Collaboration offered: non-LTR retrotransposones study

Eugene Berezikov funtik at ECOCLUB.NSU.RU
Sun Feb 9 17:34:39 EST 1997


Dr. Alexander Blinov
Head of Cell Biology Laboratory
Institute of Cytology and Genetics
prospect Lavrentjeva, 10
Novosibirsk 630090
Russia

Fax	(3832) 35-65-58
Phone:	(3832) 35-46-70
Email:	cellbiol at cgi.nsk.su

We are looking forward to establish collaboration with scientists
interested in study of regulation of expression and transposition of
non-LTR retrotransposons. As a first step we would like to get a JOINT
GRANT with somebody interested in such kind of investigations.

Here is a first draft of a project we propose:

EXPRESSION OF THE NLRCTH1 ON THE DIFFERENT DEVELOPMENTAL STAGES AND
UNDER DIFFERENT PHYSIOLOGICAL CONDITIONS.

We have previously described and characterized two non-LTR rotransposons
>From the Diptera Chironomus thummi (Blinov et al., 1993) and C.tentans
(Blinov v et al., 1997). Also we have shown that the distribution of
this elements is restricted by Chironomus genus. It has been shown that
mininimum of three different non-LTR retrotransposons are present in the
Chironomus genus. All of them contain similar nucleotide sequences in
the region of the ORF2 which encodes reverse transcriptase. We
disignated the retrotransposon as NLRCth1. 

It has been shown that a transposition of non-LTR retrotransposons
causes haemophilia and adenocarcinoma in man (Kazazian et al, 1988;
Morse et al, 1988), and hybrid dysgenesis in drosophila (Finnegan,1989).
Expression of these elements is particularly high in certain embryonal
carcinoma and teratocarcinoma cell lines (Leibold et al, 1990). It leads
to speculation that expression and transposition of non-LTR
retrotransposons may occur only in certain developmental stages.
Moreover, the synthesis of full-length transcripts has been shown only
for several organisms. 

We would like to investigate expression of the NLRCth1 on the different
larval stages of Chironomus thummi using the Nothern blot analysis. The
Chironomus is a very good subject for such kind of investigation. These
animals have several very good separated developmental stages. Also, it
will be interesting to show the influence of some kinds of stress on the
expression of this element since the role of environmental conditions in
transposition has been suggested. Heat stress, for example, can be used
as an inductor for this purpose. 

Some transcription factors have been described wich bind specifically to
non-LTR retrotransposon promoter region (Mathias and Scott, 1993). Such
proteins are candidates for factors controlling expression of
retrotransposons. Gene mobility shift assays may be used for
investigations of differential expression of NLRCth1.

The next experimental procedures in this project include:

1. Isolation of nuclear extracts from different stages of Chirnomus
thummi.
2. Constructing hybrid plasmids containing different parts of NLRCth1
promoter region.
3. Gel mobility shift assays analysis, using the nuclear extracts and
DNA probes.
4. DNAse I protection assays.
5. Constructing of Chironomus thummi expression library.
6. Screening of this library using South-West analysis.
7. Isolation and characterization of positive clones.

Using this approach we hope to get answers for two important questions:

1. Is the expression of non-LTR retrotransposons stage- and tissue-
specific; and whether physiological conditions influence their
expression or not.
2. Whether transcription factors which may control expression of the
NLRCth1 present or not.


REFERENCES

Blinov A.G., Sobanov Y.V., Bogachev S.S., Donchenko  A.P., Filippova
M.A. (1993) The Chironomus thummi genome contains a non-LTR
retrotransposon. Mol Gen Genet 237: 412-420.

Blinov A.G., Sobanov Y.V, Scherbik S.V. and Aimanova K.G. (1997) The
Chironomus (Camptochironomus) tentans genome contains two non-LTR
retrotransposons. Genome, in press.

Finnegan D.J. (1989) Eucariotic transposable elements and genome
evolution. Trends in Genet 5: 103-107.

Kazazian H.H., Wong C., Youssufian H., Scott A.F., Philips D.G.,
Antonarakis S.E. (1988) Haemophilia A resulting  from de novo insertion
of L1 sequences represents a novel mechanism for mutation in man. Nature
332: 164-166.

Leibold D.M., Swergold G.D., Singer M.F., Thayer B.A., Dombroski ,B.A.,
Fanning T.G. (1990) Translation of LINE-1 elements in vitro and in human
cells. Proc Natl Acad Sci USA 87: 6990-6994.

Mathias S.L., Scott A.F. (1993) Promoter binding proteins of an active
human L1 retrotransposon. Bioch Bioph Res Commun 191: 625-632.

Morse B., Rotherg P.G., South V.J., Spandorfer J.M., Astrin S.M. (1988)
Insertional mutagenesis of the myc locus by a LINE-1 sequence in human
breast carcinoma. Nature 333: 87-89.




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