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Three nuclear spliceosomal introns in conserved locations were amplified and sequenced from 28 strains representing 14 species and 4 genera of volvocalean green algae. Data derived from the three different introns yielded congruent results in nearly all cases. In pairwise comparisons, a spectrum of taxon-specific sequence differences ranging from complete identity to no significant similarity was observed, with the most distantly related organisms lacking any conserved elements apart from exon-intron boundaries and a pyrimidine-rich stretch near the 3' splice site. A metric (SI50), providing a measure of the degree of similarity of any pair of intron sequences, was defined and used to calculate phylogenetic distances between organisms whose introns displayed statistically significant similarities. The rate of sequences divergence in the introns was great enough to provide useful information about relationships among different geographical isolates of a single species, but in most cases was too great to provide reliable guides to relationships above the species level. A substitution rate of approximately 3 x 10(-8) per intron position per year was estimated, which is about 150-fold higher than in nuclear genes encoding rRNA and about 10-fold higher than the synonymous substitution rate in protein-coding regions. Thus, these homologous introns not only provide useful information about intraspecific phylogenetic relationships, but also illustrate the concept that different parts of a gene may be subject to extremely different intensities of selection. The intron data generated here (1) reliably resolve for the first time the relationships among the five most extensively studied strains of Volvox, (2) reveal that two other Volvox species may be more closely related than had previously been suspected, (3) confirm prior evidence that particular isolates of Eudorina elegans and Pleodorina illinoisensis appear to be sibling taxa, and (4) contribute to the resolution of several hitherto unsettled issues in Chlamydomonas taxonomy.

Today, molecular diagnostic tests are widely used in clinical medicine with polymerase chain reaction (PCR)-based techniques being of particular interest. In tissue specimens, however, false-positive and false-negative results can be obtained if pathomorphological and processing aspects are not considered. We therefore studied the impact of tissue sampling in three widely used diagnostic tests: (1) assessment of clonality in B-cell non-Hodgkin's lymphoma, (2) analysis of microsatellite instability (MSI) in colorectal neoplasia, and (3) demonstration of mycobacterium tuberculosis. Tissue sections of routinely formalin-fixed and paraffin-embedded diagnostic specimens were taken, and the significance of sampling was systematically investigated using laser microdissection or processing of the entire section. PCR analyses were done according to standard protocols. False-positive pseudo-monoclonality was obtained in small gastrointestinal biopsies and in laser microdissected lymph follicles of non-neoplastic tonsils. False negativity (pseudo-stability) could be demonstrated in a case with hereditary rectal adenoma if whole tissue sections were taken without microdissection of the most dysplastic subareas. Demonstration of mycobacteria failed in tissue sections of a formalin-fixed lymph node that was positive after complete digestion or in fresh frozen material of the same patient. In diagnostic molecular pathology, sampling error is an important source of false-positive and false-negative results, particularly if disease- and tissue-specific morphological features, such as sample size, type of fixation, and intralesional heterogeneity, are ignored.

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