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toward identifying the Hsp70partner of GlsA,we used a C. reinhardtii HSP70A fragment to screen genomic and cDNA libraries of V.carteri for hsp70genes.Among the hsp70s isolated in this way was hsp70A,a heat-shock-inducible hsp70that is the ortholog of C.reinhardtii hsp70A.Our anal-yses indicate that Hsp70A is likely to be the only cytoplasmic Hsp70in V.carteri,meaning that it is now the best candidate to be a partner of GlsA in asymmetric division.In addition,we found that an∼500-bp fragment located just upstream of the V. carteri hsp70A coding region confers a temperature-dependent Gls phenotype upon some transformants when used to drive expression of an antisense glsA cDNA transgene,suggesting that the hsp70A promoter may be useful as a tool for the molecular genetic analysis of V.carteri development.

2.Materials and methods

2.1.Volvox strains and cultivation conditions

V.carteri strains EVE,153–68(regA−nitA−),and22gls1 (regA−glsA−nitA−)were described previously(Adams et al., 1990;Miller and Kirk,1999).RegC4was isolated as a sponta-neous Reg mutant from a culture of EVE grown at24°C,and generation of transgenic strain132/116/1(which expresses Vc-Hsp70A-HA)is described below.All cultures were propagated in standard Volvox medium(SVM)with a16h light/8h dark growth regimen(Kirk and Kirk,1985),and except where noted (for heat shock or induction of the pASglsA transgene)were maintained at32°C.Heat shock conditions were essentially as described previously(Kirk and Kirk,1986;Kirk et al.,1993). Briefly,medium-density,asynchronous EVE or132/116/1cul-tures were transferred from a32°C water bath to one at42.5°C for40min and then to one at45°C for20min.In some experiments,spheroids were then harvested immediately;in other experiments spheroids were transferred back to32°C to recover for0.5–3h before they were harvested.

2.2.Nuclear transformation and DNA,RNA,and protein methods

Nuclear co-transformation of V.carteri and selection of Nit+ transformants using plasmid pVcNR15were as described(Kirk et al.,1999).Preparation of Volvox protein extracts,RNA and genomic DNA,purification of phageλDNAs,RT PCR,prep-aration of radiolabeled DNA probes,and RNA gel blot and Western analyses were as described previously(Miller et al., 1993;Miller and Kirk,1999).DNA gel blots were hybridized with probe for2h at65°C–70°C using Rapid-hyb buffer(GE Healthcare,Piscataway,NJ)and were washed at the same temperature with high-salt(3×SSC,0.1%SDS)then low-salt (0.3×SSC,0.1%SDS)buffers,each three times for15min. Protein concentrations in extracts were determined using a Bio-Rad DC assay kit with BSA as a standard.Northern blot signal intensities were quantified by phosphorimager and hsp70A band intensities were normalized to intensities measured for signals obtained after hybridization to ribosomal protein S18-encoding cDNA C38(performed without stripping the hsp70A-probed blot).Sequencing was by the dideoxy chain termination method(Sanger et al.,1977)using Big Dye sequencing mixes and an ABI Prism automated sequencer.Sequences were as-sembled using GeneTool software(Wishart et al.,2000).

A V.carteri hsp70A genomic clone and corresponding cDNAs were isolated as follows.A C.reinhardtii HSP70A fragment corresponding to bp1501to bp2310of the gene (with respect to the start codon;accession no.M76725)was generated by PCR with genomic DNA from C.reinhardtii strain c3(arg7,cw−,sr-u,mt−;kindly supplied by P.Ferris, Department of Biology,Washington University)as template using primers1–16(5′-CAACCACTTCGCCAACGAGTTC-3′)and1–14(5′-CCCTTGTCGTTGGTGATCGTG-3′).Exist-ing V.carteri genomic and juvenile-specific cDNA libraries constructed inλphage vectors DASHII andλZAP,respec-tively(Stratagene;described in Kirk et al.,1999)were screened according to standard methods(Sambrook et al., 1989).DNA from one of the most strongly hybridizing ge-nomic library clones(D1)was digested with restriction enzymes,blotted,and probed with radiolabeled fragments derived from the5′and3′ends of D10,which,at2.1kb, was the longest clone obtained from our hsp70A cDNA screen.An∼6.5-kb DNA fragment produced by Ssp I digest of D1DNA was recognized by both putative cDNA probes, and plasmid pSsp6was constructed by subcloning this frag-ment into Eco RV-digested pBluescript II KS−.The 6.5-kb insert was sequenced completely on both strands.Primers Vhsp6(5′-CATACCGGCATCCTTGGT-3′)and Vhsp22(5′-CCCGGGCATGGGTCGTGAGGC-3′)were used in a PCR with an RT template derived from total RNA purified from EVE to obtain a501-bp5′cDNA that overlaps the5′end of the D10insert.Both the5′RACE product and D10were sequenced completely on both strands to obtain the sequence of the entire hsp70A coding region plus3′UTR.Nucleotide sequences for the cDNA and the6.5-kb Ssp I–Ssp I genomic fragment were deposited into GenBank under accession numb-ers DQ059999and DQ059998,respectively.

Fragment P1used for RNA gel blot analysis was prepared by digesting vc-hsp70A cDNA clone D10with Eco RI and gel-purifying the largest cDNA fragment(containing1411bp of hsp70A sequence).Fragment P2used for DNA gel blot analysis was prepared by digesting plasmid pSsp6with Sal I and Xba I and gel-purifying the resulting3.3-kb hsp70A fragment.Plas-mid LV436,which contains a full-length glsA cDNA just up-stream of a unique Eco RI site,was generated by piecing together four overlapping,partial glsA cDNAs produced by RT PCR(Miller and Kirk,1999).The505-bp vc-hsp70A pro-moter fragment used to drive expression of the full-length glsA cDNA in the antisense orientation was produced by using pri-mers Vhsp8(5′-GAATAAAGCCTTTTGTCTTGTA-3′)and Vhsp9(5′-TAACATAATGGAAAGTCGTTAC-3′)in a PCR with genomic clone D1as template.PCR products were ligated into the Eco RV site of pBluescript KS−and the resulting clones were sequenced to identify one(pHsp-pro)that contained no PCR-induced mutations.The pHsp-pro insert was excised with Hin DIII and Eco RI,blunted with Klenow fragment,and ligated into Eco RI-digested(and Klenow-blunted)LV436to produce

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