Characterization of a heat-shock-inducible hsp70 gene of the(7)

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number of gonidia (an average of ∼14per spheroid)at both temperatures (Fig.5and data not shown)and none possessed fewer than 6gonidia at either temperature.The abundance of glsA transcripts is extremely low in wild-type strains (Miller and Kirk,1999)so we were unable to measure endogenous glsA transcript levels in the transformants by RNA gel blot or by RT PCR.4.Discussion

Our desire to clone and characterize Volvox genes encoding Hsp70s was motivated primarily by our previous discovery that an intact J domain –a potential Hsp70-binding site –is re-quired in order for the GlsA protein to play its critical role in asymmetric division and germ cell formation in V .carteri embryos (Miller and Kirk,1999).We isolated V .carteri hsp70clones by screening genomic and cDNA libraries with a probe derived from cr-hsp70A ,the heat-shock-inducible hsp70gene of the related alga C.reinhardtii .Sequencing revealed that nearly all of these clones appear to represent a single V .carteri hsp70gene that is nearly identical in exon/intron organization and in the peptide sequence that it encodes to cr-hsp70A .

4.1.Heat shock inducibility and potential regulatory elements of vc-hsp70A

vc-hsp70A transcripts were detected on Northern blots of RNA from control cultures,but they were ∼50-fold more abundant in individuals harvested immediately after a 1-h heat shock.The fact that vc-hsp70A mRNA abundance then de-clined dramatically within the next hour indicates that under these conditions the vc-hsp70A mRNA is rapidly degraded or that the response is attenuated due to autoregulation,or that both occur.vc-hsp70A contains two near-canonical HSEs (Fig.2),one that lies just upstream of the putative TATA box and is contained within the ∼500-bp vc-hsp70A promoter fragment that was used to drive the glsA antisense transgene that con-ferred temperature-dependent Gls antisense phenotypes (Sec-tion 4.2).It is likely that one or both of these HSEs are important for the heat inducibility of vc-hsp70A .

Interestingly,four of the six vc-hsp70A cDNAs for which we obtained 3′end sequence possessed markedly different poly-adenylation sites.Whereas most genes in fungi,plants,and animals probably contain one or at most two polyadenylation signals (Zhao et al.,1999),several examples of genes

whose

Fig.5.Phenotypic analysis of selected pASglsA transformants.(A)Schematic representation of construct pASglsA that was transformed into strain 153–68to generate lines that express antisense glsA.Gonidial counts of individuals from cultures of recipient strain 153–68(B)and two pASglsA transformant lines,T10–6(C)and T11–10(D),that were propagated for 3–4days at either 32°C or 37°C.(E)Representative individuals from cultures of (clockwise from upper left)153–68,22gls1,T10–6,and T11–10.Scale bar,500μM.Transformant strains T10–5and T13–8exhibited temperature-dependent Gls phenotypes that were comparable to those displayed by T10–6and T11–10but those data are not shown here.

118Q.Cheng et al./Gene 371(2006)112–120