STCU Science Research Fields Overview: Biotechnology in Natl'Academy of Sciences of Ukraine

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STCU Science Research Fields Overview
Biotechnology in Natl.Academy of Sciences of
Ukraine

• Yaroslav Blume
• Ljubljana, March 10, 2008

Thematic International Conference on Bio-, Nano-
and Space Technologies, E U Science Centers
Collaboration Ljubljana, Slovenia, March
10-12, 2008
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National policy for biotechnology

• In 1991, the Committee of Biosafety at State
  Committee of Science and Technologies was
  created.
• In 1994, all former USSR-established programs
  were transformed into the State scientific and
  technical programme Perspective
  Biotechnologies.
• In 2001, Advisory Biotechnological Council under
  the Cabinet of Ministers was created.
• In 2002, the new state priority for biotechnology
  was approved by the Parliament that were under
  operation until 2007.

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State RD Programs Microbial Biotechnologies

• 1.1. Microbial strains with high productivity
• 1.2. Microbial biotechnologies for industry and
  ecology
• 1.3. Microbial biotechnologies for medicine and
  agriculture
• 1.4. Diagnostic kits and vaccines based on
  viruses, microorganisms and their compounds.

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State RD ProgramsAnalytical and Gene
Biotechnologies

• 1. Methods for genetic therapy of
  insulin-dependent diabet and atherosclerosis
• 2. Kits for PCR diagnostics of inherited
  diseases
• 3. Genetic engineering approaches for production
  of human cytokines
• 4. Techniques for molecular genetic testing of
  cancer diseases
• 5. Biosensor systems and bioanalysators.

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State RD ProgramsPlant Biotechnology and
Biosafety

• 1. Biotechnological producing of novel plants
  with valuable traits
• 2. Molecular genetic approaches for genome
  analysis and molecular markers agriculturally
  important traits
• 3. Development of new genetic engineering
  approaches and creation of GM plants
• 4. Biosafety research new marker genes, vertical
  and horizontal gene flow, long-term monitoring of
  GM plants molecular detecting of foreign DNA.

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Plant Biotechnology and Biosafety The
projects of highest priority

• Development of the methods for preparative
  production of pharmaceutical human proteins in
  plant systems
• Modified tubulin genes as new marker system for
  selection of transgenic plants
• Producing CB-resistant potato through chloroplast
  transformation
• Development of molecular probes for foreign gene
  assessment.

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Private sector involvement in biotechnological
development in Ukraine

• Up to the present, the private sectors
  participation in biotechnological investigations
  and in their application has not been sufficient.
  This could be explained by the fact that general
  pre-conditions for the development of small and
  middle-sized business in Ukraine have not been
  favourable and the internal market of
  biotechnological products has been developing
  spontaneously.
• The easiest task in Ukraine is to identify
  biotechnological products for medical purposes.
  As a rule, these substances were obtained by
  means of bacterial synthesis and plant cell
  lines-producents.
• The process of field testing of GM plant
  varieties was postponed four years ago.

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Other Ukrainian sectoral RD Biotechnological
Programs

• National Academy of Sciences
• Genetic and cellular engineering as basis for
  new green revolution (2002-06) substituted by
  Genomics, biotechnology and biomarkers (from
  2007)
• Genome Functioning (from 2002)
• Biosensors (2003-08)
• Biofuels (2007-09).
• Ukrainian Academy of Agricultural Sciences
• - Agricultural biotechnologies (from 2002).

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BIOTECHNOLOGY R D POTENTIALIN NATL. ACADEMY
OF SCIENCES  

• In order to overview general trends of the
  development of biotechnological investigations in
  Ukraine it would be worth to demonstrate some
  institutions - leaders in this field
• Institute of Cell Biology and Genetic Engineering
  (plant molecular biology and genomics, genetic
  engineering, plant cell culture,
  phytoremediation, germplasm preservation,
  biosafety)
• Institute of Molecular Biology and Genetics
  (biotechnology of animal cells, cloning, gene
  therapy, plant cell culture)
• Institute of Microbiology and Virology (microbial
  biosynthesis, manipulations with bacterial DNA,
  environmental biotechnology)
• Institute of Biochemistry (enzymes and
  immunochemistry)
• Institute of Bioorganic Chemistry and Oil
  Chemistry (plant genetic engineering,
  environmental biotechnology).

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INSTITUTE OF CELL BIOLOGY AND GENETIC ENGINEERING

• Cell and genetic engineering
• Molecular and cell biology
• Genomics and structural bioinformatics
• Biosafety and GMOs testing

• Established in 1990
• Total staff about 175 persons, incl. 15 DSci and
  47 PhD
• Three Departments
• - Department of Genomics and Biotechnology (with
  4 Labs)
• - Department of Genetic Engineering
• - Department of Biophysics and Radiobiology
• and Lab. of Fungal Cell Biology and
  Biotechnology

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Selected topics in plant biotechnology

• Transgenic, Transplastomic and Transient
  Approaches To Express Foreign Genes in Plants
• In vitro Tissue Cultures as the Germplasm Bank

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TRANSGENOSIS

• Plants
• tobacco, potato, tomato, canola,
  flax, sugar beet, peas, bean,
  soybean, barley,
• finger millet

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New transgenic plants developed during last years

• Ukrainian breeding sugar beet line (Beta vulgaris
  L.) resistant to herbicide BASTA promoter-less
  technology
• Ukrainian breeding pea lines (Pisum sativum L.)
  resistant to herbicide BASTA
• Ukrainian breeding pea lines (Pisum sativum L.)
  resistant to herbicide PURSUIT
• Ukrainian breeding rapeseed lines (Brassica
  napus L.) resistant to herbicide BASTA
  promoter-less technology
• Chloroplast transformed Lycium barbarum L.
• Ukrainian breeding potato lines (Solanum
  tuberosum L.) resistant to herbicide BASTA
  promoter-less technology
• Dinitroaniline-resistant soybean (Glycine max L.)
  and flax original technology with
  dinitroaniline resistant tubulin gene used as
  marker gene for selection simultaneously.

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CYBRID DEVELOPMENT
PROTOPLAST FUSION BETWEEN CHLOROPHYLL DEFICIENT
RECIPIENT AND IRRADIATED 500GY CHLROPLAST
CYBRID SELECTION
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Chloroplast transformation Clipboard approach
Transformed plastids in crop plant
FUSE
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New selectible system for plant transformation
based on marker gene of plant origin

• Driving force for this approach is the public
  concern about a potential spread of antibiotic
  resistance genes in soil and intestinal bacteria.
• The approach is based on the knowledge about the
  mechanisms of microtubular resistance to
  antimitotic herbicides (dinitro-anilines and
  phosphoroamidates) - the appearance of altered
  tubulin with mutation(s) in the binding site for
  these herbicides.

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Alpha-tubulin Interdimer contact zone
Deduced Protein Modification Tua1
239 S to R Thr to Ile
a
b
Distribution of electrostatic potential on
interdimeric surface of alpha-1 tubulins from
Eleusine indica a sensitive biotype b
resistant biotype
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L1 L2 L3 P1 Co M
Southern analysis of transformed Linum lines with
a probe to nptII (A), alpha-tubulin (B) and
beta-tubulin (C) genes. L1, L3 different
transgenic lines of Linum Lk control plant
line Pl plasmid pBITUBA8. Molecular weight
markers are shown right (in bp). Specific bands
for transferred alfa- and beta-tubulin genes are
arrrowed.
Transgenic soybean (S) lines (with exception of
the line S2) possess one additional band
(arrowed) for alpha-tubulin in comparison to the
control (SK). The band length is corresponded to
transferred mutant alpha-tubulin size.
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RECOMBINANT PROTEIN PRODUCTION IN PLANTS GFP
expression in Nicotiana benthamiana infiltrated
with 35S-promotor based construct
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SDS-PAGE analysis of N. excelsior plant extracts
infiltrated with GFP VIR construct ( 60 TSP)
1- marker proteins 2 - N. excelsior infiltrated
with VIR constructs 3 - control
(non-infiltrated) plant.
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Production of the anti-tuberculosis protein at
plant cells
Gene cloning at plant cells
Transgenic plants
Protein purification
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Plant cell lines

• 2,050 plant cell lines are maintained in vitro

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Some selected topics in structural biology and
genomicsChemical design and screening of new
drugsPlant genomics and GRID potential
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Reconstruction of the space structure of fungal
?-tubulin resistant to benzymidazoles
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Docking results for the interaction of
FtsZ-protein from Mycobacterium tuberculosis and
ethylcarbamate SRI-3072 (?) and tartal (B) ..
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Arabidopsis thaliana genome scanning with
TBLASTN instrumentary

• gtP43404ZAP70_MOUSE Tyrosine-protein kinase
  ZAP-70 - Mus musculus (Mouse).
• MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSL
  VHDVRFHHFP
• IERQLNGTYAIAGGKAHCGPAELCQFYSQDPDGLPCNLRKPCNRPPGLEP
  QPGVFDCLRD
• AMVCDYVRQTWKLEGDALEQAIISQAPQVEKLIATTAHERMPWYHSSLTR
  EEAERKLYSG
• QQTDGKFLLRPRKEQGTYALSLVYGKTVYHYLISQDKAGKYCIPEGTKFD
  TLWQLVEYLK
• LKADGLIYRLKEVCPNSSASAAVAAPTLPAHPSTFTQPQRRVDTLNSDGY
  TPEPARLASS
• TDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRENLLVADIELGCGNFGS
  VRQGVYRMRK
• KQIDVAIKVLKQGTEKADKDEMMREAQIMHQLDNPYIVRLIGVCQAEALM
  LVMEMAGGGP
• LHKFLLGKKEEIPVSNVAELLHQVAMGMKYLEEKNFVHRDLAARNVLLVN
  RHYAKISDFG
• LSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWE
  AFSYGQKPYK
• KMKGPQVLDFIKQGKRMECPPECPPEMYALMSDCWIYKWEDRPDFLTVEQ
  RMRNYYYSPA
• SRAEGPPQCEQVAEAACG

TBLASTN (NCBI)
gtKinase_DOMEN_ZAP70_MOUSE(P43404) LVADIELGCGNFGSVR
QGVYRMRKKQIDVAIKVLKQGTEKADKDEMMREAQIMHQLDNPY
IVRLIGVCQAEALMLVMEMAGGGPLHKFLLGKKEEIPVSNVAELLHQVAM
GMKYLEEKNF VHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTAR
SAGKWPLKWYAPECINFRKFS SRSDVWSYGVTMWEAFSYGQKPYKKMKG
PQVLDFIKQGKRMECPPECPPEMYALMSDCWI
YKWEDRPDFLTVEQRM
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BSBA (http//www.bsbanet.org)Pannonian Biotech
Association?
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Integration of new high technological fields in
biology and biomedicine
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Thank you for attention!
Prof. Yaroslav Blume Acting Director Institute of
Cell Biology and Genetic Engineering Natl.
Academy of Sciences o Ukraine acad. Zabolotnogo
str., 148 Kiev-143, 03680 UKRAINE Phone/Fax 380
44 526 7104 E-mail cellbio_at_cellbio.freenet.viaduk
.net yablume_at_univ.kiev.ua http//www.icbge.gov.ua