The identification of pathogenic yeast strains by electrophoretic analysis of their chromosomes.

Epidemiological studies require characterisation of pathogenic yeasts at and below the species level. The chromosomes of 130 strains of four pathogenic species of the genus Candida, isolated from clinical material, were separated by pulsed field electrophoresis with the clamped homogeneous electric field (CHEF) technique. Each species was characterised by a distinct electrophoretic karyotype (EK). Furthermore, smaller variations of the EK amongst strains belonging to the same species appeared to offer a useful means of strain differentiation. A karyotyping system is proposed for C. albicans. The EKs were assigned to a code of four numbers which designated the number of bands that could be resolved in each of four sets of chromosomes. Morphotypes of the colonies of C. albicans on malt agar plates, which did not correlate with the EK, could provide a complementary means of strain characterisation in epidemiological studies.

Yeast telomere length varies in response to changes in the amount of polyC1-3A in the cell.

Yeast chromosomes terminate in a GC-rich tail of DNA. Previous investigations have shown that the length of this tail can change in response to genetic variation. Here we present data that show that the length can also alter in response to changes in the amount of the GC-rich DNA found elsewhere in the nucleus.

Characterization of telomere DNA from Neurospora crassa.

The nucleotide sequence of the telomere at the right end of linkage group V (VR) in the standard OR23-IV-A strain of the filamentous fungus, Neurospora crassa, reveals the following features. At the chromosome terminus, tandem repeats of the hexanucleotide TTAGGG are present. Immediately centromere-proximal to the simple sequence repeat is a more complex element called Pogo that is reiterated 5-10 times in the genomes of various Neurospora strains. The element possesses several features characteristic of a transposable element: direct repeats of 318 bp flank the element, there is a long internal open reading frame (ORF), and a 3-bp duplication is found at its borders. However, Pogo has other structural features that are more difficult to reconcile with the standard model of a transposable element. A second telomere from Neurospora was also cloned by screening a genomic lambda library with a synthetic oligodeoxyribonucleotide homologous to the simple sequence repeats. This telomere is entirely non-homologous with the VR telomere except for the TTAGGG repeats, has no associated copy of Pogo, and has no nearby ORFs. There are no long stretches of TTAGGG repeats present in the Neurospora genome at non-telomeric sites.

Chromatin digestion with restriction endonucleases reveals 150-160 bp of protected DNA in the centromere of chromosome XIV in Saccharomyces cerevisiae.

Isolated nuclei of Saccharomyces cerevisiae were incubated with five restriction nucleases. Out of the twenty-one recognition sequences for these nucleases in the centromere region of chromosome XIV, only five are accessible to cleavage. These sites map 11 bp and 74 bp to the left and 27 bp, 41 bp and 290 bp to the right, respectively, of the boundaries of the 118 bp functional CEN14 DNA sequence. The distance between the sites accessible to cleavage and closest to CEN14 is 156 bp, suggesting this is the maximal size of DNA protected in CEN14 chromatin. The DNA in CEN14 chromatin protected against cleavage with DNase I and micrococcal nuclease overlaps almost completely with this region. Hypersensitive regions flanking both sides are approximately 60 bp long. Analyses of other S. cerevisiae centromeres with footprinting techniques in intact cells or nucleolytic cleavages in isolated nuclei are discussed in relation to our results. We conclude that structural data of chromatin obtained with restriction nucleases are reliable and that the structure of CEN14 chromatin is representative for S. cerevisiae centromeres.