Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-09-27
2002-04-09
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S471000, C435S183000, C435S023000, C435S070100, C435S071100, C435S320100
Reexamination Certificate
active
06368830
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of this invention is the area of molecular genetics, in particular, in the area of mobile genetic elements, e.g., transposons, the transposase enzymes responsible for mobility and methods for isolating mutant transposase enzymes which mediate higher frequencies of transposition than do the naturally occurring enzymes, and uses thereof.
Transposable genetic elements are DNA sequences, found in a wide variety of prokaryotic and eukaryotic organisms, that can move or transpose from one position to another position in a genome. In vivo, intra-chromosomal transpositions as well as transpositions between chromosomal and non-chromosomal genetic material are well known. In several systems, transposition is known to be under the control of a transposase enzyme that is typically encoded by the transposable element. The genetic structures and transposition mechanisms of various transposable elements are summarized, for example, in “Transposable Genetic Elements” in “The Encyclopedia of Molecular Biology,” Kendrew and Lawrence, Eds., Blackwell Science, Ltd., Oxford (1994).
Mariner-family transposable elements are a diverse and taxonomically widespread group of transposons occurring throughout the animal kingdom [Robertson, (1993) Nature 362:241-245; Robertson and MacLeod, (1993)
Insect Mol. Biol.
2:125-139; Robertson and Asplund, (1996)
Insect Biochem. Mol. Biol.
26:945-954; Robertson, et al. (1998)
Horizontal Gene Transfer,
eds. Syvanen and Kado (Chapman & Hall, London)]. They encode transposases that belong to an extended superfamily of transposases and retroviral integrases distinguished by a conserved D,D35E (or variants thereof mariners=D,D34D) motif in the catalytic domain of the protein [Doak, et al. (1994)
Proc. Natl. Acad. Sci. USA
91:842-946]. Transposition of these elements follows a conservative cut-and-paste mechanism [Craig, (1995)
Science
270:253-254].
Most mariners are known only from their sequences obtained either through homology-based PCR screens or by the examination of sequenced genes or ESTs [Roberts (1993) supra; Robertson and Lampe, (1995)
Mol. Biol. Eval.
12:850-862]. Hundreds of different mariners have been detected in this way. Of these, only two are known to be active. The first is the canonical mariner element from
Drosophila mauritiana
discovered by its activity in that fly [Jacobson, et al. (1986)
Proc. Natl. Acad. Sci. USA.
83:8684-8688]. The most active copy of this particular element is known as MosI [Medhorn, et al. (1988)
EMBO J.
7:2185-2189]. The second is the Himar1 element discovered by using homology-based PCR in the horn fly,
Hacmatobia irritans,
and reconstructed as a consensus sequence [Robertson, et al. (1986) supra; Lampe, et al. (1996)
EMBO J.
15:5470-5479]. Both MosI and Himar1 require no host-specific factors for transposition and so have been advanced as generalized genetic tools [Loha and Hartl, (1996)
Genetics
143:3265-374; Gueiros-Filho and Beverley, (1997)
Science
276:1716-1719; Lampe, et al. (1998)
Genetics
149:179-187]. Indeed, MosI has been used as a transformation vector for chicken [Sherman, et al. (1998)
Nat. Biotechnol.
16:1050-1053], zebrafish [Fadool, et al. (1998)
Proc. Natl. Acad. Sci. USA
95:5182-5186], the yellow fever mosquito,
Aedes Aegypri
[Coates, et al. (1998)
Proc. Nation. Acad. Sci. USA
95:3748-3751],
Drosophila melanogaster [
Lidholm, et al. (1993)
Genetics
134:859-868],
Drosophila virilis
[Loha and Hartl (1996) supra], and
Leishmanla major [
Guiros-Filho, (1997) supra], with varying degrees of success. Himar1 has been used as a prokaryotic genetic tool, via in vivo transposition and subsequent homologous recombination in
Haemophilus influenzae
and
Streptococcus pneumoniae,
and in vivo in
Escherichia coli
and Mycobacterial spp. [Akerley, et al. (1998)
Proc. Natl. Acad. Sci. USA
95:8927-8932; Rubin, et al. (1999)
Proc. Natl. Acad. Sci. USA
96:1645-1650]. It is also active in human cells [Zhang, et al. (1998)
Nucleic Acids Res.
26:3687-3693].
Whereas mariner elements are becoming increasingly important tools for eukaryotic genetics, neither MosI nor Himar1 appear to be as active as would be desired to make them efficient tools, particularly for whole metazoa [Lampe, et al. (1998) supra; Fadool (1998) supra]. In fact, these transposases may have evolved to be less active in their hosts and, therefore, be less deleterious [Lampe, et al. (1998) supra; Lohe and Hartl, (1996)
Mol. Biol. Eval.
13:549-555; Hartl, (1997)
Genetics
100:177,184]. Such low transposition activity makes the use of these elements for genetic manipulations less practical. Identifying mutant transposases with higher activity might help to solve this problem but is difficult to carry out in metazoan systems [Lohe, et al. (1997)
Proc. Natl. Acad. Sci. USA
94:1293-1297].
In order to identify mutant transposases with higher activity and a broad host range, the ability of Himar1 to transpose in prokaryotes was exploited to create a genetic system for isolation of transposase mutants with altered activity in vitro and in vivo. The present invention discloses three highly active mutants that significantly improve the efficiency of transposition of Himar1-derived elements as genetic tools. Analysis of these mutants shows the locations of functional domains and amino acids within the Himar1 transposase. The hyperactive mutants of Himar1 transposase described herein are useful in generating random mutations in vivo and in vitro or in introducing a heterologous DNA into a wide range of host cells.
SUMMARY OF THE INVENTION
The present invention provides mutant Himar1 mariner transposase proteins and coding sequences therefor. These mutant transposases are such that the frequency of transposition is significantly higher than the comparison transposase which occurs in nature. By significantly higher, it is meant at least about 2-fold higher, and desirably greater than about 5-fold, and including the ranges of about 3 to about 1000-fold, and all ranges therebetween. The mutant transposases of the present invention further exhibit the useful property of being active in a wide range of prokaryotic and eukaryotic cells, including but not limited to bacteria, insects, nematodes, flatworms, and vetebrates (e.g. humans). Thus, these mutant transposases can be used to improve the efficiency of a variety of genetic manipulations which require the step of transposition of a genetic element.
The mutant Himar1 tranposases of the invention were identified as having higher transposition efficiency by a combination of the trans-papillation screen and the mating assay. The mutant transposases of the invention represent the first example of the eukaryotic transposases isolated using these assays. Using the combination of these two assays, additional transposase mutants with a varying transposition frequency can be isolated from the Himar1 transposon or any related transposable elements.
The hyperactive mutant transposases of the present invention are useful in a variety of genetic manipulations which require a transposition event in vitro and in vivo. These include but are not limited to sequencing of unknown DNA, generating random mutations in vitro or in vivo such as gene knock-outs, introducing a gene of interest, or identification of essential genes in an organism.
The hyperactive Himar1 transposase mutant proteins can be expressed and purified for use in in vitro assays using the methods known in the art. The nucleic acid coding sequences for the mutant transposases provided herein can be cloned into a transposon such as Himar1 to be used for in vivo transposition.
REFERENCES:
patent: WO 99/50402 (1999-10-01), None
Akerley et al. (1998) “Systematic identification of essential genes by in vitro mariner mutagenesis”Proc. Natl. Acad. Sci. USA95:8927-8932.
Braam et al. (1999) “A Mechanis
Akerley Brian J.
Lampe David J.
Robertson Hugh M.
Rubin Eric J.
Greenlee Winner and Sullivan P.C.
Katcheves Konstantina
President and Fellows of Harvard College
Yucel Remy
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