PNL Volume 19
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:

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
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 .

Fig. 1. Differentially stained metaphase chromosomes of Pisum
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.