Characterization of a heat-shock-inducible hsp70 gene of the

1、characterization of a heat-shock-inducible hsp70 gene of the hththfh characterization of a heat-shock-inducible hsp70gene of the green alga volvox carteri qian cheng a ,armin hallmann b ,lisseth edwards a ,stephen m.miller a,? a department of biological sciences,1000hilltop circle,university of mary

2、land,baltimore county,baltimore,md 21250,usa b department of cell and developmental biology of plants,universitt bielefeld,bielefeld,germany received 18may 2021;received in revised form 10november 2021;accepted 17november 2021 available online 14february 2021 abstract the green alga volvox carteri p

3、ossesses 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 (g onidia l es s a)is requi

4、red for these asymmetric divisions and is believed to function with an hsp70partner.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 mul

5、tiple 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

6、 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.2021elsevier b.v .all rights reserved. keywords:antisense;chlamydomonas reinhardtii ;cytoplasmic hsp70;glsa 1.introduction

7、 the order v olvocales 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 oppor-tunity to investigate the evolutionary origins of rel

8、atively sim-ple forms of cellular differentiation.at one end of the spectrum of developmental complexity within the volvocales are mem-bers of the genus chlamydomonas ,which are unicellular and have been widely used as model organisms for studying pro-cesses such as photosynthesis and assembly and f

9、unction of centrioles and flagella (rochaix,2021;silflow and lefebvre, 2021;dutcher,2021).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 an

10、d small,bi-flagellate 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

11、 j domain that is indispens-able for its asymmetric division function (miller and kirk,1999).because the only known function of a j domain is to bind and activate an hsp70partner protein,we predicted that the function of glsa in asymmetric divisions would involve an hsp70partner.all eukaryotes posse

12、ss multiple hsp70genes,and some express multiple cytoplasmic hsp70s (boorstein et al.,1994;sung et al.,2021),so identifying the hsp70partner of glsa may involve testing among several candidates. the only volvocalean hsp70genes that have been described in print so far are chlamydomonas reinhardtii hs

13、p70a and hsp70b ,which encode stress-inducible cytoplasmic and chlo-roplast hsp70s,respectively (von gromoff et al.,1989;mller et al.,1992;drzymalla et al.,1996).as a preliminary step /locate/gene abbreviations:a,adenosine;aa,amino acid(s);bp,base pair(s);bip,luminal binding protein;bsa,bovine serum

14、 albumin;cdna,dna comple-mentary 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

15、,untranslated region(s). ?corresponding author.tel.:fax:e-mail address: (s.m.miller).0378-1119/$-see front matter 2021elsevier b.v .all rights reserved.doi:10.1016/j.gene.2021.11.026 hththfh toward identifying the hsp70partner of glsa,we used a c. reinhardti

16、i 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,meani

17、ng that it is now the best candidate to be a partner of glsa in asymmetric division.in addition,we found that an500-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 antis

18、ense 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,15368(rega?nita?),and22gls1 (rega?glsa?nita?)were described pr

19、eviously(adams et al., 1990;miller and kirk,1999).regc4was isolated as a sponta-neous reg mutant from a culture of eve grown at24c,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/8

20、h dark growth regimen(kirk and kirk,1985),and except where noted (for heat shock or induction of the pasglsa transgene)were maintained at32c.heat shock conditions were essentially as described previously(kirk and kirk,1986;kirk et al.,1993). briefly,medium-density,asynchronous eve or132/116/1cul-tur

21、es were transferred from a32c water bath to one at42.5c for40min and then to one at45c for20min.in some experiments,spheroids were then harvested immediately;in other experiments spheroids were transferred back to32c to recover for0.53h before they were harvested. 2.2.nuclear transformation and dna,

22、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 phagednas,rt pcr,prep-aration of radiolabeled dna probes,and rna gel

23、blot and western analyses were as described previously(miller et al., 1993;miller and kirk,1999).dna gel blots were hybridized with probe for2h at65c70c using rapid-hyb buffer(ge healthcare,piscataway,nj)and were washed at the same temperature with high-salt(3ssc,0.1%sds)then low-salt (0.3ssc,0.1%sd

24、s)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 afte

25、r 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 softwa

26、re(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

27、c3(arg7,cw?,sr-u,mt?;kindly supplied by p.ferris, department of biology,washington university)as template using primers116(5-caaccacttcgccaacgagttc-3)and114(5-cccttgtcgttggtgatcgtg-3).exist-ing v.carteri genomic and juvenile-specific cdna libraries constructed inphage vectors dashii andzap,respec-ti

28、vely(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 the5and3ends of

29、d10,which,at2.1kb, was the longest clone obtained from our hsp70a cdna screen.an6.5-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 w

30、as 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-bp5cdna that overlaps the5end of the d10insert.both the5race product and d10were sequenced

31、completely on both strands to obtain the sequence of the entire hsp70a coding region plus3utr.nucleotide sequences for the cdna and the6.5-kb ssp issp i genomic fragment were deposited into genbank under accession numb-ers dq059999and dq059998,respectively. fragment p1used for rna gel blot analysis

32、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.p

33、las-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 t

34、he 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)th

35、at 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 113 q.cheng et al./gene371(2021)112120 hththfh plasmid pasglsa,in which the promoter fragment is oriented to

36、 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.

37、5-kb cla ibam 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

38、-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 sin

39、gle 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 west

40、ern 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 l

41、ow density(100 spheroids per300-ml medium),and maintained at either32c or37c with the standard16h light/8h dark growth regimen. three to four days later the number of gonidia present in100 randomly chosen,recently hatched asexual progeny was deter-mined by inspection under a dissection microscope.im

42、ages 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

43、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

44、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 screen300,000phage plaques from a v.ca

45、rteri 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 an13-kb insert that hybrid-ized very well with the probe fragment,was chosen for further analysis.partial sequence from an

46、 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 the5and3ends of a nearly full-length v.carteri hsp70a cdna(described below)that had been isolated con-currently with the genomic clone,we subcl

47、oned the ssp i frag-ment and sequenced it.this6.5-kb fragment contained the entire transcription unit of a gene(including2.0-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,ov

48、er 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(d11

49、1,for“dark”),twelve gave a signal intensity that was slightly weaker(m112,for“medium”),and thirty-four hybridized more weakly than members of the first two classes(l134, for“l(fā)ight”).since we wished to identify other genes that might encode cytoplasmic hsp70s,in addition to the ortholog of c. reinhar

50、dtii 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 re

51、vealed 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 s

52、tudy.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

53、 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.

54、/gene371(2021)112120 hththfh were highly similar to c.reinhardtii hsp70a contained only small inserts that corresponded to a small part of the probe. clones l1and l30both encoded proteins that were much more similar to members of the luminal binding protein (bip)family of hsp70proteins than they wer

55、e to any cytoplasmic hsp70 protein in the database.for instance,the sequenced portion of l30(183bp)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.reinhardti

56、i hsp70a .thus clones l1and l30likely correspond to a gene or genes encoding er-specific hsp70s of v .carteri .none of the hsp70cdnas isolated was long enough to encode a full-length hsp70,so we used sequence from the genomic clone and from the longest cdna (d10,2094bp)to design primers for race to

57、obtain a 501-bp cdna fragment that overlaps with cdna d10and 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 con-served in all members of the hsp70family (bukau and fig.2.putative regulatory sequences within

58、the vc-hsp70a gene.sequence of part of the 6.5-kb ssp i ssp i restriction fragment (fig.1)that spans the entire vc-hsp70a gene is represented,beginning at bp ?530with respect to the start codon,through the end of the fragment.the dots between the start codon and stop codon represent the coding seque

59、nce,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.th