Loretta Goetsch

The HOP1 gene encodes a meiosis-specific component of yeast chromosomes

The HOP1 gene in Saccharomyces cerevisiae is important for meiotic chromosomal pairing, because hop1 diploids fail to form synaptonemal complex during meiosis and are defective in crossing over between, but not within, chromosomes. We demonstrate here that the HOP1 gene is transcriptionally regulated during sporulation and that the HOP1 protein is situated along the lengths of meiotic chromosomes. Furthermore, the HOP1 protein contains a Cys2/Cys2 zinc finger motif. A mutation within this motif that changes a cysteine to serine results in the hop1 phenotype, consistent with the possibility that the HOP1 gene product acts in chromosome synapsis by directly interacting with DNA. These observations demonstrate that HOP1 encodes a component of meiotic chromosomes, perhaps serving as a constituent of the synaptonemal complex.

Cdc53p acts in concert with cdc4p and cdc34p to control the G1-to-S- phase transition and identifies a conserved family of proteins

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.

A yeast gene essential for regulation of spindle pole duplication.

In eucaryotic cells, duplication of spindle poles must be coordinated with other cell cycle functions. We report here the identification in Saccharomyces cerevisiae of a temperature-sensitive lethal mutation, esp1, that deregulates spindle pole duplication. Mutant cells transferred to the nonpermissive temperature became unable to continue DNA synthesis and cell division but displayed repeated duplication of their spindle pole bodies. Although entry into this state after transient challenge by the nonpermissive temperature was largely lethal, rare survivors were recovered and found to have become increased in ploidy. If the mutant cells were held in G0 or G1 during exposure to the elevated temperature, they remained viable and maintained normal numbers of spindle poles. These results suggest dual regulation of spindle pole duplication, including a mechanism that promotes duplication as cells enter the division cycle and a negative regulatory mechanism, controlled by ESP1, that limits duplication to a single occurrence in each cell division cycle. Tetrad analysis has revealed that ESP1 resides at a previously undescribed locus on the right arm of chromosome VII.

Preparation of yeast cells for thin-section electron microscopy

Publisher Summary This chapter describes the preparation of yeast cells for thin-section electron microscopy. Yeast cells present the electron microscopist with a variety of challenges that have been met by the modification of standard protocols suitable for higher eukaryotes. A principal difficulty is the unusually high density of the cell. A partial solution is achieved by enzymatic removal of the cell wall after an initial fixation in glutaraldehyde, before further fixation and embedding. Removing the wall not only facilitates permeation of the embedding resin but also permits the cell to expand slightly, conferring differences in density that provide for variation in visual contrast. The walls of cells fixed during logarithmic growth are easily digested with appropriate enzymes without making any special provision at the time of glutaraldehyde fixation, but the walls of meiotic cells and certain types of vegetative cells, such as those approaching stationary phase, are refractory to digestion and require a pretreatment procedure. Use of these methods permits visualization of microtubules, certain filament systems, and many other features. As an alternative, membrane systems can be accentuated by special methods, such as fixation with permanganate or the more recently applied technique of postfixation in an osmium tetroxide-ferrocyanide solution. No single method has been described to date that addresses both requirements fully, so the investigator must opt for the method that favors structures of primary concern.

The Molecular Systematics of Rhododendron (Ericaceae): A Phylogeny Based Upon RPB2 Gene Sequences

Abstract Classification of Rhododendron species based on morphology has led to a consensus taxonomy recognizing the major subgenera Azaleastrum, Hymenanthes, Pentanthera, Rhododendron, Tsutsusi, and three minor ones. To determine whether these subgenera are monophyletic and to infer phylogenetic relationships between Rhododendron sections and species, we carried out a cladistic analysis using molecular data, including all groups within the genus. For this purpose, we sequenced a large part of the nuclear gene RPB2-I, encoding a major RNA Polymerase II subunit, from 87 species and analyzed the data by maximum parsimony, maximum likelihood, and Bayesian methods. The resulting phylogenies show subgenera Azaleastrum and Pentanthera to be polyphyletic and group all Rhododendron species (except the two in section Therorhodion) into three large clades. Based upon these results, modifications in Rhododendron classification are proposed, which consolidate minor subgenera and recognize monophyletic subgenera and sections.

Reversible pachytene arrest of Saccharomyces cerevisiae at elevated temperature.

SummaryThe temperature sensitivity of sporulation in a well-characterized yeast strain lacking any known temperature sensitive genes has been investigated. Cytological observations by electron microscopy demonstrate that cells incubated in sporulation medium at a temperature inhibitory to sporulation became arrested in meiotic prophase. The stage of arrest was identified as pachytene by the presence of duplicated (but unseparated) spindle pole bodies and synaptonemal complex. Transfer of the arrested culture to lower temperature permitted resumption of meiosis and sporulation; transfer to vegetative medium resulted in reversion to mitotic division. Genetic analysis of cells that had reverted to mitosis revealed that commitment to intragenic recombination had occurred by the time of arrest. Prolonged incubation at the elevated temperature resulted in the enhancement of intragenic recombination above normal levels, suggesting that some aspect of recombination continued to occur during the pachytene arrest. Evidence is presented that DNA replication, although depressed overall in the arrested cultures, had occurred to completion in many arrested cells.

Meiotic cytology of Saccharomyces cerevisiae in protoplast lysates

SummaryThis report describes cytological features of meiosis in Saccharomyces cerevisiae prepared for electron microscopy by lysis of protoplasts or nuclei on an aqueous surface. Whereas the chromatin of cells lysed before or after meiotic prophase was widely dispersed, pachytene bivalents appeared as discrete, elongate masses of compact chromatin. These bivalents were of nearly uniform thickness; they ranged in length from about 0.6 μm to 4.0 μm, with a median of 1.6–1.8 μm. Enzymatic digestion of chromosomal DNA removed the chromatin to reveal the underlying synaptonemal complex. The lysis of partially purified nuclei was less disruptive and thereby revealed the regular association of the telomeres with fragments of the nuclear envelope. In tetraploid cells, pachytene lysates contained quadrivalents characterized by the close apposition of chromatin masses of similar length. One or more points of intimate association appear to represent sites of exchange between pairing partners. The departure of the diploid cells from pachytene was accompanied by the renewed association of spindle microtubules with the chromosomes shortly before the diplotene chromosomes decondensed. Later, the successive meiotic divisions were identified by the appearance of a single spindle for meiosis I and of two spindles for meiosis II.