Y79 cells derived from a retinoblastoma tumor have been shown to have 50 – 100 copies of the N-myc gene transcript per cell. We first tested whether N-myc is preferentially expressed during specific parts of the Y79 cell cycle. Y79 cells were synchronized with double thymidine blocks at the beginning of S, then released from the block and collected for isolation of total cytoplasmic RNA and for analysis by flow cytometry. N-myc DNA probes hybridized with dot blotted RNAs and RNAs from Northern blots ,of agarose-formaldehyde gels showed no differences in the numbers of transcripts as the cells traversed 0 1 and entered the S phase over a period of 12 hrs. Protein synthesis inhibitors added to randomly growing Y79 cells did, however, show a dramatic increase in N-myc transcript number over a period of 4 – 6 hrs; control genes showed no such increase. Inhibition of RNA synthesis by actinomycin D treatment led to a rapid decrease in N-myc transcript number which was partially prevented by protein synthesis inhibitors. Thus, N-myc transcripts have a short half life whose stability may be increased by inhibition of protein synthesis. These results confirm that similarities between c-myc and N-myc go beyond their partial sequence homology and suggest that N-myc may serve the same function in neuronal cells that c-myc plays in non-neuronal cells. The gene copy number is normally a highly conserved factor during DNA replication. It is clear that on an evolutionary scale genes have increased in number sometimes forming functional gene copies; however, often the amplification results in the formation of pseudogenes.
Recent studies have indicated that the control of gene copy number may break down on a shorter time scale. Numerous example have been found in which the number of gene copies for a particular selectible marker has increased when cells are grown in the presence of a selecting agent. Generally,hydroponic gutter after applying a selecting agent these amplified genes appear within a few cell generations in the form of extrachromosomal gene copies and may progress after long exposure to form reintegrated tandemly arranged multiple gene copies. Whether the selecting agent is inducing the amplification of genes or selecting for pre-existing cells with amplified genes is not known. It has been shown, however, that agent which damage DNA can induce either the stabilization of amplifi¢ genes, or the amplification process itself. As a model system for the study of gene amplification, we have been studying the regulation of copy number of SV 40 viral sequences present in human cells after transfonnation. Our hypothesis is that there are cellular factors that regulate the copy number of these sequences and that we can identify these factors by developing an in vitro DNA replication system. We have begun by characterizing the number of copies of SV40 DNA present in human cells transfonned ‘ by this virus. In our initial studies of two human cell lines we obtained from the American Type Tissue Culture collection that were transformed with wild type SV 40 virus we discovered that the cells contained many copies of SV 40 that were extrachromosomal elements. The cell lines we are studying are G,M637, a human fibroblast cell line from an apparently normal individu31, and XP12RO, a line from a Xeroderma pigmentosum patient containing a mutation in a DNA repair gene. Both of these cell lines were transformed with wild type SV 40 by other investigators.
The aim of these’ studies has been to determine the involvement of DNA repair processes in gene amplification. The presence of the extrachromosomal copies of SV 40 in human cells has been observed by other investigators. The origin of this extrachromosomal DNA and its relationship to the imput transforming DNA i~ not known. To understand the system better we began to characterize the DNA. We found that in an actively growing population of GM637 cells the copy number, of SV40 DNA is approximately 400 per cell. The extrachromosomal DNA is also present in XP12RO cells. Other human cells we are carrying in the lab that are not transformed with SV40 virus do not have the extrachromosomal SV40 DNA. On agarose gel electrophoresis, the extrachromosomal DNA from both cell lines has a lower molecular weight than wild type DNA. Restriction enzyme digestion indicates that the lower molecular weight is the result of a deletion in the A-gene coding for the T-antigen. In both cell lines the deletion maps near the boundary of the intervening sequence and the small exon of the T -antigen. The difference between the two DNA’s seen so far is that the size of the deletion for the DNA from GM637 is about 300 base pairs and for the DNA from the XP12RO cells is about 250 base pairs. We have cloned the extrachromosomal DNA from GM637 into a pUC vector and then subcloned a Hind III fragment containing the deletion into M13 for sequencing.
The sequence indicates that the deletion has eliminated one of the splice sites for the intervening sequence and has stopped just short of the translation termination site for the small t-antigen. The deletion stops out-of-frame so that presumably the translation machinery reads through the normal termination signal, but encounters another termination signal in the new frame just upstream from the deletion. The prediction from this data is that the mutant DNA encodes a mRNA that is similar in size to the message for wild type T-antigen, but that the premature translation termination signal would result in a protein being made that is just slightly smaller than small t-antigen. We are presently isolating the mRNA and the proteins from the cells to determine what is made from the mutant DNA. At this point we know nothing about the function of this extrachromosomal DNA,u planting gutter or about its origin from the wild type virus used to transform the cells. Its curious that two cell types that originated from separate transformation events give rise to very similar extrachromosomal deleted DNA. We have been developing a system for the study of the replication of cardnogen modified DNA in mammalian cells. For this assay we have contructed a shuttle vector that can replicate both in bacteria and mammalian cells. This vector was contructed by cloning of the SV 40 origin for DNA replication into an M13 vector. M13 virus contain a single stranded DNA which replicates efficiently in E. coli, and is ideal for sequencing DNA placed in the Messing cloning site. The construct we have made contains the SV 40 origin in the I region adjacent to the cloning site and the ,B-galactosidasegene. The assay for replication in mammalian cells has been to transfect the double stranded replication form into mammalian cells at approximately 50 ng per 106 cells. The DNA is harvested at different times after transfection, digested with Dpn I to eliminate the DNA that has not replicated, and then used to transform E. coli. The surviving and replicated DNA will form plaques on a lawn of E. coli. M13 DNA which has been mutated to block the active production of ,B-galactosidase can be distinguished from input DNA by a color assay. Our studies so far have indicated that the DNA we have constructed can indeed replicate in Cos 7 cells which provide a functional T-antigen from an integrated copy of SV 40.
So far we have not been able to demonstrate replication of our vector in human cells; however, we have only tested the GM637 cells which already contain many copies of their own extrachromosomal DNA. We plan to construct a transfection recipient of human cells by utilizing an approach similar to that used to construct the Cos 7 cell line. Wild type SV 40 will be inactivated for replication by cutting at the origin with Bgi I followed by. treatment with Bal I to digest away the ends. The deletions generated will than be transfected directly into untransformed human fibroblast cells and transformed variants will be isolated. The transformed cells will be tested for extrachromosomal DNA, and only those clones with integrated SV40 will be used as transfection recipients. We intend to carry out this technique with different human cells so that we can have recipients to test the effect of such things as differentiation state of the cells and DNA repair capacity on the apility to replicate transfected DNA both with and without carcinogen modification. UsiI1g the M13 vector containing the SV40 origin we contructed we are testing the effect of carcinogen adducts on the replication of DNA. This project has come out of our extensive work characterizing the effect of benzo[a]pyrene on the replication of SV40 viral DNA. The conclusions of these studies has been that the carcinogen causes a build up of replicated intermediates which were unable to complete the final steps in separating the daughter molecules into mature completely replicated forms. One difficulty with these studies has been the random location of the adduct and the inability therefore to locate the replication block relative to the adduct. By site specifically placing the adduct we hope to get around this difficulty and to also provide a vector for studying the kind of DNA sequence changes that occur when adducted DNA is replicated. The vector does replicate in ~onkey cells containing an active gene for production of Tantigen with a low mutation frequency as judged by the plaque assay on E. coli described above. We have treated the DNA with benzo[a]pyrene at a level which reduces the yield of plaques by 50%, and have obtained vector lacking the ability to induce ,a-galactosidase from DNA replicated in the monkey cells. We are presently characterizing these vectors to determine precisely what mutations have occurred.This work is being done in collaboration with the Prof. John Hearst’s laboratory. In our approach, we have made two M13 vectors containing the SV40 origin. One vector contains a synthetic oligomer in the cloning region, and the second lacks this region. Hybridization of these to single strands creates a double strand DNA except in the region of the cloned synthetic oligomer. The approach is then to place a psoralen adduct on another synthetic oligomer that hybridizes to the oligomer cloned into the vector and then t,o seal the fragment in place with ligase. We have completed the work to the formation of the gapped intermediate and presently are determining if the synthetic oligomer will hybridize into the gap and be ligated. Our, future work will then be to transfect this DNA into cells with different capacity to replicate and/or express the DNA and detemine how the adduct affects these processes. Our experience working with DNA transfected back into mammalian cells has shown us that the DNA is greatly modified during the transfection procedure. In order to overcome these difficulties we have begun to develope a system for conducting the DNA synthesis experiments in vitro. Until recently it was not possible to get any mammalian cell DNA replication to occur in vitro., Recent work in other laboratories have shown that it is’ possible to replicate SV 40 based DNA in complex mixtures of factors from human cell lines. Not only does elongation of preinitiated replication occur, but multiple rounds of initiation are possible. We are setting up this _ assay in our lab to look at the replication of the psorlen modified vectors described above. This system will also be used to investigate the cellular factors required for DNA synthesis and how carcinogen pretreatment of the cells affects the levels of DNA replication. Chemicals and radiation in the environment often interact adversely with DNA of living cells resulting in abnormal behavior and or death of the cells if the damage is not repaired. To understand how the damaged DNA is recognized and repaired one needs to know, among other things, the structures of the damaged molecules and their intermediates at the molecular level. Two of the most studied damaged DNAs ‘are those containing UV induced thymine dimer and psoralencross-link. Combining the structural information from the crystal structures of a double stranded DNA fragment and the psoralen-thymine monadduct we have recently constructed a molecular model for the psoralen cross-linked DNA.