Molecular Characterization and Mapping of Murine Genes Encoding Three Members of the Stefin Family of Cysteine Proteinase Inhibitors

Stefins or Type 1 cystatins belong to a large, evolutionarily conserved protein superfamily, the members of which inhibit the papain-like cysteine proteinases. We report here on the molecular cloning and chromosomal localization of three newly identified members of the murine stefin gene family. These genes, designated herein as mouse stefins 1, 2, and 3, were isolated on the basis of their relatively increased expression in moth-eaten viable compared to normal congenic mouse bone marrow cells. The open reading frames of the stefin cDNAs encode proteins of approximately 11.5 kDa that show between 50 and 92% identity to sequences of stefins isolated from various other species. Data from Southern analysis suggest that the murine stefin gene family encompasses at least 6 and possibly 10-20 members, all of which appear to be clustered in the genome. Analysis of interspecific backcross mice indicates that the genes encoding the three mouse stefins all map to mouse chromosome 16, a localization that is consistent with the recent assignment of the human stefin A gene to a region of conserved homology between human chromosome 3q and the proximal region of mouse chromosome 16.     

Evolutionary breakdown of distyly to homostyly is accompanied by reductions of floral scent in Primula oreodoxa

Reproductive traits that function in pollinator attraction may be reduced or lost during evolutionary transitions from outcrossing to selfing. Although floral scent plays an important role in attracting pollinators in outcrossing species, few studies have investigated associations between floral scent variation and intraspecific mating system transitions. The breakdown of distyly to homostyly represents a classic example of a shift from outcrossing to selfing and provides an opportunity to test whether floral fragrances have become reduced and/or changed in composition with increased selfing. Here, we evaluate this hypothesis by quantifying floral volatiles using gas chromatography-mass spectrometry in two distylous and four homostylous populations of Primula oreodoxa Franchet, a perennial herb from SW China. Our analysis revealed significant variation of volatile organic compounds (VOCs) among populations of P. oreodoxa. Although there was no difference in VOCs between floral morphs in distylous populations as predicted, we detected a substantial reduction in VOC emissions and the average number of scent compounds in homostylous compared with distylous populations. A total of 12 compounds, mainly monoterpenoids and sesquiterpenoids, distinguished homostylous and distylous morphs; of these, (E)-β-ocimene was the most important in contributing to the difference in volatiles, with significantly lower emissions in homostyles. Our findings support the hypothesis that the transition from outcrossing to selfing is accompanied by the loss of floral volatiles. The modification to floral fragrances in P. oreodoxa associated with mating system change might occur because high selfing rates in homostylous populations result in relaxed selection for floral attractiveness.     

Spatial autocorrelation invalidates the Dow-Cheverud test

Dow and Cheverud (Am. J. Phys. Anthropol. 68:367–373, 1985) have published a statistical test for comparing any three similarity matrices. Using both simulations and analytical arguments, I establish that the presence of spatial autocorrelation, a common feature of geographically based anthropological and biological data, causes this test to reject too often. Increasing the spatial autocorrelation increases the spurious rejection rate. About 20% of the papers that reference Dow and Cheverud's paper have used their test with spatially autocorrelated data. Mantel's (Cancer Res. 27:209–220, 1967) method, when used as a test of spatial autocorrelation, is unaffected by these considerations, since its null hypothesis is that the data are uncorrelated. © 1992 Wiley-Liss, Inc.     

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF PHYTOPLANKTON PIGMENTS USING A POLYMERIC REVERSED-PHASE C18 COLUMN1

A high-performance liquid chromatographic (HPLC) method is described that allows improved resolution of several chemotaxonomically significant phytoplankton pigments. The protocol, which employs two pumps and a modified Mantoura and Llewellyn (1983) solvent system, can be easily adapted for many HPLC systems currently in use. The most unique aspect of the method is the use of a polymeric C₁₈ reversed phase HPLC column (VYDAC 201TP). In comparison to the monomeric C₁₈ columns typically used in the characterization of phytoplankton pigments, polymeric C₁₈ columns offer superior selectivity for structurally similar compounds. The protocol was evaluated for the ability to resolve most of the phytoplankton pigments of diagnostic importance using algal cultures from nine classes. Pigment pairs that were resolved by the method include a) lutein and zeaxanthin, b) neoxanthin and 19′-hexanoyloxyfucoxanthin, and c) α-carotene and β-carotene, and partial resolution of chlorophyll c₁ and chlorophyll c₂.     

COMPARISON OF PLANT DNA CONTENTS DETERMINED BY FEULGEN MICROSPECTROPHOTOMETRY AND LASER FLOW CYTOMETRY

An improved procedure is reported for determining DNA amounts of plant nuclei. Nuclei stained with propidium iodide, isolated from chopped plant leaves, were passed through an Ortho Cytofluorograph with a Lexel model 95 argon laser (514 nm) and the fluorescence measured, integrated, and recorded using an Ortho 2140 Data Acquisition computer. All nuclear samples were mixed with nuclei of Sultan barley (2C DNA content = 11.12 pg [picogram]) as an internal standard. DNA contents of ten plant species, ranging from 2C = 1.7 pg to 36.1 pg measured by flow cytometry, correlated strongly (r = 0.99, slope = + 1.00) with DNA contents determined from Feulgen-stained nuclei of the same species using microspectrophotometry. The flow cytometric procedures were sufficiently sensitive to detect differences in DNA content between inbred lines of corn and their F1 hybrids. Our results obtained with improved procedures, specifically using propidium iodide as a fluorochrome and plant nuclei instead of chicken erythrocytes as an internal standard, demonstrate that laser flow cytometry can be a precise, rapid, and reliable method for determining nuclear DNA content of plants.     

Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.     

The Mouse Spam1 maps to proximal Chromosome 6 and is a candidate for the sperm dysfunction in Rb(6.16)24Lub and Rb(6.15)lAld heterozygotes

We have determined the chromosomal localization of the murine gene encoding the 68-kDa sperm adhesion molecule 1, Spam1 or Ph-20. Using two independent approaches, fluorescence in situ hybridization (FISH) and interspecific backcross analysis, we show that Spam1 maps to proximal mouse Chromosome (Chr) 6. This map position is within the conserved linkage group corresponding to human Chr 7q, where the human homolog, SPAM 1, has been shown to map previously. Genetic mapping shows the gene to be very closely linked to Met, one of the most proximal loci on MMU 6. It thus places the gene near the centromere and the junction of the Rb(6.16)24Lub and Rb(6.15)1Ald translocations. The essential role of the Spam1 sperm antigen in mouse sperm-egg interactions and its gene location provide strong support for its candidacy as the gene involved in the dysfunction of mouse sperm bearing the Rb(6.16)24Lub or Rb(6.15)1Ald translocation. Received: 16 July 1996 / Accepted: 23 September 1996     

How to do it. Run a preparation course for postgraduate examinations.

This paper provides a practical guide to running preparation courses for postgraduate examinations and is based on the authors'' experience. It is intended to be useful to organisers of proposed or existing courses as well as to potential users of courses--people in every specialty preparing for a postgraduate examination. The paper covers the practical aspects of staging and financing a course and recruiting tutors and attracting candidates, in addition to covering the structure, educational content, and evaluation of the course.