11. Summary

17.11 Summary

Yeast mating type is determined by whether the MAT locus carries the a or α sequence. Expression in haploid cells of the sequence at MAT leads to expression of genes specific for the mating type and to repression of genes specific for the other mating type. Both activation and repression are achieved by control of transcription, and require factors that are not specific for mating type as well as the products of MAT. The functions that are activated in either mating type include secretion of the appropriate pheromone and expression on the cell surface of the receptor for the opposite type of pheromone. Interaction between pheromone and receptor on cells of either mating type activates a G protein on the membrane, and sets in train a common pathway that prepares cells for sporulation. Diploid cells do not express mating type functions.

Additional, silent copies of the mating-type sequences are carried at the loci HMLα and HMRa. They are repressed by the actions of the sir loci. Cells that carry the HO endonuclease display a unidirectional transfer process in which the sequence at HMLα replaces an a sequence at MAT, or the sequence at HMRa replaces an α sequence at MAT. The endonuclease makes a double-strand break at MAT, and a free end invades either HMLα or HMRa. MAT initiates the transfer process, but is the recipient of the new sequence. The HO endonuclease is transcribed in mother cells but not daughter cells, and is under cell-cycle control. So switching is detected only in the products of a division, and the mating type has been switched in both daughter cells.

Trypanosomes carry >1000 sequences coding for varieties of the surface antigen. Only a single VSG is expressed in one cell, from an active site located near a telomere. The VSG may be changed by substituting a new coding sequence at the active site via a gene conversion process, or by switching the site of expression to another telomere. Switches in expression occur every 10⁴ 10⁶ divisions.

Agrobacteria induce tumor formation in wounded plant cells. The wounded cells secrete phenolic compounds that activate vir genes carried by the Ti plasmid of the bacterium. The vir gene products cause a single strand of DNA from the T-DNA region of the plasmid to be transferred to the plant cell nucleus. Transfer is initiated at one boundary of T-DNA, but ends at variable sites. The single strand is converted into a double strand and integrated into the plant genome. Genes within the T-DNA transform the plant cell, and cause it to produce particular opines (derivatives of arginine). Genes in the Ti plasmid allow Agrobacterium to metabolize the opines produced by the transformed plant cell.

Endogenous sequences may become amplified in cultured cells. Exposure to methotrexate leads to the accumulation of cells that have additional copies of the dhfr gene. The copies may be carried as extrachromosomal arrays in the form of double-minute "chromosomes," or they may be integrated into the genome at the site of one of the dhfr alleles. Double-minute chromosomes are unstable, and disappear from the cell line rapidly in the absence of selective pressure. The amplified copies may originate by additional cycles of replication that are associated with recombination events.

New sequences of DNA may be introduced into a cultured cell by transfection or into an animal egg by microinjection. The foreign sequences may become integrated into the genome, often as large tandem arrays. The array appears to be inherited as a unit in a cultured cell. The sites of integration appear to be random. A transgenic animal arises when the integration event occurs into a genome that enters the germ-cell lineage. A transgene or transgenic array is inherited in Mendelian manner, but the copy number and activity of the gene(s) may change in the progeny. Often a transgene responds to tissue- and temporal regulation in a manner that resembles the endogenous gene. Using conditions that promote homologous recombination, an inactive sequence can be used to replace a functional gene, thus creating a null locus. Transgenic mice can be obtained by injecting recipient blastocysts with ES cells that carry transfected DNA.