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Abstract . . iii
Introduction 1
Methods and Materials . . . . . . . . . . . . . . 3
Results and Discussion.
Acknowledgements . . . .
Financial Statement .
References . . . . . 8
5
6
7

ABSTRACT
The incorporation of the methyl group from S-methyl-Lmethionine- 14 CH 3 into the nucleic acid, DNA, in germinating winter wheat seedlings was investigated during the early germination period of 4 days.
Counts per minute per gram of tissue in the root was highest on day 1 and dropped rapidly until day 3, followed by a slight rise. In the shoot, the counts per minute per gram of tissue was also highest on day 1, dropping rapidly to its lowest point on day 4. There was no fluctuation in the counts per minute per gram of tissue in the remaining seed parts, which remained at a lower level than the other two groups.
Since any protein, which may have contained S-methyl-Lmethionine or its dissociated methyl group, was "sal ted out" using saline-EDTA, and the procedure used was for DNA isolation, the results indicate that the methyl group was incorporated into the DNA of the root and shoot of the germinating wheat seedlings.
The decrease in counts per minute per gram of tissue for the root and shoot was expected as a result of the labeled DNA being replicated and dispersed throughout the rapidly growing root and shoot, after an initial incorporation of the labeled methyl group.

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I. INTRODUCTION
Direct methyltransferase reactions have been shown by several workers to occur during the germination period in higher plants (1-3). Previous investigations of two direct methyltransferase systems in winter wheat have shown these systems to be operating at different times during the germination period and have suggested interconversions between these systems (4,5).
The production of free methionine appears essential during germination of wheat seedlings. Marcus and Feeley (6,7) showed that dry wheat embryos had very little capacity to incorporate l4C-leucine into protein; however, once water was inbibed, rapid protein synthesis occurred. An earlier study by Ingle and
Hageman (8) indicated that active synthesis of RNA occurred during the early germination phase, apparently involving all fractions of RNA. Cherry (9) indicated a doubling of RNA content during the second to sixth day of germination in peanut seed.
It was further established that methylation of the nucleic acid bases and of their associated histones not only occurred, but that activated methionine was the donor of these methyl groups (10).
Methylation of nucleic acids was first demonstrated in transfer RNA. All four nitrogen bases of RNA are known to contain substituted methyl groups (10). All of these added methyl groups have been shown to originate from methionine (11). Starr (12) and Svensson, ET Ai.., (13) have shown that methylated bases are present in both 16 Sand 23 S ribosomal RNA in E. coli. Jakob and Tal (14) have d~monstrated methylation of yicia RNA using labeled methylmethionine.

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At least two methylated bases, 6-methylpurine and 5-methylcytosine, have been found in DNA (10). These same workers have also shown that the number of methylated bases in DNA varies from organism to organism, and specifically found wheat germ to contain almost ten times as many such bases as human DNA.
These workers suggest that the rule of methylated bases may be that of punctuation determining the length of the DNA's message or possibly even a guide for copying, or not copying, a particular
DNA segment.
Kalousek and Morris (15) studied this DNA-methylase activity in pea seedlings and found S-adenosylmethionine to act as the methyl donor and 5-methylcytosine to be the product. Gold and
Hurwitz (16) conducted a similar study using
£. coli while working with a highly purified enzyme. Their reaction yielded
5-methylcytosine and 6-methylaminopurine.
The following study was undertaken to study the methylation of the nucleic acid, DNA in germinating winter wheat seedlings over a germination period of four days using radiotracers and liquid scintillation techniques. This investigation may add to the understanding of winter wheat at the molecular level, an understanding which could contribute to improvements in this important food crop.

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II. METHODS AND MATERIALS
The experimental plant used in this study, Triticum aestivum ssp. Vulgare eVill., Host) MacKey variety Gaines/Cl
13448, was stored in the seed state under vacuum at 4°C. All common laboratory chemicals used were reagent grade commercial products. Labeled compound, L-methionine-(methyl-14 C) sulfonium iodide, was purchased from ICN Pharmaceuticals, Inc., Chemical and Radioisotope Division, Irvine, California; EDTA (ethylenediamine tetraacetic acid), sodium dodecyl-sulfate, trisodium citrate, and
Tween 20 from Sigma Corp., St. Louis, MO; insta-gel scintillation cocktail from Packard Instrument Comp., Inc., Downers Grove, 1L.
Exper imen t~~ Procedure
Germination of Seedlings and Sampling Procedures
Gaines variety wheat was stored under vacuum at 4°C until ready for use. To disinfect the surfaces of the seed coat, the dry seeds were immersed for 5 minutes in a 1% sodium hypochlorite solution into which 5 drops of ZOO-fold diluted Tween 20 had been added. The seeds were then washed thorougly with sterile distilled water. After being washed, the seeds were incubated overnight (18 hours) in (0.1 p
Ci/pM/ml.) S-methyl-L-methionine-
14UI
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.
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After being incubated, the seeds were thoroughly washed with sterile distilled water. The seeds were then transferred to sterile petri dishes containing four sheets of paper towels moistened with sterile distilled water. The seeds were germinated in a Sherer growth chamber at Z6-Z8°C, 50% relative humidity, and a photoperiod of lZ hours (807 Lux).

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With planting time called zero time, seedlings were collected daily for 4 consecutive days. The seedlings were separated into root, shoot, and remaining seed material. The separated plant parts were then wrapped in several layers of Kimwipe tissue, placed in plastic bags, and frozen for later use.
DNA Extraction Procedure
To establish absorption of the radioactive label from
S-methyl-L-methionine-14CH3 into the DNA of wheat roots, shoots, and remaining seed parts, an experiment such as that done by
Lurquin, I~I J'\~., (17) was performed to obtain a re la t i ve 1y pure extract of DNA.
The separated plant parts were weighed and then ground In a mortar in the presence of dry ice. The ground up tissues were suspended in saline-EDTA (0.14 ~ NaCl and 0.1 ~ EDTA, containing
1% sodium dodecyl-sulfate, pH 8.0). Two ml of solution was used for every 250 mg of fresh tissue. For every 250 mg of fresh tissue, 2 mg of pronase was added, and the mixture incubated for 1 hour at 37°C. The extract was centrifuged at lO,OOOxg for 40 minutes at 4°C, and the nucleic acids were precipitated with 2 volumes of cold ethanol. The pellet was then resuspended in 2 ml of O. 1 x SSC (Standard Saline Citrate: 0.15 ~ NaCl,
0.015 M citric acid; pH 7 .0) . The mixture was incubated for another hour at 37°C after 2 mg of pronase was added for every
250 mg of fresh tissue.

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FIGURE A.-- Activity of system in counts per minute per gram of tissue over a 4-day germination period. The specific activity in the root tissue
is represented
Actlvi ty in the shoot tissue
the solid line. the specific is represented by the interrupted line, And the specific activity in the DNA in the remaining seed carts Is represented by the dashed line.

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After incubation, the mixture was centrifuged at lO,OOOxg for 40 minutes at 4°C, and the residual nucleic acids in the supernatant fraction were precipitated with 2 volumes of cold ethanol. The nucleic acid precipitates were pooled and placed into a scintillation vial with 10 ml of Insta-Gel scintillation cocktail. The vials were then counted in a liquid scintillation spectrometer to determine radioactive content.
III. RESULTS AND DISCUSSION
Samples of the root, shoot, and remaining seed parts were collected daily over a period of 4 days. A 10 minute count was taken for each sample. Due to the variation in the weight of each sample, the counts per minute were divided by the initial weight of each sample.
Results from this investigation are shown in Figure A.
Counts per minute per gram of tissue in the root was highest on day 1 and dropped rapidly until day 3, followed by a slight rise on day 4. In the shoot, the counts per minute per gram of tissue was also highest on day 1, dropping rapidly to its lowest point on day 4. There was no fluctuation in the counts per minute per gram of tissue in the remaining seed parts, which remained at a lower level than the other two groups.
Since any protein, which may have contained the S-methyl-Lmethionine or its dissociated methyl group, was "salted out" using the saline-EDTA, the results indicate that the methyl group was incorporated into the DNA of the root and shoot of the germinating wheat seedlings. The decrease in counts per

,.
- 6minute per gram of tissue for the root and shoot was expected as a result of the labeled DNA being replicated and dispersed throughout the rapidly growing root and shoot, after an initial incorporation of the labeled methyl group.
This study was concerned solely with the incorporation of the radiotracer into the DNA of the germinating wheat seedling.
Further analysis, involving liquid scintillation spectrometry of the protein material salted out by the saline-EDTA, could demonstrate that the methyl group from S-methyl-L-methionine is or is not incorporated into the protein (histones) of the germinating wheat seedling. It is hoped that future investigations will add to the understanding of winter wheat at the molecular level, an understanding which could contribute to improvements in this important food crop.
Acknowledgements
The researcher gratefully acknowledges a Ball State
University Undergraduate Research Grant without which this research endeavor would not have been possible.

2 grams
100
/
.u Ci
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Financial Statement
Des cr ..i£t ion
L-methionine Methyl Sulfonium
Iodide-A grade
L-methionine Sulfonium iodide,
(methyl-14C)
Total
Price
60.00
$115.00
$l75~

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REFERENCES
1. Du Vigneaud, V. 1952. A trail of research in sulfur chemistry and metabolism, and related fields.
Cornell Univ. Press.
2. Du Vigneaud, V., and J.R. Rachele. 1965. In: Transmethylation and methionine biosynthesis. CEds. S.K. Shapiro and
Schlenk) Univ. of Chicago Press, Chicago.
3. Challenger, F., and M.I. Simpson. 1948. Studies on biological methylation, Part XII. A precursor of the dimethyl sulphide evolved by Polysiphoni~ fastigiata. Dimethyl-2-carboxyethylsulphonium hydroxide and its salts. J. Chern. Soc.
43:1591-1597.
4. Bot. Gaz. 138(1):4651. 1977.
Dodd,
and R.A. Cossins. 1968. Biosynthesis of
S-adenosylmethionine in germinating pea seed.
Phytochem. 7:2143-2145.
6. Marcus, A., and J. Feeley. 1964. Activation of protein synthesis in the imbibition of seed germination.
Proc. Natl. Acad. Sci. U.S. 51:1075.
7. Marcus, A., and J. Feeley. 1966. Protein synthesis in imbibed seeds. I. Polysome formation during imbibition.
J. BioI. Chern. 240:1675-1680.
8. Ingle, J., and R.B. Hagemen. 19b5. Metabolic changes associated with the germination of corn. II. Nucleic acid metabolism. Plant Physiol. 40:48-53.
9. Cherry,
,].H.
1963. Nucleic acid changes in the storage tissue or seeds during germination. Biochem. Biophys.
Acta 68:193-198.
10. Borek, E., and P.R. Srinivasan. 1966. nucleic acids. Annu. Rev. Biochem.
The methylation of
35:275-298.
11. Mandel, L.R., and E. Borek. 1963. The biosynthesis of methylated bases in ribonucleic acid. Biochem. 2:555-560.
12. Starr, J.L. 1963. The incorporation of methyl groups into amino acid transfer ribonucleic acid. Biochem. Biophys.
Commun. 10:175-180.

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13. Svensson, I., H.G. Boman, K.G. Eriksson, and K. Kjellin. 1963.
Studies on microbial RNA.
Transfer of methyl groups from methionine to soluble RNA from Escherichia coli.
J. Mo 1. B i 01 . 7 : 254 - 271 .
14. Jakob, K.M., and I. Tal. 1973. Methylmethionine labeling of a higher plant tissue: The incorporation into RNA and pectinic acid of nucleic acid extracts. Biochem. Biophys.
Acta 308:296-309.
15. Kalousek, R., and N.R. Morris. 1969. methylase activity in pea seedlings.
Deoxyribonucleic acid
Science 164:721-722.
16. Gold, M., and f~~ch_~richia
Hurwitz. 1968. DNA methylase from coli. Methods in Enzymol. 12 (B) :491.
17. Lurquin, P.P., G. Tshitenge, G. Delaunoit, and L. Ledoux. 1975.
Isolation of DNA from plant cells by gel filtration on agarose.
Anal. Biochem. 65:1-10.