Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1987-07-23
1991-01-15
Schwartz, Richard A.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
435 91, 4351721, 4352523, 435254, 435255, 4352402, 435320, 536 27, 935 6, 935 22, 935 52, 935 54, 935 66, 935 76, 935 77, 935 79, 935 80, C12N 115, C12N 121, C12N 1510, C12N 1564
Patent
active
049853597
DESCRIPTION:
BRIEF SUMMARY
The invention concerns vectors for the construction of cDNA libraries and methods for constructing these vectors. In addition, the invention concerns methods for the construction of cDNA libraries with vectors according to the invention as well as the expression of genes in cDNA libraries constructed this way and microorganisms transformed with either the above mentioned vectors or with these vectors containing recombinant DNA.
In recent years, development of recombinant DNA methodology has permitted cloning and isolation as well as expression of genes which code for valuable products. If partial or complete primary sequence information of a gene product (e.g. protein) is available, synthetic oligodeoxynucleotide probes can be made which code for some of the known amino acid residues of the desired gene product. These oligonucleotide probes can then be used for screening of cDNA libraries for the gene coding for the protein. If the genetic information for the protein of interest is expressed in such libraries, antibodies against that protein can also be used for screening of the libraries.
Methods have been developed for cloning mRNA (messenger RNA) from cells which synthesize the protein of interest. As mRNA cannot be cloned directly, it has to be transcribed into the corresponding cDNA first. For this transcription an enzyme from a tumor virus is used, known as reverse transcriptase. Use of this enzyme and several additional procedures results in recombinant vector molecules which contain double-stranded cDNA derived from mRNA. The assembly of all these recombinant vectors which contain cDNA is called a cDNA library.
FIG. 7 illustrates the classical method for constructing cDNA libraries. In this method mRNA is first isolated from certain cells (1) and is then treated with different enzymes and alkali in successive steps of the process (steps (2) to (6)). In parallel the Yector (e.g. plasmid pBR322) is treated in such a way that insertion of the cDNA synthesized is possible. After annealing of cDNA and pretreated vector, a suitable host (e.g. strain of E. coli K12) is transformed with the vector-cDNA constructs, plated on suitable substrates and incubated. All colonies from transformed cells obtained in this way constitute a cDNA library as mentioned above.
This method for constructing cDNA libraries has the disadvantage that the mRNA isolated from the cells is subjected to several treatments (steps (1) to (6)). As an mRNA molecule is relatively labile, it is degraded to shorter pieces which lead to relatively short cDNA inserts in the order of 300 to 1000 bp (base pairs). This implies that the genetic information of proteins with more than 300 amino acids is rarely obtained completely from such libraries. Another disadvantage of the method shown in FIG. 7 results from the fact that the orientation of the cDNA in the vector cannot be directed. Therefore recombinant plasmids with cDNA in the wrong orientation cannot be detected by screening with antibodies.
The method of H. Okayama and P. Berg (Mol. Cell. Biol. 2 (1982), 161-170) allows the oriented insertion of the cDNA copies from the isolated cellular mRNA. This method is summarized in FIG. 8. This process starts with cutting the vector molecule with a restriction endonuclease and adding oligo(dT) tails to the vector with the enzyme terminal deoxynucleotidyl transferase. This results in a linearized vector molecule with oligo(dT) tails at both 3' ends. One of the (oligo)dT tails is then removed by cutting with the restriction endonuclease HpaI.
In the following step the mRNA is annealed via its poly(A) tail to the now unique oligo(dT) tail of the vector. The orientation of the annealed mRNA is predetermined as only one oligo(dT) tail is left on the manipulated vector and the mRNA contains a poly(A) tail only at its 3' end. During further steps of the procedure, the corresponding cDNA is constructed with the help of different enzymes (cf., FIG. 8), resulting finally in a circular recombinant vector molecule.
This method has the disadvantage that the add
REFERENCES:
patent: 4394443 (1983-07-01), Weissman et al.
Maniatis et al., 1982, Molecular Cloning, Cold Spring Harbor Laboratory, pp. 148, 217-229, 239 and 241.
BRL Catalogue and Reference Guide, 1985, Bethesda Research Laboratories, p. 55.
Okayama, H. and Berg, P., Molec. Cell. Biol. 2:161-170 (1982).
Coleclough, C. and Erlitz, F. L., Gene 34: 305-314 (1985).
Heidecker, G. and Messing, J., N. Acids Res. 11: 4891-4906 (1983).
Oberbaumer Ilse
Peet Richard C.
Schwartz Richard A.
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