AUTORADIOGRAPHIC STUDIES OF NUCLEIC ACIDS AND PROTEINS DURING MEIOSIS IN LILIUM LONGIFLORUM

TAYLOR, J. HERBERT (Columbia U., New York, N. Y.) Autoradiographic studies of nucleic acids and proteins during meiosis in Lilium longiflorum. Amer. Jour. Bot. 46(7): 477-484. Illus. 1959.-A study was made of the incorporation of glycine-C", orotic acid-C14 and cytidine-H3 into nucleic acids and proteins of sporogenous and tapetal cells of lily anthers preceding and during meiosis. Methods for differential extraction of nucleic acids from tissue sections, which had been frozen, dehydrated by alcohol-substitution, and fixed in hot alcohol, were tested by chromatographic analysis of extracts. Both acid and enzyme hydrolysis were shown to be useful for quantitative or, at least, semi-quantitative work. DNA synthesis was shown to occur only during premeiotic interphase in sporogenous cells, but at two intervals in tapetal nuclei, once when the microsporocytes are in zygotene and again during pachytene. Each time the synthetic period was followed by a normal mitosis. Accumulation of RNA in microsporocytes occurred at stages up to late leptotene. After this period, labeled RNA accumulated almost exclusively in their nuclei and at a slower rate than in earlier stages. DNA synthesis, as measured by incorporation of glycine-C1" and orotic acid-C14, gave the same results and confirm earlier results with inorganic phosphate-P32. For RNA, glycine-C" and orotic acid-C14 gave different results. When glycine-C" was the source of label, incorporation of C" in RNA stopped during DNA synthesis in sporogenous cells. Glycine-C" was not utilized to a significant extent at any time by tapetal cells for RNA synthesis, but extensively for DNA and protein synthesis. Orotic acid-C" was incorporated into RNA of both tapetum and sporogenous cells at various periods in development apparently including the interval of DNA synthesis. Protein synthesis as measured by incorporation of glycine is relatively rapid during premeiotic interphase and leptotene. It continues during the remainder of prophase, but at a much reduced rate. In tapetal cells the rate is rapid in the nuclei during periods of DNA synthesis, but even faster in both cytoplasm and nucleus after divisions are completed and the microsporocytes are in late prophase and division stages. This period of synthesis is perhaps necessary for the postmeiotic functioning of tapetum when it appears to secrete the wall materials for the microspores. SINCE THE PROPHASE of the first division of meiosis is a relatively long period during which genetic recombination is assumed to occur, its role in the life cycle in the higher plant has been studied extensively. However, knowledge of the physiology of the process has been difficult to obtain. Autoradiography with appropriately labeled substances provides a new approach that may contribute to a better understanding of the physiology, as well as to the genetically significant events of this period. In previous studies, phosphorus-32 has been used for studies of the metabolism of the nucleic acids of the sporogenous and tapetal tissues of anthers (Taylor, 1953; Plaut, 1953; Taylor and McMaster, 1954; Moses and Taylor, 1955; and Taylor, 1958). In an attempt to get a broader understanding of the role of both nucleic acids and proteinu during meiosis, incorporation of carbon-14 sup1 Received for publication December 21, 1958. This work was supported in part by grants from the Atomic Energy Commission, Contract AT (30-1) -1304, and from the Higgins Fund, Columbia University. The technical assistance of Miss Maria Caluya and Mrs. Toni Simon is gratefully acknowledaed. plied as orotic acid-C14 and glycine-C14 has been determined by autoradiography. Orotic acid-C14 labels the pyrimidines of both types of nucleic acid, RNA (ribonucleic acid) and DNA (deoxyribonucleic acid). Mild acid hydrolysis and enzymes were used for differential extraction of the nucleic acid, but the efficiency of these techniques was first checked by an analysis of the hydrolysates. When the enzymes ribonuclease and deoxyribonuclease were found to be reliable enough for separation of nucleic acids and proteins to make the data useful, incorporation of carbon-14 from glycine-2C14 was followed at various stages of meiosis. For autoradiography, carbon-14 has advantages over phosphorus-32. Because of the softer radiation, resolution is improved and the much longer half-life makes storage and more leisurely handling of tissues and extracts feasible. However, the long half-life makes the preparation of compounds with very high specific activities impossible. Tritium, the radioisotope of hydrogen, with its very soft beta particles has the advantage that it gives the best resolution obtainable in autoradiography. Since the half-life is 12.3 years, compounds of very [The Journal for June (46: 397-476) was issued June 18, 1959] AMERICAN JOURNAI OF BOTANY, Vol. 46, No. 7, July, 1959