Biology G4054y     Mammalian cell genetics: Syllabus     Spring, 2000         L. Chasin
Last updated: Thursday, April 20, 2000 08:56 AM
Session
Date

Discussion topic

Lecture 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
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?  

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
Date

Discussion topic

Lecture 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?  


 
Some guidelines for presentation of a paper

Terms used in the reading

Older but still useful books on Somatic Cell Genetics

Papers for presentations

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

Lecture topic: Mutation

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

Additional suggestions:

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 Oct;148(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.

Addition suggestions:

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 29;94(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 26;93(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 [CAPR, valinomycinR])
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 Oct;110 ( 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., ouaR 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., MTXR: 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 Mar;5(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 6;66(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 6;66(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 15;25(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).

>20 more since then.

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 Feb;15(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 9;279(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 15;10(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 Sep;5(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 1;89(17):7905-9  Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. 

Additional suggestions:

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 Dec;9(12):M57-60.  Genetic instability and Darwinian selection in tumors. 

Additional suggestion:   

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 Apr;17:2020-2029 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 29;400(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 17;188(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

Presentations:

David Fields
Telomerase revisited
Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg
RA.   Nature 1999 Jul 29;400(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 17;188(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 Oct;38(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 23;93(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 the 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 29;351(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:

Presentations:

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. Selection 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. Selection 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 19;283(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)

Additional suggestions:

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 16;14(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 5;252(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 3;16(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 19;283(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 1;67(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 21;269(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 15;25(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 7;94(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 1;67(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 21;269(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 1cleavage 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 28;97(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 Mar;74(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 18;94(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 20;274(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 3;92(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 28;97(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 Mar;74(6):2760-9.

Last updated: 04/20/00 08:56 AM