hththfh

plasmid pASglsA,in which the promoter fragment is oriented to drive expression of the glsA cDNA in antisense direction.

Plasmid pHsp-HA,which harbors an insert that encodes an Hsp70A variant tagged with the Influenza hemaglutinin(HA) epitope tag(Atassi and Webster,1983)at its C-terminus,was generated as follows.Plasmid pSo6was constructed by sub-cloning the6.5-kb Cla I–Bam HI genomic fragment insert from plasmid pSsp6into a pBluescript II KS+derivative whose Sal I site was inactivated.Oligonucleotides HSP-HA1(5′-TCGAC-TACCCGTACGACGTCCCGGACTACGCCG-3′)and HSP-HA2(5′-TCGACGGCGTAGTCCGGGACGTCGTACGGG-TAG-3′)were annealed(Sambrook et al.,1989)to form a small double-stranded DNA fragment that encodes the9-aa HA epitope and contains Sal I compatible ends.This fragment was inserted into the unique Sal I restriction site in pSo6located 3-bp upstream of the stop codon of the hsp70A gene to generate pHsp-HA.pHsp-HA was sequenced to determine that it con-tains a single HA-tag sequence in the correct orientation and to verify that the reading frame was intact.pHsp-HA was intro-duced into EVE along with a zeocin-resistance marker(pZeoF) as described previously(Hallmann and Rappel,1999).Zeocin resistant transformants were tested by genomic PCR,RT PCR, and by Western blot analysis using monoclonal anti-HA anti-body12CA5to identify three clonal lines that expressed the tagged protein.One of these,132/116/1,was used for the Western blot analysis studies reported here.

2.3.Phenotypic analysis of antisense transformants

Culture flasks were inoculated in SVM at low density(∼100 spheroids per300-ml medium),and maintained at either32°C or37°C with the standard16h light/8h dark growth regimen. Three to four days later the number of gonidia present in∼100 randomly chosen,recently hatched asexual progeny was deter-mined by inspection under a dissection microscope.Images of control and experimental spheroids were captured using a Nikon Microphot-SA microscope equipped with a Spot RT CCD digital camera(Diagnostic Instruments).

2.4.BLAST searches and sequence alignments

Sequences in the JGI database that are homologous to vc-hsp70A were retrieved by BLASTN(Basic Local Alignment and Search Tool,Nucleotide;(Altschul et al.,1990),and com-parisons of JGI sequences to vc-hsp70A and Vc-Hsp70A were performed using“BLAST2Sequences”software(Tatusova and Madden,1999).Percent identities were calculated as the number of identical base pairs divided by the total number of base pairs compared.Protein sequence alignments were per-formed by CLUSTAL W(Higgins,1994).

3.Results

3.1.Isolation of V.carteri hsp70A genomic and cDNA clones

A probe generated from a portion of the coding region of the C.reinhardtii HSP70A gene was used to screen∼300,000λphage plaques from a V.carteri genomic library.Over50 hybridization-positive plaques were picked and the inserts from a subset of these were isolated and restriction mapped. One phage,D1,which contained an∼13-kb insert that hybrid-ized very well with the probe fragment,was chosen for further analysis.Partial sequence from an internal region of the D1 insert revealed that it was homologous to part of C.reinhardtii HSP70A,and since a6.5-kb Ssp I fragment of D1hybridized with both the5′and3′ends of a nearly full-length V.carteri hsp70A cDNA(described below)that had been isolated con-currently with the genomic clone,we subcloned the Ssp I frag-ment and sequenced it.This6.5-kb fragment contained the entire transcription unit of a gene(including∼2.0-kb of5′-and0.85kb of3′-non-coding sequence)potentially encoding a protein nearly identical to C.reinhardtii HSP70A(Fig.1).

In parallel with the screen for genomic hsp70clones,over 250,000plaques from a juvenile-specific cDNA library were screened for V.carteri hsp70cDNAs using the same C.rein-hardtii HSP70A probe described above.Fifty-seven plaques that hybridized at above-background level were initially chosen for further study.Eleven of these hybridized very strongly(D1–11,for“dark”),twelve gave a signal intensity that was slightly weaker(M1–12,for“medium”),and thirty-four hybridized more weakly than members of the first two classes(L1–34, for“light”).Since we wished to identify other genes that might encode cytoplasmic Hsp70s,in addition to the ortholog of C. reinhardtii HSP70A,members of all three classes were ana-lyzed further.The inserts from representative clones were re-striction mapped and sized,and then portions of ten L clones and the largest M and D clones were sequenced.Inspection of the sequences and comparison with other hsp70sequences in GenBank revealed that all but two of the sequenced clones,L1 and L30,corresponded to the same gene.All eight L clones

that Fig.1.Structure of the V.carteri hsp70A plete sequences for vc-hsp70A genomic and cDNA clones were used to determine exon/intron borders and restriction site landmarks pertinent to this study.Filled boxes represent exons and inverted carets represent introns.Asterisks underneath inverted carats indicate introns that are absent from cr-hsp70A,and the solid vertical line within the fifth filled box indicates the position at which the corresponding region in cr-hsp70A is interrupted by an intron.The regions spanned by genomic DNA probe P2and cDNA probe P1are indicated by the open boxes below the lines depicting the cloned genomic(upper)and cDNA(lower)fragments,respective-ly,that are described in this study.Abbreviations:A,Apa I;N,Nhe I;Nr,Nru I;S, Ssp I;X:Xho I.

114Q.Cheng et al./Gene371(2006)112–120