A NEW PROCEDURE WILL BE EMPLOYED TO ISOLATE CELLULAR ONCOGENES WITH THE AIM TO RETRIEVE THE TNO ONCOGENE AND TO PROPAGATE THIS GENE IN BACTERIA. THIS PROCEDURE INVOLVES THE FOLLOWING: MOUSE DNA FRAGMENTS, PREPARED WITH A RESTRICTION ENZYME, ARE LIGATED TO A PLASMID CONTAINING A TRANSCRIPTION-ENHANCING SEQUENCE OF RETROVIRAL ORIGIN AND PROKARYOTIC GENE CAUSING RESISTANCE TO NEOMYCIN. AFTER TRANSFECTION THE RECIPIENT CELLS ARE KEPT UNDER CONTINUOUS SELECTION PRESSURE WITH NEOMYCIN. THIS WILL LEAD TO THE MAINTENANCE OF THE PLASMID SIGNAL AND THE NEOPLASTIC STATUS OF THE CELLS. A LIBRARY WILL BE PREPARED OF DNA OF THE RESULTING TRANSFORMED CELL LINES AFTER PARTIAL DIGESTION WITH ECORI, USING THE CHARON 4A PHAGE. THE PHAGE CLONES, WHICH CARRY A PLASMID SIGNAL, ARE SELECTED BY MEANS OF IN SITU HYBRIDIZATION WITH P-32 LABELLED PLASMID DNA. THE CLONES ARE THEN TESTED FOR TRANSFORMING ACTIVITY ON NIH/3T3 CELLS.
The T-neo 1 cell line is the result of transfection of NIH/3T3 cells with a mixture containing irradiated fragmented BALB/c mouse deoxyribonucleic acid (DNA), the plasmid pM1sp which harbours a Mo-MLV long terminal repeat (LTR) and pKO-neo, a plasmid which can confer G 418 resistance as a selectable marker.
These experimentsyielded many G 418 resistant cells (3 to 5 clones per ng plasmid) almost all of which were not morphologically transformed. One clone gave rise to the T-neo 1 line which has a transformed phenotype, presumably caused by the action of a cellular oncogene associated with an acquired copy of the Mo-MLV LTR. Numerous copies of integrated plasmid of DNA were found in the genome of T-neo 1 cells. Southern blot hybridisation with a probe that can discriminate between endogenous and exogenous Mo-MLV LTRs, also revealed the presence of at least 6 copies of newly inserted LTR.
DNA FROM NORMAL MOUSE CELLS WILL BE PREPARED IN SUCH A WAY THAT IT CONTAINS MANY SINGLE STRAND BREAKS; FOR INSTANCE BY ISOLATION WITH ACID PHENOL, BY IRRADIATION OR BY TREATMENT WITH DNASE I. THEN NIH/3T3 CELLS ARE TRANSFECTED WITH SUCH A DNA PREPARATION AFTER THIS HAS BEEN FRAGMENTED BY ECORI DIGESTION AND LIGATED TO THE PLASMID WITH TRANSCRIPTION-ENHANCERS AND NEOMYCIN RESISTANCE GENE. THE TRANSFORMING GENE WILL THEN BE ISOLATED FROM SEVERAL TRANSFORMED CELL LINES. THESE GENES WILL BE COMPARED BY 2-D DNA BLOT HYBRIDIZATION PROCEDURES AS WELL AS BY NUCLEOTIDE SEQUENCING WITH THE NATIVE TNO GENE IN ORDER TO ESTABLISH THE NATURE AND THE SITE OF MUTATION.
ANOTHER APPROACH WILL BE TO INDUCE DNA REPAIR BY A LOW DOSE OF GAMMA IRRADIATION OF THE NIH/3T3 CELLS OR TRANSFECTION OF THE CELLS WITH IRRADIATED LOW-MOLECULAR-WEIGH DNA (LESS THAN 2 KBP) AND INCUBATION OF THE PRETREATED CELLS WITH ECORI MOUSE DNA FRAGMENTS WITHOUT SINGLE-STRAND BREAKS. FURTHERMORE, THE EFFECT OF CAFFEINE ON THE TRANSFORMATION BY TNO WILL BE INVESTIGATED.
TRANSFECTION STUDIES WITH THE MODIFIED PROCEDURE AS OUTLINED ABOVE (THE LIGATION OF CELLULAR DNA FRAGMENTS TO A PLASMID CONTAINING THE NEOMYCIN RESISTANCE GENE) WILL BE CARRIED OUT WITH DNA FROM RADIATION-INDUCED TUMOURS. BY MEANS OF SOUTHERN BLOTTING IT WILL BE FIRST INVESTIGATED WHETHER IN THE TRANSFORMED NIH/3T3 CELLS THE RAS OR TNO ONCOGENES ARE AMPLIFIED. IF NOT, A LIBRARY WILL BE PREPARED AND THE TRANSFORMING GENE WILL BE ISOLATED AS DESCRIBED BEFORE. THEREAFTER, IT WILL BE INVESTIGATED BY MEANS OF SOUTHERN BLOTTING WHETHER THIS ONCOGENE HAS BEEN TRANSLOCATED OR AMPLIFIED. THE LATTER WOULD BE IN SUPPORT OF THE GENE TRANSFER-MISREPAIR HYPOTHESIS.
PRIMARY RAT FIBROBLASTS WILL BE ONCOGENICALLY TRANSFORMED BY A MIXTURE OF DNA OF SEVERAL RADIATION-INDUCED RAT TUMOURS WITH A CLONED ACTIVATED RAS ONCOGENE, LIKE THE T24 GENE FROM HUMAN BLADDER CARCINOMA CELLS. IN CASE TUMOUR DNA WOULD FACILITATE THE TRANSFORMATION BY T24, IT WILL THEN BE LIGATED TO THE NEOMYCIN-RESISTANCE GENE, AND SO ON. ALSO IN THIS CASE, IT WILL BE INVESTIGATED WHETHER THE CO-TRANSFORMING GENE HAS BEEN TRANSLOCATED OR AMPLIFIED IN THE ORIGINAL RAT TUMOUR.