Yeast chromosome, separation

Schwartz, D. C., and C. R. Cantor, Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37 67-75, 1984. 

Figure 31.14. Yeast Chromosomes. Pulsed-field electrophoresis allows the separation of 16 yeast chromosomes. [From G. Chu, D. Wollrath, and R. W. Davis. Science 234(1986) 1583.]...

Clones are auxotrophically selected in top agar and screened for completeness by multiplex PCR. Those that amplify all PCR products are screened for size by restriction digestion and either pulsed-field or field inversion gel electrophoresis (PFGE or FIGE), followed if necessary by Southern blot. Clones can also be sequenced (6), although it is likely to be difficult to isolate significant qnantities of clone DNA separately from yeast chromosomal DNA. 

By using two unequal fields and two unequal pulse durations, Lalande et al. 2 showed that it is indeed possible to separate yeast chromosomes (200-2200 kbp) in order of molecular size by using Just a few pulse durations instead of a full ramp. A quantitative analyzis of the experimental results helped to understand the electrophoretic processes involved as well as to optimize the separation. In this section, we discuss field-inversion electrophoresis, and compare the predictions of our model with available experimental results. 

In terms of evolutionary biology, the complex mitotic process of higher animals and plants has evolved through a progression of steps from simple prokaryotic fission sequences. In prokaryotic cells, the two copies of replicated chromosomes become attached to specialized regions of the cell membrane and are separated by the slow intrusion of the membrane between them. In many primitive eukaryotes, the nuclear membrane participates in a similar process and remains intact the spindle microtubules are extranuclear but may indent the nuclear membrane to form parallel channels. In yeasts and diatoms, the nuclear membrane also remains intact, an intranuclear polar spindle forms and attaches at each pole to the nuclear envelope, and a single kinetochore microtubule moves each chromosome to a pole. In the cells of higher animals and plants, the mitotic spindle starts to form outside of the nucleus, the nuclear envelope breaks down, and the spindle microtubules are captured by chromosomes (Kubai, 1975 Heath, 1980 Alberts et al., 1989). 

---