Information

16.5: Micro-report 4- Complementation analysis - Biology

16.5: Micro-report 4- Complementation analysis - Biology



We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Figure: The figure at the heart of this micro-report is multi-panel figure showing replica plates of strains that have been transformed with overexpression plasmids. Refer to the guidelines for Micro-report 1 to see how to set up figures with multiple panels.

Materials and Methods: Provide information on the transformation and replica plating procedures, as well as the media used in the experiments. When possible, reference the lab manual, noting any changes that you made to procedures.

Strains and plasmids: See micro-reports 1 and 3.

Media: See micro-report 1.

Transformation: Someone trying to reproduce your results will need to know details about the transformation procedure, because transformation efficiencies vary widely and show a strong dependence on reagents and incubation conditions. You can reference the lab manual.

Replica plating: This is a standard procedure. You can also reference the lab manual.

Results and Discussion: Your figure with the scanned plates is the focal point of this section. Include a single data table that with the calculated transformation efficiency with each plasmid and the ability of transformed strains (Y/N or +/-) to grow on the various replica plates.Transformation efficiencies should be expressed in number of transformed cells/μg plasmid DNA.

The R&D section should tell a story of how the replica plates allowed you to decide if your
Met protein is conserved between the two yeast species. You may or may not have observed complementation. Failure to observe complementation is not necessarily due to experimental error. (Which plates serve as a control against experimental error?) Complementation is a functional assay that depends on both the expression of the fusion protein and the ability of the fusion protein to catalyze a reaction in Met biosynthesis. The fusion proteins have large C-terminal extensions that might affect their normal enzymatic functions. Negative results can be just as important as positive results in advancing scientific understanding.

If you did not observe complementation, discuss possible reasons that this may have happened and propose future experiments that could help to answer these questions. You may want to suggest new plasmid constructs for additional experiments. You may also want to bring in information from your BLASTP analyses. (What is the E-value?) Be sure to include enough justification that your proposed experiments will provide useful data.


Gary Ruvkun

Massachusetts General Hospital Department of Molecular Biology home page:

Major Research Interests

microRNA and RNA interference mechanisms, bacterial and animal genetic analysis of microbiome interactions, neuroendocrine control of detoxification, immunity, and aging, life on other planets.

Academic trajectory

1985 - present Asst, Assoc., Professor of Genetics, Harvard Medical School
1982 ‑ 1985 Junior Fellow, Society of Fellows, Harvard University
1982 Ph.D. Harvard University (Biophysics)
1973 A.B. University of California at Berkeley (Biophysics)

2004 Rosenstiel Award, Brandeis University (with Victor Ambros, Andy Fire, Craig Mello)
2008 National Academy of Sciences
2008 Benjamin Franklin Medal, Franklin Institute (with Victor Ambros and David Baulcombe)
2008 Albert Lasker Award for Basic Medical Research (with Victor Ambros and David Baulcombe)
2008 Canada Gairdner International Award (with Victor Ambros)
2008 Warren Triennial Prize, Massachusetts General Hospital (with Victor Ambros)
2009 Louisa Gross Horwitz Prize, Columbia University (with Victor Ambros)
2009 American Academy of Arts and Sciences
2009 Shaul and Meira Massry Prize (with Victor Ambros)
2010 Académie Royale des Sciences, des Lettres et des Beaux-Arts de Belgique
2009 National Academy of Medicine
2011 Dan David Prize (with Cynthia Kenyon)
2012 Paul Janssen Award for Biomedical Research (with Victor Ambros)
2013 Ipsen Foundation Longevity Prize
2013 Irving Wright Award, American Foundation for Aging Research
2014 Wolf Prize in Medicine (with Victor Ambros)
2014 Gruber Genetics Prize (with Victor Ambros and David Baulcombe)
2015 Breakthrough Prize in Life Sciences (with Victor Ambros)
2016 March of Dimes Prize in Developmental Biology (with Victor Ambros)
2019 American Philosophical Society

Professional Activities

2004 - present Harvard Origins of Life Initiative
2006 - present Broad Institute Associate
2015 - present Chair, Scientific Review Committee, Smith Family Awards Program in Biomedical Research
2015 - present Paul Glenn Medical Foundation Scientific Advisory Board
2018 - present California Life Sciences Company (Calico) Scientific Advisory Board

Past
2003 - 2011 Harvard Microbial Sciences Initiative Organizing committee
2004 - 2007 NIH National Advisory Council on Aging
2012 - 2014 Ellison Medical Foundation Scientific Advisory Board
2015 Vallee Foundation Visiting Professorship
2012 - 2017 NAS Space Studies Board, Committee on Astrobiology and Planetary Science
2017 - 2018 NAS Space Studies Board, Review of Planetary Protection Policy Development Processes
2015 – 2019 Chair, Genetics Section of the National Academy of Sciences
2018 - 2019 NASA Mars Sample Return Sterilization Working group, Jet Propulsion Laboratory, Caltech

Honorific invited lectures

1998 Nippon Telephone and Telegraph Science Forum Public Lecture, Tokyo
2003 Harvey Lecturer, Rockefeller University
2008 Jean Mitchell Watson Lecture, University of Chicago
2012 Mendel 190th Birthday Symposium, Brno, Czech Republic
2013 60th anniversary of the Double Helix, Cold Spring Harbor

Bibliography

1. Ruvkun G, Ausubel FM 1980 Interspecies homology of nitrogenase genes. Proc. Natl. Acad. Sci. USA 77: 191‑195. PMCID: PMC348234

2. Ruvkun G, Ausubel FM 1981. A general method for site‑directed mutagenesis in prokaryotes. Nature 289: 85‑88. PMID: 6256652

3. Ruvkun G, Long, SR, Meade, HM, Ausubel, FM 1981. Molecular genetics of symbiotic nitrogen fixation. Cold Spring Harbor Symposium on Quantitative Biology 45: 492‑500.

4. Meade HM, Long SR, Ruvkun G, Brown SE, Ausubel FM 1982. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149: 114‑122. PMCID: PMC216598

5. Ruvkun G, Sundaresan V, Ausubel FM 1982. Specific protection of nucleotides in the lac operator from dimethyl sulfate (DMS) methylation of DNase I nicking by crude bacterial extracts. Gene 18: 245‑255. PMID: 6290328

6. Ruvkun G, Sundaresan V, Ausubel FM 1982. Directed transposon Tn5 mutagenesis and complementation analysis of the Rhizobium meliloti symbiotic nitrogen fixation (nif) genes. Cell 29: 551‑559. PMID: 6288262

7. Ruvkun G, Long SR, Meade HM, van den Bos RC, Ausubel FM 1982. ISRml: a Rhizobium meliloti insertion sequence which preferentially transposes into nitrogen fixation (nif) genes. J Mol Appl Genet 1: 405‑418. PMID: 6296251

8. Finney M, Ruvkun, G, and Horvitz, HR 1988. The Caenorhabditis elegans cell lineage and differentiation gene unc-86 encodes a protein containing a homeodomain and extended similarity to mammalian transcription factors. Cell 55: 757‑769. PMID: 2903797

9. Herr W, Sturm, RA, Clerc, RG, Corcoran, LM, Baltimore, D, Sharp, PA, Ingraham, HA, Rosenfeld, MG, Finney, M, Ruvkun, G, and Horvitz, HR 1988 The POU domain: a large conserved region in the mammalian pit-1, oct-1, oct-2, and Caenorhabditis elegans unc-86 gene products. Genes and Development 2: 1513‑1516. PMID: 3215510

10. Ruvkun G, Ambros, V, Coulson, A, Waterston, R, Sulston, J, and Horvitz, HR 1989. Molecular genetics of the Caenorhabditis elegans heterochronic gene lin-14. Genetics 121: 501‑516. PMCID: PMC1203636

11. Ruvkun G and Giusto, J 1989 The Caenorhabditis elegans heterochronic gene lin-14 encodes a nuclear protein that forms a temporal switch during development. Nature 338: 313‑319. PMID: 2922060

12. Bürglin TR, Finney, M, Coulson, A, and Ruvkun, G 1989. C. elegans has scores of homeobox-containing genes. Nature 341: 239‑243. PMID: 2571091

13. Ruvkun G, Gilbert, W, Horvitz, HR 1990. Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization. Nucleic Acids Research 18: 809‑815. PMCID: PMC330331

14. Finney M and Ruvkun, G 1990. The unc-86 gene product couples cell lineage and cell identity in Caenorhabditis elegans. Cell 63: 895‑905. PMID: 2257628

15. Wightman B, Bürglin, TR, Gatto, J, Arasu, P, G Ruvkun 1991. Negative regulatory sequences in the lin-14 3' untranslated region are necessary to generate a temporal switch during C. elegans development. Genes and Development 5: 1813‑1824. PMID: 1916264

16. Arasu P, Wightman, B, G Ruvkun 1991. Temporal regulation of lin-14 by the antagonistic action of two other heterochronic genes, lin-4 and lin-28. Genes and Development 5: 1825‑1833. PMID: 1916265

17. Ruvkun G, Wightman, B, Bürglin, TR, and P Arasu. 1991. Dominant gain of function mutations that lead to misregulation of the C. elegans heterochronic gene lin-14, and the evolutionary implications of dominant mutations in pattern‑formation genes. Development (Supplement): 47‑54. PMID: 1742500

18. Bürglin TR, Ruvkun, G, Coulson, A, Hawkins, N, McGhee, J, Schaller, D, Wittmann, C, Müller, F, and Waterston, R 1991. Nematode homeobox cluster. Nature 351: 703. PMID: 1676487

19. Miller D, Shen, M, Shamu, C, Bürglin, T, Ruvkun, G, Ghee, M Dubois, L Wilson 1992 The C. elegans unc-4 gene encodes a homeodomain protein that determines the pattern of synaptic input to specific motor neurons. Nature 355: 841‑845. PMID: 1347150

20. Xue DM, Finney, M, Ruvkun, G and M Chalfie 1992. Regulation of the mec-3 gene by the C. elegans homeoproteins Unc-86 and Mec-3. EMBO J. 11: 4969‑4979. PMCID: PMC556975

21. Bürglin TR and G Ruvkun 1992. New motif in PBX genes. Nature Genet 1: 319‑320. PMID: 1363814

22. Wightman B, I Ha, and G Ruvkun 1993. Post-transcriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75: 855-862. PMID: 8252622

23. Gottlieb S and G Ruvkun 1994. daf-2, daf-16, daf-23: genetically interacting genes controlling dauer formation in C. elegans. Genetics 137: 107-120. PMCID: PMC1205929

24. Greenstein D, Hird, S Plasterk, R, Andachi, Y, Kohara, Y, Wang, B, Finney, M, and G Ruvkun 1994. Targeted mutations in the C. elegans POU-homeobox gene ceh-18 cause defects in oocyte cell cycle arrest, gonad migration, and epidermal differentiation. Genes and Development 8: 1935-1948. PMID: 7958868

25. Baumeister R, Liu, Y, and G Ruvkun 1996. Lineage specific regulators couple cell lineage asymmetry to the transcription of the Caenorhabditis elegans POU gene unc-86 during neurogenesis. Genes and Development 10: 1395-1410. PMID: 8647436

26. Morris JZ, Tissenbaum, HA and G Ruvkun 1996. A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans. Nature 382: 536-538. PMID: 8700226

27. Barnes TM, Jin, Y, Horvitz, HR, Ruvkun, G, and S Hekimi 1996. The C. elegans behavioral gene unc-24 encodes a novel bipartite protein similar to both erythrocyte band 7.2 (stomatin) and non-specific lipid transfer protein. J. of Neurochemistry 67: 46-57. PMID: 8667025

28. Ha I, Wightman, B and G Ruvkun 1996. A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation. Genes and Development 10: 3041-3050. PMID: 8957004

29. Sze JY, Liu, Y and G Ruvkun 1997. VP16-activation of the C. elegans neural specification transcription factor UNC-86 suppresses mutations in downstream genes and causes defects in neural migration and axon outgrowth. Development 124: 1159-1168. PMID: 9102303

30. Sluder AE, Lindblom, T and G. Ruvkun 1997. The Caenorhabditis elegans orphan nuclear hormone receptor gene nhr-2 functions in early embryonic development. Developmental Biology 184: 303-319. PMID: 9133437

31. Hobert O, Mori, I, Yamashita, Y, Honda, H, Ohshima, Y, Liu, Y and G Ruvkun 1997. Regulation of interneuron function in the C. elegans thermoregulatory pathway by the ttx-3 LIM homeobox gene. Neuron 19: 345-357. PMID: 9292724

32. Patterson G I, Koweek, A, Wong, A, Liu, Y, and G Ruvkun 1997. The DAF-3 Smad protein antagonizes TGF-beta-related receptor signaling in the C. elegans dauer pathway. Genes and Development 11: 2679-2690. PMCID: PMC316611

33. Kimura KD, Tissenbaum, HA, Liu, Y and G Ruvkun 1997. daf-2, an insulin receptor-like gene that regulates longevity and diapause in C. elegans. Science 277: 942-946. PMID: 9252323

34. Ogg S, Paradis, S , Gottlieb, S, Patterson, GI, Lee, L, Tissenbaum, HA, and G Ruvkun 1997. The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389: 994-999. PMID: 9353126

35. Tissenbaum HA and G Ruvkun 1998. An insulin-like signaling pathway affects both longevity and reproduction in C. elegans. Genetics 148: 703-717. PMCID: PMC1459840

36. Hobert O, Liu, Y, D’Alberti, T, and G Ruvkun 1998. Control of neural development and function in a thermoregulatory network by the LIM homeobox gene lin-11. J. Neuroscience 18: 2084-2096. PMID: 9482795

37. Paradis S and G Ruvkun 1998. Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes and Development 12: 2488-98. PMCID: PMC317081

38. Ruvkun G and O Hobert 1998. The taxonomy of developmental control in Caenorhabditis elegans. Science 282: 2033-41. PMID: 9851920

39. Ogg S and G Ruvkun 1998. The C. elegans PTEN homolog DAF-18 acts in the insulin receptor-like metabolic signaling pathway. Mol Cell 2: 887-93. PMID: 9885576

40. Hobert O and G Ruvkun 1998. A common theme for LIM homeobox gene function across phylogeny? Biol. Bull. 195: 377-80. PMID: 9924780

41. Slack F and G Ruvkun 1998. A novel repeat domain that is often associated with RING finger and B-box motifs. Trends Biochem Sci 23:474-5. PMID: 9868369

42. Hobert O, Moerman DG, Clark KA, Beckerle MC, and G Ruvkun 1999. A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in Caenorhabditis elegans. J Cell Biol 144: 45-57. PMCID: PMC2148118

43. Hobert O, Tessmar K and G Ruvkun 1999. The C. elegans lim-6 LIM homeobox gene regulates neurite outgrowth and function of particular GABAergic neurons. Development 126, 1547-1562. PMID: 10068647

44. Paradis S, Ailion, M, Toker A, Thomas, JH, and G Ruvkun 1999. A PDK1 homolog is necessary and sufficient to Transduce AGE-1 PI3 kinase signals that regulate diapause in C. elegans. Genes and Development 13: 1438-1452. PMCID: PMC316759

45. Sagasti A, Hobert O, Troemel ER, Ruvkun G, Bargmann CI 1999. Alternative olfactory neuron fates are specified by the LIM homeobox gene lim-4. Genes Dev 13: 1794-80. PMCID: PMC316880

46. Tissenbaum HA Hawdon, J, Perregaux, M , Hotez, P, Guarente, L, and G Ruvkun 2000. A common muscarinic pathway for diapause recovery in the distantly related nematode species Caenorhabditis elegans and Ancylostoma caninum. Proc. Natl. Acad. Sci. 97: 460-465. PMCID: PMC26685

47. Sze JY, Victor, M, Loer, C, Shi, Y, and G Ruvkun 2000. Food and metabolic signaling defects in a C. elegans serotonin-synthesis mutant, Nature 403: 560-4. PMID: 10676966

48. Reinhart BJ, Slack, FA., Basson, M, Pasquinelli, AE, Bettinger, JC, Rougvie, AC, Horvitz, HR, and G Ruvkun 2000. The 21 nucleotide let-7 RNA regulates developmental timing in C. elegans. Nature, 403: 901 - 906. PMID: 10706289

49. Slack FJ, Basson, M, Liu, Z, Ambros, V, Horvitz, HR, and G Ruvkun 2000. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the lin-29 transcription factor. Mol. Cell 5: 659-69. PMID: 10882102

50. Kagoshima H, Sommer, R, Reinhart, B , Ruvkun, G, Cassata, G, and TR Bürglin 2000. Graded expression of ceh-14 reporters in the hypodermis is induced by a gonadal signal. Development Genes and Evolution 210:564-569. PMID: 11180807

51. Nasrin N, Ogg, S, Cahill, C, Biggs, W, Nui, S, Dore, J, Calvo, D, Shi, Y, Ruvkun, G, and M Alexander-Bridges 2000. DAF-16 recruits the CREB-binding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells. Proc. Natl. Acad. Sci., 97: 10412-7. PMCID: PMC27038

52. Pasquinelli A, Reinhart, B, Slack F , Maller, B, Kuroda, M, Martindale, M, Srinivasan, A, Fishman, M, Hayward D, Ball E, Degnan, B, Müller, P , Spring, J, Finnerty, J, Corbo, J, Levine, M, Leahy, P , Davidson, E, and G Ruvkun 2000. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 408: 86-89. PMID: 11081512

53. Wolkow CA, Kimura, KD, Lee, M and G Ruvkun 2000. Regulation of C. elegans life-span by insulinlike signaling in the nervous system. Science 290:147-50. PMID: 11021802

54. Cahill CM, Tzivion, G, Nasrin, N, Ogg, S, Dore, J, Ruvkun, G, and M Alexander-Bridges 2001. Phosphatidylinositol 3-kinase signaling inhibits DAF-16 DNA binding and function via 14-3-3-dependent and 14-3-3-independent pathways. J. Biol. Chem. 276: 13402-10. PMID: 11124266

55. Reinhart BJ and G Ruvkun 2001. Isoform-specific mutations in the Caenorhabitis elegans heterochronic gene lin-14 affect stage-specific patterning. Genetics 157: 199-209. PMCID: PMC1461488

56. Bürglin TR and G Ruvkun 2001. Regulation of ectodermal and excretory cell function by the C. elegans POU homeobox gene ceh-6 Development 128: 779-790. PMID: 11171402

57. Pierce SB, Costa, M, Wisotzkey, R , Devadhar, S, Homburger, SA, Buchman, AR, Ferguson, KC, Heller, J, Platt, DM , Liu, LX , Pasquinelli, AE, Doberstein, SK, G Ruvkun 2001. Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes and Development 15: 672-686. PMCID: PMC312654

58. Grishok A, Pasquinelli, AE, Conte, D, Li, N, Parrish, S, Ha, I, Baillie, DL, Fire, A, Ruvkun, G, and Mello, CC 2001. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control Caenorhabditis elegans developmental timing. Cell 106: 23-34. PMID: 11461699

59. Lee RYN, Hench, J, and G Ruvkun 2001. Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr Biol. 11: 1950-1957. PMID: 11747821

60. Sze JY, Zhang, S, Li, J, and G Ruvkun 2002. The C. elegans POU-domain transcription factor UNC-86 regulates the tph-1 tryptophan hydroxylase gene and neurite outgrowth in specific serotonergic neurons. Development, 129:3901-11. PMID: 12135927

61. Ruvkun G, M Finney, G Church, M Zuber, W Gilbert 2002. A Robotic-PCR Detector for DNA-based Life on Other Planets, in Signs of Life: A Report based on the April 2000 Workshop on Life Detection Techniques. Space Sciences Board, NASA.

62. Wolkow CA, Munoz MJ, Riddle DL, G Ruvkun 2002. Insulin receptor substrate and p55 orthologous adaptor proteins function in the Caenorhabditis elegans daf-2/insulin-like signaling pathway. J Biol Chem. 277:49591-7. PMID: 12393910

63. Lee SS, Lee, RY, Fraser, AG, Kamath, RS, Ahringer, J and G Ruvkun 2002. A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nature Genetics, 33:40-8. PMID: 12447374

64. Ashrafi K, Chang, FY,Watts, JL, Fraser, AG, Kamath, RS, Ahringer, J and G Ruvkun 2003. Genome-wide RNAi analysis of C. elegans fat regulatory genes. Nature, 421:268-72. PMID: 12529643

65. Li W and G Ruvkun 2003. daf-28 encodes a C. elegans insulin superfamily member that is regulated by environmental cues and acts in the DAF-2 signaling pathway. Genes and Development, 17: 844-58. PMCID: PMC196030

66. Bashirullah A, Pasquinelli, AE Kiger, A, Perrimon, N, Ruvkun, G, and C S Thummel 2003. Coordinate regulation of small temporal RNAs at the onset of Drosophila metamorphosis. Developmental Biology, 259:1-8. PMID: 12812783

67. Pasquinelli AE, McCoy, A Jimenez, E Salo, G Ruvkun, M Martindale, and J Baguñà 2003. Expression of the 22 nucleotide let-7 heterochronic RNA throughout the Metazoa: A role in life history evolution? Evolution and Development, 5:372-378. PMID: 12823453

68. Aspöck G, G Ruvkun, and T Bürglin 2003. The Caenorhabditis elegans ems class homeobox gene ceh-2 is required for M3 pharynx motoneuron function. Development, 130:3369-3378. PMID: 12810585

69. Lee SS, Kennedy,S, Tolonen, AC and G Ruvkun 2003. DAF-16 target genes that control C. elegans life-span and metabolism. Science. 300:644-7. PMID: 12690206

70. Grad Y, Aach, J, Hayes, GD, Reinhart, B, Church, GM, Ruvkun, G, and J Kim. 2003. Computational and experimental identification of C. elegans microRNAs. Molecular Cell, 11:1253-63. PMID: 12769849

71. Garsin DA. JM Villanueva, J Begun, DH Kim, CD Sifri, SB Calderwood, G Ruvkun, and FM Ausubel 2003. Long-Lived Caenorhabditis elegans daf-2 mutants are resistant to bacterial pathogens, Science, 300:1921. PMID: 12817143

72. Ruvinsky I and G Ruvkun 2003. Functional tests of enhancer conservation between distantly related species, Development, 130:5133-42. PMID: 12944426

73. Sze JY and G Ruvkun 2003. Activity of the Caenorhabditis elegans UNC-86 POU transcription factor modulates olfactory sensitivity, Proc. Natl. Acad. Sci., 100:9560-5. PMCID: PMC170957

74. Kim J, Krichevsky, A, Grad, Y, Hayes, GD, Kosik, KS, Church, GM, and G Ruvkun 2004. Identification of many microRNAs that copurify with polyribosomes in mammalian neurons. Proc. Natl. Acad. Sci.101: 360-5. PMCID: PMC314190

75. Kennedy SK, D Wang, and G Ruvkun 2004. A conserved siRNA degrading RNase negatively regulates RNA interference in C. elegans. Nature 427: 645-9. PMID: 14961122

76. Tewari M, Hu PJ, Ahn JS, Ayivi-Guedehoussou N, Vidalain PO, Li S, Milstein S, Armstrong CM, Boxem M, Butler MD, Busiguina S, Rual JF, Ibarrola N, Chaklos ST, Bertin N, Vaglio P, Edgley ML, King KV, Albert PS, Vandenhaute J, Pandey A, Riddle DL, Ruvkun G, Vidal M 2004. Systematic interactome mapping and genetic perturbation analysis of a C. elegans TGF-beta signaling network. Molecular Cell 13:469-82. PMID: 14992718

77. Mak HY and G Ruvkun 2004. Intercellular signaling of reproductive development by the C. elegans DAF-9 cytochrome P450. Development, 131:1777-86. PMID: 15084462

78. Mansfield JH, Harfe BD, Nissen R, Obenauer J, Srineel J, Chaudhuri A, Farzan-Kashani R, Zuker M, Pasquinelli AE, Ruvkun G, Sharp PA, Tabin CJ, McManus MT 2004. MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression. Nat Genet. 36:1079-83. PMID: 15361871

79. Wang D and G Ruvkun 2004. The insulin pathway regulates RNAi in C. elegans. Cold Spring Harbor Symposium on Quantitative Biology, 69th edition, 69: 429-433.

80. Kim JK, HW Gabel, RS Kamath, M Tewari, A Pasquinelli, S Kennedy, M Dybbs, N Bertin, M Vidal, JM Kaplan, and G Ruvkun 2005. Functional genomic analysis of RNA interference in C. elegans. Science 308: 1164-7. PMID: 15790806

81. Efimenko E, Bubb K, Mak HY, Holzman T, Leroux MR, Ruvkun G, Thomas JH, Swoboda P 2005. Analysis of xbx genes in C. elegans. Development. 132:1923-34. PMID: 15790967

82. Hamilton B , Y Dong, M Shindo, W Liu, G Ruvkun, and SS Lee 2005. A systematic RNAi screen for longevity genes in C. elegans. Genes and Development 19:1544-55. PMCID: PMC1172061

83. Sieburth D, Q Ch ng, M Dybbs, M Tavazoie, S Kennedy, D Wang, D Dupuy, J Rual, D Hill, M Vidal, G Ruvkun, and J Kaplan 2005. Systematic analysis of genes required for synapse structure and function. Nature 436: 510-517. PMID: 16049479

84. Wang DS, S Kennedy, D Conte, J Kim, H Gabel, R Kamath, C Mello, and G Ruvkun 2005. Somatic misexpression of germline P granules and enhanced RNA interference in Retinoblastoma pathway mutants. Nature 436: 593-597. PMID: 16049496

85. Frand AR, S Russel, G Ruvkun 2005. Functional genomic analysis of C. elegans molting. PLoS Biol. 2005 Oct3(10):e312. doi: 10.1371/journal.pbio.0030312. Epub 2005 Aug 30.PMID: 16122351

86. Duchaine TF, JA Wohlschlegel, S Kennedy, Y Bei, D Conte, K Pang, DR Brownell, S Harding, S Mitani, G Ruvkun, JR Yates, and CC Mello 2006. Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways. Cell 124:343-54. PMID: 16439208

87. Mak HY, LS Nelson, M Basson, CD Johnson and G Ruvkun 2006. Polygenic control of C. elegans fat storage. Nature Genetics 38:363-8. PMID: 16462744

88. Sandoval GM, JS Duerr, J Hodgkin, JB Rand and G Ruvkun 2006. A genetic interaction between the vesicular acetylcholine transporter VAChT/UNC-17 and synaptobrevin/SNB-1 in C. elegans. Nature Neuroscience, 9:599-601. PMID: 16604067

89. Hu PJ, J Xu, and G Ruvkun 2006. Two membrane-associated tyrosine phosphatase homologs potentiate C. elegans AKT-1/PKB signaling. PLoS Genet. 2006 Jul2(7):e99. PMCID: PMC1487177

90. Hayes GD, AR Frand, and G. Ruvkun 2006. The mir-84 and let-7 paralogous microRNA genes of Caenorhabditis elegans direct the cessation of molting via the conserved nuclear hormone receptors NHR-23 and NHR-25. Development 133: 4631-4641. PMID: 17065234

91. Ruvinsky I, U Ohler, CB Burge, and G Ruvkun 2006. Detection of broadly expressed neuronal genes in C. elegans. Dev Biol. 302: 617-26. PMID: 17046742

92. Hayes GD and G Ruvkun 2006. Misexpression of the C. elegans miRNA let‑7 is sufficient to drive developmental programs. Cold Spring Harbor Symp Quant Biol. 71: 21-7. Regulatory RNAs. PMID: 17381276

93. Curran SP and G Ruvkun 2007. Lifespan regulation by evolutionarily conserved genes essential for viability. PloS Genetics 3(4):e56. PMCID: PMC1847696

94. Kim N, Dempsey C, Kuan CJ, Zoval J, O'Rourke E, Ruvkun G, Madou M, J Sze 2007. Gravity force transduced by the MEC-4/MEC-10 DEG/ENaC channel modulates DAF-16/FoxO activity in C. elegans. Genetics. 177:835-45. PMCID: PMC2034647

95. Samuelson AV, Carr, CE, and G Ruvkun 2007. Gene activities that mediate increased lifespan of C. elegans insulin-like signaling mutants. Genes and Development 21:2976-94. PMCID: PMC2049198

96. Parry DH, Xu, J and G Ruvkun 2007 A whole-genome RNAi screen for C. elegans miRNA pathway genes. Current Biology 17: 2013-22. PMCID: PMC2211719

97. Isenbarger TA, M Finney, C Ríos-Velázquez, J Handelsman, and G Ruvkun 2007. Miniprimer PCR, a new lens for viewing the microbial world. Appl Environ Microbiol. 2008 Feb74(3):840-9. PMCID: PMC2227730

98. Pierce ML, Weston MD, Fritzsch B, Gabel HW, Ruvkun G, Soukup GA 2008. MicroRNA-183 family conservation and ciliated neurosensory organ expression. Evol Dev. 10(1):106-13. PMCID: PMC2637451

99. Samuelson AV, RR Klimczak, D Thompson, CE Carr, and G Ruvkun 2008. Identification of C. elegans genes regulating longevity using enhanced RNAi-sensitive strains. Cold Spring Harb Symp Quant Biol. 200772:489-97. PMID: 18419309

100. Gabel HW and G. Ruvkun 2008. The exonuclease ERI-1 has a conserved dual role in 5.8S rRNA processing and RNAi. Nat Struct Mol Biol. 15: 531-3. PMCID: PMC2910399

101. Patel DS, Fang LL, Svy DK, Ruvkun G, Li W 2008. Genetic identification of HSD-1, a conserved steroidogenic enzyme that directs larval development in Caenorhabditis elegans. Development. 135: 2239-49. PMID: 18495818

102. Simon DJ, Madison JM, Conery AL, Thompson-Peer KL, Soskis M, Ruvkun G, Kaplan JM, Kim JK 2008. The microRNA miR-1 regulates a MEF-2-dependent retrograde signal at neuromuscular junctions. Cell 133: 903-15. PMCID: PMC2553566

103. Fischer SEJ, MD Butler, Q Pan and G Ruvkun 2008. Trans-splicing in C. elegans generates the negative RNAi regulator ERI-6/7. Nature 455: 491-6. PMCID: PMC2756026

104. Wang MC, E O’Rourke and G Ruvkun 2008. Fat metabolism links germline stem cells and longevity in C. elegans. Science 322: 957-60. PMCID: PMC2760269

105. Isenbarger TA, CE Carr, SS Johnson, M Finney, GM Church, W Gilbert, MT Zuber, and G Ruvkun 2008. The most conserved genome segments for life detection on Earth and other planets, Origin of Life and Evolution of Biospheres, 38(6):517-33. Oct 14. PMID: 18853276

106. Soukas AA, EA Kane, CE Carr, JA Melo, and G Ruvkun 2009. Rictor/TORC2 regulates fat metabolism, feeding, growth, and lifespan in Caenorhabditis elegans. Genes and Development 23: 496-511. PMCID: PMC2648650

107. Butcher RA, JR Ragains, G Ruvkun, J Clardy, HY Mak. 2009 Biosynthesis of the C. elegans dauer pheromone. Proceedings of the National Academy of Sciences 106(6):1875-9. PMCID: PMC2631283

108. Curran SP, X Wu, C Riedel, and G Ruvkun 2009. A soma-to-germline transformation in long-lived C. elegans mutants. Nature 459: 1079-84. PMCID: PMC2716045

109. O'Rourke EJ, Soukas AA, Carr CE, G Ruvkun 2009. C. elegans major fats are stored in vesicles distinct from lysosome-related organelles. Cell Metab.10(5):430-5. PMCID: PMC2921818

110. Sebastiani P, Montano M, Puca A, Solovieff N, Kojima T, Wang MC, Melista E, Meltzer M, Fischer SE, Andersen S, Hartley SH, Sedgewick A, Arai Y, Bergman A, Barzilai N, Terry DF, Riva A, Anselmi CV, Malovini A, Kitamoto A, Sawabe M, Arai T, Gondo Y, Steinberg MH, Hirose N, Atzmon G, Ruvkun G, Baldwin CT, Perls TT 2009. RNA editing genes associated with extreme old age in humans and with lifespan in C. elegans. PLoS One. 2009 Dec 144(12):e8210. PMCID: PMC2788130

111. Meli VS , Osuna B, Ruvkun G, Frand AR 2010. MLT-10 Defines a Family of DUF644 and Proline-rich Repeat Proteins Involved in the Molting Cycle of Caenorhabditis elegans. Mol Biol Cell. May 1521(10):1648-61. PMCID: PMC2869372

112. Zhang C, TA Montgomery, HW Gabel, SE Fischer, CM Phillips, N Fahlgren, C Sullivan, JC Carrington, and G Ruvkun 2011. mut-16 and other mutator-class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans. Proc. Natl. Acad. Sci., Jan 25108(4):1201-8. Epub Jan 18. PMCID: PMC3029761

113. Wang MC, W Min, G Ruvkun, XS Xie 2011. RNAi screening for fat regulatory genes with SRS microscopy. Nature Methods, Feb8(2):135-8. Epub Jan 16. PMCID: PMC3061290

114. Hayes GD, C Riedel, and G Ruvkun 2011. The Caenorhabditis elegans SOMI-1 zinc finger protein and SWI/SNF promote regulation of development by the mir-84 microRNA. Genes Dev. Oct 125(19):2079-92. PMCID: PMC3197206

115. Fischer SEJ, TA Montgomery, C Zhang, N Fahlgren, A Hwang, CM Sullivan, JC Carrington and G Ruvkun 2011. The ERI-6/7 helicase acts at the first stage of an siRNA amplification pathway that targets recent gene duplications. PLoS Genet. Nov7(11):e1002369. Epub Nov 10. PMCID: PMC3213143

116. Russel S, AR Frand, and G Ruvkun 2011. Regulation of the C. elegans molt by pqn-47. Developmental Biology 360: 297-309. PMCID: PMC3618673

117. Kimura KD, Riddle DL, and G. Ruvkun 2011 The C. elegans DAF-2 insulin-like receptor is abundantly expressed in the nervous system and regulated by nutritional status. Cold Spring Harb Symp Quant Biol. 76:113-20. Epub Nov 28. PMID: 22123849

118. Wu X, Z Shi, M Cui, M Han and G. Ruvkun 2012. Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes. PLoS Genet. Mar8(3):e1002542. Epub 2012 Mar 8. PMCID: PMC3297578

119. Shi Z and G. Ruvkun 2012. The mevalonate pathway regulates microRNA activity in Caenorhabditis elegans. Proc Natl Acad Sci U S A. Mar 20109(12):4568-73. Epub Mar 6. PMCID: PMC3311396

120. Montgomery TA, Rim, E, Zhang, C, Dowen RH, Phillips CM, Fischer, SEJ and G. Ruvkun 2012. PIWI associated siRNAs and piRNAs specifically require the C. elegans HEN1 ortholog henn-1. PLoS Genetics, Apr8(4):e1002616. Epub Apr 19. PMCID: PMC3334881

121. Melo JA and G Ruvkun 2012. Inactivation of conserved C. elegans genes engages pathogen- and xenobiotic-associated defenses. Cell 149:452-66. PMCID: PMC3613046

123. Zhang C, TA Montgomery, SE Fischer, SM Garcia, CG Riedel, N Fahlgren, CM Sullivan, JC Carrington and G Ruvkun 2012. The Caenorhabditis elegans RDE-10/RDE-11 complex regulates RNAi by promoting secondary siRNA amplification. Current Biology 22:881-90. Epub Apr 26. PMCID: PMC3371361

124. Phillips CM, PC Breen, TA Montgomery and G Ruvkun 2012. MUT-16 promotes formation of perinuclear mutator foci required for RNA silencing in the C. elegans germline. Genes and Development 26:1433-44. Epub Jun 19. PMCID: PMC3403012

125. Shore DE, CE Carr and G Ruvkun 2012. Induction of cytoprotective pathways is central to the extension of lifespan conferred by multiple longevity pathways. PloS Genetics 8(7):e1002792. PMCID: PMC3400582

126. Wählby C, Kamentsky L, Liu ZH, Riklin-Raviv T, Conery AL, O'Rourke EJ, Sokolnicki KL, Visvikis O, Ljosa V, Irazoqui JE, Golland P, Ruvkun G, Ausubel FM, Carpenter AE 2012. An image analysis toolbox for high-throughput C. elegans assays Nat Methods 9:714-6. doi: 10.1038/nmeth.1984. PMCID: PMC3433711

127. Tacutu R, Shore DE, Budovsky A, de Magalhães JP, Ruvkun G, Fraifeld VE, Curran SP. Prediction of C. elegans Longevity Genes by Human and Worm Longevity Networks 2012. PLoS One 20127(10):e48282. doi: 10.1371/journal.pone.0048282. Epub 2012 Oct 29. PMCID: PMC3483217

128. Carr CE, H Rowedder, C Vafadari, CS Lui, E Cascio, MT Zuber, and G Ruvkun 2013. Radiation resistance of biological reagents for in-situ life detection. Astrobiology Jan13(1):68-78. doi: 10.1089/ast.2012.0869. PMID: 23330963

129. Tabach Y, A Billi, G Hayes, O Zuk, H Gabel, R Kamath, M Newman, K Yacoby, B Chapman, M Borowsky, J Kim, and G Ruvkun 2013. Identification of small RNA pathway genes using patterns of phylogenetic conservation and divergence. Nature Jan 31493(7434):694-8. doi: 10.1038/nature11779. Epub 2012 Dec 23. PMCID: PMC3762460

130. Shi Z, T Montgomery, Y Qi and G Ruvkun 2013. High-throughput sequencing reveals extraordinary fluidity of miRNA, piRNA and siRNA pathways in nematodes. Genome Research Mar23(3):497-508. doi: 10.1101/gr.149112.112. Epub 2013 Jan 30. PMCID: PMC3589538

131. O’Rourke EA, P Kuballa, R Xavier, and G Ruvkun 2013. w-6 Polyunsaturated fatty acids extend life span through the activation of autophagy. Genes and Development Feb 1527(4):429-40. doi: 10.1101/gad.205294.112. Epub Feb 7. PMCID: PMC3589559

132. Riedel CG, G Lima, NV. Kirienko, T Heimbucher, JA West, A Dillin, J Asara, and G Ruvkun 2013. DAF-16 employs the chromatin remodeller SWI/SNF to promote stress resistance and longevity. Nature Cell Biology, 2013 May15(5):491-501. doi: 10.1038/ncb2720. Epub Apr 21. PMCID: PMC3748955

133. Kirienko NV, DR Kirienko, J Larkins-Ford, C Wählby, G Ruvkun, FM Ausubel 2013 Pseudomonas aeruginosa disrupts Caenorhabditis elegans iron homeostasis, causing a hypoxic response and death. Cell Host and Microbe, 13(4):406-16. doi: 10.1016/j.chom.2013.03.003. PMCID: PMC3641844

134. O’Rourke EA and G Ruvkun 2013. MXL-3 and HLH-30 transcriptionally link lipolysis and autophagy to nutrient availability. Nature Cell Biology, 2013 Jun15(6):668-76. doi: 10.1038/ncb2741. Epub 2013 Apr 21. PMCID: PMC3723461

135. Carr CE, Rowedder H, Lui CS, Zlatkovsky I, Papalias CW, Bolander J, Myers JW, Bustillo J, Rothberg JM, Zuber MT, and G Ruvkun 2013. Radiation Resistance of Sequencing Chips for in situ Life Detection. Astrobiology. 2013 Jun 4. PMID: 23734755

136. Shi Z, G Hayes, and G Ruvkun 2013. Dual regulation of the lin-14 target mRNA by the lin-4 miRNA. PloS One, Sep 138(9):e75475. doi: 10.1371/journal.pone.0075475. PMCID: PMC3772890

137. Tabach Y, T Golan, A Hernández-Hernández AR Messer, T Fukuda, A Kouznetsova, JG Liu, I Lilienthal, C Levy, and G Ruvkun 2013. Human disease locus discovery and mapping to molecular pathways through phylogenetic profiling. Molecular Systems Biology, Oct 19:692. doi: 10.1038/msb.2013.50. PMCID: PMC3817400

138. Soukas AA, CE Carr, and G Ruvkun 2013. Genetic Regulation of C. elegans Lysosome Related Organelle Function. PloS Genetics, Oct9(10):e1003908. doi: 10.1371/journal.pgen.1003908. Epub Oct 24. PMCID: PMC3812091

139. Fischer SEJ, Q Pan, PC. Breen, Y Qi, Z Shi, C Zhang, TA Montgomery, and G Ruvkun 2013. Multiple small RNA pathways regulate the silencing of repeated and foreign genes in C. elegans. Genes and Development, 2013 Dec 1527(24):2678-95. doi: 10.1101/gad.233254.113, PMCID: PMC3877757

140. Schwartz S, Agarwala SD, Mumbach MR, Jovanovic M, Mertins P, Shishkin A, Tabach Y, Mikkelsen TS, Satija R, Ruvkun G, Carr SA, Lander ES, Fink GR, Regev A. 2013. High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis. Cell. 2013 Dec 5155(6):1409-21. doi: 10.1016/j.cell.2013.10.047. Epub 2013 Nov 21. PMCID: PMC3956118

141. Liu Y, BS Samuel, PC Breen and G Ruvkun 2014. Caenorhabditis elegans pathways that surveil and defend mitochondria. Nature. 2014 Apr 17508(7496):406-10. doi: 10.1038/nature13204. Epub 2014 Apr 2. PMCID: PMC4102179

142. Phillips CM, Rim Y, Roovers, E, Breen PC, Ohsumi, TK, van Wolfswinkel, JC, Ketting, RF, Ruvkun, G, TA Montgomery 2014. MUT-14 and SMUT-1 DEAD box RNA helicases have overlapping roles in germline RNAi and endogenous siRNA formation. Curr Biol. 2014 Apr 1424(8):839-44. doi: 10.1016/j.cub.2014.02.060. Epub 2014 Mar 27. PMCID: PMC4010136

143. Garcia SMDA, Y Tabach, G Lourenço, and G Ruvkun 2014. Identification of genes in toxicity pathways of trinucleotide-repeat RNA in C. elegans. Nature Structural and Molecular Biology, 2014 Aug21(8):712-20. doi: 10.1038/nsmb.2858. Epub 2014 Jul 20.PMID: 25038802.

144. Wang MC, HD Oakley, CE Carr, JN Sowa, G Ruvkun 2014. Gene pathways that delay Caenorhabditis elegans reproductive senescence. PLoS Genet. 2014 Dec 410(12):e1004752. doi: 10.1371/journal.pgen.1004752. eCollection 2014 Dec. PMID: 25474471

145. Kirienko NV, FM Ausubel, and G Ruvkun 2015. Mitophagy confers resistance to siderophore-mediated killing by Pseudomonas aeruginosa. Proc Natl Acad Sci U S A., 2015 Feb 10112(6):1821-6. doi: 10.1073/pnas.1424954112. Epub 2015 Jan 26. PMID:25624506

146. Sadreyev IR, Ji F, Cohen E, Ruvkun G, Tabach Y. 2015. PhyloGene server for identification and visualization of co-evolving proteins using normalized phylogenetic profiles. Nucleic Acids Res. Jul 143(W1):W154-9. doi: 10.1093/nar/gkv452. Epub 2015 May 9. PMID: 25958392

147. Phillips CM, Brown KC, Montgomery BE, Ruvkun G, Montgomery TA. 2015. piRNAs and piRNA-dependent siRNAs protect conserved and essential C. elegans genes from misrouting into the RNAi pathway. Dev Cell. 2015 Aug 2434(4):457-65. doi: 10.1016/j.devcel.2015.07.009. Epub 2015 Aug 13.PMID:26279487

148. Govindan JA, E Jayamani, X Zhang, P Breen, J Larkins-Ford, E Mylonakis and G Ruvkun. 2015. Lipid signalling couples translational surveillance to systemic detoxification in Caenorhabditis elegans. Nat Cell Biol. 2015 Oct17(10):1294-303. doi: 10.1038/ncb3229. Epub 2015 Aug 31.PMID: 26322678

149. Govindan JA, Jayamani E, Zhang X, Mylonakis E, and G Ruvkun. 2015. Dialogue between E. coli free radical pathways and the mitochondria of C. elegans. Proc Natl Acad Sci U S A. 2015 Sep 21. pii: 201517448. PMID: 26392561

150. Rettberg P, Anesio AM, Baker VR, Baross JA, Cady SL, Detsis E, Foreman CM, Hauber E, Ori GG, Pearce DA, Renno NO, Ruvkun G, Sattler B, Saunders MP, Smith DH, Wagner D, Westall F. 2016. Planetary Protection and Mars Special Regions-A Suggestion for Updating the Definition. Astrobiology. 2016 Feb16(2):119-25. doi: 10.1089/ast.2016.1472. PMID: 26848950.

151. Samuel BS, H Rowedder, C Braendle, MA Félix, G Ruvkun. 2016. Caenorhabditis elegans responses to bacteria from its natural habitats. Proc Natl Acad Sci U S A., 2016.. pii: 201607183. PMID:27317746

152. Dowen RH, PC Breen, T Tullius, AL Conery, and G Ruvkun. 2016. A microRNA program in the C. elegans hypodermis couples to intestinal mTORC2/PQM-1 signaling to modulate fat transport. Genes Dev. 2016 Jul 130(13):1515-28. doi: 10.1101/gad.283895.116. PMID:27401555

153. Lehrbach NJ and G Ruvkun. 2016. Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1. bioRxiv preprint posted online May. 12, 2016 doi: http://dx.doi.org/10.1101/052936. Elife. 2016 Aug 165. pii: e17721. doi: 10.7554/eLife.17721. PMID:27528192

154. Mojarro A, Ruvkun G, Zuber MT, Carr CE. Nucleic Acid Extraction from Synthetic Mars Analog Soils for in situ Life Detection. Astrobiology. 2017 Jul 13. doi: 10.1089/ast.2016.1535. PMID:28704064

155. Pontefract A, TF Zhu, VK Walker, H Hepburn, C Lui, MT Zuber, G Ruvkun, and CE Carr. 2017. Microbial Diversity in a Hypersaline Sulfate Lake: A Terrestrial Analog of Ancient Mars. Frontiers in Microbiology, section Extreme Microbiology, Front Microbiol. 2017 Sep 268:1819. doi: 10.3389/fmicb.2017.01819. eCollection 2017. PMID:29018418

156. Mojarro A, J Hachey , G Ruvkun, MT Zuber, & CE Carr. 2018. CarrierSeq: a sequence analysis workflow for low-input nanopore sequencing. BMC Bioinformatics. 2018 Mar 2719(1):108. doi: 10.1186/s12859-018-2124-3. PMID:29587645

157. Newman MA, Ji F, Fischer SEJ, Anselmo A, Sadreyev RI, G Ruvkun. 2018. The surveillance of pre-mRNA splicing is an early step in C. elegans RNAi of endogenous genes. Genes Dev. 2018 May 132(9-10):670-681. doi: 10.1101/gad.311514.118. Epub 2018 May 8. PMID: 29739806

158. Alexander JK, JR Casani, L Chiao, DP Fidler, J Gabrynowicz, GS Hubbard, EH Levy, N Noonan, K Olden, F Raulin, G Ruvkun, MP Saunders, BA Simmons, PD Stabekis, A Steele, DH Smith. 2018. Review and Assessment of Planetary Protection Policy Development Processes. Committee on the Review of Planetary Protection Policy Development Processes, Space Studies Board. https://doi.org/10.17226/25172.

159. Carr CE, NC Bryan, KN Saboda, SA Bhattaru, G Ruvkun, MT Zuber. 2018. Acceleration profiles and processing methods for parabolic flight NPJ Microgravity. 2018 Aug 74:14. doi: 10.1038/s41526-018-0050-3. eCollection 2018. PMID: 30109261

160. Pontefract A, Hachey J, Zuber MT, Ruvkun G, Carr CE 2018. Sequencing nothing: Exploring failure modes of nanopore sensing and implications for life detection. Life Sci Space Res (Amst). 2018 Aug18:80-86. doi: 10.1016/j.lssr.2018.05.004. Epub 2018 May 16. PMID: 30100151

161. Tillman EJ, Richardson CE, Cattie DJ, Reddy KC, Lehrbach NJ, Droste R, Ruvkun G, Kim DH. 2018. Endoplasmic Reticulum Homeostasis Is Modulated by the Forkhead Transcription Factor FKH-9 During Infection of Caenorhabditis elegans.Genetics. 2018 Dec210(4):1329-1337. doi: 10.1534/genetics.118.301450. Epub 2018 Oct 4. PMID:30287474

162. Lin XX, Sen I, Janssens GE, Zhou X, Fonslow BR, Edgar D, Stroustrup N, Swoboda P, Yates JR 3rd, Ruvkun G, Riedel CG. DAF-16/FOXO and HLH-30/TFEB function as combinatorial transcription factors to promote stress resistance and longevity. Nat Commun. 2018 Oct 239(1):4400. doi: 10.1038/s41467-018-06624-0. PMID: 30353013

163. Kniazeva M and G Ruvkun. 2019. Rhizobium induces DNA damage in Caenorhabditis elegans intestinal cells. Proceedings of the National Academy of Sciences, Proc Natl Acad Sci U S A. 2019 Feb 26116(9):3784-3792. doi: 10.1073/pnas.1815656116. Epub 2019 Feb 11. PMID: 30808764

164. Mao K, F Ji, PC Breen, A Sewell, M Han, R Sadreyev, and G Ruvkun. 2019. Mitochondrial dysfunction in C. elegans activates mitochondrial relocalization and nuclear hormone receptor-dependent detoxification genes. Cell Metab. 2019 Feb 14. pii: S1550-4131(19)30022-1. doi: 10.1016/j.cmet.2019.01.022. [Epub ahead of print] PMID: 30799287

165. Warnhoff K and G Ruvkun. 2019. Molybdenum cofactor transfer from bacteria to nematode mediates sulfite detoxification. Nat Chem Biol. 2019 May15(5):480-488. doi: 10.1038/s41589-019-0249-y. Epub 2019 Mar 25. PMID: 30911177

166. Lehrbach NJ and G Ruvkun. 2019. ER-associated SKN-1A/Nrf1 mediates a cytoplasmic unfolded protein response and promotes longevity. Elife. 2019 Apr 118. pii: e44425. doi: 10.7554/eLife.44425. PMID: 30973820

167. Lehrbach NJ, PC Breen, and G Ruvkun. 2019. Protein sequence editing of SKN-1A/Nrf1 by peptide:N-glycanase controls proteasome gene expression. Cell. 2019 Apr 18177(3):737-750.e15. doi: 10.1016/j.cell.2019.03.035. PMID: 31002798

168. Ast T, Meisel JD, Patra S, Wang H, Grange RMH, Kim SH, Calvo SE, Orefice LL, Nagashima F, Ichinose F, Zapol WM, Ruvkun G, Barondeau DP, Mootha VK. 2019 Hypoxia Rescues Frataxin Loss by Restoring Iron Sulfur Cluster Biogenesis. Cell. 2019 Apr 23. pii: S0092-8674(19)30346-0. doi: 10.1016/j.cell.2019.03.045. [Epub ahead of print] PMID: 31031004

169. Mojarro A, J Hachey, R Bailey, M Brown, R Doebler, G Ruvkun, MT Zuber, and CE Carr. Nucleic acid extraction and sequencing from low-biomass synthetic Mars analog soils for in situ life detection. Astrobiology, 2019 Jun 11. doi: 10.1089/ast.2018.1929. [Epub ahead of print] PMID:31204862

170. Armakola M and G Ruvkun 2019. Regulation of Caenorhabditis elegans neuronal polarity by heterochronic genes. Proc Natl Acad Sci U S A. 2019 Jun 4. pii: 201820928. doi: 10.1073/pnas.1820928116. [Epub ahead of print] PMID:31164416

171. Govindan, JA, E Jayamani, and G Ruvkun. 2019. ROS-based lethality of C. elegans mitochondrial electron transport mutants grown on E. coli siderophore iron release mutants Proc Natl Acad Sci U S A. 2019 Oct 7. pii: 201912628. doi: 10.1073/pnas.1912628116. [Epub ahead of print] PMID:31591219

172. Fischer, SEJ and G Ruvkun. 2020. Caenorhabditis elegans ADAR editing and the ERI-6/7/MOV10 RNAi pathway silence endogenous viral elements and LTR retrotransposons. Proc Natl Acad Sci U S A. 2020 Mar 17117(11):5987-5996. doi: 10.1073/pnas.1919028117. Epub 2020 Mar 2. PMID: 32123111

173. Wei W and Gary Ruvkun 2020. Lysosomal activity regulates Caenorhabditis elegans mitochondrial dynamics through vitamin B12 metabolism bioRxiv 2020.04.20.049502 doi: https://doi.org/10.1101/2020.04.20.049502. Proc Natl Acad Sci U S A. 2020 Aug 18117(33):19970-19981. doi: 10.1073/pnas.2008021117. Epub 2020 Jul 31.PMID: 32737159

174. Carr, CE, NC Bryant, KN Saboda, SA Bhattaru, G Ruvkun MT Zuber 2020. Nanopore sequencing at Mars, Europa, and microgravity conditions npj Microgravity 6: 24

175. Govindan, JA, E Jayamani, V Lelyveld, J Szostak, and G Ruvkun. 2020. Bacterial carotenoids suppress Caenorhabditis elegans surveillance and defense of translational dysfunction. bioRxiv 2020.01.08.898668 doi: https://doi.org/10.1101/2020.01.08.898668

176. Mao K, P Breen, G Ruvkun 2020. Mitochondrial dysfunction induces RNA interference in C. elegans through a pathway homologous to the mammalian RIG-I antiviral response. PLoS Biol. 2020 Dec 218(12):e3000996. doi: 10.1371/journal.pbio.3000996. Online ahead of print.PMID: 33264285

177. Charlesworth, AG, NJ Lehrbach, U Seroussi, MS Renaud, RI Molnar, JR Woock, MJ Aber, AJ Diao, G Ruvkun, JM Claycomb. 2020. Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility through distinct small RNA classes. bioRxiv 2020.07.20.212050.

178. Warnhoff, K, TW. Hercher, RR Mendel, G Ruvkun Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans, in press Genes and Development. bioRxiv 2020.10.08.332338 doi: https://doi.org/10.1101/2020.10.08.332338

Research support

5 R01GM44619-27 NIH Control of C. elegans lineage by heterochronic genes. 05/01/1991 - 11/30/20. Direct Costs $379,000 $648,090 Total costs per year, 30% effort.

5 R01 AG16636-21 NIH Genetic and molecular basis of longevity. 04/01/1999 - 02/28/21. 20% effort. $262,835 Direct costs, $457,333 Total costs.

5 R01 AG043184-23 NIH Inositol signaling in C. elegans senescence and diapause. 20% effort. 05/01/1996 - 03/31/2022 Budget Period: 09/01/2017 – 03/31/2018 Project Period: 08/15/2012 – 03/31/2022 Direct Costs $276,999 Total costs $473,668.

Grace Science Foundation: Regulation of the proteasome by PNG-1/NGLY1 Principal Investigator: Gary Ruvkun Total Grant Amount: $100,000 Start Date: October 14, 2016 End Date: Dec. 31, 2020

Friedreich's Ataxia Accelerator Supports the research on the iron sulfur cluster assembly factor gene frh-1 research.
PROPOSAL NUMBER: 2020A011058 09/01/2020 to 07/14/2023

NASA NNH14ZDA001NMATISSE. A Search for Extraterrestrial Genomes (SETG): An in-situ Detector for life on Mars Ancestrally Related to Life on Earth. PI: Maria Zuber 5% effort. Ruvkun co-I, 5% effort. Mature SETG in preparation for future flight. Demonstrate extraction and purification of nucleic acids followed by single-molecule nanopore sequencing of DNA. Year 1 $970,392.25 May 1 2015 to Apr 30 2015. Year 2 $958,467.88 May 1 2016 to Apr 30 2016, year 3 $998,199.79 May 1 2017 to Apr 30 2018.

Postdoctoral fellowship funding to Ruvkun lab:

1. Kurt Warnhoff, Damon Runyon Postdoctoral Fellowship (until 6/21)

2. Josh Meisel, Jane Coffin Childs Postdoctoral Fellowship (until 8/21) plus a K99 about to start.

Spring 2007 Genetics 206 (with Perrimon, Vidal). 6 lectures and discussion.

Fall and Spring 2007, 2008 Life Sciences 190, Course director, with Schrag, Kolter, Cavanaugh.

2004 to 2012 Co-director, Biology of Aging summer course, Marine Biological Laboratory, Woods Hole

2016 Four lectures Astronomy 305, Topics in Origin of Life Research.

Invited Presentations 2017 to 2020

2017 Jet Propulsion Laboratory, Caltech

2017 Salk Institute RNA symposium

2017 Gordon Conference on Insulin Signaling, Ventura

2017 University of California, Davis

2017 University of California, Santa Cruz

2017 University of Alabama, Birmingham

2017 Grace Foundation, Palo Alto

2017 University of Texas Southwestern Medical Center

2017 University of California, San Francisco, Biochemistry and Biophysics

2017 Canadian Institute for Advanced Research, Toronto

2018 Sanford Burnham Rare Genetic Disease Symposium, San Diego

2018 James Watson 90th Birthday Cold Spring Harbor

2018 RNA bench to bedside, University of Massachusetts

2018 National Cancer Institute Distinguished Lecture Series

2018 Buck Institute 30 years of aging research symposium

2018 Breakthrough Discuss, Migration of Life across the Galaxy, Center for Astrophysics, Harvard

2019 Mars Sample Return Planetary Protection meeting, Jet Propulsion Laboratory, Pasadena Caltech

2019 Breakthrough Discuss, Migration of Life across the Galaxy, Berkeley

2019 Canadian Institute for Advanced Research Santa Cruz meeting

2019 Keystone Meeting on Inflammation and Metabolism, Vancouver BC

2019 Gordon Research Conference on Aging, Sunday River Maine

2019 Ketsen Lecture, Andrus Aging Center, USC

2020 University of Toronto, Donnelly Genome Centre

Patents issued

1. Issued 5/1/2001 US Patent # 6,225,120 Therapeutic and Diagnostic Tools for Impaired Glucose Tolerance Conditions.

2. Issued 10/8/2002 US Patent # 6,461,854 Methods of Screening Compounds Useful For Prevention of Infection or Pathogenicity.

3. Issued 8/19/2008 US Patent # 7,414,169 Therapeutic and Diagnostic Tools for Impaired Glucose Tolerance Conditions

Patent applications

WO-2020162959-A1

Carotenoids for treating or preventing nausea

US-2019255124-A1

Compositions and methods for improving mitochondrial function

EP-3677909-A1

Methods and compositions for inhibiting detoxification response

US-2019024080-A1

Methods and compositions for inhibiting detoxification response

US-2017023551-A1

Tools and Methods for Targeting Oligonucleotide Repeat RNA Toxicity

US-2007087378-A1

Polynucleotide and polypeptide fat metabolism regulators and uses thereof

US-2005260652-A1

Compositions and methods that modulate RNA interference

WO-2005074560-A3

Compositions and methods that enhance rna interference

WO-2004113501-A2

Methods and compositions relating to lipid accumulation

WO-2004061087-A2

Methods and compositions of ecdysozoan molt inhibition

US-2003086871-A1

Methods of screening compounds useful for prevention of infection or pathogenicity

US-2003181364-A1

Therapeutic and diagnostic tools for impaired glucose tolerance conditions

US-7414169-B2

Therapeutic and diagnostic tools for impaired glucose tolerance conditions

Methods for screening compounds useful for the prevention of infection or pathogenicity

HU-0002199-A2

Therapeutic and diagnostic tools for impaired glucose tolerance conditions

CA-2287839-A1

Therapeutic and diagnostic tools for impaired glucose tolerance conditions

Testing procedures of substances useful for prevention of infection or pathogenicity

HU-0002781-A2

Methods of screening compounds useful for prevention of infection or pathogenicity

US-2001016332-A1

Age-1 polypeptides and related molecules and methods

EP-0828520-A1

Methods of screening compounds useful for prevention of infection or pathogenicity


Introduction

The mobilization of free fatty acids (FFA) from stored triglyceride is a fundamental cellular process that is mediated in many tissues by the functional interaction of alpha-beta hydrolase domain-containing 5 (ABHD5) with adipose triglyceride lipase (ATGL). ABHD5 null mutations disrupt lipolysis and lead to ectopic lipid accumulation in Arabidopsis 1 , C. elegans 2,3 , mice 4,5 , and humans (Chanarin-Dorfman syndrome 6 ). ABHD5 is essential for ATGL activation in fat and muscle where it integrates extracellular and intracellular signals in the control of lipolysis 7,8,9 . Additionally, lipase activation by ABHD5 suppresses colon cancer progression 10,11 and promotes hepatitis C viral replication 12 .

The structural determinants of ABHD5 activation of ATGL are poorly understood. Although ABHD5 is a member of the alpha-beta hydrolase family, it lacks the serine nucleophile of the consensus catalytic triad, and thus lacks hydrolytic activity 8 . In adipocytes, the lipase-activating function of ABHD5 is repressed by its binding to perilipin 1 (PLIN1), a lipid droplet (LD) scaffold. Extracellular signals that activate protein kinase A (PKA) lead to phosphorylation of PLIN1 and ABHD5 and stimulate release of ABHD5, which then activates ATGL 7,13,14 . Furthermore, ABHD5 is the direct target of endogenous and synthetic ligands that modulate its lipase-activating function by regulating its interactions with inhibitory PLIN proteins 9 . Although ABHD5 binds ATGL 8,15 and PLIN proteins indirectly regulate that interaction 13,16,17,18 , the molecular basis of ATGL activation by ABHD5 remains unclear.

To gain insights into the mechanism of ATGL activation, we performed comparative structural and evolutionary analysis of ABHD5 and ABHD4, a functionally-distinct paralog present in mammals and bony fishes that shares 50–55% sequence identity with ABHD5. ABHD4 hydrolyzes n-acyl phosphatidylserine and n-acyl phosphatidylethanolamine and does not promote ATGL activity 19,20 . We identified two highly conserved ABHD5 amino acids (R299 and G328) that are necessary for ATGL activation by ABHD5 and sufficient to enable that activity in ABHD4 (ABHD4 N303R/S332G). Importantly, analysis of lipolysis-inactive ABHD5 mutants demonstrated that ATGL activation was dissociable from ATGL translocation to the LD surface and from ABHD5 interactions with PLIN proteins and synthetic ABHD5 ligands. Structural modeling based on shape analysis identified a novel functional surface in ABHD5 and gain-of-function ABHD4 that contains the residues critical for ATGL activation.


Discussion

The phenotype of human Fanconi anemia patients indicates that the FA pathway is important for normal embryonic development, the maintenance of genomic stability, and the preservation of several types of stem cells (Auerbach et al. 2001). The recent discovery that the breast cancer gene, BRCA2, is an FA gene (Howlett et al. 2002) has connected this rare disorder to a common form of cancer. Nonetheless, the precise function(s) of the FA/BRCA pathway remains unknown (D'Andrea and Grompe 2003). We and others have previously created strains of mice with targeted deletions of the murine Fanca, Fancc, and Fancg genes, all components of the FA nuclear complex. These mutants have very similar phenotypes (Chen et al. 1996 Whitney et al. 1996 Cheng et al. 2000 Yang et al. 2001 Koomen et al. 2002 Noll et al. 2002), supporting a model in which the components of the complex participate in a common function. To determine whether Fancd2 participates only in this function in vivo or has additional roles, we generated a strain of mice with a null allele in this gene.

Earlier studies have suggested that FANCD2 may have unique roles that are distinct from the other FA proteins. First, it acts downstream of the FA nuclear complex and is the target of the monoubiquitination mediated by the complex (Garcia-Higuera et al. 2001 Timmers et al. 2001). Hence, the integrity of the FA nuclear complex is not perturbed in FA-D2 cells, unlike in other complementation groups (Garcia-Higuera et al. 1999 de Winter et al. 2000c). Second, FANCD2 is the only FA protein known to form nuclear foci after DNA damage and to colocalize with the repair proteins BRCA1 and RAD51 (Garcia-Higuera et al. 2001 Taniguchi et al. 2002a). Finally, FANCD2 is directly phosphorylated by ATM in response to IR but not in response to ICLs (Nakanishi et al. 2002 Taniguchi et al. 2002b). Other FA proteins are not known to be targets of the ATM kinase.

Despite these biochemical differences, human FA-D2 patients do not differ significantly clinically from FA patients belonging to other complementation groups (Timmers et al. 2001). However, FA-D2 is a rare complementation group, and all human patients reported to date have at least one mutant allele that could have some residual function, and, therefore, the phenotype of a true FANCD2-null mutation remained uncertain. The Fancd2 mutant mice reported here display all the features of previously reported strains of FA knockout mice, but also have important differences.

Fancd2, ionizing radiation, and the ATM signaling pathway

Ser 222 of human FANCD2 is phosphorylated by ATM in response to IR, and this posttranslational modification has been correlated to increased IR sensitivity in immortalized fibroblasts (Taniguchi et al. 2002b). In addition, phosphorylation of Ser 222 was associated with radiation-induced arrest of DNA synthesis. Therefore, to test whether Fancd2 played an important role in the response to IR, the survival of isogenic, primary cells from Fancc mutant and Fancd2 mutant cells was compared. Interestingly, Fancd2-null cells were not differentially sensitive to IR or ICLs compared with Fancc-null cells. Furthermore, although mild hypersensitivity to IR was observed in vivo (LD50 = ∼9.5 Gy), this sensitivity was also not greater than that observed for Fancc mutant mice (Noll et al. 2001).

In addition, and in contrast to Atm mutants, Fancd2-deficient cells did not display radiation-resistant DNA synthesis. Therefore, in murine primary cells, Fancd2 is not necessary for the ATM-mediated S-phase arrest following IR. Overall, the phenotype of Fancd2 mutant mice is significantly different from that of Atm-null mice, which display marked radiation sensitivity, immune deficiency, and hematologic tumors (Xu et al. 1996). Together, our observations suggest that Fancd2 does not play a significant role in ATM-mediated physiologic responses to IR. Perhaps human FANCD2 and murine Fancd2 differ in this aspect of their function, although the proteins are highly homologous and Ser 222 is conserved between the species. Alternatively, Fancd2 may play a role as a mediator of ATM function in only some specific circumstances, for example, T-antigen immortalized fibroblasts.

Novel phenotypes in Fancd2 knockout mice

Although Fancd2 mutant mice do not differ measurably from Fancc mutants in terms of their response to DNA damage, they have some features not seen in Fanca, Fancc, or Fancg mutant animals. These phenotypes include microphthalmia, perinatal lethality, more severe hypogonadism, and tumor development. Normal function of the FA nuclear complex is necessary for the monoubiquitination of FANCD2 in response to DNA damage or DNA replication (Garcia-Higuera et al. 2001 Taniguchi et al. 2002a). Therefore, two basic models exist to explain the divergence of phenotypes between mutants in Fancd2 and nuclear complex genes. First, nonubiquitinated Fancd2 protein, as present in Fanca, Fancc, and Fancg knockout mice, may have some residual activity in the function common to the FA pathway. In this model, knockouts of the nuclear complex genes would be similar to hypomorphic mutations of the Fancd2 gene. The phenotypes would be the result of deficiency in the same biochemical function, but they would be more severe in Fancd2 mutant mice. In the second model, FANCD2 is a multifunctional protein with a domain that functions in the FA pathway and other domains that mediate unrelated functions. The additional phenotypes of Fancd2 mutants would then be caused by deficiency of these additional functions.

Most of the features observed in Fancd2-null mice can be interpreted as more severe manifestations of the qualitatively similar defects seen in other FA knockouts. This is most obvious with the germ-cell defects. Although testicular weight is clearly more affected in Fancd2 mutant mice, the histology is similar to other FA models. Similarly, we have observed microphthalmia in the mutant offspring of some Fancc breeding pairs (M. Grompe, unpubl.), indicating that this is not a truly novel phenotype but a more complete manifestation of a defect common to all FA mice. The increased susceptibility of FA cells to apoptotic cell death after DNA damage could provide a common explanation for the observed germ-cell deficiency, microphthalmia, small size, and perinatal lethality. In the case of these developmental defects, the cells would be responding to spontaneous DNA damage. Overall, the phenotype of Fancd2 mutant mice does not convincingly establish the existence of additional functional domains in the Fancd2 protein.

Fancd2, Brca2, and carcinomas

Although human patients with FA develop a variety of cancers, tumors have never been reported in the other FA knockout mice even when followed to very late ages. In contrast, it is well established that mice with a truncating mutation in Brca2 are at an increased risk of a variety of neoplasms (Connor et al. 1997 McAllister et al. 2002). Interestingly, the tumor spectrum of the Brca2 hypomorphic mice was comparable to that seen in Fancd2 mutants (McAllister et al. 2002). In both cases, a predisposition toward epithelial cancers was seen, and the age of onset was similar. Brca2 hypomorphic mice also have other similarities such as an FA-like germ-cell defect, small size, and perinatal lethality. Taken together, this extensive phenotypic overlap is consistent with the hypothesis that the C-terminal domain of Brca2 functions in the same pathway as Fancd2.

Human BRCA2 is known to interact with and modulate the activity of RAD51, a central player in homologous recombination, and Brca2 mutant cells are known to have defects in error-free recombination (Moynahan et al. 2001b Tutt et al. 2001). Therefore, the identification of Brca2 as an FA gene supports a role for the FA pathway in recombination DNA repair. This hypothesis is strengthened by the observation that chromosomes in the pachytene stage of meiosis mispair, as described here. One potential function for the FA pathway could be to control the interaction between Rad51 and Brca2 and thereby modulate homologous recombination events at sites of DNA damage. However, Western blot analysis of Fancd2 mutant tissues showed normal Rad51 levels as well as normal interaction of the Brca2 and Rad51 proteins as determined by coimmunoprecipitation. Furthermore, Rad51 foci formation following DNA damage was normal in Fancd2 –/– MEFs. Thus, our data do not support a simple model in which the FA pathway controls the stability/rate of Brca2/Rad51 interaction.

In humans, heterozygosity for inactivating BRCA2 germ-line mutations is associated with breast, ovarian, and pancreatic cancer, all tumors of epithelial origin. It is unclear at present why these tissues are particularly affected, but it is interesting to note that epithelial tumors in rodents have been associated with telomere shortening (Artandi et al. 2000 Chang et al. 2001). Human FA patients are known to display significantly shortened telomeres in hematopoietic cells (Leteurtre et al. 1999 Adelfalk et al. 2001 Brummendorf et al. 2001 Hanson et al. 2001), and it is therefore interesting to speculate that the FA/BRCA pathway could be involved in telomere maintenance in some tissues.

The high incidence of epithelial tumors observed in Fancd2 mutants here also raises the issue whether FANCD2 could be an important gene in some human epithelial cancers, especially of the breast and ovary. Importantly, some cases of familial ovarian cancer have been linked precisely to the region where FANCD2 resides in the human genome (Sekine et al. 2001 Simsir et al. 2001 Zhang and Xu 2002). Future studies of human patients with these disorders will be needed to determine whether FA genes play a role in sporadic or inherited human cancers.


16.5: Micro-report 4- Complementation analysis - Biology

1) Description: The genetic manipulation of cultured mammalian cells represents a major modern experimental approach to questions of cell differentiation, gene regulation, and cell structure and function as well as bona fide genetic processes such as mutation and recombination. By far the most common such genetic manipulation is the transfection of mammalian cells with cloned genes. The power of this procedure has turned almost every laboratory working with cultured mammalian cells into a mammalian cell genetics laboratory, and no course could deal with such experiments in any unified way. This course will include some consideration of the transfection process per se as well transfections that are inherent in most recent work. However, emphasis will also be given to three other distinct genetic manipulations: (1) mutation and the isolation and exploitation of mutants (2) genome juxtaposition using heterokaryons and hybrids formed by cell fusion and (3) homologous recombination. These manipulations most often represent genetic tools rather than processes being investigated in their own right, and they will for the most part be discussed in that sense. As a result, the readings will include diverse biological questions as the subjects of these approaches. The readings and lectures are designed to provide a conceptual and historical background to these approaches and a sampling of current work of this type. If time permits, we will survey some of the molecular biological methods used in these studies.

2) Format: The weekly 2-2.5 hour meetings (Thursdays 2 - 4 or 4:30 in Room 800) will include both lectures and student presentations of papers in the classic and current literature. After the first few meetings, which will be mostly lectures, there will be a discussion of the week's reading. Students will be asked at random to comment on one of the readings or to present a short summary. The comment could be a detailed question, a criticism or qualification of a conclusion, or about a finding that was particularly interesting or important. There will also be a prearranged presentation of a paper in detail (see below). After the discussion and student presentation, there will be a lecture (

30 minutes) that provides an overview/background for the reading assigned for the coming week.

3) Student presentations: Each student will be responsible for 1 to 2

thirty-minute presentations of papers during the semester. Some background reading will be necessary to make a good presentation. Students may substitute their own choice for a paper rather than the one listed for a session, with the approval (well in advance) by the instructor. Students may work in teams for these presentations if they wish a team presentation counts as one-half of a presentation for each team member.

4) Exams and grading: There will be a short paper as a midterm assignment. The subject is the proposal of an experiment based on the topics covered to that point. The proposal could be an extension of the work described in the papers read. The papers should be e-mailed and will be published on the course Web site for possible discussion later in the course, time permitting. There will be written final take-home exam, designed to be answered in one hour. Grades will be based on presentations (25%), midterm paper (20%), final exam (25%) and the ability to provide a meaningful comment/question on the reading (30%, based on proportion of successful comments (on an all-or-none basis: 0 or 1) per query. The last parameter is obviously designed to promote timely reading and class participation.

5) Preparation: This course is intended for graduate students in biology, but is open to undergraduates or post-graduates who have a knowledge of biochemistry, genetics, and molecular biology at the intermediate undergraduate level and some familiarity with cell biology. All reading will be from the scientific literature.

6) Schedule of Lecture and Discussion Topics (subject to change)

Session DateLecture topic
1 1/20 Cell lines. Mutation. Spontaneous mutation rates. The problem of diploidy. Mutagenesis.
2 1/27 Cell lines. Mutation. Mutagenesis. The problem of diploidy. No student presentation. Selection of mutants. Exploitable metabolic pathways. Drug resistance. FACS. Antibodies.
3 2/3 Selection of mutants. Cell fusion. Heterokaryons.
4 2/10 Cell fusion. Heterokaryons. Hybrid cells: Complementation. Dominance/ recessiveness. Extinction of differentiated phenotypes
5 2/17 Hybrid cells: Extinction of differentiated phenotypes Transfection. Co-transfection. Cloning transfected genes.
6 2/24 Transfection. Cloning transfected genes. Recombination. Gene targeting, knockout, replacement. Gene position effects. Gene boundary elements.
7 3/2 Gene targeting. Gene knockout. Gene replacement. Gene position effects. Genetic instability. Cancer cell genetics. Tumor suppressor genes. Genetic instability.
8 3/9 Cancer cell genetics. Tumor suppressor genes. Genetic instability. Gene amplification. Co-amplification of transfected genes. No assignment, but a midterm paper is due at the next meeting.
3/16 Spring vacation Spring vacation
9 3/23 Methods discussion and catch-up. More genetic instability. Mutants cells: signal transduction mutants pre-mRNA splicing mutants mutants of cholesterol metabolism
10 3/30 Mutants cells Gene identification by transfection: triggers of muscle cell differentiation - (1) MyoD (2) 3' UTRs
11 4/6 Gene identification by transfection: triggers of muscle differentiation - (1) MyoD (2) 3' UTRs Transfection-mediated phenotypic blocking
12 4/13 Transfection-mediated phenotypic blocking Isolation of mutant molecules by SELEX.
13 4/20 Isolation of mutant molecules by SELEX. DNA shuffling. Mutant characterization by microarrays?
14 4/27 DNA shuffling. Mutant characterization by microarrays?

Biol. G4054y Week 1 Jan. 20, 2000 Session 1

Mammalian cell lines
The problem of diploidy and heteroploidy
Measurement of spontaneous mutation rates. Rate vs. frequency. Fluctuation analysis.
Mutagenesis. Chemical and physical agents. Dosage. Expression period. Metabolic cooperation.
Dominant vs. recessive mutations
Mutagen specificity. Mutational spectra. Strand specificity.

Reading to be discussed next time:

1. Most loci are diploid even in CHO cells
Siciliano, M.J., J. Siciliano, and R.M. Humphrey. 1978. Electrophoretic shift mutants in Chinese hamster ovary cells: Evidence for genetic diploidy. Proc. Natl.Acad.Sci. USA 75: 1919-1923.

2. The two DNA strands are differentially susceptible to mutagenesis
Carothers, A.M., J. Mucha, and D. Grunberger. 1991. DNA strand-specific mutations induced by (")-3?,4?-dihydroxy-1?,2?-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene in the dihydrofolate reductase gene. Proc. Natl. Acad. Sci. USA 88: 5749-5753

3. Mutations occur randomly, and how to measure the spontaneous rate of mutation .
Luria, S.E. and M. Delbrück. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 8:491-511. Reprinted in "Papers on Bacterial Genetics", E. Adelberg, ed. Little Brown, Boston, 1960. pp. 3-24. See alternatively a shorter description in G. Stent, "Molecular Genetics," Freeman, San Francisco, 1971, pp. 148-157. In addition to being a truly classic paper in genetics that shows the random nature of mutation, this paper describes the use of fluctuation analysis as a means to measure spontaneous mutation rate, which it turns out is not easy to do. You need not follow all the math to see what was being done here so read it over anyway.

4. A biochemical basis for the immortalization of cultured cell lines.
A. G. Bodnar, M. Ouellette, M. Frolkis, S. E. Holt, C. Chiu, G. B. Morin, C. B. Harley, J. W. Shay, S. Lichtsteiner, W. E. Jan. 16, 1998. Science 279: 349-352. Extension of Life-Span by Introduction of Telomerase into Normal Human Cells. http://www.sciencemag.org/feature/data/telomerase/telomerase.shl

Unstable alleles
Adair, G.M., R.S. Nairn, K.A. Brotherman, and M.J. Siciliano. 1989. Spontaneous CHO APRT heterozygotes reflect high-frequency, allele-specific, deletion of the chromosome Z4 APRT gene. Somat. Cell Molec. Genet. 15: 535-544

Loss of heterozygosity via chromosome loss
Li-C-Y. Yandell-D-W. Little-J-B. 1992 . Molecular mechanisms of spontaneous and induced loss of heterozygosity in human cells in vitro. Somat-Cell-Mol-Genet. 18: 77-87.

Multiple mutations may not occur independently
Li-C-Y. Yandell-D-W. Little-J-B. 1992. Evidence for coincident mutations in human lymphoblast clones selected for functional loss of a thymidine kinase gene. Mol. Carcinogenesis. 5: 270-7. (at P&S library)

A sub-population of mutagen-treated cells continues to mutate at high frequency for many generations
Little JB, Nagasawa H, Pfenning T, Vetrovs H. Radiat Res 1997 Oct148(4):299-307. Radiation-induced genomic instability: delayed mutagenic and cytogenetic effects of X rays and alpha particles.

Dealing with the ploidy problem for isolating recessive mutants: brute force selection of the double mutants
Chasin, L.A., 1974. Mutations affecting adenine phosphoribosyltransferase activity in Chinese hamster cells. Cell 2: 37-41

Ploidy can be a problem for isolating recessive mutants importance of cell density and expression time.
Chasin, L.A., 1973. The effect of ploidy on chemical mutagenesis in cultured Chinese hamster cells. J. Cell. Physiol. 82: 299-308

What kinds of DNA changess occur spontaneously in mammalian cells?
Zhang, L-H., H. Vrieling, A. A. van Zeeland, and D. Jenssen. 1992. Spectrum of spontaneously occurring mutations in the hprt gene of V79 Chinese hamster cells. J. Mol. Biol. 223:627-635.

Mutants affected in the mitochondrial genome can also be selected.
Hofhaus, G. and G. Attardi. 1995. Efficient selection and characterization of human cell lines which are defective in mitochondrial DNA-encoded subunits of NADH dehydrogenase. Mol. Cell. Biol. 15:964-974 (correction 15:3461)

G4054y Week 2 Jan. 27, 2000 Session 2

Lecture: Selection of mutants

Exploitable metabolic pathways: purine and pyrimidine biosynthesis. Amino acid biosynthesis.
Drug resistance: 6-thioguanine (TG), 5-bromodeoxyunridine (BrdU, BUdR), ouabain
FACS (Fluorescence-activated cell sorter)
Antibodies. Lysis with complement. Auxotrophs. Temperature-sensitive mutants: tritium suicide.
Sib selection, replica plating
Selection of revertants
Expression period
Cell density effects: cross-feeding, metabolic cooperation.

Reading to be discussed next time:

1. Auxotrophs via BrdU suicide
Kao FT, Puck TT. Proc Natl Acad Sci U S A 1968 60: 1275-81. Genetics of somatic mammalian cells, VII. Induction and isolation of nutritional mutants in Chinese hamster cells.

2.The mutation-indicative stain kills the mutant, which can be isolated by "sib selection"
Rosenstraus M, Chasin LA. Proc Natl Acad Sci U S A 1975 72: 493-7. Isolation of mammalian cell mutants deficient in glucose-6-phosphate dehydrogenase activity: linkage to hypoxanthine phosphoribosyltransferase.

3. Identification of mutants by detection of secreted product (failure to secrete).
Coffino P, Scharff MD. Proc Natl Acad Sci U S A 1971 J 68: 219-23 Rate of somatic mutation in immunoglobulin production by mouse myeloma cells.

4. Tour de force of several modern techniques (many not yet covered, but do the best you can).
Rice GC, Goeddel DV, Cachianes G, Woronicz J, Chen EY, Williams SR, Leung DW. Proc Natl Acad Sci U S A 1992 89: 5467-71. Random PCR mutagenesis screening of secreted proteins by direct expression in mammalian cells.

Drug-resistance, with fine manipulation of dosage
Jones, GE and Sargent, PA. 1974. Cell 2: 43-54. Mutants of cultured Chinese hamster cells deficient in adenine phosphoribosyltransferase.

Selection for genotype instead of phenotype.
Khrapko K, Coller H, Andre P, Li XC, Foret F, Belenky A, Karger BL, Thilly WG Nucleic Acids Res 1997 25: 685-693 Mutational spectrometry without phenotypic selection: human mitochondrial DNA.

Replica plating of mammalian cells.
Stamato TD, Jones C Somatic Cell Genet 1977 3: 639-47. Isolation of a lactic dehydrogenase-A-deficient CHO-K1 mutant by nylon cloth replica plating.

Fancier mutant selection involving colony screening
Nagan N, Hajra AK, Das AK, Moser HW, Moser A, Lazarow P, Purdue PE, Zoeller RA Proc Natl Acad Sci U S A 1997 Apr 2994(9):4475-4480 A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.

Fancier drug-resistance selection
Nohturfft A, Hua X, Brown MS, Goldstein JL Proc Natl Acad Sci USA 1996 Nov 2693(24):13709-13714. Recurrent G-to-A substitution in a single codon of SREBP cleavage-activating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines.

Biol. G4054y Week 3 Feb. 3, 2000 Session 3

Lecture 3: Cell fusion - Heterokaryons.

Fusogenic agents: PEG, Sendai virus (syncytia promoting, as HIV).
Heterokaryons. Hybrids.
Cytoplasts (cytochalasin enucleated cells), karyoplasts, reconstructed cells, mitochondrial inheritance [CAP R , valinomycin R ])
Microcells (via colcemid-induced micronuclei + cytochalasin) and fusion. See below for mapping)

Edidin: plasma membrane proteins motility

Complementation (e.g., X. pigmentosum unscheduled DNA syn. [repair])

Gene regulation studies: Cell cycle control (Rao and Johnson):

G2 x S = pulverization of S, G2 --> delayed M no DNA syn in G2 nuc.
G1 x S = S in both. G1 chromosomes are good substrates for DNA syn (need not wait)
G1 x G2 = G1 --> normal S, G2 --> delayed M, G2 does not inhibit DNA synthesis of G1

Reactivation of pycnotic nuclei (Henry Harris, hen erthyrocytes x HeLa)
RBC=negligible cytoplasm nucleus swells Hu nuc. Ag nucleoli enlarge, syn RNA chick products=surface Ag HPRT DNA synthesis (H. Harris)

HIV viral fusion simulation: CD4+ mouse cell + GP120/GP41+ CHO cell. Test therapeutic reagents (e.g., sCD4 competitor proteins)

Activation of specialized genes (muscle genes in non-muscle cells - preview) (paper to be discussed next time)

Transient fusion for biochemical analysis (FB x GH3 -> PRL-CAT in fibroblasts) (paper to be discussed next time)

Reading to be discussed next time: 1. Turning specialized genes on in heterokaryons
Blau, H., C.-P. Chiu, and C. Webster. 1983. Cytoplasmic activation of human nuclear genes in stable heterokaryons. Cell 32: 1171-1180.

2. Using heterokaryons to prove that some hnRNPs move out of and then back into the nucleus
Pinol-Roma S, and G. Dreyfuss. 1992. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature 355:730-2

3. A transient fusion experiment to show the presence of specialized gene activators in pituitary cells
Lufkin, T. and Bancroft, C. 1987. Identification by cell fusion of gene sequences that interact with positive trans-acting factors. Science 237: 283-286 Additional suggestions :

Using heterokaryons to do complementation analysis between DNA repair mutants
Kraemer et al. 1975. Genetic heterogeneity in Xeroderma pigmentosum : complementation groups and their relationship to DNA repair rates. P.N.A.S. 72: 59-63

Cell cycle control in heterokaryons formed between cells in different stages of the cell cycle
Rao, P. and R.T. Johnson 1970. Mammalian cell fusion: I. Studies on the regulation of DNA synthesis and mitosis. Nature 225: 159-164

Trans-activation of dormant genes by exposure to a different cytoplasm in heterokaryons
Harris, H. 1965. Behavior of differentiated nuclei in heterokaryons of animal cells from different species. Nature 206: 583-588.

The fluidity of the cell membrane is demonstrated as heterokaryons are formed by cell fusion
Frye, L.D., and M. Edidin. 1970. The rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons.

The most extensive use of heterokaryons to probe the determinants of cell differentiation
Blau, H. 1985. Review of her work on myoblast heterokaryons. Science 230: 758

Using heterokaryons to reveal the presence of muscle specific positive regulatory factors
Miller, S.C., G.K. Pavlath, B.T. Blakely, and H.M. Blau. 1988. Muscle cell components dictate hepatocyte gene expression and the distribution of the Golgi apparatus in heterokaryons. Genes and Dev. 2: 330-340.

Onset of extinction soon after cell fusion
Junker S, Lamm M, Nielsen V, Matthias P. J Cell Sci 1997 Oct110 ( Pt 20):2579-2587 Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner.

(Biol G4054 Reading list 2: Weeks 4 and 5 : Feb. 10 and 17, 2000)

Biol. G4054y Week 4 : Feb. 10, 2000

Lecture: Cell fusion - cell hybrids
Selection of true hybrid cells (nuclear fusion)
For: mapping complementation analysis dom./recess. and differentiated gene control.
HAT selection (TK - x HPRT - ) Universal hybridizer (e.g., oua R HPRT - x WT) exog. genes: neoR=G418R
Frequencies: heterokaryons = 10%, hybrids = 0.1% (cell cycle? synchronization helps)
Selection without markers (poisons: ricin x dipth. toxin iodoacetamide x DEPC?)
Cybrids reconstructed cells
Assessment of dominance vs. recessiveness (e.g., MTX R : permeat=recess, others=dom :e.g., smarter enzymes, amplified loci).
Complementation tests (gly [4] , ade [-9] auxotrophy)
Genetic mapping
Intraspecific hybrids and rapid chromosome segregation
Concordant segregation. Follow via: chromosome banding, chromosome painting, isozymes, PCR.
Mouse-human and hamster-human permanent panels (different human combos).
(5 hybrids can tell all synteny: 5 bits=32: 1-6 1-8,17-24 1-4,9-12,17-20 12, 56,etc odd)
Single human chromosome panels microcell fusion
Sub-chromosomal mapping: natural translocations radiation hybrids (in reading for next time)
Extinction or activation of tissue-specific genes:
melanomas and pigmentation (Ephrussi, Davidson)
hepatomas: albumin, liver enzymes (Weiss). Independence (via segregation) vs. programmatic (dedifferentiated variants)
gene (nuclear) dosage and activation (Darlington)
mutual extinction (melanoma X hepatoma) (Weiss)
extinction = gene repression, activator repression, activator dilution ??
De novo methylation
Karin example: pituitary x FB = prolactin: extinction correlates with lack of pit-1 activator (in reading):
Mapping the extinguishers (Fournier [2 papers])
Defining cis target for extinction: Eckhardt paper to be discussed next time

Cell hybridization: Mechanisms of extinction

Reading to be discussed next time:
1. How to select cell hybrids
Littlefield, J. 1964. Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145: 709-710.

2. An example of intra-chromosomal gene mapping using cell hybridization
Gyapay G, Schmitt K, Fizames C, Jones H, Vega-Czarny N, Spillett D, Muselet
D, Prud'Homme JF, Dib C, Auffray C, Morissette J, Weissenbach J, Goodfellow
PN (1996) A radiation hybrid map of the human genome. Hum Mol Genet 1996 Mar5(3):339-46

3. Extinction can involve the regulation of transcription factor expression.
McCormick, A., D. Wu, J.L.Castrillo, S. Dana, J. Strobl, E.B. Thompson, and M. Karin. 1988. Extinction of growth hormone expression in somatic cell hybrids involves repression of the specific trans-activator GHF-1.

4. Use of selectable transgenes to show extinction affecting classical enhancer elements,
Yu, H., Porton, B., Shen, L.Y., and Eckhardt, L.A. 1989. Role of the octamer motif in hybrid cell extinction of immunoglobulin gene expression: extinction is dominant in a two enhancer system. Cell. 58: 441-8l 55: 379-389. Additional suggestions :

Genome-wide map construction using radiation hybrids.
Stewart EA, et al. Genome Res 1997 May 7:5 422-33. An STS-based radiation hybrid map of the human genome.

Dedifferentiated variant hepatoma cells can be isolated, and they extinguish liver-specific genes
Deschatrette, J. and M.C. Weiss. 1975. Extinction of liver-specific functions in hybrids between differentiated and dedifferentiated rat hepatoma cells. Somatic Cell Genet. 1: 279-292

Extinction can be a two-way street
Fougere, C. and M.C. Weiss. 1978. Phenotypic exclusion in mouse melanoma-rat hepatoma hybrids cells: pigment and albumin production are not reexpressed simultaneously

Extinction of liver-specific genes can occur via a high level of protein kinase A catalytic activity
Jones KW, Shapero MH, Chevrette M, Fournier RE. Cell 1991 Sep 666(5):861-872. Subtractive hybridization cloning of a tissue-specific extinguisher: TSE1 encodes a regulatory subunit of protein kinase A.
and
Boshart M, Weih F, Nichols M, Schutz G. Cell 1991 Sep 666(5):849-859 The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-dependent protein kinase.

Extinction affects many transcription factors .
Nitsch, D., M. Boshart, and G. Schutz. 1993. Extinction of tyrosine aminotransferase gene activity in somatic cell hybrids involves modification and loss of several essential transcriptional activators. Genes & Devel. 7: 308-319

Extinction of liver-specific transcription factors
Bulla GA. Nucleic Acids Res 1997 Jun 1525(12):2501-2508. Hepatocyte nuclear factor-4 prevents silencing of hepatocyte nuclear factor-1 expression in hepatoma x fibroblast cell hybrids.

Extinction of liver-specific genes studied with a selectable transgene
Keherly MJ, Hsieh CC, McCombs JL, Merryman LS, Papaconstantinou, J., Somat Cell Mol Genet 1996. 22(2):119-134. Characterization of somatic cell hybrids exhibiting extinction of AFP, albumin and an AFP-HPRT transgene.

Biology G4054y Week 5 Feb. 17, 2000

Lecture topic: Transfection and gene transfer

CaPO4, Electroporation, Lipofection
Must traverse cytoplasm. Much engulfed in lysosomes. Inhibition of lysosomal function often helps (chloroquin)
Pechelosome
2000 KB co-integration (Robins)
Separate transfections -> separate locations
Random or semi-random (many) integration sites (unless targeted)
Low homologous recombination (prelude to next week's reading)
Cf. yeast: 50% homologous rec'n, 1/100th the DNA. So if illegitimate recombination proportional to false sites, expect 50%/100 = 0.5% homologous in mammalian cells (

what you get).
Transient transfection vs. permanent: cloned genes -> 10-50% transient (stain)
Permanents more like 0.001 (per ?g DNA per cell). i.e., 106 -> 1000 colonies
One the most dramatic first applications of gene transfection from total DNA: Transfer of the growth-transformed phenotype: ability to grow in multilayers or in suspension in soft agar: (Weinberg, Wigler)
DNA from tumor transfected into growth controlled mouse 3T3 cells. Look for foci (focus).
Make a library from growth-transformed transfectant.
Screen for human Alu repeat.
Verify cloned DNA yields high frequency of focus-forming transfectants.
Isolate cDNA by hybridization.
Sequence.
Identify gene: = a dominant oncogene. Ras, a signaling protein (in transducing pathway for sensing growth factors).

And so now: Add genes, make a cell. Make a mouse.

Reading be discussed next time:

1. First DNA transfer of a single defined gene
Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y.-C., and Axel, R. 1977. Cell 11:223-232. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

2. First well-characterized transfer of a single defined gene from total genomic DNA
Wigler M. Pellicer A. Silverstein S. Axel R. 1978. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 14: 725-31.

3. Retroviruses as vectors for gene transfer to mammalian cells.
Whitehead I, Kirk H, Kay R. Mol Cell Biol 1995 Feb15(2):704-710. Expression cloning of oncogenes by retroviral transfer of cDNA libraries.

4. A more complex selection to clone a gene by selection for function
Evans, C.J., D.E. Keith, Jr., H. Morrison, K. Mogandzo and R.H. Edwards. 1992. Cloning of a delta opioid receptor by functional expression. Science 258:1952-1955.

Additional suggestions:

Refinement and characterization of stable transfer of a defined gene into mammalian cells
Pellicer A. Wigler M. Axel R. Silverstein S. 1978. The transfer and stable integration of the HSV thymidine kinase gene into mouse cells Cell. 14:133-41.

One of the first cellular genes cloned by selection for function
Lowy, I., A. Pellicer, J.F. Jackson, G-K. Sim, S. Silverstein, and R. Axel. 1980. Isolation of transforming DNA: cloning the hamster aprt gene. Cell 22: 817-823.

Adenovirus as a gene transfer vector: Zhao H, Ivic L, Otaki JM, Hashimoto M, Mikoshiba K, Firestein S. Science 1998 Jan 9279(5348):237-24.Functional expression of a mammalian odorant receptor.

Biology G4054y Week 6 Feb. 24, 2000

Recombination gene targeting
Non-homologous and homologous recombination
Mitotic recombination between homologous chromosomes
Relation to cancer through the loss of tumor suppressor genes
(unmasked through recombination leading to loss of heterozygosity (LOH)(Cavanee)).
Recombination of transfected genes: homologous vs. non-homologous recombination.
Gene conversion vs. reciprocal recombination.
Example: Recombination between tandem inserts (Liskay)
Gene knockouts via homologous recombination.
ES cells and transgenic mice.
Selection for homologous recombinants via loss of viral TK gene (Capecchi paper to be discussed next time)
Allele replacements in cultured cell lines. Example: APRT gene replacement (Adair)
Position effects. Boundary elements. SARs/MARs.

Reading to be discussed next time:
1. Selection against non-homologous recombinants and therefore for gene targeting.
Mansour SL, Thomas KR, and Capecchi MR.1988.Disruption of the proto-oncogene int2 in mouse embryo derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336:348-52

2. KO of a splicing gene in a chicken cell line exhibiting high levels of homologous recombination
Wang J, Takagaki Y, Manley JL Genes Dev 1996 Oct 1510(20):2588-2599 Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability.

3. Finding new genes via insertional mutagenesis
Friedrich G, Soriano P. Genes Dev. 1991 Sep5(9):1513-23. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.

4. Engineered site-specific integration.
Fukushige S, Sauer B. Proc Natl Acad Sci U S A 1992 Sep 189(17):7905-9 Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells.

Influence of local chromatin structure on transfected genes and vice versa
Pikaart, M., Feng, J.A.U., and Villeponteau, B. 1992. The polyomavirus enhancer activates chromatin accessibility on integration into the HPRT gene. Molecular & Cellular Biology 12:5785-92

Effect of gene position on mutation in an integrated transgene
Lichtenauer-Kaligis, E.G., van der Velde-van Dijke, I.,den Dulk, H., van de Putte, P., Giphart-Gassler, M.,Tasseron-de Jong, J.G. 1993. Genomic position influences spontaneous mutagenesis of an integrated retroviral vector containing the hprt cDNA as target for mutagenesis. Human Molecular Genetics 2:173-82

Lichtenauer-Kaligis EG, Thijssen J, den Dulk H, van de Putte P, Tasseron-de Jong JG, Giphart-Gassler M. Comparison of spontaneous hprt mutation spectra at the nucleotide sequence level in the endogenous hprt gene and five other genomic positions. Mutat Res 1996 351:147-155.

Cis-acting elements that promote position independence
Talbot, D., Descombes, P., Schibler, U. 1994. The 5' flanking region of the rat LAP (C/EBP beta) gene can direct high-level, position-independent, copy number-dependent expression in multiple tissues in transgenic mice. Nucleic Acids Research 22:756-66

K.O. of Oct2 still allows Ig gene expression, but decreases action of artificial enhancers . Feldhaus AL, Klug CA, Arvin KL, Singh H. EMBO J 1993 7:2763-72. Targeted disruption of the Oct-2 locus in a B cell provides genetic evidence for two distinct cell type-specific pathways of octamer element-mediated gene activation.

Biology G4054y Week 7 Mar. 2, 2000

Cancer cell genetics
Oncogenes and proto-oncogenes
Dominant oncogenes vs. tumor suppressor genes
Retinoblastoma (Knudson)
Cell cycle control
Genetic instability and cancer susceptibility

Reading to be discussed next time

1. Classic original molecular genetic analysis of tumor suppressors
Cavenee, W.K,. Dryja , T.P., Phillips, R.A., Benedict, W.F., Godbout, R., Gallie, B.L., Murphree, A.L., Strong, L.C., and White, R.L. 1983. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature. 305: 779-84

2. Mutations affecting DNA repair contribute to cancer susceptibility
Leach et al., 1993. Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell 75: 1215-1225.

3. Global genomic instability in tumors
Stoler DL, Chen N, Basik M, Kahlenberg MS, Rodriguez-Bigas MA, Petrelli NJ, Anderson GR The onset and extent of genomic instability in sporadic colorectal tumor progression. Proc Natl Acad Sci U S A (1999) 96:15121-6

4. Tumor mutations and tumor evolution
Cahill DP, Kinzler KW, Vogelstein B, Lengauer C . Trends Cell Biol 1999 Dec9(12):M57-60. Genetic instability and Darwinian selection in tumors.

Selection of mutants resistant to p53-mediated growth inhibition.
Jennifer A. Pietenpol, Christoph Lengauer, Jan Jordan, Kenneth W. Kinzler, Bert Vogelstein. Proceedings of the National Academy of Sciences. Volume 93: 8390-8394. Mammalian cells resistant to tumor suppressor genes.

Biochemical basis of repair deficiency associated with colon cancer
Parsons, R., Li, G.-M., Longely, M.J., Fang, W., Papadopoulos, N., Jen, J., de la Chapelle, A., Kinzler, K.W., Vogelstein, B., and Modrich, P. 1993. Hypermutability and mismatch repair deficiency in RER+ tumor cells. Cell 75: 1227-1236.

No genetic instability is associated with polyoma-induced tumors in mice (against the trend).
Jakubczak JL, Merlino G, French JE, Muller WJ, Paul B, Adhya S, Garges S. Proc Natl Acad Sci U S A 1996. 93(17):9073-9078. Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target.

Dissecting a cancer-susceptibility and DNA repair gene associated with a human disease
Morgan SE, Lovly C, Pandita TK, Shiloh Y, Kastan. Mol Cell Biol 1997 Apr17:2020-2029 Fragments of ATM (the ataxia-telangiectasia gene) which have dominant-negative or complementing activity.

Biology G4054y Week 8 Mar. 9, 2000

No reading assignment, paper proposing an experiment due next meeting (Mar. 23).

You may want to look at the papers to be presented next time:

David Fields
Telomerase revisited
Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg
RA. Nature 1999 Jul 29400(6743):464-8. Creation of human tumour cells with defined genetic elements.

Dan Crossman
Immunoglobulin gene hypermutation
Storb U, Klotz EL, Hackett J Jr, Kage K, Bozek G, Martin TE A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript. J Exp Med 1998 Aug 17188(4):689-98

Gene amplification (no reading assigned on this topic) (see more extensive notes on Web)
Historically: Methotrexate resistance (Littlefield): High dihydrofolate reductase (DHFR) enzyme activity, protein, protein synthetic rate, translatable mRNA. (Schimke): mRNA level, DNA level.
Homogeneously staining, expanded chromosomal regions (HSRs): Biedler
Nunberg: = dhfr genes.
Double minute chromosomes.
Amplicons.
Models: over-replication, unequal sister chromatid exchange. Latter is supported.
Gene amplification and genetic instability.
Tltsy: normal cells don't amplify p53- cells do.
In nature: rDNA in oocytes, Drosophila chorion genes.
In medicine: chemotherapy resistance cancer: N-src in neuroblastoma.
In biotechnology: high level recombinant protein production in mammalian cells.

Readings on gene amplification:

Cytogenetic study of changes accompanying early gene amplification events
Trask, B.J., and Hamlin, J.L. 1989. Early dihydrofolate reductase gene amplification events in CHO cells usually occur on the same chromosome arm as the original locus. Genes & Development. 3: 1913-25 (background for #2).
and
Ma, C., Martin,S., Trask, B., Hamlin , J.L. 1993. Sister chromatid fusion initiates amplification of the dihydrofolate reductase gene in Chinese hamster cells. Genes & Development 7: 605-20.

Gene amplification only occurs in genetically unstable tumor cells
Tlsty, T.D. 1990. Normal diploid human and rodent cells lack a detectable frequency of gene amplification. Proc. Natl. Acad. Sci. USA 87: 3132-6, 1990 (background for #4).

Gene amplification is repressed by p53
Livingstone, L.R., White, A ., Sprouse, J., Livanos, E ., Jacks, T., and Tlsty, T.D .1992. Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 70: 923-35.

Classic paper demonstrating drug-resistance could be caused by gene amplification
Alt FW, Kellems RE, Bertino JR, Schimke RT J Biol Chem 1978: 253:1357-1370 Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells.

Classic demonstration of co-amplification of transfected genes
Wigler, M., Perucho, M., Kurtz, D., Dana, S., Pellicer, A., Axel, R, Silverstein, S. Proc Natl Acad Sci USA 1980.77:3567-3570 Transformation of mammalian cells with an amplifiable dominant-acting gene.

Co-amplification of transfected genes for high-level recombinant protein production
Page MJ, Sydenham MA. Biotechnology (1991) 9:64-68 High level expression of the humanized monoclonal antibody Campath-1H in Chinese hamster ovary cells.

Biology G4054y Week 9 Mar. 23, 2000. Isolation of mutant cells : examples of interesting phenotypes

David Fields
Telomerase revisited
Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg
RA. Nature 1999 Jul 29400(6743):464-8. Creation of human tumour cells with defined genetic elements.

Dan Crossman
Immunoglobulin gene hypermutation
Storb U, Klotz EL, Hackett J Jr, Kage K, Bozek G, Martin TE A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript. J Exp Med 1998 Aug 17188(4):689-98

Lecture: examples of some interesting mutants

Receptors: LDL, aryl hydrocarbon hydroxylase (resistant to polycyclic aromatic hydrocarbons)
Signal transduction: cyclic AMP (resistant: Regulatory subunit Km, catalytic subunit-negatives)
interferon (to be discussed next time)
Regulation of biosynthesis: cholesterol (to be discussed next time)
Transcriptional regulation: glucocorticoid receptor (Dexamethasone-resistant lymphoid cells)
Organelle biogenesis: peroxisomes (pyrene-alcohol resistant), mitochondria (Attardi)
Cis-acting pre-mRNA splicing mutations (screening natural gene negatives transgene with killer exon)

Reading to be discussed next time: 1. A clever push-pull double selection for mutants in the interferon-response signal transduction pathway
Pellegrini, S. et al. 1989. Use of a selectable marker regulated by interferon to obtain mutations in the signaling pathway Mol. Cell. Biol. 9: 4605-4612. ( Wayne Devonish)

2. Metabolic pathway mutants include those affected n regulatory genes
Rawson RB, Cheng D, Brown MS, Goldstein JL J Biol Chem 1998 273:28261-9. Isolation of cholesterol-requiring mutant Chinese hamster ovary cells with defects in cleavage of sterol regulatory element-binding proteins at site 1.( Jinshi Shen )

3. Selection for dihydrofolate reductase enzyme negative mutants yields many splicing mutants
Carothers, A.M., G. Urlaub, D. Grunberger, and L.A. Chasin. 1993. Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells. Mol. Cell. Biol. 13: 5085-5098.

4. Setting up an engineered gene to reveal cis-acting splicing mutations
Chen, I-T. and L.A. Chasin. 1993. Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene. Mol. Cell. Biol. 13: 289-300. Additional suggestion:

Characterization of mutants selected in the interferon-response signal transduction pathway
Leung, S.A., Qureshi, S.A., Kerr, I.M., Darnell, J.E. Jr., Stark, G.R. 1995. Role of STAT2 in the alpha interferon signaling pathway. Molecular & Cellular Biology 15:1312-7

Selection for hprt enzyme negative mutants yields many splicing mutants
Steingrimsdottir, H., G. Rowley, G. Dorado, J. Cole, and A.R. Lehmann. 1992. Mutations which alter splicing in the human hypoxanthine guanine phosphoribosyltransferase gene. Nucleic Acids Res. 20: 1201-1208.

Isolation and characterization of mutants defective in peroxisome formation
Tateishi K, Okumoto K, Shimozawa N, Tsukamoto T, Osumi T, Suzuki Y, Kondo ,N, Okano I, Fujiki. Newly identified Chinese hamster ovary cell mutants defective in peroxisome biogenesis represent two novel complementation groups in mammals. Eur J Cell Biol 1997 Aug 73:4 352-9

Isolation and characterization of cholesterol metabolism mutants: one of several types
Jacobs NL, Andemariam B, Underwood KW, Panchalingam K, Sternberg D, Kielian M, Liscum L. J Lipid Res 1997 Oct38(10):1973-1987 Analysis of a Chinese hamster ovary cell mutant with defective mobilization of cholesterol from the plasma membrane to the endoplasmic reticulum.

TGF-beta pathway mutants isolated in a manner similar to the interferon pathway mutants above.
Hocevar BA, Howe PH. Proc Natl Acad Sci U S A 1996 Jul 2393(15):7655-7660. Isolation and characterization of mutant cell lines defective in transforming growth factor beta signaling.

Review of the JAK/STAT pathway probed by th e interferon-response mutants above.
Briscoe J, Guschin D, Rogers NC, Watling D, Muller M, Horn F, Heinrich P, Stark GR, Kerr I. Philos Trans R Soc Lond B Biol Sci 1996 Feb 29351(1336):167-171. JAKs, STATs and signal transduction in response to the interferons and other cytokines.

Biology G4054y. Week 10. March 30, 2000

Isolation of interesting cell mutants affected in transcriptional regulation or signal transduction :

Wayne Devonish
10A. Pellegrini S, John J, Shearer M, Kerr IM, Stark GR, Mol Cell Biol 1989. 9: 4605-4612. Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway.

Jinshi Shen
10B. Rawson RB, Cheng D, Brown MS, Goldstein JL J Biol Chem 1998 273:28261-9. Isolation of cholesterol-requiring mutant Chinese hamster ovary cells with defects in cleavage of sterol regulatory element-binding proteins at site 1.

Reading to be discussed next time:

Gene identification by transfection: triggers of muscle cell differentiation

1. Identification of the myoD gene by transfection-induced phenotypic change
Davis RL. Weintraub H. Lassar AB. 1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51: 987-1000.

2. Mammalian cell genetic characterization of deregulation of differentiation in muscle tumor cells
Fiddler TA, Smith L, Tapscott SJ, Thayer MJ. 1996. Amplification of MDM2 inhibits MyoD-mediated myogenesis. Mol Cell Biol 16:5048-57

3. S election for factors that can trigger myogenic differentiation in transfectants yields a surprise
Rastinejad, F. and H.M. Blau. 1993. Genetic complementation reveals a novel regulatory role for 3' untranslated regions in growth and differentiation. Cell. 72:903-917.

Additonal suggestions:
Follow-up to 3' UTR regulatory RNA paper above by same first author
Rastinejad F, Conboy MJ, Rando TA, Blau HM. 1993. Tumor suppression by RNA from the 3' untranslated region of alpha-tropomyosin. Cell 75:1107-1117

Exploring extinction of myoD action
Thayer MJ. Weintraub H. 1990. Activation and repression of myogenesis in somatic cell hybrids: evidence for trans-negative regulation of MyoD in primary fibroblasts. Cell 63:23-32

Biology G4054y. Week 11. April 6, 2000

Gene identification by transfection

Presentations and discussion:

1. Identification of the myoD gene by transfection-induced phenotypic change
Davis RL. Weintraub H. Lassar AB. 1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51: 987-1000.

2. Mammalian cell genetic characterization of deregulation of differentiation in muscle tumor cells
Fiddler TA, Smith L, Tapscott SJ, Thayer MJ. 1996. Amplification of MDM2 inhibits MyoD-mediated myogenesis. Mol Cell Biol 16:5048-57

3. S election for factors that can trigger myogenic differentiation in transfectants yields a surprise
Rastinejad, F. and H.M. Blau. 1993. Genetic complementation reveals a novel regulatory role for 3' untranslated regions in growth and differentiation. Cell. 72:903-917.

Transfection-mediated phenotypic blocking (selection of genetic suppressor elements)

Antisense cDNAs + selection for inhibition of a biological pathway (Roninson)
Truncated sense cDNAs used the same way.
Also: Kimchi: resistance to interferon Beach: yeast

Reading for discussion next time: 1. Very satisfying demonstration of the feasibility of the system using lambda genes and E. coli.
Holzmayer, T.A., Pestov, D.G., Roninson, I.B. 1992. Isolation of dominant negative mutants and inhibitory antisense RNA sequences by expression selection of random DNA fragments. Nucleic Acids Research 20:711-7

2. General genetic suppressor strategy with examples from yeast.
Hannon GJ, Sun P, Carnero A, Xie LY, Maestro R, Conklin DS, Beach D. 1999. MaRX: an approach to genetics in mammalian cells. Science. 1999 Feb 19283(5405):1129-30.

3. Different p53-based genetic suppressor elements block different p53 functions.
Valeria S. Ossovskaya, Ilya A. Mazo, Michail V. Chernov, Olga B. Chernova, Zaklina Strezoska, Roman Kondratov, George R. Stark, Peter M. Chumakov, Andrei V. Gudkov. Proceedings of the National Academy of Sciences USA 93:10309-10314. Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains. (Kristi McKinney)

4. Gene amplification is repressed by p53
Livingstone, L.R., White, A ., Sprouse, J., Livanos, E ., Jacks, T., and Tlsty, T.D .1992. Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 70: 923-35.
( Wayne Devonish)

Application to mammalian cells
Gudkov, A.V., Kazarov, A.R., Thimmapaya, R., Axenovich, S.A., Mazo, I.A., and Roninson, I.B. 1994. Cloning mammalian genes by expression selection of genetic suppressor elements: association of kinesin with drug resistance and cell immortalization. Proc. Natl. Acad. Sci. USA 91:3744-8

Blocking of p53 by genetic suppressor elements coupled with selection for drug resistance
Gallagher WM, Cairney M, Schott B, Roninson IB, Brown R. Oncogene 1997 Jan 1614(2):185-193. Identification of p53 genetic suppressor elements which confer resistance to cisplatin

Selecting antisense cDNA fragments that confer resistance to interferon-mediated growth inhibition
Deiss LP, Kimchi A. Science 1991 Apr 5252(5002):117-120. A genetic tool used to identify thioredoxin as a mediator of a growth inhibitory signal.
Follow-up to the above
Cohen O, Feinstein E, Kimchi A. EMBO J 1997 Mar 316(5):998-1008. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity

Another application to mammalian cells by the Roninson lab
Gudkov, A.V., Zelnick, C.R., Kazarov, A.R., Thimmapaya, R., Suttle, D.P., Beck, W.T., and Roninson, I.B. 1993. Isolation of genetic suppressor elements, inducing resistance to topoisomerase II-interactive cytotoxic drugs, from human topoisomerase II cDNA. Proc. Natl. Acad. Sci. USA 90:3231-5

Biology G4054y. Week 12. April 13, 2000

Transfection-mediated phenotypic blocking

Presentations and discusssion:

1. Very satisfying demonstration of the feasibility of the system using lambda genes and E. coli.
Holzmayer, T.A., Pestov, D.G., Roninson, I.B. 1992. Isolation of dominant negative mutants and inhibitory antisense RNA sequences by expression selection of random DNA fragments. Nucleic Acids Research 20:711-7

2. General genetic suppressor strategy with examples from yeast.
Hannon GJ, Sun P, Carnero A, Xie LY, Maestro R, Conklin DS, Beach D. 1999. MaRX: an approach to genetics in mammalian cells. Science. 1999 Feb 19283(5405):1129-30.

3. Different p53-based genetic suppressor elements block different p53 functions.
Valeria S. Ossovskaya, Ilya A. Mazo, Michail V. Chernov, Olga B. Chernova, Zaklina Strezoska, Roman Kondratov, George R. Stark, Peter M. Chumakov, Andrei V. Gudkov. Proceedings of the National Academy of Sciences USA 93:10309-10314. Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains. (Kristi McKinney)

4. Gene amplification is repressed by p53
Livingstone, L.R., White, A ., Sprouse, J., Livanos, E ., Jacks, T., and Tlsty, T.D .1992. Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 70: 923-35. (
Wayne Devonish)

SELEX: selection of nucleic acid sequence targets

Sequence space
Selection for protein ligands
Selection for small molecules ligands
Selection for ribozymes
In vivo SELEX
Genomic SELEX

Reading for discussion next time:

1. Selection of RNA sequences that bind best to the HIV Rev protein
Bartel DP, Zapp ML, Green MR, Szostak Cell 1991 Nov 167(3):529-536. HIV-1 Rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA. (Igor Shuryak)

2. Selection of sequences that can catalyze RNA ligation
Ekland EH, Szostak JW, Bartel DP. Science 1995 Jul 21269(5222):364-370 Structurally complex and highly active RNA ligases derived from random RNA sequences

3. In vivo SELEX for splicing enhancer sequences
Coulter LR, Landree MA, Cooper TA. Mol Cell Biol 1997 Apr 17:4 2143-50 Identification of a new class of exonic splicing enhancers by in vivo selection.

Additional suggestions
Genomic SELEX ideas
Singer BS, Shtatland T, Brown D, Gold L. Nucleic Acids Res 1997 Feb 1525(4):781-786. Libraries for genomic SELEX.
and
Gold L, Brown D, He Y, Shtatland T, Singer BS, Wu Y. Proc Natl Acad Sci U S A 1997 Jan 794(1):59-64. From oligonucleotide shapes to genomic SELEX: novel biological regulatory loops.

Biology G4054y. Week 13. April 20, 2000

Discussion of papers read last week:

1. Selection of RNA sequences that bind best to the HIV Rev protein
Bartel DP, Zapp ML, Green MR, Szostak Cell 1991 Nov 167(3):529-536. HIV-1 Rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA. (Igor Shuryak)

2. Selection of sequences that can catalyze RNA ligation
Ekland EH, Szostak JW, Bartel DP. Science 1995 Jul 21269(5222):364-370 Structurally complex and highly active RNA ligases derived from random RNA sequences

3. In vivo SELEX for splicing enhancer sequences
Coulter LR, Landree MA, Cooper TA. Mol Cell Biol 1997 Apr 17:4 2143-50 Identification of a new class of exonic splicing enhancers by in vivo selection.

4. Not assigned for reading:
Rawson et al.: Cloning the sterol regulatory element-binding protein site 1 cleavage enzyme. (Jingshi Shen)

New directions in mutagenesis and mutant analysis

Reading for discussion next time:

1. DNA shuffling
Crameri A, Raillard SA, Bermudez E, Stemmer WP Nature 1998 39:288-91 DNA shuffling of a family of genes from diverse species accelerates directed evolution. ( Adam Meshel)

2. Characterization of mutant phenotypes by DNA microarray analysis
Sudarsanam P, Iyer VR, Brown PO, Winston F. Proc Natl Acad Sci U S A 2000 Mar 2897(7):3364-3369. Whole-genome expression analysis of snf/swi mutants of Saccharomyces
cerevisiae. (David Fields)

3. Genetic footprinting.
Laurent LC, Olsen MN, Crowley RA, Savilahti H, Brown PO. Functional characterization of the human immunodeficiency virus type 1 genome by genetic footprinting. J Virol. 2000 Mar74(6):2760-9.

Additional related papers:
High-resolution functional mapping of a cloned gene by genetic footprinting.
Proc Natl Acad Sci U S A. 1997 Feb 1894(4):1304-9.

Smith V, Chou KN, Lashkari D, Botstein D, Brown PO. Functional analysis of the genes of yeast chromosome V by genetic footprinting.Science. 1996 Dec 20274(5295):2069-74.

Smith V, Botstein D, Brown PO. Genetic footprinting: a genomic strategy for determining a gene's function given its sequence. Proc Natl Acad Sci U S A. 1995 Jul 392(14):6479-83.

Goryshin IY, Miller JA, Kil YV, Lanzov VA, Reznikoff WS. Proc Natl Acad Sci U S A. 1998 95(18):10716-21. Tn5/IS50 target recognition.

Biology G4054y. Week 14. April 27, 2000

Last class, no assignment.

Discussion of papers assigned last time.

1. DNA shuffling
Crameri A, Raillard SA, Bermudez E, Stemmer WP Nature 1998 39:288-91 DNA shuffling of a family of genes from diverse species accelerates directed evolution.- ( Adam Meshel)

2. Characterization of mutant phenotypes by DNA microarray analysis
Sudarsanam P, Iyer VR, Brown PO, Winston F. Proc Natl Acad Sci U S A 2000 Mar 2897(7):3364-3369. Whole-genome expression analysis of snf/swi mutants of Saccharomyces cerevisiae.

3. Genetic footprinting.
Laurent LC, Olsen MN, Crowley RA, Savilahti H, Brown PO. Functional characterization of the human immunodeficiency virus type 1 genome by genetic footprinting. J Virol. 2000 Mar74(6):2760-9.