Reference Work Entry

Encyclopedia of Genetics, Genomics, Proteomics and Informatics

pp 84-85

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Amplification

Amplification is the temporary synthesis of extra, functional copies of some genes, in vivo or in vitro, by some forms of the polymerase chain reaction. Bacteriophage l can be amplified by a series of nitrocellulose filter transfers after in situ hybridization. The addition of chloramphenicol (10–20 μg/mL) to pBR322 and pBR327 may amplify plasmid yield, if the synthesis of protein is not completely prevented. Cosmid libraries may be amplified by starting on solid plates followed by liquid cultures. Replica-plating can amplify animal cell cultures. Approximately 5 × 104 colonies can be accommodated on a 138-mm filter, and this way about 30 filters are required to obtain a representative library of overlapping fragments. DNA amplification can occur in a genetically programmed and predetermined manner in eukaryotes. For example, in the ovarian follicle of Drosophila, large quantities of an egg-shell protein is needed during oogenesis. The need is met by a disproportionately favorable replication of the chorion gene clusters in the X-chromosome and chromosome 3. DNA replication is initiated bidirectionally at a replicational origin, and generates multiple copies of the genes needed. The replication tapers off after a distance and the flanking regions are amplified less and less in proportion to the distance from the origin. Similar programmed amplification takes place in the ribosomal genes of amphibia during intense periods of protein synthesis in embryogenesis. The approximately 500–600 genomic copies of rRNA genes may thus be increased by a factor of 1000. The replication of detached DNA sequences follows a rolling circle type process, and the new DNAs (in about 100 rDNA repeats) are separately localized in micronuclei. The replicates of these nuclei are structurally similar, indicating that they are the clonal products of a single replicating unit; but the new micronuclei generated in different cells may not be the same as judged by the differences in length in the intergenic spacers. Ribosomal DNA amplification takes place during the amitotic divisions of the protozoon, Tetrahymena. Here again, the macronuclear rDNA copies may be selectively amplified in the 104 range, whereas the micronuclear DNA contains only a single rDNA gene. A genetically non-programmed amplification takes place in several mutant cell lines to correct mutational defects. Producing multiple copies of gene-controlling low-efficiency enzymes may compensate for enzyme deficiencies. Transfection of ADA genes to mammalian cells may be amplified in the presence of dCF (see adenosine deaminase). Mammalian cells can be amplified if they are co-transfected with the dhfr (conveying methotrexate resistance) gene and other desired sequences. In the presence of methotrexate, the dhfr genes, as well as the flanking DNA, may be amplified (1000) fold. The amplified DNA, in stable lines, is integrated into the chromosome in homogeneously stained regions (HSRs). In unstable cell lines, dhfr occurs in autonomously replicating elements, called double-minute chromosomes (DMs), which have no centromeres and can be maintained only in cultures that contain methotrexate. Amplification may generate fragile sites in the chromosomes by integration of DMs sequences. Hypoxia may be a factor inducing such integration. Some general features of amplification are: (i) expansion of a particular locus and flanking regions, or the generation of small supernumerary chromosomes called double minutes that contain the critical gene, (ii) possible rearrangements of the amplified unit (iii) the amplified sequences are not all identical and may change, but these changes are somewhat unusual because a larger number of copies may be altered simultaneously in an identical manner. In vivo amplification of genes during evolution may account for the presence of gene families. Some amplified genes, in which production of a larger number of copies was no longer advantageous, may have acquired new functions without entirely losing their structural similarity to the ancestral sequences. Other members of the amplified group lost their function(s) through deletions and mutations and became pseudogenes. Carcinogenesis commonly involves amplification of some oncogenes and genes involved with the cell cycle (cyclins). Fragile sites in some chromosomes aid amplification. PCR, MDA, nitrocellulose filter, in situ hybridization, chloramphenicol, pBR322, cosmid library, oogenesis, chorion, bidirectional replication, rolling circle, micronucleus, ADA, HSR, methotrexate, fragile sites, pseudogene, unequal crossing over, DM chromosome, adaptive amplification, breakage–bridge–fusion cycles, translocation heterozygote; Romero D, Palacios R 1997 Annu Rev Genet 31:91; Monni O et al 2001 Proc Natl Acad Sci USA 98:5711; Dean FB et al 2002 Proc Natl Acad Sci USA 99:5261; Tower J 2004 Annu Rev Genet 38:273.

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