Characterization of a heat-shock-inducible hsp70 gene of the

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Characterization of a heat-shock-inducible hsp70gene of the

green alga Volvox carteri

Qian Cheng a ,Armin Hallmann b ,Lisseth Edwards a ,Stephen M.Miller a,⁎

a

Department of Biological Sciences,1000Hilltop Circle,University of Maryland,Baltimore County,Baltimore,MD 21250,USA

b

Department of Cell and Developmental Biology of Plants,Universität Bielefeld,Bielefeld,Germany

Received 18May 2005;received in revised form 10November 2005;accepted 17November 2005

Available online 14February 2006

Abstract

The green alga Volvox carteri possesses several thousand cells,but just two cell types:large reproductive cells called gonidia,and small,biflagellate somatic cells.Gonidia are derived from large precursor cells that are created during embryogenesis by asymmetric cell divisions.The J domain protein GlsA (G onidia l es s A)is required for these asymmetric divisions and is believed to function with an Hsp70partner.As a first step toward identifying this partner,we cloned and characterized V .carteri hsp70A ,which is orthologous to HSP70A of the related alga Chlamydomonas reinhardtii .Like HSP70A ,V .carteri hsp70A contains multiple heat shock elements (HSEs)and is highly inducible by heat shock.Consistent with these properties,Volvox transformants that harbor a glsA antisense transgene that is driven by an hsp70A promoter fragment express Gls phenotypes that are temperature-dependent.hsp70A appears to be the only gene in the genome that encodes a cytoplasmic Hsp70,so we conclude that Hsp70A is clearly the best candidate to be the chaperone that participates with GlsA in asymmetric cell division.©2006Elsevier B.V .All rights reserved.

Keywords:Antisense;Chlamydomonas reinhardtii ;Cytoplasmic Hsp70;GlsA

1.Introduction

The order V olvocales encompasses a group of green algae that range in complexity from unicellular to multicellular with a division of labor between fully differentiated cell types (Starr,1980;Kirk,1998).Therefore they provide an excellent oppor-tunity to investigate the evolutionary origins of relatively sim-ple forms of cellular differentiation.At one end of the spectrum of developmental complexity within the volvocales are mem-bers of the genus Chlamydomonas ,which are unicellular and have been widely used as model organisms for studying pro-cesses such as photosynthesis and assembly and function of centrioles and flagella (Rochaix,2001;Silflow and Lefebvre,

2001;Dutcher,2003).The best studied of the multicellular volvocaleans is Volvox carteri ,a spherical organism composed of several thousand cells of two completely different types:large,asexual reproductive cells called gonidia and small,bi-flagellate somatic cells.Progenitors of the two cell types are set aside by a series of stereotyped asymmetric cell divisions that are temporally and spatially regulated in the cleaving embryo.We discovered that glsA ,a gene required for those asymmetric divisions,encodes a protein with a J domain that is indispens-able for its asymmetric division function (Miller and Kirk,1999).Because the only known function of a J domain is to bind and activate an Hsp70partner protein,we predicted that the function of GlsA in asymmetric divisions would involve an Hsp70partner.All eukaryotes possess multiple hsp70genes,and some express multiple cytoplasmic Hsp70s (Boorstein et al.,1994;Sung et al.,2001),so identifying the Hsp70partner of GlsA may involve testing among several candidates.

The only volvocalean hsp70genes that have been described in print so far are Chlamydomonas reinhardtii HSP70A and HSP70B ,which encode stress-inducible cytoplasmic and chlo-roplast Hsp70s,respectively (von Gromoff et al.,1989;Müller et al.,1992;Drzymalla et al.,1996).As a preliminary

step

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Abbreviations:A,adenosine;aa,amino acid(s);bp,base pair(s);BiP,luminal binding protein;BSA,bovine serum albumin;cDNA,DNA comple-mentary to RNA;°C,degree Celsius;ER,endoplasmic reticulum;HSE,heat shock element;Gls,gonidialess;HA,Influenza hemaglutinin;h,hour;Hsp70,Heat shock protein 70;kb,kilobase(s);kDa,kilodalton(s);min,minute;Nit,nitrate-utilizing;Reg,somatic regenerator;RT,reverse transcriptase;UTR,untranslated region(s).

⁎Corresponding author.Tel.:+14104553381;fax:+14104553875.E-mail address:stmiller@umbc.edu (S.M.Miller).0378-1119/$-see front matter ©2006Elsevier B.V .All rights reserved.doi:10.1016/j.gene.2005.11.026