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THE COUNCIL FOR ToBAcco REBEARCH-U.S.A., INC. Sept:enber 28, 1982 MEVORANDLM M: Drs. F3ockett, Lisanti and Samers FROM: Drs. Ford and Stone SUS7DCA': Site visit to Dr. E. Bresnick, Department Vermont, Burlington, Vermont "Expression of C.ytochrane P450c" Grant No. 1369R1 DAM OF VISIT: September 8, 1982 H KO1 1 6808' of Bioahenistry, University of PImPO.SFiL: Drs. Bresnick, Clmiecinski and Hines as well as Ms. Joan Levin took part in the presen- tation. Dr. Bresnick briefly reviewed their interest in cytochrome P450c in terms of metabolism of polycyclic hydrocarbons to produce diol epoxides which are the proximate carcinogens at organ sites (See Figure). The cytlochrane P450 enzymes consist of a family of iso;~yaes which may be induced by various substances: phenobarbital induces P450 b and polycyclic hydrocarbons induce P450 c, etc. There may be as few as 8 isozymes of the P450 family or as many as 100. Dr. Bresnick is essentially interested in the mechanisms which regulate the synthesis of these various P450s at the level of DM transcription. Also to investigate the appearance of mRNA for P450c in fetal, neonatal and adult rat liver, and to deter- mine whnther specific 3NC binding protein is involved. Finally, the group intends to screen a rat genanic library for the P450c gene and to undertake studies on the 1M sequence. The latter specific aim has received the initial attention of the group. The reason for this is that it is easier to obtain infoanatipn on, base sequences fraa small amrnmts of nucleic acid, than to obtain amino acid seguences from small quantities of protpin (ie. P450 c). Knowing the sequence of bases then the sequence of the amino acids in the translation-pro3uct can be deduoad. In brief, the plan is to obtain in pure fonn, and in sufficient amount, the messenger MR wlaich translates for the specific protein in question. Zb do this, the message is anplified,ie the - MM of interest is increased (by one means or another) in coirQarison to the other RNAtnolecule$ in the cell. Oopy DN41 moleeules are then made, inserted into bacteria and their gene-products examined. Cloning of the r.artant of interest

. ....2/~.~a...aq....... i. .. . ...:..F:~Sa~Oi..~.`w.~~ .. . -2 - HKP1168083 leads to enonrous anplification of the desired gene and message (and pYoduct). Essentially, polysomes rich in m.RtM for P450.c(by 3Nr induction in rat liver) are immniopreci.pitated to concentrate the RNh message. Copy DiVA is obtained by reverse transcriptase., a plasmid then constructed fron double-stranded DNA for inclusion into E. coli HB10). Location of the specific DM sequence is monitored by inclusion between two marker sequences (ie tetracycline and ampicillin resistarces). Now employ'ing a variety of restriction enzymes which have been shown to snip the base chain at a variety of specific sites a series of overlapping base sequences can be obtained which allows the investigator to construct the actual sequence ira the gene under investiaation. 1(eg. Cbnsider a simple example in the hidden sequence: A. B. C. D. E. F. G. H. I- etc. If snips are made and the following pieces beoome identifiable C D E BCDEF FGHI ABC then the original hidden sequence can easily be deduoe-1. / In simple terms, this is what Bresnick and his group are~iindertaking. Bresnick will be working in collaboration with Dr. Wayne Levin, who is interested in determining if there are cross reactions between P450s between different species, and with a Dr. R. K)eni, who has a lymphocyte bank for P450s in relation to patient his- tories. Dr. R. Hines: EsTloying the method of Sanger, is developing the technique to'determine the DI4F1 sequence. He will later eanpare the various harologous forms of P450. So far has sequenced about 750 base pairs, but the data does not yet make sufficient sense. As he utilizes his probes for the DNA code, he will eventually have the entire cDNA to code for the mRNA for P450. From this they will be able to determine the amino acid structure of the enzyme. As mentioned above, this is apparently easier than the direct sequencing amino acid of the P450. Dr. C. Cmiecinski is eoncerned with studies dealing with the microheterogeneit~ of q-to- chrare P450 b (for phenobarbital). Has found that there are four immunocytocT~nically identical forms of P450b linduced by phenabarbital) which exist in various ca~nbinations characteristic of different strains or colonies of rat. These do not appear to be post- translational modifications but to be derived from sPparat'e mM P450-eodes. Feels that the translation of the DNA oode may depend on various factors which mask or urnuask dif- ferent areas of the eode. The factors influencing regulation at this level eoald include hoznanes (ie, thyroid, which acts at the level of the nucleus) or an interaction with same cytoplasmic receptor which could influence histones coating the DM or factors which pennit various regions of the code to ooine togPther or separate, pvF . .. ,~- r..-am .. Am 'up .~ .__..-_ ... 9W .;,-M ~'~l..~G. ~- n~,,,.- - -,._+ _.-, FORM A F=o ~CM $

3 HK51168084 Ms. Joan Lev~ (graduate student in 4th year) is working at screening the gencanic library of Bonner and Sargent, searching for specific genes for the cDMA message for the P450 c and b mM. The bonner/Sarqent library is based on Sprague Dawley rat liver, using a high rroleculas weight DIaA; cut with restriction enzymes. ECORI was used and the result sedimented through a 10-30% sucrose gradient. Frac•nts were recovered of beta,wee.n 10 and 20 kilo bases which were cloned in a 4A virus, which can then infect a host strain to provide a genomiic library. She has used cloning proce-dures to isolate the cD61A code for P450 b and c. one clone, which she calls mc16 con- tains the information for both P450s. She has tested this result several tirtes with several restriction enzymes and feels that as they cut the length of the base pair segment into smaller units that they will find that the two P450s will probably nctc have the same base message, but will differ by 1 or 2 kb pairs. If this proves not . to be so, and the two P450s have the same code, it may be that the code is read diffe- rently under different circuostances to provide the two different P450s. She has developed explanations for this eventually, which would of course have to be proveai. Comrent This team nauc~le c acid codes for P450 b andand for developing techniques for determining the beginning to under- stand the basic biological mechanisms which regulate the expression of these induced enzymes. Vdule there may be one level of regulation at the level of translation, with 3 meyhyl cholanthrene, there appears to be a reaction with a cytosolic protein before influencing the nucleus. Age, transuterine, postnatal and hornnnal factors in regula- tion appear to be scheduled for the future. This is good solid basic genetic biologi- cal chenistry and merits continued support. Attacthmmnts First pages of CVs for C. Ctniecinski and R. N. Hines; 1 reprint and 1 manuscript. (The reprint acknowledges CPR). Also attached, a discussion of the "dideoxy" chain tenninator sequencing procedure of Sanger. - D. MD D.SZCNE DF:DS:am