The Mosquito Dihydrofolate Reductase Gene Functions as a Dominant Selectable Marker in Transfected Cells F R A N K A. SHOTKOSKI,*t:~ ANN MARIE FALLON*
Received 2 November 1992; revised and accepted 25 March t993
An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10-15 days in the presence of selective medium containing 1 / i M methotrexate. The transformed clones contained an estimated 100-500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, hut in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.
Gene amplification Methotrexate Gene transfer Aedes albopictus
INTRODUCTION
Gene transfer provides an important tool for exploring genetic, developmental, and regulatory processes that ultimately may contribute to the genetic manipulation of pest insects. Molecular approaches with direct bearing on pest control, ranging from disruption of reproduction by expression of antisense RNA molecules (Qian et al., 1988) to modification of behavior (Wheeler et al., 1991), have been demonstrated in Drosophila melanogaster using transformation vectors based on the transposable element P.
Although mosquito embryos have been transformed with P element-based vectors encoding neomycin- resistance as a dominant selectable marker (Miller et al., 1987; McGrane et al., 1988; Morris et al., 1989), few transformed individuals were recovered, and the trans- formation events were attributed to illegitmate recombi- nation rather than P element activity. In contrast to these efforts at the level of the organism, transfection of cells in culture offers the opportunity to examine rela- tively large numbers of transformation events, and to test the function of DNA constructs that may eventually
*Department of Entomology, University of Minnesota, 1980 Folwell Avenue, St Paul, MN 55108, U.S.A.
+Author for correspondence. ~Present address: Department of Entomology, University of Wiscon-
sin, Madison, WI 53706, U.S.A.
be useful for genetic manipulation of vector species. In the first report of stably-transformed mosquito (C6/36) cells, Monroe et al. (1992) characterized 45 clones that had been stably transformed with a vector encoding hygromycin phosphotransferase. Considerable variabil- ity was observed among these C6/36 clones with respect to gene copy number, which ranged from a few up to several thousand copies, location of exogenous DNA within chromosomes or as double minute extrachromo- somal elements, and distribution of transfected genes within the karyotype. In one clone, c. 60,000 copies of the plasmid were organized in a large array that re- sembled and functioned as a normal segregating chromosome.
The present study focuses on the development and testing of gene transfer vectors encoding the dihydrofo- late reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP* oxidoreductase, EC 1.5.1.3) gene as a dominant selectable marker for direct recovery of stably transfected mosquito cells. We have recently described the structure of an amplified dhfr gene from methotrexate (mtx)-resistant Aedes albopictus cells in which the gene copy number approached 1200 per nucleus (Shotkoski and Fallon, 1991). The presence of a single 56 nucleotide intron in the A. albopictus dhfr gene facilitated use of this gene and its associated regulatory sequences to design plasmids that could be used to recover stably transformed mosquito cells using selective medium containing mtx.
883
884 FRANK A. SHOTKOSKI and ANN MARIE FALLON
M A T E R I A L S A N D M E T H O D S
Plasmid constructs
The plasmid p F S D H F R 9 (Fig. 1) was constructed by ligating a 1.8 kb A c c I fragment containing the entire 617 bp mosqui to dhfr gene and flanking regulatory D N A from p F S D H F R 6 (Shotkoski and Fallon, 1991) into the A c c I site o f pBluescript II SK ÷ (Stratagene, La Jolla, Calif.). To construct p D H F R : c a t - 1 , p F S D H F R 9 was digested at the B s s H I I sites in the pBluescript polylinker, and the resulting 2 kb fragment containing the dhfr gene was purified by electroelution. Ends were made blunt using Klenow fragment, and the 2 kb fragment was ligated into hsp-cat 1 (Di Nocera and Dawid, 1983) that had been digested at a unique A a t I site and blunt-ended with T4 D N A polymerase (Fig. 1). The orientation o f the 2 kb D N A fragment was determined by restriction analysis. Plasmids were purified by a single cycle o f centrifugation in cesium chloride (Fallon, 1989), and D N A concentra t ions were measured spectrophotometr i - cally.
Transfection conditions
The A. albopictus cells used in this study were f rom the phenotypically 'wild type' methotrexate-sensitive clone, C7-10. The C7-10 cells were maintained at 28°C in Eagle's minimal medium (Eagle, 1959) containing non- essential amino acids, glutamine and 5% heat-inacti- vated fetal bovine serum (E-5 medium) (Fallon, 1989). Cells were transfected in 60 m m culture dishes using polybrene (12.5/xg/ml) and plasmid D N A at a final concentra t ion o f 3/xg per ml, essentially as described previously (Fallon, 1989). For co-transfection, plasmid D N A s were added at a molar ratio o f l, at a total D N A concentra t ion o f 3 #g per ml. Following a 48 h post- transfection recovery period, t ransformed cells were selected by addit ion o f mtx to a final concentra t ion o f 1/xM. The selective medium was replaced every 5 days until clones appeared. Non-transfected control C7-10 cells were exposed to similar selective condit ions to determine the frequency at which resistant clones ap- peared spontaneously. Stock solutions o f mtx [ ( + )
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FIGURE 1. Construction of plasmids pFSDHFR9 and pDHFR:cat-l. To construct pFSDHFR9, the 1.8 kb AccI (Ac) fragment containing the entire dhfr gene and flanking regulatory sequences was purified from the 8.5 kb EcoRI insert in pFSDHFR6 (shown on top), and ligated into the AccI site of pBluescript II SK+ vector. To construct pDHFR:cat-1, a fragment containing the dhfr gene was removed from pFSDHFR9 by digestion with BssHIl (B), and ligated into hsp-cat 1 at the unique AatI (Aa) site as detailed in the Materials and Methods; Aa/B designates the location of the blunt-end ligation. The dhfr and cat coding regions are indicated by solid areas and the single intron in the dhfr gene is indicated by the hatched region. The shaded region represents the hsp70 promoter in pDHFR:cat-1. The direction of transcription of the cat and dhfr genes is indicated by arrows. Other diagnostic restriction sites: DraI (D), EcoRI (E), SmaI (S), Xhol (X) and XmnI (Xm), are shown. It should be noted that for each of these enzymes, all sites are not indicated; a more complete restriction map of
the plasmid constructs can be obtained from the authors.
MOSQUITO DIHYDROFOLATE REDUCTASE GENE 885
amethopterin Sigma Chemical Co., St Louis, Mo] were prepared in 8 mM NaOH, and concentrations were determined spectrophotometrically as described pre- viously (Fallon, 1984).
Mtx-resistant clones were transferred to plastic tissue culture tubes containing 2 ml of selective medium. Upon reaching confluency, cells were transferred from tubes to 25 cm 2 culture flasks for continuous maintenance. To verify acquisition and retention of plasmid DNA, trans- formed cell lines cultured in the presence or absence of 1 p M mtx were assayed for chloramphenicol acetyltrans- ferase (CAT) activity by thin layer chromatography (TLC) or by immunostaining. CAT activity was induced by a 4 h heat shock at 41°C, followed by a 2 h recovery period at 28°C, essentially as described previously (Gerenday et al., 1989).
DNA purification and analysis
Genomic DNA was isolated from mosquito cells essentially as described previously (Fallon, 1986), di- gested with various restriction enzymes, and DNA concentrations were determined using a fluorometer. BstEII and HindlII-digested 2 DNA (1 ng) were used as size markers. Southern blots were probed with DNA representing the entire pDHFR:cat-1 plasmid, the dhfr-specific 637 bp XmnI fragment, or the hsp70 promoter/cat-specific 450 bp XhoI/EcoRI fragment from pDHFR:cat-1 (Fig. 1), and with 2 DNA to detect the size markers. The restriction fragments were purified by electroelution from 1% agarose gels. Probes were labeled with [~-32 P]dATP ( ~ 3000 Ci/mmol; Amersham, Arlington Heights, I11.) by random priming using a kit from United States Biochemical (Cleveland, Ohio) to a specific activity of 0.5-1 × 109cpm/#g, and 1-6 x 107cpm/ml were used in a typical hybridization reaction.
The copy number of transfected genes was estimated by dot-blot hybridization to genomic DNA from trans- formed cells in two independent experiments. Since pDHFR:cat-1 measured 7.2kb, and the A. albopictus haploid genome contains 1.3 x 10 6kb, 5.5 pg of pDHFR:cat-1 per #g genomic DNA was equivalent to 1 gene copy per haploid genome. Samples were com- pared to known standards, corresponding to 10-2500 gene copies (0.055-14ng) per 0.1 pg genomic DNA, based on the hybridization signal from pDHFR: cat-1 DNA, labeled by random priming using [0t-3:p]dATP.
Karyotype and in situ hybridization
Cells were grown to 70% confluency in 60 mm tissue culture dishes and treated with colcemid (1 ~tg/ml for 4 h) to accumulate metaphase chromosomes. Chromo- some spreads were prepared on slides as described previously (FaUon, 1984). For in situ hybridization (Pardue, 1985), the chromosome spreads were denatured in 2 x SSC containing 70% (v/v) formamide at 70°C for 2 min. The chromosomes were hybridized simul- taneously with both the 637 bp dhfr-specific probe and the 450 bp cat-specific probe in 2 x SSC containing 50%
(v/v) formamide, 10% dextran sulfate, 40mM sodium phosphate, pH6.5, and 10mg/ml sonicated salmon sperm DNA at 37°C for 16 h. Following hybridization, the slides were rinsed once in 2 x SSC containing 50% formamide at 39°C for 15 min, and twice in 2 x SSC for 1 h. The dried slides were coated with Kodak NTB- 2 nuclear track emulsion (Eastman Kodak Co., Rochester, N.Y.), and exposed for 4 days at 4°C. Following development, the chromosomes were stained with Giemsa and examined microscopically for silver grains.
RESULTS
Plasmid constructs
An intact mosquito dhfr gene including single 56 bp intron (Shotkoski and Fallon, 1991) and c. 800bp of upstream regulatory sequences containing multiple tran- scription initiation sites (Park and Fallon, 1993) was incorporated into transfection vectors to test the func- tion of dhfr as a dominant selectable marker gene under standard and co-transfection conditions. Since high levels of DHFR activity can be recovered from cells that contain amplified dhfr genes, we anticipated that total DHFR activity would also increase when the cloned dhfr gene, regulated by its native promoter, was introduced into wild-type cells by transfection. The 7.2 kb plasmid pDHFR: cat-1 was constructed from the 5.2 kb hsp-cat 1 by insertion of a genomic copy of the mosquito dhfr gene (Fig. 1). In this composite plasmid, expression of the Escherichia coli chloramphenicol acetyltransferase (cat) gene was regulated by the temperature-inducible promoter from the Drosophila heat shock protein (hsp) 70 gene (Di Nocera and Dawid, 1983). Transient ex- pression of CAT activity provided a rapid assay to ensure that mtx-resistant cells were in fact transformed, and did not result from spontaneous resistance ac- companied by amplification of endogenous dhfr alleles. The 5.0 kb plasmid pFSDHFR9 contained the selectable mosquito dhfr gene and flanking regulatory sequences, but lacked the cat reporter gene (Fig. 1). In cells co-transfected with pFSDHFR9 and hsp-cat 1, ex- pression of CAT activity by mtx-resistant clones confirmed acquisition and retention of the nonselectable hsp-cat 1 plasmid.
Selection of stably transformed cells
10o15 days after addition of 1.0/~M mtx, discrete clones of mtx-resistant cells appeared in plates contain- ing cells transfected with either of the dhfr plasmid constructs. In contrast, no mtx-resistant clones appeared spontaneously when non-transfected C7-10 control cells were maintained under similar selective conditions. When transfected cells were treated with 0.5 ~tM mtx, discrete colonies were difficult to isolate, suggesting that selective conditions were inadequate. No transfected cells survived when mtx was added at a concentration of 5.0#M. 26 pDHFR:cat-1 transfected clones, and 5
886 FRANK A. SHOTKOSKI and ANN MARIE FALLON
p F S D H F R 9 / h s p - c a t 1 co-transfected clones were recov- ered at 1.0/~M mtx and expanded into populat ions for further analysis.
Cells f rom each o f the p D H F R : c a t - I transfected clones were first screened for expression of C A T activity by histochemical staining to confirm that the cells were in fact t ransformed. Each of the 26 clones expressed C A T activity, confirming the presence and function o f the composi te plasmid (data not shown). Cells f rom the single clonal popula t ion with the highest level o f C A T activity, as estimated by visual inspection (Mtx-T, Transfected cells) were maintained in cont inuous cul- ture. Similarly, cells f rom the five co-transfected clones were assayed for C A T activity by thin layer chromatog- raphy (TLC) to determine whether the hsp-cat 1 plasmid had been successfully acquired by the cells in conjunc- tion with the selectable p F S D H F R 9 . Cells f rom each of the five clones were positive for C A T activity, suggesting that the non-selectable hsp-cat 1 plasmid had in fact been stably transfected and maintained in the
mtx-resistant t ransformants (data not shown). Cells f rom one clonal popula t ion (Mtx-CT, Co-Transfected cells) were maintained cont inuously in selective medium.
Stability of transfected DNA in the absence of selection
To test the stability o f the transfected D N A , subpopu- lations o f the Mtx-T (passage 4) and Mtx-CT cells (passage 5) (Mtx-T0 and Mtx-CT0 cells, where the '0 ' refers to medium lacking mtx) were transferred into non-selective E-5 medium. After an additional 11-12 passages, C A T activity was compared to that in Mtx-T and Mtx-CT cells maintained under cont inuous selec- tion. Expression of C A T activity was comparable in cells maintained in the presence or absence o f mtx [Fig. 2(A) compare lanes 2 and 4; 6 and 8] and remained heat inducible, showing negligible basal levels o f C A T activity at 28°C in control cells (lanes 3, 5, 7 and 9). Lanes I0 and l 1 demonstra te the absence o f activity in untrans- fected C7-10 cells. After 60 passages, the Mtx-T0 cells cont inued to express heat-inducible C A T activity at
C m
1 2 3 4 5 6 7 8 9 10 11
6
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FIGURE 2. Expression of CAT activity in transformed mosquito cells. Autoradiograms of thin-layer chromatograms are shown. In panels A and B, lane l is a positive control showing CAT activity from commercially-available enzyme. The positions of 1-acetate chloramphenicol (lower arrow) and 3-acetate chloramphenicol (upper arrow) are indicated; Cm represents unaltered substrate. Panel A shows CAT activity in cells at the 16th passage, after a 4 h heat shock at 41°C and 2 h recovery period at 28°C (lanes 2, 4, 6, 8) and in non-heat shocked controls (lanes 3, 5, 7, 9). Lanes 2 and 3 are from Mtx-T cells, 4 and 5 are from Mtx-T0 cells, 6 and 7 are from Mtx-CT cells, and 8 and 9 are from Mtx-CT0 cells. Lanes 10 and 11 show the absence of CAT activity in C7-10 cells with (lane 10) or without (lane 11) heat shock treatment. Panel B shows activity in cells that had been maintained under selective or non-selective conditions for an extended period of time. Lanes 2 and 3 show activity in Mtx-T, Mtx-T0 cells at passages 66 and 60, respectively, and lanes 4 and 5 show activity from Mtx-CT and Mtx-CT0 cells at passages 50 and 45, respectively. Lane 6 shows the absence of activity in nontransfected control C7-10 cells.
MOSQUITO DIHYDROFOLATE REDUCTASE GENE 887
levels comparable to those of the Mtx-T cells maintained in selective medium for 66 passages [Fig. 2(B), lanes 2 and 3]. Similarly, after 45 passages in the absence of mtx, the Mtx-CT0 cells expressed CAT activity at levels similar to those in Mtx-CT cells maintained for 50 passages in the presence of mtx [Fig. 2(B), compare lanes 4 and 5].
DNA analysis Stable expression of CAT activity after prolonged
maintenance under nonselective conditions suggested that the transfected DNA was likely integrated in to chromosomal DNA. To investigate the copy number and arrangement of transfected genes, DNA from trans- fected cells was used in dot-blot and Southern analyses. Dot-blot hybridization indicated that at passage 16, both Mtx-T and Mtx-T0 cells contained an estimated 50-100 copies of pDHFR:cat -1 DNA per haploid genome (Fig. 3, rows 1 and 2). When the same DNA preparations from the Mtx-T and Mtx-T0 cells were digested with AatI, which does not cleave the 7.2kb pDHFR:ca t - I DNA, and hybridized with a 32 P-labeled p D H F R : cat- 1 DNA probe, the signals were confined to high molecular weight DNA [Fig. 4(A), lanes 7 and 8], which migrated more slowly than the (concatemerized) covalently closed circular plasmid DNA (see lower band in lane 6), suggesting that the transfected DNA was either inte- grated into chromosomal DNA or maintained as extra- chromosomal plasmid in multimeric structures. No hybridization signal was detected in DNA from untrans- formed C7-10 cells (lane 9), confirming the absence of endogenous sequences that cross-hybridized with the probe.
To better understand the arrangement of transfected DNA in these Mtx-T and Mtx-T0 cells, the DNA samples were digested with PvuI, which linearizes pDHFR:ca t - I plasmid [Fig. 4(A), lanes 2-5]. For both Mtx-T (lane 3) and Mtx-T0 cells (lane 4), some of the transfected DNA co-migrated with linearized 7.2 kb pDHFR:ca t - I DNA (lane 2), suggesting that this DNA may be arranged as head-to-tail repeats that could occur extrachromosomally or integrated into the mosquito chromosomes. However, a large proportion of the transfected DNA migrated more slowly than the 7.2 kb band, and a minor proportion was detected in smaller fragments (lanes 3 and 4), suggesting integration as single molecules and/or as rearranged molecules at multiple chromosome sites. Note, however, the the hybridization pattern was similar for both Mtx-T and Mtx-T0 cell lines, reflecting their common clonal origin, and suggesting that transfected DNA did not undergo substantial rearrangement under nonselective con- ditions.
To determine whether the dhfr and cat coding regions remained intact in the transfected DNA molecules, DNA from the Mtx-T and Mtx-T0 cells was digested with Sinai [Fig. 4(A), lanes 10-13], which cleaves pDHFR:cat -1 into a 1.4 kb fragment containing the dhfr coding and regulatory sequences and a 5.8 kb band
2500
1000
500
250
100
10
1 2 3 4
FIGURE 3. Estimated copy number of transfected DNA in trans- formed mosquito cells. Vertical lanes 3 and 4 show a standard curve based on hybridization of pDHFR:cat-1 probe to pDHFR:cat-I DNA corresponding to the copy numbers/haploid genome indicated on the right. The DNA was labeled with [ct-3:P]dATP by random priming to a specific activity of 0.5-1 x 10 9 cpm/#g, and 1 x 108 cpm/ml was used in a typical reaction. In lanes 1 and 2, horizontal rows 1-4 show hybridization of the pDHFR:cat-1 probe to genomic DNA (100 ng) extracted at the 16th passage: row I, Mtx-T cells; row 2, Mtx-T0 cells; row 3, Mtx-CT cells; row 4, Mtx-CT0 cells; row 5, Mtx-T cells, passage 64; row 6, Mtx-T0 cells, passage 58; row
7, C7-10 cells.
containing the cat coding region [Fig. 1; Fig. 4(A), lane 10]. DNA from transfected cells yielded these two ex- pected SrnaI fragments (lanes 11 and 12), as well as additional bands corresponding predominantly to frag- ments >5.8 kb. When the SmaI-digested DNAs were hybridized separately with the dhfr-specific probe [Fig. 4(B), lanes 2-4] most of the signal migrated at 1.4 kb, indicating that among transfected genes, the dhfr coding region remained intact. With the cat-specific probe [Fig. 4(B), lanes 5-8], however, DNA larger than the 5.8 kb band was detected, suggesting that in Mtx-T and Mtx- TO cells, the nonselectable cat gene had undergone more extensive rearrangement than the dhfr domain of pDHFR:cat -1 . Note, however, that as in panel A the hybridization patterns in panel B are the same for cells maintained in the presence or absence of selective medium, suggesting that rearrangements likely occurred
888 F R A N K A. SHOTKOSK1 and A N N M A R I E F A L L O N
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F I G U R E 4. Southern analysis o f genomic D N A from C7 I0, Mtx-T and Mtx-T0 cells. Genomic D N A was isolated from transfected cells at passage 16. Panel A shows an autoradiogram of a Southern blot that had been hybridized with a 32 P-labeled pDHFR:ca t -1 probe. Samples of D N A (1/~g) digested with PvuI (lanes 2-5), AatI (lanes 6-9), or Sinai (lanes 10-13) were electrophoresed in a 1% agarose gel and transferred to a nitrocellulose filter. EcoRI-digested C7 10 D N A (1 pg) was used as carrier for p D H F R : c a t - I D N A (1 ng; lanes 2, 6, and 10) and for the BstElI or HindlII-digested 2 D N A markers (2 ng; lanes 1 and 14). Sizes of 2 D N A markers are shown in kb. Lanes 3, 7, and 11 contain Mtx-T DNA; lanes 4, 8, and 12 contain Mtx-T0 DNA; and lanes 5, 9, and 13 contain C7-10 DNA. Panel B shows an autoradio- gram of a Southern blot which was cut in half and hybridized separately with either a dhfr (lanes 1-4) or cat-specific (lanes 5-8) 32p-labeled probe. EcoRI-digested C7-10 D N A (1/~g) was used as carrier for p D H F R : c a t - I D N A (1 ng; lanes 2 and 6) and for the HindlII or BstEII-digested 2 D N A markers (2 ng; lanes 1 and 5). Cellular D N A samples were digested with SmaI; lanes 3 and 7 contain
Mtx-T DNA; lanes 4 and 8 contain Mtx-T0 DNA.
within the first 4-5 passages, before subpopulations of cells were removed from selective culture medium.
Based on the dot-blot analysis, the cotransfected lines Mtx-CT and Mtx-CT0 contained 250-500 copies of total plasmid DNA per haploid genome (Fig. 3, rows 3 and 4; the individual copy numbers of pFSDHFR9 relative to those of hsp-cat 1 were not determined). When genomic DNA from the Mtx-CT and Mtx-CT0 cells was digested with ApaI, which linearized hsp-cat 1 [Fig. 5(A), lanes 2-5], and hybridized to a cat-specific probe, a proportion of the DNA co-migrated with the linearized
5.2 kb hsp-cat 1 DNA (lane 3), but much of the trans- fected DNA was present in larger fragments (lanes 4 and 5). When the same DNA samples were digested with SmaI, for which there are no recognition sites in hsp-cat 1 [Fig. 5(A), lanes 6-9], and hybridized to the same cat-specific probe, most of the DNA was present in high molecular weight fragments that migrated more slowly than undigested, concatemerized (compare lane 7 with lane 2) plasmid DNA. When Mtx-CT and Mtx-CT0 DNAs were digested with Eco RI, which linearized pFS- DHFR9 [Fig. 5(B), lanes 2-5], and hybridized with a dhfr-specific probe, the hybridization signal was dis- tributed over a broad size range, with a minor pro- portion of bands that coelectrophoresed with the linearized 5.0 kb pFSDHFR9 DNA (lane 3). After treat- ment with AatI, which does not have recognition sites in pFSDHFR9 [Fig. 5(B), lanes 6-9], the dhfr-specific probe hybridized primarily to high molecular weight DNA (lanes 8 and 9), rather than to DNA that co-mi- grated with undigested pFSDHFR9 DNA (lane 7). The presence of both DNA constructs in high molecular weight DNA suggested that the transfected DNA mol- ecules were integrated into the chromosomal DNA. Moreover, the complex banding patterns obtained from the Southern blots of DNA that had been digested with enzymes that linearize hsp-cat 1 and pFSDHFR9 suggested that both transfected DNA molecules had become integrated at multiple sites, and even though some of the DNA appeared to have been integrated as head-to-tail tandem repeats, much of the DNA may be randomly integrated, with possible rearrangements. In both panels A and B, neither probe hybridized to C7-10 DNA (lanes 2 and 6), confirming that the DNA detected by the cat and dhfr-specific probes was of exogenous origin [see also Fig. 4(A), lanes 5, 9 and 13].
To assess the stability of transfected DNA after an extended period in the absence of selection, DNA was isolated from Mtx-T and Mtx-T0 cells after passages 64 and 58, respectively, digested and assayed by dot- blotting and Southern analysis. As shown in Fig. 3 (row 5), after 64 passages in selective medium the Mtx-T cells contained at least as many, and possibly somewhat more copies of pDHFR:cat - I DNA than the Mtx-T cells at passage 16 (row 1). In contrast, the Mtx-T0 cells appeared to have lost most of the transfected DNA (Fig. 3, compare row 6 with row 2) during 58 passages under non-selective conditions. Based on an over-ex- posed film, we estimated that after 58 passages in nonselective medium, the Mtx-T0 cells retained approxi- mately five dhfr gene copies per haploid genome, com- pared to one in the C7-10 cells (data not shown). When genomic DNA from Mtx-T (passage 64) and Mtx-T0 (passage 58) cells was digested with PvuI, which lin- earizes pDHFR:cat-1, and hybridized to a dhfr-specific probe, a significant signal was obtained with DNA from the Mtx-T cells [Fig. 6(A), lane 1], whereas hybridization to DNA from the Mtx-T0 cells was minimal [Fig. 6(A), lane 2], consistent with the dot-blot data (Fig. 3, com- pare rows 5 and 6). In Mtx-T cells, DNA that co-
M O S Q U I T O D I H Y D R O F O L A T E R E D U C T A S E GENE 889
F I G U R E 5. Southern analysis of genomic D N A from C7 10, Mtx-CT and Mtx-CT0 cells. Genomic D N A was isolated from Mtx-CT and Mtx-CT0 cells at passage 16. Panel A shows an autoradiogram of a Southern blot after hybridization with a 32p-labeled cat-specific probe. Samples (1 #g) were digested with ApaI (lanes 2 5) or Sinai (lanes 6-9), electrophoresed in a 1% agarose gel, and transferred to a nitrocellulose filter. EcoRI-digested C7 10 D N A (1/~g) was used as carrier for hsp-cat 1 D N A (1 ng) and for the BstElI-digested lambda D N A markers (2 ng). Lane 1 shows the sizes of BstEII-digested 2 D N A in kb. Lanes 2 and 6 contain C7-10 DNA; lanes 3 and 7 contain hsp-cat 1 DNA; lanes 4 and 8 contain Mtx-CT DNA; and lanes 5 and 9 contain Mtx-CT0 DNA. Panel B shows an autoradiogram of a Southern blot after hybridization with a dhfr-specific 32P-labeled probe. Samples (1/~g) were digested with EcoRI (lanes 2 5) or AatI (lanes 6-9), electrophoresed in a 1% agarose gel, and transferred to a nitrocellulose filter. Lane 1 shows BstEII-digested 2 DNA, the sizes of which are indicated in panel A. Lanes 2 and 6 contain C7 10 DNA; lanes 3 and 7 contain p F S D H F R 9 DNA; lanes 4 and 8 contain
Mtx-CT DNA; and lanes 5 and 9 contain Mtx-CT0 DNA.
migrated with the 7.2kb pDHFR:cat-1 was still present [compare the starred band in Fig. 6(A), lane 1 to Fig. 6(B), lanes 2 and 3], but most of the transfected DNA was distributed over a broad size range, consistent with random integration as intact molecules (>7.2 kb) or as rearranged molecules (<7.2 kb). When the same PvuI-digested DNA samples were hybridized to a cat- specific probe [Fig. 6(B), lanes 2~] , again signal was detected in DNA from the Mtx-T cells, but not from the Mtx-T0, passage 58 cells. Note that for these Mtx-T, passage 64 cells, the combined hybridization signal from dhfr and cat-specific probes to PvuI digested genomic DNA was qualitatively similar to that at passage 16 [Fig. 4(A), lane 3] with the pDHFR:cat - I probe.
When DNA from passage 48 Mtx-CT and Mtx-CT0 cells was digested with EcoRI [Fig. 6(A), lanes 4-5], which linearized pFSDHFR9, and hybridized to a dhfr- specific probe, the hybridization signal in both cell lines was of comparable intensity, including bands that may correspond to the linearized 5.0 kb pFSDHFR9 DNA (lane 3). Similarly, when DNA from passage 48 Mtx-CT and Mtx-CT0 cells was digested with ApaI [Fig. 6(B), lanes 4-7], which linearized hsp-cat 1 (lane 5), and hybridized to a cat-specific probe, the hybridization
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signal for both lanes was of comparable intensity, in- cluding the bands that may represent linearized hsp-cat 1 DNA. These data with the cat-specific probe were consistent with the data obtained using the dhfr-specific probe, suggesting that both co-transfected DNA mol- ecules were integrated into the mosquito chromosomes and were stable in the absence of mtx-selection. As noted above for Mtx-T cells, ApaI digested Mtx-CT and Mtx-CT0 DNA at passages 16 and 48 also produced consistent banding patterns with the cat-specific probe [compare Fig. 5(A), lanes 4 and 5 to Fig. 6(B), lanes 6 and 7]. These data reinforce the conclusion that minimal rearrangement of the transfected DNA occurred once integration had taken place, whether in the presence or absence of mtx selection.
These data suggest that in the absence of continuous selective pressure, there may be clonal variability in retention of integrated DNA. Specifically, for the Mtx- TO cells, after many generations in the absence of selection, the transfected pDHFR:cat - I DNA was largely lost. Surprisingly, after 60 and 81 passages in the absence of mtx-selection, heat-inducible CAT activity in the Mtx-T0 cells was roughly comparable to that of the Mtx-T cells maintained in the presence of 1.0 #M mtx
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FIGURE 6. Southern analysis of genomic DNA from Mtx-T, Mtx-T0, Mtx-CT and Mtx-CT0 cells after extended maintenance in selective or nonselective medium. Genomic DNA was isolated from Mtx-T cells, passage 64; Mtx-T0 cells, passage 58, Mtx-CT and Mtx-CT0 cells, passage 48. Panel A shows an autoradiogram of one half of a Southern blot after hybridization with a 32p-labeled dhfr-specific probe. Samples (1 #g) were digested with PvuI (lanes I and 2) or EcoRI (lanes 3-5), electrophoresed in a 1% agarose gel, and transferred to a zetaprobe (Bio-Rad Laboratories, Richmond, Calif.) filter. EcoRl-digested C7 10 DNA (1 #g) was used as carrier for the pFSDHFR9 DNA (1 ng) and for the BstEII-digested 2 DNA markers (2 ng). Lane 6 in panel A and lane 1 in panel B show the sizes of Bst Ell-digested 2 DNA in kb. In panel A, lane 1 contains Mtx-T DNA; lane 2, Mtx-T0 DNA; lane 3 pFSDHFR9 DNA; lane 4, Mtx-CT DNA; and lane 5, Mtx-CT0 DNA. Panel B shows an autoradiogram of the other half of the Southern blot after hybridization with the 32P-labeled cat-specific probe. DNA samples (1/~g) were digested with PvuI (lanes 2-4) or ApaI (lanes 5 7). Lane 2 contains pDHFR:cat-I DNA; lane 3, Mtx-T DNA; lane 4, Mtx-T0 DNA; lane 5, hsp-cat 1 DNA; lane 6, Mtx-CT DNA; and lane 7, Mtx-CT0 DNA.
for 66 and 86 passages, respectively, despi te the appa ren t loss o f gene copies [Fig. 2(B), lanes 2 and 3, o ther da t a not shown]. This observa t ion raises quest ions a b o u t the cor re la t ion between gene copy number , site(s) o f inte- g ra t ion and the level o f gene expression.
Karyotype analysis
Al though the mtx- res i s tan t Mtx-5011-256 cells exhib- i ted complex changes in ka ryo type (Shotkosk i and Fal lon , 1990), the dhfr-transformed cell lines ma in ta ined a no rma l ka ryo type , with a m o d a l c h r o m o s o m e number o f 6 and < 10% te t rap lo idy and an absence o f ma jo r c h r o m o s o m a l aber ra t ions . When c h r o m o s o m e spreads for the M t x - T cells were analyzed by s imul taneous in situ hybr id iza t ion (Fig. 7, panels 1 and 2) with the dhfr and cat-specific probes , a consis tent signal hybr id ized near the end o f a single ch romosome . A second, somewha t weaker signal appea red in cen t romer ic D N A . Similarly, in spreads f rom M t x - C T cells the p robes consis tent ly hybr id ized near the end o f a single c h r o m o s o m e (Fig. 7, panels 3 and 4). These da t a confi rm tha t at least a p r o p o r t i o n o f the t ransfected D N A resides within ch romosomes , consis tent with results f rom Southern
analyses. We note that single copy genes, which may be d is t r ibu ted t h roughou t the ch romosomes , would not have been detected in this analysis.
DISCUSSION
Gene t ransfer vectors con ta in ing dhfr c D N A s as d o m i n a n t selectable marke r s were p ioneered in mam- mal ian cell cul ture systems (Kucher l apa t i and Shoultchi , 1986). In ear ly studies, it was shown that dhfr expression f rom c D N A cons t ruc ts (Lee et al., 1981; Gasse r et al., 1982; K a u f m a n and Sharp, 1982; Crouse et al., 1983) could be used rel iably to recover t r ans fo rmed clones f rom m u t a n t dhfr - recipient cells. In cont ras t , these expression vectors failed to p rov ide a consis tent ly effec- tive d o m i n a n t selectable m a r k e r with dhfr + recipient cells (Thillet and Pictet, 1990; Hussa in et al., 1992) unless the cons t ruc t con ta ined a s t rong viral p r o m o t e r ( M u r r a y et al., 1983). Al terna t ive ly , vectors con ta in ing a l tered dhfr genes, in which rep lacement o f a conserved Leu at pos i t ion 22 by Arg or Phe resulted in D H F R enzyme with reduced affinity for mtx, were shown to funct ion as d o m i n a n t selectable marke r s in bo th dhfr- cells and in
MOSQUITO DIHYDROFOLATE REDUCTASE GENE
&
891
FIGURE 7. In situ hybridization of cat and dhfr-specific probes to metaphase chromosomes. The specific activity of the probes, labeled by random priming with [3H]dTTP (~ 119 Ci/mmol; Amersham, Arlington Heights, Ill.), was 0.5-1.0 × 108 cpm/pg, and 1 .ffqS.0 x 107 cpm/ml of mixed probe were used in a typical in situ hybridization as detailed in the Materials and Methods. Panels 1 and 2 show the hybridization of probes to metaphase chromosomes from Mtx-T cells. Panels 3 and 4 show the hybridization of probes to chromosomes from Mtx-CT cells. The arrows indicate the location of strong hybridization signals
that were seen consistently in multiple spreads.
wild-type, dhfr + cells (Simonsen and Levinson, 1983; Hussain et al., 1992). Similarly, mouse dhfr mutants in which the Phe at codon 31 was replaced by Trp were also functional in wild type cells after transfection (McIvor and Simonsen, 1990).
In contrast to these results with mammalian cells, we have successfully used a mosquito dhfr gene as a domi- nant selectable marker to transform wild-type dhfr + recipient cells. The vectors we describe contain a ge- nomic copy of the entire mosquito dhfr gene, including a 56bp intron, and native transcriptional regulatory sequences. It is possible that the mosquito vectors con- tain important regulatory elements, absent from chimeric cDNA constructs used in many mammalian systems, that provide for more efficient expression of the transfected dhfr gene. Support for this possibility derives from the early studies of Gasser et al. (1982) as well as from more recent studies in which the first of five introns in the mouse dhfr gene, which corresponds to the only intron conserved in the mosquito dhfr gene (Shotkoski and Fallon, 1991), was shown to contain sequences with potential transcriptional regulatory and enhancer ac- tivity (Farnham and Means, 1990; Schmidt et al., 1990).
An alternative explanation for the efficient expression of mosquito dhfr constructs in wild type recipient cells relates to recent studies that implicate both mtx affinity and catalytic activity as factors that affect D H F R func- tion as a dominant selectable marker (Hussain et al.,
1992). The mosquito dhfr gene was cloned from mtx- resistant cells that contained c. 300 copies of the gene per haploid genome. Despite the fact that the 12 amino acids common to all D H F R proteins are conserved in the mosquito sequence, including the leucine at position 22, which in vertebrate DHFRs is known to affect affinity for mtx, overall there is only 43-48% amino acid similarity between mosquito and vertebrate D HFR s (Shotkoski and Fallon, 1991). Thus, it remains possible that the cloned mosquito gene encodes a D H F R protein with reduced affinity for mtx, relative to vertebrate dhfr genes. Support for this possibility derives from a recent kinetic analysis of D H F R from Drosophila melanogaster (Rancourt and Walker, 1990), in which the Kd for mtx was reported to be higher than that reported for other, non-insect DHFRs. A careful study comparing the kinetic properties of D H F R enzyme from wild-type and Mtx-5011-256 cells will be required to address this possibility. Regardless of the molecular basis for their expression in wild type mosquito cells, the dhfr vectors described in this study promise to be particularly useful, since a number of mosquito cell mutants are already available for genetic studies (Fallon and Stollar, 1987), and it will not be necessary to convert these cells to dhfr derivatives.
The present report is the second description of stably transformed A. albopictus cells, and it remains to be learned how the variability among transformed clones
892 FRANK A. SHOTKOSKI and ANN MARIE FALLON
relates to the properties of a particular cell line, the nature of the dominant selectable marker gene and the role of its product in cellular metabolism, and method of introducing foreign D N A into the cells. Particularly striking in the present study was the apparent 20-100- fold reduction in pDHFR:ca t -1 copy number in Mtx-T0 cells during maintenance under non-selective conditions. Even though the majority of the pDHFR:ca t -1 copies had been lost, the cells still expressed CAT activity at levels comparable to those in Mtx-T cells. This obser- vation indicates that the correlation between gene copy number and expression may be indirect, supports the likelihood that site(s) of D N A integration influence the level of gene expression, and suggests that the importance of maintaining transfected lines in the con- tinuous presence of methotrexate may vary among clonal isolates.
Additional studies will also be needed to determine the extent to which the properties of the Mtx-T and Mtx-CT cells described here are representative of the 29 other clones obtained during initial selection, the relation between transfected D N A copy number and dhfr gene expression levels, and the extent to which vectors con- taining the mosquito dhfr gene are amplifiable with stepwise increases in mtx selection. In particular, with prolonged maintenance in the presence of mtx, the dhfr gene copy-number in Mtx-T cells may have increased c. 2-fold. In mammalian systems, dhfr vectors have been of tremendous value as vehicles for amplification of co-linear or co-transfected genes, including the glucocor- ticoid receptor and a secreted form of human thyroid peroxidase (Bellingham et al., 1992; Kaufman et al., 1991). These results from mammalian systems provide a basic framework for extending the present studies with mosquito cells.
Mtx-resistant clones were successfully obtained from wild type cells that had been transfected with the com- posite plasmid, pDHFR:ca t -1 , or co-transfected with p F S D H F R 9 and hsp-cat 1 as separate molecules. Since CAT activity can be easily assayed, we used a cat reporter gene regulated by the Drosophila hsp70 pro- moter to confirm stable transformation of transfected cells. For both transformed cell lines, expression of CAT activity was dependent on heat induction of the hsp70 promoter, which had limited constitutive activity at 28°C, regardless of the copy number or pattern of integration. In contrast, with the p U C h s h y g vector used for stable transformation of C6/36 mosquito cells, hy- gromycin phosphotransferase activity was constitutively expressed from the Drosophila heat shock promoter (Monroe et al., 1992). None of the 31 clones initially examined showed evidence of spontaneous amplification of the endogenous D H F R genes, nor were chromosome anomalies characteristic of mtx-resistant cells that arose from amplification of the endogenous gene (Shotkoski and Fallon, 1990) observed.
In Mtx-T and Mtx-CT cells maintained under con- tinuous mtx selection, the transfected D N A was mostly confined to high molecular weight species. The complex
banding patterns on Southern blots suggested that some of the D N A was integrated as head-to-tail tandem repeats, and that much of the D N A was integrated at multiple sites. In contrast to what we have observed here, in an earlier study using Drosophila Kc cells transfected with a construct containing the E. coli dhfr gene under the regulation of the copia promoter, Bourouis and Jarry (1983) observed large oligomeric structures consisting of 1000-3000 vector molecules per cell, organized within chromosomes as head-to-tail repeats with minimal re- arrangement.
In situ hybridization studies provided additional evi- dence that in the present study, transfected D N A was at least in part integrated into chromosomal DNA. In both cell lines examined, a measurable hybridization signal was detected near the telomeric end of a single chromo- some. In future studies, we hope to learn whether the signal detected at" this location represents tandemly reiterated genes that integrated into a native dhfr locus. Since the in situ hybridization conditions used in the present study would be expected only to detect high copy number genes, we speculate that additional transfected D N A may also occur as single gene copies distributed throughout the genome and/or as extrachromosomal elements. In future studies, we plan to focus on early events in the transformation process, and to evaluate in more detail using a variety of different transformation vectors the rate at which transfected D N A becomes integrated into chromosomal material. We anticipate that these and subsequent studies with transformed cells in culture will eventually be extended to vector insects at the organism level.
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Acknowledgements--This work was supported by the USDA grant 90-37263-5521 and by the University of Minnesota Agricultural Exper- iment Station. This is contribution 20,141 from the University of Minnesota Experiment Station, St Paul, Minn. F. A. Shotkoski was the recipient of a University of Minnesota Doctoral Dissertation fellowship. We thank Jacqueline Larson for typing the manuscript.
