Evidence for multiple mitochondrial lineages of Fasciola hepatica (liver fluke) within infrapopulations from cattle and sheep.

The economic, veterinary, and medical impact of the parasite Fasciola hepatica, liver fluke, is difficult to alleviate due to increasing incidences of resistance to the principal anthelmintic drugs. These have occurred in widely separated regions. The rate of response to selection imposed by such drugs will be dependent on the genetic variation present in the F. hepatica gene pool, but this is at present unknown. We have assessed the genetic diversity of mitochondrial haplotypes found in the infrapopulation of flukes recovered from a calf of known provenance and from six other cattle and sheep hosts located in Ireland and four from elsewhere. Our results revealed that at least ten different mitochondrial composite PCR-restriction fragment length polymorphism haplotypes had been acquired by a single animal in 1 year, and there was comparable diversity in six other definitive hosts carrying field-acquired infections. The extent of divergence between these fluke lineages suggests that they predate the last ice age and, thus, cannot have developed in Northern Europe. A consequence of this high level of diversity is that there will be frequent selection for anthelmintic resistance and rapid responses to climatic changes.

The occurrence and significance of triploidy in the liver fluke, Fasciola hepatica.

Karyotyping of Fasciola hepatica samples from Britain and Ireland has identified a triploid isolate which is effectively aspermic, rendering it necessarily asexually reproducing. Considering the extensive presence of asexually reproducing diploid and triploid Fasciola in Asia it is suggested that facultative gynogenesis is widespread in this parasite. This has important implications for the population genetics and evolution of Fasciola, especially in relation to the development and spread of drug resistance, and must be considered in the mathematical modelling of this process.

Possible loss of length conservation and reciprocity during recombination or conversion in tandem arrays.

Recombination or conversion between arrays of repeated sequences need not be conservative for length because two single strands of DNA from one chromatid may form heteroduplexes in different registers on the other participating duplex. This can cause an overall change in repeat number. Loss of length conservation is equally possible whether models of recombination initiated by single-strand transfer or double-strand break are applicable. Length changing conversion will frequently produce a characteristic insertion within a deletion, often appearing as a double deletion such as are frequently found in new variants of human minisatellite MS32. There is no apparent means of preserving parity during length-changing conversion or recombination, and if the changes are biased then the overall copy number will increase or decrease according to that bias. The observation that arrays persist suggests that any bias in these arrays will be toward gains. An equilibrium may be reached where gains, which may be largely independent of array length, equal losses from the array-length-dependent processes of intrachromatid recombination and repeated unequal sister chromatid exchanges.

The radial positions of metaphase chromosomes may be a consequence of the relative strength of their interaction with the spindle and their size.

Microtubule oriented forces acting on chromosomes on spindles in mitosis and meiosis will produce a radial component of force in the plane of the metaphase plate. The strength of this vector will depend on the angle at which the microtubule meets the plate. Radial forces will tend to segregate chromosomes to peripheral or central positions, depending on their size, and also on the strength of the activity of individual centromeres. In prometaphase, forces pushing chromosomes from the poles will tend to force them to the periphery of the metaphase plate, as seen in radial metaphases. Tension towards the poles at late metaphase will pull smaller chromosomes and those with more powerfully active kinetochores towards the centre of the plate. If the two genomes in a hybrid cell have different centromeric activities, their chromosomes will be segregated. Microtubule assembly and disassembly, and motor proteins such as the kinesins and dynein which haul organelles along microtubules, can provide forces in both directions.

The effects of unequal sister chromatid exchange on length of arrays of repeated sequences.

Models of homogenization of repeated sequences by unequal sister chromatid exchange (USCE) assume that no significant changes in array length occur. The effects of successive USCEs on the lengths of arrays of repeated sequences has been examined mathematically and by simulation, assuming misalignment to be greater in longer arrays, a condition necessary for homogenization. The series of duplications and deletions gives a wide, asymmetric variation in copy number. Frequencies follow a binomial distribution, but if misalignment increases with array length, lengths become logarithmically distributed, most arrays being shorter than the original, counterbalanced by a few very long arrays. The median length, which is also the modal length, decreases exponentially or asymptotically. Given a proportional misalignment of a, the median (mode) decreases by a2/2 per unequal crossover. Since drift is a random transmission of available alleles in the population, it follows that fixation of common short arrays is much more probable than fixation of rare long arrays. This process will continue inexorably until each array is too short to undergo further unequal crossing over, and no more variation is generated. The number of unequal crossovers required by some published models of homogenization would almost certainly cause dramatic reduction in number, or complete loss, of repeats. Frequent unequal sister chromatid exchanges are not compatible with survival of arrays unless counteracted by an independent amplification mechanism or selection, so are unlikely to be important as a long-term homogenization mechanism in non-essential repeated sequences.

A cell surface antigen associated with meiosis in male Staurdoderus scalaris (Orthopteran).

Immunofluorescence has identified seven monoclonal antibodies reactive with the surface of meiotic cells and absent in premeiotic cells. Analysis by immunogold electron microscopy indicated that these antigens were present on the external surface of the cells and were coincident with the presence of synaptonemal complexes in the nucleus. On immunoblots a common glycosylated protein of 205 kDa was recognized, in addition to smaller subunits, suggesting the presence of a protein complex comprised of smaller peptides.

Intracellular antigenic changes associated with meiosis in the orthopteran Stauroderus scalaris.

Monoclonal antibodies have been prepared against purified pachytene cells from grasshopper testes. Immunoblotting and immunofluorescence analyses identified those monoclonal antibodies which showed specificity for antigens in pachytene cells. Several antigenic changes were found to be associated with meiotic cells. Five monoclonal antibodies detected antigens which were located in the cytoplasm of premeiotic cells but were nuclear during meiosis. One monoclonal antibody showed a discrete cytoplasmic fluorescent pattern in meiotic, but not in premeiotic, cells. Another bound specifically to the nuclei of some epithelial cells at the base of follicles in mature testes.

Study of grasshopper meiosis-associated proteins using two-dimensional polyacrylamide gel electrophoresis.

Premeiotic and meiotic whole testes from grasshoppers were compared for the presence of meiosis associated proteins using one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. One-dimensional sodium dodecyl sulphate-polyacrylamide gels detected differences between premeiotic and meiotic samples but two-dimensional gels gave more precise results. Isoelectric focusing revealed only one meiosis-associated protein, while nonequilibrium pH gradient electrophoresis detected five more. It is not known whether these proteins relate to the nuclear aspects of meiosis, or associated cellular changes. These proteins have been electrophoretically purified and monoclonal antibodies are being prepared.

A search for synaptonemal complexes in Ustilago maydis.

Germinating spores, germ-tubes (promycelia), gall tissue and spores developing in the gall were examined. No synaptonemal complexes (SCs) were found in any of these cell types. There are 3 possible explanations for this: (1) Ustilago maydis does not have SCs. This is the case in Schizosaccharomyces pombe (Olson, Eden, Egel-Mitani & Egel, 1978) and in many strains of Saccharomyces cerevisiae (e.g. see Byers & Goetch, 1975). (2) The SC might occur in spores either in the gall or before germination when the spore wall was too solid to allow examination of the contents by the methods used. (3) The SC was present, but for a very short time, and most of the cells examined were in any case not undergoing meiosis, so the SCs were not seen. (Large numbers of spores were examined, but few spores germinate and undergo normal meiotic division; also a minority of the cells in the gall are forming spores at any one time.) The gall was found to contain uninucleate single cells (i.e. it is a yeast, as it is in artificial culture medium) and virtually all these cells were haploid, 1 in 3000 recovered cells was diploid. It appears that the haploids fuse to form a heterokaryotic or diploid cell immediately before spore formation. A heterokaryotic phase is presumed to exist to establish and maintain the infection.