Characterization of a heat-shock-inducible hsp70 gene of the(8)

hththfh

transcripts are polyadenylated at more than two locations have been reported(Cheng and Tsai,1999;Zhao et al.,1999;Jin and Bian,2004).In some cases mRNAs produced from the same gene but polyadenylated at different sites have been found to accumulate differentially under at least certain conditions (Skadsen and Knauer,1995;Cheng and Tsai,1999;Jin and Bian,2004),but differential polyadenylation may not always lead to or be indicative of differential accumulation(Magnotta and Gogarten,2002).It will be very interesting to determine whether the four classes of vc-hsp70A transcripts that contain different polyadenylation sites display any developmental or temperature-dependent differences in their accumulation patterns.

4.2.The vc-hsp70A promoter as a tool to express transgenes in V.carteri

Previously it was demonstrated that expression of a portion of the volvoxopsin(vop)gene in antisense orientation can reduce the accumulation of V olvoxopsin protein(Ebnet et al., 1999),but our studies constitute the first report of the condi-tional expression of an antisense phenotype in V.carteri.We have yet to test additional genes to determine whether the vc-hsp70A promoter fragment may function as well in the context of other antisense transgenes,and it may well be that efficient generation of temperature-dependent antisense phenotypes will be limited to experiments with genes that,like glsA,are poorly expressed.Nevertheless,with the nearing completion of the first draft of the V.carteri genome sequence(D.Rokhsar, personal communication),the availability of an inducible pro-moter should help facilitate the analysis of many candidate genes by an antisense approach.

For years the promoters of hsp70genes from other organ-isms have been used to drive high-level expression of trans-genes under normal growth conditions or in response to heat shock,and it is likely that the promoter of vc-hsp70A may be adapted similarly.In fact,essentially the same upstream frag-ment that we used was recently placed upstream of a259-bp rbcS3(RUBISCO small subunit-encoding gene3)promoter from V.carteri to create a hybrid promoter that when used to drive a dominant selectable marker gene permitted an∼3-fold increase in the number of transformants that could be obtained relative to the same reporter driven by the V.carteriβ2-tubulin gene promoter(Jakobiak et al.,2004).A follow-up to these preliminary investigations should help to optimize the use of this promoter region as an important addition to the molecular toolbox for V.carteri.

4.3.vc-hsp70A appears to encode the only cytoplasmic Hsp70 in V.carteri

All eukaryotes that have been examined to date possess multiple Hsp70species,with different paralogs apparently spe-cialized for different functions within the cytoplasm,mitochon-dria,and endoplasmic reticulum(Bukau and Horwich,1998; Gething,1999;V oos and Rottgers,2002).Photosynthetic eukaryotes possess a fourth type of Hsp70that functions within the chloroplast(Gray and Row,1995).Many(but not all) organisms also possess multiple Hsp70paralogs that localize within the same cellular compartment and that may carry out non-redundant functions(Boorstein et al.,1994;Sung et al., 2001).

However,C.reinhardtii possesses only one cytoplasmic Hsp70(von Gromoff et al.,1989plus our unpublished results), and we have found that V.carteri also appears to have only one hsp70gene that encodes a cytoplasmic Hsp70.This may turn out to be typical of volvocalean algae,which would seem somewhat surprising,since these algae are at least as complex as yeast(which has four differentially regulated cytoplasmic Hsp70s),and often live in temporary ponds and puddles that frequently undergo rapid and extreme environmental fluctua-tions.The existence of just one cytoplasmic Hsp70in V.carteri would,however,simplify the analysis of Hsp70and GlsA function in mediating asymmetric division of the embryo,be-cause it would diminish the possibility that the effects of modifying Hsp70structure or expression might be masked by an Hsp70of partially redundant function.In any event the next step will be to determine whether Vc-Hsp70A and GlsA inter-act physically,and if so,how these two chaperones contribute to the spatial and temporal control over asymmetric division in V.carteri.

Acknowledgements

We are very grateful to Eric Balzer,Norbert Eichner,Laura Satkamp,and Mark Womer for important technical contribu-tions and P.Ferris for providing C.reinhardtii genomic DNA. We also thank Leonard Duncan,David Kirk,Valeria Pappas, and Cynthia Wagner for insightful discussions and advice before and/or during preparation of this manuscript.This work was supported by research grants from the National Science Foundation(grant#0077535)and the National Re-search Initiative of the USDA Cooperative State Research, Education and Extension Service(grant#2003-35304-13385) to SMM and the Deutsche Forschungsgemeinschaft(SFB521) to AH.

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