A Guide to the Russian Academy of Sciences - University of Texas ...

Golgi network and reached the cytosol. The crystal structure of Shiga toxin, an
AB5 hexamer, has been determined at 2.5 resolution. The A subunit interacts with
the B pentamer via C-terminal helix and a four-stranded mixed -sheet. The fold of
the rest of the A subunit is similar to that of A chain of plant toxin ricin; both are Nglycosidases.
However, the active site in the bacterial holotoxin is blocked by a
segment of polypeptide chain. These residues of A subunit would be released as a
part of the toxin activation mechanism.
1997 update: CELL ENGINEERING
Head Vladimir S. Prassolov, Ph.D, D.Sc.
In 1995 our efforts were focused on the creation of new vectors with the cloned gene
regulated expression in mammalian cells, including human ones. A self-inactivating
retroviral vector containing polylinker and conveying the resistance to antibiotic
G418 was constructed. Based on this construct, a retroviral vector has been made
that contains p53-responsive element modulating the hygromycin resistance gene
expression. We suppose that the introduction of this vector into the cells bearing the
wild type p53 will provide the resistance to hygromycin, whereas the cells carrying
mutant p53 will die. The development of this approach seems to be promising for
gene therapy. The analysis of p53-binding elements from p53-responsive genes has
been carried out. Recombinant plasmids were constructed in which expression of a
reporter gene of chloramphenicol-acetyl transferase is under control of a promoter
containing p53-responsive elements. The p53-responsive elements from different
genes were shown to display very different p53-dependent enhancer activity in
CAT assays. The p53-responsive elements from human WAF1 and ADA genes
exhibited the highest enhancer activity. This work was done in collaboration with
the Laboratory of cell proliferation. As a part of the project dealing with the study of
neuropathologic lesions induced by different murine leukemia viruses the cell lines
were created able to produce recombinant retroviruses. These viruses were
constructed on the basis of Moloney murine leukemia virus and contained a mutant
LTR and an amphotropic env gene. The kinetics of the CNS lesion development is
modulated by Freund leukemia virus in the case of co-infection with the abovementioned
viruses. These studies are carried out in cooperation with the Department
of Virus and Cell Genetics, Heinrich-Pette Institute of Experimental Virology and
Immunology, University of Hamburg, Germany. In 1995 studying the mechanism
of eukaryotic cell electrotransfection continued in cooperation with E. S.
Tsymbalyuk, A. N. Frumkin Institute of Electrochemistry, RAS, Moscow. The
method of electrotransfection of the support-anchored cells by the double-impulse
electrotransfection technique was further developed, and this allowed us to increase
the effectivity till standard parameters of the single-impulse electrotreatment. On the
other side, the earlier described electrotransfection by the low-voltage (nonporing)
impulse action on the preliminarily electroporated anchored cells was demonstrated
both for COS-1, and CHO, and HeLa cells. In this case the electrotransfection level
comparable with that in control (electrotransfection by the exponentially lowering
impulse) allows us to state that we were the first in the world who managed to
separate electrotransfection process into two steps: 1). electroporation of the cells
and 2). plasmid DNA seizure into electroporated cells caused by the low-voltage
field (this step is equivalent to the low-voltage part of exponential impulse). The
above results were presented at the 23d FEBS Meeting, August 13-18, Basel,
Switzerland.
1997 update: CELL PROLIFERATION
Head Peter M. Chumakov, M.D., Ph.D., D.Sc.
522