Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-06-10
2001-12-04
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S463000, C435S468000, C435S477000
Reexamination Certificate
active
06326206
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for in vivo recombination of homologous DNA sequences. The method is a forced artificial evolution resulting in a DNA sequence encoding a polypeptide having an advantageous property.
BACKGROUND OF THE INVENTION
Homologous recombination between DNA sequences placed on the same plasmid is well known to the skilled artisan (Weber and Weissmann, Nucl. Acid. Res. (1983) 11, 5661-5669). EP 252666 B1 (Novo Nordisk A/S), WO95/22625 A1 (Affymax Technologies N.V.) and WO9101087 discloses in vivo recombination of genes placed on the same plasmid.
EP 449923 B1 (Setratech) discloses a process of intergenic recombination in vivo of partially homologous DNA sequences. The recombination takes place in cells of which the enzymatic mismatch repair system is defective.
J. Biotechnol. (1991) 19, 221-240 discloses a process for inactivating a gene in the genome of
B. Amyloliquefaciens.
The process involves integration and excision of a pE194 mutant.
In Chemical Abstracts 118:161925 (1993) the minimal sequence length of homology necessary for recombination is determined. The method of determination involves integration of a pE194 derivative carrying a gluconase gene.
In J. Bacteriol. (1982) 152, 524-526 a plasmid pBD9, which comprises the two plasmids from
Staphylococcus aureus,
pE194 and pUB110, is disclosed. Other plasmids based on pE194 or pUB110 are disclosed in Molecular and General Genetics (1988), 213, 465-70, Molecular and General Genetics (1984), 195, 374-7, Plasmid (1981), 6, 67-77 and Genetika, (1986) 22, 2750-7.
In Yichuan Xuebao (1993), 20, 272-8 (see Chemical Abstracts 120:1670 (1994)), Chen et. Al. disclose the plasmid pNW102, a pE194 derivative, in which the thermostable &agr;-amylase gene from
Bacillus licheniformis
is inserted. pNW102 was transformed into the
Bacillus subtilis
strain BF 7658, followed by incubation at non-permissive temperature, allowing homologous recombination between the &agr;-amylase genes of pNW102 and BF 7658. The &agr;-amylase produced of the recombinant strains shows the same characteristics as the &agr;-amylase from
Bacillus licheniformis.
However, in the related art there is no indication of that DNA sequences can be recombined in vivo by repeating integration and cross-out in order to obtain a gene encoding a polypeptide with improved characteristics, such as increased stability, improved specificity or higher activity.
Accordingly, a problem of the invention is to provide an improved in vivo process of generating new DNA sequences.
BRIEF DESCRIPTION OF THE INVENTION
The present invention is directed to a in vivo process of generating new DNA sequences. It has turned out that new DNA sequences with improved properties can be generated in a fast and efficient way from at least two homologous DNA sequences by a process involving in vivo exchange of DNA between at least two vectors, each containing a homologous DNA sequence and an origin of replication.
The process of the invention is characterized by, that said exchange of said homologous sequences is done by repeating in vivo at least once, said integration and excision of one vector into the other.
Accordingly, in a first aspect the invention relates to process for in vivo recombination of homologous DNA sequences, comprising the following steps
(a) incubating a cell containing at least two DNA structures, each comprising a DNA sequence and an origin of replication, under conditions which favour integration of one of the DNA-structures into one of the others, thereby forming a hybrid DNA structure;
(b) incubating the cell under conditions which favour crossing out from said hybrid DNA structure, thereby forming novel DNA structures, each comprising a recombined DNA sequence and an origin of replication;
(c) repeating steps a)-b) at least once.
One of the advantages of the in vivo recombination process of the invention is by repeating the exchange in vivo between the DNA sequences it is possible to obtain numerous different recombination patterns between said DNA sequences.
This is illustrated in
FIG. 1
herein, which shows the result of in vivo recombination between the two DNA sequences Savinase and Savisyn performed as described in example 1.
In
FIG. 1
Savinase/Savisyn recombined clones such as clone no. 3, 8 12 are derived from more than one in vivo recombination event between the two DNA sequences.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1
illustrates the recombination of two DNA sequences, placed on the plasmids pTR, temperature resistant since origin of replication is functional at 50° C., and pTS, temperature sensitive since origin of replication not functional over 45° C. Under non-permissive conditions (i.e. at 50° C.), pTS integrates into pTR, forming a hybrid plasmid.
FIG. 2
illustrates the cross out from the hybrid plasmid, forming two new DNA sequences, each placed on a plasmid. Repeated crossing in and out may be done by e.g. temperature cycling or simply by keeping temperature pressure and selecting for resistance of pTS (i.e. Erm
R
).
FIG. 3
illustrates the plasmids which are obtained after repeated integration and crossing out events.
Further,
FIGS. 1 and 2
illustrate a DNA structure system suitable for in vivo measurement of hybrid structure formation, and suitable for performing a forced cross out of the DNA hybrid structure.
FIG.
1
:
DNA structure pTS:
Suitable active promoter directing expression of active protein
Erm
R
: antibiotic resistance gene
DNA structure pTR comprises:
Not any active promoter to direct transcription of GFP
GFP: Green Fluorescent Protein
The Erm
R
antibiotic resistance gene in pTS is active due to the active promoter.
The GFP gene in pTR is inactive (i.e. the gene is non-transcribed) due to there is not an active promoter to drive expression of the gene.
FIG.
2
: After formation of the hybrid structure according to the invention GFP is now active (driven by the promoter). This makes it possible to in vivo measure the hybrid structure formation (see below for further details).
In contrary the Erm
R
antibiotic resistance gene is now inactive (i.e. the gene is non-transcribed) due to there is not an active promoter to drive expression of the gene. Forced cross out of the DNA hybrid structure is performed by incubating the cells under Erm
R
antibiotic pressure, thereby forcing a cross out of the DNA hybrid structure, since the cell is only able to express the Erm
R
antibiotic resistance gene after the crossing out of the DNA structure has taken place.
FIGS.
4
and
5
: Illustrate the two vectors pSX120 (
FIG. 4
) and pMB430 (
FIG. 5
) used as described in example 1 to in vivo recombine the two DNA sequences Savinase and Savisyn.
pSX120 comprises i) an open reading frame encoding Savinase, ii) an active promoter directing transcription of Savinase, and iii) a gene conferring resistance to chloramphenicol (Cam
R
).
pMB430 comprises i) a Savizyn DNA fragment, ii) a terminator (resulting in no transcription through the regions following this), iii) a gene conferring resistance to erythromycin (Erm
R
), and iv) an open reading frame encoding Green Fluorescent Protein (GFP).
FIG.
6
: Illustrates the results of Savinase/Savisyn in vivo recombination performed as described in example 1.
The schematic representation shows 12 of the shuffled/recombined clones.
Horizontally are given the positions of the restriction sites unique for Savizyn.
The letter “S” denotes that the sequence at this position is identical to the Savinase sequence and the letter “Z” denotes that the sequence stems from Savizyn. All of the sequenced clones are different in their pattern of recombination.
Definitions
As used herein, the following terms have the following meanings:
The term “DNA sequence” includes any DNA sequence which is desirable to modify according to the invention e.g. a DNA sequence encoding, a polypeptide, for example an enzyme, pharmaceutically active polypeptides, e.g. insulin growth hormone human hormones or growth regulators, and sequences such as promoters, transcription or translation re
Bjornvad Mads Eskelund
Borchert Torben Vedel
Ehrlich Stanislas Dusko
Jorgensen Per Lina
Rasmussen Michael Dolberg
Garbell Jason I.
Lambiris Elias J.
McKelvey Terry
Novozymes A/S
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