Molecular phylogeny of the Praomys complex (Rodentia: Murinae): a study based on DNA/DNA hybridization experiments.

Within the Murinae (Muridae: Rodentia), the African rats of the Praomys group, whose systematics has been studied through different approaches, have raised numerous taxonomic problems. Different taxa related to Praomys have successively been described, among which Mastomys, Myomys and Hylomyscus were considered either as separate genera or subgenera of Praomys. In order to clarify the relationships within the Praomys group, we conducted a series of DNA/DNA hybridization experiments involving different species of Praomys, Mastomys, Myomys and Hylomyscus plus other Murinae and a Cricetomyinae. This study indicates that the Praomys complex is a monophyletic entity clearly separated from the other African and Asian Murinae. If Mastomys and Hylomyscus appeared to be independent genera, the taxonomic situation of Praomys and Myomys is more difficult to ascertain. Indeed, Praomys tullbergi appears more closely related to Myomys daltoni than to another species of Praomys , namely P. jacksoni , suggesting paraphyly for Praomys. Furthermore, P. jacksoni is as distant from P. tullbergi as from any species of Mastomys. Additional species of Praomys and, especially, of Myomys , are needed for reaching a definitive conclusion on these latter taxa. The Praomys group is more related to Mus than to Rattus. To calibrate our molecular distances with geological time, we used a dating of 10 Myr for the Musi Rattus dichotomy. The inferred rate of molecular evolution suggests a dating of c. 8 Myr for the separation of the Praomys group from the Mus lineage.     

Reversible cytoplasmic rearrangements precede wall apposition,hypersensitive cell death and defense-related gene activation in potato/Phytophthora infestans interactions

We used video microscopy techniques as a tool for live examination of the dynamic aspects of plant/fungus interactions. Early, dynamic responses of epidermal midrib cells of leaves from a potato cultivar (Solanum tuberosum L. cv. Datura) carrying resistance gene R1 to Phytophthora infestans (race 1: compatible interaction, race 4: incompatible interaction) were monitored. Similar responses were observed in both types of interaction, ranging from no visible reaction of invaded plant cells to hypersensitive cell death. The overall defense response of each individual cell exhibited a highly dynamic behavior that appeared to be tightly coordinated with the growth of the fungus. Initial localized reactions, including major rearrangements within the cytoplasm, occurred directly at the fungal penetration site, where rapid apposition of autofluorescent material and callose took place. If fungal invasion stopped at this stage, the host cell restored its normal cytoplasmic activity and survived. Hypersensitive cell death occurred only when fungal growth had proceeded to the formation of a clearly identifiable haustorium. In such cases, cytoplasm and nucleus conglomerated around the intracellular fungal structure, followed by a sudden collapse of the whole conglomerate and an instantaneous collapse of the fungal haustorium. Only small quantitative differences between the compatible and incompatible interactions of the two fungal races were observed for these early responses of epidermal cells. In the incompatible interaction, a slightly larger number of epidermal cells responded to fungal attack. More pronounced quantitative differences between compatible and incompatible interactions occurred upon fungal invasion of the mesophyll. These differences in the number of responding cells were not reflected at the level of gene expression: the spatial and temporal activation patterns of two defense-related genes, encoding phenylalanine ammonia-lyase and pathogenesis-related protein 1, were similar in both types of interaction.Dedicated to Professor Peter Sitte, Freiburg, Germany, on the occasion of his 65th birthday     

Identification of aneuploid cells in cytological specimens by combined in situ hybridization and immunocytochemistry

The purpose of our study was the application of non-isotopic in situ hybridization with chromosome-specific repetitive DNA probes for the determination of cytogenetically aberrant cells in routine cytological materials, such as cervical smears and breast tumour aspirates. Hyperdiploid cells in fine needle aspirates (FNA) of breast tumours could be visualized by in situ hybridization with a chromosome l-specific repetitive DNA probe. However, for the evaluation of a specific cell type in heterogeneous cell populations, i.e. cervical smears, a procedure combining immunocytochemistry and in situ hybridization can be required. Therefore, we developed a combination protocol using β-galactosidase/ ferri-ferrocyanide (blue-green) for immunocytochemistry and peroxidase/DAB (brown-black) for detection of the DNA probe. the described protocol enabled us to distinguish squamous epithelial cells within heterogeneous cell populations. By combining the chromosome 1 DNA probe with a specific cytokeratin marker it was possible to identify the chromosomal abnormal cells within cervical smears.     

Association of the catalytic subunit of aspartate transcarbamoylase with a zinc-containing polypeptide fragment of the regulatory chain leads to increases in thermal stability.

The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stability to the manifold interchain interactions among the 12 polypeptide chains. With the availability of a recombinant 70-amino acid zinc-containing polypeptide fragment of the regulatory chain of ATCase, it has become possible to analyze directly the interaction between catalytic and regulatory chains in a complex of simpler structure independent of other interactions such as those between the 2 C trimers, which also contribute to the stability of the holoenzyme. Also, the effect of the interaction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured directly. Differential scanning microcalorimetry experiments demonstrated that the binding of the zinc domain to the C trimer leads to a complex of markedly increased thermal stability. This was shown with a series of mutant forms of the C trimer, which themselves varied greatly in their temperature of denaturation due to single amino acid replacements. With some C trimers, for which tm varied over a range of 30 degrees C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 degrees C. The values of tm for a variety of complexes of mutant C trimers and the wild-type zinc domain were similar to those observed when the holoenzymes containing the mutant C trimers were subjected to heat denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The impact of hybrids between genetically modified crop plants and their related species: biological models and theoretical perspectives

Forces affecting the rate of spread and increase of hybrids between genetically modified crop plants and their related species remain qualitatively similar, irrespective of whether genetic modification was achieved using traditional methods, those of biotechnology or as a result of the natural evolutionary process. However, the precise magnitude of the forces and, consequently, the likely environmental impact of such hybrids, may depend strongly on the nature of the gene or genes introduced into the native species. While many classes of transgenes are similar to those manipulated by conventional breeding techniques or evolution, biotechnology offers the potential to introduce genes into crops which are novel both from the point of view of function and origin. The qualitative similarity between transgenes and the products of conventional or evolutionary modification suggests that a historical view of the environmental impact of hybrids between traditionally produced crops or exotic species and their relatives would be of use in estimating the probable fate of hybrids containing transgenes in the environment. However, with certain classes of transgenes for which there are no existing analogues, there will need to be greater care in assessing the possible risks associated with release into the environment.     

Insertion sequence (IS) hybridization supports classification of Rhizobium meliloti by phage typing

Sixty-one isolates of Rhizobium meliloti from two field sites which had been previously classified into 15 phage types on the basis of sensitivity to 16 typing phages, were subjected to insertion sequence (IS) hybridization using DNA probes for ISR m 3 and ISR m 5. Isolates from all but one phage type contained ISR m 3 (apparent copy no. 1–11) and all isolates contained ISR m 5 (apparent copy no. 3–11). The isolates were placed into 24 IS classes based on differences in their respective ISR m 3 and ISR m 5 hybridization profiles. At either field site, isolates representing different phage types possessed IS hybridization profiles that differed from each other, while those comprising a specific type had identical or closely related profiles. Isolates from one phage type were unusual since they did not react with any of the typing phages and were shown by IS hybridization to constitute a heterogeneous group. Evidence for spatial effects were provided by isolates from two of six types present at both sites which fell into separate IS classes on the basis of their site of origin. These data have ecological implications and suggest that for a particular site, phage typing may be employed for the rapid assessment of the genetic diversity among field isolates.     

Assessing the risks of transgene escape through time and crop-wild hybrid persistence

Transgenes introduced into crops can escape in time, as well as space, via the seed bank. For annual plants, especially ruderals, seed bank behaviour may be the most important factor determining population persistence. Crop seeds may exhibit some dormancy and germination cueing in the soil but are expected to be less able to persist than their wild relatives, which often have considerable dormancy and longevity, as well as effective germination cueing responses. Crop-wild hybrids may have seed bank characteristics more suited to persistence, and maternal effects may favour persistence of hybrids having wild plants for their female parent. Escape of transgenes via crop-wild hybrids presents unique concerns not present for crops. Hybrids can undergo natural selection and may back-cross with wild plants. We suggest methods that can be used in conjunction with evaluation of the relative fitness of crop-wild hybrids that will determine the likelihood of back-crossing. Accurate assessment of escape in time and transgene persistence via crop-wild hybrids requires proper plant materials. We emphasize the use of null segregants as controls for transgenic crops and for generating crop-wild hybrid controls for transgenic hybrids. Since good empirical and theoretical understanding of how individual genes influence the fate of plants in different environments is lacking, evaluation of escape in time and the persistence of transgenes via crop-wild hybrids should be on a case-by-case basis.     

Opportunities for gene transfer from transgenic oilseed rape (Brassica napus) to related species

Before novel transgenic plant genotypes are grown outside containment facilities and evaluated under field conditions, it is necessary to complete a risk assessment to consider the possible consequences of that release. An important aspect of risk assessment is to consider the likelihood and consequences of the transgene being transferred by cross-pollination to related species, including other crops, weeds and ruderal populations. The purpose of this report is to review the literature to assess the ease with whichBrassica napus can hybridize with related species. The evidence for hybridization is considered at three levels: a) by open pollination, b) by hand pollination and c) by the use ofin vitro ovule and embryo rescue techniques; and also examines the fertility and vigour of the F₁, F₂ and backcross generations. Four species are reported to hybridize withB. napus by open pollination:B. rapa andB. juncea using fully fertile parents; andB. adpressa andR. raphanistrum using a male-sterileB. napus parent. Seventeen species are reported to form hybrids (including the four species above) withB. napus when pollination is carried out manually. At least 12 of these species were unable to form F₂ progeny, and eight were unable to produce progeny when the F₁ was backcrossed to one of the parental species. Many factors will influence the success of hybridization under field conditions, including: distance between the parents, synchrony of flowering, method of pollen spread, specific parental genotypes used, direction of the cross and the environmental conditions. Even where there is a possibility of hybridization betweenB. napus and a related species growing in the vicinity of a release, poor vigour and high sterility in the hybrids will generally mean that hybrids and their progeny will not survive in either an agricultural or natural habitat.