Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-01-04
2001-01-30
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...
C435S252300, C435S252330, C435S320100, C435S480000, C435S488000
Reexamination Certificate
active
06180407
ABSTRACT:
SUBJECT-MATTER OF THE INVENTION
The invention relates to a cloning and/or sequencing vector which enables recombinant clones to be selected directly.
The invention also relates to the procaryote cell which is transformed by this vector and to the procaryote host cell for this vector, as well as to the use of this cloning and sequencing vector for selecting and sequencing recombinant clones.
STATE OF THE ART AND TECHNOLOGICAL BACKGROUND UNDERLYING THE INVENTION
Phage (the M13 series) and plasmid (the pUC series) cloning vectors, containing numerous unique cloning sites, were constructed by Messing et al (P.N.A.S. USA, 79, pp. 3642-3646 (1977), by Norrander et al (Gene, 26, pp. 101-106 (1983) and Yanisch-Perron et al (Gene, 33 pp. 103 to 119) (1985))
The multiple cloning sites (MCS—multiple cloning sites) of these vectors are located in the coding sequence of the LacZ gene.
Discrimination between the transformed cells which harbour a recombinant vector and the cells which harbour a non-recombinant vector is achieved using the “blue screen” technique described by Gronenborn and Messing (Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites, Nature, 272, pp. 375-377 (1978)).
However, this “blue screen” technique suffers from the disadvantage of using a screening procedure (discrimination) rather than a procedure for selecting the clones.
Discrimination by screening is based on identifying a clone within a population of clones on the basis of a characteristic (color) which differentiates it. Selection has no need of this characteristic, since it is only recombinant clones which are isolated by this method.
The screening procedure is based on the color of the recombinant clones (white color) and of the non-recombinant clones (blue color). This color is based on inactivation of the marker beta-galactosidase, preventing cleavage of X-gal (5-bromo-4-chloro-3-indolyl &bgr;-galactoside). The cell colonies harbouring a non-recombinant vector produce a functional beta-galactosidase and, by hydrolysing the X-gal substrate, produce a blue coloration. In general, the insertion of a DNA fragment into the &bgr;-galactosidase gene prevents cleavage of the X-gal. For this reason, the cells harbouring a recombinant vector have a white color.
Moreover, this complex procedure requires the use of the substrate X-gal which is a product which is very expensive, unstable and awkward to use.
On the other hand, various cloning vectors permitting direct selection (positive selection) of recombinant strains have been described in the scientific literature.
Pierce et al (Proc. Natl. Acad. Sci., vol 89. No. 6, 1992, pp. 2056-2060) describe a vector which comprises the lethal gene sacB from Bacillus amylolique-faciens, integrated into a plasmid derived from the bacteriophage P1 and under the control of a specific
E. coli
promoter.
The promoter of this vector includes a region having several specific cloning sites (cleavage site for a restriction enzyme).
Since the gene sacB encodes levan sucrase, which catalyses the hydrolysis of sucrose into products which are toxic for
E. coli
, direct selection of the mutants which incorporate a recombinant plasmid is effected on a culture medium containing sucrose. Since the levan sucrase is toxic, even in the absence of sucrose, it is essential, consequently, to repress its synthesis if one wishes to obtain a large number of plasmid copies in the bacterial cytoplasm.
However, it is difficult, if not impossible, to repress the cytotoxic gene completely, particularly if a large number of copies of the vector are required.
Therefore, the impossibility of repressing the cytotoxic gene leads, in phases of producing the plasmid, to the death of the cell and, as a consequence, to selective pressure towards mutated strains (characterised by an inactive lethal gene).
In this case, in order to ensure that the enzyme encoded by the sacB gene does not kill the host cell, it is necessary to incorporate a CI repressor, which regulates the expression of this gene, into the cloning vector.
Furthermore, since sucrose is often incorporated into bacterial culture media, it will be essential to prepare media which are totally free of sucrose in order to carry out these manipulations.
Henrich et al (Gene, vol 42, No. 3, 1986, pp. 345-349) describe a vector which includes the E gene from the bacteriophage &PHgr;X174, the said E gene being incorporated into the plasmid pUH84 under the control of the Lac promoter.
In this case, the E gene includes six unique restriction sites (located over the whole of the E gene sequence) and encodes gpE, which causes lysis of the
E.coli
cell. In this case, positive selection is effected when a foreign recombinant gene has been inserted into one of the restriction sites.
However, this insertion of a foreign gene into a restriction site located in the sequence of the E gene, encoding gpE, makes it more difficult to sequence the foreign gene and/or amplify it by PCR since, in this case, portions of useless sequences belonging to the E gene encoding gpE are also sequenced, amplified and characterised.
Kuhn et al (Gene, vol 42, No. 3, 1986, pp. 253-263) describe a vector which includes a large gene encoding a restriction enzyme which kills by cleaving the genome of the bacterium, the said gene being incorporated into the plasmid pKG2 under the control of the LacUV5 promoter.
The cloning vectors of the state of the art suffer from the disadvantage of having to be maintained in a host strain which includes the LacI
q
repressor in episomal form, or the CI repressor, in order to inactivate the promoter and prevent expression of the killer gene, leading to the death of the host strain.
In addition, if it is desired to use this strain to produce a large number of copies of the cloning vectors, the repressor will not be adequate for preventing either a selective pressure which modifies the cytotoxic activity of the vector or a “genetic leakage”, that is to say expression of certain copies of the vector and death of the host strain.
Consequently, none of the documents of the state of the art describes a cloning vector which can incorporate large nucleotide fragments, which is easy to manipulate and which can be produced by a micro-organism on an industrial scale; that is to say, which can be produced in a large number of copies by a micro-organism without bringing about the death of the latter.
OBJECTS OF THE INVENTION
The present invention aims to supply a novel cloning and/or sequencing vector, and also its host strain, which are simple and relatively inexpensive to construct and produce, and which enable recombinant clones to be selected directly, without suffering from the disadvantages of the above-mentioned state of the art.
A particular object of the present invention is to obtain a vector which permits specific and certain selection of the recombinant clones.
Another object of the present invention is directed towards obtaining a vector which permits the sequencing, amplification and/or characterisation, using the same primer, of any foreign DNA fragment (whatever its size) in the recombinant clones.
An additional object of the present invention is directed towards obtaining a vector which also permits simple extraction of this foreign DNA fragment from the recombinant clone.
A final object of the present invention is directed towards obtaining a host strain for the said vector which allows a large number of copies of the said vector to be produced without bringing about selective pressure which modifies the cytotoxic activity of the said vector or causing the death of the host strain.
CHARACTERISTIC ELEMENTS OF THE INVENTION
The invention relates to a novel cloning and/or sequencing vector which includes, incorporated into an autonomously replicating vector, at least one promoter nucleotide sequence and at least one nucleotide sequence encoding a fusion protein which is active as a poison, the said nucleotide sequence being obtained by fusing a coding nucleotide sequence which includes several unique cloning site
Bernard Philippe
Gabant Philippe
Knobbe Martens Olson & Bear LLP
Schwartzman Robert A.
Universite Libre De Bruxelles
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