Genomic subtractive hybridization to isolate species-specific DNA sequences in insects.

Selective enrichment has been used in a number of instances for the isolation of species-specific sequences in prokaryotes. This paper reports the successful application of the technique to insects. Genomic probes were derived to the target species D. funebris and D. simulans. The method involves the biotinylation of non-target 'driver' DNA prepared from the closely related species D. melanogaster and its hybridization to homologous sequences in the target DNA. Hybrid molecules were removed from the reaction by incubation with streptavidin followed by phenol extraction, leaving a preparation enriched for target fragments. All DNA fragments isolated in the D. funebris experiments proved to be specific to that species. Five out of twenty-four fragments screened in the D. simulans experiments were specific when screened with homologous DNA and genomic DNA from its sibling species, D. melanogaster.

Random amplified polymorphic DNA (RAPD) markers for genetic analysis inAllium.

RAPD analysis was applied to onion (Allium cepa) and otherAllium species in order to assess the degree of polymorphism within the genus and to investigate if this approach was suitable for genetic studies of onion. Seven cultivars ofA. cepa, including shallot, and single cultivars of Japanese bunching onion (A. fistulosum), chive (A. schoenoprasum), leek (A. ampeloprasum), and a wild relative of onion (A. roylei), were evaluated for variability using a set of 20 random 10-mer primers. Seven out of the twenty primers revealed scorable polymorphisms between cultivars ofA. cepa and these will be further evaluated for use in genetic mapping. Wide variations in banding profiles between species were observed with nearly every primer tested. These were assessed for use in systematic studies within the genus. Ninety-one band positions were scored (+/-) for all the cultivars studied. Genetic distances between each of the cultivars were calculated and cluster analysis was used to generate a dendrogram showing phylogenetic relationships between them. The resulting analysis was in broad agreement with previous classifications of the species studied, confirming the validity of the method. However, amongst the species studied, it placedA. roylei as the closest relative ofA. cepa, questioning the current classification of the former species in the section Rhizideum.

Isolation of plant nuclei.

The isolation of plant nuclei is a useful first step in many experiments concerned with the mechanism and control of gene expression in plants. For example, isolated nuclei can be used for the isolation of nuclear components such as chromosomal proteins (1), for the study of the processing of primary transcripts (2,3), for the assay and characterization of RNA polymerase activities (4-7), or for the measurement of transcription rates of specific genes (8) (see Chapter 37 ). A method is described here for the isolation of a crude preparation of intact plant nuclei, with an additional protocol for nuclei purification on a discontinuous gradient of Percoll (9) (see Note 1 in section 4). Centrifugation of crude nuclei preparations through Percoll gradients removes much of the contaminating cytoplasmic material such as starch grains (10), and the Percoll step appears to reduce the ribonuclease activity associated with nuclei (10). It is therefore recommended for transcription experiments. The crude nuclei preparation alone may be adequate for some work, for example, when attempting RNA polymerase assays for the first time, when very small amounts of tissue are involved, for preliminary experiments on the characterization of enzyme activities, or for the isolation of nuclear components that may be lost during purification.

In vitro transcription in plant nuclei.

Isolated plant nuclei can be used for fundamental studies on the transcription apparatus. Total RNA polymerase activity can be measured using plant nuclei, and by using different α-amanitin concentrations in the enzyme assay, the individual RNA polymerase I, II, and in activities can be measured (1-5). The assay procedure involves the incubation of nuclei in the presence of the four substrates for RNA synthesis: ATP, GTP, CTP, and UTP. If one of these precursors is supplied as a radio-labeled molecule, transcription can be detected as incorporation of radioactivity into acid-insoluble material. Following incubation, transcription products are precipitated with TCA, collected and washed on glass fiber filter discs, and counted by liquid scintillation counting.

The extraction of total RNA by the detergent and phenol method.

Successful extraction of RNA depends on the quantitative recovery of pure nucleic acids in an undegraded form. In practice, this means that a selective extraction process is required to remove all the unwanted cellular material in a manner that minimizes degradation of the RNA by hydrolysis or ribonuclease activity. The method described here relies on cell homogenization in an aqueous medium containing a strong detergent (sodium tri-isopropylnaphthalene sulfonate) and a chelating agent (sodium 4-aminosalicylate) to solubilize the cell components. An immiscible solution of phenol is then added to selectively extract hydophobic components and to denature protein. Following phase separation, the RNA is recovered by precipitation from the aqueous phase by the addition of absolute alcohol, thereby separating the RNA from small molecular weight contaminants such as carbohydrates, amino acids, and nucleotides.

The purification of poly(a)-containing RNA by affinity chromatography.

The vast majority of eukaryotic mRNA molecules contain tracts of poly(adenylic) acid, up to 250 bases in length, at the 3' end. This property is very useful from the point of view of mRNA extraction because it forms the basis of a convenient and simple affinity chromatography procedure (1). Under high salt conditions (0.3-0.5M NaCl or KCl), poly(A) will hybridize to oligo(dT)-cellulose or poly(U)-Sepharose. These commercially available materials consist of polymers of about 10-20 nucleotides, covalently bound to a carbohydrate support, and bind RNA containing a poly(A) tract as short as 20 residues. Ribosomal and transfer RNAs do not possess poly(A) sequences and will not bind (see Note 1).

The estimation of mRNA content by poly(u) hybridization.

The sequence of poly(adenylic) acid, present at the 3' end of the majority of eukaryotic mRNA molecules, forms the basis of a sensitive technique for the estimation of mRNA content in nucleic acid samples. Under suitable conditions, poly(A) will form RNA-RNA hybrids with poly(U) in vitro. The poly (A) content of RNA samples can therefore be detected by hybridization with saturating amounts of (3)H-poly(U) (1,2). Following the removal of excess (3)H-poly(U) by ribonuclease treatment, the hybrids can be collected by TCA precipitation and quantified by scintillation counting. If the results are compared with data obtained from a parallel experiment using known amounts of poly (A), a value for the poly (A) content of any number of RNA preparations can be obtained. The technique can be used to detect less than 10(-10)g of poly(A).

Field testing of a minimal record of disability in multiple sclerosis: the United States and Canada.

The IFMSS Minimal Record of Disability (MRD) in Multiple Sclerosis was field tested at eight medical centers in the U.S. and Canada. The goals were to conduct a qualitative and quantitative evaluation of the MRD. Assessment were completed on 249 patients with definite MS by neurologists and allied health professionals. Effective administration required some study and practice. Refinement of some unclear wording and awkward format will improve ease of administration. The MRD fit well into clinic routines and was accepted by staff and patients. Scoring presented few problems and these were related to overlap among the MRD scales, poor wording, and content not appropriate to MS. Quantitative evaluation of the MRD indicated that Incapacity Status primarily reflects disability in mobility and self-care when used as a composite score. Heterogeneity of content in Incapacity Status suggests that summed scores be used cautiously. Both Incapacity and Environmental Status had high levels of reliability and high correlations with established measures of impairment in MS. Inter-rater agreement of the ISS and ESS were also high. Once some necessary revisions are made, the MRD should be well on its way to achieving the IFMSS goal of developing a brief, reliable, valid, and appropriate instrument acceptable to a wide variety of workers in MS.

Criteria and uses of the Minimal Record of Disability in multiple sclerosis.

A Minimal Record of Disability of Multiple Sclerosis has been developed by collaborative studies over 5 years among twelve countries in nine languages. The purpose is to provide a simple data set reflecting the profile of personal disability and environmental situations of individuals with MS so that local, regional and national planning and evaluation of resource allocations can be developed as well as to permit international comparisons of system effectiveness. The MRD is patterned on the World Health Organization taxonomy for disability and is now ready for implementation in service studies.

A model of care: matching human services to patients' needs.

Multiple sclerosis(MS) provides a paradigm of the problems of management in many forms of chronic neurologic disability. Many problems stem from the fragmentation and difficulty of access to the medical and psychosocial support systems, lack of coordination among the mix of services, and economic instability. The evolving role of voluntary agencies is to coordinate services for the complex problem-solving required by patients and families. The achievement of this role requires greater attention from physicians and other professionals who must interface in these complicated situations. Increasing governmental cost containment and regulation could erode the capability to provide personal, flexible, and timely responses to complex human predicaments, many of which can become costly, long-term, and catastrophic situations.

Characterization of ribonucleic acid synthesis by nuclei isolated from Zea mays.

Nuclei were isolated from the shoots of Zea mays and assayed for endogenous RNA polymerase activity in vitro. Maximum incorporation from radioactive precursors (70 pmol [(3)H]uridine 5' monophosphate/100 µg DNA) was reached after incubation for 1 h at 25°C. The RNA product, analysed by polyacrylamide gel electrophoresis, was polydisperse in size with an upper limit of 2x10(6) daltons. Discrete peaks of rRNA were not detected, probably because of endogenous ribonuclease activity. The inclusion of α-amanitin (4 µg/ml) in the incubation reduced the total incorporation by approximately 40% but did not significantly alter the size of the RNA product. Although 40% of the total activity could be attributed to RNA polymerase II, [(3)H]RNA synthesised in vitro was found not to contain long sequences of poly (A).