9. Selection of amplified genomic sequences

17.9 Selection of amplified genomic sequences

Key terms defined in this section

Amplification refers to the production of additional copies of a chromosomal sequence, found as intrachromosomal or extrachromosomal DNA.

The eukaryotic genome has the capacity to accommodate additional sequences of either exogenous or endogenous origin. Endogenous sequences may be produced by amplification of an existing sequence. The additional sequences often take the form of a tandem array, containing many copies of a repeating unit. A gene that is contained within the repeating unit is not necessarily expressed in every copy, but expression tends to increase with the number of copies.

A tandem array of multiple copies may exist in either of two forms in a cell. If it takes the form of an extrachromosomal unit, it is inherited in an irregular manner: there is no equivalent in animal cells to the ability of a bacterial plasmid to be segregated evenly at cell division, so the entire unit is lost at a high frequency. If the array is integrated into the genome, however, it becomes a component of the genotype, and is inherited like any other genomic sequence.

Amplification of endogenous sequences is provoked by selecting cells for resistance to certain agents. The best-characterized example of amplification results from the addition of methotrexate (mtx) to certain cultured cell lines. This reagent blocks folate metabolism. Resistance to it is conferred by mutations that change the activity of the enzyme dihydrofolate reductase (DHFR). As an alternative to change in the enzyme itself, the amount of enzyme may be increased. The cause of this increase is an amplification of the number of dhfr structural genes. Amplification occurs at a frequency greater than the spontaneous point mutation rate, generally ranging from 10 ^V4 V10 ^V6. Similar events now have been observed in >20 other genes.

A common feature in most of these systems is that highly resistant cells are not obtained in a single step, but instead appear when the cells are adapted to gradually increasing doses of the toxic reagent. So gene amplification may require several stages. Amplification generally occurs at only one of the two dhfr alleles; and increased resistance to methotrexate is accomplished by further increases in the degree of amplification at this locus (for review see Schimke, 1981; Stark and Wahl, 1984).

Figure 17.28 The dhfr gene can be amplified to give unstable copies that are extrachromosomal (double minutes) or stable (chromosomal). Extrachromosomal copies arise at early times.

The number of dhfr genes in a cell line resistant to methotrexate varies from 40 V400, depending on the stringency of the selection and the individual cell line. The mtx^r lines fall into two classes, distinguished by their response when the selective pressure for high levels of DHFR activity is relieved by growth in the absence of methotrexate. The basis for the difference is illustrated in Figure 17.28.

• In stable lines, the amplified genes are retained, because they reside on the chromosome, at the site usually occupied by the single dhfr gene. Usually the other chromosome retains its normal single copy of dhfr.
  
• In unstable lines, the amplified genes are at least partially lost when the selective pressure is released, because the amplified genes exist as an extrachromosomal array.

Figure 17.29 Amplified copies of the dhfr gene produce a homogeneously staining region (HSR) in the chromosome. Photograph kindly provided by Robert Schimke.

Gene amplification has a visible effect on the chromosomes. In stable lines, the dhfr locus can be visualized in the form of a homogeneously staining region (HSR). An example is shown in Figure 17.29. The HSR takes its name from the presence of an additional region that lacks any chromosome bands after treatments such as G-banding. This change suggests that some region of the chromosome between bands has undergone an expansion.

Figure 17.30 Amplified extrachromosomal dhfr genes take the form of double-minute chromosomes, as seen in the form of the small white dots. Photograph kindly provided by Robert Schimke.

In unstable cell lines, no change is seen in the chromosomes carrying dhfr. However, large numbers of elements called double-minute chromosomes are visible, as can be seen in Figure 17.30. In a typical cell line, each double-minute carries 2 V4 dhfr genes. The double minutes appear to be self-replicating; but they lack centromeres. As a result, they do not attach to the mitotic spindle and therefore segregate erratically, frequently being lost from the daughter cells. Notwithstanding their name, the actual status of the double minutes is regarded as extrachromosomal.

The irregular inheritance of the double minutes explains the instability of methotrexate resistance in these lines. Double minutes are lost continuously during cell divisions; and in the presence of methotrexate, cells with reduced numbers of dhfr genes will die. Only those cells that have retained a sufficient number of double minutes will appear in the surviving population.

The presence of the double minutes reduces the rate at which the cells proliferate. So when the selective pressure is removed, cells lacking the amplified genes have an advantage; they generate progeny more rapidly and soon take over the population. This explains why the amplified state is retained in the cell line only so long as cells are grown in the presence of methotrexate.

Because of the erratic segregation of the double minutes, increases in the copy number can occur relatively quickly as cells are selected at each division for progeny that have gained more than their fair share of the dhfr genes. Cells with greater numbers of copies are found in response to increased levels of methotrexate. The behavior of the double-minutes explains the stepwise evolution of the mtx^r condition and the incessant fluctuation in the level of dhfr genes in unstable lines.

Both stable and unstable lines are found after long periods of selection for methotrexate resistance. What is the initial step in gene amplification? After short periods of selection, most or all of the resistant cells are unstable. The formation of extrachromosomal copies clearly is a more frequent event than amplification within the chromosome. At very early times in the process, amplified dhfr genes can be found as (small) extrachromosomal units before double minutes or any change in chromosomes can be detected. This suggests that the acquisition of resistance is most often due to generation of extrachromosomal repeats.

The amplified region is longer than the dhfr gene itself. The gene has a length of ~31 kb, but the average length of the repeated unit is 500 V1000 kb in the chromosomal HSR. The extent of the amplified region is different in each cell line. The amount of DNA contained in a double minute seems to lie in a range of 100 V1000 kb.

How do the extrachromosomal copies arise? We know that their generation occurs without loss of the original chromosomal copy. There are two general possibilities. Additional cycles of replication could be initiated in the vicinity of the dhfr gene, followed by nonhomologous recombination between the copies. Or the process could be initiated by nonhomologous recombination between alleles. The extra copies could be released from the chromosome, possibly by some recombination-like event. Depending on the nature of this event, it could generate an extrachromosomal DNA molecule containing one or several copies. If the double minutes contain circular DNA, recombination between them in any case is likely to generate multimeric molecules.

Some information about the events involved in perpetuating the double minutes is given by an unstable cell line whose amplified genes code for a mutant DHFR enzyme. The mutant enzyme is not present in the original (diploid) cell line (so the mutation must have arisen at some point during the amplification process). Despite variations in the number of amplified genes, these cells display only the mutant enzyme. So the wild-type chromosomal genes cannot be continuously generating large numbers of double minutes anew, because these amplified copies would produce normal enzyme.

Once amplified extrachromosomal genes have arisen, therefore, changes in the state of the cell are mediated through these genes and not through the original chromosomal copies. When methotrexate is removed, the cell line loses its double minutes in the usual way. On re-exposure to the reagent, normal genes are amplified to give a new population of double minutes. This shows that none of the extrachromosomal copies of the mutant gene had integrated into the chromosome.

Another striking implication of these results is that the double minutes of the mutant line carried only mutant genes Xso if there is more than one dhfr gene per double minute, all must be of the mutant type. This suggests that multicopy double minutes can be generated from individual extrachromosomal genes.

A major question has been whether amplified chromosomal copies arise by integration of the extrachromosomal copies or by an independent mechanism. We do not know whether intrachromosomal amplification simply proceeds less often as a de novo step or requires extrachromosomal amplification to occur as an intermediate step. The form taken by the amplified genes is influenced by the cell genotype; some cell lines tend to generate double minutes, while others more readily display the HSR configuration.

The type of amplification event also depends upon the particular locus that is involved. Another case of amplification is provided by resistance to an inhibitor of the enzyme transcarbamylase, which occurs by amplification of the CAD gene. (CAD is a protein which has the first three enzymatic activities of the pathway for UMP synthesis.) Amplified CAD DNA is always found within the chromosome. In this case, the amplified genes are found in the form in the form of several dispersed amplified regions, often involving more than one chromosome.

Reviews

Schimke, R. T (1981). Chromosomal and extrachromosomal localization of amplified DHFR genes in cultured mammalian cells. Cold Spring Harbor Symp. Quant. Biol. 45, 785-797.

Stark, G. R. and Wahl, G. M. (1984). Gene amplification. Ann. Rev. Biochem 53, 447-491.