Collaboration offered: non-LTR retrotransposones study

Alexander Blinov blinov at BENPC.BIONET.NSC.RU
Wed Feb 12 09:57:34 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

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 
retransposons 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. (Genome, Febriary 1997)

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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