Peter R. Baverstock

Phylogenetic relationships of Cryptosporidium determined by ribosomal RNA sequence comparison

Reverse transcription of total cellular RNA was used to obtain a partial sequence of the small subunit ribosomal RNA of Cryptosporidium, a protist currently placed in the phylum Apicomplexa. The semi-conserved regions were aligned with homologous sequences in a range of other eukaryotes, and the evolutionary relationships of Cryptosporidium were determined by two different methods of phylogenetic analysis. The prokaryotes Escherichia coli and Halobacterium cuti were included as outgroups. The results do not show an especially close relationship of Cryptosporidium to other members of the phylum Apicomplexa.

Blastocystis hominis: Phylogenetic affinities determined by rRNA sequence comparison

In 1912 Blastocystis hominis was identified as a new species and classified as a yeast (Brumpt 1912). In the early 1920s several groups confirmed its classification as a yeast, specifically a member of the genus Schizosaccharomyces (discussed by Zierdt et al. 1967). Apart from an occasional case report, the classification of B. hominis and its role as a harmless intestinal yeast was not questioned for another 50 years. Then, Zierdt (1967) suggested that it should be classified in the phylum Protozoa, subphylum Sporozoa, and that it should be considered as a potential pathogen. The likely role of B. hominis as a human pathogen has recently become more firmly established (Garcia et al. 1984; Sheehan et al. 1986) and its classification has been changed. Although the classification of B. hominis as a protozoon was assumed widely, classification as a sporozoon was not accepted, and the most recent definitive classification of the Protozoa did not even list B. hominis (Lee et al. 1985). Then, based essentially on a review of the known characteristics of the organism, it was recently reclassified into the subphylum Sarcodina (Zierdt 1988). Clearly, the phylogeny of this emerging human pathogen needs definitive analysis (Mehlhorn 1988).

A genetic approach to species criteria in the amoeba genus Naegleria using allozyme electrophoresis

The present study employs allozyme electrophoresis to characterize and inter-relate 61 isolates of Naegleria. Diploidy was confirmed, with heterozygotes observed at 29 of the 33 loci established and in all but two isolates. With a single exception, isolates clustered at two levels of similarity, either below 21% or above 52%. It is argued that such a major discontinuity provides a sound biological basis for a species concept in Naegleria. On this basis the present species-level taxonomy does not reflect the genetic diversity of the genus. The study recognized 18 genetic groups of species rank. The subspecies N. australiensis italica deserves specific rank; additional thermophilic species not closely related to N. fowleri and N. lovaniensis are recognized; and N. gruberi as currently conceived is a complex of 10 species, at least five of which are represented in the formal culture collections. Most species are genetically too different for relationships to be elucidated by allozyme electrophoresis, supporting the view that some of the times of divergence within the genus are extremely ancient.

Genetic characterization of Leishmania isolates at 37 enzyme loci

Abstract Andrews R. H., Handman E., Adams M., Baverstock P. R. and Mitchell G.F. 1988. Genetic characterization of Leishmania isolates at 37 enzyme loci. International Journal for Parasitology 18 : 445–452. Genetic characterization of medically important parasites by allozyme electrophoresis has great potential as a routine analytical tool. Studies to date however have usually been based on a limited number of loci, and have therefore failed to provide the resolution necessary for comprehensive characterization. We herein report the successful typing of 37 enzyme loci in 20 isolates of six recognized species of the protozoan parasite Leishmania . The results showed that (i) there were at least five distinct species differing at 60–91 % of loci, (ii) the L. major group of isolates exhibited allelic variation at a number of loci, (iii) virulent and avirulent clones were genetically identical to the parental isolate and (iv) several of the isolates examined were contaminated. The results illustrate the power of allozyme electrophoresis for delineating species, classifying isolates and strains to species, providing a basis for understanding the emergence of virulence, monitoring efficacy of cloning of isolates, providing quality control of laboratory cultures for cross-contamination and determining the state of ploidy. Moreover, the data highlight the need for routine genetic screening in laboratories and world reference centres where investigations centre on devising molecularly defined vaccines, diagnostic tests or typing reagents for medically important parasites.

Identification of life cycle stages of the nematode Echinocephalus overstreeti by allozyme electrophoresis

Data presented in this study highlight the potential of allozyme electrophoresis in providing unequivocal genetic evidence for the identification of life cycle stages, particularly where species have complex life cycles. Adults of the nematode Echinocephalus overstreeti parasitize the elasmobranch Heterodontus portusjacksoni. The putative larval form which is morphologically dissimilar is found in two species of marine molluscs, Chlamys bifrons and Pecten albus. Electrophoretic analysis indicated that the adult and larval forms shared alleles at all of the 34 enzyme loci established. Furthermore, there were no fixed allelic differences between larval forms from different mollusc species.

Genetic characterization of three species of Onchocerca at 23 enzyme loci

The technique of allozyme electrophoresis was applied to species of Onchocerca from cattle to increase the number of enzyme loci established and therefore provide a genetic basis for a rational species-level taxonomy. Twenty-three enzyme loci were established and provided unequivocal genetic evidence for the taxonomic validity of Onchocerca gibsoni, O. gutturosa and O. lienalis. Furthermore, the diagnostic enzyme markers detected form the basis for identification of life-cycle stages, individuals and species and population structure analyses.

Genetic characterization of three strains of Hymenolepis diminuta at 39 enzyme loci

The technique of allozyme electrophoresis was applied to three strains of Hymenolepis diminuta to distinguish between three hypotheses [(1) multiple species, (2) genetically distinct founder stocks and (3) response to differential selection among similar stocks] proposed to account for metabolic differences among strains. There was no evidence from the 39 enzyme loci established that the three strains represented more than one species. In the absence of knowledge of the population structure of H. diminuta in the wild, electrophoretic data herein could not distinguish between the latter hypotheses. Nevertheless, all three strains were distinguishable on electrophoretic profiles and allelic similarities between strains question the view of their proposed origins.

The structure and evolution of immunoglobulin kappa chain constant region genes in the genus Rattus

We have cloned and sequenced the C-kappa (Ck) genes from seven species and subspecies of rats which have diverged over the past few million years in Australia. Comparisons of these sequences with each other and the Ck genes of the laboratory rat, Rattus norvegicus, indicate noncoding regions have accumulated fewer mutations than adjacent coding sequences, and amino acid replacing nucleotide substitutions in the coding regions have accumulated at a rate at least as great as silent changes. Exactly the opposite of both of these findings is observed when comparisons are made between Ck or other genes from more distantly related species, indicating that these features may be characteristic of Ck short-term evolutionary gene divergence. Changes in the coding regions of these genes result in a non-random distribution of amino acid substitutions on the three-dimensional alpha-carbon backbone of the Ck domain in the most serologically distinct forms of Ck. While phylogenetic relationships inferred from the Ck nucleotide sequences are in general agreement with those derived from other data, considerable differences are seen in rates of accumulation of Ck gene nucleotide substitutions vs rates of accumulation of enzyme polymorphisms.

Duplication of Jκ genes within genus Rattus

We have isolated a region containing the immunoglobulin kappa chain joining segments from a liver DNA library of the Australian rat Rattus villosissimus, and determined its nucleotide sequence. While the laboratory rat (Rattus norvegicus) had previously been shown to contain three recently duplicated copies of Jκ2, R. villosissimus has only two. Furthermore, all three copies of Jκ2 in R. norvegicus share an 11 by deletion in their 5′ flanking regions which is not evident in either copy of Jκ2 in R. villosissimus. This suggests that the initial duplication events occurred separately in the two lineages, and were followed by a second duplication in R. norvegicus, all three duplications having occurred within the last 6–12 million years (although more complicated schemes involving gene conversion events cannot be excluded). These results indicate that there is a high degree of plasticity in this region of the genome, and that selective forces must exist which have maintained the number of expressible Jκ segments in humans (5) and rodents (4–6) within their narrow range.