Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-01-15
2001-05-08
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C536S023100
Reexamination Certificate
active
06228647
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of this invention is molecular biology, particularly in the area of retrotransposons, nucleotide sequence encoding integrase therefrom, and molecular genetic methods based thereon. In particular, the present invention relates to the insertion of heterologous DNA into eukaryotic genomes at specific locations and to protein domains targeting insertion at particular genomic sites.
Retroelements, which include the retroviruses and retrotransposons, insert cDNA copies of themselves into the host genome as part of their replication cycle. The selection of integration sites is not random. While a target bias for the retroviruses is not apparent from the genomic distribution of insertions, they often show local target preferences and tend to integrate into transcriptionally active or DNase I hypersensitive regions [Sandmeyer et al. (1990)]. This suggests that integration sites are not determined by a preference for specific DNA sequences. Rather, target choice is likely mediated by higher order structural features of the target sites (e.g. chromatin) [Curcio and Morse (1997)].
Target biases are clearer for the retrotransposons, particularly those of
Saccharomyces cerevisiae.
The Ty3 elements preferentially integrate upstream of genes transcribed by RNA polymerase III (pol III), usually within 1-2 bases of transcription start sites [Chalker and Sandmeyer (1993); Chalker and Sandmeyer (1992)]. The use of in vitro Ty3 transposition assays has shown that loading of transcription factors TFIIIB and TFIIIC onto tRNA gene promoters is sufficient for targeting [Kirchner et al. (1995)]. Ty1 elements typically integrate within a one kb window upstream of genes transcribed by pol III, and pol III transcription is required for Ty1 target choice [Devine and Boeke (1996)]. The data for both Ty1 and Ty3, therefore, indicate that targeting occurs as a consequence of interactions between a component of the retrotransposon integration complex and a host factor localized to sites of pol III transcription. HIV integrase has been found to interact by two-hybrid assays with a transcription factor homolog called ini1 [Kalpana et al. (1994)], implying that selection of target sites through interactions with chromosomal proteins may be a general feature of retroelements. Ini1, however, is not known to target HIV integration.
The yeast Ty5 retrotransposons integrate almost exclusively into telomeric regions and near the silent mating loci HMR and HML [Zou et al. (1996a); Zou et al. (1995)]. These regions are bound in unique chromatin, called silent chromatin, which represses the expression of adjacent genes and plays a role in telomere maintenance [Laurenson and Rine (1992)]. A large number of factors make up silent chromatin, including proteins involved in DNA replication (ORC), transcription factors (RAP1, ABF1) and silent information regulatory proteins (SIR2-SIR4). Mutations in cis-acting sequences that disrupt the assembly of silent chromatin at HMR also abolish Ty5 integration to this locus [Zou and Voytas (1997)]. This indicates that silent chromatin directs Ty5 target choice.
Despite the evidence that retroelements select target sites through interactions with chromosome-localized proteins, neither retroelement nor specific host factors required for targeted integration have been identified heretofore. In contrast to Ty1 and Ty3, for which there are multiple genomic targets (e.g. 274 tRNA genes; http://genome-www.stanford.edu/Saccharomyces), the number of known Ty5 targets are limited to the 32 telomeres and the two silent mating loci. To identify Ty5-encoded proteins required for targeting, we took advantage of this limited number of targets to devise an assay that monitors the frequency of integration to a single plasmid-borne locus. This assay was used to screen for Ty5 mutations that disrupt targeting, and as a consequence, a region near the C-terminus of integrase was identified as the determinant of target choice.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a retrotransposon protein, termed an integrase herein, which determines the site specificity (or lack thereof) for insertion of the retrotransposon into the genomic DNA of a eukaryotic cell. Specifically, the present invention provides the protein from Ty5 which determines the specificity of that retrotransposon for silent DNA including, but not limited to, telomeres and the yeast silent mating type loci HMR and HML. The coding sequence of this insertion-specificity determining protein, i.e., the integrase is given in Table 3 (SEQ ID NO:1, nucleotides 2852-4827), and the deduced amino acid sequence is given in Table 3 (SEQ ID NO:2). The specificity determining domain of the integrase protein is located from about amino acid residue 1070 to about 1110, or as more narrowly defined, from about amino acid residues 1074 to about 1105, or as further more narrowly defined, from amino acids 1092 to 1097, as given in Table 3 (SEQ ID NO:2).
The present invention further provides a specificity-determining domain of the Ty5 integrase; this domain is from about amino acid 1070 to about 1110 as given in SEQ ID NO:2 (Table 3). Modifications to the amino acid sequence of this domain results in altered insertion site specificity. As specifically exemplified, a replacement of the serine residue at position 1094 of SEQ ID NO:2 results in a loss of insertion site specificity; i.e., insertion into genomic DNA by this mutated Ty5 derivative is substantially random. Identification of this insertion specificity-determining domain (or peptide portion) allows its substitution for the corresponding domain of the integration protein of other retrotransposons or of retroviruses or for other peptides that interact with components of chromatin, including DNA or DNA-associated proteins such that retrotransposon or retrovirus derivatives can be constructed by one of ordinary skill in the art of molecular biology to generate retrotransposon or retroviruses with variations from the normal insertion site specificity (or lack thereof).
The present invention provides a method for target insertion of heterologous (i.e., retroviral or retrotransposon) DNA into silent chromatin. This is accomplished by replacing the wild-type coding region of a portion or by inserting into a wild-type integrase coding sequence a nucleic acid fragment encoding a peptide portion that interacts with chromatin (or a protein bound to chromatin) at a specific location. One example of such a peptide portion is that naturally encoded by Ty5 (determining SEQ ID NO:3). The skilled artisan understands how to select from among synonymous codons according to the prevalent codon usage for the organism in which use of the modified retrotransposon or retrovirus is intended. For example, the Ty4 integrase could be targeted to silent chromatin by replacing the sequence LERDKKRSKKNR (amino acids 2940 of SEQ ID NO:6) with LDSSPP, by mutating the coding sequence for that portion of the protein with the portion of Ty5 encoding SEQ ID NO:3. Similarly, the sequence encoding LEPPRSKKR (amino acids 32 to 40 in SEQ ID NO:5) can be replaced with the nucleotide sequence encoding SEQ ID NO:3 to give a silent chromatin-specific derivative of Ty2 or Ty1.
The present invention further provides a peptide determining the interaction of one protein with another (target) protein (which in turn interacts with a specific region of DNA) can be substituted for SEQ ID NO:3 within the Ty5 integrase to re-direct the integration of the modified Ty5p element to the region of the genome to which the target protein is bound. The substitution is achieved by oligonucleotide site-directed mutagenesis, for example, of the integrase coding sequence to replace the Ty5 targeting domain coding sequence (for amino acids 1092 to 1097 of SEQ ID NO:2, i.e., nucleotides 4714 to 4731 of SEQ ID NO:1.
In addition, any retrovirus or retrotransposon can be modified to alter integration site specificity.
Gai Xiaowu
Voytas Daniel F.
Davis Katharine F
Greenlee Winner and Sullivan P.C.
Iowa State University & Research Foundation, Inc.
Schwartzman Robert A.
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