An approach to overcoming regeneration recalcitrance in genetic transformation of lupins and other legumes

For pulse legume research to fully capitalise on developments in plant molecular genetics, a high throughput genetic transformation methodology is required. In Western Australia the dominant grain legume is Lupinus angustifolius L. (narrow leafed lupin; NLL). Standard transformation methodology utilising Agrobacterium tumefaciens on wounded NLL seedling shoot apices, in combination with two different herbicide selections (phosphinothricin and glyphosate) is time consuming, inefficient, and produces chimeric shoots that often fail to yield transgenic progeny. Investigation of hygromycin as an alternative selection in combination with expression of green fluorescent protein indicated that transformation of NLL apical cells was not the rate limiting step to achieve transgenic shoot materials. In this research it was identified that despite ready transformation, apical cells were not competent to regenerate. However a deep and broad wounding procedure to expose underlying axillary shoot and vascular cells to Agrobacterium, in combination with delayed selection proved successful, increasing initial explants transformation efficiency up to 75?% and generating axillary shoots with significant transgenic content. Based on knowledge gained from studies of plant chimeras, further subculture of these initial axillary shoots will result in development of low chimeric transgenic materials with heritable content. Furthermore, the method was also tested successfully on other Lupinus species, faba bea and field pea. These results demonstrate that development of a high yielding transformation methodology for pulse legume crops is achievable.     

An approach to the prediction of temperate freshwater fish communities

The organization and structure of temperate fish communities is discussed. A measure of organization based on the Shannon measure of entropy is applied. It is concluded that the way organization changes with species number reflects the underlying structure of the community. The prediction of fish assemblages by the analysis of individual species requirements is also investigated using fish data sets from regions considered to be undergoing acidification. The species set within a habitat is generated by a set of rules termed the operational niche. It was concluded that an analysis based on physico-chemical conditions within the habitats gave reasonable correspondence between observed and predicted.     

An approach to early genetic alterations in precancerous cells

To investigate the potential role of the PTEN tumor-suppressor gene in the carcinogenesis of ovarian endometrioid carcinoma and its related subtype, clear cell carcinoma, we examined 20 ovarian endometrioid carcinomas, 24 clear cell carcinomas and 34 solitary endometrial cysts of the ovary for LOH at 10q23.3 and point mutations of the PTEN gene, using a laser-assisted microdissection method. LOH was found in 8 of 19 ovarian endometrioid carcinomas (42.1%), 6 of 22 clear cell carcinomas (27.3%) and 13 of 23 solitary endometrial cysts (56.5%). Somatic mutations in the PTEN gene were identified in 4 of 20 ovarian endometrioid carcinomas (20.0%), 2 of 24 clear cell carcinomas (8.3%) and 7 of 34 solitary endometrial cysts (20.6%). In 5 endometrioid carcinomas with endometriosis, 3 displayed LOH events common to both the carcinoma and the endometriosis. In 7 clear cell carcinomas with endometriosis, 3 displayed LOH events common to both the carcinoma and the endometriosis. In no cases there were LOH events in the endometriosis only. These results indicate that inactivation of the PTEN gene is an early event in the development of both endometrioid and clear cell carcinoma of the ovary. A laser-assisted microdissection method enables us to collect target cells without contamination by non-tumor cells. We expect that this technique will be very useful for investigating genetic alterations in cancerous or precancerous lesions. Early genetic alterations in various precancerous cells detected by light microscopy can be readily identified by the tissue-microdissection method.     

An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis

A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basislayer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l⁻¹ kanamycin or 5 mg l⁻¹ paromomycin. Single resistant cells can be recovered from about 10 000 sensitive cells in one alginate layer. Injection of theneo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.     

An alternative approach to evaluating the intraspecific genetic variability of parasites

Analysis of DNA polymorphisms provides important information for the molecular characterization of parasite strains and clones. Because we still know little about the genomes of parasites, such analysis has to rely on methods applicable to any eukaryotic genome, such as DNA fingerprinting with multilocal minisatellite probes and the polymerase chain reaction (PCR)-based random amplified polymorphic DNA technique (RAPD). However, DNA fingerprinting is cumbersome and needs large amounts of parasite DNA, and RAPD can exhibit low reproducibility and spurious bands, both of which appear to be related to the low stringency of the PCR procedure. Riva Oliveira, Andréa Macedo, Egler Chiari and Sérgio Pena here evaluate the applicability to parasites of a technique described two years ago called simple sequence repeat-anchored PCR amplification (SSR-PCR), in which a single primer is needed [the (CA)(8)RY primer] and highstringency conditions are applied.     

An alternative method for the genetic transformation of sweet organce

Summary An alternative method for transforming sweet organe [Citrus sinensis (L.) Osbeck] has been developed. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein gene was introduced using polyethylene glycol into protoplasts of ‘Itaborai’ sweet organe isolated from an embryogenic nucellar-derived suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of the green fluorescent protein, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots. To expedite transgenic plant recovery, regenerated shoots were also micrografted onto sour orange seedling rootstocks. Presence of the transgene in calluses and regenerated sweet organe plants was verified by gene amplification and Southern analyses. Potential advantages of this transformation system over the commonly used Agrobacterium methods for citrus are discussed.     

An extension of the probability approach to genetic relationships: One locus

A path-counting method has been presented for calculating the nine phylotype pair probabilities (k-coefficients of this paper) for a diploid autosomal locus. Cockerham (1971) has provided a somewhat more elegant method for arriving at these probabilities. The procedure explained herein is more akin to the familiar calculation of Wright's inbreeding coefficient and is easier to apply to specific complex pedigrees such as arise in human genetics problems. Cockerham's more general formulation is superior for the investigation of regular systems of inbreeding.     

An integrated epigenetic and genetic approach to common human disease

Epigenetic information is heritable during cell division but is not contained within the DNA sequence itself. Despite increasing evidence for and interest in the role of epigenetics in human disease, particularly in cancer, virtually no epigenetic information is routinely or systematically measured at the genome level. The current population-based approach to common disease relates common DNA sequence variants to either disease status or incremental quantitative traits contributing to disease. Although this purely genetic approach is powerful and general, there is currently no conceptual framework to integrate epigenetic information. In this article, we propose an approach to common human disease that incorporates epigenetic variation into genetic studies. Epigenetic variation might also help to explain the late onset and progressive nature of most common diseases, the quantitative nature of complex traits and the role of environment in disease development, which a purely sequence-based approach might not.     

An integrated exploratory approach to examining the relationships of environmental stressors and fish responses

The Chesapeake Bay is one of the mostproductive systems in the world. It is theNation's largest estuary (64,000 square miles)and is home to about 13 million people. Itsupports a variety of aquatic resources offlora and fauna. However, for the past 350years and especially in the last two to threedecades, there has been substantialdeterioration of the natural resources. Manyspecies of submerged aquatic vegetation andbenthic invertebrates have been diminished orbecome extinct. Commercial harvests of fish,crab and shell fish have also declined.In 1983, a Chesapeake Bay Agreement was signedby Pennsylvania, Maryland, the District ofColumbia, Virginia and the Bay Commission. Itwas subsequently amended in 1987 and 1992. TheAgreement identified the improvement andmaintenance of water quality as the mostcritical elements in the overall restorationand protection of the Chesapeake Bay. In orderto restore the Bay area and to conserve thefish resources, the causal relationshipsbetween the environmental stressors and thecomposition and health of the fish communitiesmust be understood.Multivariate ordination techniques are usefulexploratory tools to help elucidate latentenvironmental relationships, define specificbiocriteria and to generate hypotheses. Geographical information systems (GIS) is ananalytical technique for identifying spatialrelationships. In this project, an integratedmethodology involving the use of multivariateordination, statistical, and GIS techniques wasadopted. A non-metric multi-dimensionalscaling (NMDS) ordination technique wasemployed in conjunction with other statisticaltechniques (such as correlation analysis) andArcView GIS to analyze a huge data set from theMaryland Biological Stream Survey (MBSS). Theobjectives were to elucidate the intricaterelationships between a suite of environmentalfactors and fish conditions in the riverinesystem in the Chesapeake Bay and to evaluatethe effectiveness of this approach inexploratory analyses.The results showed that landuse issignificantly related to nutrient loading. Toa large extent, landuse and nitrates are alsoaffecting the composition and health of thefish communities in some subwatersheds in theChesapeake Bay. It was also found that theapproach adopted in this study is flexible,requiring few model assumptions. But it iscomprehensive and reliable, capable ofrevealing the impacts of environmentalstressors on the ecology, structure,composition and health of the fishcommunities.     

An updated meta-analysis approach for genetic linkage

We present a meta-analysis procedure for genome-wide linkage studies (MAGS). The MAGS procedure combines genome-wide linkage results across studies with possibly distinct marker maps. We applied the MAGS procedure to the simulated data from the Genetic Analysis Workshop 14 in order to investigate power to detect linkage to disease genes and power to detect linkage to disease modifier genes while controlling for type I error. We analyzed all 100 replicates of the four simulated studies for chromosomes 1 (disease gene), 2 (modifier gene), 3 (disease gene), 4 (no disease gene), 5 (disease gene), and 10 (modifier gene) with knowledge of the simulated disease gene locations. We found that the procedure correctly identified the disease loci on chromosomes 1, 3, and 5 and did not erroneously identify a linkage signal on chromosome 4. The MAGS procedure provided little to no evidence of linkage to the disease modifier genes on chromosomes 2 and 10.     

An approach to measuring the in vivo transformation of glycerol to a very low density lipoprotein triglyceride.

This paper describes a new method which permits measurement of the steady-state rate of transformation of serum glycerol to a very low density lipoprotein (VLDL) triglyceride in vivo in dogs. Although the turnover of glycerol and the turnover of VLDL triglyceride glycerol have both been previously measured, the rate of transformation of the former into the latter has not. While there is considerable dog-to-dog variation in the absolute turnover and transformation rates, the relationship between the various rates is quite constant. Thus, 13% of the serum glycerol which normal fasting dogs utilize is converted to VLDL triglyceride. The remaining 87% is converted to other products. Also, 28% of VLDL triglyceride glycerol in these dogs is derived from serum glycerol. The balance, 72%, is derived from other sources. The procedure described here can be used to quantitate the contribution of glycerol to VLDL in a number of conditions in which glycerol and (or) VLDL triglyceride metabolism is altered, thereby providing another way to gain insight into the metabolism of VLDL. Even more generally, the principles developed here can be applied to estimate the transformation of other precursors to other products in vivo.     

An attempt at genetic transformation in chickens through cock sperm irradiation

Summary Two experiments were conducted to verify the real possibility for use of the genetic transformation technique as described by Pandey and Patchel in chickens in commercial poultry breeding. Multiple recessive and multiple dominant marker stocks were employed, as well as a tester and a donor line. Recipient tester females were first inseminated with dominant donor semen which was irradiated with doses of ⁶⁰Co gamma irradiation ranging from 100 to 800 Gy (control group) and 24 h later were reinseminated with unirradiated, normal semen of the recipient strain (experimental group). One genetic transformed chicken was found in the first experiment; no genetic transformed event occurred in the second experiment but one embryo was present in the 600 Gy irradiated group with parthenogenetic development capable of giving a live chick. One hundred Gy was observed not to be enough to destroy completely sperm fertilizing ability. An increased frequency of parthenogenetic development was found in all groups after insemination with irradiated semen. There were 11 individuals with developmental abnormalities from the total of 1264 analysed embryos which died after the 18th day of incubation. We concluded that egg transformation is a rare event in domestic fowl and further research for use of this technique in commercial poultry breeding is needed.     

Evolutionary mechanisms in behaviour: An intraspecific genetic approach

The study of the evolutionary mechanisms in behaviour and of the biological grounds of individual variability may be based on the comparative phylogenetic approach or on an intraspecific genetic analysis. The discontinuous behavioural progression evident across species is due to the assumption that the phylogenetic “scale” may be adopted in place of the phylogenetic trees and to the fact that today's existing species have evolved in parallel and may not be used to represent an evolutionary sequence. However, despite the existence of different motor and perceptual abilities the comparative approach has shown that many analogies exist between some basic brain mechanisms across species.The existence of outstanding individual differences within the same species must be regarded as a powerful potential tool since quantitative differences between the behaviours of different individuals belonging to the same species might later result in qualitatively different phenotypes. By using different strains and mutations of mice and different genetic approaches it has been possible to assess the mode of inheritance, to calculate estimates of heritability and of the number of segregating units for different behavioural traits ranging from avoidance to maze learning and activity. The existence of clear genetic correlations between some of these behavioural patterns has also allowed the identification of some single-gene mutants affecting these traits and the characterization of a gene responsible for a major effect on activity. This psychogenetic approach shows that there are behaviour differences which are the products of evolutionary adaptive processes and that the knowledge of the genetic systems that underlie these differences is a basic step for understanding the brain mechanisms in Man.     

An approach to study the evolution of theDrosophila 5S ribosomal genes using P-element transformation

Summary The P-element-mediated gene transfer system was used to introduceDrosophila teissieri 5S genes into theDrosophila melanogaster genome. Eight transformedD. melanogaster strains that carryD. teissieri 5S mini-clusters consisting of 9–21 adjacent 5S units were characterized. No genetic exchanges betweenD. melanogaster andD. teissieri 5S clusters were detected over a 2-year survey of the eight strains. The occurrence of small rearrangements within theD. melanogaster 5S cluster was demonstrated in one of the transformed strains.     

An approach to intercalibrate ecological classification tools using fish in transitional water of the North East Atlantic

A simple procedure to harmonise and intercalibrate eight national methods classifying the ecological status using fish in transitional waters of the North East Atlantic is described. These methods were initially intercalibrated and a new method recently developed was added to this exercise. A common human pressure index pre-classified the status of each water body in an independent way. Ecological class boundaries values were established according to the level of anthropogenic pressure using regression analyses. A simulated dataset was used to assess the level of agreement between the fish classification methods. Fleiss’ multi-rater kappa analysis indicated that boundary harmonisation was achieved; all classifications fell within one class of each other and class agreement between methods exceeded 70%. The use of a pressure index to establish boundary thresholds provides a practical method of defining and harmonizing the quality classes associated with human pressures, as required by the European Water Framework Directive.     

An optimal stopping approach for onset of fish migration

Comprehending life history of migratory fish, onset of migration in particular, is a key biological and ecological research topic that still has not been clarified. In this paper, we propose a simple mathematical model for the onset of fish migration in the context of a stochastic optimal stopping theory, which is a new attempt to our knowledge. Finding the criteria of the onset of migration reduces to solving a variational inequality of a degenerate elliptic type. As a first step of the new mathematical modeling, mathematical and numerical analyses with particular emphasis on whether the model is consistent with the past observation results of fish migration are examined, demonstrating reasonable agreement between the theory and observation results. The present mathematical model thus potentially serves as a simple basis for analyzing onset of fish migration.