Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
1998-04-27
2001-05-01
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C435S455000, C435S456000, C435S462000, C435S465000
Reexamination Certificate
active
06225121
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. Members belonging to the Tc-1-like family have been characterized in nematodes, diptera, fish and amphibians: 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.
Transposable elements are natural components of genomes ranging from bacteria to vertebrate organisms (Lewin,
Genes VI,
Chapter 18, Oxford University Press, (1997)). Thus, due to their widespread phylogenetic distribution, evolutionary conservation and genomic mobility, transposons are valuable tools for genetic manipulations, such as, for example, the integration of nucleic acids in germ cells for the production of transgenic animals, and genetic transformation and insertional mutagenesis in somatic cells and viral vectors for use as therapeutics.
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 is 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 nucleic acid sequence of interest into a 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 nucleic acid sequence of interest flanked by the termini of the isolated transposable element. This modified transposable element is introduced into the cell in the presence of a transposase protein, or a nucleic acid sequence or a virus encoding a transposase protein. The role of the transposase protein is to catalyze the transposition of the modified transposable element containing the nucleic acid sequence of interest into the genome of the cell. Also envisioned are cells produced by this method.
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.
In a fourth aspect, the invention relates to a transgenic animal. The transgenic animal is produced by a method which involves the use of an isolated transposable element characterized by the ability to hybridize to the DNA sequence of Minos 1, the isolated transposable element being modified to include the nucleic acid sequence of interest flanked by the termini of the isolated transposable element. This modified transposable element is introduced into a cell in the presence of a transposase protein, or a DNA sequence or a virus encoding a transposase protein.
In a fifth aspect, the invention relates to methods of integrating a nucleic acid sequence of interest into a chromosome of a 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 nucleic acid sequence of interest flanked by the termini of the isolated transposable element. This modified transposable element is introduced into the cell in the presence of a transposase protein, or a nucleic acid sequence or a virus encoding a transposase protein. Also envisioned are cells produced by this method.
In a sixth aspect, the invention relates to a transgenic plant. The transgenic plant is produced by a method which involves the use of an isolated transposable element characterized by the ability to hybridize to the DNA sequence of Minos-1, the isolated transposable element being modified to include the nucleic acid sequence of interest flanked by the termini of the isolated transposable element. This modified transposable element is introduced into a plant cell in the presence of a transposase protein, or a nucleic acid sequence or a virus encoding a transposase protein.
In a seventh aspect, the invention relates to insertional mutagenesis and gene tagging. In this approach, the Minos-transposable elements are inserted into a nucleic acid (e.g., a gene) to induce a mutation in the nucleic acid which produces a phenotypic alteration. The location of the nucleic acid is identified by the presence of the Minos transposon sequence. The Minos transposon sequence can be identified, for example, using standard molecular hybridization techniques, such as in situ hybridization, Southern blotting, and colony hybridization. The terms “transposable element” and “transposon” are used interchangeably herein.
In a particular embodiment, this aspect of the invention relates to methods for inducing a mutation of interest in a cell (a Minos transposon-induced mutation), and identifying and isolating a gene of interest which includes the mutation from the cell. The methods involve a the use of an isolated transposable element of the type described above which is introduced into a cell in the presence of a transposase protein, or a nucleic acid sequence or a virus encoding the transposase protein. In a particular embodiment, the transposable element is modified to include a promoter operably linked to an indicator gene (such as a reporter gene or a selectable marker gene) flanked by the inverted terminal repeats of the isolated transposable element. In a further embodiment, expression of the indicator gene is detected, thereby identifying cells in which the transposable element has integrated into the genome of the cells. Cells which have a mutation of interest can then identified and selected by looking for a particular phenotype conferred by the mutation but not the corresponding endogenous gene. These cells are referred to herein as cells including a Minos transposon-induced (or Minos transposable element-induced) mutation. The location of the gene which includes the mutation can then be identified by the presence of the Minos transposon sequence and then isolated.
In a second embodiment, this aspect of the invention relates to methods for selecting an insertional mutation in a gene (a Minos transposon-induced mutation). The methods comprise introducing a transposable element of the type described above, modified to include a minimal promoter or a splice acceptor site operably linked to an indicator gene flanked by the inverted terminal
Franz Gerald H.
Klinakis Apostolos G.
Loukeris Athanasios
Savakis Charalambos
Hamilton Brook Smith & Reynolds P.C.
Institute of Molecular Biology and Biotechnology/FORTH
Nashed Nashaat T.
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