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

poly[d(A-T)].poly[d(A-T)], only the 1:1 complex is formed when the ligand binds
to the homopolymer poly(dA)úpoly(dT). The DNA binding to peptides and
netropsin-peptide coniugates using a double-stranded motif for sequence
recognition in the minor groove was studied. The studies on the DNA binding with
peptides implicated in the DNA-binding domains of regulatory proteins continued.
Effects of sibiromycin, distamycin A, and its analogs on DNA and
poly(dA).poly(dT) binding by a synthetic 23-residue zinc-binding peptide, a part of
the DNA-binding domain of the transcription activator Gal-4 were studied. Using
circular dichroism and fluorimetric methods, we have found that the synthetic
peptide and two distamicyn A analogs compete for binding sites on DNA and
poly(dA).poly(dT). The antibiotic sibiromycin forming a covalent bond with the
guanine 2-amino group in the minor DNA groove can displace the peptide from the
19 bp self-complementary oligonucleotide, a specific target site for Gal-4 protein.
The peptide binds to the glucosylated phage T2 DNA, but its affinity to this DNA is
lower, than that to calf thymus DNA under the same conditions. The binding
isoterms of distamycin analogs obtained in the absence and in the presence of
synthetic peptide were used to determine both binding constant and site size for the
peptide to poly(dA).poly(dT). The binding constant is found to very from 1.4.107
to 1.8.107 M-1 in the presence of 0.1 M NaCl.
1997 update: ANTIBODY ENGINEERING
Head Serguei M. Deyev, Ph.D., D.Sc.
The laboratory was organized in 1995 to design of unnatural antibodies and to develop
appropriate systems for their biosynthesis. In 1995 the experiments were carried
out in two main directions. The first is the full size chimeric mouse/human
immunoglobulin synthesis in eucaryotic (lymphoid and nonlymphoid) cells. The
second one deals with the design of single-chain antibodies and their production in
procaryotic cells. A new solid phase immunoanalysis method, group selective
immunoassay was developed.
1997 update: PROTEIN INHIBITORS OF CELLULAR PROCESSES Head
Iuri V. Kozlov, Ph.D., D.Sc., Professor
Our studies have focused on the structure of the ribosome-inactivating toxins and their
transport into the cytosol of target cells. Like some plant ribosome-inactivating
toxins, Shiga toxin causes death of eukaryotic cells upon penetrating into their
cytosol. The enzymatically active part of the toxin (A subunit), acting as a highly
specific N-glycosidase, removes the sole adenine from ribosomal 28S RNA,
leading to the protein synthesis inhibition and following cell death. Cellular
proteases split the Shiga toxin A subunit into two disulfide-bound polypeptides
during its transport and translocation. To elucidate the effect of A subunit splitting
on toxin transport to the target ribosome and, as a result, on the cytotoxicity, the
substitutions and deletions were inserted into the site of hypothetical splitting of A
subunit. The wild-type toxin was much more efficient than the mutants in
provoking rapid intoxication of the cells. However, ultrastructural analysis of toxin
tagged with horseradish peroxidase demo nstrated that the wild-type and mutant
toxins were transported from endosomes to the trans-Golgi network and further
through the Golgi cisterns to the endoplasmic reticulum. The cells were able to
cleave both wild-type Shiga toxin and mutants, but cellular location for cleavage
appears to differ. The wild-type toxin was cleaved in the endosomes or in the trans-
Golgi network. The mutant toxin (where the trypsin- sensitive site was replaced by
that sensitive to cytosolic endoprotease calpain) was not cleaved in the presence of
brefeldin A, indicating that the cleavage can occur only when the toxin left the trans-
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