Separation of chromosomal DNA molecules fromC.albicansby pulsed field gel electropboresis

Abstract

Modifications have been made to standard pulse field gel electrophoresis (PFGE) systems to enable very large DNA molecules to be resolved. The single most important modification was to elevate the temperature of electrophoresis to 35 degrees C. This enabled the largest Saccharomyces cerevisiae chromosome to be reproducibly resolved. More impressively, it enabled the DNA of Candida albicans to be clearly resolved into six bands, a feat which was very difficult at lower temperatures. Even so, optimal resolution could only be obtained by carefully adjusting field voltages and switching times. The DNA from the two largest C. albicans chromosomes, which was estimated to be at least 5-10Mbp in size, ran somewhat anomalously, giving fuzzy bands which did not migrate in the direction of the average electric field. That the highest molecular weight band was a distinct chromosome was demonstrated by specific hybridisation to the C. albicans ADE2 gene probe. With further fine tuning, the PFGE system described here should be capable of resolving DNA from the smallest human chromosomes.

The use of karyotyping in the systematics of yeasts

“…Medically important Candida species have been extensively studied (Asakura et al 1991;Iwaguchi et al 1990;Mahrous et al 1990;Rustchenko-Bulgac 1991;Snell & Wilkins 1986;Suzuki et al 1988). Chromosomal length polymorphisms have been observed in numerous species, and Rustchenko-Bulgac (1991) considers this phenomenon to be an important mechanism for increasing genetic variability of apomictic species.…”

Antigens and immune responses in Candida albicans infection

“…Recently, separation of C albicans chromosomal DNA has been achieved by pulsefield gel electrophoresis (PFGE) (14). The karyotype of C. albicans is species-specific (15), and it can by this means readily be distinguished from that of other Candida strains and variants.…”

Pulsed-field gel electrophoresis studies of Rhizobiaceae

“…PF6 experiments have permitted the resolution of entire chromosomes of lower eucaryotes, such as a variety of fungi (Orbach et al, 1988;Carle and Olson, 1985;Schwartz and Cantor, 1984;Smith et al, 1987b;Vollrath and Davis, 1987;Snell and Wilkins, 1986;Brody and Carbon, 1989) and parasitic protozoa ( Van der Ploeg et al, 1984). Techniques have been developed that allow the manipulation of DNAs from various procaryotic and eucaryotic organisms .…”

“…albicans (Snell and Wilkins, 1986;Lasker et al, 1989), Aspergillus nidulans (Brody and Carbon, 1989), trypanosomes (Van der Ploeg et al, 1984;Bernards et al, 1986), leishmania (Giannini et al, 1986;Spithill and Samaras, 1985), Plasmodium falciparum (Corcoran et al, 1986;Van der Ploeg et al, 1985), Tetrahvmena thermofila (Conover and Brunk, 1986), giardia (C. L. Smith, Department of Genetics, Columbia University, NY, personal communication), and…”

“…Research using pulsed-field gel electrophoresis (PFG) has yielded complete physical maps for large regions of mammalian chromosomes (Fulton et al, 1989;Pohl et al, 1988;Shaw, 1986;Wallace et al, 1989), has permitted the resolution of entire chromosomes of lower eucaryotes, such as a variety of fungi (Orbach et al, 1988;Carle and Olson, 1985;Schwartz and Cantor, 1984;Smith et al, 1987b;Vollrath and Davis, 1987;Snell and Wilkins, 1986;Brody and Carbon, 1989) and parasitic protozoa (Van der Ploeg et al, 1984), and has produced physical maps of the entire chromosome of some procaryotes (Smith et al, 1987a;Ventra and Weiss, 1989;Lee and Smith, 1988;Kauc et al, 1989;Canard and Cole, 1989;Bautsch, 1988;Suwant andKaplan, 1989a, 1989b;Bancroft et al, 1989;Ely and Gerardot, 1988). PFG can also be used to monitor genetic rearrangements .…”