Eukaryotic Genes Can Be Amplified or Rearranged During Development or in Response to Drugs

During early development of metazoans, there is an abrupt increase in the need for specific molecules such as ribosomal RNA and messenger RNA molecules for proteins that make up such organs as the eggshell. One way to increase the rate at which such molecules can be formed is to increase the number of genes available for transcription of these specific molecules. Among the repetitive DNA sequences are hundreds of copies of ri-bosomal RNA genes and tRNA genes. These genes preexist repetitively in the genomic material of the gametes

Table39-4. Gene expression is regulated by transcription and in numerous other ways in eukaryotic cells.

• Gene amplification

• Gene rearrangement

• RNA processing

• Alternate mRNA splicing

• Transport of mRNA from nucleus to cytoplasm

• Regulation of mRNA stability and thus are transmitted in high copy numbers from generation to generation. In some specific organisms such as the fruit fly fdrosophilaj, there occurs during oogenesis an amplification of a few preexisting genes such as those for the chorion (eggshell) proteins. Subsequently, these amplified genes, presumably generated by a process of repeated initiations during DNA synthesis, provide multiple sites for gene transcription (Figures 36-16 and 39-18).

As noted in Chapter 37, the coding sequences responsible for the generation of specific protein molecules are frequently not contiguous in the mammalian genome. In the case of antibody encoding genes, this is particularly true. As described in detail in Chapter 50, immunoglobulins are composed of two polypeptides, the so-called heavy (about 50 kDa) and light (about 25 kDa) chains. The mRNAs encoding these two protein subunits are encoded by gene sequences that are subjected to extensive DNA sequence-coding changes. These DNA coding changes are integral to generating the requisite recognition diversity central to appropriate immune function.

IgG heavy and light chain mRNAs are encoded by several different segments that are tandemly repeated in the germline. Thus, for example, the IgG light chain is composed of variable (VL), joining (JL), and constant (CL) domains or segments. For particular subsets of IgG light chains, there are roughly 300 tandemly repeated VL gene coding segments, five tandemly arranged JL coding sequences, and roughly ten CL gene coding segments. All of these multiple, distinct coding regions are located in the same region of the same chromosome, and each type of coding segment (VL, JL, and CL) is tandemly repeated in head-to-tail fashion within the segment repeat region. By having multiple VL, JL, and CL segments to choose from, an immune cell has a greater repertoire of sequences to work with to develop

Unamplified s36 s38

s36 s38

s36 s38

Figure 39-18. Schematic representation of the amplification of chorion protein genes s36 and s38. (Reproduced, with permission, from Chisholm R: Gene amplification during development. Trends Biochem Sci 1982;7:161.)

both immunologic flexibility and specificity. However, a given functional IgG light chain transcription unit— like all other "normal" mammalian transcription units—contains only the coding sequences for a single protein. Thus, before a particular IgG light chain can be expressed, single VL, JL, and CL coding sequences must be recombined to generate a single, contiguous transcription unit excluding the multiple nonutilized segments (ie, the other approximately 300 unused VL segments, the other four unused JL segments, and the other nine unused CL segments). This deletion of unused genetic information is accomplished by selective DNA recombination that removes the unwanted coding DNA while retaining the required coding sequences: one VL, one JL, and one CL sequence. (VL sequences are subjected to additional point mutagenesis to generate even more variability—hence the name.) The newly recombined sequences thus form a single transcription unit that is competent for RNA polym-erase II-mediated transcription. Although the IgG genes represent one of the best-studied instances of directed DNA rearrangement modulating gene expression, other cases of gene regulatory DNA rearrangement have been described in the literature. Indeed, as detailed below, drug-induced gene amplification is an important complication of cancer chemotherapy.

In recent years, it has been possible to promote the amplification of specific genetic regions in cultured mammalian cells. In some cases, a several thousand-fold increase in the copy number of specific genes can be achieved over a period of time involving increasing doses of selective drugs. In fact, it has been demonstrated in patients receiving methotrexate for cancer that malignant cells can develop drug resistance by increasing the number of genes for dihydrofolate reductase, the target of methotrexate. Gene amplification events such as these occur spontaneously in vivo—ie, in the absence of ex-ogenously supplied selective agents—and these unscheduled extra rounds of replication can become "frozen" in the genome under appropriate selective pressures.

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Diabetes 2

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