Chimeric proteins activating polymerase III transcription, use t

Details Chimeric proteins activating polymerase III transcription, use t Chimeric proteins activating polymerase III transcription, use t

1997-11-26

1999-05-18

Degen, Nancy

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

530350, 536 234, C07K14/00

Patent

active

059050250

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to new means for detecting and analysing the interactions between proteins
2. Discussion of the Related Art
Protein/protein interactions are fundamental cellular mechanisms which are involved in the formation of multimeric complexes responsible both for functions such as transcription and translation, and for the transmission of signals, the response to pathogenic agents and the like.
To analyse these interactions, conventional biochemical techniques (cross-linking, co-immunoprecipitation, co-fractionation by chromatography) which are intended to isolate the proteins interacting with a target protein are generally difficult to use, especially when the interacting proteins are in a small quantity. In addition, they make it possible to identify the interacting proteins, but not to directly obtain the genes corresponding to the said proteins.
One procedure called: "double-hybrid method", which makes it possible to detect interactions between 2 proteins, has been developed by FIELDS and No. 5,283,173!.
This procedure is based on the co-expression, in the same yeast cell, of the following genes:
one reporter gene expressing a detectable protein, whose level of expression depends on transcriptional activation by a polypeptide domain; and
two chimeric genes, encoding two hybrid proteins comprising respectively the sequences of the two proteins whose interaction it is desired to detect: one comprises, in addition, a transcription activation domain regulating the expression of the reporter gene, the other comprises, in addition, a DNA-binding domain, which recognizes a binding site situated on the reporter gene in the host cell.
When the two chimeric genes are expressed in the same cell, if an interaction occurs between the two proteins, it causes the transcription activation domain and the reporter gene to come into contact due to the attachment of the DNA-binding domain to its site situated on the reporter gene. The transcription of the latter is activated, and an increase in its expression product can therefore be observed.
In the system described in Patent U.S. Pat. No. 5,283,173, FIELDS and SONG exploited more particularly the properties of GAL4, a transcription activator in Saccharomyces cerevisiae.
GAL4 activates the transcription by RNA polymerase II (PolII) of genes encoding enzymes involved in the metabolism of galactose. This protein comprises 2 functionally independent and physically separable domains: one DNA-binding domain, represented by the N-terminal domain (amino acids 1-147) which binds to specific sequences of the DNA (UAS.sub.G, for Upstream Activating Sequence for Galactose), and a transcription activation domain represented by the C-terminal domain (amino acids 768-881), which activates the transcription by PolII.
Two types of hybrid proteins can thus be produced from GAL4: one contains the GAL4(1-147) domain fused to a first test protein, the other contains the GAL4(768-881) domain fused to a second test protein. If the 2 test proteins are capable of interacting, they bring the two domains of GAL4 closer and trigger the transcription of the reporter gene (for example the lacZ gene encoding the .beta.-galactosidase of E. coli).
It is possible to simultaneously test several proteins with the aim of investigating their interactions with a given protein. For example, the protein whose partners are sought is fused with the binding domain GAL4(1-147) and it is tested against a library of proteins fused with the activator domain GAL4(768-881).
Numerous improvements have been made to the technique initially used by FIELDS and SONG: for example a system for cloning into .lambda. phages subsequently convertible to plasmids by recombination of lox sites, has been developed; a second reporter gene, consisting of a marker for auxotrophy, the HIS3 gene (involved in the metabolism of histidine), was used in combination with the lacZ gene in order to eliminate the false 555-569, (1993)!.
Variants of this technique have also

REFERENCES:
Marsolier,M-C et al : "The antirepression role of general class III transcription factors, " Journal of Cellular Biochemistry Supplement 0 (21B); 1995; 150. Abstract No. J8-001.
B. Le Douarin et al, "A new version of the two-hybrid assay for detection of protein-protein interactions, " Nucleic Acids Research, vol. 23, No. 5, Mar. 11, 1995, pp. 876-878.
Marsolier, M-C et al, "Directing transcription of an RNA polyerase III gene via GAL4 sites, " Proceedings of the National Academy of Sciences of the U.S., 91 (25). 1994. 11938-11942.
P. Legrain and C. Chapon, "Interaction betweenn PRP11 and SPP91 yeast splicing factors and characterization of a PRP9-PRP11-SPP91 complex", Science, vol. 262, Oct. 1, 1993, AAAS, Wash. D. C., pp. 108-110.
M. Werner et al, "Interaction between a complex of RNA polymerase III subunits and the 70-kDa component of transcription factor IIIB", J. Biol. che., vol. 268, No. 28, Oct. 5, 1993, Am. Soc. Biochem. Mol. Biol., Inc., Baltimore, US., pp. 20721-20724.
J.E.Arenas and J.N. Abelson, "The Saccharomyces cerevisiae PRP21 gene product is an integral component of the spliceosome, " Proc. Natl. Acad Sci., vol. 90, Jul. 1990, Natl. Acad Sci., Wash. D.C., pp. 6771-6775.
M-C Marsolier et al, "reciprocal interferences between nucleosomal organization and transcriptional activity of the yeast SNR6 gene, " Genes & Development, vol. 9, No. 4, Feb. 15, 1995, pp. 410-422.

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