Eukaryotic transposable element

Details Eukaryotic transposable element Eukaryotic transposable element

1994-05-09

2002-10-22

Nguyen, Dave T. (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C800S025000

Reexamination Certificate

active

06469228

ABSTRACT:

BACKGROUND OF THE INVENTION
The Tc1-like family of transposons and the retroviral-like transposons are unique for their wide dispersion in diverse organisms. Six members belonging to the Tc-1-like family have been characterized in nematodes, diptera and fish: Tc1 in
Caenorhabditis elegans,
TCb1 in
Caenorhabditis briggsae,
HB1 in
Drosophila melanogaster,
Uhu in
Drosophila heteroneura,
Minos in
Drosophila hydei
and Tes1 in the Pacific hagfish
Eptatetrus stouti.
All are characterized by a relative short length (1.6 to 1.8 kb), the presence of inverted terminal repeats, and significant sequence similarity in the region between the repeats.
The Minos-1 transposable element has been identified as a 1775 bp dispersed repetitive sequence inserted within the transcribed spacer in one of the repeats of
Drosophila hydei
(Franz and Savakis,
Nucl. Acids Res.
19: 6646 (Dec. 11, 1991)). The element is characterized by 255-bp long perfect inverted repeats and the presence of two long, non-overlapping open reading frames (ORFs) on the same strand. The longest of the ORFs shows approximately 30% sequence identity with TcA, but does not begin with an ATG codon. It appears, therefore, that the cloned element represents a defective member of the Minos family, as is the case with all previously sequenced Tc1-like elements, with the possible exceptions of Tc1 and TCb1.
SUMMARY OF THE INVENTION
The invention relates to an isolated transposable element, or an isolated DNA sequence which encodes a transposase protein (or a portion of a transposase protein). The isolated transposable element or the isolated DNA sequence being characterized by the ability to hybridize to the DNA sequence of Minos 1 under stringent hybridization conditions. The invention also relates to a purified transposase protein, or peptide fragments thereof, encoded by such DNA sequences.
In another aspect, the invention relates to a method for the stable introduction of a DNA sequence of interest into a eukaryotic cell. This method involves the use of an isolated transposable element of the type described in the preceding paragraph, the isolated transposable element being modified to include the DNA sequence of interest flanked by the termini of the isolated transposable element. This modified transposable element is introduced into the eukaryotic cell in the presence of a transposase protein, or a DNA sequence encoding a transposase protein. The role of the transposase protein is to catalyze the transposition of the modified transposable element containing the DNA sequence of interest into the genomic DNA of the eukaryotic cell.
In a third aspect, the invention relates to a method for isolating members of the Tc-1 family of transposable elements from genomic DNA of a eukaryote of interest. According to this method, oligonucleotide primers are provided which are complementary to a sequence of at least about 12 consecutive nucleotides which encode amino acids which are highly conserved in aligned sequences of nematode Tc-1 family members and Minos family members. These oligonucleotide primers are used to prime amplification by the polymerase chain reaction (PCR). The amplification products are then used to isolate DNA encoding the entire Tc-1 family member from the eukaryote of interest by conventional methods.


REFERENCES:
Minos-2 DNA sequence submitted to EMBL data library by Charalambos Savakis, Accession No. x61695. Released by EMBL data library on Sep. 12, 1991.*
Franz, Gerald and Savakis, Charalambos “Minos, a new transposable element fromDrosophila hydei, is a member of the TC1-like family of transposons” Nucleic Acid Research 19 (23): 6646, 19991.*
Ashburner M. (Drosophia, a Laboratory Handbook, CSH, 1989) pp. 1049, 1051 only.
Crampton J. M. (Symp. R. Entomol. Soc. London, 1992, vol. Date 1991, 16th, 3-20).
Franz et al (1991) Nucleic Acids Research 19: 6646.
Savakis (1991) EMBL Accession No. X61695.
D O'Brochta et al (1996) Insect Biochemistry and Molecular Biology 26: 739-753 (abstract only).
RC Brand et al (1989) Mol Gen Genet 215: 469-477.
ST Seo et al (1990) J Econ Entomol 83: 1949-1953.
M Ashburner (1995) Science 270: 1941-1942.

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