Verification of intermediate hosts in the life cycles of microsporidia by small subunit rDNA sequencing.

Small subunit rDNA sequences were obtained from field-collected Amblyospora connecticus (Microsporida: Amblyosporidae) spores isolated from the mosquito, Aedes cantator (Diptera: Culicidae), and from field collected spores isolated from the putative intermediate host, Acanthocyclops vernalis (Copepoda: Cyclopidae). The ribosomal DNA sequences of the spores isolated from the two hosts were identical. These findings corroborate previous laboratory transmission studies and validate the intermediary role of A. vernalis in the life cycle of this microsporidium. These data represent the first comparative sequence analysis of a microsporidium isolated from its definitive and intermediate hosts. The results demonstrate the effectiveness of using rDNA sequence data for screening potential intermediate hosts. Unlike laboratory transmission tests, which can take months or years to complete, this technique can be completed in days and can be performed on a single infected organism.

Restriction analysis of the ribosomal DNA internal transcribed spacer region of Culex restuans and mosquitoes in the Culex pipiens complex.

Members of the Culex pipiens Linn. complex in the eastern, southern, and central United States are the primary vectors of St. Louis encephalitis virus. Although species and subspecies in the complex can be identified as 4th-instar larvae and by characters on the male genitalia, adult females cannot be identified accurately. In this study a ribosomal DNA (rDNA) segment that includes the internal transcribed spacer region (ITS) was amplified from Culex pipiens pipiens Linn., Culex quinquefasciatus Say, and Culex restuans Theobald. The DNA was amplified from single abdomens or single legs. The amplified rDNA segment from Cx. restuans is 90 base pairs smaller than those from members of the Cx. pipiens complex. Ribosomal DNA was amplified separately from 3 individuals for each population of Cx. pipiens and analyzed by restriction digestion. Intrapopulation variation is seen, because for each population, bands are present that are common to all 3 individuals within the population, but are also unique to that population. These results indicate that this method may provide a means for distinguishing among the mosquitoes in the Cx. pipiens complex.

Characterization of the highly divergent U2 RNA homolog in the microsporidian Vairimorpha necatrix.

An RNA homologous to U2 RNA and a single copy gene encoding the RNA homolog have been characterized in the microsporidian, Vairimorpha necatrix. The RNA which is 165 nucleotides in length possesses significant similarity to U2 RNA, particularly in the 5' half of the molecule. The U2 homolog contains the highly conserved GUAGUA branch point binding sequence seen in all U2 RNAs except those of the trypanosomes. A U2 RNA sequence element implicated in a U2:U6 RNA intermolecular pairing is also present in the U2 homolog. The V. necatrix U2 RNA homolog differs at positions previously found to be invariant in U2 RNAs and appears to lack an Sm binding site sequence. The RNA can be folded into a secondary structure possessing three of the four principal stem-loops proposed for the consensus U2 RNA structure. A cis-diol containing cap structure is present at the 5' end of the U2 homolog. Unlike the cap structures seen in U-snRNAs and mRNAs it is neither 2,2,7-trimethylguanosine, gamma-monomethyl phosphate, nor 7-methylguanosine.

Ribosomal DNA sequences of Encephalitozoon hellem and Encephalitozoon cuniculi: species identification and phylogenetic construction.

A segment of ribosomal DNA, about 1,350 base pairs long, was amplified from the microsporidian species Encephalitozoon hellem, isolated from AIDS patients, and Encephalitozoon cuniculi. The amplified DNA segment extends from position 530 in the small ribosomal RNA subunit to position 580 in the large ribosomal RNA subunit. A comparison of sequence data from this region for Encephalitozoon hellem and Encephalitozoon cuniculi shows relatively high sequence similarity, supporting the placement of these two organisms in the same genus. At the same time, sequence differences between the two organisms confirm that they are not the same species. Three separate isolates of E. hellem were sequenced in the highly variable intervening spacer region. The sequence was identical for all three isolates. Within the amplified DNA segment, regions were sequenced which yield highly variable, moderately variable and highly conserved sequence information, appropriate for comparison with other species in the phylum Microspora at all taxonomic levels. We suggest that sequence data from these regions be included in future species descriptions for the purposes of species identification and phylogenetic analysis. Restriction digests of the amplified region are presented and give a rapid method for distinguishing between the two Encephalitozoon species.

Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes.

The microsporidia are a group of unusual, obligately parasitic protists that infect a great variety of other eukaryotes, including vertebrates, arthropods, molluscs, annelids, nematodes, cnidaria and even various ciliates, myxosporidia and gregarines. They possess a number of unusual cytological and molecular characteristics. Their nuclear division is considered to be primitive, they have no mitochondria, their ribosomes and ribosomal RNAs are reported to be of prokaryotic size and their large ribosomal subunit contains no 5.8S rRNA. The uniqueness of the microsporidia may reflect their phylogenetic position, because comparative sequence analysis shows that the small subunit rRNA of the microsporidium Vairimorpha necatrix is more unlike those of other eukaryotes than any known eukaryote 18S rRNA sequence. We conclude that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.

Gram-positive bacteria: possible photosynthetic ancestry.

A 16S ribosomal RNA gene has been sequenced from Heliobacterium chlorum, the recently discovered photosynthetic bacterium that contains a novel form of chlorophyll. Comparisons with other 16S ribosomal RNA sequences show that the organism belongs to the Gram-positive bacteria (one of ten eubacterial "phyla")--more precisely to the so-called low G + C (G, guanine; C, cytosine) subdivision thereof. This brings to five the number of such phyla that contain photosynthetic species, the other four being the purple bacteria and relatives, the green sulfur bacteria, the green nonsulfur bacteria, and the cyanobacteria. The finding suggests that Gram-positive bacteria may be of photosynthetic ancestry, and it strengthens the case for a common photosynthetic ancestry for all eubacteria.

Isolation and sequence analysis of three cloned cDNAs for rabbit liver proteins that are related to rabbit cytochrome P-450 (form 2), the major phenobarbital-inducible form.

We have isolated from rabbit liver three cDNA clones of 1400-1800 base pairs that hybridize selectively to RNA from animals treated with phenobarbital. The nucleotide sequences of the cDNAs have been determined. In the protein coding region the nucleotide sequences of two of the cDNAs are 88% homologous, and the third cDNA is about 72-74% homologous to the other two. All three are 55-60% homologous to rat liver cytochrome P-450b cDNA. The amino acid sequences derived from the cDNA sequences are about 50% homologous to those of rat liver cytochrome P-450b and rabbit liver cytochrome P-450 (form 2). The degree of homology differs substantially in different regions of the protein. The hydrophobicity profiles of these five mammalian cytochromes P-450 are very similar and contain up to eight regions of hydrophobicity that are long enough to span a membrane. These results indicate that these three cDNAs code for rabbit liver cytochromes P-450 which are different from any rabbit liver cytochrome P-450 for which amino acid sequence information is published. These cDNAs are part of a family of genes that are related to rabbit liver cytochrome P-450 (form 2) and rat liver cytochrome P-450b which are the major phenobarbital-inducible forms. The divergence of amino acid sequence between the rat and rabbit forms and the divergence of nucleotide sequences of silent sites in the two most closely related rabbit forms suggest that cytochromes P-450 have a relatively high rate of amino acid divergence compared to many other vertebrate proteins.