1022-7954/03/3903- $25.00 © 2003
Russian Journal of Genetics, Vol. 39, No. 3, 2003, pp. 283–287. Translated from Genetika, Vol. 39, No. 3, 2003, pp. 357–361.
Original Russian Text Copyright © 2003 by Mazheika, Kolomiets.
The method for preparing total synaptonemal com-
plexes (SCs) by spreading nuclei on the surface of a
hypotonic solution was ﬁrst proposed for analysis of
chromosome behavior in
spermatocytes at the
meiotic prophase I stage . In recent years, this
method has been widely applied in cytogenetics of
are fungus species best investigated
by now by the method of spreading nuclei. All studies
dedicated to this method can be arbitrarily divided into
two groups. The ﬁrst group includes in situ hybridiza-
tion experiments concerning the behavior of chromo-
somes during the nucleus cycle [2–5]. Studies of the
second group analyze formation of SC-speciﬁc nucle-
oprotein chromosomal structures at the meiotic
prophase I stage [6–10]. In both cases, spreading of
fungal nuclei includes two steps. At the ﬁrst step, pro-
toplasts are isolated by degradation of the cell walls of
basidia with lytic enzymes in an isotonic medium
[6, 11, 12]. The second step is spreading of the proto-
plast nuclei. At the third step, preparations for electron
microscopy are made.
Several methods proposed for protoplast nucleus
spreading are applied to yeast species. One of them
consists in application of protoplast suspension onto
the convex meniscus of a hypotonic medium drop .
Another method involves transferring protoplast sus-
pension into a hypotonic solution (1 : 25) followed by
an application of burst nuclei onto the surface of a glass
covered by an electron-transparent ﬁlmy support .
In recent years, a third method for fungal protoplast
spreading has gained general acceptance. A drop of
protoplast suspension is loaded onto a plastic-covered
glass and is supplemented with a detergent and a ﬁxa-
tive [13, 14].
Independent of the method for obtaining spread pro-
toplast nuclei, preparations for microscopic examina-
tion are stained, as a rule, with silver nitrate. Many
researchers use an express method with the use of silver
nitrate and gelatin .
So far, few papers have been dedicated to the elec-
tron microscopy of SCs in spread nuclei of ﬁlamentous
fungi. In contrast to the aforementioned method for
yeast SCs, SC preparations from the ﬁlamentous fungi
were obtained by mechanical crush of basidium cells.
The aim of the present study is to develop a method for
obtaining total preparations of synaptonemal complexes
of meiotic chromosomes from meiosporangium proto-
plasts of cultivated white button mushroom
This is the ﬁrst experience of developing a method
for preparation of spread nuclei of ﬁlamentous fungi
(white button mushroom) involving the main steps of the
corresponding method for yeasts. In addition, we used
the results of other authors concerning preparation of
basidial protoplasts of white button mushroom [21, 22].
MATERIALS AND METHODS
Experiments were carried
out with the Bs94 strain, which was a descendant of the
Kerrigan et Callac holotype
, characterized by predominance of four-spored
basidia. The Bs94 strain was kindly provided by P. Cal-
lac (France). In addition, we investigated bispore vari-
eties grown in the Zarech’e state farm (Moscow region).
A Method for Preparation of Synaptonemal Complexes
of Meiotic Chromosomes from Basidial Protoplasts
of the White Button Mushroom
I. S. Mazheika
and O. L. Kolomiets
Department of Mycology and Algology, Moscow State University, Moscow, 119899 Russia;
Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia; e-mail: email@example.com
Received August 8, 2002
—For the ﬁrst time, preparations of synaptonemal complexes (SCs) were made from meiotic chromo-
somes of white button mushroom (
) basidia. It is the ﬁrst experience of obtaining SC prepa-
rations of ﬁlamentous fungi from isolated meiosporangium protoplasts. Previously, only yeast SC preparations
were obtained following this approach. The method includes four major stages: isolation of basidium proto-
plasts by treatment of basidia with lytic enzymes, spreading of protoplast nuclei on a ﬁlmy support by osmotic
shock, staining the preparations with silver nitrate, and examination under light and electron microscopes. The
structures of spread premeiotic nuclei, axial elements of chromosomes, SCs, chromatin, and nucleoli were stud-
ied at the leptotene–diplotene stage of meiotic prophase I.