Some guidelines for presentation of a paper
| Session |
|
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 |
|
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
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
Loss of heterozygosity via chromosome loss
Multiple mutations may not occur independently
A sub-population of mutagen-treated cells continues to mutate at
high frequency for many generations
Dealing with the ploidy problem for isolating recessive mutants:
brute force selection of the double mutants
Ploidy can be a problem for isolating recessive mutants; importance
of cell density and expression time.
What kinds of DNA changess occur spontaneously in mammalian cells?
Mutants affected in the mitochondrial genome can also be selected.
G4054y Week 2 Jan. 27, 2000 Session 2
Lecture: Selection of mutants
Exploitable metabolic pathways: purine and pyrimidine biosynthesis.
Amino acid biosynthesis.
Reading to be discussed next time:
1. Auxotrophs via BrdU suicide
2.The mutation-indicative stain kills the mutant, which can be isolated
by "sib selection" 3. Identification of mutants by detection of secreted product (failure
to secrete).
4. Tour de force of several modern techniques (many not yet covered,
but do the best you can).
Addition suggestions:
Drug-resistance, with fine manipulation of dosage
Selection for genotype instead of phenotype.
Replica plating of mammalian cells.
Fancier mutant selection involving colony screening
Fancier drug-resistance selection
Biol. G4054y Week 3 Feb. 3, 2000 Session 3
Lecture 3: Cell fusion - Heterokaryons.
Fusogenic agents: PEG, Sendai virus (syncytia promoting, as HIV).
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.
Reactivation of pycnotic nuclei (Henry Harris, hen erthyrocytes x HeLa) 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:
2. Using heterokaryons to prove that some hnRNPs
move out of and
then back into the nucleus
3. A transient fusion experiment to show the presence of specialized
gene activators in pituitary cells
Using heterokaryons to do complementation analysis between DNA repair
mutants
Cell cycle control in heterokaryons formed between cells in different
stages of the cell cycle
Trans-activation of dormant genes by exposure to a different cytoplasm
in heterokaryons
The fluidity of the cell membrane is demonstrated as heterokaryons
are formed by cell fusion
The most extensive use of heterokaryons to probe the determinants
of cell differentiation
Using heterokaryons to reveal the presence of muscle specific positive
regulatory factors
Onset of extinction soon after cell fusion
(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
Cell
hybridization: Mechanisms of extinction
Reading to be discussed next time: 2. An example of intra-chromosomal gene mapping using cell hybridization
3. Extinction can involve the regulation of transcription factor
expression.
4. Use of selectable transgenes to show extinction affecting classical
enhancer elements,
Genome-wide map construction using radiation hybrids. Dedifferentiated variant hepatoma cells can be isolated, and they
extinguish liver-specific genes Extinction can be a two-way street
Extinction of liver-specific genes can occur via a high level of
protein kinase A catalytic activity
Extinction affects many transcription factors.
Extinction of liver-specific transcription factors
Extinction of liver-specific genes studied with a selectable transgene
Biology G4054y Week 5 Feb. 17, 2000
Lecture topic: Transfection and gene transfer
CaPO4, Electroporation, Lipofection
>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 2. First well-characterized transfer of a single defined gene from total
genomic DNA
3.Retroviruses as vectors for gene
transfer to mammalian cells.
4. A more complex selection to clone a gene by selection
for function
Additional suggestions: One of the first cellular genes cloned by selection for function
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. Recombination; gene targeting Reading to be discussed next time: 2. KO of a splicing gene in a chicken cell line exhibiting high
levels
of homologous recombination 3. Finding new genes via insertional mutagenesis
Additional suggestions:
Influence of local chromatin structure on transfected genes and vice
versa
Effect of gene position on mutation in an integrated transgene
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
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 Reading to be discussed next time
1. Classic original molecular genetic analysis of tumor suppressors
2. Mutations affecting DNA repair contribute to cancer susceptibility
3. Global genomic instability in tumors 4. Tumor mutations and tumor evolution Selection of mutants resistant to p53-mediated growth inhibition.
Biochemical basis of repair deficiency associated with colon cancer
No genetic instability is
associated with polyoma-induced tumors in mice (against the trend).
Dissecting a cancer-susceptibility and DNA repair gene associated
with a human disease
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 Dan Crossman Gene amplification (no reading assigned on this topic) (see more
extensive notes on Web) Readings on gene amplification: Gene amplification only occurs in genetically unstable tumor cells
Gene amplification is repressed by p53
Classic paper demonstrating drug-resistance could be caused by
gene amplification
Classic demonstration of co-amplification of transfected genes
Co-amplification of transfected genes for high-level recombinant protein
production
Biology G4054y Week 9 Mar. 23, 2000. Isolation of mutant cells: examples of interesting phenotypes
Presentations:
David Fields Dan Crossman Lecture: examples of some interesting mutants Receptors: LDL, aryl hydrocarbon hydroxylase
(resistant to polycyclic aromatic hydrocarbons)
Reading to be discussed next time:
2. Metabolic pathway mutants include those affected n regulatory genes 3. Selection for dihydrofolate reductase enzyme negative mutants
yields many splicing mutants
4. Setting up an engineered gene to reveal cis-acting splicing mutations
Characterization of mutants selected in the interferon-response
signal transduction pathway
Selection for hprt enzyme negative mutants yields many splicing mutants
Isolation and characterization of mutants defective in peroxisome
formation
Isolation and characterization of cholesterol metabolism mutants: one of
several types
TGF-beta pathway mutants isolated in a manner similar to the interferon
pathway mutants above.
Review of the JAK/STAT pathway probed by the
interferon-response mutants above.
Biology G4054y. Week
10. March 30, 2000
Isolation of interesting cell mutants affected in transcriptional regulation
or signal transduction:
Presentations:
Wayne
Devonish Jinshi Shen 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
2. Mammalian cell genetic characterization of deregulation of
differentiation in muscle tumor cells 3. Selection for factors that can trigger myogenic differentiation
in transfectants yields a surprise
Additonal suggestions:
Exploring extinction of myoD action
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
2. Mammalian cell genetic characterization of deregulation of
differentiation in muscle tumor cells 3. Selection for factors that can trigger myogenic differentiation
in transfectants yields a surprise
Transfection-mediated phenotypic blocking (selection of genetic
suppressor elements)
Antisense cDNAs + selection for inhibition of a biological pathway (Roninson)
Reading for discussion next time:
2. General genetic suppressor strategy with examples from yeast. 3. Different p53-based genetic suppressor elements block different
p53 functions.
4. Gene amplification is repressed by p53
Application to mammalian cells
Blocking of p53 by genetic
suppressor elements coupled with selection for drug resistance
Selecting antisense cDNA fragments that confer resistance to interferon-mediated
growth inhibition
Transfection-mediated phenotypic blocking Presentations and discusssion: 2. General genetic suppressor strategy with examples from yeast.
3. Different p53-based genetic suppressor elements block different
p53 functions.
4. Gene amplification is repressed by p53
SELEX: selection of nucleic acid sequence targets Sequence space
Reading for discussion next time: 1. Selection of RNA sequences that bind best to the HIV Rev protein
2. Selection of sequences that can catalyze RNA ligation 3. In vivo SELEX for splicing enhancer sequences Discussion of papers read last week: 1. Selection of RNA sequences that bind best to the HIV Rev protein
2. Selection of sequences that can catalyze RNA ligation 3. In vivo SELEX for splicing enhancer sequences 4. Not assigned for reading: New directions in mutagenesis and mutant analysis Reading for discussion next time: 1. DNA shuffling 2. Characterization of mutant phenotypes by DNA microarray analysis 3. Genetic footprinting. Last class, no assignment. Discussion of papers assigned last time. 1. DNA shuffling 2. Characterization of mutant phenotypes by DNA microarray analysis 3. Genetic footprinting. Last updated: 04/20/00 08:56 AM
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
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.
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)
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.
Chasin, L.A., 1974. Mutations affecting adenine phosphoribosyltransferase
activity in Chinese hamster cells. Cell 2: 37-41
Chasin, L.A., 1973. The effect of ploidy on chemical mutagenesis in
cultured Chinese hamster cells. J. Cell. Physiol. 82: 299-308
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.
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)
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.
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.
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.
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.
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.
Jones, GE and Sargent, PA. 1974. Cell 2: 43-54. Mutants of cultured
Chinese hamster cells deficient in adenine phosphoribosyltransferase.
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.
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.
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.
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.
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)
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
RBC=negligible cytoplasm; nucleus swells; Hu nuc. Ag; nucleoli enlarge,
syn RNA; chick products=surface Ag; HPRT; DNA synthesis (H. Harris)
Blau, H., C.-P. Chiu, and C. Webster. 1983. Cytoplasmic activation
of human nuclear genes in stable heterokaryons. Cell 32: 1171-1180.
Pinol-Roma S, and G. Dreyfuss. 1992. Shuttling of pre-mRNA binding
proteins between nucleus and cytoplasm. Nature 355:730-2
Lufkin, T. and Bancroft, C. 1987. Identification by cell fusion of
gene sequences that interact with positive trans-acting factors. Science
237: 283-286
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
Rao, P. and R.T. Johnson 1970. Mammalian cell fusion: I. Studies on
the regulation of DNA synthesis and mitosis. Nature 225: 159-164
Harris, H. 1965. Behavior of differentiated nuclei in heterokaryons
of animal cells from different species. Nature 206: 583-588.
Frye, L.D., and M. Edidin. 1970. The rapid intermixing of cell surface
antigens after formation of mouse-human heterokaryons.
Blau, H. 1985. Review of her work on myoblast heterokaryons. Science
230: 758
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.
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.
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
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.
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
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.
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:
Stewart EA, et al. Genome Res 1997 May 7:5 422-33. An STS-based radiation
hybrid map of the human genome.
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
Fougere, C. and M.C. Weiss. 1978. Phenotypic exclusion in mouse melanoma-rat
hepatoma hybrids cells: pigment and albumin production are not reexpressed
simultaneously
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.
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
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.
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.
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).
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.
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.
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.
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.
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.
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.
Biology G4054y Week 6 Feb. 24, 2000
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.
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
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.
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.
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
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
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
Oncogenes and proto-oncogenes
Dominant oncogenes vs. tumor suppressor genes
Retinoblastoma (Knudson)
Cell cycle control
Genetic instability and cancer susceptibility
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
Leach et al., 1993. Mutations of a mutS homolog in hereditary nonpolyposis
colorectal cancer. Cell 75: 1215-1225.
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
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:
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.
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.
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.
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.
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.
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
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.
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.
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).
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.
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.
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.
Page MJ, Sydenham MA. Biotechnology (1991) 9:64-68 High
level expression of the humanized monoclonal antibody Campath-1H in Chinese
hamster ovary cells.
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.
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
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)
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)
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)
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.
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.
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
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.
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
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.
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.
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.
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.
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.
Davis RL. Weintraub H. Lassar AB. 1987. Expression of a single transfected
cDNA converts fibroblasts to myoblasts. Cell 51: 987-1000.
Fiddler TA, Smith L, Tapscott SJ, Thayer MJ. 1996. Amplification of MDM2 inhibits MyoD-mediated myogenesis.
Mol Cell Biol 16:5048-57
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.
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
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
Davis RL. Weintraub H. Lassar AB. 1987. Expression of a single transfected
cDNA converts fibroblasts to myoblasts. Cell 51: 987-1000.
Fiddler TA, Smith L, Tapscott SJ, Thayer MJ. 1996. Amplification of MDM2 inhibits
MyoD-mediated myogenesis.
Mol Cell Biol 16:5048-57
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.
Truncated sense cDNAs used the same way.
Also: Kimchi: resistance to interferon; Beach: yeast
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
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.
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)
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)
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
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
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
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
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.
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)
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)
Selection for protein ligands
Selection for small molecules ligands
Selection for ribozymes
In vivo SELEX
Genomic SELEX
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)
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
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.
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
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)
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
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.
Rawson et al.: Cloning the sterol regulatory element-binding protein site
1cleavage enzyme. (Jingshi Shen)
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)
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)
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
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)
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.
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.