1022-7954/02/3809- $27.00 © 2002
Russian Journal of Genetics, Vol. 38, No. 9, 2002, pp. 1009–1014. Translated from Genetika, Vol. 38, No. 9, 2002, pp. 1196–1202.
Original Russian Text Copyright © 2002 by Ladygin, Boutanaev.
Unicellular green alga
Dang. provides a convenient model for studying vari-
ous biological processes, such as chloroplast function,
cell interaction, mating type switching, cell cycle, and
photosynthesis. Owing to the capability of autotrophic
growth on inexpensive mineral media, this alga is con-
sidered as a promising producer of foreign proteins.
However, such production is still unfeasible because of
unstable expression of heterologous genes in
The problem of heterologous gene expression is
among the major problems of
engineering. In most relevant works, the
nuclear genome has been transformed with the bacte-
rial neomycin phosphotransferase (
) gene used as
a selectable marker . However, this is a rather poor
choice because of the high rate of spontaneous kanamy-
cin resistance mutations in
view of this, the foreign hygromycin phosphotrans-
) gene seems to have more promise.
Genetic transformation of
reported for the ﬁrst time as early as in 1982 . How-
ever, progress has been made only recently owing to
new cell transformation techniques elaborated for
microorganisms, plants, and animals [5–7]. For
instance, a bioballistic technique has been employed in
chloroplasts . Efﬁcient
transformation of the nuclear genome has been
achieved with vigorous agitation of a cell suspension in
the presence of glass beads and plasmid DNA . Sta-
ble transformants have been obtained using restoration
of the wild-type sequence in mutant chloroplast genes,
This approach is based on homologous recombination
between a mutant gene and the wild-type sequence.
Transformation of the nuclear genome has been carried
out with the homologous
[9, 15], and
Heterologous gene expression in
difﬁcult to achieve. Stable expression has been reported
only for two genes,
gene fused with the 5'-untranslated region of the
gene  and for the
aminoglycoside adenine transferase (
) gene con-
ferring spectinomycin and streptomycin resistance on
cells . Notwithstanding these exam-
ples, the complex problem of foreign gene expression
still remains to be solved.
Early works on
gene used as a selectable marker had two
major drawbacks, low (
) transformation efﬁciency
 and high rate of spontaneous mutations to kanamy-
cin resistance [3, 13].
The objectives of this work were to enhance the
transformation efﬁciency by using the CW-15 mutant
lacking the cell wall and to avoid the effect of sponta-
neous mutations by using the hygromycin phospho-
) gene as a selectable marker.
MATERIALS AND METHODS
Cell suspensions of the
mutant were used for transformation. In contrast to the
wild-type cells of strain 137C mt+, the CW-15 mutant
does not produce the cell wall .
with the Hygromycin Phosphotransferase Gene
as a Selectable Marker
V. G. Ladygin and A. M. Boutanaev
Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia;
Received August 20, 2001; in ﬁnal form, January 9, 2002
Dang. cells, plasmid pCTVHyg was constructed with the
use of the
hygromycin phosphotransferase gene (
) controlled by the SV40 early promoter.
Cells of the CW-15 mutant strain were transformed by electroporation, with the yield reaching 10
) clones per 10
recipient cells. The exogenous DNA integrated in the
genome showed stable transmission for approximately 350 cell generations, while hygromycin resistance was
expressed as an unstable character. Codon usage was compared for the
genes. The results testiﬁed that codon usage bias, which is characteristic of
, is not the major fac-
tor affecting foreign gene expression. The advantages of the selective system for studying
formation with heterologous genes are discussed.