Analysis of transcription during the cell cycle in toluenized Chlamydomonas reinhardi cells.

Abstract

A toluene-permeabilized cell system was established to examine the transcription of certain RNAs regulated during the cell cycle in Chlamydomonas reinhardi.The incorporation of [a-32P]UTP into RNA which hybridizes to specific cloned cDNA, such as P-tubulin, indicates that the cell cycle pattern of RNA accumulation may be controlled, in part, by differential transcription.The cell cycle in light-dark-synchronized cultures of Chlamydomonas reinhardi is marked by the periodic synthesis of certain cell cycle-regulated proteins (4) and the stage-specific appearance of their corresponding mRNAs. Of note is the burst of a-and P-tubulin synthesis (5, 9) and the coordinate appearance of the tubulin mRNAs (1, 2) at about the time of division. Also, the 32-kd precursor of the light-harvesting chlorophyll a/b-binding protein (LHCP), a major chloroplast membrane component, is synthesized, and its mRNA is accumulated in a wavelike manner during the mid-light phase of the cycle (4, 6), at a time when cells are actively producing components of the photosynthetic apparatus.These 1A). At 150 mM NH4+, total nucleotide incorporation was reduced to 60% of that lacking NH4', but 45% of the remaining incorporation was aamanatin sensitive (Fig. 1B). Nucleotide incorporation at 150 mM NH4+ was further optimized for Mg2+ (Fig. 1C) and K+ (Fig. 1D) concentrations, which in the standard reaction were 20 and 50 mM, respectively.The efficiency of UTP incorporation at different stages during the cell cycle was examined in light-dark-synchronized cultures. Incorporation expressed on a per cell basis declined progressively from L2, the beginning of the light period, to D2, the time of nuclear and cell division, and then rose sharply (Fig. 2). The incorporation pattern appears to reflect a reduced transcriptional capacity during division.The transcription of specific genes was examined during the cell cycle by hybridizing RNA labeled in cells toluenized at different cell cycle stages to various cloned cDNAs. Plasmid pMAD251 is a Chlamydomonas cDNA apparently derived from a multigene family and hybridizes to two major abundant RNA species of 950 and 820 bases (M. Ares, Ph.D. thesis, University of California, San Diego, 1982). These RNAs are not cell cycle regulated but are present in relatively unchanging amounts during the cell cycle when expressed relative to total RNA (Ares, Ph.D. thesis). The pattern of hybridization of [32P]UTP-labeled RNA to pMAD251 DNA (Fig. 3) reflects, by and large, the overall efficiency of RNA synthesis (Fig. 2). RNA hybridizable to pMAD251 is synthesized extensively in late-dark-phase to early-light-phase cells (GI cells) and declines as cells move into 1537 on May 11, 2018 by guest

Regulation of genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase and the photosystem II polypeptides D-1 and D-2 during the cell cycle of Chlamydomonas reinhardtii.

“…Previous studies of the cell cycle expression of LHCII, tubulin, and other nuclear-encoded genes of Chlamydomonas have demonstrated control of mRNA levels (1). Accumulation of these mRNAs may be determined by transcriptional and/or posttranscriptional controls in the nucleus or posttranscriptional control over mRNA stability in the cytoplasm (3,13). In contrast, the present analysis of three major chloroplast-encoded genes (rbcL, psbA, and psbD) shows that these mRNAs are abundant throughout the cell cycle.…”

Rapid changes in tubulin RNA synthesis and stability induced by deflagellation in Chlamydomonas.

“…That is, if rRNA synthesis is actually somewhat depressed after deflagellation (12), then the 2.9-fold average increase in tubulin RNA synthesis we have measured would be an overestimate. DaUman et al (7) have made a related observation in Chlamydomonas. They reported that an approximate 2-fold increase in transcription of beta tubulin sequences preceding mitosis in permeabilized Chlamydomonas cells is insufficient to account for the 8-10fold increase in abundance which occurs at this time.…”

Transcription of alpha-tubulin and histone H4 genes begins at the same point in the Physarum cell cycle.

“…Our data are the first clear example of cell cycle regulation of tubulin mRNA transcription. Earlier reports of periodicity in Chlamydomonas (2,10,36) appear to be synchronization artifacts, since the periodicity disappears when cultures are returned to normal growth conditions (31). There is precedent in Chlamydornonas for the direct induction of tubulin gene transcription during flagellar regeneration.…”