We have examined patterns of variation of several kinds of molecular markers (isozymes, RFLPs of ribosomal DNA and anonymous low-copy number DNA, RAPDs and microsatellites) and an adaptive trait [date of bud set in Scots pine (Pinus sylvestris L.)]. The study included Finnish Scots pine populations (from latitude 60°N to 70°N) which experience a steep climatic gradient. Common garden experiments show that these populations are adapted to the location of their origin and genetically differentiated in adaptive quantitative traits, e.g. the date of bud set in first-year seedlings. In the northernmost population, bud set took place about 21 days earlier than in the southernmost population. Of the total variation in bud set, 36.4% was found among the populations. All molecular markers showed high levels of within-population variation, while differentiation among populations was low. Among all the studied markers, microsatellites were the most variable (He=0.77). Differences between populations were small, GST was less than 0.02. Our study suggests that molecular markers may be poor predictors of the population differentiation of quantitative traits in Scots pine, as exemplified here by bud-set date.

Protein disulfide isomerase (PDI) is a multifunctional polypeptide that acts as a subunit in the animal prolyl 4-hydroxylases and the microsomal triglyceride transfer protein, and as a chaperone that binds various peptides and assists their folding. We report here that deletion of PDI sequences corresponding to the entire C-terminal domain c, previously thought to be critical for chaperone activity, had no inhibitory effect on the assembly of recombinant prolyl 4-hydroxylase in insect cells or on the in vitro chaperone activity or disulfide isomerase activity of purified PDI. However, partially overlapping critical regions for all these functions were identified at the C-terminal end of the preceding thioredoxinlike domain aЈ. Point mutations introduced into this region identified several residues as critical for prolyl 4-hydroxylase assembly. Circular dichroism spectra of three mutants suggested that two of these mutations may have caused only local alterations, whereas one of them may have led to more extensive structural changes. The critical region identified here corresponds to the C-terminal α helix of domain aЈ, but this is not the only critical region for any of these functions.

We describe a method for determination of pregnenolone, progesterone, 17alpha-hydroxyprogesterone, testosterone, and 5alpha-dihydrotestosterone in 1-2 ml of serum from male or female. Using microcolumns of Lipidex-5000 (hydroxyalkoxypropyl Sephadex, 0.5 g) and light petroleum/chloroform (97/3) as the solvent during chromatography, we resolved these five steroids into four fractions, with pregnenolone and 5alpha-dihydrotestosterone eluting together. By use of selected antibodies, the latter two steroids were also determined specifically. Use of microcolumns allowed minimization of solvent volumes and sample transfers. Consequently, blank values for all the five steroids were negligible. Lowest measureable concentrations (in ng/liter) were: pregnenolone 100, progesterone 25, 17alpha-hydroxyprogesterone 50, testosterone 25, and 5alpha-dihydrotestosterone 25. Intra-assay and inter-assay coefficients of variation ranged from 5 to 9% and 10 to 15%, respectively, for the five steroids. Serum concentrations of these steroids are given for women in the follicular and luteal phases of the menstrual cycle and for women on oral contraceptives of the combination type, as well as for normal men.

Variability in the ribosomal DNA (rDNA) gene family was surveyed in Scots pine (Pinus sylvestris L.) populations from southern and northern Finland. A total of 97 trees were studied with three restriction enzymes, revealing 13 variable rDNA phenotypes. Each rDNA phenotype represents the combined genotype of the eight rDNA loci (NORs, nucleolus organizer regions) that are present in the Scots pine genome. The studied Scots pine populations were equally variable and there was no evidence of geographic differentiation. Of the total rDNA diversity of the species, 86 per cent was found within populations (including within-individual variability) and 14 per cent was found between populations. Within individual trees, one to four rDNA repeat types were distinguished. No variation was found in the coding regions but variable restriction sites were identified in the IGS and transcribed spacer regions. The inheritance pattern of an rDNA variant carrying a 0.4 kb deletion in the transcribed spacer region was studied. The deletion-carrying rDNA variants were distributed non-randomly across the NOR loci and showed regular Mendelian segregation in the progeny. The observed distribution pattern of rDNA variability suggests that the rate of intrachromosomal homogenization is greater than that of interchromosomal homogenization in Scots pine.

Protein-disulfide isomerase (PDI) is a catalyst of folding of disulfide-bonded proteins and also a multifunctional polypeptide that acts as the ␤-subunit in the prolyl 4-hydroxylase ␣ 2 ␤ 2 -tetramer (P4H) and the microsomal triglyceride transfer protein ␣␤-dimer. The principal peptide-binding site of PDI is located in the b domain, but all domains contribute to the binding of misfolded proteins. Mutations in the C-terminal part of the a domain have significant effects on the assembly of the P4H tetramer and other functions of PDI. In this study we have addressed the question of whether these mutations in the C-terminal part of the a domain, which affect P4H assembly, also affect peptide binding to PDI. We observed a strong correlation between P4H assembly competence and peptide binding; mutants of PDI that failed to form a functional P4H tetramer were also inactive in peptide binding. However, there was also a correlation between inactivity in these assays and indicators of conformational disruption, such as protease sensitivity. Peptide binding activity could be restored in inactive, protease-sensitive mutants by selective proteolytic removal of the mutated a domain. Hence we propose that structural changes in the a domain indirectly affect peptide binding to the b domain. Protein-disulfide isomerase (PDI),1 an abundant protein of the eukaryotic endoplasmic reticulum, is a catalyst of disulfide bond formation and rearrangement in the course of protein folding (for review see Ref. 1). A molecular interpretation of PDI activity is made difficult by the fact that PDI is multifunctional in the cell. In addition, it has a wide range of actions in vitro and consists of multiple domains. Furthermore, there is as yet no high resolution structure of full-length PDI. Current efforts are focused on analyzing the domain structure of PDI and in establishing the roles of specific domains in specific functional activities.It is now clear (2, 3) that PDI has a structural organization based on duplicated sequence modules (Fig. 1). The full-length protein is constructed of four structural domains with homologous thioredoxin folds plus a C-terminal acidic extension. The homologous a and a sequence modules contain the active site motif -WCGHC-and show significant sequence identity to thioredoxin; a high resolution NMR analysis of the recombinant a domain confirms that it has the thioredoxin fold. The homologous b and b modules do not show significant sequence similarity to the a domain, but NMR analysis has revealed that the b domain also exhibits the thioredoxin fold (2, 4). Analysis of the properties of individual domains as catalysts of simple thiol:disulfide oxidoreduction and of more complex protein folding linked to disulfide isomerization shows that the a and a domains function effectively as simple thiol:disulfide oxidoreductases but that the remaining domains are required for full activity in catalyzing protein folding associated with the formation of native disulfide bonds (5). No specific function has yet been ascribed...