Cell Biology International Reports, Vol. 16, No. 11, 1992 1077

DIFFERENTIAL EXPRESSION OF THREE ACTIN GENES IN THE CELL CYCLE OF PHYSARUM POLYCEPHALUM

Olga L. Arellano.Dominick Pallotta and Helmut W. Sauer.

Department of Biology, Texas A & M University, College Station, Texas 77843, USA. and "D6partement de Biochimie et Biologie, Unlversit6 Lava~ Qudbec, Q.

C. Canada.

ABSTRACT

Regulation of expression of 3 isocoding genes of the actin gene family of Physarum was analyzed with gene specific probes. At steady state level, ardB and ardC seem to fluctuate coordinately during the cell cycle; ardA transcripts were not detected. Nuclear run-on assays suggested different transcription rates for ardB and attiC. Late G2 phase nuclei revealed hybridization signals of in v/tro synthesized RNA to ardA sequences, indicating transient activation of this late replicating gene.

INTRODUCTION

Four unlinked actin loci, designated ardA (containing 2 genes: A2-7 and A2-17), ardB, ardC, and ardD have been identified in the Physarum genome (Schedl and Dove, 1982) Sequence analysis has revealed that 3 actin genes, ardA2-17, ardB, and ardC, if expressed, would encode identical protein sequences (Hamelin et aL 1988). Expression of the isocoding ardB and ardC genes has been shown by Northern analysis, but no activity of the ardA locus has been detected. In this study, steady state levels of ardB and ardC mRNA were estimated by Northern blot analysis of total RNA from different points of the cell cycle and transcriptional activity of all 3 actin genes was analyzed by nuclear run-on assays.

MATERIALS AND METHODS

Cell culture: Physarum microplasmodia (TU291) were grown as a suspension (Daniel and Baldwin, 1964), then allowed to fuse into macroplasmodia. Time points of the 8 to 9 hr. synchronous cell cycle refer to metaphase (0 min.); S- phase begins in telophase and lasts about 3 hrs.

Steady State RNA Analysis: Total RNA was isolated from plasmodia according to Hamelin et al. (1988); 1.8 ttg were glyoxylated, separated on 1.2% agarose gels, and transferred onto nylon membrane (Maniafis, et al, 1982). Actin-specific clones were 32p labelled with the Amersham multiprime DNA

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1078 Cell Biology lntemational Reports, Vol. 16, No. 11, 1992

labelling system, and hybridized as in previous work with these probes (Hamelin, et al, 1988). All Northerns were standardized to rRNA content. For RNase protection assays, the actin gene specific sequences were transcribed from the T-7 promoter in the presence of 32p aUTP as recommended by the supplier (Pharmacia), and hybridized in liquid to 10 ttg total RNA. Protected RNAs were separated on 7% denaturing polyacrylamide gels (O.L. Arellano, Thesis, Texas A&M Univ., 1989).

Nuclear Run-on Transcription Assays: Nuclear RNA was elongated in the presence of 32p aIYI~ and isolated from approximately 107 nuclei per cell cycle time point (Nothacker and Hildebrandt, 1985). These transcripts were then used to probe sequences specific for ardA, ardB, or ardC, which had been separated on a 1.2% agarose gel and transferred onto nylon membranes. These "master blots" were hybridized and washed at optimal (50°C) or high (55°C) stringencies (O.L. Arellano, Thesis, Texas A&M Univ., 1989).

RESULTS

Northern blots containing RNA from each cell cycle time point were hybridized to ardB (fig. 1) and ardC (fig. 2) specific actin DNAs. Both sequences hybridized to 1.4 kb messages, as previously shown. Densitometric analysis of the autoradiographs allowed us to determine the relative levels of the transcripts at different time points. Although the absolute mounts of ardB and ardC RNAs cannot be compared, it is obvious from the graph in figure 3 that the biphasic fluctuations of the relative RNA levels parallel each other throughout the cell cycle. No hybridization signal was observed with the ardA hybridization probe (data not shown).

To support the validity of the Northern blot analysis, a quantitative RNase protection assay was performed in which RNA from each cell cycle time point was hybridized in liquid to a greater than 20X excess of ardB probe DNA. As shown in fig.5, the relative levels of ardB mRNA are very similar in both assays.

Steady state levels represent the sum of regulation via transcription, degradation, and stabilization. To estimate transcription levels exclusively, run-on assays were performed with nuclei isolated from the different time points of the cell cycle. Two sample "master blots", show the autoradiographs obtained with 32P-labelled run-off RNA from the +40 rain (fig. 6) and +360 rain time points (fig.7) A summary of all hybridization values is given on fig. 8. In contrast to the closely paralleled steady state levels of ardB and ardC, the transcript ion levels of ardB and ardC seem less coordinated. Although both genes appear to be transcribed biphasically, it seems that in S-phase, ardB is more active than ardC. Also, a small peak of hybridization to the ardA specific sequences occurred at the +360 and +420 time points in late G2 phase.

Cell Biology lnternational Reports, VoL 16. No+ 11, 1992 1079

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Fig.1 Northern Analysis of ardB Specific mRNA in the Cell Cycle.

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CELL CYCLE T IME POIWrS (WN)

Fig.3 Graph depicting the Relative Intensities of Hybridization Signals in Figs.1 and 2 as determined by Densitometric analysis.

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Fig.2 Northern Analysis of ardC Specific mRNA in the Cell Cycle.

- 10 +10 + 4 0

+ 9 0 + 1 8 0 + 3 6 0 + 4 8 0

Fig.4 RNase Protection Analysis of ardB Specific mRNA in the Cell Cycle.

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0m

u~

° ~ , 0 0 + ' ; " ,.:'+ " , . : o " , ; o ' " , ; o • ,,,,+

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Fig.5 Graph Depicting the Relative Intensifies of Hybridization Signals of Northern Blots and RNase Protection Assays As Determined by Densitometric Analysis of Figs. 1 and 4.

+ 4 0

A B C + 3 6 A B C

Fig.6 Autoradiography after Hybridization of Nuclear Run-on RNA from 40 rain After Mitosis, to an Actin Gene "Master Blof'.

Fig.7 Autoradiography after Hybridization of Nuclear Run-on RNA from 360 rain after Mitosis, to an Actin Gene "Master Blot".

1 0 8 0 Cell Biology lnternational Reports, VoL 16, No. 11, 1992

Pig.8 Graph depicting the Relative ="

Intensities of the hybridization signals as de t e r mine d by ~ ~. Densitometric Analysis of Nuclear Run-on RNA from Different Time Points of the Cell Cycle, to Actin ~ '" Gene "Master Blots". =

DISCUSSION

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CELL CYCLE TiME pOINTS (MIN)

Actin gene family members are differentially expressed in embryonic development (Lee et al., 1984). However, ardB and ardC transcripts were detected throughout the life cycle of Physarum, whereas ardA2-17 seemed to be always repressed (Hamelin et al., 1988) This study was focused on the expression of these actin genes in the cell cycle. Their transcription was undetectable immediately after mitosis. During S-phase (there is no G l phase in the Physarum plasmodium) transcription of ardB is more intense than that of ardC. Also, activity of ardC is increased in (32 phase as compared to S-phase. These differences are not apparent from the analysis of the steady state levels of the 2 genes, which suggested coordinate biphasic fluctuations. It seems therefore, that the expression of ardB and ardC is modulated at both the transcriptional and post-transcriptional levels during the cell cycle.

A study establishing the invariant chronology of replication of the 4 actin loci had shown that ardA replicates last (Pierron et al., 1982), and it was suggested that late replication of ardA might be the cause" for its transcriptional repression. On the other hand, it could be argued that ardA expression was transiently required at some critical time point since mutations would have accumulated in a permanently inactivated gene (Hamelin et al., 1988) The small but significant hybridization signal obtained with the ardA specific target sequence at high stringency suggests that this actin gene is expressed in late G2 phase. The absence of a hybridization signal in Northern blot analyses would indicate that the transcripts are unstable, perhaps because of the presence of 8 AU-rich regions in the 3'untranslated region (3'UTR) of the ardA2-17 gene (Hamelin et al. 1988) Moreover, the apparent activity of ardA coincides with the transient period of expression of the also late replicating Lay1-4 gene (Pierron et al. 1989) for which stable transcripts have been observed throughout the cell cycle (Flanagan, Thesis, Texas A&M Univ., 1988) Interestingly, run-on transcripts of ardA appear at a G2 transition point known to be essential for preparation of mitosis and S-phase of the Physarum cell cycle (Laffier and Tyson, 1986)

Cell Biology International Reports, VoL 16, No. 11, 1992 1081

ACKNOWLEDGEMENT This research was in part supported by a grant from NSF (DCB 8608017) and the Texas Advanced Research Project (No 10366-078).

REFERENCES

Daniel, J. W. and Baldwin, H. H. (1964). Methods of culture of plasmodial myxomycetes, in: Prescott, D. M. (ed.) Methods Cell Physiology VoL 1, pp. 9-41. New York: Academic Press

Hamelin, M., Adam, L, Lemieux, G. and Pallotta, D. (1988). Expression of the three unlinked isocoding actin genes of Physarum polycephalum. DNA 7, 317-328.

Laffier, T. G. and Tyson, J. J. (1986). The Physarum cell cycle, in: W. F. Dove et al. (eds.) The Molecular Biology of Physarum polycephalum, Vol. 1, pp. 79-109. New York: Plenum.

Lee, J. J., Shott IL J. and Davison, E. H. (1984). Sea urchin actin gene sub-types: gene number, gene linkage, and evolution. J. Mol. Biol. 172, 149-176.

Maniatis, T., Fritsch E. F. and Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory)

Nothacker, K. and Hildebrandt, A. (1985). Isolation of highly purified and more native nuclei of Physarum polycephalum utilizing Surfynol, hexylene glycol, and Percoll. Eur. J. Cell Biol. 39, 278-282.

Pierron, G., Durica, D. S., and Sauer, H. W. (1984). Invariant temporal order of replication of the four actin loci during naturally synchronous mitotic cycles-of Physarum polycephalum. Proe. Natl. Acad. SeX. 81, 6393-6397.

Pierron, G., Bernard, M., Puvion, E., Fianagan, R., Sauer, H. W. and Pallotta, D. (1989). Replication timing of ten developmentally regulated genes in Physarum polycephalum. Nucleic Acids Ree. 7, 553-566.

Schedl, T. and Dove, W. F. (1982). Mendelian analysis of the organization of actin sequences in Physarum polycephalum. J. Mol. Biol. 160, 41-57.