Restriction enzyme produces linear plasmid



Linear plasmid lacking TEL is unstable

Linear plasmids containing ARS and CEN behave like normal chromosomes if genomic fragment TEL is added to both ends

▲ EXPERIMENTAL FIGURE 10-32 Yeast transfection experiments identify the functional chromosomal elements necessary for normal chromosome replication and segregation. In these experiments, plasmids containing the LEU gene from normal yeast cells are constructed and introduced into leu~ cells by transfection. If the plasmid is maintained in the leu~ cells, they are transformed to LEU+ by the LEU gene on the plasmid and can form colonies on medium lacking leucine. (a) Sequences that allow autonomous replication (ARS) of a plasmid were identified because their insertion into a plasmid vector containing a cloned LEU gene resulted in a high frequency of transformation to LEU+. However, even plasmids with ARS exhibit poor segregation during mitosis, and therefore do not appear in each of the daughter cells. (b) When randomly broken pieces of genomic yeast DNA are inserted into plasmids containing ARS and LEU, some of the subsequently transfected cells produce large colonies, indicating that a high rate of mitotic segregation among their plasmids is facilitating the continuous growth of daughter cells. The DNA recovered from plasmids in these large colonies contains yeast centromere (CEN) sequences. (c) When leu~ yeast cells are transfected with linearized plasmids containing LEU, ARS, and CEN, no colonies grow. Addition of telomere (TEL) sequences to the ends of the linear DNA gives the linearized plasmids the ability to replicate as new chromosomes that behave very much like a normal chromosome in both mitosis and meiosis. [See A. W. Murray and J. W. Szostak, 1983, Nature 305:89, and L. Clarke and J. Carbon, 1985, Ann. Rev. Genet. 19:29.]

Even though circular ARS-containing plasmids can replicate in yeast cells, only about 5-20 percent of progeny cells contain the plasmid because mitotic segregation of the plasmids is faulty. However, plasmids that also carry a CEN sequence, derived from the centromeres of yeast chromosomes, segregate equally or nearly so to both mother and daughter cells during mitosis (see Figure 10-32b).

If circular plasmids containing an ARS and CEN sequence are cut once with a restriction enzyme, the resulting linear plasmids do not produce LEU+ colonies unless they contain special telomeric (TEL) sequences ligated to their ends (see Figure 10-32c). The first successful experiments involving transfection of yeast cells with linear plasmids were achieved by using the ends of a DNA molecule that was known to replicate as a linear molecule in the ciliated protozoan Tetrahymena. During part of the life cycle of Tetrahy-mena, much of the nuclear DNA is repeatedly copied in short pieces to form a so-called macronucleus. One of these repeated fragments was identified as a dimer of ribosomal DNA, the ends of which contained a repeated sequence (G4T2) n. When a section of this repeated TEL sequence was ligated to the ends of linear yeast plasmids containing ARS and CEN, replication and good segregation of the linear plasmids occurred.

Centromere Sequences Vary Greatly in Length

Once the yeast centromere regions that confer mitotic segregation were cloned, their sequences could be determined and compared, revealing three regions (I, II, and III) conserved between them (Figure 10-33a). Short, fairly well conserved nucleotide sequences are present in regions I and III. Although region II seems to have a fairly constant length, it contains no definite consensus sequence; however, it is rich in A and T residues. Regions I and III are bound by proteins that interact with a set of more than 30 proteins that bind the short sS. cerevisiae chromosome to one microtubule of the spindle apparatus during mitosis. Region II is bound to a nu-cleosome that has a variant form of histone H3 replacing the usual H3. Centromeres from all eukaryotes similarly are bound by nucleosomes with a specialized, centromere-specific form of histone H3 called CENP-A in humans.

sS. cerevisiae has by far the simplest centromere sequence known in nature.

In the fission yeast ,S. pombe, centromeres are «40 kb in length and are composed of repeated copies of sequences similar those in ,S. cerevisiae centromeres. Multiple copies of proteins homologous to those that interact with the ,S. cerevisiae centromeres bind to these complex ,S. pombe centromeres and in turn bind the much longer ,S. pombe chromosomes to several microtubules of the mitotic spindle apparatus. In plants and animals, centromeres are megabases in length and are composed of multiple repeats of simple-sequence DNA. One Drosophila simple-sequence DNA, which comes from a centromeric region, has a repeat unit that bears some similarity to yeast CEN regions I and III (see Figure 10-33b). In humans, centromeres contain 2- to 4-megabase arrays of a 171-bp simple-sequence DNA called alphoid DNA that is bound by nucleosomes with the CENP-A histone H3 variant, as well as other repeated simple-sequence DNA.

In higher eukaryotes, a complex protein structure called the kinetochore assembles at centromeres and associates with multiple mitotic spindle fibers during mitosis. Homologs of most of the centromeric proteins found in the yeasts occur in humans and other higher eukaryotes and are thought to be components of kinetochores. The role of the centromere and proteins that bind to it in the segregation of sister chromatids during mitosis is described in Chapters 20 and 21.

Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes

Sequencing of telomeres from a dozen or so organisms, including humans, has shown that most are repetitive oligomers with a high G content in the strand with its 3' end at the end of the chromosome. The telomere repeat sequence in humans and other vertebrates is TTAGGG. These simple sequences are repeated at the very termini of chromosomes for a total of a few hundred base pairs in yeasts and protozoans and a few thousand base pairs in vertebrates. The 3' end of the G-rich strand extends 12-16 nucleotides beyond the 5' end of the complementary C-rich strand. This region is bound by specific proteins that both protect the ends of

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