The Sda1 Protein Is Required for Passage through Start

We have used affinity chromatography to identify proteins that interact with Nap1, a protein previously shown to play a role in mitosis. Our studies demonstrate that a highly conserved protein called Sda1 binds to Nap1 both in vitro and in vivo. Loss of Sda1 function causes cells to arrest uniformly as unbudded cells that do not increase significantly in size. Cells arrested by loss of Sda1 function have a 1N DNA content, fail to produce the G1 cyclin Cln2, and remain responsive to mating pheromone, indicating that they arrest in G1 before Start. Expression of CLN2 from a heterologous promoter in temperature-sensitive sda1 cells induces bud emergence and polarization of the actin cytoskeleton, but does not induce cell division, indicating that the sda1 cell cycle arrest phenotype is not due simply to a failure to produce the G1 cyclins. The Sda1 protein is absent from cells arrested in G0 and is expressed before Start when cells reenter the cell cycle, further suggesting that Sda1 functions before Start. Taken together, these findings reveal that Sda1 plays a critical role in G1 events. In addition, these findings suggest that Nap1 is likely to function during G1. Consistent with this, we have found that Nap1 is required for viability in cells lacking the redundant G1 cyclins Cln1 and Cln2. In contrast to a previous study, we have found no evidence that Sda1 is required for the assembly or function of the actin cytoskeleton. Further characterization of Sda1 is likely to provide important clues to the poorly understood mechanisms that control passage through G1.

Isolation of cDNA of an auxin-regulated gene encoding a G protein beta subunit-like protein from tobacco BY-2 cells

The addition of 2,4-dichlorophenoxyacetic acid to tobacco BY-2 cells that had been cultured in modified Linsmaier and Skoog medium deprived of auxin for 3 days induced cell division, whereas without 2,4-dichlorophenoxy-acetic acid application, no such induction of cell division was seen. When differential cDNA screening for auxin was done at 4 hr after the addition of 2,4-dichlorophenoxyacetic acid, the cDNA of an auxin-responsive gene designated arcA was isolated. The predicted gene product of arcA is a polypeptide with a M(r) of 35,825. arcA, thus, is a plant hormone-regulated gene that encodes a protein structurally related to the beta subunit of a guanine nucleotide-binding regulatory protein, which is composed of seven repetitive segments of Trp-Asp 40-aa repeats. The possibility that arcA gene products induce cell division is discussed.

Protoplast isolation from Ulmusamericana L. pollen mother cells, tetrads, and microspores

Meiotic protoplasts of Ulmusamericana (American elm) are potentially valuable for producing interspecific elm hybrids through protoplast fusion. Meiotic cells (pollen mother cells, tetrads, and microspores) were incubated in either a cellulase, hemicellulase, and pectinase enzyme solution or a β-1,3-glucanase (laminarinase) solution. Respective protoplast isolation frequencies for the three meiotic cell types were 100, 50, and 10%. Exclusion staining with 0.2% Evans blue and 0.1% methyl blue suggested protoplast viability. Some of the microspore protoplasts were vacuolated, which is an important condition for cell division. Although attempts to regenerate cell walls and induce cell division were unsuccessful, these two problems may be superceded by protoplast fusion with more regenerative protoplasts. To our knowledge this is the first report of protoplast isolation from meiotic cells of a tree species.