transfection and heat-inducible expression of molluscan promoter

Am. J. Trop. Med. Hyg., 59(3), 1998, pp. 414–420 Copyright q 1998 by The American Society of Tropical Medicine and Hygiene

TRANSFECTION AND HEAT-INDUCIBLE EXPRESSION OF MOLLUSCAN PROMOTERLUCIFERASE REPORTER GENE CONSTRUCTS IN THE BIOMPHALARIA GLABRATA EMBRYONIC SNAIL CELL LINE TIMOTHY P. YOSHINO, XIAO-JUN WU, AND HONG-DI LIU Department of Pathobiological Sciences, University of Wisconsin, Madison, Wisconsin

Abstract. Studies were initiated to begin developing a genetic transformation system for cells derived from the freshwater gastropod, Biomphalaria glabrata, an intermediate host of the human blood fluke Schistosoma mansoni. Using a 70-kD heat-shock protein (HSP70) cDNA probe obtained from the B. glabrata embryonic (Bge) cell line, we cloned from Bge cells a complete HSP70 gene including a 1-kb genomic DNA fragment in its 59-flanking region containing sequences indicative of a HSP promoter. Identified in the 59-half (416 nucleotides) of this genomic fragment were TATA and CAAT boxes, two putative transcription initiation sites, and a series of palindromic DNA repeats with shared homology to the heat-shock element consensus sequence (Bge HSP700.5k promoter). The 39-half of this upstream flanking region was comprised of a 508-base intron located immediately 59 of the ATG start codon. To determine the functionality of the putative snail promoter sequence, Bge HSP promoter/luciferase (Luc) reporter gene constructs were introduced into Bge cells by N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP)-mediated transfection methods, and assayed for Luc activity 48 hr following a 1.5-hr heat-shock treatment (408C). Compared with control vectors or the Bge HSP700.5k/1.0k promoter constructs at 268C, a 10- to 300-fold increase in Luc expression was obtained only in the Bge HSP70 promoter/Luc-transfected cells following heat-shock. Results of transfection experiments demonstrate that the Bge HSP700.5k DNA segment contains appropriate promoter sequences for driving temperature-inducible gene expression in the Bge snail cell line. This report represents the first isolation and functional characterization of an inducible promoter from a freshwater gastropod mollusc. Successful transient expression of a foreign reporter gene in Bge cells using a homologous, inducible promoter sequence now paves the way for development of methods for stable integration and expression of snail genes of interest into the Bge cell line. In insect vectors of parasitic disease, the technology for DNA-mediated gene transfer is well established using gene constructs driven by heterologous promoters, transposable elements, or viruses.1,2 Application of these methods in mosquitoes have resulted in successful stable transfection of both cell lines3–6 and whole organisms.5,7,8 Although from a technological perspective, this continues to be a rapidly evolving field, it is clear that current advancements in developing genetic transformation systems in medically important insects will provide invaluable tools for assessing the function of transgenes and their products, testing the effects of transgene expression on host-parasite compatibilities, and potentially, may lead to development of novel genetic approaches to controlling pathogen transmission through disruption of vector competence.2,9,10 In contrast, advances in gene transfer technology for molluscan intermediate hosts of human and domestic animal parasites have not kept pace with the insects. Using lipofectin-mediated transfection methods, Lardans and others11 were the first to introduce and express luciferase (Luc) reporter gene constructs in snail cells. In their experiments, Luc gene expression in the Biomphalaria glabrata embryonic (Bge) cell line was driven by Drosophila heat-shock protein (HSP70) and cytomegalovirus (CMV) promoters, and appeared to be transient in nature. In the present study, as a next step in developing a gene transfer system with stable expression in B. glabrata, we have continued our investigations of a HSP70 cDNA derived from the Bge cell line12 by cloning the Bge HSP70 gene, characterizing a putative HSP promoter region in its 59 untranslated flanking region, and demonstrating heat-inducible homologous promoter activity for this region through Bge cell transfection experiments.

MATERIALS AND METHODS

Biomphalaria glabrata embryonic cell line. The Bge cell line, originally isolated from B. glabrata embryos by Hansen,13 was obtained from the American Type Culture Collection (Rockville, MD) (ATCC #CRL 1494) and maintained in Hansen’s Bge medium as described by Laursen and others.12 Genomic library construction and screening. Total DNA was isolated from 108 Bge cells by digestion in proteinase K (1 mg/ml of proteinase, 0.5% sodium dodecyl sulfate, 10 mM EDTA in 50 mm Tris-HCl, pH 8.0) overnight at 508C, followed by phenol-chloroform extraction, ethanol precipitation and DNA resuspension in TE buffer (10 mM Tris-HCI, 1 mM EDTA; pH 8.0).14 Southern blot analysis. Fifteen micrograms of Bge cell genomic DNA were digested with Bam HI, Pst I, Hind III, Eco RI, or Xba I, electrophoretically separated in a 0.6% agarose gel, and blotted to nitrocellulose. The DNA fragments were probed with a 32P-labeled 720-base sequence of the Bge HSP70 cDNA located near its 59 end (cDNA sequence position 1295 to 11014).12 Biomphalaria glabrata embryonic cell line HSP70 gene cloning and sequencing. Fragments of Bam HI-digested genomic DNA in the size range of . 9 kb were electrophoretically separated on a gel, cloned into the EMBL-3 vector (Promega Corp., Madison, WI), and packaged according to the manufacturer’s protocols. Approximately 105 plaque-forming units were screened with the above described 720-base Bge HSP70 cDNA probe. One hybridizing recombinant clone contained a large (approximately 15 kb) Bam HI DNA insert, which upon further digestion with Sal I produced positive fragments of 1 and 6

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kb. We subsequently found that the 720-nucleotide HSP70 cDNA probe sequence overlapped the 59 end of the 1-kb Sal I DNA fragment and the 39 end of the 6-kb upstream fragment. Therefore, further analysis of the putative HSP70 promoter region focused on the 6-kb Sal I fragment, although complete sequencing of the 1-kb segment also was performed. The 6-kb Sal I fragment was subcloned into pGEM3z (Promega Corp.), followed by digestion with Eco RI, which yielded DNA segments of 1, 2.5, and 3 kb. Based on sequence information for the 1-kb Eco RI fragment showing overlap of its 39 end with the 59end of the Bge HSP70 cDNA,12 and the observed size-ordering pattern of all Eco RI fragments, it was determined that 39 to 59 order of these gene segments was 1, 2.5, and 3 kb. Because we were interested in the region immediately upstream of the ATG start codon, the 1- and 2.5-kb fragments were then subcloned into the Eco RI site of pGEM3z and sequenced according to the dideoxy chain termination method of Sanger and others15 using the Sequenase 2.0 kit (Amersham Life Sciences, Cleveland, OH). In addition, to confirm that the genomic sequence contained in the original 15-kb Bam HI fragment represented the Bge HSP70 gene and to determine the possible presence of additional intragenic introns, various oligonucleotide primers were synthesized based on the previously published Bge HSP70 cDNA sequence,12 and the polymerase chain reaction (PCR) was used to produce a series of overlapping genomic HSP70 DNA segments. These segments were cloned into pGEM-T Easy (Promega Corp.) and sequenced using the Sequenase 2.0 kit (Amersham Life Sciences). Primer extension. Primer extension was used to map the site(s) of transcription initiation in the putative Bge HSP70 promoter region.16 Heat-shocked Bge cells served as the source of template mRNA and the oligomer 59GCGCTTAGTAATCCTTGA-39, complementing a sequence of the Bge HSP70 cDNA (2122 to 2139),12 was used to prime first-strand synthesis. Isolated cDNA was heated to 1008C for 3 min, chilled on ice, and then loaded onto an 8% polyacrylamide sequencing gel. A dideoxy sequencing ladder from M13mp18 (Amersham Life Sciences) was used to determine the size of the oligonucleotide extension products. Isolation of putative HSP70 promoter sequences. Sequence analysis of the 1-kb region immediately upstream of the HSP70 gene ATG start codon revealed the presence of a 508 nucleotide intron, followed by TATA and CAAT boxes, and several consensus heat shock elements (see Results for detailed analysis). These nucleotide motifs were contained within a 416-base gene segment located upstream of the intron, and appeared to represent the HSP70 gene promoter region. Therefore, two DNA sequences, one corresponding to the 416-base promoter region and the other composed of this promoter region and the intron (1068 nucleotides in total length), were constructed as follows. 1) The 416-base promoter sequence (Bge HSP700.5k). The 59[59-CTTTAAGCTTCTAGATCTATAAACTAT-39] and 39[59GCGTTGCGCTTAGGGATCCTTGATTAC-39] primers were synthesized and used to PCR-amplify the 416-base promoter region from isolated Bge genomic DNA. Two nucleotide changes from the genomic sequence were engineered into each primer (see boldfaced bases) that introduced Hind III and Bam HI restriction enzyme sites (under-

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lined) into the 59 and 39 ends, respectively, of the final PCR promoter product. The PCR amplification was carried out using Bge cell genomic DNA as template, Taq DNA polymerase (Promega Corp.), and the following amplification schedule: six cycles at 948C for 1 min, 408C for 2 min, and 728C for 2 min, followed by 30 cycles at 948C for 1 min, 508C for 2 min, and 728C for 2 min. The PCR products were isolated by digestion with Hind III and Bam HI and cloning into pGEM3Z (Promega Corp.). 2) The 1068-base promoter (Bge HSP701.0k). The same 59 primer described above was used in conjunction with a synthesized 39 primer [59-CGTCCTGACATCTGAAAAAATA39] to PCR-amplify the 1068-base promoter/intron segment to be used in reporter constructs. The PCR amplification products were complemented with Klenow (DNA polymerase I large fragment; Promega Corp.), blunt-end ligated into the Eco RV site of pbluescript II SK(1) (Stratagene, La Jolla, CA), and isolated following digestion with Bam HI and Hind III. Construction of HSP70 expression vectors. To evaluate the ability of putative Bge cell HSP70 promoter sequences to regulate HSP70 gene expression, two promoter-reporter constructs were made based on modification of the pcDNA3 (Stratagene) and pSP-Luc1 (Promega Corp.) vectors. 1) The Bge HSP700.5k promoter construct. The CMV promoter (PCMV) of pcDNA3 was first excised by digestion with Nur I and Hind III, followed by ligation of the 416base Bge HSP700.5k fragment into the Bam HI-Hind III locus of the pcDNA3 multicloning site (MCS). The Luc gene sequence was then digested from the pSP-Luc1 plasmid using Bgl II and Xho I and ligated into the Bam HI-Xho I site of the pcDNA3 MCS, just downstream of the Bge HSP700.5k promoter sequence. 2) The Bge HSP701.0k promoter construct. The 1068-base promoter/intron fragment was ligated into the Bgl II-Hind III MCS of pSP-Luc1 just 59 of the Luc gene. Following plasmid digestion with Kpn I-Xho I, the HSP701.0k promoter/Luc gene construct was ligated into the MCS of pcDNA3 previously modified by deletion of the PCMV promoter sequence. Bge cell transfection. Following previously described methods,11 two expression vectors incorporating the putative Bge cell HSP70 promoter-Luc reporter gene constructs were used in transient transfection experiments to determine whether the 416- and 1068-nucleotide sequences possessed promoter activity. The Bge cells were introduced into wells of a six-well tissue culture plate (approximately 106 cells/ well) and maintained for 24 hr at 268C to permit cell attachment and spreading. They were washed twice with Chernin’s balanced salt solution (CBSS),17 followed by exposure to a 1:5 mixture of DNA and N-(1-(2,3-dioleoyloxy)propyl)N,N,N-trimethylammonium methylsulfate (DOTAP; Waisman Center, University of Wisconsin, Madison, WI) for 6 hr at 268C. The DNA-DOTAP solution was then removed, complete Bge medium was added, and cultures were incubated for an additional 2 hr at 268C. Separate cultures were then placed either at 408C for 90 min (heat-shock test) or maintained at 268C for 90 min (heat-shock control) before bringing all cultures to 268C and incubating them for an additional 48 hr. Cells were then harvested by first solublizing monolayers in 100 ml of luciferase cell lysis buffer (Promega Corp.), transferring lysates to microcentrifuge tubes,

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FIGURE 2. Southern blot analysis of a cloned 15-kb Bam HI fragment of Biomphalaria glabrata embryonic (Bge) cell genomic DNA before (B) and after (S) digestion with Sal I. Right panel: following hybridization with the 32P-labeled, 720-base Bge heat-shock protein (HSP70) cDNA probe. Arrowheads indicate the probe-reactive 1and 6-kb Sal I restriction enzyme fragments. Left panel: ethidium bromide–stained gel of the 15-kb Bam HI fragment before (B) and after (S) digestion with Sal I and prior to Southern blot analysis. Numbers on left represent DNA size markers in kilobases.

FIGURE 1. Southern blot hybridization of restriction enzyme-digested Biomphalaria glabrata embryonic (Bge) cell genomic DNA probed with a 720-base Bge heat-shock protein (HSP70) cDNA fragment. Numbers on the left represent DNA size markers in kilobases.

and immediately freezing at 2808C. To assay for enzyme activities, samples were thawed by warming to 228C, followed by centrifugation at 14,000 rpm for 5 min, and addition of 60 ml of sample to 100 ml of substrate (luciferase assay reagent; Promega Corp.). Light emission was measured using a TC 20/20 luminometer (Turner Designs Instruments, Sunnyvale, CA) and data were expressed as relative light units/mg of protein/min. Sample protein concentrations were determined using a MicroBCAy protein asay reagent kit (Pierce, Rockford, IL) with bovine serum albumin as the protein standard. RESULTS

Southern blot analyses and isolation of the 59 region of the HSP70 gene. The Bam HI restriction enzyme digestion of Bge cell genomic DNA and probing with the 32P-labeled 720-bp HSP70 cDNA fragment revealed strong hybridization with a broad 9–20-kb band of genomic DNA (Figure 1). This region was cloned into EMBL-3, and the resulting library was screened with the above HSP70 cDNA probe. One probe-reactive clone containing a 15-kb Bam HI fragment was further digested with Sal I producing positive restriction fragments of 1 and 6 kb (Figure 2). Partial sequence analysis of the 1-kb and 6-kb fragments showed that the 720 nucleotide HSP70 cDNA probe sequence exactly overlapped the 59 and 39 ends of the 1-kb and 6-kb fragments, respec-

tively. Therefore, the 6-kb sequence was subcloned into pGEM3z for further analysis by digestion with Eco RI. The Bge HSP70 gene sequence and identification of putative promoter region. Digestion of the 6-kb Sal I genomic fragment with Eco RI yielded three DNA segments of approximately 1, 2.5, and 3 kb. Nucleotide sequence analysis of the 39 end of the 2.5-kb fragment (approximately 500 bases) revealed complete identity with the 59-end of the HSP70 cDNA described by Laursen and others,12 and an additional 2-kb region upstream of the ATG start codon. Further analysis demonstrated the presence of a 508-base intron located immediately 59 of the ATG start codon that split the Bge HSP70 gene into two exons, a 182-base noncoding upstream region (Exon I) and a second gene segment of 2294 bases containing the HSP70 gene coding region (Exon II) (Figure 3). Intron identification was based on the presence of flanking splice sites (ATAAG/gt. . .ttcag/AT),18 and the absence of this nucleotide region from the corresponding cDNA.12 Other features of the upstream genomic DNA region included a TATA box at position 2715, CAAT boxes at positions 2824, 2886 (inverted CAAT), and 2962, and a series of partial palindromic DNA repeats with shared homology to the consensus sequence characterizing heat shock elements (CnnGAAnnTTCnnG).19 Six such elements were identified; two upstream of the TATA box, two distal to the first CAAT box, and one each upstream of the second and third CAAT boxes (Figure 3). Primer extension of Bge HSP70 mRNA transcripts generated two extension products of 59 and 61 nucleotides (Figure 4), placing them at positions 2178 and 2180 upstream of the mRNA start codon and 31 bases 39 of the TATA box. However, when taking into consideration the intervening intron sequence, the actual genomic locations of these putative transcription start sites are at position 2588 and 2690 from the ATG codon. Finally, the nucleotide sequence of the genomic HSP70 open reading frame (ORF) of Exon II and the 39 untranslated region were nearly identical to that reported previously for the Bge HSP70 cDNA. Three nucleic acid substitutions (CG → A,


417

FIGURE 3. Biomphalaria glabrata embryonic (Bge) cell line heat-shock protein (HSP70) gene promoter sequence and HSP70 genomic organization. A, the 59-untranslated flanking DNA segment includes 1) a putative promoter region with a TATA box (double underlined), CAAT boxes (single underlined), heat-shock elements (HSE1-6; boldfaced caps, italics), and two transcription initiation sites (boldfaced caps); 2) a 182-nucleotide exon (Exon 1); and 3) an intron (sequence in lower case letters). The arrow indicates the position and sequence of the primer used in generating primer extension products from Bge mRNA. The complete Bge HSP70 gene sequence can be found in GenBank under accession number AF025477. B, diagram of genomic organization of the Bge cell line HSP70 gene. Indicated from left to right are heat-shock elements (HSE), CAAT boxes, TATA box, Exon I, intron, and Exon II (open reading frame). nt 5 nucleotides. C, location of the major restriction enzyme sites in the 59-untranslated region representing the putative Bge HSP70 promoter region. Numbers represent nucleotide positions upstream of the ATG start codon. A5 Ase I; B 5 Bgl II, R 5 Eco RI; X 5 Xba I.

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ciferase activity in nonheat-shocked Bge cells in comparison with other controls. Promoter activity of the Bge HSP700.5k construct was also clearly heat-inducible, as shown by a . 300-fold increase in Luc gene expression in heat-treated Bge cells compared with construct-transfected cells maintained at 268C (nonheat-shocked control). In addition, little Luc expression was seen in Bge cells exposed to pcDNA3 constructs containing the HSP700.5k promoter only (no Luc control), the Luc gene only (no promoter control), or cells exposed to DOTAP only. DISCUSSION

FIGURE 4. Putative transcription start sites identified by primer extension reactions. Extension products (arrowheads) of 59 and 61 nucleotides in length (M13 dideoxy sequencing ladder) and terminating at two adenines (heat-shock protein [HSP] Sal I sequence) were generated by an HSP oligonucleotide primed-reverse transcription reaction of Biomphalaria glabrata embryonic cell mRNA.

G → A, and G → A at positions 1236, 1252, and 1261, respectively) and one base insertion (A at position 1240) resulted in changes in three amino acids (S → V, I → V, and S → G at AA positions 413, 418 and 421, respectively) compared with the previously reported Bge HSP70 protein sequence.12 The complete genomic sequence has been submitted to GenBank under accession number AF025477. In summary, the genomic organization of the Bge HSP70 gene consists of a putative 59 promoter region (consisting of a TATA box, three CAAT boxes, and six homologous HSEs), followed sequentially by a 182-base 59 untranslated flanking region (Exon 1), a 508-base intron, and finally, a second exon (Exon II) comprised of the HSP70 ORF and 383 untranslated nucleotides in the 39 flanking region. A schematic representation of the Bge HSP70 gene, including a restriction map of the 59 promoter segment, is shown in Figure 3. Biomphalaria glabrata embryonic cell line transfection. Although there was considerable variation between replicates in transfection experiments, significant luciferase expression was detected only in Bge cells transfected with the Bge HSP700.5k/Luc or HSP701.0k/Luc constructs following heat-shock at 408C (Table 1). Maximum promoter activity of the HSP700.5k region was not significantly affected by inclusion of the 508-base intron (Bge HSP701.0k), although the HSP701.0k construct consistently yielded higher levels of lu-

As demonstrated for arthropod vectors of disease, development of efficient whole-animal genetic transformation systems initially should incorporate the use of species-related cell lines to establish effective transfection protocols and develop homologous promoter-driven expression gene constructs.9 It is fortuitous that the only true cell line currently available for molluscs, the Bge cell line,13 originated from B. glabrata, an important intermediate host of the human blood fluke S. mansoni in the New World. Due to recent success in culturing early larval stages of S. mansoni and S. japonicum20,21 in the presence of Bge cells, it has been proposed that this cell line could serve as an effective research tool for identifying and evaluating host cell factors involved in early intramolluscan schistosome development.11,20 In addition, the ability to genetically transform Bge cells through homologous promoter-driven stable transfection would further permit the introduction and expression of foreign genes of interest in Bge cells, thereby providing a means for biochemically characterizing their products and functionally assessing their interactions with larval schistosomes under in vitro conditions. The results of the present study bring us closer to this goal by identifying a Bge cell HSP70 gene promoter and demonstrating its ability to drive transient expression of a foreign reporter gene in Bge cells. Lipofectinmediated transfection of Bge cells11 and molluscan heart primary culture cells22 with DNA constructs containing a luciferase reporter gene recently has been achieved. However, in these studies heterologous promoters (e.g., CMV or Drosophila HSP70) were used in functional gene constructs. The present study is the first to demonstrate a homologous promoter capable of driving foreign gene expression in molluscan cells. The fact that Luc gene expression in cells transfected with the Bge HSP70 promoter sequence was heatinducible further attests to the normal functioning and regulation of the introduced promoter/reporter gene constructs. Although, the relative efficiencies of the different promoters (homologous versus heterologous) used in the present and previous molluscan studies11,22 cannot be compared directly,

TABLE 1 Specific luciferase (Luc) activity (relative light units or RLU/mg of protein) in Biomphalaria glabrata embryonic (Bge) cells under normal (268C) and heat-shock (408C) conditions*

268C 408C

HSP0.5k

HSP1.0k

HSP only

Luc only

DOTAP

0.04 6 0.02 14.9 6 11.9

0.95 6 0.76 10.6 6 1.4

0.003 6 0.005 0.13 6 0.15

0.006 6 0.01 0.008 6 0.04

0.025 6 0.043 0.016 6 0.014

* Cells had been transfected with gene constructs consisting of the 416-base Bge heat-shock protein (HSP promoter 1 Luc) gene (HSP0.5k), the 1068-base Bge HSP promoter 1 Luc gene (HSP1.0k), Bge HSP0.5k promoter only (HSP only), luciferase gene only (Luc only), or cells exposed only to N-(1-(2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium methylsulfate(DOTAP). Mean 6 SD values were calculated from three independent replicate experiments.


based on the fold increase in reporter expression under heatshock and nonheat-shock conditions, homologous Bge HSP0.5k promoter activity appeared to be greater than that of the Drosophila HSP70, CMV, or simian virus early promoters11,22 under similar experimental conditions. Now that methods for introducing and transiently expressing foreign DNA in molluscan cells have been achieved, the next critical step towards establishing a stable genetic transformation system will involve development of methods for stable integration, replication, and expression of desired genes and identification of selectable markers for efficient recovery of transformed cells.9 The Bge HSP70 promoter described in the present study may now provide basic information for building selectable gene constructs that can be targeted to the Bge cell HSP70 locus for stable transfection and whose expression is under temperature control.4,23 Integration and stable transformation of Bge cells with Bge HSP70 promoter-reporter constructs also could be facilitated by incorporating such constructs into infective pantropic viral vectors such as vesicular stomatitis virus-pseudotyped retrovirus,24,25 or through cotransfection with broadly reactive transposable elements.26 In this regard, further investigations into a B. glabrata long interspersed nuclear element– like transposon exhibiting significant homology to reverse transcriptase27 or other insect transposable elements, as functional vehicles for insertion of heterologous gene segments into the Bge cell genome should be pursued. Although the HSP70 genes represent one of most widespread and extensively characterized of this gene family, with the exception of the marine opisthobranch Aplysia californica,28 very few HSP genes have been described in the phylum Mollusca. Moreover, to our knowledge, no information currently exists regarding the structure of promoter regions regulating inducible expression of the HSPs in molluscs. Thus, the present structural and functional characterization of a putative HSP70 promoter sequence in Bge cells represent the first for cells derived from gastropod molluscs, and in particular, cells related to the medically important snail species B. glabrata.13 Results of Bge transfection experiments demonstrate that both promoter activity and structural elements regulating heat-inducible HSP70 expression are located in the distal 416 nucleotides (Bge HSP700.5k) of the 1068-base DNA segment immediately upstream of the ATG start codon. Among these include a consensus TATA box sequence, common to most HSP promoters,29 and located 29 and 31 nucleotides upstream of two adenines, identified by primer extension as putative mRNA termini. This location is typical of eukaryotic transcription initiation sites and represents a conserved feature of HSP gene promoter regions.30 A second structural feature of this gene segment is the presence of a series of partial palindromic repeats (Figure 5) with shared homology to the consensus heat-shock element (HSE) C--GAA--TTC-G.19,31 The first of these HSEs is located 28 nucleotides upstream of the TATA box, which falls in the range of distance (14–28 bases)29 reported for the proximal-most HSE. Previous studies have shown that the basic HSE dyad unit, GAA--TTC, serves as the binding site for heat-shock factors32 and is responsible for temperature-mediated induction of HSP transcription in most organisms.29,33,34 Although the Bge cell HSEs are not identical to the basic eight-nucleotide

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FIGURE 5. Sequence comparison of Biomphalaria glabrata embryonic (Bge) heat-shock elements (HSEs) with the palindromic consensus HSE sequence.19 The Bge cell HSE base-matches with the consensus sequence are indicated by boldfaced capital letters. *Number of nucleotides between the end of HSE sequences and the TATA box.

consensus sequence (Figure 5), most exhibit seven of eight consensus base-matches, which is still considered to be a strong HSE by Bienz and Pelham.29 Furthermore, studies investigating the ability of synthetic HSE sequences to induce reporter-gene expression in transfected Drosophila cells have shown that a GAG substitution for selected GAA trimers still yield significant induction of gene expression.34 A similar case was suggested for a TTC-to-CTC trimer substitution. Based on these findings, it is speculated that the Bge HSEs 1, 2, 3, and 5 most likely are serving as binding sites for snail cell heat-shock factors and thus are responsible for induction of Luc reporter gene expression. Finally, consensus CAAT and inverted CAAT promoter sequences also are located in the 416-base Bge promoter region within 14-27 nucleotides downstream of several HSEs (HSE3-6). These CAAT sequences, in conjunction with HSEs, can function as enhancers in vertebrate cell HSP gene induction,29,35 and may be functioning in a similar capacity in Bge cells. One feature, apparently unique to the HSP70 gene family, was the presence of an intron just 59 of the ATG start codon, which divides the Bge gene into two exons (a short 182base Exon I and a 2295-base ORF-containing Exon II). This intron does not appear to play a significant role in Bge HSP70 gene induction since transfection of cells with the Bge HSP701.0k promoter construct, containing both the 416base promoter and intron, resulted in a similar maximum level of luciferase activity as the 416-base promoter alone. However, one consistent result obtained with the Bge HSP701.0k construct was a higher basal level of luciferase in nonheat-shocked Bge cells, suggesting the possible presence of weak constitutive promoter activity associated with some, yet unknown, intron sequence(s). A similar gene arrangement has been described in the HSP83 gene of Drosophila,30 although no mention was made of the potential involvement of upstream intron sequences in HSP promoter function. Clearly, further work is needed to expand both our knowledge of the inducible molluscan HSP promoters, and our repertoire of alternative snail promoters capable of driving high level constitutive gene expression in Bge cells.

Acknowledgment: We thank Laura Johnston for technical assistance and expertise with computer graphics.

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Financial support: This research was supported by NIH grant AI15503 and NIAID supply contract AI-02656. 16. Authors’ addresses: Timothy P. Yoshino and Xiao-Jun Wu, Department of Pathobiological Sciences, University of Wisconsin, School of Veterinary Medicine, 2015 Linden Drive West, Madison, WI 53706. Hong-Di Liu, Institute of Microbiology, Academia Sinica, Beijing, People’s Republic of China. Reprint requests: Timothy P. Yoshino, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706.

17. 18. 19. 20.

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