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Cellulose-bound DNA complementary to ovalbumin mRNA was used in a continous hybridization system to isolate single-stranded DNA molecules containing the ovalbumin gene. Fragmented DNA segments containing the ovalbumin gene were enriched 300-350 fold in one cycle of purification. Two cycles of purification resulted in a DNA fraction which was enriched 2300 fold in the ovalbumin sequence. The method is suitable for purification of the ovalbumin sequence from both sheared DNA fragments, as well as larger molecular weight DNA containing more than twice the number of nucleotides necessary to code for ovalbumin mRNA. The chromatographic procedures were specific and reproducible. In addition, the recovery of ovalbumin DNA was essentially quantitative (80-100%), even when large amounts of starting DNA (70-75 mg) were used. This purification scheme should also be useful for the enrichment of other unique sequence gened form eucaryotic DNA.

Methotrexate-resistant Leishmania tropica contain two separate regions of DNA amplification, one encoding the bifunctional thymidylate synthetase-dihydrofolate reductase (TS-DHFR) characteristic of protozoans and the other of yet unknown function. The amplified DNAs are initially found as extrachromosomal closed circular forms, which are unstable in the absence of selection. After prolonged culture in methotrexate the amplified DNAs are found as repetitive arrays associated with the chromosomal DNA fraction after CsCl-ethidium bromide density gradient centrifugation, and are stable once selection is removed. The molecular description of gene amplification in Leishmania thus closely parallels the cytological features of gene amplification in cultured mammalian cells.

We have studied a line of 3T6 mouse fibroblasts grown in progressively increasing concentrations of methotrexate. Initially, drug resistance results from amplification of the gene encoding the normal dihydrofolate reductase. Growth of these methotrexate-resistant populations at higher methotrexate concentrations results in the emergence of cells expressing high levels of dihydrofolate reductase with a reduced methotrexate affinity. Using the fluorescence-activated cell sorter, we demonstrate that the variant gene is not present in the population of cells resistant to lower levels of methotrexate, and hence we postulate that the mutational event occurred in cells already containing multiple normal dihydrofolate reductase genes. Growth of the variant cells in the absence of selection is associated with the permanent loss of the altered genes and the disappearance of double-minute chromosomes, on which these genes reside. The pattern of accumulation and loss of double-minute chromosomes is reproduced following transformation of methotrexate-sensitive cells with the altered genes. Our results are consistent with autonomous replication of double-minute chromosomes and a selective advantage of cells with the smallest number of extrachromosomal elements necessary for survival at a given methotrexate concentration.

We have examined in detail the RNA coding for dihydrofolate reductase (DHFR) in methotrexate-resistant mouse cells. We find four distinct DHFR messengers, ranging in size from 750 to 1600 nucleotides. All four are polyadenylated and polysomal and can be translated in vitro to produce a 21,000 dalton protein co-migrating with purified dihydrofolate reductase on SDS polyacrylamide gels. The major difference in these RNAs is the length of 3' untranslated regions, varying from about 80 nucleotides in the smallest mRNA to about 930 nucleotides in the largest. The RNAs are also present in methotrexate-sensitive murine cells and mouse liver. Multiple DHFR RNAs are found in the poly(A)+ RNA of methotrexate-resistant Chinese hamster ovary cells but are of different molecular weights than the mouse messengers. We discuss these results in terms of the function of 3' untranslated regions of eucaryotic mRNAs and the possible origin and significance of multiple messenger RNAs for a single protein.

Restriction endonucleases can be used to define the structure and position of genetic loci for which specific molecular hybridization reagents are available. We have used this approach to compare 18 chicken embryos with respect to several cellular genes; endogenous viral DNA related to the replicative genes of avian sarcoma virus (ASV) or to RAV-O, an endogenous virus of chickens; and sequences related to the transforming (src) gene of ASV. Each cellular gene eas remarkably homogeneous within our test population. We found little or no variation in globin and ovomucoid genes; ovalbumin and transferrin (with one exception) showed variation which is probably allelic in nature. The endogenous viral DNA which has homology with RAV-O was found at several different positions in host DNA and its structure resembled that of proviruses acquired by experimental infection, with sequences from both ends of viral RNA repeated near both ends of viral DNA. Within the population of 18 chickens, one endogenous provirus was always present, whereas the several other proviruses were each found in only a few members of this group. However, screening of additional chickens identified individuals lacking the provirus common to the initial 18 animals surveyed; in at least one embryo no RAV-O-related DNA was detected. These findings suggest that the endogenous RAV-O-related sequences have entered the germ line by relatively recent infection and are still segregating in several contemporary chicken flocks. The sequences in the chicken genome which have homology with the src gene of ASV are invariant from bird to bird and in this sense resemble a cellular gene rather than a viral sequence.

The genomic organization of the mouse dihydrofolate reductase gene has been determined by hybridization of specific cDNA sequences to restriction endonuclease-generated fragments of DNA from methotrexate-resistant S-180 cells. The dihydrofolate reductase gene contains a minimum of five intervening sequences (one in the 5' untranslated region and four in the protein-coding region) and spans a minimum of 42 kilobase pairs on the genome. Genomic sequences at the junction of the intervening sequence and mRNA-coding sequence and at the polyadenylation site have been determined. A similar organization is found in independently isolated methotrexate-resistant cell lines, in the parental sensitive cell line and in several inbred mouse strains, indicating that this organization represents that of the natural gene.