research article characterization of expressed sequence...

Comparative and Functional GenomicsComp Funct Genom 2005; 6: 301–306.Published online 11 July 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cfg.484

Research Article

Characterization of expressed sequence tagsfrom a Gallus gallus pineal gland cDNA library

Stefanie Hartman1, Greg Touchton1, Jessica Wynn1, Tuoyu Geng1, Nelson W. Chong2 and Ed Smith1*1Comparative Genomics Lab, Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA2Division of Cardiology, Department of Medicine, University of Leicester, Leicester, UK

*Correspondence to:Ed Smith, 2250 Litton ReavesHall, Blacksburg, VA24061, USA.E-mail: [email protected]

Received: 12 January 2005Revised: 22 May 2005Accepted: 1 June 2005

AbstractThe pineal gland is the circadian oscillator in the chicken, regulating diversefunctions ranging from egg laying to feeding. Here, we describe the isolation andcharacterization of expressed sequence tags (ESTs) isolated from a chicken pinealgland cDNA library. A total of 192 unique sequences were analysed and submittedto GenBank; 6% of the ESTs matched neither GenBank cDNA sequences nor thenewly assembled chicken genomic DNA sequence, three ESTs aligned with sequencesdesignated to be on the Z random, while one matched a W chromosome sequence andcould be useful in cataloguing functionally important genes on this sex chromosome.Additionally, single nucleotide polymorphisms (SNPs) were identified and validatedin 10 ESTs that showed 98% or higher sequence similarity to known chicken genes.Here, we have described resources that may be useful in comparative and functionalgenomic analysis of genes expressed in an important organ, the pineal gland, in amodel and agriculturally important organism. Copyright 2005 John Wiley & Sons,Ltd.

Keywords: Gallus gallus; pineal gland ESTs, SNPs

Introduction

Circadian rhythm is a general characteristic of liv-ing organisms. Both physiological and genetic fac-tors involved in this process continue to be verywidely investigated in different organisms. In mam-malian and avian systems, it is a general consensusthat the physiological and genetic processes of bio-logical rhythms occur in a loop. The molecularmechanisms that control the loop appear to be con-served among diverse species. The avian circadianrhythm is unique as it involves multiple organswhose inputs and interactions influence the oscilla-tory patterns of rhythmic behaviour (Ebihara et al.,1987). Some of the positive and negative regulatorgenes involved in the autoregulatory feedback loopmechanism for the circadian oscillator in the pinealgland have been described in diverse birds, includ-ing the quail (Yoshimura et al., 2000) and chicken(Okano et al., 2001).

The chicken pineal gland is an important modelfor vertebrate circadian clock systems becauseof its ability to retain circadian rhythm in cul-ture. Several important genes have been iden-tified in the pineal gland. One important com-ponent of the autoregulatory feedback loop ofthe circadian oscillator is the negative regula-tor gene, cPer2 ; the gene products of cBmal1,cBmal2 and cClock form heterodimers that bindto a promoter sequence of cPer2 and activatetranscription (Okano et al., 2001). The photore-ceptor pinopsin has been shown to be present,although its expression responds exclusively tolight and not circadian patterns. The arylalkylamineN -acetyltransferase (AA-NAT) gene product, how-ever, has been directly linked to melatonin produc-tion in a circadian rhythm (Takanaka et al., 1988).In addition, GCAP1, GCAP2 and GC, genes thatare important in resetting rods and cones after lightexposure, have been identified in the pineal gland(Semple-Rowland, 1999).

Copyright 2005 John Wiley & Sons, Ltd.

302 S. Hartman et al.

Since the chicken is considered an excellentmodel for further understanding the genetic andmolecular basis of rhythmic behaviour, here weinvestigated the characteristics of expressed se-quence tags (ESTs) isolated from the chicken pinealgland. While previous work by Hubbard et al.(2005) has yielded a number of ESTs in suchimportant functional tissues as the liver, pancreas,heart, cerebellum, kidney and ovary, none hasbeen described to date from the pineal gland.Bailey et al. (2003) used microarray technologyto evaluate pineal genes expressed in periodsof light and darkness with a focus on functionrather than sequence comparisons. The primarygoal of our study was to identify novel genes thatcould be useful in comparative genome analysis ofthe molecular mechanisms that underlie rhythmicbehaviour. Additionally, we evaluated the level ofvariation in selected ESTs that matched knownchicken genes using in silico analysis followed byPCR-based resequencing for validation.

Materials and methods

Sequence analysis

The ESTs were obtained from a previously descri-bed chicken pineal gland-cDNA library (Chonget al., 2000). Briefly, the library was establishedfrom 10–11 day-old White Leghorn birds under12 h light. The ESTs were produced from single-pass sequencing of randomly selected clones, pro-cessed by a modification of the toothpick PCRdescribed by Smith et al. (2001). The modificationinvolved first converting the original library fromHybriZAP2.1 into phagemid, using the manufac-turer’s (Stratagene, La Jolla, CA92037) recommen-dation. The ESTs were characterized using BLAT(http://genome.ucsc.edu/cgi-bin/hgBlat?com-mand=start&org=Chicken&db=galGal2&hgsid=30295 885) and BLAST to identify data-base matches corresponding to the recently releasedchicken genomic DNA sequence and known genesin GenBank, respectively.

The chicken radiation hybrid panel (Morissonet al., 2002) was used to map VTEST71 in order tovalidate the in silico chromosomal location of theEST. Forward and reverse primers specific for theEST, designed using Primer 3 (Rozen and Skalet-sky, 1997), were used for the genotyping. The

forward and reverse primers were 5′-GAT TTCAAA ACG GAC TTG AG-3′ and 5′-TGA GCAGTC ACT TTT AGC ATT-3′, respectively. ThePCR was carried out in a final volume of 10 µlcontaining 1.5 mM Mg2+ Buffer (Eppendorf, West-bury, NY), 200 µM dNTPs, 70 µg primer (MWGBiotech), 1 U Taq (Eppendorf), and 5 ng template.The cycling was performed using a Mastercycler(Brinkmann, Westbury, NY) with the followingprogram: initial denaturation at 95 ◦C for 5 min fol-lowed by 95 ◦C for 45 s, 55 ◦C for 45 s, 72 ◦C for45 s for a total of 38 cycles of denaturation, anneal-ing and extension, respectively. A final extension at72 ◦C was carried out for 7 min. The PCR productwas run on a 2% agarose gel stained with ethidiumbromide, and scored as 0, 1 or 2 for absent, present,or ambiguous, respectively. Mapping results weredetermined by the Morisson lab from these data.

SNP analysis

An in silico analysis of 10 ESTs that closelymatched chicken genes was used to identify can-didate SNPs in the ESTs according to the pipelineprotocol of Buetow et al. (1999). Validation of thecandidate SNPs for three of the ESTs was carriedout by PCR-based resequencing of amplicons from10 unrelated commercial birds, using previouslydescribed protocols (Smith et al., 2001).

Results and discussion

Of the 200 clones sequenced, a total of 192sequences exceeded a Phred quality score of 30(Ewing et al., 1998). These 192 sequences weresubmitted to GenBank and have been assignedaccession numbers (Table 1; and at http://filebox.vt.edu/users/esmith/Hartman Va Tech CFGsupplement/Hartman Va Tech Table 1.doc). Atotal of 17 ESTs (9%) matched neither GenBankcDNA sequences nor the newly assembled chickengenomic DNA sequence. Additionally, only 80ESTs matched known chicken gene or cDNAsequences. All but 28 ESTs aligned with genomicDNA sequences assigned to chicken chromo-somes. Ninety-one (about 47%) ESTs aligned withsequences assigned to macrochromosomes (GGA)1–6, and four sequences aligned to genomic DNAsequences assigned to the Z chromosome. An addi-tional three ESTs aligned with sequences desig-nated to be on the Z random, while one matched

Copyright 2005 John Wiley & Sons, Ltd. Comp Funct Genom 2005; 6: 301–306.

http://genome.ucsc.edu/cgi-bin/hgBlat?com-mand=start&org=Chicken&db=galGal2&hgsid=30295885

http://filebox.vt.edu/users/esmith/Hartman_Va_Tech_CFG_supplement/Hartman_Va_Tech_Table 1.doc

http://genome.ucsc.edu/cgi-bin/hgBlat?com-mand=start&org=Chicken&db=galGal2&hgsid=30295885


Comparative analysis of chicken ESTs 303

Tab

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347

564

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240

166

2724

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1152

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358

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513

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a W chromosome sequence. The highest numberof ESTs, 30, matched sequences from chromo-some 1, while none aligned with sequences frommicrochromosomes 16, 19, 21, 25 and 26. Nine-teen ESTs (11.7%) aligned with sequences desig-nated ‘unknown,’ which are reported by the Inter-national Chicken Genome Sequencing Consortium(2004) to be about 12% of the chicken genome.Seven ESTs matched sequences assigned either tomore than one region of a chromosome or ondifferent chromosomes. A few ESTs aligned withsequences from Escherichia coli, which could bedue to bacterial contamination or simply to con-served sequences. The chromosomal assignmentsof some of the ESTs should be considered putative,as there are still many errors in the draft chickengenomic DNA sequence. The incompleteness of theGallus gallus DNA sequence may also account forthe relatively high percentage of ESTs that showedno significant sequence similarity to known chickensequences.

The chromosomal assignment of VTEST71 tochromosome 18, based on the sequence alignment

with the recently released genomic DNA, was con-firmed by radiation hybrid mapping. VTEST71 isdesignated as locus VTC08 on the chicken radi-ation hybrid map and is flanked by MCW0217and ADL0290, with LOD scores of 10.7 and 13.1,respectively. VTEST71 showed 99% sequence sim-ilarity to chicken histone protein H3 and 95% iden-tity with human Histone H3.3 (AK130772). Previ-ously, chicken H3 was also mapped to chromosome18 by RFLP, while the human H3 was linked tochromosome 17 (Levin et al., 1994).

A total of 22 SNPs were identified and vali-dated in the three ESTs scanned (data not pre-sented). Eight of the SNPs were non-synonymousand are described in Table 2. All the SNPs appearto be novel and have not been previously described(Smith et al., 2002; Wong et al., 2004). There-fore, these SNPs, although few, may be useful inefforts to assign phenotypes to genotypes and iden-tifying the effects of the three genes on differentchicken traits, e.g. knowledge of the function ofcofilin, an essential protein for depolymerization ofactin filaments, is still limited (Arber et al., 1998).

Table 2. Sequence contexts of SNPs validated by resequencing

ID of VTESTMatched gene/Accession No./%similarity/length of match (bp) EST-SNP sequence context/position∗

VTEST 85 Gallus gallus cofilin mRNA/M55659/98% ccatggct(tct, S → tgt, W)ggagtaacag/49†

648/661 tttaatgac(atg, M, → ttg, L)aaagtaa/93†

aagaaag(c/g)cgttctcttctgct/145gagacaaagga(a/g)tctaag(aag, K → agg, R)ga/320, 328†

aggtattaaacatga(g/a)tggcaagta/443cagacaagt(g/a)ccatctggatcta/557tggaatagt(a/g)ttagtctcccttt/610cctggtagtttta(t/c)gtaggatccaa/734ggtgggatgg(t/c)agactctatac/900gcacacaaca(c/t)tatgcatttaa/1018catatctta(t/c)aaatgaagtagct/1150aaacatcgg(t/c)catgatggca/1270

VTEST 56 Gallus gallus collapsin response mediatorprotein/U17 277/99%

gtgggaagatc(gtc, V → gcc, A)aatgacgac/375†

639/642 tgattac(tcc, S → tgc, C)ctgcacgtggac/708†

tggctttagc(ttg, L → atg, M)tctggcgcact/1922†

VTEST 137 Gallus gallus elongation factor 1α

mRNA/L00677/99% 643/649ctaaagacca(t/c)ccgaaatgggaa/55

aggaccatcg(a/g)gaagttcg/aagaa/179,187cttttgtgcca(a/g)tctctggttggaacgg/637caactgaca(a/g)acctctgcgtct/791gaaagatgtccgc(cgt,R → ggt,G)ggtaacgttg/1024†

∗ Each sequence context is followed by the position or locus of the SNP in the GenBank sequence of the matched Gallus gallusgene. Within each sequence context, the two alleles at the SNP locus are shown in parentheses. Each allele was observed in aminimum of two chromosomes or a frequency of 10% in a commercial population previously described (Smith et al., 2002)† Represents a non-synonymous change. The amino acid and codon changes are both indicated.


Comparative analysis of chicken ESTs 305

The three non-synonymous SNPs described may beuseful in further defining its role in skeletal func-tion and the dynamics of actin filaments. Similarly,the recently discovered collapsin response media-tor gene product is thought to have a role in theincidence and/or severity of Alzheimer’s disease(Yoshida et al., 1998). The SNPs described in thisgene in Gallus gallus may be useful in investigat-ing the role of this apparently important gene thatis also expressed in the chicken pineal gland.

It is not surprising that only 45% of the ESTsaligned to GGA1-6 DNA sequences, which com-prise approximately 65% of the chicken genome.In their analysis of the draft sequence, the Inter-national Chicken Genome Sequencing Consortium(2004) reported that the density of CpG islandsshowed a strong negative correlation with chro-mosome length. This distribution supports ear-lier studies by McQueen et al. (1998) and Smithet al. (2000) of a higher density of genes onthe microchromosomes than on the macrochromo-somes. Several explanations are possible for the9% of ESTs that did not match known sequencesin GenBank, including novelty in vertebrates, tooshort to match known sequences, or contamination.In a recent comparative gene analysis between thechicken and human genomes, Castelo et al. (2005)predicted that the undiscovered genes in the humangene set may be very low, at a predicted lower limitof about 0.2%.

The number of ESTs and SNPs described in thepresent work are small relative to the total num-bers of both genomic reagents currently available inGenBank and other databases. That they are poten-tially useful, however, is evident by the novelty ofsome of the sequences. Since a significant fractionmatched mammalian genes and/or DNA sequences,they can be used as resources for comparativegenome analysis of genes expressed in the pinealgland. Such comparative analysis may be useful inassigning function to chicken sequences. A simi-lar impact on chicken biology is also likely withthe SNPs described. Finally, it is worthy of notethat one of the ESTs matched a W chromosome-assigned sequence. Currently, the number of genesassigned to this chromosome is limited. As effortssuch as ours, even though limited in scope, iden-tify additional ESTs, it will provide the genomicreagents essential to further increase our under-standing of a chromosome that continues to be littleunderstood.

Acknowledgements

We thank Dr Alain Vignal of the National Institute of Agri-cultural Research, France, for providing the RH panel, andKwaku Gyenai in the Comparative Genomics Laboratoryat VT for the EST homology data from BLAST analysis.We are also grateful to three anonymous reviewers whosecomments were used to make an extensive revision to theResults and Discussion.

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Nucleic AcidsJournal of

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Enzyme Research


International Journal of

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research article characterization of expressed sequence...

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