Josephine Bowles

Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing

The pattern of species and strain variation within the genus Echinococcus is complex and controversial. In an attempt to characterise objectively the various species and strains, the sequence of a region of the mitochondrial cytochrome c oxidase subunit I (CO1) gene was determined for 56 Echinococcus isolates. Eleven different genotypes were detected, including 7 within Echinococcus granulosus, and these were used to categorise the isolates. The 4 generally accepted Echinococcus species were clearly distinguishable using this approach. In addition, the consensus view of the strain pattern within E. granulosus, based on a variety of criteria of differentiation, was broadly upheld. Very little variation was detected within Echinococcus multilocularis. Remarkable intra-strain homogeneity was found at the DNA sequence level. This region of the rapidly evolving mitochondrial genome is useful as a marker of species and strain identity and as a preliminary indication of evolutionary divergence within the genus Echinococcus.

NADH dehydrogenase 1 gene sequences compared for species and strains of the genus Echinococcus

Nucleotide sequences of a 471 bp region of the mitochondrial NADH dehydrogenase 1 gene were obtained for 59 Echinococcus isolates including representatives of each of the 4 recognised species. Ten distinct genotypes were detected among these isolates, including 6 within E. granulosus. This information complements and extends knowledge of inter- and intraspecific variation within Echinococcus and should prove useful in phylogenetic studies.

Retinoic acid, meiosis and germ cell fate in mammals.

Although mammalian sex is determined genetically, the sex-specific development of germ cells as sperm or oocytes is initiated by cues provided by the gonadal environment. During embryogenesis, germ cells in an ovary enter meiosis, thereby committing to oogenesis. By contrast, germ cells in a testicular environment do not enter meiosis until puberty. Recent findings indicate that the key to this sex-specific timing of meiosis entry is the presence or absence of the signaling molecule retinoic acid. Although this knowledge clarifies a long-standing mystery in reproductive biology, it also poses many new questions, which we discuss in this review.

Rapid Discrimination of Echinococcus Species and Strains Using a Polymerase Chain Reaction-Based Rflp Method

Echinococcus species and genetically distinct strains of Echinococcus granulosus can be rapidly and reliably identified using a polymerase chain reaction (PCR)-linked restriction fragment length polymorphism (RFLP) method which surveys the sequence of a rapidly evolving region of the ribosomal DNA (rDNA) unit. Internal transcribed spacer 1 (ITS1) of the rDNA repeat was amplified from various isolates and the product was digested with one of a number of 4-base cutting restriction enzymes. Characteristic patterns were produced when samples within various species and strain groups were analysed. This method offers an objective, simple, highly sensitive and rapid approach for the discrimination of Echinococcus isolates and for study of other parasite complexes.

Molecular genetic approaches to parasite identification: Their value in diagnostic parasitology and systematics

A wide range of approaches is available to parasitologists to aid in specific parasite identification and to formulate phylogenetic relationships. This review emphasises the usefulness of molecular genetic techniques, especially DNA-based procedures, in addressing problems of identification, characterisation and phylogeny of parasites. It should be stressed that an understanding of the various DNA approaches, techniques and target genes most likely to be effective in addressing key issues in diagnostic parasitology and systematics is still developing. Nevertheless, DNA methods clearly have great potential with regard to specificity and sensitivity, and applications will increase further with technological advance. Indeed, because of the minimal requirements for material, PCR-based methods especially should prove of immense value in future studies with parasites.

FGF9 suppresses meiosis and promotes male germ cell fate in mice.

Sex determination of mammalian germ cells occurs during fetal development and depends on signals from gonadal somatic cells. Previous studies have established that retinoic acid (RA) triggers ovarian germ cells to enter meiosis and thereby commit to oogenesis, whereas in the developing testis, the enzyme CYP26B1 degrades RA and germ cells are not induced to enter meiosis. Using in vitro and in vivo models, we demonstrate that fibroblast growth factor 9 (FGF9) produced in the fetal testis acts directly on germ cells to inhibit meiosis; in addition, FGF9 maintains expression of pluripotency-related genes and upregulates markers associated with male germ cell fate. We conclude that two independent and mutually antagonistic pathways involving RA and FGF9 act in concert to determine mammalian germ cell sexual fate commitment and support a model in which the mitosis/meiosis switch is robustly controlled by both positive and negative regulatory factors.

A Molecular Phylogeny of the Genus Echinococcus

Three nucleotide data sets, two mitochondrial (COI and ND1) and one nuclear (ribosomal ITS1), have been investigated in order to resolve relationships among species and strains of the genus Echinococcus. The data have some unusual properties in that mitochondrial heteroplasmy was detected in one strain of E. granulosus, and more than one class of ITS1 sequence variant can occur in a single isolate. The data failed to support the hypothesis that E. granulosus, as it is currently viewed, is a single valid species. Rather, the strains of E. granulosus seem to comprise at least three evolutionarily diverse groups, the sheep strain group, bovine strain group and horse strain group. Molecular distances between them are comparable to, or greater than, molecular evolutionary distances observed between recognized species. The affinities of the cervid strain of E. granulosus are unclear because of ambiguous data, but this strain does not appear to be ancestral to others. E. multilocularis may not be distinct from E. granulosus. However, the remaining two species. E. vogeli and E. oligarthrus appear distinct and rather distant from the first two. Based on the results presented here, taxonomic revision of the genus is clearly warranted.

Nuclear and Mitochondrial Genetic-Markers Highly Conserved Between Chinese and Philippine Schistosoma-Japonicum

Geographical isolates of S. japonicum, and particularly isolates from China and the Philippines, were examined at the molecular level for genetic divergence. Sequences from both nuclear and mitochondrial genomes were selected as markers of evolutionary divergence and S. mekongi and S. mansoni were included in the study for comparison purposes. Restriction fragment length polymorphism (RFLP) and PCR-RFLP analysis of the rDNA repeat unit and sequence analysis of the second internal transcribed spacer region (ITS2) within the rDNA repeat and the cytochrome c oxidase I (COI) gene of the mitochondrial genome were performed. No intra-specific variation in S. japonicum was found in the rDNA repeat and only very slight variation was detected within the COI sequence. A survey of the entire genome, using random amplified polymorphic DNA (RAPD) analysis, again showed that Chinese and Philippine S. japonicum are remarkably similar at the DNA sequence level. We were thus unable to obtain direct molecular evidence in support of previous findings, particularly those based on isoenzyme analysis, that a very high level of intra-specific variation exists in S. japonicum.

Sry requires a CAG repeat domain for male sex determination in Mus musculus.

SRY, the mammalian Y-chromosomal sex-determining gene, encodes a protein characterized by a DNA-binding and -bending domain referred to as the HMG box. Despite the pivotal role of this gene, only the HMG box region has been conserved through evolution, suggesting that SRY function depends solely on the HMG box and therefore acts as an architectural transcription factor. In mice (genus Mus) Sry also includes a large CAG trinucleotide repeat region encoding a carboxy-terminal glutamine-rich domain that acts as a transcriptional trans-activator in vitro. The absence of this or any other potential trans-activating domain in other mammals, however, has raised doubts as to its biological relevance. To test directly whether the glutamine-rich region is required for Sry function in vivo, we created truncation mutations of the Mus musculus musculus Sry gene and tested their ability to induce testis formation in XX embryos using a transgenic mouse assay. Sry constructs that encode proteins lacking the glutamine-rich region were unable to effect male sex determination, in contrast to their wild-type counterparts. We conclude that the glutamine-rich repeat domain of the mouse Sry protein has an essential role in sex determination in vivo, and that Sry may act via a fundamentally different biochemical mechanism in mice compared with other mammals.

Mice null for sox18 are viable and display a mild coat defect.

ABSTRACT We have previously shown that Sox18 is expressed in developing vascular endothelium and hair follicles during mouse embryogenesis and that point mutations in Sox18 are the underlying cause of cardiovascular and hair follicle defects inragged (Ra) mice. Here we describe the analysis of Sox18 −/− mice produced by gene targeting. Despite the profound defects seen in Ra mice,Sox18 −/− mice have no obvious cardiovascular defects and only a mild coat defect with a reduced proportion of zigzag hairs. A reduction in the amount of pheomelanin pigmentation in hair shafts was also observed; later-forming hair follicles showed a reduced subapical pheomelanin band, giving Sox18 −/−mice a slightly darker appearance than Sox18 +/+and Sox18 +/− siblings.Sox18 −/− mice are viable and fertile and show no difference in the ability to thrive relative to littermates. Because of the mild effect of the mutation on the phenotype ofSox18 −/− mice, we conclude that the semidominant nature of the Ra mutations is due to atrans-dominant negative effect mediated by the mutant SOX18 proteins rather than haploinsufficiency as has been observed for otherSOX genes. Due to the similarity of SOX18 to other subgroup F SOX proteins, SOX7 and −17, and the overlap in expression of these genes, functional redundancy amongst these SOX proteins could also account for the mild phenotype of Sox18 −/−mice.