By J. P. J., H. P. S. (auth.), Dr. Jean-Pierre Jost, Dr. Hans-Peter Saluz (eds.)
A safeguard issues Many strategies defined right here contain a couple of dangers, comparable to excessive electric present and voltage, radioactivity and hugely poisonous chemical substances. it really is totally crucial that the directions of apparatus brands be undefined, and that individual cognizance be paid to the neighborhood and federal safeguard rules. B creation The expression of prokaryotic and eukaryotic genes has been proven frequently to be regulated on the point of mRNA synthesis. due to the quick improvement of equipment for dissecting DNA sequences, cis-acting regulatory components similar to promoters and enhancers were regarded. extra lately, the commonly expressed instinct that discrete sequences inside those parts represent binding websites for sequence-specific binding proteins has been proven, specially by utilizing "footprinting" assays (for examples, Galas and Schmitz, 1978). This and comparable assays have already ended in the popularity, isolation and research of DNA-bind ing proteins for a number of genes. very good studies exist of the structural experiences on those transcription regulatory proteins and similar DNA components (for instance, Glover, 1989 and Johnson and McKnight, 1989), to which the reader is referred for special details. To set the scene for functions of the innovations defined during this quantity, in simple terms the barest define of past reports is gifted the following. Protein-DNA interactions are depending on very particular tertiary configurations of the binding protein which enable the nearest touch with the DNA helix.
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Additional info for A Laboratory Guide to In Vitro Studies of Protein-DNA Interactions
P', The ubiquitous nuclear protein, NHPI binds with high affinity to different sequences of the chicken vitellogenin II gene. , 17 (1989) 8511-8519. , Ruet, A, Moenne A. and Sentenac, A, Selective proteolysis defines two DNA binding domains in yeast transcription factor tau. Nature, 323 (1986) 176-178. D. and Klug, A, Repetitive zinc-binding domains in the protein transcription factor IlIA from Xenopus oocyte. , 4 (1985) 1609-1614. M. , 7 (1988) 4221-4229). , Domains of the positive transcription factor specific for the Xenopus 5S RNA gene.
Incubate at 3TC for 20 min. > Extract once with phenol and once with chloroform. > Add 3 volumes of ethanol and chill at -80T for 10 min. > Centrifuge 10 min at 15000 rpm in a SS-34 Sorvall rotor. > Wash sample by centrifugation with cold 90% ethanol, 10% water. > Dry sample in a SpeedVac concentrator. 2 DNA Size Standards Take the G+C and the A+ T sequence reaction product of the end-labelled oligonucleotide as DNA size standards. 3 Separation of the Reaction Product on a Sequencing Gel For the separation of 40-mer templates we use a 40-cm-Iong 20% polyacrylamide sequencing gel (Maxam and Gilbert, 1980).
Add 30 III of 3 M NaOAc solution to each sample. > Add 750 III of 95% ethanol and vortex. > Chill in an ethanol-dry ice bath as before to precipitate the DNA. Then repeat the spin, rinse, and dry steps as described above. Deamination Reaction > Resuspend purified, ethanol-precipitated DNA pellets in 300 III of deamination buffer. > Incubate the DNA samples in a water bath at 50 C for 5 h D in the dark. > Add 30 III of 3 M NaOAc and 750 III of 95% ethanol to precipitate the DNA. The precipitation is carried out as described above.
A Laboratory Guide to In Vitro Studies of Protein-DNA Interactions by J. P. J., H. P. S. (auth.), Dr. Jean-Pierre Jost, Dr. Hans-Peter Saluz (eds.)