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- Bergö, Martin, 1970, et al.
(author)
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Genetic analyses of the role of RCE1 in RAS membrane association and transformation.
- 2008
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In: Methods in enzymology. - 0076-6879. ; 438, s. 367-89
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Journal article (peer-reviewed)abstract
- Proteins terminating with a CAAX motif, such as the nuclear lamins and the RAS family of proteins, undergo post-translational modification of a carboxyl-terminal cysteine with an isoprenyl lipid--a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by an endoprotease of the endoplasmic reticulum. RCE1 is responsible for the endoproteolytic processing of the RAS proteins and is likely responsible for endoproteolytic processing of the vast majority of CAAX proteins. Prenylation has been shown to be essential for the proper intracellular targeting and function of several CAAX proteins, but the physiologic importance of the endoprotease step has remained less certain. Here, we will review methods that have been used to define the physiologic importance of the endoproteolytic processing step of CAAX protein processing.
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| 2. |
- Yang, Shao H., et al.
(author)
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Caution! Analyze transcripts from conditional knockout alleles.
- 2009
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In: Transgenic research. - : Springer Science and Business Media LLC. - 1573-9368 .- 0962-8819. ; 18:3, s. 483-9
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Journal article (peer-reviewed)abstract
- A common strategy for conditional knockout alleles is to "flox" (flank with loxP sites) a 5' exon within the target gene. Typically, the floxed exon does not contain a unit number of codons so that the Cre-mediated recombination event yields a frameshift and a null allele. Documenting recombination within the genomic DNA is often regarded as sufficient proof of a frameshift, and the analysis of transcripts is neglected. We evaluated a previously reported conditional knockout allele for the beta-subunit of protein farnesyltransferase. The recombination event in that allele-the excision of exon 3-was predicted to yield a frameshift. However, following the excision of exon 3, exon 4 was skipped by the mRNA splicing machinery, and the predominant transcript from the mutant allele lacked exon 3 and exon 4 sequences. The "Deltaexon 3-4 transcript" does not contain a frameshift but rather is predicted to encode a protein with a short in-frame deletion. This represents a significant concern when studying an enzyme, since an enzyme with partial function could lead to erroneous conclusions. With thousands of new conditional knockout alleles under construction within mouse mutagenesis consortiums, the protein farnesyltransferase allele holds an important lesson-to characterize knockout alleles at both the DNA and RNA levels.
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