hththfh

the JGI genomes database (/cgi-bin/runAlignment?db=chlre2),and identified 20records with e values of 10−5or 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 pre-dicted coding regions.The remaining 13hits 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 vc-hsp70A and the cytoplasmic-Hsp70encoding sequences prob-ably result from errors in the shot-gun sequencing,because most of the reads were N 900bp 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 nucle-otide 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 4different 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

there is a gene that is nearly identical in coding sequence to vc-hsp70A ,the regions flanking its coding region (∼7kb upstream and ∼4kb downstream)must also be similar enough to the vc-hsp70A 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.

e 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 expres-sion 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 −522to bp −17with respect to the start codon;Fig.2),inserted it upstream of an antisense-oriented,full-length glsA cDNA,and co-transformed the con-struct (pASglsA)along with the nitA -containing plasmid pVcNR15into 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 se-lected and their progeny propagated at 32°C and examined in wells of microtitre plates.Under these conditions,spheroids of the recipient strain 153–68typically produce 9–18gonidia,and most of the Nit +transformants were indistinguishable from 153–68in 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 trans-gene 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,1998plus 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 N 100individuals from each of the transfor-mant and control cultures that had been grown at each tem-perature.All four transformant strains produced significantly fewer gonidia than 153–68at 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.5and data not shown).For instance,75%of T10–6spheroids pro-duced 4or fewer gonidia at 37°C,but only 10%did so at 32°C,and 27%of T11–10spheroids made 4or fewer gonidia at 37°C but only 2%did so at 32°C.153–68individuals made the

same

Fig.4.Southern analysis of vc-hsp70A copy number.Genomic DNA prepared from strain RegC4was digested with the indicated restriction enzymes,electro-phoresed,transferred to a nylon membrane,and hybridized under stringent annealing conditions with radiolabeled probe fragment P2(Fig.1).

117

Q.Cheng et al./Gene 371(2006)112–120