Characterization of a heat-shock-inducible hsp70 gene of the green alga Volvox carteri

Gene 371 (2006) 112 – 120 www.elsevier.com/locate/gene

Characterization of a heat-shock-inducible hsp70 gene of the green alga Volvox carteri Qian Cheng a , Armin Hallmann b , Lisseth Edwards a , Stephen M. Miller a,⁎ a

Department of Biological Sciences, 1000 Hilltop Circle, University of Maryland, Baltimore County, Baltimore, MD 21250, USA b Department of Cell and Developmental Biology of Plants, Universität Bielefeld, Bielefeld, Germany Received 18 May 2005; received in revised form 10 November 2005; accepted 17 November 2005 Available online 14 February 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 (Gonidialess A) is required for these asymmetric divisions and is believed to function with an Hsp70 partner. 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Antisense; Chlamydomonas reinhardtii; Cytoplasmic Hsp70; GlsA

1. Introduction The order Volvocales 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 opportunity to investigate the evolutionary origins of relatively simple forms of cellular differentiation. At one end of the spectrum of developmental complexity within the volvocales are members of the genus Chlamydomonas, which are unicellular and have been widely used as model organisms for studying processes such as photosynthesis and assembly and function of centrioles and flagella (Rochaix, 2001; Silflow and Lefebvre, Abbreviations: A, adenosine; aa, amino acid(s); bp, base pair(s); BiP, luminal binding protein; BSA, bovine serum albumin; cDNA, DNA complementary 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.: +1 410 455 3381; fax: +1 410 455 3875. E-mail address: [email protected] (S.M. Miller). 0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.11.026

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, biflagellate 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 indispensable for its asymmetric division function (Miller and Kirk, 1999). Because the only known function of a J domain is to bind and activate an Hsp70 partner protein, we predicted that the function of GlsA in asymmetric divisions would involve an Hsp70 partner. All eukaryotes possess multiple hsp70 genes, and some express multiple cytoplasmic Hsp70s (Boorstein et al., 1994; Sung et al., 2001), so identifying the Hsp70 partner of GlsA may involve testing among several candidates. The only volvocalean hsp70 genes that have been described in print so far are Chlamydomonas reinhardtii HSP70A and HSP70B, which encode stress-inducible cytoplasmic and chloroplast Hsp70s, respectively (von Gromoff et al., 1989; Müller et al., 1992; Drzymalla et al., 1996). As a preliminary step

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toward identifying the Hsp70 partner of GlsA, we used a C. reinhardtii HSP70A fragment to screen genomic and cDNA libraries of V. carteri for hsp70 genes. Among the hsp70s isolated in this way was hsp70A, a heat-shock-inducible hsp70 that is the ortholog of C. reinhardtii hsp70A. Our analyses indicate that Hsp70A is likely to be the only cytoplasmic Hsp70 in 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−), and 22gls1 (regA−glsA−nitA−) were described previously (Adams et al., 1990; Miller and Kirk, 1999). RegC4 was isolated as a spontaneous Reg mutant from a culture of EVE grown at 24 °C, and generation of transgenic strain 132/116/1 (which expresses VcHsp70A-HA) is described below. All cultures were propagated in standard Volvox medium (SVM) with a 16 h light/8 h dark growth regimen (Kirk and Kirk, 1985), and except where noted (for heat shock or induction of the pASglsA transgene) were maintained at 32 °C. Heat shock conditions were essentially as described previously (Kirk and Kirk, 1986; Kirk et al., 1993). Briefly, medium-density, asynchronous EVE or 132/116/1 cultures were transferred from a 32 °C water bath to one at 42.5 °C for 40 min and then to one at 45 °C for 20 min. In some experiments, spheroids were then harvested immediately; in other experiments spheroids were transferred back to 32 °C to recover for 0.5–3 h 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 pVcNR15 were as described (Kirk et al., 1999). Preparation of Volvox protein extracts, RNA and genomic DNA, purification of phage λ DNAs, RT PCR, preparation 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 for 2 h at 65 °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 for 15 min. Protein concentrations in extracts were determined using a BioRad 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 S18encoding cDNA C38 (performed without stripping the hsp70A-

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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 assembled 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 bp 1501 to bp 2310 of 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 primers 1–16 (5′-CAACCACTTCGCCAACGAGTTC3′) and 1–14 (5′-CCCTTGTCGTTGGTGATCGTG-3′). Existing V. carteri genomic and juvenile-specific cDNA libraries constructed in λ phage vectors DASHII and λZAP, respectively (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 genomic library clones (D1) was digested with restriction enzymes, blotted, and probed with radiolabeled fragments derived from the 5′ and 3′ ends of D10, which, at 2.1 kb, was the longest clone obtained from our hsp70A cDNA screen. An ∼6.5-kb DNA fragment produced by SspI digest of D1 DNA was recognized by both putative cDNA probes, and plasmid pSsp6 was constructed by subcloning this fragment into EcoRV-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 a 501-bp 5′ cDNA that overlaps the 5′ end of the D10 insert. Both the 5′ RACE product and D10 were sequenced completely on both strands to obtain the sequence of the entire hsp70A coding region plus 3′UTR. Nucleotide sequences for the cDNA and the 6.5-kb SspI–SspI genomic fragment were deposited into GenBank under accession numbers DQ059999 and DQ059998, respectively. Fragment P1 used for RNA gel blot analysis was prepared by digesting vc-hsp70A cDNA clone D10 with EcoRI and gelpurifying the largest cDNA fragment (containing 1411 bp of hsp70A sequence). Fragment P2 used for DNA gel blot analysis was prepared by digesting plasmid pSsp6 with SalI and XbaI and gel-purifying the resulting 3.3-kb hsp70A fragment. Plasmid LV436, which contains a full-length glsA cDNA just upstream of a unique EcoRI site, was generated by piecing together four overlapping, partial glsA cDNAs produced by RT PCR (Miller and Kirk, 1999). The 505-bp vc-hsp70A promoter fragment used to drive expression of the full-length glsA cDNA in the antisense orientation was produced by using primers Vhsp8 (5′-GAATAAAGCCTTTTGTCTTGTA-3′) and Vhsp9 (5′-TAACATAATGGAAAGTCGTTAC-3′) in a PCR with genomic clone D1 as template. PCR products were ligated into the EcoRV 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 HinDIII and EcoRI, blunted with Klenow fragment, and ligated into EcoRI-digested (and Klenow-blunted) LV436 to produce

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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 pSo6 was constructed by subcloning the 6.5-kb ClaI–BamHI genomic fragment insert from plasmid pSsp6 into a pBluescript II KS+ derivative whose SalI site was inactivated. Oligonucleotides HSP-HA1 (5′-TCGACTACCCGTACGACGTCCCGGACTACGCCG-3′) and HSPHA2 (5′-TCGACGGCGTAGTCCGGGACGTCGTACGGGTAG-3′) were annealed (Sambrook et al., 1989) to form a small double-stranded DNA fragment that encodes the 9-aa HA epitope and contains SalI compatible ends. This fragment was inserted into the unique SalI restriction site in pSo6 located 3-bp upstream of the stop codon of the hsp70A gene to generate pHsp-HA. pHsp-HA was sequenced to determine that it contains a single HA-tag sequence in the correct orientation and to verify that the reading frame was intact. pHsp-HA was introduced 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 antibody 12CA5 to 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 per 300-ml medium), and maintained at either 32 °C or 37 °C with the standard 16 h light/8 h dark growth regimen. Three to four days later the number of gonidia present in ∼ 100 randomly chosen, recently hatched asexual progeny was determined 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).

phage plaques from a V. carteri genomic library. Over 50 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 hybridized 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 a 6.5-kb SspI fragment of D1 hybridized with both the 5′ and 3′ ends of a nearly full-length V. carteri hsp70A cDNA (described below) that had been isolated concurrently with the genomic clone, we subcloned the SspI fragment and sequenced it. This 6.5-kb fragment contained the entire transcription unit of a gene (including ∼ 2.0-kb of 5′and 0.85 kb of 3′-non-coding sequence) potentially encoding a protein nearly identical to C. reinhardtii HSP70A (Fig. 1). In parallel with the screen for genomic hsp70 clones, over 250,000 plaques from a juvenile-specific cDNA library were screened for V. carteri hsp70 cDNAs using the same C. reinhardtii 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 analyzed further. The inserts from representative clones were restriction 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 hsp70 sequences in GenBank revealed that all but two of the sequenced clones, L1 and L30, corresponded to the same gene. All eight L clones that

2.4. BLAST searches and sequence alignments Sequences in the JGI database that are homologous to vchsp70A were retrieved by BLASTN (Basic Local Alignment and Search Tool, Nucleotide; (Altschul et al., 1990), and comparisons of JGI sequences to vc-hsp70A and Vc-Hsp70A were performed using “BLAST 2 Sequences” 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 performed 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 ∼ 300,000 λ

Fig. 1. Structure of the V. carteri hsp70A gene. Complete sequences for vchsp70A 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 P2 and cDNA probe P1 are indicated by the open boxes below the lines depicting the cloned genomic (upper) and cDNA (lower) fragments, respectively, that are described in this study. Abbreviations: A, ApaI; N, NheI; Nr, NruI; S, SspI; X: XhoI.

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were highly similar to C. reinhardtii HSP70A contained only small inserts that corresponded to a small part of the probe. Clones L1 and L30 both encoded proteins that were much more similar to members of the luminal binding protein (BiP) family of Hsp70 proteins than they were to any cytoplasmic Hsp70 protein in the database. For instance, the sequenced portion of L30 (183 bp) encodes a polypeptide that is 82% identical to part of an Arabidopsis thaliana BiP (locus BAA12348, GenBank accession number D84414.1), but is only 54% identical to the corresponding region of C. reinhardtii HSP70A. Thus clones

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L1 and L30 likely correspond to a gene or genes encoding ERspecific Hsp70s of V. carteri. None of the hsp70 cDNAs isolated was long enough to encode a full-length Hsp70, so we used sequence from the genomic clone and from the longest cDNA (D10, 2094 bp) to design primers for RACE to obtain a 501-bp cDNA fragment that overlaps with cDNA D10 and encodes the N-terminal part of the protein. The complete cDNA encodes a deduced protein that contains a 395-aa ATPase domain, which is highly conserved in all members of the Hsp70 family (Bukau and

Fig. 2. Putative regulatory sequences within the vc-hsp70A gene. Sequence of part of the 6.5-kb SspI–SspI restriction fragment (Fig. 1) that spans the entire vc-hsp70A gene is represented, beginning at bp − 530 with respect to the start codon, through the end of the fragment. The dots between the start codon and stop codon represent the coding sequence, which is not shown. Two imperfect HSE-like sequences are underlined and in bold, the predicted TATA box is in bold, alternative polyadenylation sites are indicated by arrowheads, and typical algal polyadenylation signals (TGTAA, TGAAA) that precede three of the polyadenylation sites are marked by dashed underlines. The predicted transcription start site is marked by a solid diamond below the sequence.

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Horwich, 1998), and that is 94% 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/VcHsp70A, respectively. Comparison of the complete vc-hsp70A genomic and cDNA sequences revealed that the vc-hsp70A transcription unit consists of 9 exons and 8 introns (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 intron 5) interrupt the coding sequence at the same positions as their vc-hsp70A counterparts do (Fig. 1). The nucleotide sequence of the 700 bp just upstream of the start codon of vchsp70A is ∼ 70% identical to the corresponding region of crhsp70A. While there are two canonical heat shock elements (HSEs; GAANNTTCNNGAA or TTCNNGAANNTTC) within the 400-bp region just upstream of the start codon of crhsp70A (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 located 665 bp downstream of the stop codon, both with single mismatches (Fig. 2). Interestingly, of the 6 vc-hsp70A cDNAs for which 3′-end sequence was obtained, poly A tracts were found to start at 4 different positions: 225–228, 490, 606–607, and 654–658 bp 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 as 429 bp in the lengths of their 3′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 spheroids 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). Normalization with respect to a constitutively expressed mRNA indicated that vc-hsp70A was induced ∼ 50-fold by the heat shock treatment (data not shown). To determine whether Vc-Hsp70A protein levels follow a similar induction pattern, we heat shocked cultures of a transgenic 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 VcHsp70A was only modestly induced by heat shock, and peaked ∼30 min after the mRNA did (Fig. 3B). We conclude that vchsp70A, 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 hsp70 genes in each organism that potentially encode known and/or suspected cytoplasmic Hsp70s (Heschl and Baillie, 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 cytoplasmic Hsp70 in 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 sequence to the ∼ 450,000 V. 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 a 1-h heat shock treatment (0), or harvested 45 or 90 min after completion of heat shock treatment was electrophoresed on a denaturing gel, blotted, and probed with vc-hsp70A cDNA fragment P1 and control S18 cDNA 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 strain 132/116/1 that were either grown continuously at 32 °C (0), harvested immediately after completion of 1-h heat shock treatment (0), or harvested 30, 60, 120, or 180 min after heat shock were subjected to SDS PAGE and Western analysis using anti-HA antibody 12CA5. kDa, kilodaltons.

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the JGI genomes database (http://genome.jgi-psf.org/cgi-bin/ runAlignment?db=chlre2), and identified 20 records with e values of 10− 5 or better. Of these, the seven with the lowest e values encode polypeptides that are (a) more closely related to cytoplasmic Hsp70s than to organellar ones, (b) N 94% identical in nucleotide sequence to vc-hsp70A, and (c) just as similar to vc-hsp70A in presumptive non-coding regions as in their predicted coding regions. The remaining 13 hits encode portions of Hsp70s that are much more similar to ER, chloroplast, and/ or mitochondrial Hsp70s than to cytoplasmic Hsp70s (data not shown). We believe that most of the non-identities between vchsp70A and the cytoplasmic-Hsp70 encoding sequences probably result from errors in the shot-gun sequencing, because most of the reads were N 900 bp and most of the non-identities were near the ends of the reads. A translated BLAST search returned the same seven hits as were identified by the nucleotide BLAST search. These findings indicate that if there are multiple cytoplasmic Hsp70-encoding genes in V. carteri, they must be extremely highly conserved with respect to each other. As an alternative approach to determining whether there is another V. carteri gene that is very similar to vc-hsp70A, we used a vc-hsp70A fragment that spans the coding region of the gene to probe DNA gel blots of genomic DNAs digested with 4 different restriction enzymes that cut near one end of the gene but not the other. In each such digest just a single hybridizing band of moderate or high intensity could be detected (Fig. 4). If

Fig. 4. Southern analysis of vc-hsp70A copy number. Genomic DNA prepared from strain RegC4 was digested with the indicated restriction enzymes, electrophoresed, transferred to a nylon membrane, and hybridized under stringent annealing conditions with radiolabeled probe fragment P2 (Fig. 1).

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there is a gene that is nearly identical in coding sequence to vchsp70A, the regions flanking its coding region (∼7 kb upstream and ∼ 4 kb downstream) must also be similar enough to the vchsp70A flanking sequences for all four kinds of restriction sites to be conserved. While this is formally possible, the simplest interpretation of these results is that the V. carteri genome encodes a single cytoplasmic Hsp70. 3.4. Use of a putative regulatory element of vc-hsp70A to drive conditional expression of a glsA antisense transgene Because vc-hsp70A is highly inducible by heat shock, we wondered whether its promoter might be able to drive expression of antisense transgenes to produce conditional RNA “knockdown-like” phenotypes in V. carteri. As a preliminary test of this idea, we cloned a fragment of vc-hsp70A that corresponds to the heat-shock-inducible regulatory region of cr-hsp70A (spanning bp − 522 to bp − 17 with respect to the start codon; Fig. 2), inserted it upstream of an antisenseoriented, full-length glsA cDNA, and co-transformed the construct (pASglsA) along with the nitA-containing plasmid pVcNR15 into Nit− Reg− strain 153–68. Inhibition of glsA function can be readily monitored by counting the number of gonidia per spheroid. Eighty-six Nit+ transformants were selected and their progeny propagated at 32 °C and examined in wells of microtitre plates. Under these conditions, spheroids of the recipient strain 153–68 typically produce 9–18 gonidia, and most of the Nit+ transformants were indistinguishable from 153–68 in this regard. However, four transformants generated significant numbers of progeny with noticeably fewer gonidia than are made by individuals of the recipient strain, and genomic PCR confirmed that the antisense transgene had been incorporated into each of these transformants (data not shown). So these four strains and the parental strain, 153–68, were all propagated in 500-ml bubbler flasks at 32 °C and at 37 °C to provide better quantitation of the phenomenon, and to determine whether the gonidial-production phenotype might be modulated by temperature. V. carteri does not grow well at temperatures above 37 °C, and heat shock causes embryos to cleave aberrantly (Kirk, 1998 plus our unpublished observations), so continuous growth at 37 °C was the most stringent heat stress we could apply to the transformants for this purpose. Three to four days after inoculation, gonidia were counted in N100 individuals from each of the transformant and control cultures that had been grown at each temperature. All four transformant strains produced significantly fewer gonidia than 153–68 at both temperatures. Interestingly, while the Gls phenotype exhibited by these transformants was not as severe as that of glsA mutant 22gls1 (which never produces more than four gonidia per spheroid and averages one gonidium/spheroid; Miller and Kirk, 1999), in each of the transformant strains the gonidial deficiency was significantly greater in cultures grown at 37 °C than at 32 °C (Fig. 5 and data not shown). For instance, 75% of T10–6 spheroids produced 4 or fewer gonidia at 37 °C, but only 10% did so at 32 °C, and 27% of T11–10 spheroids made 4 or fewer gonidia at 37 °C but only 2% did so at 32 °C. 153–68 individuals made the same

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Fig. 5. Phenotypic analysis of selected pASglsA transformants. (A) Schematic representation of construct pASglsA that was transformed into strain 153–68 to 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 3–4 days 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, 500 μM. Transformant strains T10–5 and T13–8 exhibited temperature-dependent Gls phenotypes that were comparable to those displayed by T10–6 and T11–10 but those data are not shown here.

number of gonidia (an average of ∼ 14 per spheroid) at both temperatures (Fig. 5 and data not shown) and none possessed fewer than 6 gonidia 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 required 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 hsp70 clones by screening genomic and cDNA libraries with a probe derived from cr-hsp70A, the heat-shock-inducible hsp70 gene of the related alga C. reinhardtii. Sequencing revealed that nearly all of these clones appear to represent a single V. carteri hsp70 gene 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 declined 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 conferred temperature-dependent Gls antisense phenotypes (Section 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 polyadenylation 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

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transcripts are polyadenylated at more than two locations have been reported (Cheng and Tsai, 1999; Zhao et al., 1999; Jin and Bian, 2004). In some cases mRNAs produced from the same gene but polyadenylated at different sites have been found to accumulate differentially under at least certain conditions (Skadsen and Knauer, 1995; Cheng and Tsai, 1999; Jin and Bian, 2004), but differential polyadenylation may not always lead to or be indicative of differential accumulation (Magnotta and Gogarten, 2002). It will be very interesting to determine whether the four classes of vc-hsp70A transcripts that contain different polyadenylation sites display any developmental or temperature-dependent differences in their accumulation patterns. 4.2. The vc-hsp70A promoter as a tool to express transgenes in V. carteri Previously it was demonstrated that expression of a portion of the volvoxopsin (vop) gene in antisense orientation can reduce the accumulation of Volvoxopsin protein (Ebnet et al., 1999), but our studies constitute the first report of the conditional expression of an antisense phenotype in V. carteri. We have yet to test additional genes to determine whether the vchsp70A promoter fragment may function as well in the context of other antisense transgenes, and it may well be that efficient generation of temperature-dependent antisense phenotypes will be limited to experiments with genes that, like glsA, are poorly expressed. Nevertheless, with the nearing completion of the first draft of the V. carteri genome sequence (D. Rokhsar, personal communication), the availability of an inducible promoter should help facilitate the analysis of many candidate genes by an antisense approach. For years the promoters of hsp70 genes from other organisms have been used to drive high-level expression of transgenes under normal growth conditions or in response to heat shock, and it is likely that the promoter of vc-hsp70A may be adapted similarly. In fact, essentially the same upstream fragment that we used was recently placed upstream of a 259-bp rbcS3 (RUBISCO small subunit-encoding gene 3) promoter from V. carteri to create a hybrid promoter that when used to drive a dominant selectable marker gene permitted an ∼ 3-fold increase in the number of transformants that could be obtained relative to the same reporter driven by the V. carteri β2-tubulin gene promoter (Jakobiak et al., 2004). A follow-up to these preliminary investigations should help to optimize the use of this promoter region as an important addition to the molecular toolbox for V. carteri. 4.3. vc-hsp70A appears to encode the only cytoplasmic Hsp70 in V. carteri All eukaryotes that have been examined to date possess multiple Hsp70 species, with different paralogs apparently specialized for different functions within the cytoplasm, mitochondria, and endoplasmic reticulum (Bukau and Horwich, 1998; Gething, 1999; Voos and Rottgers, 2002). Photosynthetic eukaryotes possess a fourth type of Hsp70 that functions within

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the chloroplast (Gray and Row, 1995). Many (but not all) organisms also possess multiple Hsp70 paralogs that localize within the same cellular compartment and that may carry out non-redundant functions (Boorstein et al., 1994; Sung et al., 2001). However, C. reinhardtii possesses only one cytoplasmic Hsp70 (von Gromoff et al., 1989 plus our unpublished results), and we have found that V. carteri also appears to have only one hsp70 gene that encodes a cytoplasmic Hsp70. This may turn out to be typical of volvocalean algae, which would seem somewhat surprising, since these algae are at least as complex as yeast (which has four differentially regulated cytoplasmic Hsp70s), and often live in temporary ponds and puddles that frequently undergo rapid and extreme environmental fluctuations. The existence of just one cytoplasmic Hsp70 in V. carteri would, however, simplify the analysis of Hsp70 and GlsA function in mediating asymmetric division of the embryo, because it would diminish the possibility that the effects of modifying Hsp70 structure or expression might be masked by an Hsp70 of partially redundant function. In any event the next step will be to determine whether Vc-Hsp70A and GlsA interact physically, and if so, how these two chaperones contribute to the spatial and temporal control over asymmetric division in V. carteri. Acknowledgements We are very grateful to Eric Balzer, Norbert Eichner, Laura Satkamp, and Mark Womer for important technical contributions and P. Ferris for providing C. reinhardtii genomic DNA. We also thank Leonard Duncan, David Kirk, Valeria Pappas, and Cynthia Wagner for insightful discussions and advice before and/or during preparation of this manuscript. This work was supported by research grants from the National Science Foundation (grant # 0077535) and the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service (grant # 2003-35304-13385) to SMM and the Deutsche Forschungsgemeinschaft (SFB 521) to AH. References Adams, C.R., Stamer, K.A., Miller, J.K., McNally, J.G., Kirk, M.M., Kirk, D.L., 1990. Patterns of organellar and nuclear inheritance among progeny of two geographically isolated strains of Volvox carteri. Curr. Genet. 18, 141–153. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Atassi, M.Z., Webster, R.G., 1983. Localization, synthesis, and activity of an antigenic site on influenza virus hemagglutinin. Proc. Natl. Acad. Sci. U. S. A. 80, 840–844. Boorstein, W.R., Ziegelhoffer, T., Craig, E.A., 1994. Molecular evolution of the HSP70 multigene family. J. Mol. Evol. 38, 1–17. Bukau, B., Horwich, A.L., 1998. The Hsp70 and Hsp60 chaperone machines. Cell 92, 351–366. Cheng, C.P., Tsai, C.H., 1999. Structural and functional analysis of the 3′ untranslated region of bamboo mosaic potexvirus genomic RNA. J. Mol. Biol. 288, 555–565. Drzymalla, C., Schroda, M., Beck, C.F., 1996. Light-inducible gene HSP70B encodes a chloroplast-localized heat shock protein in Chlamydomonas reinhardtii. Plant Mol. Biol. 31, 1185–1194.

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