Test for temporal or spatial restrictions in gene product function during the cell division cycle

The ability of a functional gene to complement a nonfunctional gene may depend upon the intracellular relationship of the two genes. If so, the function of the gene product in question must be limited in time or in space. CDC (cell division cycle) gene products of Saccharomyces cerevisiae control discrete steps in cell division; therefore, they constitute reasonable candidates for genes that function with temporal or spatial restrictions. In an attempt to reveal such restrictions, we compared the ability of a CDC gene to complement a temperature-sensitive cdc gene in diploids where the genes are located within the same nucleus to complementation in heterokaryons where the genes are located in different nuclei. In CDC X cdc matings, complementation was monitored in rare heterokaryons by assaying the production of cdc haploid progeny (cytoductants) at the restrictive temperature. The production of cdc cytoductants indicates that the cdc nucleus was able to complete cell division at the restrictive temperature and implies that the CDC gene product was provided by the other nucleus or by cytoplasm in the heterokaryon. Cytoductants from cdc28 or cdc37 crosses were not efficiently produced, suggesting that these two genes are restricted spatially or temporally in their function. We found that of the cdc mutants tested 33 were complemented; cdc cytoductants were recovered at least as frequently as CDC cytoductants. A particularly interesting example was provided by the CDC4 gene. Mutations in CDC4 were found previously to produce a defect in both cell division and karyogamy. Surprisingly, the cell division defect of cdc4 nuclei is complemented by CDC4 nuclei in a heterokaryon, whereas the karyogamy defect is not.

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DIPLOID SPORE FORMATION AND OTHER MEIOTIC EFFECTS OF TWO CELL-DIVISION-CYCLE MUTATIONS OF SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/96.4.859 ◽

1980 ◽

Vol 96 (4) ◽

pp. 859-876 ◽

Cited By ~ 3

Author(s):

David Schild ◽

Breck Byers

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Division ◽

Spindle Pole Body ◽

Spindle Pole ◽

Cell Division Cycle ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Single Spore ◽

Diploid Cells ◽

Meiosis I

ABSTRACT The meiotic effects of two cell-division-cycle mutations of Saccharomyces cerevisiae (cdc5 and cdc14) have been examined. These mutations were isolated by L. H. Hartwell and his colleagues and characterized as defective in mitosis, causing a temperature-sensitive arrest in late nuclear division. When subjected to the restrictive temperature in meiosis, diploid cells homozygous for either of these mutations generally proceeded through premeiotic DNA synthesis and commitment to meiotic levels of recombination, but then arrested at a stage following spindle pole body (SPB) duplication and separation. The two SPBs lacked the interconnection by spindle microtubules typical of the complete meiosis I spindle. Challenge of these homozygotes by a semi-restrictive temperature often caused the production of asci containing two diploid spores. Genetic analysis of the viable pairs of spores revealed that each spore had become homozygous for centromere-linked markers significantly more frequently than for distal markers, indicating that the two spores each contained pairs of sister centromeres that had co-segregated in the reductional division of meiosis I. Ultrastructural analysis of the cdc5 homozygote demonstrated that these cells had completed meiosis I and formed two meiosis II spindles, but that the latter remained unusually short. This resulted in the encapsulation of both poles of each spindle within a single spore wall. These mutations therefore are defective in both meiotic divisions, as well as in the mitotic division described originally.

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GENETIC CONTROL OF THE CELL DIVISION CYCLE IN YEAST: V. GENETIC ANALYSIS OF cdc MUTANTS

Genetics ◽

10.1093/genetics/74.2.267 ◽

1973 ◽

Vol 74 (2) ◽

pp. 267-286

Author(s):

Leland H Hartwell ◽

Robert K Mortimer ◽

Joseph Culotti ◽

Marilyn Culotti

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Nuclear Gene ◽

Temperature Shift ◽

Time Lapse ◽

Cell Division Cycle ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Gene Products ◽

Diploid Cells

ABSTRACT One hundred and forty-eight temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae have been isolated and characterized. Complementation studies ordered these recessive mutations into 32 groups and tetrad analysis revealed that each of these groups defines a single nuclear gene. Fourteen of these genes have been located on the yeast genetic map. Functionally related cistrons are not tightly clustered. Mutations in different cistrons frequently produce different cellular and nuclear morphologies in the mutant cells following incubation at the restrictive temperature, but all the mutations in the same cistron produce essentially the same morphology. The products of these genes appear, therefore, each to function individually in a discrete step of the cell cycle and they define collectively a large number of different steps. The mutants were examined by time-lapse photomicroscopy to determine the number of cell cycles completed at the restrictive temperature before arrest. For most mutants, cells early in the cell cycle at the time of the temperature shift (before the execution point) arrest in the first cell cycle while those later in the cycle (after the execution point) arrest in the second cell cycle. Execution points for allelic mutations that exhibit first or second cycle arrest are rather similar and appear to be cistron-specific. Other mutants traverse several cycles before arrest, and its suggested that the latter type of response may reveal gene products that are temperature-sensitive for synthesis, whereas the former may be temperature-sensitive for function. The gene products that are defined by the cdc cistrons are essential for the completion of the cell cycle in haploids of a and α mating type and in a/α diploid cells. The same genes, therefore, control the cell cycle in each of these stages of the life cycle.

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THE ROLE OF S. CEREVISIAE CELL DIVISION CYCLE GENES IN NUCLEAR FUSION

Genetics ◽

10.1093/genetics/100.2.175 ◽

1982 ◽

Vol 100 (2) ◽

pp. 175-184

Author(s):

Susan K Dutcher ◽

Leland H Hartwell

Keyword(s):

Cell Division ◽

Parent Strain ◽

Nuclear Fusion ◽

Cell Division Cycle ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Gene Products ◽

Sensitive Cell ◽

Single Lesion

ABSTRACT Forty temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae were examined for their ability to complete nuclear fusion during conjugation in crosses to a CDC parent strain at the restrictive temperature. Most of the cdc mutant alleles behaved as the CDC parent strain from which they were derived, in that zygotes produced predominantly diploid progeny with only a small fraction of zygotes giving rise to haploid progeny (cytoductants) that signalled a failure in nuclear fusion. However, cdc4 mutants exhibited a strong nuclear fusion (karyogamy) defect in crosses to a CDC parent and cdc28, cdc34 and cdc37 mutants exhibited a weak karyogamy defect. For all four mutants, the karyogamy defect and the cell cycle defect cosegregated, suggesting that both defects resulted from a single lesion for each of these cdc mutants. Therefore, the cdc 4, 28, 34 and 37 gene products are required in both cell division and karyogamy.

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THE SELECTION OF S. CEREVISIAE MUTANTS DEFECTIVE IN THE START EVENT OF CELL DIVISION

Genetics ◽

10.1093/genetics/95.3.561 ◽

1980 ◽

Vol 95 (3) ◽

pp. 561-577 ◽

Cited By ~ 4

Author(s):

Steven I Reed

Keyword(s):

Cell Division ◽

Genetic Analysis ◽

Linkage Map ◽

Nuclear Genes ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Mating Pheromone ◽

Preliminary Characterization ◽

Complementation Groups ◽

Selection Of

ABSTRACT Thirty-three temperature-sensitive mutations defective in the start event of the cell division cycle of Saccharomyces cereuisiae were isolated and subjected to preliminary characterization. Complementation studies assigned these mutations to four complementation groups, one of which, cdc28, has been described previously. Genetic analysis revealed that these complementation groups define single nuclear genes, unlinked to one another. One of the three newly identified genes, cdc37, has been located in the yeast linkage map on chromosome IV, two meiotic map units distal to hom2.—Each mutation produces stage-specific arrest of cell division at start, the same point where mating pheromone interrupts division. After synchronization at start by incubation at the restrictive temperature, the mutants retain the capacity to enlarge and to conjugate.

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Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

Genetics ◽

10.1093/genetics/134.1.63 ◽

1993 ◽

Vol 134 (1) ◽

pp. 63-80 ◽

Cited By ~ 9

Author(s):

T A Weinert ◽

L H Hartwell

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Dna Replication ◽

Cell Cycle Control ◽

Dna Lesions ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

A Cell ◽

G2 Phase ◽

Rad Mutants

Abstract In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

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RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4314 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4314-4326 ◽

Cited By ~ 21

Author(s):

C Mann ◽

J Y Micouin ◽

N Chiannilkulchai ◽

I Treich ◽

J M Buhler ◽

...

Keyword(s):

Cell Division ◽

Amino Acid ◽

Rna Polymerase ◽

Essential Gene ◽

Temperature Sensitive ◽

G1 Arrest ◽

Restrictive Temperature ◽

Amino Acid Residues ◽

Gene Encoding ◽

Carboxy Terminal

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

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