- Email: [email protected]
cDNA array analysis of Japanese quail lines divergently selected for four-week body weight1
cDNA Array Analysis of Japanese Quail Lines Divergently Selected for Four-Week Body Weight1 I. W. Mott2 and R. Ivarie3 Department of Genetics, University of Georgia, Athens, Georgia 30602-7223 were confirmed by Northern blot analysis. The 35-kD quail EB1 protein, previously unidentified, was shown to have elevated transcripts in the L line and decreased transcripts in the H and P lines, compared with the C line. It was also widely expressed in embryonic and adult tissues by BLASTN analysis of chicken expressed sequence tag (EST) libraries. Two other cDNA clones are novel sequences expressed at higher levels in the L line and at lower levels in the H and P lines, one of which was more selectively expressed in embryos and adult tissues by BLASTN analysis. These limited findings suggest that anonymous cDNA array analysis is a productive means to identify differentially expressed genes in growth-selected poultry.
(Key words: differential expression, DNA array, EB1, growth-selected poultry, Japanese quail) 2004 Poultry Science 83:1524–1529
INTRODUCTION Japanese quail (Coturnix japonica) lines have been divergently selected for high and low BW at 4 wk posthatch (Anthony et al., 1996; Marks, 1996). A major advantage of these lines over other growth-selected poultry is the randombred C line that provides a genetic control for comparison. The P line has been selected for high 4-wk BW for >110 generations. Currently, P line birds are nearly 3-fold larger (251 g) than the C line birds (88 g) at 4 wk of age. Two other lines, L and H, were later selected for low (54 generations) and high (52 generations) 4-wk BW, respectively. The L line individuals average less than 40 g, and H line birds average more than 190 g at 4 wk of age. The dramatic increase in P line BW is primarily pleiotropic, resulting in increased size and mass of all body organs except for brain. At 9 wk of age, the major differ-
2004 Poultry Science Association, Inc. Received for publication February 25, 2004. Accepted for publication May 27, 2004. 1 The nucleotide sequence data reported in this paper have been submitted to the NCBI nucleotide sequence database (http:// www.ncbi.nlm.nih.gov/BankIt/) and have been assigned Accession Numbers AY353854 (EB1), AY353856 (F3-15), and AY353855 (D10-15). 2 Present address: USDA-ARS, Forage and Range Research Laboratory, Utah State University, 695 N. 1100 E, Logan, UT 84322. 3 To whom correspondence should be addressed: [email protected].
ences in these lines were in the relative percentages of the pectoralis muscle (24 vs. 19%), head (2.8 vs. 3.4%), and brain (0.18 vs. 0.39%) for P and C lines, respectively (Ricklefs and Marks, 1985). Muscle comprises 40% of birth weight in vertebrates. The increase in muscle mass in these lines is largely from myofiber hyperplasia and, to a lesser extent, myofiber hypertrophy (Burke and Henry, 1999). Anatomical responses to selection under varying diets (Ricklefs and Marks, 1985), the relationships between egg weight, hatch weight, and growth rates (Marks, 1975), survival rates (Aggrey, 2002), and the satellite cell content of semimembranous muscles (Campion et al., 1982) of the different lines have all been documented. Surprisingly, expression of myostatin, a negative regulator of muscle mass, is not altered in these lines of quail (Mott and Ivarie, 2002). To date, no specific genetic factors have been identified that can account for the increased or decreased growth rates in these lines of quail, including any that explain the increased muscle mass. DNA macro- and microarrays have been used to assess differences in gene expression in many organisms and under different experimental conditions. In yeast, DNA
Abbreviation key: C = control line of randombred quail; EST = expressed sequence tags; H = quail line selected for 52 generations for high 4-wk BW; L = quail line selected for 54 generations for low 4-wk BW; P = quail line selected over >110 generations for high 4-wk BW.
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ABSTRACT Decades of divergent selection for 4-wk BW has produced 3 lines of growth-selected Japanese quail. P line quail have been selected for >110 generations for 4-wk posthatch BW and are nearly 3-fold larger than the randomly bred control C line. The H line has been selected for high 4-wk BW for 52 generations and the L line has been selected for low 4-wk BW for 54 generations. To identify differentially expressed genes that may play a role in defining the differences in these lines, a DNA array containing 4,704 random anonymous cDNA clones from 8-d C line embryos was screened using isotopicallylabeled cDNA from the different quail lines. Array analysis yielded 3 differentially expressed cDNA clones that
EXPRESSION PROFILING OF QUAIL LINES
MATERIALS AND METHODS RNA and Sample Preparation Eggs from lines L, C, H, and P quail (Coturnix japonica) were obtained from the Southern Regional Poultry Genetics Laboratory at the University of Georgia. Eggs were incubated at 37.5°C and 55% humidity with rocking through 60° for indicated times. Tissues from 3 embryos for each sampling were pooled, snap-frozen in liquid nitrogen, ground to fine powder, and stored in liquid nitrogen until use. Total RNA was extracted from samples using RNA STAT-604 according to the manufacturer’s protocol. RNA concentrations were measured by absorption at an optical density of 260 nm in a Spectronic Genesys 2 spectrophotometer,5 and were stored at −80°C until used. Isolation of mRNA was carried out using MACS mRNA Isolation Kit6 according to the manufacturer’s protocol.
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Tel-Test, Inc., Friendswood, TX. Spectronic Instruments, Rochester, NY. 6 Miltenyi Biotec, Auburn, CA. 7 Clontech Laboratories, Inc., Palo Alto, CA. 8 Stratagene, La Jolla, CA. 9 Matrix Technologies, Hudson, NH. 10 Amersham Pharmacia, Piscataway, NJ. 11 Fisher Scientific, Suwanee, GA. 12 Ambion, Austin, TX. 13 Bellco Glass Inc., Vineland, NJ. 5
cDNA Library Construction Control quail eggs were incubated for 8 d, after which total RNA was extracted from whole embryos. Synthesis of cDNA from total RNA (1 µg) was carried out according to the LD-PCR protocol in the Smart cDNA Library Construction Kit.7 Size-selected cDNA (0.1 to 4.0 kb) was ligated into λ TriplEx2 phage and packaged using Gigapack Packaging Extracts8 according to the manufacturer’s protocol. The cDNA library contained 1.9 × 106 recombinants and was amplified according to the manufacturer’s protocol,7 and stored at −70° or 4°C until used.
cDNA Arrays Phage from the 8-d C line embryo λ library were converted to plasmids in E. coli BM25.8 cells7 according to the manufacturer’s protocol, then plated on LB plates containing carbenicillin, and grown overnight at 37°C. Colonies (4,704) were robotically picked to 49 96-well plates in duplicate containing 180 µL of freezing medium (LB supplemented with 36 mM K2HPO4, 13.2 mM KH2PO4, 1.7 mM sodium citrate, 0.4 mM MgSO4, 6.8 mM (NH4)2SO4, and 4.4% glycerol) and grown overnight without shaking at 37°C. They were robotically arrayed with a BioRobotics BioPick9 in a 7 × 7 pattern on HybondXL nylon membranes,10 overlaid on LB agar containing ampicillin, and grown for 8 h at 37°C. After incubation, membranes were stored at 4°C. The membranes were treated for 5 min on heavy chromatography paper11 saturated with denaturation solution (1.5 M NaCl, 0.5 N NaOH, and 10% SDS), and then rinsed for 5 min each in neutralization solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.4), and 2× saline sodium citrate. Membranes were then air-dried and baked at 80°C for 2 h. Before prehybridization, membranes were washed (2× saline sodium citrate, 0.1% SDS) overnight with gentle agitation, and the cell debris was wiped off.
cDNA Probe Synthesis and Array Hybridization Filters were incubated in prehybridization buffer (0.5 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA) overnight at 65°C. Labeled cDNA probes were prepared using the Strip-EZ RT kit12 according to the manufacturer’s protocol. Briefly, individual cDNA synthesis reactions from P and C line mRNA (250 ng) from 14-d embryos were primed with random decamers and incubated with 20 µCi of [α-32P]-dATP10 at 37°C for 90 min. Reactions were stopped by addition of 10× stop buffer (2 M NaOH, 2 mM EDTA), and a fraction of each reaction was precipitated with trichloroacetic acid to quantitate labeled cDNA probes. Each probe (1.0 × 106 cpm/mL in prehybridization buffer) was hybridized to membranes at 65°C overnight in Autoblot glass tubes in a hybridization oven.13 Membranes were washed twice with wash buffer (0.25× saline-sodium phosphate-EDTA, 0.25% SDS) at 65°C for 30 min, and once with 2× saline sodium citrate at room
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arrays have been used to assess general differences between strains (Shalon et al., 1996), to identify expression changes after temperature shock (Lashkari et al., 1997), and to evaluate responses to growth on different carbon sources (Lashkari et al., 1997). Gene arrays were used to compare expression profiles between activated and resting murine T cells (Teague et al., 1999) and to characterize expression patterns of signaling pathways (Fambrough et al., 1999; Madhani et al., 1999). They have been used to identify genes involved in human disease states such as inflammatory disease (Heller et al., 1997), and cancer (DeRisa et al., 1996; Trent et al., 1997), and in hematopoietic differentiation (Tamayo et al., 1999). In poultry, arrays have been used to identify host genes in chicken fibroblasts induced by infection with oncogenic Marek’s disease virus (Morgan et al., 2001) as well as candidate genes underlying Marek’s disease virus-resistance in resistant and susceptible chicken lines (Liu et al., 2001). To identify genes that are differentially expressed in these growth-selected lines of quail, we have performed a preliminary screen of arrayed cDNA. A DNA array containing 4,704 random unidentified clones from a C line quail whole embryo cDNA library was screened with isotopically-labeled cDNA from P and C lines. Subsequent Northern blot analysis was performed on all 4 lines to confirm the results of 2 of the most profoundly differentially expressed cDNA clones on the array, a homolog of EB1 and a novel sequence.
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temperature before being exposed to Kodak imaging film14 or a phosphorimager for 24 to 48 h.
DNA Sequencing Plasmids containing cDNA were prepared for sequencing using Qiagen Plasmid Maxi or Mini Kits.15 Cycle sequencing was done using 1/8 dilutions of BigDye Terminator10 v3.1 with the following program in a Genamp 9600 thermal cycler: 96°C for 1 min, 25 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Purified reactions were analyzed on an ABI/PE 3700 sequencer.
Northern Blot Analysis
RESULTS AND DISCUSSION To identify cDNA clones that are differentially expressed in the growth-selected lines of Japanese quail (Coturnix japonica), arrayed cDNA clones were screened with 32P– labeled cDNA synthesized from either P or C line whole embryo RNA. The results are shown in Figure 1. Three differentially expressed cDNA clones (C2-08, F3-15, and D10-15) were identified (Figure 1B), all of which were down-regulated in the P line quail. The selected cDNA clones were retrieved from the library for sequencing and further characterized via Northern blots to confirm differential expression.
Quail EB1 Is Differentially Expressed in Growth-Selected Quail Embryos As shown in Figure 2A, Northern analysis identified C208 as a 2,300 nucleotide transcript expressed at significantly higher levels in L line, and at the lowest levels in H and P lines, compared with control C line. As an internal control for Northern analysis, expression of myostatin (MSTN) was used because MSTN transcripts are expressed at low levels
Two Novel cDNAs Differentially Expressed in High and Low BW Quail As shown for EB1, Northern analysis of the F3-15 clone revealed a transcript of 1.8 kb (Figure 2B) that was expressed at highest levels in the L line, and at lower levels in the H and P lines, compared with the control C line. One of several open reading frames in this cDNA can encode a 108 amino acid peptide with no conserved domains but with 82% identity to annotated mouse and human protein sequences (accession numbers XM 351675.1 and AK013986, respectively). When blasted against chicken EST databases (BLASTN), F3-15 EST were found only in adult brain (2 hits) and small intestine (4 hits), as well as in stage 36 limbs (1 hit) and stage 20-21 whole chick embryo (2 hits). In contrast to C2-08/EB1, this gene may be more selectively expressed in the embryo and the adult. Annotation of a contig hit (UD.EU.final.Contig20103) in the University of Delaware EST database suggests that the predicted protein is a target of phosphorylation by protein kinase C. D10-15 was differentially expressed between P and C quail (Figure 1B) with the lower expression levels detected in the P line. In 3 attempts, no D10-15 transcript was detectable in Northern blots. D10-15 mRNA codes for a predicted 133 amino acid peptide that contains 5 VGQ repeats with 38% identity to Plasmodium falciparum phosphatidylcholinesterol acyltransferase precursor protein. BLASTN analysis revealed no hits in any chicken EST database. It is not possible to speculate as to how these 2 cDNA clones might contribute to differences in BW in these quail lines until the proteins and their functions are identified and characterized in model organisms.
Role of EB1 in BW Phenotypes of the Growth-Selected Lines of Quail 14
Eastman Kodak Company, Rochester, NY. Qiagen, Valencia, CA. Roche Applied Science, Indianapolis, IN.
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EB1 proteins have been identified in many organisms, from yeast to humans (Tirnauer and Bierer, 2000). The
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Digoxigenin (DIG)-labeled probes were synthesized from BamH1-linearized plasmids using T7 RNA polymerase or by PCR according to the manufacturer’s protocol.16 RNA samples were electrophoresed on 1% agarose / 2% formaldehyde gels and transferred to nylon membranes. Hybridization and digoxigenin luminescent detection were carried out according to the manufacturer’s protocol. Both the original RNA used for the cDNA library array and a freshly extracted RNA preparation were used to avoid preparation-specific artifacts in the Northern blots. Quantitative analysis of transcripts was determined by densitometry. Bands (n = 3) representing cDNA were scanned and normalized to background on the autoradiograph. Student’s t-test was used to compare band intensities between the lines of quail.
and do not differ among the lines (Mott and Ivarie, 2002). The cDNA clone was sequenced to produce a 1,038 base sequence containing a 789 nucleotide open reading frame (nucleotides 106 to 894) coding for a 263 amino acid protein having 87% identity to the human/mouse APC-binding protein, EB1. Given the size (2.3 kb) of the quail transcript, our cDNA clone is lacking most of the long 3′ untranslated region found in the human EB1 mRNA. Human EB1 is a 35kDa, mildly acidic leucine zipper protein first discovered in a yeast 2-hybrid screen as an interactor with the human adenomatous polyposis coli (APC) tumor suppressor protein (Su et al., 1995). Analysis by BLASTN searches of chicken EST databases indicates that the avian EB1 gene is probably ubiquitously expressed throughout the chicken embryo and in many adult tissues. Of 72 hits, 46 occurred in whole embryos at various stages as well as in embryonic trunk, brain, head, heart, and limbs; of the remaining hits, 26 occurred in adult heart, liver, muscle, ovary, brain, chondrocytes, small intestine, and kidney/adrenal gland.
EXPRESSION PROFILING OF QUAIL LINES
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FIGURE 1. Autoradiographs of the total cDNA array duplicates (A) and 3 selected regions (B) showing 3 differentially expressed genes in P and C line embryos. A total of 4,704 random and anonymous cDNA clones from a C line 8-d embryo were screened with 32P-labeled cDNA synthesized from total RNA from 14-d embryos. Spots highlighted by boxes identify 3 clones whose expression differed between the 2 lines. C208 refers to the cDNA arrayed in the C2 well of plate 8, F3-15 to that in the F3 well of plate 15, and D10-15 to the D10 well of plate 15.
budding yeast EB1 homolog BIM1p localizes at the plus ends of microtubules where it increases their dynamic instability (Tirnauer et al., 1999), facilitates orientation of the spindle toward the bud site (Korinek et al., 2000; Lee et al., 2000), and indirectly participates in a checkpoint delaying cytokinesis until mitotic exit (Muhua et al., 1998). It plays a similar role in Drosophila (Lu et al., 2001; Rogers et al., 2002). Interestingly, overexpression of Mal3, the fission yeast EB1 homolog, led to severe growth inhibition (Beinhauer et al., 1997). In vertebrates, EB1 localizes to the growing (plus) ends of microtubules, and targets APC to the tips of microtubules (Mimori-Kiyosue et al., 2000a,b). Although little is known about the function of EB1 proteins in verte-
brates, it is noteworthy that quail EB1 mRNA expression was negatively correlated with BW. Perhaps higher expression in the L line leads to reduced numbers of proliferating precursor cells, thus yielding smaller animals. Conversely, the reduced levels of expression in C and L birds might give rise to larger animals by relaxing the growth inhibition of developing stem cells as suggested by the fact of EB1’s widespread pattern of expression and that loss of heterozygosity and APC function via truncation of the C-terminal EB1 binding domain in mutants leads to tumor formation via uncontrolled cell replication (Kinzler et al., 1996; Polakis, 1997). Hence, EB1 may be one of many quantitative trait loci affecting BW in these lines.
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REFERENCES
In summary, we have demonstrated that anonymous cDNA array analysis can be an effective tool to identify differentially expressed genes in growth-selected quail embryos. The use of anonymous libraries is advantageous for organisms that are not targeted for whole genome sequencing and lack large EST databases because only those clones that are differentially expressed need to be sequenced. As reported here, 3 clones out of 4,704 (0.06%) were identified in an initial screen of un-normalized whole embryo cDNA clones. Additional arrays printed with un-normalized and anonymous cDNA may be expected to yield one differentially expressed cDNA for every 1,500 random sequences, based on our limited findings. Normalizing the library would lead to the identification of a larger number of genes with altered expression by eliminating redundancy in the library. Also, other genes may be found by screening such arrays with other tissue- or stage-specific populations of RNA. Finally, with the release of the first draft of the chicken genome sequence, high-resolution gene-chip arrays will ultimately enable the identification of the collection of genes that have undergone changes in expression in growth-selected lines of poultry.
ACKNOWLEDGMENTS The authors thank Henry Marks of the Poultry Science Department for providing fertilized quail eggs, and James Griffith of the Genetics Department, UGA, Athens, Georgia for help in printing the arrays.
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FIGURE 2. Northern blot analysis of clone C2-08 (A) and F3-15 (B) expression in L, C, H, and P line embryo RNA. Digoxigenin-labeled DNA probes from each clone were used to screen total RNA from 14d embryos of each line (left panel). Myostatin (MSTN) and ethidium bromide-stained 18S rRNA served as internal loading controls. Three separate Northern blots probing the original RNA used in the cDNA library construction, as well as another RNA sample were used to quantify the differences among the lines (right panel, a–cP < 0.05).
Aggrey, S. E., and H. L. Marks. 2002. Analysis of censored survival data in Japanese quail divergently selected for growth and their control. Poult. Sci. 81:1618–1620. Anthony, N. B., K. E. Nestor, and H. L. Marks. 1996. Short-term selection for four-week body weight in Japanese quail. Poult. Sci. 75:1192–1197. Beinhauer, J. D., I. M. Hagan, J. H. Hegemann, and U. Fleig. 1997. Mal3, the fission yeast homologue of the human APCinteracting protein EB-1 is required for microtubule integrity and the maintenance of cell form. J. Cell Biol. 139:717–728. Burke, W. H., and M. H. Henry. 1999. Growth and muscle characteristics of a growth selected line of Japanese quail (Coturnix japonica), a control line and reciprocal crosses between them. Growth Dev. Aging 63:33–47. Campion, D. R., H. L. Marks, and L. R. Richardson. 1982. An analysis of satellite cell content in the semimembranous muscle of Japanese quail (Coturnix japonica) selected for rapid growth. Acta Anat. 112:9–13. DeRisi, J., L. Penland, P. O. Brown, M. L. Bittner, P. S. Meltzer, M. Ray, Y. Chen, Y. A. Su, and J. M. Trent. 1996. Use of a cDNA microarray to analyze gene expression patterns in human cancer. Nat. Genet. 14:457–460. Fambrough, D., K. McClure, A. Kazlauskas, and E. S. Lander. 1999. Diverse signaling pathways activated by growth factor receptors induce broadly overlapping, rather than independent, sets of genes. Cell 97:727–741. Heller, R. A., M. Schena, A. Chai, D. Shalon, T. Bedilion, J. Gilmore, D. E. Woolley, and R. W. Davis. 1997. Discovery and analysis of inflammatory disease-related genes using cDNA microarrays. Proc. Natl. Acad. Sci. USA 94:2150–2155. Korinek, W. S., M. J. Copeland, A. Chaudhuri, and J. Chant. 2000. Molecular linkage underlying microtubule orientation toward cortical sites in yeast. Science 287:2257–2259. Lashkari, D. A., J. L. DeRisi, J. H. McCusker, A. F. Namath, C. Gentile, S. Y. Hwang, P. O. Brown, and R. W. Davis. 1997. Yeast microarrays for genome wide parallel genetic and gene expression analysis. Proc. Natl. Acad. Sci. USA 94:13057– 13062. Lee, L., J. S. Tirnauer, J. Li, S. C. Schuyler, J. Y. Liu, and D. Pellman. 2000. Positioning of the mitotic spindle by a corticalmicrotubule capture mechanism. Science 287:2260–2262. Liu, H. C., H. H. Cheng, V. Tirunagaru, L. Sofer, and J. Burnside. 2001. A strategy to identify positional candidate genes conferring Marek’s disease resistance by integrating DNA microarrays and genetic mapping. Anim. Genet. 32:351–359. Lu, B., F. Roegiers, L. Y. Jan, and Y. N. Jan. 2001. Adherens junctions inhibit asymmetric division in the Drosophila epithelium. Nature 409:522–525. Madhani, H. D., T. Galitski, E. S. Lander, and G. R. Fink. 1999. Effectors of a developmental mitogen-activated protein kinase cascade revealed by expression signatures of signaling mutants. Proc. Natl. Acad. Sci. USA 96:12530–12535. Marks, H. L. 1975. Relationship of embryonic development to egg weight, hatch weight, and growth in Japanese quail. Poult. Sci. 54:1257–1262. Marks, H. L. 1996. Longterm selection for body weight in Japanese quail under different environments. Poult. Sci. 75:1198–1203. Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000a. Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells. J. Cell Biol. 148:505–518. Mimori-Kiyosue, Y., N. Shiina, and S. Tsukita. 2000b. The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules. Curr. Biol. 10:865–868. Morgan, R. W., L. Sofer, A. S. Anderson, E. L. Bernberg, J. Cui, and J. Burnside. 2001. Induction of host gene expression following infection of chicken embryo fibroblasts with oncogenic Marek’s disease virus. J. Virol. 75:533–539.
EXPRESSION PROFILING OF QUAIL LINES Mott, I., and R. Ivarie. 2002. Expression of myostatin is not altered in lines of poultry exhibiting myofiber hyper- and hypoplasia. Poult. Sci. 81:799–804. Muhua, L., N. R. Adames, M. D. Murphy, C. R. Shields, and J. A. Cooper. 1998. A cytokinesis checkpoint requiring the yeast homologue of an APC-binding protein. Nature 393:487–491. Polakis, P. 1997. The adenomatous polyposis coli (APC) tumor suppressor. Biochim. Biophys. Acta 1332:F127–F147. Ricklefs, R. E., and H. Marks. 1985. Anatomical response to selection for four-week body mass in Japanese quail. Auk 102:323–333. Shalon, D., S. J. Smith, and P. O. Brown. 1996. A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res. 6:639–645. Su, L. K., M. Burrell, D. E. Hill, J. Gyuris, R. Brent, R. Wiltshire, J. Trent, B. Vogelstein, and K. W. Kinzler. 1995. APC binds to the novel protein EB1. Cancer Res. 55:2972–2977.
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Tamayo, P., D. Slonim, J. Mesirov, Q. Zhu, S. Kitareewan, E. Dmitrovsky, E. S. Lander, and T. R. Golub. 1999. Interpreting patterns of gene expression with self-organizing maps: Methods and application to hematopoietic differentiation. Proc. Natl. Acad. Sci. USA 96:2907–2912. Teague, T. K., D. Hildeman, R. M. Kedl, T. Mitchell, W. Rees, B. C. Schaefer, J. Bender, J. Kappler, and P. Marrack. 1999. Activation changes the spectrum but not the diversity of genes expressed by T cells. Proc. Natl. Acad. Sci. USA 96:12691–12696. Tirnauer, J. S., and B. E. Bierer. 2000. EB1 proteins regulate microtubule dynamics, cell polarity, and chromosome stability. J. Cell Biol. 149:761–766. Trent, J. M., M. Bittner, J. Zhang, R. Wiltshire, M. Ray, Y. Su, E. Gracia, P. Meltzer, J. De Risi, L. Penland, and P. Brown. 1997. Use of microgenomic technology for analysis of alterations in DNA copy number and gene expression in malignant melanoma. Clin. Exp. Immunol. 107(Suppl. 1):33–40.
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(Key words: differential expression, DNA array, EB1, growth-selected poultry, Japanese quail) 2004 Poultry Science 83:1524–1529
INTRODUCTION Japanese quail (Coturnix japonica) lines have been divergently selected for high and low BW at 4 wk posthatch (Anthony et al., 1996; Marks, 1996). A major advantage of these lines over other growth-selected poultry is the randombred C line that provides a genetic control for comparison. The P line has been selected for high 4-wk BW for >110 generations. Currently, P line birds are nearly 3-fold larger (251 g) than the C line birds (88 g) at 4 wk of age. Two other lines, L and H, were later selected for low (54 generations) and high (52 generations) 4-wk BW, respectively. The L line individuals average less than 40 g, and H line birds average more than 190 g at 4 wk of age. The dramatic increase in P line BW is primarily pleiotropic, resulting in increased size and mass of all body organs except for brain. At 9 wk of age, the major differ-
2004 Poultry Science Association, Inc. Received for publication February 25, 2004. Accepted for publication May 27, 2004. 1 The nucleotide sequence data reported in this paper have been submitted to the NCBI nucleotide sequence database (http:// www.ncbi.nlm.nih.gov/BankIt/) and have been assigned Accession Numbers AY353854 (EB1), AY353856 (F3-15), and AY353855 (D10-15). 2 Present address: USDA-ARS, Forage and Range Research Laboratory, Utah State University, 695 N. 1100 E, Logan, UT 84322. 3 To whom correspondence should be addressed: [email protected].
ences in these lines were in the relative percentages of the pectoralis muscle (24 vs. 19%), head (2.8 vs. 3.4%), and brain (0.18 vs. 0.39%) for P and C lines, respectively (Ricklefs and Marks, 1985). Muscle comprises 40% of birth weight in vertebrates. The increase in muscle mass in these lines is largely from myofiber hyperplasia and, to a lesser extent, myofiber hypertrophy (Burke and Henry, 1999). Anatomical responses to selection under varying diets (Ricklefs and Marks, 1985), the relationships between egg weight, hatch weight, and growth rates (Marks, 1975), survival rates (Aggrey, 2002), and the satellite cell content of semimembranous muscles (Campion et al., 1982) of the different lines have all been documented. Surprisingly, expression of myostatin, a negative regulator of muscle mass, is not altered in these lines of quail (Mott and Ivarie, 2002). To date, no specific genetic factors have been identified that can account for the increased or decreased growth rates in these lines of quail, including any that explain the increased muscle mass. DNA macro- and microarrays have been used to assess differences in gene expression in many organisms and under different experimental conditions. In yeast, DNA
Abbreviation key: C = control line of randombred quail; EST = expressed sequence tags; H = quail line selected for 52 generations for high 4-wk BW; L = quail line selected for 54 generations for low 4-wk BW; P = quail line selected over >110 generations for high 4-wk BW.
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ABSTRACT Decades of divergent selection for 4-wk BW has produced 3 lines of growth-selected Japanese quail. P line quail have been selected for >110 generations for 4-wk posthatch BW and are nearly 3-fold larger than the randomly bred control C line. The H line has been selected for high 4-wk BW for 52 generations and the L line has been selected for low 4-wk BW for 54 generations. To identify differentially expressed genes that may play a role in defining the differences in these lines, a DNA array containing 4,704 random anonymous cDNA clones from 8-d C line embryos was screened using isotopicallylabeled cDNA from the different quail lines. Array analysis yielded 3 differentially expressed cDNA clones that
EXPRESSION PROFILING OF QUAIL LINES
MATERIALS AND METHODS RNA and Sample Preparation Eggs from lines L, C, H, and P quail (Coturnix japonica) were obtained from the Southern Regional Poultry Genetics Laboratory at the University of Georgia. Eggs were incubated at 37.5°C and 55% humidity with rocking through 60° for indicated times. Tissues from 3 embryos for each sampling were pooled, snap-frozen in liquid nitrogen, ground to fine powder, and stored in liquid nitrogen until use. Total RNA was extracted from samples using RNA STAT-604 according to the manufacturer’s protocol. RNA concentrations were measured by absorption at an optical density of 260 nm in a Spectronic Genesys 2 spectrophotometer,5 and were stored at −80°C until used. Isolation of mRNA was carried out using MACS mRNA Isolation Kit6 according to the manufacturer’s protocol.
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Tel-Test, Inc., Friendswood, TX. Spectronic Instruments, Rochester, NY. 6 Miltenyi Biotec, Auburn, CA. 7 Clontech Laboratories, Inc., Palo Alto, CA. 8 Stratagene, La Jolla, CA. 9 Matrix Technologies, Hudson, NH. 10 Amersham Pharmacia, Piscataway, NJ. 11 Fisher Scientific, Suwanee, GA. 12 Ambion, Austin, TX. 13 Bellco Glass Inc., Vineland, NJ. 5
cDNA Library Construction Control quail eggs were incubated for 8 d, after which total RNA was extracted from whole embryos. Synthesis of cDNA from total RNA (1 µg) was carried out according to the LD-PCR protocol in the Smart cDNA Library Construction Kit.7 Size-selected cDNA (0.1 to 4.0 kb) was ligated into λ TriplEx2 phage and packaged using Gigapack Packaging Extracts8 according to the manufacturer’s protocol. The cDNA library contained 1.9 × 106 recombinants and was amplified according to the manufacturer’s protocol,7 and stored at −70° or 4°C until used.
cDNA Arrays Phage from the 8-d C line embryo λ library were converted to plasmids in E. coli BM25.8 cells7 according to the manufacturer’s protocol, then plated on LB plates containing carbenicillin, and grown overnight at 37°C. Colonies (4,704) were robotically picked to 49 96-well plates in duplicate containing 180 µL of freezing medium (LB supplemented with 36 mM K2HPO4, 13.2 mM KH2PO4, 1.7 mM sodium citrate, 0.4 mM MgSO4, 6.8 mM (NH4)2SO4, and 4.4% glycerol) and grown overnight without shaking at 37°C. They were robotically arrayed with a BioRobotics BioPick9 in a 7 × 7 pattern on HybondXL nylon membranes,10 overlaid on LB agar containing ampicillin, and grown for 8 h at 37°C. After incubation, membranes were stored at 4°C. The membranes were treated for 5 min on heavy chromatography paper11 saturated with denaturation solution (1.5 M NaCl, 0.5 N NaOH, and 10% SDS), and then rinsed for 5 min each in neutralization solution (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.4), and 2× saline sodium citrate. Membranes were then air-dried and baked at 80°C for 2 h. Before prehybridization, membranes were washed (2× saline sodium citrate, 0.1% SDS) overnight with gentle agitation, and the cell debris was wiped off.
cDNA Probe Synthesis and Array Hybridization Filters were incubated in prehybridization buffer (0.5 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA) overnight at 65°C. Labeled cDNA probes were prepared using the Strip-EZ RT kit12 according to the manufacturer’s protocol. Briefly, individual cDNA synthesis reactions from P and C line mRNA (250 ng) from 14-d embryos were primed with random decamers and incubated with 20 µCi of [α-32P]-dATP10 at 37°C for 90 min. Reactions were stopped by addition of 10× stop buffer (2 M NaOH, 2 mM EDTA), and a fraction of each reaction was precipitated with trichloroacetic acid to quantitate labeled cDNA probes. Each probe (1.0 × 106 cpm/mL in prehybridization buffer) was hybridized to membranes at 65°C overnight in Autoblot glass tubes in a hybridization oven.13 Membranes were washed twice with wash buffer (0.25× saline-sodium phosphate-EDTA, 0.25% SDS) at 65°C for 30 min, and once with 2× saline sodium citrate at room
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arrays have been used to assess general differences between strains (Shalon et al., 1996), to identify expression changes after temperature shock (Lashkari et al., 1997), and to evaluate responses to growth on different carbon sources (Lashkari et al., 1997). Gene arrays were used to compare expression profiles between activated and resting murine T cells (Teague et al., 1999) and to characterize expression patterns of signaling pathways (Fambrough et al., 1999; Madhani et al., 1999). They have been used to identify genes involved in human disease states such as inflammatory disease (Heller et al., 1997), and cancer (DeRisa et al., 1996; Trent et al., 1997), and in hematopoietic differentiation (Tamayo et al., 1999). In poultry, arrays have been used to identify host genes in chicken fibroblasts induced by infection with oncogenic Marek’s disease virus (Morgan et al., 2001) as well as candidate genes underlying Marek’s disease virus-resistance in resistant and susceptible chicken lines (Liu et al., 2001). To identify genes that are differentially expressed in these growth-selected lines of quail, we have performed a preliminary screen of arrayed cDNA. A DNA array containing 4,704 random unidentified clones from a C line quail whole embryo cDNA library was screened with isotopically-labeled cDNA from P and C lines. Subsequent Northern blot analysis was performed on all 4 lines to confirm the results of 2 of the most profoundly differentially expressed cDNA clones on the array, a homolog of EB1 and a novel sequence.
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temperature before being exposed to Kodak imaging film14 or a phosphorimager for 24 to 48 h.
DNA Sequencing Plasmids containing cDNA were prepared for sequencing using Qiagen Plasmid Maxi or Mini Kits.15 Cycle sequencing was done using 1/8 dilutions of BigDye Terminator10 v3.1 with the following program in a Genamp 9600 thermal cycler: 96°C for 1 min, 25 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Purified reactions were analyzed on an ABI/PE 3700 sequencer.
Northern Blot Analysis
RESULTS AND DISCUSSION To identify cDNA clones that are differentially expressed in the growth-selected lines of Japanese quail (Coturnix japonica), arrayed cDNA clones were screened with 32P– labeled cDNA synthesized from either P or C line whole embryo RNA. The results are shown in Figure 1. Three differentially expressed cDNA clones (C2-08, F3-15, and D10-15) were identified (Figure 1B), all of which were down-regulated in the P line quail. The selected cDNA clones were retrieved from the library for sequencing and further characterized via Northern blots to confirm differential expression.
Quail EB1 Is Differentially Expressed in Growth-Selected Quail Embryos As shown in Figure 2A, Northern analysis identified C208 as a 2,300 nucleotide transcript expressed at significantly higher levels in L line, and at the lowest levels in H and P lines, compared with control C line. As an internal control for Northern analysis, expression of myostatin (MSTN) was used because MSTN transcripts are expressed at low levels
Two Novel cDNAs Differentially Expressed in High and Low BW Quail As shown for EB1, Northern analysis of the F3-15 clone revealed a transcript of 1.8 kb (Figure 2B) that was expressed at highest levels in the L line, and at lower levels in the H and P lines, compared with the control C line. One of several open reading frames in this cDNA can encode a 108 amino acid peptide with no conserved domains but with 82% identity to annotated mouse and human protein sequences (accession numbers XM 351675.1 and AK013986, respectively). When blasted against chicken EST databases (BLASTN), F3-15 EST were found only in adult brain (2 hits) and small intestine (4 hits), as well as in stage 36 limbs (1 hit) and stage 20-21 whole chick embryo (2 hits). In contrast to C2-08/EB1, this gene may be more selectively expressed in the embryo and the adult. Annotation of a contig hit (UD.EU.final.Contig20103) in the University of Delaware EST database suggests that the predicted protein is a target of phosphorylation by protein kinase C. D10-15 was differentially expressed between P and C quail (Figure 1B) with the lower expression levels detected in the P line. In 3 attempts, no D10-15 transcript was detectable in Northern blots. D10-15 mRNA codes for a predicted 133 amino acid peptide that contains 5 VGQ repeats with 38% identity to Plasmodium falciparum phosphatidylcholinesterol acyltransferase precursor protein. BLASTN analysis revealed no hits in any chicken EST database. It is not possible to speculate as to how these 2 cDNA clones might contribute to differences in BW in these quail lines until the proteins and their functions are identified and characterized in model organisms.
Role of EB1 in BW Phenotypes of the Growth-Selected Lines of Quail 14
Eastman Kodak Company, Rochester, NY. Qiagen, Valencia, CA. Roche Applied Science, Indianapolis, IN.
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EB1 proteins have been identified in many organisms, from yeast to humans (Tirnauer and Bierer, 2000). The
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Digoxigenin (DIG)-labeled probes were synthesized from BamH1-linearized plasmids using T7 RNA polymerase or by PCR according to the manufacturer’s protocol.16 RNA samples were electrophoresed on 1% agarose / 2% formaldehyde gels and transferred to nylon membranes. Hybridization and digoxigenin luminescent detection were carried out according to the manufacturer’s protocol. Both the original RNA used for the cDNA library array and a freshly extracted RNA preparation were used to avoid preparation-specific artifacts in the Northern blots. Quantitative analysis of transcripts was determined by densitometry. Bands (n = 3) representing cDNA were scanned and normalized to background on the autoradiograph. Student’s t-test was used to compare band intensities between the lines of quail.
and do not differ among the lines (Mott and Ivarie, 2002). The cDNA clone was sequenced to produce a 1,038 base sequence containing a 789 nucleotide open reading frame (nucleotides 106 to 894) coding for a 263 amino acid protein having 87% identity to the human/mouse APC-binding protein, EB1. Given the size (2.3 kb) of the quail transcript, our cDNA clone is lacking most of the long 3′ untranslated region found in the human EB1 mRNA. Human EB1 is a 35kDa, mildly acidic leucine zipper protein first discovered in a yeast 2-hybrid screen as an interactor with the human adenomatous polyposis coli (APC) tumor suppressor protein (Su et al., 1995). Analysis by BLASTN searches of chicken EST databases indicates that the avian EB1 gene is probably ubiquitously expressed throughout the chicken embryo and in many adult tissues. Of 72 hits, 46 occurred in whole embryos at various stages as well as in embryonic trunk, brain, head, heart, and limbs; of the remaining hits, 26 occurred in adult heart, liver, muscle, ovary, brain, chondrocytes, small intestine, and kidney/adrenal gland.
EXPRESSION PROFILING OF QUAIL LINES
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FIGURE 1. Autoradiographs of the total cDNA array duplicates (A) and 3 selected regions (B) showing 3 differentially expressed genes in P and C line embryos. A total of 4,704 random and anonymous cDNA clones from a C line 8-d embryo were screened with 32P-labeled cDNA synthesized from total RNA from 14-d embryos. Spots highlighted by boxes identify 3 clones whose expression differed between the 2 lines. C208 refers to the cDNA arrayed in the C2 well of plate 8, F3-15 to that in the F3 well of plate 15, and D10-15 to the D10 well of plate 15.
budding yeast EB1 homolog BIM1p localizes at the plus ends of microtubules where it increases their dynamic instability (Tirnauer et al., 1999), facilitates orientation of the spindle toward the bud site (Korinek et al., 2000; Lee et al., 2000), and indirectly participates in a checkpoint delaying cytokinesis until mitotic exit (Muhua et al., 1998). It plays a similar role in Drosophila (Lu et al., 2001; Rogers et al., 2002). Interestingly, overexpression of Mal3, the fission yeast EB1 homolog, led to severe growth inhibition (Beinhauer et al., 1997). In vertebrates, EB1 localizes to the growing (plus) ends of microtubules, and targets APC to the tips of microtubules (Mimori-Kiyosue et al., 2000a,b). Although little is known about the function of EB1 proteins in verte-
brates, it is noteworthy that quail EB1 mRNA expression was negatively correlated with BW. Perhaps higher expression in the L line leads to reduced numbers of proliferating precursor cells, thus yielding smaller animals. Conversely, the reduced levels of expression in C and L birds might give rise to larger animals by relaxing the growth inhibition of developing stem cells as suggested by the fact of EB1’s widespread pattern of expression and that loss of heterozygosity and APC function via truncation of the C-terminal EB1 binding domain in mutants leads to tumor formation via uncontrolled cell replication (Kinzler et al., 1996; Polakis, 1997). Hence, EB1 may be one of many quantitative trait loci affecting BW in these lines.
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REFERENCES
In summary, we have demonstrated that anonymous cDNA array analysis can be an effective tool to identify differentially expressed genes in growth-selected quail embryos. The use of anonymous libraries is advantageous for organisms that are not targeted for whole genome sequencing and lack large EST databases because only those clones that are differentially expressed need to be sequenced. As reported here, 3 clones out of 4,704 (0.06%) were identified in an initial screen of un-normalized whole embryo cDNA clones. Additional arrays printed with un-normalized and anonymous cDNA may be expected to yield one differentially expressed cDNA for every 1,500 random sequences, based on our limited findings. Normalizing the library would lead to the identification of a larger number of genes with altered expression by eliminating redundancy in the library. Also, other genes may be found by screening such arrays with other tissue- or stage-specific populations of RNA. Finally, with the release of the first draft of the chicken genome sequence, high-resolution gene-chip arrays will ultimately enable the identification of the collection of genes that have undergone changes in expression in growth-selected lines of poultry.
ACKNOWLEDGMENTS The authors thank Henry Marks of the Poultry Science Department for providing fertilized quail eggs, and James Griffith of the Genetics Department, UGA, Athens, Georgia for help in printing the arrays.
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FIGURE 2. Northern blot analysis of clone C2-08 (A) and F3-15 (B) expression in L, C, H, and P line embryo RNA. Digoxigenin-labeled DNA probes from each clone were used to screen total RNA from 14d embryos of each line (left panel). Myostatin (MSTN) and ethidium bromide-stained 18S rRNA served as internal loading controls. Three separate Northern blots probing the original RNA used in the cDNA library construction, as well as another RNA sample were used to quantify the differences among the lines (right panel, a–cP < 0.05).
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