| Genome analysis in the triticinae using isoenzymes
of phosphodiesterase1) G. WOLF and B. LERCH Institut fur Pflanzenpathologie der Universitat Gottingen, and Institut fur Biochemie der Biologischen Bundesanstalt, Brauschweig, Deutschland Introduction The hitherto accepted concept of the origin of the three different genomes of hexaploid wheat has been questioned in recent times because the origin of the B genome is not clear (see the review by SEARS, 1969). This concept was based mainly on cytological and morphological investigations. In recent years electrophoretical investigation have been used increasingly to elucidate the relationships in the Triticinae. In particular the spectra of the seed storage proteins (JOHNSON, 1972) and of several enzymes of different species (JAASKA, 1971 ; MITRA and BHATIA, 1971) have been compared by electrophoresis. However, efforts so far to elucidate the relationships in the Triticinae by comparing the isoenzymes of twelve different enzymes have been unsuccessful (BREWER et al., 1969). While working on nucleic-acid-degrading enzymes in wheat, we found three isoenzymes of phosphodiesterase (PDE) in Triticum aestivum ssp. vulgare using disc electrophoresis in polyacrylamide gels (WOLF, 1968). We presumed that the three enzymes could be traced back to the three genomes of the hexaploid wheat. In the work presented here we compared the patterns of the isoenzymes of phosphodiesterase of 18 species or subspecies of Triticum and Aegilops, hoping that by this means we might obtain information about the donors of the three genomes of the hexaploid wheat. Materials and Methods Extraction Leaves (about 1-8 g fresh weight) were ground at 4C with sand in a mortar with 3x the amount (v,w) of 0.05 M Tris-HCl buffer (pH 7.5), containing 0.5 M KCl. The extracts were centrifuged at 20,000 g for 10 minutes and concentrated to about 1/3 of the original volume by polyethylene glycol (Aquacide, Calbiochem). For some experiments the enzyme was partially purified by slowly heating the crude extract to 60C in a water bath. Under these conditions about 50% of the proteins were precipitated without loss of PDE activity or changes of electrophoretic mobility (LERCH and WOLF, 1972). Electrophoresis Disc electrophoresis was performed in 7% Polyacrylamide gels (0.6 x 9 cm) at 4C according to DAVIS (1964). Two hundred V were applied until the proteins had entered the running gel (after about 30 min) and 300 V until the bromphenolblue band had reached the bottom of the gel. Under these standard conditions the electrophoresis was completed after about 3 1/2 hours. Detection of Enzyme Each gel was stained for PDE (orthophosphoric diester phosphohydrolase, EC 3.1. 4.1) according to LERCH (1968) by incubation in a solution of 2.5 mg 2'-deoxythymidine-5'-naphthylphosphate (Merck, Darmstadt) and 15 mg fast red RC (Serva, Heidelberg) in 25 ml 0.05 M Tris-HCl (pH 7.5) for 15 minutes to one hour at room temperature. After staining, the gels were washed in a solution of 7% acetic acid and scanned in a Joyce-Loeble densitometer at 460 nm. |
| 1) We are grateful to Mrs. U. OULEVEY for technical assistance and Dr. Ch. LEHMANN, Gatersleben, Dr. E.R. SEARS, Columbia, Dr. E. FUCHS, Braunschweig, and Dr. G. ROBBELEN, Gottingen, for supplying seeds. We thanks Dr. E. R. SEARS, Columbia, for helpful discussions. Copied from the Proceedings of the 4th Wheat Genetics Symposium by the kind permission of the Editors and Organizing Committee. |
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