Reagents and methods for diversification of DNA

Details Reagents and methods for diversification of DNA Reagents and methods for diversification of DNA

1997-11-24

2001-05-15

Houtteman, Scott W. (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

Reexamination Certificate

active

06232112

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of molecular biology and a method and reagents for the diversification of DNA sequences. The present invention allows the generation of variant DNA sequences, which can provide variants of sequences that regulate gene expression, code for proteins, control the export of gene products from cells and the localization of gene products within cells. In particular, the present invention provides a method and reagents for efficiently generating new variant sequences in heterologous DNA (DNA foreign to the cell) by placing two sequences, that differ at multiple sites, into the same cell at a location that permits the expression of the new variant sequence after diversification and which provides for the generation of new variants as a result of the exchange of parts of the two sequences within the living cell.
BACKGROUND OF THE INVENTION
Diversification of DNA molecules provides a way to generate new proteins with properties not found in nature. This can be achieved in vitro (by manipulation outside living cells) by a variety of methods such as the insertion of specific novel and random sequence oligonucleotides (Gold, L et al 1997 Proceedings of the National Academy of Science, 94:59-97) in selected regions of genes, or the cleavage of variant DNA molecules (two sequences that have similar functions but differ in one or more sites) and reassembly of the fragments in new combinations (Stemmer, WPC 1994 Proceedings of the National Academy of Science, 91: 10747-10751), or by amplification of DNA by the polymerase chain reaction under conditions where the polymerase is error prone (Leung, D W et al 1989 Technique 1: 11-15). Screening for desired properties of any protein coded by the resultant novel nucleotide sequence requires transcription and translation of the sequence to yield the corresponding peptide and the appropriate post-translational modification. In these cases, this is usually achieved by introducing each new construct into a cell by transfection or electroporation (hereafter both processes will be covered by the term transfection) to form a transformed cell, a complex and time consuming procedure since it is necessary to check each construct to ensure it is correctly inserted and is complete. This difficulty is compounded where the new construct codes for one component of a multimeric protein since the procedure must be done twice for each new combination. That problem can be reduced by the use of fungal heterokaryons to lower the number of transfections required to one per component of a combinatorial array (U.S. Pat. No. 5,643,745, to Stuart, issued Jul. 1, 1997). However, the number of transfections required is still large and each must be checked to ensure the DNA insert is correctly placed and complete.
Genetic recombination
Genetic recombination in eukaryotes, higher organisms that have a true nucleus, occurs during the prophase of the reduction division that converts a diploid cell having two complete sets of homologous chromosomes to a tetrad (or sometimes an octad) of haploid cells each with one complete set of chromosomes. Two manifestations of recombination events are recognized: crossing over in which genes located at different sites (loci) on the same chromosome are recombined by reciprocal exchange of chromosome sections between a pair of homologous chromosomes and gene conversion in which the number of copies of a pair of allelic genes, ie genes that occupy the same locus on homologous chromosomes, is unequal in the tetrad or octad. Instead of a two:two segregation of the parental alleles, the tetrad comprises three haploid cells carrying one of the parental versions of the gene and one carrying the other parental version of the gene. Crossing over was first discovered in the fruit-fly Drosophila (Morgan Proc. Soc. Exp. Biol. Med. 8:17 1910) and gene conversion in the fungus Neurospora (M B Mitchell Proc. Natl. Acad. Sci. USA 41: 216-220 1955). There is now evidence that both crossing over and gene conversion occur universally in species that reproduce sexually and that a process having similar outcomes occurs in bacteria and their viruses and plasmids.
Genetic recombination in eukaryotes occurs in diploid cells (cells that contain two complete sets of homologous chromosomes) that are undergoing meiosis. Prior to the division, each of the two chromosome sets is replicated, generating two pairs of identical sister chromatids. The process of genetic recombination involves the establishment of joints between two homologous but not necessarily identical DNA sequences, one located on one chromatid of one sister pair and the other in a homologous chromatid which is a member of the other sister pair. The joints establish regions in one or both chromatids where one strand of the DNA duplex has the sequence of one homologue and the second strand has the sequence of the other homologue. Where the DNA sequences of the homologues differ, bases will be in mismatched pairs, that is pairs which are not A:T or G:C (A=deoxyadenine, T=deoxythymine, C=deoxycytidine and G=deoxyguanine). Enzymatic machinery corrects mismatched base pairs and the joints between the molecules are resolved, separating the two chromatids once more. For each site of mismatch, in half of the cases, the base pair present in one chromatid is now replaced by the base pair originally present in the other homologous chromatid. This accounts for gene conversion. In some cases the joints between molecules are resolved such that there is a reciprocal exchange of the regions each side of the joint. This process is called crossing over and also leads to novel combinations of DNA sequence information by which the parental homologues differed. Each chromatid is incorporated into one of the haploid cells (cells having only one set of chromosomes) that arise from meiosis, becoming a member of the complete set of chromosomes present in each cell.
The molecular processes of crossing over and gene conversion are yet to be fully understood. In the most widely accepted model for the molecular events of recombination (
FIG. 3
) (H Sun et al Cell 64: 1155-1161, 1991) it is supposed that one of the two homologous chromatids suffers a break in both strands of the DNA molecule and that the strands that end with a 5′ phosphate are resected, leaving a single strand tail of several hundred bases that ends with a 3′ hydroxyl group. It is proposed that the single strand tail pairs with the complementary strand of the unbroken chromosome to initiate the joint. The joint is thought to be completed by DNA synthesis from the 3′ ends to provide a replacement strand for the DNA lost in the initial resection followed by rejoining of the breaks. This will form a double junction between the molecules in the manner shown in FIG.
3
. Each junction is free to move. This leads to strand exchange between the two DNA molecules forming heteroduplex DNA. It is supposed that recombination is completed by scission of the junctions and correction of mispaired bases. Scission of the junctions can occur by breaks in either the “inner” or “outer” strands with equal probability (FIG.
3
). Due to the limitations of a two dimensional representation of the junctions, the expectation of an equal frequency of these two modes of scission is not self evident. However in reality, the two pairs of complementary strands, both the inner and outer pair, are identically juxtaposed. If the resolution of both junctions occurs in the inner strands or alternatively in the outer strands, only gene conversion can occur. If the resolution of one junction is by scission of the inner strands and the other junction by scission of the outer strands, the flanking regions are reciprocally exchanged and there is both a crossover event and also the possibility of gene conversion.
There is direct evidence that recombination is initiated by two strand breaks in the yeast
Saccharomyces cereviseae
(A Schwacha and N Kleckner, Cell 83: 1-20 1995). However, the exact seri

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