|
|||||
86 |
PNL Volume 19 |
1987 RESEARCH REPORTS |
|||
|
|||||
SISTER CHROMATID EXCHANGE (SCE) IN PISUM
SATIVUM |
|||||
|
|||||
Wolff, G.,
A. Neumann, and R. Rapp |
Institute of Genetics, University
of Bonn Federal Republic of Germany |
||||
|
|||||
After application of BrdU
(Bromdesoxyuridine) spontaneous
sister chromatid exchange (SCE) was first demonstrated by Latt (2) in human chromosomes. This method subsequently was improved, and SEC was observed in a great number of mammalian chromosomes as well as in plant cells (Vicia faba [1], Allium cepa [3], and others). Recently, we could show SCE in Pisum sativum root tip chromosomes. We applied the method of Kihlman and Kronberg (1) with a few modifications. Root tips were exposed to BrdU concen- trations of 10-4 m and 10-6 m in tap water. To increase the number of metaphases isovalerylurea was used instead of colchi- cine. The tissue was macerated in a solution of pectinase (5%) and cellulase (1%). After staining with Hoechst 33258 and Giemsa the two chromatids of the metaphase chromosomes were differen- tially stained and SCE's could be detected (Fig. 1.1 and 1.2-1.4, arrows indicating SCE's). We investigated two series of prepara- tions : 1. Root tips were exposed to BrdU during one
cell cycle,
followed by one cycle in Thd (Thymidine). 2. Root tips were exposed to BrdU during two
cell cycles,
followed by one cycle in Thd. All solutions were made with tap
water, Schematically, the incor- poration of BrdU results in the following chromosomes: |
|||||
|
|||||
|
|||||
|
||||
PNL Volume 19 1987 RESEARCH REPORTS |
87 |
|||
|
||||
The above scheme is based on the
assumption that a chromosome is built up on one DNA element. Metaphase chromosomes constructed according to the scheme labelled with (*) should show differential staining and SCE's; in those labelled with (+) we should find the results of an SCE occurring during the preceeding cell cycle, schematically as follows: |
||||
|
||||
Metaphase chromosome after two
cell-cycles in BrdU, both chromatids differentially stained (see above) |
||||
The same chromosome after a terminal SCE |
||||
|
||||
After mitosis this chromosome
gives two types of chromosome, and after a further cycle in Thd, these result in metaphase chro- mosomes which show a discontinuous staining in only one chromatid (cf. Fig. 1.5), and schematically: |
||||
|
||||
|
||||
The incorporation of BrdU
influences the length of the chromatids
as Fig. 1.5 shows. We did not make any measurements so we cannot decide whether it shortens or lengthens the respective element. Several questions result from the
application of this method.
Because of a low mitotic index in Pisum sativum root tip meristems and the fact that only a small number of well stained chromosomes is available, we are not yet able to count the SCE's per meta- phase. For the same reasons, we are not able to decide whether homologous chromosomes have an identical SCE pattern, or whether a special pattern is characteristic for a specific chromosome. Yet the available material seems to point to the fact that the homo- logues are not identical in this respect, and this would mean that the pattern is random. In addition, the results could provide a basis to
investigate the question whether a metaphase chromosome of Pisum sativum is composed of only two chromatids or of two chromatids which are subdivided into half-chromatids, a question currently under dis- cussion. C-banded metaphase chromosomes of this material exhibit a subdivision of certain heterochromatin blocks, which suggest that the chromatids are subdivided (Fig. 2, arrows). If this is true, then we should find in connection with our SCE experiments exchanges within a single chromatid. Some of the patterns found agree with this assumption (Fig. 1.6). Further investigations are required. |
||||
|
||||
1. Kihlman, B. A. and D. Kronberg.
1975. Chromosoma 51:1-10.
2. Latt, S. H. 1973. Proc. Nat. Acad. Sci.
70:3395-3399.
3. Schwartzman, J.
B. and F. Cortes.
1977. Chromosoma 62:119- 131 . |
||||
|
||||
|
||||
88 |
PNL Volume 19 1987 RESEARCH REPORTS |
|||
|
||||
|
||||
Fig. 1. Differentially stained
metaphase chromosomes of Pisum sativum after BrdU incorporation; arrows point to SCE's. See text for details. |
||||
|
||||
Fig. 2. C-banded
metaphase
chromosomes of Pisum sativum;
arrows point to
subdivided
heterochromatin
blocks. |
||||
|
||||
***** |
||||
|
||||