Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
2000-08-02
2002-06-18
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S004000, C435S019000, C435S440000
Reexamination Certificate
active
06406896
ABSTRACT:
BACKGROUND OF THE INVENTION
Bacterial transppsons such as Tn5 evolved within the cell by maintaining a low mobility level. While necessary for the transposon to survive, the low mobility level has inhibited the ability of researchers to detail the molecular transposition process and to exploit the transposition process for use, e.g., in the development of new diagnostic and therapeutic resources. Tn5 is a conservative “cut and paste” transposon of the IS4 family (Rezsohazy, R., Hallet, B., Delcour, J., and Mahillon, J, “The IS4 family of insertion sequences: evidence for a conserved transposase motif,”
Mol Microbiol
. 9:1283-1295 (1993)) that encodes a 53 kD transposase protein (Tnp) that is responsible for its movement. The wild-type Tn5 transposase amino acid and nucleic acid sequences are known. Ahmed, A. and Podemski, L. The Revised Sequence of Tn5. Gene 154(1), 129-130(1995), incorporated by reference as if set forth herein in its entirety. A nucleic acid sequence that encodes wild-type Tn5 transposase is attached as SEQ ID NO:1. A polypeptide sequence encoded by SEQ ID NO:1 which corresponds to wild-type Tn5 transposase is attached as SEQ ID NO:2.
The Tnp protein facilitates movement of the entire element by binding initially to each of two 19 bp specific binding sequences termed outside end (OE; SEQ ID NO:3), followed by formation of a nucleoprotein structure termed a synapse, blunt ended cleavage of each end, association with a target DNA, and then strand transfer (Rezikoff, W. S., Bhasin, A., Davies, D. R., Goryshin, I. Y., Mahnke, L. A., Naumann, T., Rayment, I., Steiniger-White, M., and Twining, S. S., “Tn5: A molecular window on transposition,”
Biochem. Biophys. Res. Commun
. 266:729-34 (1999)). Tn5 transposase can also promote movement of a single ingertion sequence by using a combination of OE and inside end (IE; SEQ ID NO:4) sequences. The IE is also 19 bp long and is identical to OE at 12 of 19 positions (FIG.
1
). In vivo, Tn5 transposase exhibits a marked preference for OE in
E. coli
. Transposase recognition and binding to IE is inhibited in
E. coli
by the presence of two dam miethylation sites (CATC palindromes) which add four methyl groups per inside end sequence (IE
ME
; also depicted as SEQ ID NO:4, methylation not shown) (Yin, J. C. P., Krebs, M. P., and Reznikoff, W. S., “Effect of dam Methylation on Tn5 Transpositlion,”
J. Mol Biol
., 199:35-45 (1988), incorporated by reference as if set forth herein in its entirety). This methylation reduces transposition by reducing protein-DNA primary recognition (Jilk, R. A., York, D., and Reznikoff, W. S., “The organization of the outside end of transposon Tn5
, ” J. Bacteriol
. 178:1671-1679 (1996)).
A principal roadblock to understanding how Tn5 transposes is the fact that purified wild-type Tnp has no detectable activity in vitro. Recently, a double mutant hyperactive form of tansposase (“Tnp EK/LP”) that promotes all steps of the transposition reaction in vitro was developed. The Tnp EK/LP protein differs from wild-type Tn5 Tnp at position 54 (Glu to Lys mutation) and at position 372 (Leu to Pro mutation), in addition to a non-essential but advantageous change at position 56 that prevents production of a so-called inhibitor protein. The modified hyperactive Tnp protein retains the dramatic preference for OE (or OE-like) termini of wild-type Tn5 transposase. Tnp EK/LP has clarified many aspects of Tn5 transposition that were not previously adequately addressable in vivo.
In vitro polynucleotide transposition is a powerfull tool for introducing random or targeted mutations into a genome, Useful in vitro transposition systems based upon the Tn5 tansposon are disclosed in U.S. Pat. No. 5,925,545 and International Publication No. WO 00/17343, both of which are incorporated herein by reference in their entirety as if set forth herein.
A Tnp protein having an ability to discriminate between IE and OE and having a preference for binding IE is desired to permit directed nucleic acid transposition and to facilitate more complex transposition and genetic engineering strategies of the type disclosed in the above-mentioned patent and application than are available using a Tnp having a single specificity for OE. A Tnp having an enhanced preference for IE
ME
is also desired because methylation of DNA in common dam+bacterial hosts inhibits binding of existing Tn5 transposases and reduces the ability of existing transposases to facilitate movement of IE-defined transposons.
BRIEF SUMMARY OF THE INVENTION
The present invention is summarized in that a transposase protein modified relative to wild-type Tn5 Tnp as disclosed herein preferentially promotes transposition of a target sequence flanked with wild-type Tn5 transposon inside ends (IE) rather than outside ends (OE) without regard to whether the IE sequences are methylated.
In a related aspect, the present invention is also summarized in that a transposase modified relative to wild-type Tn5 Tnp as disclosed herein has a preference for IE over OE and is hyperactive with regard to transposition frequency.
In another related aspect, the present invention is also summarized in that a transposase modified relative to wild-type Tn5 Tnp as disclosed herein has a preference for IE over OE and catalyzes transposition at a high level even when the IE sequences are methylated. In contrast, wild-type Tn5 transposase does not efficiently recognize methylated IE sequences.
In yet another related aspect, the present invention is summarized in that a transposase according to the invention includes a mutation relative to wild type Tn5 transposase that either (1) alters binding of the transposase to the DNA termini or (2) enhances transposition or (3) both. The mutation can be end-sequence-specific (as in the exemplified embodiments that alter DNA binding) or non-specific (as in the exemplified embodiment that enhances transposition:
In still another related aspect, the present invention is summarized in that a transposase according to the invention has (1) a greater preference for IE than OE and (2) differs from wild-type Tnp in at least one an amino acid selected from the group consisting of amino acid 58, amino acid 344 and amino acid 372.
In still another related aspect, the present invention is summarized in that a transposase according to the invention differs from wild-type Tnp in that it contains at least one of a mutation from glutamic acid to valine at amino acid 58, a mutation from gultamine to lysine at amino acid 344, and a mutation from leucine to gultamine at amino acid 372.
In another related aspect the invention is summarized in that the transposase of the invention can also exhibit a greater preference for IE by reducing the preference for OE. A mutation at amino acid position 8 relative to wild-type transposase can reduce the preference of a transposase for OE, and thereby increase the apparent preference for IE. A mutation from arginine to cysteine can accomplish this modification.
A transposae protein of the invention can promote more transposition of an IE-flanked target sequence in vivo or in vitro than wild-type Tn5 transposase does. A suitable method for determining transposase enzyme activity in vitro is disclosed herein and in U.S. Pat. No. 5,925,545, incorporated herein by reference in its entirety. A suitable method for determining transposase activity in vivo is disclosed herein.
The modified Tn5 Tnp of the present invention differs from wild-type Tn5 Tnp by virtue of at least one change to an amino acid position, where the change is selected from the group consisting of (1) a change at amino acid position 58 that reduces or eliminates a negative interaction between the Tnp and a methylated DNA residue and (2) a change at amino acid position 344 that alters DNA binding. In addition to the changes noted herein, the modified Tnp's of the invention can also include a change at position 56 (such as a Met to Ala change) that prevents production of the so-called inhibitor protein that interferes with transposition. Moreover, the mutant Tn5 transposa
Naumann Todd A.
Reznikoff William S.
Achutamurthy Ponnathapu
Quarles & Brady LLP
Walicka Malgorzata A.
Wisconsin Alumni Research Foundation
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