hththfh

Horwich,1998),and that is94%identical in amino acid sequence to C.reinhardtii HSP70A.Because it appears to be the closest V.carteri homolog of C.reinhardtii HSP70A (see below),we have named the newly isolated gene hsp70A. For the sake of clarity,throughout the remainder of this report we refer to the C.reinhardtii and V.carteri hsp70A genes/proteins as cr-hsp70A/Cr-Hsp70A and vc-hsp70A/Vc-Hsp70A,respectively.

Comparison of the complete vc-hsp70A genomic and cDNA sequences revealed that the vc-hsp70A transcription unit con-sists of9exons and8introns(Fig.1).As is the case for other V. carteri and C.reinhardtii ortholog pairs examined,most but not all of the exon/intron borders are perfectly conserved between vc-hsp70A and cr-hsp70A.Five of the six cr-hsp70A introns(all but intron5)interrupt the coding sequence at the same positions as their vc-hsp70A counterparts do(Fig.1).The nucleotide sequence of the700bp just upstream of the start codon of vc-hsp70A is∼70%identical to the corresponding region of cr-hsp70A.While there are two canonical heat shock elements (HSEs;GAANNTTCNNGAA or TTCNNGAANNTTC)with-in the400-bp region just upstream of the start codon of cr-hsp70A(one perfect and one with a single mismatch;Müller et al.,1992),there is one HSE-like motif within the corresponding region of vc-hsp70A,plus another that is located665bp down-stream of the stop codon,both with single mismatches(Fig.2). Interestingly,of the6vc-hsp70A cDNAs for which3′-end sequence was obtained,poly A tracts were found to start at4 different positions:225–228,490,606–607,and654–658bp after the stop codon(ranges reflect the occurrence of one or more As at the position in the UTR where the poly A tract begins;Fig.2),so that use of alternate polyadenylation signals yields a population of messages that vary by as much as429bp in the lengths of their3′UTRs.

3.2.vc-hsp70A encodes a heat-shock-inducible Hsp70

Since cr-hsp70A is strongly induced by exposure to elevated temperatures(Kropat et al.,1995),we predicted that vc-hsp70A would be also,especially since it contains HSE-like motifs.To test this notion,we used Northern blots to assay vc-hsp70A mRNA abundance in extracts of control spheroids and spher-oids that had been heat shocked.While vc-hsp70A transcripts were detectable in control spheroids,they were much more abundant in spheroids immediately after exposure to heat shock,but then they declined almost to background levels within an hour following the heat shock(Fig.3A).Normaliza-tion with respect to a constitutively expressed mRNA indicated that vc-hsp70A was induced∼50-fold by the heat shock treat-ment(data not shown).

To determine whether Vc-Hsp70A protein levels follow a similar induction pattern,we heat shocked cultures of a trans-genic strain(132/116/1)that expresses an HA-tagged version of Vc-Hsp70A and used Western blots to assay the abundance of HA-tagged proteins at intervals after the cessation of heat shock.In contrast to vc-hsp70A mRNA,HA-tagged Vc-Hsp70A was only modestly induced by heat shock,and peaked ∼30min after the mRNA did(Fig.3B).We conclude that vc-hsp70A,like cr-hsp70A,is a heat-shock-inducible gene.

3.3.Copy number of genes that encode cytoplasmic Hsp70s in V.carteri

Surveys of the genomes of Saccharomyces cerevisiae,A. thaliana,Caenorhabditis elegans,Drosophila melanogaster, Homo sapiens,and other sequenced species have revealed multiple hsp70genes in each organism that potentially encode known and/or suspected cytoplasmic Hsp70s(Heschl and Bail-lie,1990;Boorstein et al.,1994;Sung et al.,2001;Nikolaidis and Nei,2004).On the other hand,bioinformatic and molecular genetic analyses indicate that cr-hsp70A encodes the only cy-toplasmic Hsp70in C.reinhardtii(von Gromoff et al.,1989, plus our unpublished results).

In an effort to determine whether vc-hsp70A might also be the only gene in its genome to encode a cytoplasmic Hsp70 we first used BLASTN to compare the hsp70A genomic se-quence to the∼450,000V.carteri genomic sequence reads

in Fig.3.Expression analysis of vc-hsp70A mRNA and protein in response to heat shock stress.(A)Total RNA isolated from EVE cultures either grown continuously at 32°C(no HS),harvested immediately after a1-h heat shock treatment(0),or harvested45or90min after completion of heat shock treatment was electrophoresed on a denaturing gel,blotted,and probed with vc-hsp70A cDNA fragment P1and control S18cDNA fragment kb,kilobases.The blot was intentionally underexposed to highlight differences in vc-hsp70A mRNA levels in these samples.(B)Approximately equal amounts of protein prepared from individuals of strain132/116/1that were either grown continuously at32°C(0),harvested immediately after completion of1-h heat shock treatment(0),or harvested30,60,120,or180min after heat shock were subjected to SDS PAGE and Western analysis using anti-HA antibody12CA5.kDa,kilodaltons.

116Q.Cheng et al./Gene371(2006)112–120