Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-04-28
2002-05-21
Elliott, George C. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S069100, C435S091500, C435S440000, C435S455000
Reexamination Certificate
active
06391641
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of mutagenesis and mutation detection. The invention is also related to a DNA construct in which at least two genes are translationally coupled. The invention is also related to a method of detecting missense mutations and eliminating nonsense mutations after a random mutagenesis procedure.
BACKGROUND OF THE INVENTION
Random mutagenesis of a cloned gene followed by phenotypic selection is a powerful tool for identifying amino acid residues or protein domains that are critical for protein function, particularly when specific residues that are likely to be mechanistically important cannot be identified by other means. Random mutations can be introduced into a specific gene or portion of a gene by a variety of methods, including PCR amplification under mutagenic conditions (Fromant, M., Blanquet, S., and Plateau, P. (1995)
Anal. Biochem
. 224, 347-353; Vartanian, J.-P., Henry, M., and Wain-Hobson, S. (1996)
Nucl. Acids Res
. 24, 2627-2631). However, a problem that can arise with this approach is that nonsense or frameshift mutations that give rise to a truncated protein may give the selected phenotype, but they are otherwise uninformative as to the specific residues that are required for activity. Moreover, a truncated mutant protein may be susceptible to proteolytic degradation in the cell (Gottesman, S., Wickner, S., and Maurizi, M. R. (1997)
Genes
&
Devel
. 11, 815-823) and thus may not be isolatable for further study. In some studies a large majority of mutants (80-90%) produced by random procedures encoded truncated or unstable proteins (Friedrich, T., Roth, M., Helm-Kruse, S., and Jeltsch, A. (1998)
Biol. Chem
. 379, 475-480; Kostelidou, K., Jagura-Burdzy, G.; and Thomas, C. M. (1998)
J. Mol. Biol
. 281, 453-463; van den Ent, F. M., Vos, A., and Plasterk, R. H. (1998)
J. Virol
. 72, 3916-3924; Shibata, H., Kato, H., and Oda, J. (1998)
Protein Eng
. 11, 467-472). Thus hundreds of colonies that expressed mutant protein had to be screened by relatively time consuming and labor intensive methods such as SDS-PAGE and/or western blot analysis to identify those that expressed full-length protein and were potential missense point mutants.
We sought an efficient way to winnow out nonsense mutations that produce truncated protein in the course of a random mutagenesis study of the RecD subunit of the RecBCD enzyme from
Escherichia coli
. For this purpose, we constructed a synthetic operon in which a reporter gene that encodes a selectable marker, the gene for kanamycin resistance (Oka, A., Sugisaki, H., and Takanami, M. (1981)
J. Mol. Biol
. 147, 217-226), is downstream of the recD gene. Translation of the kanamycin resistance gene, and therefore generation of a kanamycin resistant colony, should be dependent on complete translation of the upstream recD gene ()as, A., and Yanofsky, C. (1989)
Nucl. Acids Res
. 17, 9333-9340; Spanjaard, R. A., and van Duin, J. (1989)
Nucl. Acids Res
. 17 5501-5507). More than 90% of the random mutants obtained using this synthetic operon expressed full-length RecD protein. This approach may be of general use to practitioners of random mutagenesis.
SUMMARY OF THE INVENTION
The present invention has met the hereinbefore-described need.
It is an object of the present invention to provide a method of selecting for missense mutants that express a protein comprising:
constructing an operon comprising an upstream gene and a downstream reporter gene, wherein said upstream gene and said reporter gene are translationally coupled; wherein the reporter protein is expressed after the translation of the upstream gene,
expressing the downstream reporter gene after introducing at least one mutation into the upstream gene, and
screening for the mutant that expresses the reporter protein, which is indicative of said missense mutant.
In a preferred embodiment, the downstream reporter gene and the upstream gene are coupled via a ribosome-binding site. The reporter gene may encode a protein having virtually any detective property, which may include, but not limited to, ones that have fluorescent property, calorimetric property, or enzymatic property. Furthermore, reporter gene can be a selective marker gene, such as an antibiotic resistance gene or an auxotrophic marker.
The reporter gene, upstream gene, or both can be from any source expressed in any suitable vector in a transformed cell, such as an eucaryotic cell, including, but not limited to, mammalian or plant cell, or bacterial cell.
The mutation can be introduced by polymerase chain reaction or chemical mutagens, among other conventionally known methods.
The ribosome binding site can include but is not limited to the sequence 5′-AGGAGGU, 5′-GAGGGG, 5′-GGAG, 5′-GGUGGU, 5′-GGAGG or 5′-UAAGGAGGU. In addition, the ribosome binding site may form part of a stem and loop structure with a nearby sequence, wherein the stability of said stem and loop structure is optimized by arranging the complementarity in the stem structure so that a maximum number of base pairs is formed.
Another object of the invention is to provide a method for identifying and eliminating nonsense mutations in the upstream gene after mutagenizing comprising:
constructing an operon comprising a downstream reporter gene and an upstream gene, wherein the said reporter gene and the upstream gene are translationally coupled, wherein the reporter protein is expressed after the translation of the upstream gene,
generating the random mutations in the upstream gene before or after said operon construct is made,
expressing the downstream reporter gene after introducing at least one mutation into the upstream gene, and
screening for the mutants by expressing the proteins.
It is still another object of this invention to provide a kit for determining the presence or absence of missense or nonsense mutations present on an upstream gene in a translationally coupled construct as desired. Each component of the kit(s) may be individually packaged in its own suitable container. The individual containers may also be labeled in a manner, which identifies the contents. Moreover, the individually packaged components may be placed in a larger container capable of holding all desired components. Associated with the kit may be instructions, which explain how to use the kit. These instructions may be written on or attached to the kit. Thus, the invention provides a kit for generating random mutations and for eliminating those that encode a truncated protein, comprising a vector constituting the translationally coupled operon described above, and instructions for carrying out the detection method.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.
REFERENCES:
Sergey M. Podkovyrov et al, A new vector-host system for construction of lac Z transcriptional fusions where only low-level gene expression is desirable, Gene, 156 (1995) 151-152.*
New England Biolabs Catalog. Copyright 1996. p. 164.*
New England Biolabs Catalog. Copyright 1996. p. 204.*
Bernasconi et al. 1994. Functional expression of Arabidopsis thaliana anthranilate synthase subunit inEscherichia coli. Plant Physiol. 106: 353-358.*
Ishioka et al. 1997. Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast. Proc. Natl. Acad. Sci. U.S.A. 94: 2449-2453.*
Korangy et al. 1992. Alteration by Site-directed mutagenesis of the conserved lysine residue in the ATP-binding consensus sequence of the RecD subunit of theEscherichia coliRecBCD enzyme. J. Biol. Chem. 267:1727-1732.*
Translational Reinitiation in the presence and absence of a shine and dalgarno sequence, Spanaard, Remco et al.,Nucleic Acids Research, vol. 17, No. 14, pp.: 5501-5507, 1998.
Translation Through an uncDC mRNA Secondary Structure Governs the Level of uncC Expression inEscherichia Coli, Dallman, H. Garry, et al., Journal of Bacteriology, vol. 176, No. 5
Julin Douglas A.
Wang Jingdi
Wang Ruiwu
Elliott George C.
Loeb Bronwen M.
McDermott & Will & Emery
University of Maryland College Park
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