| It was confirmed by a DNase digestion test that the material within chloroplasts
stained by DAPI is really DNA. Chlorophyll autofluorescence showed no interference
with fluorescent images of chloroplast DNA stained with DAPI. These fluorescent
particles are rather regular in shape and are not located ubiquitously within
chloroplast architechture as far as Fig. 1 shows.
The number of fluorescent particles of chloroplast DNA by DAPI per chloroplast is 18.5 on the average in mesophyll protoplasts released from the mature region of the first foliage leaves. Since a single chloroplast fluorescent dot appears to contain at least as much as 120 x 106 daltons of DNA contained in T4 phage11), it seems reasonable to say that the number of chloroplast DNA per chloroplast, i.e. the copy number is about 20 or more. About 135 chloroplasts are present in a single mesophyll cell in this species12. So, a single mature mesophyll cell appears to contain at least 2500 chloroplast DNA molecules. Chloroplast DNA of higher plants is a double-stranded, circular, covalently closed molecules with a contour length of 43-55 microm and a molecular weight of 85-100 x 106 daltons13). The amount of chloroplast DNA per cell is estimated to be at least 5 x 10-13g, which is about 1% of the nuclear DNA amount. It seems reasonable to suppose that DAPI-staining should permit detection of a single full sized chloroplast DNA molecule. The fluorescence microscopy for detecting chloroplast DNA in situ reported here is believed to be very useful to see details of the organization, replication and segregation, and relative amounts of DNA per chloroplast in the wheat genus and its related genus from ontogenetic and phylogenetic interests. The study was partly supported by Grant Aids for Fundamental Scientific Research from the Ministry of Education, Science and Culture. The author wishes to thank Dr. H. NIEDDERER, Serva-Feinbiochemica, Heidelberg, Fed. Rep. of Germany, for his generous gift of DAPI. References 1) SAGER, R., 1972 Cytoplasmic Genes and Organelles. Academic Press, New York. 2) KIHARA, H., 1951 Cytologia 16: 177-193. 3) FUKASAWA, H., 1953 Cytologia 18: 167-175. 4) SUEMOTO, H., 1968 Proc. III Int. Wheat Genet. Symp. 141-152. 5) TSUNEWAKI, K., Y. MUKAI, T. Ryu ENDO, S. TSUJI and M. MURATA, 1976 Japan J. Genet. 51: 175-191. 6) VEDEL, F., F. QUEITER, F. DOSBA and G. DOUSSINAULT, 1978 Plant Science Letters 13: 97- 102. 7) WILLIAMSON, D.H. and D.J. FENNELL, 1975 Methods Cell Biol. 12: 335-351. 8) RUSSELL, W.C., C. NEWMAN and D.H. WILLIMSON, 1975 Nature 253: 461-462. 9) HAJDUK, S.L., 1976 Science 191: 858-859. 10) COLEMAN, A.W., 1978 Exp. Cell Res. 114: 95-100. 11) JAMES, T.W. and C. JOPE, 1978 J. Cell Biol. 79: 623-630. 12) IKUSHIMA, T., 1980 Abstracts of the 45th Ann. Meeting of the Botanical Society of Japan, 222. 13) POSSINGHAM, J.V., 1980 Ann. Rev. Plant Physiol. 31: 113-129. |
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