1. The discovery, in 1978-1979, of the first
nucleosome-depleted, nuclease-hypersensitive regions in
chromosomes. Such "exposed" chromosomal segments are characteristic of transcriptional promoters, recombination hotspots, and the origins of DNA replication. 4. A verifiable conjecture about molecular basis of sleep causation, termed the fragment generation (FG) hypothesis. According to the FG hypothesis, a molecular cause of sleep stems from production, during wakefulness, of numerous extracellular and intracellular protein-sized protein fragments that can be transiently beneficial but can also perturb, through their diverse and cumulative effects, the functioning of the brain and other organs. The FG hypothesis posits that sleep evolved, at least in part, to counteract overproduction (owing to an insufficiently fast elimination) of hundreds of different protein fragments during wakefulness. The FG hypothesis is consistent with available experimental evidence. It remains to be verified. 5. Inventions of genetic and biochemical methods (1980-2017) (see references and references therein): (i) A method for two-dimensional electrophoretic mapping of DNA replication/multicatenation intermediates, in 1980-1981. (ii) Nucleosome mapping using a two-dimensional hybridization method, in 1982. (iii) The ubiquitin fusion technique, in 1986. This method makes it possible to expose, in vivo, a desired N-terminal residue in a protein of interest. Owing to the mechanics of the genetic code, all nascent proteins bear the N-terminal Met residue, which is either retained in or removed from mature proteins. The ubiquitin fusion technique makes it possible to "bypass" the endogenous rules of N-terminal Met removal and retention. (iv) Chromatin immunoprecipitation (ChIP) assay, in 1988. Advanced versions of ChIP are being used for mapping in vivo locations of chromosomal proteins. (v) Mutations in many (most) genes that cause a hypersensitivity to heavy water (
D2O), a novel and generally applicable conditional phenotype, in 1988. (vi) Heat-activated N-degron for producing temperature-sensitive mutants, in 1994. (vii) Split-ubiquitin method for detecting protein interactions in vivo, in 1994. The central idea of the split-ubiquitin technique opened the field of single-subunit split proteins, such as split-GFP, split lactamase, split Cas9
CRISPR nuclease, and many other split protein sensors and effectors. (viii) Ubiquitin translocation assay, in 1994, for analyzing, in vivo, specific mechanisms and kinetics of protein translocation across cellular membranes. (ix) Ubiquitin sandwich technique, in 2000. It uses ubiquitin fusions and multiple tandem reporters to detect and measure cotranslational proteolysis in vivo. (x) Subunit decoy technique, in 2013, for analyzing the in vivo regulation of subunit stoichiometries in oligomeric proteins. (xi) Promoter reference technique, in 2017. This reference-based method for measuring the in vivo protein degradation uses RNA aptamers and bypasses the necessity of global translation inhibitors in a chase-degradation assay. 1.
Alexander Varshavsky, California Institute of Technology (Caltech) (https://www.bbe.caltech.edu/people/alexander-varshavsky).
2. Varshavsky, A. "(2022) Interview about life and work, to David Zierler, Caltech Heritage Project".
. (https://heritageproject.caltech.edu/interviews-updates/alexander-varshavsky)
. 3. Bachmair, A.,Finley, D., Varshavsky, A. (1986))
In vivo half-life of a protein is a function of Its N-terminal residue. Science 234: 179–186.
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10.1126/science.3018930. 4.
Varshavsky, A. (2008) Discovery of cellular regulation by protein degradation. Journal of Biological Chemistry 283: 34469-34489.
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10.1074/jbc.x800009200. 5.
Varshavsky, A. (2019)) N-degron and C-degron pathways of protein degradation. Proceedings of the National Academy of Sciences 116 : 358–366.
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10.1073/pnas.1816596116. 6.
Jentsch, S., McGrath, J. P., Varshavsky, A. (1987) The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature 329: 131-134.
doi:
10.1038/329131a0. 7.
Johnson, E. S., Gonda, D.K., Varshavsky, A. (1990) Cis-trans recognition and subunit-specific degradation of short-lived proteins. Nature 346: 287-291.
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10.1038/346287a0. 8.
Varshavsky, A. (2014). "Discovery of the biology of the ubiquitin system". Journal of the American Medical Association (JAMA) 311: 1969.
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10.1001/jama.2014.5549. 9.
Hershko, A. Ciechanover, A., Varshavsky, A. (2000) The ubiquitin system. Nature Medicine 6: 1073-1081.
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10.1038/80384. 10.
Oh, J.H., Hyun, J.Y., Chen, S. J., Varshavsky, A. (2020) "Five enzymes of the Arg/N-degron pathway form a targeting complex: the concept of superchanneling". Proceedings of the National Academy of Sciences 117 (20): 10778-10788.
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10.1073/pnas.2003043117. 11.
Vu, T. T. M., Mitchell, D. C., Gygi, S. P., Varshavsky, A. (2020) "The Arg/N-degron pathway targets transcription factors and regulates specific genes". Proceedings of the National Academy of Sciences 117: 31094-31104. '''' 12.
Varshavsky, A. (2007) Targeting the absence: homozygous DNA deletions as immutable signposts for cancer therapy. Proceedings of the National Academy of Sciences 104: 14935-14940.
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10.1073/pnas.0706546104. 13.
Varshavsky, A., Lewis, K., Chen, S. J. (2023). Deletions of DNA in cancer and their possible uses for therapy. BioEssays 45.
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10.1002/bies.202300051. 14.
Varshavsky, A. (2019) On the cause of sleep: protein fragments, the concept of sentinels, and links to epilepsy. Proceedings of the National Academy of Sciences 116: 10773-10782.
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10.1073/pnas.1904709116. ==References==