box snoRNAs and introduces a complementary RNA–RNA hybridization test. We identify three previously unknown methylation guide snoRNAs targeting ribosomal 18S and 28S RNAs, and two snoRNAs targeting serotonin receptor 2C mRNA. We show that the three snoRNA candidates likely possess methylation strands complementary to, and form stable complexes with, human ribosomal RNAs. Our screen also identifies 8 other snoRNA candidates that do not pass the rRNA-complementarity and/or hybridization tests. Two of these candidates have extensive sequence similarity to HBII-52, a snoRNA that regulates the alternative splicing of serotonin receptor 2C mRNA. Six out of our eleven candidate snoRNAs are also predicted by other existing methods.     

Nucleotide Sequence Homology Exists between the Chloroplast and Nuclear Ribosomal DNAs of Euglena gracilis

The nuclear and chloroplast ribosomal DNAs from Euglena were shown to have specific regions of nucleotide sequence homology. The regions of homology were identified by hybridization of restriction endonuclease DNA fragments of cloned chloroplast and nuclear ribosomal DNAs to one another. The regions of homology between these two ribosomal DNAs were in that part of the genes that code for the 3′ end of the small rRNAs (16S and 19S) and near or at the DNA sequences coding for the 5S RNAs. The nucleotide sequence homology between these regions was estimated to be approximately 94% by the melting point depression of a hybrid formed between the two ribosomal DNAs.     

The mitochondrial tRNAs of Trypanosoma brucei are nuclear encoded

The mitochondrial DNA of Trypanosoma brucei is organized as a catenated network of maxicircles and minicircles. The maxicircles are equivalent to the typical mitochondrial genome except that the genes for the mitochondrial tRNAs have not been identified by sequence analysis of the maxicircle DNA. The apparent absence of tRNA genes in the maxicircle DNA suggests that the mitochondrial tRNAs are encoded by either the minicircle or the nuclear DNA. In order to determine their genomic origin, we isolated and identified the mitochondrial tRNAs of T. brucei. We show that these mitochondrial tRNAs are truly mitochondrially located in vivo and that they are free from detectable contamination by cytosolic RNAs. By hybridization analysis, using mitochondrial tRNAs as the probe, we determined that the mitochondrial tRNAs are encoded by nuclear DNA. This implies that RNAs, like proteins, are imported into the mitochondria. We investigated the relationship between the cytosolic and the mitochondrial tRNA genes and show that there are unique cytosolic tRNA genes, unique mitochondrial tRNA genes, and tRNA genes which appear to be shared and whose products are therefore targeted to both the cytosol and the mitochondrion.     

Organization of tRNA and rRNA genes in N. crassa mitochondria: intervening sequence in the large rRNA gene and strand distribution of the RNA genes.

Through analysis of cloned fragments of N. crassa mitochondrial DNA, we have derived a physical map for the region of the mitochondrial genome which encodes the ribosomal RNAs and most of the tRNAs. We have located RNA genes on this map by hybridization of purified 32P end-labeled RNA probes, and our findings are as follows. First, the gene for the large ribosomal RNA contains an intervening sequence of approximately 2000 bp. Second, the genes for the small and large ribosomal RNAs are not adjacent, as previously reported, and the region between them contains a number of tRNA genes, including that for the mitochondrial tRNATyr, which is located close to the small rRNA gene on the same strand of the mitochondrial DNA. Third, there is a second cluster of tRNA genes on the mitochondrial DNA following the large ribosomal RNA gene, but there is no evidence for the presence of tRNA genes in the intervening sequence of the large ribosomal RNA. Fourth, hybridization of labeled ribosomal and transfer RNAs to the separated strands of a cloned 16 kbp DNA fragment covering this region indicates that the two ribosomal RNAs and most, if not all, of the mitochondrial tRNAs are encoded on one strand of the mitochondrial DNA.