Following are links to packets of information to be used on various days. Dates are approximate.

January 29, 2013: Course syllabus, schedule, why E. coli?, glossary of genetic terms, reactions catalyzed by beta-galactosidase, table of galactosides.

January 31, 2013: Lederberg's genetic cross showing that lac and pro are linked.

February 5, 2013: Alternative explanations for the effect of removing inducer and for the effect of transferring uninduced cells grown in labeled medium to unlabeled medium containing IPTG.

February 7, 2013: Starting Pardee et al. F-duction, formation of Hfr and conjugation; genetic mapping by "interrupted mating". properties of diploids in negative and positive control systems; 3-factor cross to determine the order of lacZ and lacI with respect to lacY (the two possible orders are shown and the reciprocal cross is also given).

February 12, 2013. Pardee et al. continued (2). Determination of whether all lacZ mutations are recessive; comparison of complementation and recombination.

February 14, 2013. Pardee et al., continued (3). Questions for the future; model of lac operon regulation based on Pardee et al.

February 19, 2013: This diagram shows how F' episomes are formed. The availability of the F'lac episome enabled Jacob et al. to use stable partial diploids in their analysis of the dominance of the lacOc mutations.

February 21, 2013: This packet provides supplementary information for the paper by Scaife and Beckwith, including a description of lacOo mutants, properties of nonsense mutations and their suppressors, cartoons of the coupling of transcription and translation, translation on polycistronic mRNAs, the role of rho factor in transcription termination, and the mechanism of polarity; also given are the reasons the lac promoter was thought to lie between lacO and lacZ, and mapping of the lac promoter mutations with respect to lacZ and lacI.

February 28, 2013: This packet provides supplementary information for the paper by Beckwith et al., including the steps in formation of specialized transducing phages by bacteriophage lambda, the steps in formation of strains in which lac has been transposed to a site near the integration site of phage 80, and the steps in formation of specialized transducing phages carrying lac in both possible orientations. Also, you may be interested in reading "Cloning with F80lac: The French Connection". It is a short reminescence by Signer and Beckwith about the time when they carried out the first genetic engineering experiment that is described in the paper you will be reading for this class, i.e., when they developed the method for directing the transposition of the lac operon to the T1 rec locus so that they could isolate a specialized transducing phage carrying it.

March 5, 2013: This packet on deletion mapping will be used in the discussion of the paper by Ippen et al.

March 7, 2013: A packet for use with the paper by Gilbert and Muller-Hill.

March 12, 2013: A packet providing information relevant to the isolation of the lac repressor. The sucrose gradient centrifugation experiment first demonstrating that lactose repressor binds the lac operator is described and as is the nitrocellulose filter binding assay, which is used to quantify protein-DNA interactions and determining the half-life of these complexes. Also, the methods for isolation of pure lac operator and determining its DNA sequence is described.

April 4, 2013: This set of handouts contains data demonstrating how the entire lac regulatory region was initially sequenced and how genetics helped define the boundaries of the various regulatory regions. Also included are handouts describing selected laboratory methods used to study protein--DNA interactions, e.g., methylation protection, ethylation interference, DNase I footprinting, and gel mobility shift assay.

April 9, 2013: Handouts supplementing the paper by Ogata and Gilbert. One handout shows how UV-irradiation of BU-dR substituted DNA leads to strand-breakage of cross-linking to the side group of a closely bound protein, e.g., lactose repressor. The other describes the steps to prepare a DNA fragment labeled on one end of one strand.

April 11 , 2013: Handouts describing the mechanism of repression by lactose repressor. Included are DNase I footprinting data from a paper by Galas and Schmidtz.

April 16, 2013: Handouts supplementing the 1984 paper by Ebright et al. by providing a cartoon depicting the DNA-binding, helix-turn-helix motif of lambda repressor. Images of the three-diminensional structures of lambda cro protein and CAP are given as is the consensus sequence of the DNA bound by CAP, which shows its dyad symmetry.

April 18, 2013: A second set of handouts providing descriptions of molecular biology methods (Methods II). Included are: handouts of the chain-termination (Sanger) method of DNA sequencing; PCR and applications of it to cloning; the original and current methods for automated DNA sequencing; the QuikChange method of site-directed mutagenesis; "SOEing"; and the "Gibson Assembly" method of cloning.

April 23, 2013: Handout providing a supplement to the 1987 paper by Ebright et al.

April 25, 2013: Handouts for the first paper by Zhou et al. One describes the abortive initiation assay and the other provides better copies of the last two figures.

May 2, 2013: A handout summarizing the purification of the subunits of RNAP and the method for using them to reconstitute holo-RNAP.

May 7 , 2013: Handout summarizing information from the Busby and Ebright papers of 1994 and 1999 on the UP element as a third promoter recognition element and the protein-protein interactions that mediate CAP-dependent activation at class I and class II promoters.

May 7, 2013: Handout summarizing the paper by Niu et al. in which mutants specifically defective in activation of class II CAP-dependent promoters are isolated and characterized.

May 9 , 2013: Handout showing crystal structure of the ternary complex of DNA-bound CAP and the alphaCTD, as published by Benoff et al and a summary of the conclusions drawn from the structure.

May 9, 2013: A figure depicting schematically the ChIP and ChIP-chip approaches used by Grainger et al.

May 11, 2013: A figure comparing the recruitment and pre-recruitment mechanisms of transcription activation and a figure providing genetic evidence for pre-recruitment. For use with the paper by Grainger et al.

May 14, 2013: A packet containing background information for the paper by Griffith and Wolf on the genetic evidence for prerecruitment. It also contains recent experiments using "pull-out" experiments that demonstrate that wild type SoxS and DNA-binding mutations of it form binary complexes with RNAP in vivo but positive control mutations do not. This experiment provides additional, very strong support for the hypothesis that SoxS activates transcription by pre-recruitment.

No Longer Used: Action of chemical mutagens, e.g., hydroxlylamine.

No Longer Used: Summary of the data describing the binding of AraC protein to araI as obtained by Schleif and by Lee et al.

No Longer Used: Handout illustrating supercoiled DNA

No Longer Used: Handout summarizing the forms of AraC during repression and induction with respect to the light switch mechanism.

No Longer Used: Handout describing some remaining unanswered questions about the ara system and also some suggestions for new mutants to isolate.

No Longer Used: Handout describing the action of CAP at Class II CAP-dependent promoters.

No Longer Used: Handouts introducing gene regulation in phage lambda including its genetic map, a table summarizing the stages of transcription, a description of the assembly and action of the N protein anti-termination complex, a "scorecard" of the genes involved in lambda development, a figure describing the regulatory circuits involved in lambda development, and a figure showing the action of repressor and Cro at the rightward lambda operator/promoter region.

No Longer Used: Handout of electron microscopy experiments demonstrating DNA looping by lambda repressor.

No Longer Used: Handout illustrating the effect of DNA looping by lambda repressor on expression from the Prm promoter and use of this system to isolate mutants defective in the protein-protein interactions underlying repressor-mediated cooperativity.

No Longer Used: Handout describing the isolation of new specificity mutants of lambda 434 repressor.

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