Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1994-09-01
1998-05-26
Ketter, James
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
536 232, C12N 1501, C12N 1512
Patent
active
057563233
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for generating structural or functional diversity in a peptide sequence by introducing insertions or deletions of nucleotides in the nucleotide sequence which codes for the said peptide sequence.
The present invention also relates to pharmaceutical compositions, drugs or diagnostic reagents containing proteins or peptides obtained by this method.
The mature genes coding for the constituent chains of immunoglobulins and T cell receptors are assembled early during lymphocyte development from gene segments termed variability (V), linking, or junction (J), and in some cases diversity (D).
Seven loci are able to be rearranged by recombination of these fragments.
The recombination signal sequences (RSS) adjacent to each gene supply the targets for the recombination. These sequences are composed of a palindromic heptamer and a nonamer rich in adenosine and thymidine, separated by a sequence of 12 or 23 base pairs. The rearrangements are made between RSS with separation sequences of different lengths.
Two types of junction or linking are formed during the recombination: coding junctions created by the juxtaposition of gene segments and noncoding junctions formed by contiguous RSS. In the latter case, the heptamers are generally joined without nucleotide insertions or deletions. The coding junctions themselves are liable to substantial modifications.
The variations in the junctions during the rearrangement of the gene segments coding for the immunoglobulins represent a major source of diversity. Several nucleotides can thus be eliminated and two types of insertion can occur.
The random addition of nucleotides results in the insertion of regions termed N regions in the immunoglobulin heavy chains. The hypothesis has been advanced (Randau et al., Molecular and Cellular Biology, 1987, 3237-3243) that the deoxynucleotidyl transferase (TdT) is responsible for this random addition of nucleotides.
The type P nucleotides insertions correspond to the inverse repetition of sequences adjacent to those of the coding sequences. The hypothesis has been advanced that their addition represents a necessary step in the recombination mechanism.
Transfection experiments with genomic DNA have enabled the isolation of two genes actively involved in the recombination: the Rag-1 and Rag-2 genes (Oettinger et al., Science, Volume 248, 1517-1523, 1990).
It has been shown that the Rag-1 and Rag-2 genes are responsible for the site-specific recombination.
Nevertheless, the combination of the products of the Rag-1 and Rag-2 genes does not restore the diversity of antibodies found in vivo, in other words does not allow to add the N sequences.
Various methods have been developed to attempt to modify the immunoglobulin heavy and light chains or the receptors.
EP patent No-368.684 relates to a method for cloning nucleotide sequences corresponding to the variable regions of the molecules in the immunoglobulin family. This method consists of producing a DNA complementary to the variable region of the immunoglobulin.
EP patent No-328.444 relates to a method for modifying the structure of an antibody while retaining its functional specificity. Thus, the constant regions in particular are modified by a classical genetic engineering technique.
To the knowledge of the applicant, there is no method for efficiently obtaining structurally modified immunoglobulins with a wide diversity in the modifications.
The applicant has thus aimed to demonstrate recombination mechanisms which allow the organism to achieve a wide diversity in the immunoglobulins such as the IgG, the IgM, the IgA, the IgE, and in the lymphoid cell receptors.
The applicant has also aimed to develop a general method for randomly obtaining a very diversified range of mutations, in particular by random insertion, in the nucleotide sequence corresponding to proteins with various structures and functions.
The applicant has also found that the combination of the products of expression of the Rag-1 and Rag-2 genes leads unexpectedly to a site-specific rec
REFERENCES:
Watson et al. Molecular Biology of the Gene, Fourth Editior pp. 313-338 Benjamin Cummings Pub. Co. (1987).
Kallenbach et al. Proc. Natl. Acad. Sci. USA 89 2799-2803 (1992) Three Lymphoid Specific Factors Account for all . . .
Oettinger et al. Science 248 1517-1523 (1990).
Rag-1 and Rag-2, Adjacent Genes that Synergistically . . .
Kallenbach et al. Nucleic Acids Research 18 6730 (1990) A Rapid Test for U(O)J Recombinase Activity.
Kowai et al. Nucleic Acids Research 14 5777-5792 (1986) Isolation and Characterization of Bovine and Mouse Terminal . . .
Landau et al. Molecular and Cellular Biology 7 3237-3243 (1987) Increased Frequency of N-region Insertion in a Murine Pre-B-cell . . . .
Huse et al. Science 246 1275-1281 (1989) Generation of a Large Combinatorial Library of the Immunoglobulin . . . .
Davis Annual Review of Biochemistry 59 475-496 (1990) T Cell Receptor Gene Diversity and Selection.
Hasty et al. Human neutrophil collagenase J. Biol. Chem. vol. 265 11421-11424, 1990.
Doyen Noelle
Kallenbach Sacha
Rougeon Francois
Brusca John S.
Institut Pasteur
Ketter James
LandOfFree
Method for generating structural and functional diversity in a p does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for generating structural and functional diversity in a p, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for generating structural and functional diversity in a p will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1959008