Investigating if transformation of the Sciara coprophila II/9A locus into Drosophila melanogaster induces ectopic DNA amplification

[A] Background and Significance

Every diploid nucleus within a eukaryotic organism generally has the same amount of DNA because the genome is replicated once and only once per cell cycle. By what mechanism is this “rule of DNA constancy” (Boivin et al., 1948; Mirsky and Ris, 1951; Swift, 1950) enforced? One way to answer this question is to study exceptions to the rule where the usual regulatory pathways are bypassed and DNA amplification occurs without intervening mitoses. Two naturally occurring exceptions in lower dipteran insects have been described, the first occurring in the larval salivary glands of the fungus fly Sciara coprophila and the second occurring in the ovarian follicle cells of the fruit fly Drosophila melanogaster (reviewed in Claycomb and Orr-Weaver, 2005). In both cases, DNA amplification occurs, producing multiple copies of select loci superimposed over an endoreduplicated genome. We study the former exception in the Gerbi lab.

Toward the end of the fourth larval instar of Sciara, endoreduplication of the entire genome occurs (to 4096C in males and 8192C in females) (Rasch 1970). The replicated chromatids of both homologous chromosomes remain tightly synapsed together, forming giant polytene chromosomes that can be visualized easily by chromosome squashes and microscopy. During the last endocycle, select loci amplify up to 17-fold higher than the rest of the genome (Rasch 1970; Foulk et al. 2006). Subsequent transcription of the amplified DNA results in localized distension along the polytene chromosomes, resulting in gigantic DNA puffs (Gabrusewycz-Garcia, 1964; Wu et al., 1993). DNA amplification in Sciara is presumably a strategy to produce sufficient template for the production of large quantities of cocoon protein in a short time span that is needed for formation of the puparium during the next developmental stage. Sciarid flies avoid the burden of carrying “extra” copies of cellular DNA because their salivary glands undergo histolysis during pupation (reviewed in Claycomb and Orr-Weaver, 2005). In researching this phenomenon, we have focused our attention on a DNA puff that is the largest and the earliest to form in late fourth larval instar larvae: position 9A on chromosome II (II/9A).

What regulates DNA puffing in Sciara? Ecdysone, the molting (steroid) hormone and the master regulator of insect development, induces premature formation of DNA puffs on polytene chromosomes when injected into pre-amplification stage larvae (Crouse, 1968). More recently, ecdysone was shown by real-time PCR to induce premature amplification in DNA puff II/9A when injected into pre-amplification stage larvae (Foulk et al., 2006), providing molecular evidence of its involvement in DNA amplification. Additionally, a putative ecdysone response element (EcRE) was discovered to be adjacent to the origin recognition complex (ORC)-binding site within the amplification origin of II/9A (Bielinsky et al., 2001; Foulk et al., 2006; Liang et al., 1993; Liang and Gerbi, 1994). This EcRE is capable of being bound by the ‘A’ isoform of the ecdysone receptor that predominates in Sciara salivary glands (EcR-A) (Foulk et al. 2006; Foulk et al. in prep). The ecdysone receptor was originally identified in Drosophila as a DNA transcription factor, directing the expression of ecdysone-responsive genes (Koelle et al., 1991). Binding of EcR-A to the EcRE within the amplification origin of II/9A during amplification would thus suggest a new role for the ecdysone receptor in regulating DNA replication. By conducting immunofluorescence on chromosome squashes of staged late fourth instar larvae with a Sciara-specific antibody, I have shown that EcR-A—which appears at II/9A only during amplification—binds across the genome to other amplified loci at the same time that the process is just initiating at II/9A (Liew et al., in prep).

Taken together, these results suggest a possible mechanism for activation of DNA amplification. Ecdysone could first upregulate EcR-A levels, following which EcR-A would bind to the ORI EcRE where it interacts with components of the replication machinery to initiate DNA amplification (Foulk et al., 2006). A logical experimental approach to test this hypothesis would be to remove or mutate the putative EcRE sequence in II/9A and ask if amplification can still occur at that locus (Foulk et al., 2006). However, this loss-of-function approach must await the development of a transformation system for Sciara, akin to the ones that currently exist for Drosophila.

Given the above constraint, I suggest using a gain-of-function approach instead. I propose transforming the II/9A locus from Sciara into Drosophila and asking if ectopic amplification and puffing occur in both the follicle cells and salivary glands of Drosophila. Previous work in the Gerbi lab has demonstrated that a promoter from the II/9A locus (II/9-1 promoter fragment) can direct in vivo expression of a CAT P-element construct in the salivary glands of transgenic Drosophila during the pre-pupal stage (Bienz-Tadmor et al., 1991), suggesting that some elements of the ecdysone response are conserved between Sciara and Drosophila. Thus, detection of amplification and/or puffing with Drosophila transformed with the entire II/9A locus would provide strong support for our hypothesis. Given the differences in amplification biology between Sciara and Drosophila, however, co-transformation and ectopic expression of the EcR-A gene in Drosophila might be necessary as well.

Numerous controls (not all of which are well-understood) exist in eukaryotic cells to ensure that the genome is replicated only once per cell cycle. These controls are overridden in some forms of cancer, resulting in multiple copies of cancer-causing genes in the genome (reviewed in Wintersberger, 1994). Unfortunately it is not possible to study the mechanistic basis for this in humans because the results of DNA amplification in cancer cells are observed only after it has occurred, precluding a study of the process itself. Besides furthering our knowledge of how the “rule of DNA constancy” is implemented, an understanding of the developmental relationship between EcR-A and DNA amplification in Sciara might provide us with a model for the link between steroid hormone receptors and cancers, most notably the estrogen receptor and breast cancer.

[B] Hypotheses and Specific Aims

The main hypothesis guiding this set of experiments is that there is a minimum genetic sequence in the II/9A locus that the ecdysone receptor can bind to and activate amplification. A schematic of the entire II/9A locus with the DNA-hypersensitive site, 1-kb amplification origin (ORI) and two genes (with 85% homology to each other) is shown below in Fig. 1. The presence of a bona fide EcRE next to the ORC-binding site within the 1-kb ORI suggests that upon binding, EcR-A could interact with components of the replication machinery within the 1-kb ORI to subsequently activate amplification (Foulk et al., 2006). If induction of ectopic amplification and puffing is successful in Drosophila, then the next step would be to sequentially delete portions of the transformed II/9A locus in order to find the minimum genetic sequence that is required for amplification.

• Aim 1: Transform II/9A locus from Sciara into Drosophila; assay for amplification and puffing in follicle cells and salivary glands
• Aim 2: Transform EcR-A gene (with HSP70 promoter for control) from Sciara into Drosophila if necessary
• Aim 3: Systematically delete regions from the Sciara DNA puff II/9A transgene inserted into Drosophila to find minimum sequence required for amplification/puffing

Fig. 1: Schematic of Sciara DNA puff II/9A (adapted from Foulk et al., 2006) which is about 8.9 kb in length (Wu et al., 1993)

[C] Experimental Plan

Aim 1: Transform II/9A locus from Sciara into Drosophila; assay for amplification and puffing in follicle cells and salivary glands

Various Drosophila transformation methods were considered—including Cre/loxP (Siegal and Hartland, 1996) and FLP/FRT (Rong and Golic, 2000)—but the one that is most suitable for my application is the method of site-specific transformation with ΦC31 integrase-mediated cassette exchange (Bateman et al., 2006). The site-specific nature of this method (termed recombinase-mediated cassette exchange—RMCE) avoids positional effects that might otherwise complicate analyses of Drosophila transformants. In addition, the use of cassette exchange allows swapping of different transgenic constructs, facilitating deletion of regions of Sciara DNA puff II/9A in order to find the minimum sequence required for amplification and puffing. Finally, transformation rates using the ΦC31 system have been reported to be much higher than the other methods (Groth et al., 2004).

Transformation of Drosophila will be performed as described by Bateman et al. (2006). First, a plasmid containing the 8.9kb Sciara DNA puff II/9A sequence (Fig. 1 and Wu et al., 1993) and attB will be constructed as described. This will then be co-injected into Drosophila embryos together with mRNA encoding the ΦC31 integrase. Bateman et al. (2006) created four y- w- Drosophila parental lines carrying a mini-white target RMCE cassette (mini-white gene flanked by inverted attP sites) introduced by standard P-element transformation. The four lines carry this target cassette at different genomic sites—three are found on chromosome II at polytene positions 25C1, 42A13, and 52D9, while one is found on chromosome III at polytene position 82F7 (Bateman et al., 2006). I will inject only embryos from the parental lines carrying the target cassette at positions 52D9 on chromosome II and 82F7 on chromosome III, because the other polytene positions where the target cassette is located—25C1 and 42A13 on chromosome II—correspond to Drosophila puff loci (see Ashburner, 1972 for a list of Drosophila puff loci), which would complicate analyses of ectopic puffing at those sites. As the integration event is not marked by a visible marker, RMCE events can first be identified among the progeny by loss of the mini-white eye color. To confirm that the entire II/9A locus integrated into the genome by RMCE, PCR and Southern analyses can then be performed. Each PCR primer will be designed to cover both the attB-specific region of attR and the P-element end in such a way that PCR products will only result if the RMCE event occurred as predicted (Bateman et al., 2006). Following restriction digests customized according to the structure of the II/9A locus (Fig. 1), Southern analysis can be performed with appropriate probes to ensure that the integrated locus is of the right size. I do not anticipate that introduction of the II/9A locus will cause any undesired outcomes, and will confirm this by screening the progeny that successfully incorporated the II/9A transgene for morphological defects. A positive control that can be performed initially to optimize the transformation protocol would be to transform the y- w- Drosophila parental lines with a plasmid carrying the yellow gene and attB. Integration of this construct would be checked by assaying for white eye color and brown body, and then verified by PCR and Southern analyses.
After successful transformation of the II/9A locus, I will assess ectopic amplification of the II/9A transgene in both the salivary glands and follicle cells of Drosophila. Ectopic amplification in the salivary glands of Drosophila can be conveniently assayed at the cytological level by conducting chromosome squashes prepared from different stages of third instar larvae and then seeing if ectopic puffing occurs at the two target sites (52D9 on chromosome II and 82F7 on chromosome III), using the polytene chromosome maps of Bridges (1935) for reference. The presence of the II/9A transgene at these target sites can be confirmed by conducting FISH with an II/9A-specific probe on the same chromosome squashes and observing if the FISH signal localizes to those positions. To provide evidence of amplification at the molecular level, I will perform real-time PCR with primers specific to the II/9A locus and an unrelated locus (unamplified control) on extracts derived from either the follicle cells or salivary glands of Drosophila. The extent of amplification for the II/9A transgene at different loci can be obtained by computing the ratio of signal obtained from the II/9A transgene with that from the unamplified locus.

Based on the literature, transformation of Drosophila via the RMCE method should be generally successful. However, the transformation efficiency could vary based on the size of the construct. Groth et al. (2004) reported a transformation efficiency of 55% using single attP and attB sites and the white gene (5 kb) as a donor, while Bateman et al. (2006) was only able to achieve a RMCE efficiency of 9% using the yellow gene (5 kb) as a donor, and an efficiency of 24% using a GFP cassette (2kb). Because the II/9A locus is 8.9 kb in length, transformation rates might be too inefficient to render this method viable.

Bateman et al. (2006) speculated that the integration rate could be having a higher concentration of ΦC31 integrase in Drosophila. This can be achieved by co-injecting a higher concentration of ΦC31 integrase mRNA. An alternative approach would be to use Drosophila with ΦC31 integrase already transformed into the germline. Bischof et al. (2007) have generated twenty-five attP lines of Drosophila which are homozygous viable and have the attP landing site precisely mapped to intergenic regions. Several of these lines have also been transformed with constitutively expressing ΦC31 integrase, greatly facilitating experimentation by obviating the need for co-injections of ΦC31 integrase mRNA. This germ-line specific ΦC31 integrase does not appear to affect chromosomal stability in Drosophila lines with integrated attP sites (Bischof et al., 2007). In fact, the presence of endogenous ΦC31 integrase enhances integration of transgenic attB constructs (Bischof et al., 2007).

Aim 2: Transform EcR-A gene (with HSP70 promoter for control) from Sciara into Drosophila if necessary

The amplification biology in Sciara and Drosophila differs in two important aspects: (i) Amplification in Sciara occurs in the salivary glands, while amplification in Drosophila occurs in the ovarian follicle cells (reviewed in Claycomb and Orr-Weaver, 2005); and (ii) the ecdysone receptor isoform that predominates in Drosophila follicle cells and salivary glands is the B1-isoform (EcR-B1) (Talbot et al., 1993; Romani et al., 2009), while the isoform that predominates in Sciara salivary glands is the A isoform (EcR-A) (Foulk et al., in prep). Although a low level of EcR-A (required for subsequent histolysis of the salivary glands during pupation) is present in Drosophila salivary glands (Davis et al., 2005), it is not known if Drosophila-specific EcR-A can activate transcription at the Sciara II/9A locus.

In light of the above, it is possible that ectopic amplification and puffing might occur at a very low level or not at all in transformed Drosophila. To overcome this problem, I propose conducting a P-element mediated transformation of the Sciara-specific EcR-A gene (sequence unpublished, Gerbi lab) under the control of a HSP70 promoter into Drosophila lines already carrying the II/9A locus. Positional effects will be minimized by flanking this transposon with suppressor of hairy-wing binding site (SHWBS) insulators, as described by Xie and Orr-Weaver (2008). Again, integration of the EcR-A gene can be assessed by PCR and Southern blotting. Production of EcR-A protein can be assessed by heat shock and Western blotting with our Sciara-specific EcR-A antibody. I will activate ectopic expression of Sciara EcR-A in both follicle cells and salivary glands at the appropriate times during development and then assay for amplification and puffing as described in Aim 1.

Aim 3: Systematically delete regions from Sciara DNA puff II/9A to find minimum sequence required for amplification/puffing

Provided that ectopic amplification and puffing are observed in Drosophila with the integrated II/9A construct, I can now ask if there is a minimum sequence in the II/9A locus required for amplification and/or puffing. Using the method described by Bateman et al. (2006), I can construct plasmids with various combinations of deletions in II/9A and the attB site. Subsequent RMCE transformations, controls, and enhancements can be performed as described in Aim 1.

[D] Conclusions

Multiple controls exist in eukaryotic cells to ensure that the genome is replicated only once per cell cycle. These controls are overridden in some forms of cancer, resulting in multiple copies of cancer-causing genes in the genome (Wintersberger, 1994). Unfortunately it is not possible to study the mechanistic basis for this amplification in humans. Sciara is one of two model systems that exist to study the phenomenon of DNA amplification. Because no transformation system exists for Sciara yet, it is not possible to directly test our hypothesis that EcR-A activates DNA amplification by binding to the EcRE besides the ORC-binding site in the 1-kb ORI of the II/9A locus. Hence, transformation of the Sciara II/9A locus into Drosophila will allow us to ask the same question in a more genetically tractable organism. Besides furthering our knowledge of how the “rule of DNA constancy” is implemented, my proposed experiments will enhance our understanding of the developmental relationship between EcR-A and DNA amplification in Sciara. Additionally, they might provide us with a model for the link between steroid hormone receptors and cancers, most notably the estrogen receptor and breast cancer. Finally, amplification of the II/9A transgene in Drosophila will suggest a novel link between certain types of transcription factors and DNA replication in general.

[E] References

Ashburner, M, 1972. Puffing patterns in Drosophila melanogaster and related species. Results and problems in cell differentiation 4, 101-151.

Bateman, J.R., Lee, A.M., Wu, C, 2006. Site-specific transformation of Drosophila via ΦC31 integrase-mediated cassette exchange. Genetics 173, 769–777.

Bielinsky, A.K., Blitzblau, H., Beall, E.L., Ezrokhi, M., Smith, H.S., Botchan, M.R., Gerbi, S.A., 2001. Origin recognition complex binding to a metazoan replication origin. Curr. Biol. 11, 1427-1431.

Bienz-Tadmor, B., Smith, H.S., Gerbi, S.A., 1991. The promoter of DNA puff gene II/9-1 of Sciara coprophila is inducible by ecdysone in late prepupal salivary glands of Drosophila melanogaster. Cell Regul. 2, 875–888.

Bischof, J., Maeda, R.K., Hediger, M., Karch, F., Basler, K., 2007. An optimized transgenesis system for Drosophila using germ-line specific ΦC31 integrases. Proc. Natl. Acad. Sci. U. S. A. 104, 3312–3317.

Boivin, A., Vendrely, R., Vendrely, C., 1948. L'acide désoxyribonuleicue du noyau cellulaire dépositaire des caractères héréditaires: Arguments d'ordre analytique. Compt. Rend. 226, 1061–1063.

Bridges, C. B., 1935. Salivary chromosome maps with a key to the banding of the chromosomes of Drosophila melanogaster. J. Hered. 26, 60–64.

Claycomb, J.M., Orr-Weaver, T.L., 2005. Developmental gene amplification: insights into DNA replication and gene expression. Trends Genet. 21, 149–162.

Crouse, H.V., 1968. The role of ecdysone in DNA-puff formation and DNA synthesis in the polytene chromosomes of Sciara coprophila. Proc. Natl. Acad. Sci. U. S. A. 61, 971–978.

Davis, M.B., Carney, G.E., Robertson, A.E., Bender, M., 2005. Phenotypic analysis of EcR-A mutants suggests that EcR isoforms have unique functions during Drosophila development. Dev. Biol. 282, 385–396.

Foulk, M.S., Liang, C., Wu, N., Blitzblau, H.G., Smith, H., Alam, D., Batra, M., Gerbi, S.A., 2006. Ecdysone induces transcription and amplification in Sciara coprophila DNA puff II/9A. Dev. Biol. 299, 151–163.

Gabrusewycz-Garcia, N., 1964. Cytological and autoradiographic studies in Sciara coprophila salivary gland chromosomes. Chromosoma 15, 12–344.

Groth, A.C., Fish, M., Nusse, R., Calos, M.P., 2004. Construction of transgenic Drosophila by using the site-specific integrase from phage ΦC31. Genetics 166, 1775–1782.

Koelle, M.R., Talbot, W.S., Segraves, W.A., Bender, M.T., Cherbas, P., Hogness, D.S., 1991. The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67, 59–77.

Liang, C., Gerbi, S.A., 1994. Analysis of an origin of DNA amplification in Sciara coprophila by a novel three-dimensional gel method. Mol. Cell. Biol. 14, 1520-1529.

Liang, C., Spitzer, J.D., Smith, H.S., Gerbi, S.A., 1993. Replication initiates at a confined region during DNA amplification in Sciara DNA puff II/9A. Genes Dev. 7, 1072-1084.

Mirsky, A.E., Ris, H., 1951. The deoxyribonucleic acid content of animal cells and its evolutionary significance. J. Gen. Physiol. 34, 451–462.

Rasch, E.M., 1970. DNA cytophotometry of salivary gland nuclei and other tissue system in dipteran larvae. In: Wied, G.L., Bahr, G.F. (Eds.), Introduction to Quantitative Cytochemistry. Academic Press, New York, pp. 357–397.

Romani, P., Bernardi, F., Hackney, J., Dobens, L., Gargiulo, G., Cavaliere, V., 2009. Cell survival and polarity of Drosophila follicle cells require the activity of ecdysone receptor B1 isoform. Genetics 181, 165-175.

Rong, Y.S., Golic, K.G., 2000. Gene targeting by homologous recombination in Drosophila. Science 288, 2013–2018

Siegal, M.L., Hartl, D.L., 1996. Transgene coplacement and high efficiency site-specific recombination with the Cre/loxP system in Drosophila. Genetics 144, 715–726.

Swift, H.H., 1950. The deoxyribose nucleic acid content of animal nuclei. Physiol. Zool. 23, 169–198.

Talbot, W.S., Swyryd, E.A., Hogness, D.S., 1993. Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell 73, 1323–1337.

Wintersberger, E., 1994. DNA amplification: new insights into its mechanism. Chromosoma 103, 73–81.

Wu, N., Liang, C., DiBartolomeis, S.M., Smith, H.S., Gerbi, S.A., 1993. Developmental progression of DNA puffs in Sciara coprophila: amplification and transcription. Dev. Biol. 160, 73–84.

Xie, F., Orr-Weaver, T., 2008. Isolation of a Drosophila amplification origin developmentally activated by transcription. Proc. Natl. Acad. Sci. U. S. A. 105, 9651–9656.

MATERIALS AND METHODS

Fly stocks
Sciara (Bradysia) coprophila stock 7298 (wild type with wavy wing marker linked to the X' inversion chromosome) and 7298 derivative stocks (“Holo”) were raised in the laboratory at 21°C. Only female fourth instar larvae from these stocks were used for immunofluorescence studies because they undergo an additional endoreduplication cycle (Rasch 1970), resulting in larger polytene chromosomes. Well-fed larvae grown in medium density conditions yielded chromosome squashes of significantly better quality. The expansion of puff morphology coincides with development of the eye anlage from mid to late fourth larval instar, allowing larvae to be staged by counting their eyespot granules under a 16X objective. Each developmental stage is specified by the number of eyespot granules in the longest row multiplied by the number of rows minus one (Gabrusewycz-Garcia 1964, Wu et al. 1993). At DNA puff II/9A, amplification initiates at 10 x 5 eyespot stage and continues through 12 x 6, while maximal puffing resulting from a burst of transcription occurs at the 14 x 7 stage (Gerbi et al. 1993, Wu et al. 1993, Foulk et al. 2006).

Antibodies
Isoform A of the ecdysone receptor (EcR-A) predominates in the salivary glands of fourth larval instar Sciara coprophila (Foulk et al. in prep). A polyclonal antibody to this isoform was produced against the unique EcR-A 5' nucleotide sequence as described elsewhere (Foulk et al. in prep). The antibody preparation was then affinity purified with Affi-Gel® 10 Activated Affinity Media (BioRad, Hercules, Calif.) coupled to the EcR-A specific protein fragment, and the specificity of this antibody was checked by immunoblotting. Alexa Fluor 488 dye-conjugated goat anti-rabbit IgG was obtained from Molecular Probes, Invitrogen (Eugene, Oregon).

Preparation of chromosome squashes and immunofluorescent detection of EcR-A
Polytene chromosome squashes and immunostaining were performed as described by Zink et al. (1991) with modifications. Briefly, the anterior portions of salivary glands were excised from staged larvae in dissecting buffer (PBS pH 7.4, 0.1% Triton X-100) [PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4] and immediately fixed for 25 s in freshly prepared solution I (3.7% formaldehyde, 1% Triton X-100, PBS pH 7.4). Glands were then transferred to a 35 μl drop of solution II (3.7% formaldehyde, 50% acetic acid) on a siliconized coverslip for 70 s. The 37% formaldehyde stock solution used for solutions I and II was made fresh before each experiment by boiling paraformaldehyde (Sigma, St. Louis, Missouri) in 15 mM KOH until dissolved. After picking up the coverslip with a poly-L-lysine coated slide, the entire preparation was flipped over [slide coating: clean slides were soaked for 5 min in 0.1% poly-L-lysine (Sigma, St. Louis, Missouri) and oven-dried at 80°C for 1 h]. Chromosomes were spread by firmly tapping the coverslip around the glands with the eraser end of a pencil. The chromosomes were then flattened by compression of the slide between several sheets of blotting paper with thumb pressure. After freezing the preparation in liquid nitrogen, the coverslip was pried off with a razor blade and the exposed chromosome squash was washed in PBS (2 x 15 min, agitating). Slides were then blocked (PBS pH 7.4, 3% nonfat dried milk, 0.3% Triton X100) for 1 h at room temperature or overnight at 4°C, and washed (2 x 10 min, agitating) with PBT (PBS pH 7.4, 0.1% BSA, 0.2% Tween 20).

Chromosome preparations were reacted sequentially with anti-EcR-A (1:2500 in PBT) and then Alexa Fluor 488 dye-conjugated goat anti-rabbit IgG (1:1000 in PBT) for 1 h each. Each immunostaining step was followed by a PBT wash (2 x 10 min, agitating). Slides were then counterstained with DAPI (200 ng/ml in H2O) for 10 min. All incubation steps were carried out at room temperature in a darkened humid chamber. After a brief rinse in PBT, slides were mounted with ProLong® Gold (Invitrogen, Carlsbad, Calif.) and stored in the dark at 4°C. Reacted preparations were examined under a Carl Zeiss Axiovert 200 M microscope and photographed with the 40X objective (dry). The accompanying AxioVision software was used to optimize exposure times automatically according to the immunostaining intensity of each chromosome preparation. Captured images were then exported into Adobe Photoshop® for resizing and cropping.

GRE Analyze an Argument Practice #2

The following appeared in a letter from a department chairperson to the president of Pierce University.

"Some studies conducted by Bronston College, which is also located in a small town, reveal that both male and female professors are happier living in small towns when their spouses are also employed in the same geographic area. Therefore, in the interest of attracting the most gifted teachers and researchers to our faculty and improving the morale of our entire staff, we at Pierce University should offer employment to the spouse of each new faculty member we hire. Although we cannot expect all offers to be accepted or to be viewed as an ideal job offer, the money invested in this effort will clearly be well spent because, if their spouses have a chance of employment, new professors will be more likely to accept our offers."

The notion that offering employment to the spouses of newly hired faculty members would make job offer acceptance more likely seems at first glance an obvious conclusion from a well-presented argument. After all, the studies conducted by Bronston College, a college with the same small town locality as Pierce University, show that professors of both sexes are happier when their spouses are working in the same geographical area. However, a closer examination reveals that this argument is not thoroughly researched enough.

First of all, the author’s premise is that professors of both sexes are happier when their spouses are employed in the same geographical area. This point is then used to justify the offering of employment to the spouses of newly-hired faculty members in order to attract the most talented teachers and researchers to the university. In building this argument, however, the author neglects to take into consideration other factors that might attract new professors to Pierce University as well. Such factors include salaries, the rate of award of tenure, proximity to amenities and shopping, or the general safety of the area. Because people respond to different motivations, the argument would be considerably bolstered if the author shows some statistics to justify why spouse employment should be valued above all other factors not mentioned.

Second, no details are given on the studies conducted by Bronston College, except that the college is located in a small town just like Pierce University. It is possible that there are other factors influencing the happiness of professors whose spouses are employed in the same geographical area, such as quality of students, adequate research funding, or even the presence of affordable child-care. The phrase “some studies conducted by Bronston College” in the first line of the argument suggests that the studies were not exhaustive in nature. It is thus appropriate to expect more information from the author regarding these studies in order to ensure that the results of these studies can be properly extrapolated to the faculty at Pierce University.

Finally, the author commits several errors in logic while constructing this argument. The premise of the argument rests on the assumption that all newly hired faculty members will be married – something that may not be true. Furthermore, the author writes that offering employment to the spouse of each newly hired professor will “improve the morale of [their] entire staff.” Given the content of the current argument, it is difficult to see how employing the spouse of a new faculty member will enhance the morale of personnel already working in the university, e.g. a tenured professor with a spouse who just got laid off.

Overall, the argument presented above seems logical as presented above, given that the author appears genuinely interested in attracting the best talents to Pierce University, presumably for the benefit of improving quality of instruction and research. However, before any final decisions are made on job offers for spouses of newly hired professors, the president and faculty search committees should consider all possible alternatives that might increase the appeal of the university to potential faculty members.