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Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1–q12.2
Gene 348 (2005) 73 – 81 www.elsevier.com/locate/gene
Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1–q12.2 Anne Wfhlke*, Cord Drfgemqller, Heidi Kuiper, Tosso Leeb, Ottmar Distl Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bu¨nteweg 17 p, 30559 Hannover, Germany Received 30 August 2004; received in revised form 19 November 2004; accepted 22 December 2004 Received by M. D’Urso
Abstract The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report. D 2004 Elsevier B.V. All rights reserved. Keywords: GART; Cattle; Gene structure; BAC clone; FISH; SNP
1. Introduction The glycinamide ribonucleotide formyltransferase (GART) gene is part of a gene family that encodes multifunctional enzymes (Aimi et al., 1990). These enzymes have been found in several metabolic pathways in eukaryotes. In mammals and birds, the GART gene encodes Abbreviations: BAC, bacterial artificial chromosome; bp, base pair; BLASTN, basic local alignment search tool nucleotide; BTA, bovine chromosome; E2F, E2 transcription factor; EST, expressed sequence tag; FISH, fluorescence in situ hybridization; GART, glycinamide ribonucleotide formyltransferase; HSA, human chromosome; kb, kilobase; kDa, kilodalton; MMU, mouse chromosome; nt, nucleotide; Rb, retinoblastoma; RH, radiation hybrid; RT-PCR, reverse transcriptase polymerase chain reaction; SNP, single nucleotide polymorphism; Sp1, stimulating protein 1. * Corresponding author. Tel.: +49 511 9538872; fax: +49 511 9538582. E-mail address: [email protected] (A. Wfhlke). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.12.038
enzymes of purine synthesis, which catalyze three steps of this pathway (glycinamide ribonucleotide synthetase (GARS), glycinamide ribonucleotide formyltransferase (GART) and aminoimidazole ribonucleotide synthetase (AIRS)) (Kan et al., 1993). The enzymes GARS, GART and AIRS catalyze the second, third and fifth step of the de novo purine biosynthetic pathway, respectively (Daubner et al., 1985). The human gene encodes not only the trifunctional protein of 110 kDa, but also a monofunctional GARS protein of 50 kDa (Brodsky et al., 1997). Previous studies on the human and mouse GART locus demonstrated that the mRNA for monofunctional GARS is produced by the use of polyadenylation signals present in intron 11 (Kan and Moran, 1995, 1997). The expression of the monofunctional and trifunctional proteins is regulated during development of the human cerebellum (Brodsky et al., 1997). All three proteins are expressed at a high level in the cerebellum during normal prenatal development and become undetect-
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able in the cerebellum shortly after birth. In individuals with Down syndrome the expression of these proteins continues in the postnatal development of the cerebellum (Brodsky et al., 1997). The human GART locus maps to human chromosome (HSA) 21q22.1 (Chadefaux et al., 1984). The human GART gene consists of 22 exons and spans 38.1 kb (NCBI map viewer, human genome build 34.3). The murine Gart gene also consists of 22 exons spanning about 25.5 kb on chromosome MMU 16C3-C4 (NCBI map viewer, mouse genome build 32.1). Because the bovine genome represents an evolutionary clade distinct from primate or rodent genomes we selected the bovine GART gene for sequencing. Until now no sequence information of the orthologous bovine gene has been reported. In this report, we provide the cloning, chromosomal assignment, genomic organization and the complete sequence of the bovine GART gene, respectively. Additionally, we present data on new single nucleotide polymorphisms (SNPs) in this gene.
2. Materials and methods
Table 1 Sequences for annotation of bovine GART exons Exon no.
EST accession nos.
1 2–4 5 6 7 8–11 12–14 15–17 18–20 21 22 23
BE487216 BE487216 BI681173 CN789820 CN789820 BM967887 CK837096 CB457481 CB457481
Experimental bovine cDNAs
Human mRNA isoform 1
AJ783707 AJ783707
NM_000819 NM_000819 NM_000819 NM_000819 NM_000819 NM_000819 NM_000819 NM_000819 NM_000819 NM_000819
AJ783709 AJ783709
genomic alignment program Spidey (http://www.ncbi. nlm.nih.gov/IEB/Research/Ostell/Spidey/index.html) was used. The putative promoter sequence was analyzed in silico with the program MatInspector from Genomatix (http:// www.genomatix.de/cgi-bin/matinspector_prof/). GC content was calculated with the EBI toolbox CpG Plot/CpGreport (http://www.ebi.ac.uk/Tools/sequence.html).
2.1. Cloning and sequencing of the bovine GART gene 2.2. cDNA synthesis, RT-PCR For the isolation of a bovine BAC clone with the GART gene the bovine BAC library RPCI-42 (Warren et al., 2000) was initially screened with a 32P-labeled insert of a human IMAGE cDNA clone (IMAGp998J037162Q2) provided by the German Human Genome Resource Center/Primary Database (http://www.rzpd.de/) from the orthologous human gene. DNA from the clone RP42564N14 was isolated using the Qiagen Large Construct kit (Qiagen, Hilden, Germany). BAC DNA was mechanically sheared to obtain fragments of approximately 2 kb. Sheared BAC DNA was used to construct a shotgun plasmid library. Plasmid subclones were sequenced with the ThermoSequenase kit (Amersham Biosciences, Freiburg, Germany) and a LICOR 4200 automated sequencer (MWG Biotech, Ebersberg, Germany). Sequence data were analyzed with Sequencher 4.1.4 (GeneCodes, Ann Arbor, MI). Remaining gaps were closed by a primer walking strategy until both strands were completely sequenced. Repetitive elements were detected with Repeatmasker 2 (http://www.repeatmasker.genome. washington.edu/). The genomic structure of the bovine GART gene was determined by using the genomic DNA sequence as query in BLASTN (basic local alignment search tool nucleotide) analyses of the bovine expressed sequence tag (EST) databases (http://www.ncbi.nlm.nih.gov/BLAST/). The ESTs which were used to determine the exon organization of the bovine GART gene are given in Table 1. Only when no corresponding bovine EST could be detected the human GART mRNA (Table 1) was used to annotate the GART exons on the genomic sequence. For the exact localization of the exon/intron boundaries the mRNA-to-
RNeasyk 96 Universal Tissue kit (Qiagen) was used to extract total RNA from bovine liver, heart muscle, spleen, kidney, brain, small intestine, pituitary, lung and skin, respectively, from a normal German Holstein calf according to the manufacturer’s protocol. Aliquots of 1 Ag total RNA were reverse transcribed into cDNA using 20 pmol (T)24V primer and Omniscriptk Reverse Transcriptase (Qiagen) in 20 Al reactions. One microliter of the cDNA was used as template in a reverse transcriptase polymerase chain reaction (RT-PCR) reaction. The reaction was performed in a total of 25 Al containing 100 AM dNTPs, 25 pmol of each primer, the reaction buffer supplied by the manufacturer (Qiagen) and 1 U Taq polymerase. After a 5 min initial denaturation at 94 8C, 35 cycles of 30 sec at 94 8C, 1 min at 58 8C and 45 sec at 72 8C were performed in a MJ Research thermocycler (Biozym, Hess. Oldendorf, Germany). To detect the two possible splice variants, we created a single forward primer overlapping the exon 10/11 junction (Ex10-11_F 5V-AGA TAA CAG GGT TTC CTG AG-3V). The GART isoform 1 was detected by a combination of this forward primer with a reverse primer situated at the junction of exon 13/14 (Ex13-14_R 5V-ACT GCT GGG CAA TCT TCA GT-3V) and the GART isoform 2 by a combination with a reverse primer located in intron 11 (GEx11_R 5V-CCT TTG TTC AGG TTC AGT GG-3V). For detection of the 3V end of GART isoform 1, we used a forward primer situated at the junction of exon 21/22 (Ex21-22_F ACT TTG TAG CTG AAG ATG TAG ATG C) and a reverse primer (poly A_R ACC ATC TGT GTG GGT TTT CC) near the predicted polyadenylation signal.
A. Wo¨hlke et al. / Gene 348 (2005) 73–81
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lation using a Nick-Translation-Mix (Boehringer Mannheim, Mannheim, Germany). Fluorescence in situ hybridization (FISH) on GTG-banded bovine chromosomes (ISCNDB, 2000) was performed using 750 ng of digoxigenin-labelled BAC DNA. One Ag sheared total bovine DNA and 10 Ag salmon sperm were used as competitors in this experiment. After hybridization over night, signal detection was performed using a Digoxigenin-FITC Detection Kit (Quantum Appligene, Heidelberg, Germany). The chromosomes were counterstained with 4,6-diamino-2phenylidole (DAPI) and propidium iodide and embedded in antifade. Thirty metaphases that were previously photographed were re-examined after hybridization with a Zeiss Axioplan 2 microscope equipped for fluorescence.
The obtained RT-PCR products were directly sequenced using the PCR primers as described below. 2.3. Mutation analysis To identify variations within the bovine GART sequence, exons with flanking regions were PCR amplified and sequenced from 24 unrelated animals from the German Fleckvieh (17), German Holstein (3) and Pinzgauer (4) breed. PCR primers and conditions for the amplification of GART exons with flanking sequences are given in Table 2. PCR primers were developed with the Primer 3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). PCR assays were performed as described above. The obtained PCR products were directly sequenced with the DYEnamic ET Terminator kit (Amersham Biosciences) and a MegaBACE 500 capillary sequencer (Amersham Biosciences), using the PCR primers as sequencing primers.
3. Results and discussion
2.4. Fluorescence in situ hybridization
3.1. Analysis of the genomic organization of the bovine GART gene
The 120 kb bovine BAC clone containing the bovine GART gene was labelled with digoxigenin by nick trans-
A human GART cDNA clone was used to screen a genomic bovine BAC library and five positive clones were
Table 2 PCR primers for the amplification of GART exons Exon
Primer
Sequence (5V–3V)
Exon 1
GEx1_F GEx1_R GEx2_F GEx2_R GEx3_F GEx3_R GEx4_F GEx4_R GEx5_F GEx5_R GEx6-7_F GEx6-7_R GEx8_F GEx8_R GEx9-10_F GEx9-10_R GEx11_F GEx11_R GEx12-14_F GEx12-14_R GEx15-16_F GEx15-16_R GEx17_F GEx17_R GEx18_F GEx18_R GEx19-20_F GEx19-20_R GEx21-22_F GEx21-22_R GEx23_F GEx23_R
TGA CAA GCC TGC ATG AAC TCT CAA TGG GCA GAA CCA TGA ACT ATT GAT TCA CCT GGT CTT TGT CCA CAG TAG AGT AAA GCT ATC GAC AAA CCC TGA
Exon 2 Exon 3 Exon 4 Exon 5 Exons 6–7 Exon 8 Exons 9–10 Exon 11 Exons 12–14 Exons 15–16 Exon 17 Exon 18 Exons 19–20 Exons 21–22 Exon 23
GAG CAC ACA TAT GTG TTT GCC ATG TAT GTG CCT AAG TAA CAA CAT GGC CCC TTG ACT ATA TTG CAA ATG ACC TGA GTG TGT ACT AAT CCT AGA ATG
Product length (bp) TAA ACA TTT AAC AAA TCC ATC TAT GCT AGA GGA TGA CAT GGT GGA CTC TAG TTC CTT CGC CAG GAA GAT CTG AGT GAA TCC GGG GAA GAA TTT ACT
ATC AGC GTC CTG CTC CAA TGC CCT CTC CCC AAG GGC GGT TTT GGA ACA AGC AGG TCC AGG AAT GTC CCC AGG TGG GGA TCA TCA AAC AGA CAG TGG
CGC AGC TCT AAC TCC GGT CTT TTC AGG AGC GAG CAT GCC GGA AGT CCC AGA TTC TTC GCT ACA CAA CAG CAG CAG CTG TCC TTC ACC CCA CTT TCA
CAA TGA TGT AAC TGC AAT ATC CCC GTA ACA GCT AAA CTT ATG TTA TTA ACA AGT CCA GAA GCT ATG AAA AAA TGT AGG TTC TCT CAA CTG CAC GAG
AC AC CC AGG A AA GTT G TC CTA GG AC AG TC ACC CAG C C GG GG TAC C AC TCC ATG G TG CC ATG AG GG CC GAG AG GAC
Annealing temperature (8C)
451
58
872
58
373
58
513
58
659
58
1092
58
500
58
976
58
518
58
1422
58
956
58
533
58
506
58
934
55
623
55
888
58
76
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Table 3 Exon/intron boundaries of the bovine GART gene
3'-Splice site
5'-Splice site
Exon
Intron phase
Intron size
... (exon 1,
>38 bp) ...
-42 CGCCGGgtgagtttcctatgc
-41 gttttctttctgcagCTTTCA ... (exon 2,
186 bp) ...
+145 ATACTGgtaatgacttttttt
1
1082 bp
+146 tttctcttttcacagACATCT ... (exon 3,
96 bp) ...
+241 CTGCTGgtaaagttcatttct
1
477 bp
+242 tattttcatcactagGAATTG ... (exon 4,
175 bp) ...
+416 TATGAGgtagatagaagacag
2
3244 bp
+417 cctctgtcttcacagTGCAGA ... (exon 5,
112 bp) ...
+528 ATGCAGgtaagttgatccttt
0
969 bp
+529 tttatctccaataagGGTAAA ... (exon 6,
69 bp) ...
+597 GTGTCTgtatgtatatccatc
0
502 bp
+598 ttgttttttctgcagTGTCTG ... (exon 7,
126 bp) ...
+723 CCTCAGgtagccatcatttgg
0
1077 bp
+724 ctctgtattccccagGTTTCT ... (exon 8,
88 bp) ...
+811 ACACAGgtaagcacatggatg
1
489 bp
+812 tcttttttaatgaagGTGTCC ... (exon 9,
86 bp) ...
+897 TGCCAAgtgagtaaaaaaaga
0
164 bp
+898 ttgtgtgttttccagGTGATC ... (exon 10, 169 bp) ...
+1066 TAACAGgtgaacatcagggaa
1
2972 bp
2686 bp
Isoform 2 only +1067 +1302 tgctttctcttacagGGTTTC ... (exon 11, 236 bp) ... GCCCAGGTAAACTT ... TTCCAATAAAAAA Isoform 1 only +1067 tgctttctcttacagGGTTTC ... (exon 11, 232 bp) ...
+1298 GCCCAGgtaaacttgtgaccc
2
1879 bp
+1299 ttttgaactttttagGGGCCT ... (exon 12,
+1393 GACCAGgtaagtcagatcctg
1
294 bp
+1394 ttttctctccaacagGCTGTG ... (exon 13, 110 bp) ...
+1503 GCTGAAGgtaatcagactta
0
342 bp
+1504 tatacccttcggtagATTGCC ... (exon 14, 199 bp) ...
+1702 TCCTCGgtatgcacttccatt
1
1042 bp
+1703 ttctcttcctgccagGAGGTG ... (exon 15, 252 bp) ...
+1954 CCTTAGgtacacacgctcacg
1
292 bp
+1955 cttcccaatatacagGTGACT ... (exon 16, 153 bp) ...
+2107 ATTTAGgtaagatcactaaaa
1
2003 bp
+2108 cttgcctgtttttagATGCCC ... (exon 17, 207 bp) ...
+2314 CCAAGGgtaacctcttccttc
1
1085 bp
+2315 acatttgcaccaaagGTTCTC ... (exon 18, 138 bp) ...
+2452 GGACAGgtgagacatggctcc
1
993 bp
+2453 tttgccttttttcagGATCAA ... (exon 19, 131 bp) ...
+2583 ACGAGAgtaagttactttgaa
0
241 bp
+2584 ccatttctcttctagGTAATT ... (exon 20, 142 bp) ...
+2725 GGAATGgtaagtaaaaaactg
1
657 bp
+2726 ttaattaatttccagGGAAAA ... (exon 21, 116 bp) ...
+2841 GTAGCTgtgagtatggctcgt
0
82 bp
+2842 tgctttcttttccagGAAGAT ... (exon 22, 230 bp) ...
+3071 AGCATTgtaggtgctgcaggg
+3072 tctttgtgtttgaagGCCTCC ... (exon 23, 378 bp) ...
+3449 GATGAATAAAAGACTTCCTAA
95 bp) ...
765 bp
A. Wo¨hlke et al. / Gene 348 (2005) 73–81
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GART (A) AATAAA
AATAAA
] 1
2
3 4
5
6 7
8 9 10
11
12 13 14
15 16
17
18
19 20 21 22 23
(B)
1kb SINE LINE
(C)
ATG
isoform 2
(D)
2
3 4
5
6 7
8 9 10
11
1
ATG 2 3 4
5
6 7
8 9 10
TAA 11
]
12 13 14
1516
17
18
19 20 21 22 23
] CpG Island
isoform 1
TGA
1
GC content
60%
40%
20%
Fig. 1. Genomic structure of the bovine GART gene. (A) Translated exons are shown as solid boxes. Untranslated regions of exons are shown as open boxes. Polyadenylation sites are indicated. (B) Repetitive sequences are indicated beneath the genomic structure. (C) In this part of the figure, the splicing of transcript isoforms 1 and 2 is illustrated. (D) GC content and CpG islands are shown in the lower part of the figure. For the calculation of the GC content, a 300 bp window was used.
isolated. Comparative BLAST analysis of the bovine BAC clone end sequences with respect to the human genome suggested that the clone RP42-564N14 contained the entire open reading frame of the GART gene. This BAC clone was selected for sequencing and the complete 120,377 bp DNA sequence was determined. As the last untranslated exon of the bovine GART gene was not contained on the BAC clone, the genomic sequence was extended for another 1581 bp using cattle whole genome shotgun sequences from the trace archive (Trace Archive identifier TI171578883, TI168408439, TI75419957, TI80262915). Thus, a contiguous sequence of 121,958 bp harbouring the complete GART gene was submitted to the EMBL nucleotide database (accession no. AJ780930). Using EST sequences and
RT-PCR analyses for cDNA-genomic sequence comparisons we detected that the bovine GART gene consists of 23 exons with exon/intron boundaries that conform perfectly to the GT/AG rule (Table 3). During cDNA sequence analyses it was determined that the bovine GART gene consists of 23 exons contrary to the human and murine GART genes, which only consist of 22 exons. This is caused by an additional intron in the 3VUTR of the bovine GART gene. The exon sizes range from 69 to 252 bp, the introns between these exons span between 82 and 3244 bp, the total size of the bovine GART gene is approximately 27 kb. However, the sequence homology between the human, murine and bovine GART gene is limited to the coding sequence of exons 2 to 22. In the coding sequence of isoform 1, the
Notes to Table 3: Exon sequences are shown in uppercase letters and intron sequences in lowercase letters. Untranslated regions are shown in italics. The conserved GT/AG exon/intron junctions are shown in boldface type. For exons 11 and 23, the polyadenylation signals are shown underlined instead of an exon/intron junction. Position +1 corresponds to the adenine of the translation initiation codon ATG.
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1 10
11 12 13 14
10
11
2
ntc liv mus spl 1 2 1 2 1 2 1 2
TAA
kid brn int pit lng 1 2 1 2 1 2 1 2 1 2
skn 1 2
M 459 bp 328 bp
Fig. 2. RT-PCR amplification of two GART mRNA isoforms. RNA of nine different bovine tissues (liv: liver, mus: heart muscle, spl: spleen, kid: kidney, brn: brain, int: small intestine, pit: pituitary, lng: lung, skn: skin) was reverse transcribed and amplified using a forward primer located at the exon 10/11 junction in combination with two alternative reverse primers located at the exon 13/14 junction (1) or in intron 11 (2), respectively. (M: 100 bp ladder, ntc: no template control).
bovine GART gene displays 88.0% similarity to the human GART gene and 81.9% similarity to the murine Gart gene, respectively. The coding sequence of isoform 2 displays 89.0% and 82.7% similarity to the human and murine GART gene, respectively. In the untranslated regions including the entire first exon the sequence similarity between human, mouse and cattle is rather low. The repeat content in the 27 kb region of the bovine GART gene is 27.1% (Fig. 1). The fraction of the SINE (11.7%) and LINE (13.8%) elements are nearly balanced. Other repetitive elements constitute 1.7%. The entire bovine GART gene has an overall GCcontent of 40.3% which is in the range of the mammalian average of 41% (Fig. 1). The bovine GART gene contains one CpG island (Gardiner-Garden and Frommer, 1987) in the region of the first exon (Fig. 1) and the putative promoter (GC content of 58.6% over 483 bp). Sequence analysis of the genomic region upstream of the putative transcription start site indicated the absence of TATA and CCAAT boxes, but the presence of one Sp1 and one E2F binding site. E2F is involved in cell cycle regulation such as DNA synthesis and plays a critical role in the expression of genes involved in differentiation and development (Neuman et al., 1996). E2F interacts with the retinoblastoma (Rb) family member p107 protein. Previous studies described that the p107 protein is required for normal cerebellar development (Marino et al., 2003), which seems consistent with the postulated role of GART in cerebellar development. A possible regulation of the GART gene by E2F seems plausible as an upregulation of GART during S phase of the cell cycle could be required for sufficient purine biosynthesis during DNA replication. 3.2. Analysis of the bovine GART cDNA The bovine mRNA to genomic alignment indicated the existence of two transcript isoforms produced by the bovine
GART gene. In order to confirm the existence of the two transcripts in cattle, cDNA was synthesized. Expression studies of the gene in nine different organs by RT-PCR amplification showed that both GART transcripts are widely expressed in bovine tissues (Fig. 2). The three obtained RTPCR products were sequenced and the generated sequence data were submitted to the EMBL nucleotide database (accession nos. AJ783707, AJ783708, AJ783709). The bovine GART gene showed an additional non coding exon no. 23 at the 3V end. The transcript of isoform 1 is encoded by the exons 1–23 and the shorter transcript of isoform 2 by the exons 1–11, respectively (Fig. 1; accession no. AJ783708). The cDNA of bovine GART isoform 1 contains an open reading frame of 3033 nt coding for a protein of 1010 amino acids and the GART isoform 2 transcript contains an open reading frame of 1302 nt coding for a protein of 433 amino acids, respectively. The translation start codon was assigned based on the homology to the human ortholog (Fig. 1). The polyadenylation signal AAUAAA of the longer isoform 1 is located approximately 1.2 kb downstream of the stop codon (Fig. 1). The second shorter GART transcript is terminated by a polyadenylation signal AAUAAA 504 bp downstream of the stop codon in intron 11 (Fig. 1). The bovine GART isoform 1 protein displays 88.3% and 84.6% similarity to the human and murine GART proteins (Fig. 3). The bovine GART isoform 2 protein displays 89.8% similarity to the human GART protein and 84.8% to the murine Gart protein, respectively (Fig. 3). 3.3. Polymorphisms within the bovine GART gene The search for sequence variations within the GART gene revealed a total of 11 SNPs shown in Table 4. One out of these 11 SNPs located in exon 17 affect the amino acid sequence at position 771 of bovine GART protein. This SNP causes an amino acid change from lysine to glutamic
Fig. 3. Alignment of the bovine GART isoform 1 protein (1010 amino acids) with known orthologous GART isoform 1 protein sequences. The sequences were derived from GenBank entries with the accessions NP_000810 (human GART) and NP_034386 (mouse Gart). The bold amino acid indicates the last position of the shorter GART isoform 2 protein at position 433. Identical residues are indicated by asterisks beneath the alignment, while colons and dots represent very similar and similar amino acids, respectively.
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cattle human mouse
MAARVLVIGNGGREHTLAWKLAQSTHVKQVLVTPGNAGTACSEKISNTDISISDHTALAQ MAARVLIIGSGGREHTLAWKLAQSHHVKQVLVAPGNAGTACSEKISNTAISISDHTALAQ MAARVLVIGSGGREHTLAWKLAQSPQVKQVLVAPGNAGTAGAGKISNAAVSVNDHSALAQ ****** **:**************::****** *******: :**** : * :** ****
60 60 60
cattle human mouse
FCKDEKIEFVVVGPEAPLAAGIVGNLNSVGVRCFGPTAQAAQLESSKRFAKEFMDRHGIS FCKEKKIEFVVVGPEAPLAAGIVGNLRSAGVQCFGPTAEAAQLESSKRFAKEFMDRHGIP FCKDEKIELVVVGPEAPLAAGIVGDLTSAGVRCFGPTAQAAQLESSKKFAKEFMDRHEIP *** :***:***************:*:*:**:******:******** *********:*:
120 120 120
cattle human mouse
TARWRAFTKPKEACDFIMSADFPALVVKASGLAAGKGVIVAKSKEEACEAVREIMQGKAF TAQWKAFTKPEEACSFILSADFPALVVKASGLAAGKGVIVAKSKEEACKAVQEIMQEKAF TAQWRAFTNPEDACSFITSANFPALVVKASGLAAGKGVIVAKSQAEACRAVQEIMQEKSF **:* ***:*: **:** **:**********************::***:**:****:* *
180 180 180
cattle human mouse
GEAGETVVIEELLEGEEVSCLCFTDGRTVAPMPPAQDHKRLLEGDEGPNTGGMGAYCPAP GAAGETIVIEELLDGEEVSCLCFTDGKTVAPMPPAQDHKRLLEGDGGPNTGGMGAYCPAP GAAGETVVVEEFLEGEEVSCLCFTDGKTVAEMPPAQDHKRLLDGDEGPNTGGMGAYCPAP *:**** * **:* ************ ***:*********** **:**************
240 240 240
cattle human mouse
QVSKDLLLKIKNNILQRTVDGMQEEGMPYTGVLYAGIMLTKNGPKVLEFNCRFGDPECQV QVSNDLLLKIKDTVLQRTVDGMQQEGTPYTGILYAGIMLTKNGPKVLEFNCRFGDPECQV QVSKDLLVKIKNTILQRAVDGMQQEGAPYTGILYAGIMLTKDGPKVLEFNCRFGDPECQV ***:*** ***:: *** *****:**:**** *********:******************
300 300 300
cattle human mouse
ILPLLKSDLYEVIQSILDGLLCTSLPVWLDNCAAVTVVMASKGYPGDYTKGVEITGFPEA ILPLLKSDLYEVIQSTLDGLLCTSLPVWLENHTALTVVMASKGYPGDYTKGVEITGFPEA ILPLLKSDLYEVMQSTLGGLLSASLPVWLENHSAVTVVMASKGYPGAYTKGVEITGFPEA ************ **:*:***: ****** *: * ***********:*************
360 360 360
cattle human mouse
QALGLEVFQAGTALKDGKVVTNGGRVLTVTAIRENLISALEEARKGLAAIKFEGAVYRKD QALGLEVFHAGTALKNGKVVTHGGRVLAVTAIRENLISALEEAKKGLAAIKFEGAIYRKD QALGLQVFHAGTALKDGKVVTSGGRVLTVTAVQENLMSALAEARKGLAALKFEGAIYRKD *****:**:******:*****:***** *** :*** ***:** ***** ***** ****
420 420 420
cattle human mouse
IGFRAIAFLQQPRGLTYKESGVDIAAGNMLVQKIKPLAKATSRPGCDVDLGGFAGLFDLK VGFRAIAFLQQPRSLTYKESGVDIAAGNMLVKKIQPLAKATSRSGCKVDLGGFAGLFDLK IGFRAVAFLQRPRGLTYKDSGVDIAAGNMLVKKIQPLAKATSRPGCSVDLGGFAGLFDLK **** ****:**:**** ************:**:********:**:*************
480 480 480
cattle human mouse
AAGFTDPLLACGTDGVGTKLKIAQQCSKHDTIGQDLVAMCVNDILAQGAEPLFFLDYFSC AAGFKDPLLASGTDGVGTKLKIAQLCNKHDTIGQDLVAMCVNDILAQGAEPLFFLDYFSC AAGFKDPLLASGTDGVGTKLKIAQLCNKHDSIGQDLVAMCVNDILAQGAEPLFFLDYFSC ****:*****:*************:*:*** *****************************
540 540 540
cattle human mouse
GKLDLRTTEAVITGIAKACKKAGCALLGGETAEMPDMYPPGEYDLAGFAVGAMERDQKLP GKLDLSVTEAVVAGIAKACGKAGCALLGGETAEMPDMYPPGEYDLAGFAVGAMERDQKLP GKLDLSTTEAVIAGIAAACQQAACALLGGETAEMPNMYPPGEYDLAGFAVGAMERHQKLP *****::**** ***:**::*:************:*******************:****
600 600 600
cattle human mouse
QLERITEGDAVIGIASSGLHSNGFSLVRKIVAKSSLEYSSPAPGGCGDQTLGDLLLTPTK HLERITEGDVVVGIASSGLHSNGFSLVRKIVAKSSLQYSSPAPDGCGDQTLGDLLLTPTR QLERITEGDAVIGVASSGLHSNGFSLVRKIVERSSLQYSSPAPGGCGDQTLGDLLLTPTR :********:* * *****************: ***:******:***************
660 660 660
cattle human mouse
IYSRSLLPVLRSGRVKAVAHITGGGLLENIPRVLPQKLGVNLDAQTWRVPRIFSWLQQEG IYSHSLLPVLRSGHVKAFAHITGGGLLENIPRVLPEKLGVDLDAQTWRIPRVFSWLQQEG IYSHSLLPIIRSGRVKAFAHITGGGLLENIPRVLPQKFGVDLDASTWRVPKVFSWLQQEG ***:**** ***:***:*****************:*:**:***:*** * ********
720 720 720
cattle human mouse
HLSEEEMARTFNCGIGAALVVSEDLVKQTLQDIEQHQEEACVIGRVVACPKGSPRVKVEH HLSEEEMARTFNCGVGAVLVVSKEQTEQILRDIQQHKEEAWVIGSVVARAEGSPRVKVKN ELSEEEMARTFNCGIGAALVVSKDQAEQVLHDVRRRQEEAWVIGSVVACPEDSPRVRVKN :************* **:****: :::*:*:* ::::***:***:***::::**** *::
780 780 780
cattle human mouse
LIETMQINGSVLENGTLRNHFSVQPKKARVAVLISGTGSNLQALIDSTREPSSLAHIVIV LIESMQINGSVLKNGSLTNHFSFEKKKARVAVLISGTGSNLQALIDSTREPNSSAQIDIV LIETIQTNGSLVANGFLKSNFPVQQKKARVAVLISGTGSNLQALIDSTRDPKSSSHIVLV *** *:*** :** *:::*::::************************ *:*: :*: *
840 840 840
cattle human mouse
ISNKAAVAGLDKAEKAGIPTRVINHKLYKNRAAFDTAIDEVLEEFSTDIVCLAGFMRILS ISNKAAVAGLDKAERAGIPTRVINHKLYKNRVEFDSAIDLVLEEFSIDIVCLAGFMRILS ISNKAAVAGLDRAERAGIPTRVINHKLSKNRVEFDNAVDHVLEEFSVDIVCLAGFMRILS *********** ** ************:***::** * *:******:*************
900 900 900
cattle human mouse
GPFVRKWNGKMLNIHPSLLPSFKGSNAHEQVLDAGVTVTGCTVHFVAEDVDAGQIILQEA GPFVQKWNGKMLNIHPSLLPSFKGSNAHEQALETGVTVTGCTVHFVAEDVDAGQIILQEA GPFVRKWDGKMLNIHPSLLPSFKGSNAHEQVLEAGVTITGCTVHFVAEDVDAGQIILQEA ****:**:**********************:* *** *********************
960 960 960
cattle human mouse
VPVKRGDTVETLSERVKLAEHKIFPSALQLVASGAVRLGENGRICWVTED VPVKRGDTVATLSERVKLAEHKIFPAALQLVASGTVQLGENGKICWVKEE VPVRRGDTVATLSERVKVAEHKIFPAALQLVASGAVQLREDGKIHWAKEQ **** *****:******* ******* ******* *:*:*:* *:*::*
1010 1010 1010
80
A. Wo¨hlke et al. / Gene 348 (2005) 73–81
Table 4 Single nucleotide polymorphisms within the bovine GART gene Location of polymorphic site
Positiona
Intron 4 Intron 5 Intron 5 Intron 5 Intron 11 Intron 15c Exon 17 Intron 18c Exon 19c Exon 19c Intron 20c
3054 245 253 837 1689 9 204 898 5 95 84
a b c
Bovine cDNA position
+2311 +2457 +2547
Nucleotide polymorphism TYA TYC TYC CYT CYG GYA AYG insA AYG GYC CYT
Amino acid substitution
771
LysY771Glu
silent silent
Allele frequencies
Genotype frequenciesb
0.90/0.10 0.79/0.21 0.92/0.08 0.85/0.15 0.42/0.58 0.98/0.02 0.67/0.33 0.83/0.17 0.94/0.06 0.94/0.06 0.96/0.04
19/5/0 19/0/5 20/4/0 17/7/0 6/8/10 23/1/0 12/8/4 20/0/4 21/3/0 21/3/0 22/2/0
Numbering refers to the position of the polymorphic nucleotide within the given exon or intron, respectively. Genotypes are given as number of animals [homozygous for allele 1/heterozygous/homozygous for allele 2]. These SNPs showed only a polymorphism in the German Fleckvieh animals.
acid. In the human and murine GART proteins the corresponding amino acid is glutamic acid. Six out of the observed 11 SNPs were polymorphic in all three examined breeds (Table 4).
bovine GART gene. Consistent with the human–cattle comparative map the localization of four genes on the sequenced BAC clone confirmed syntenic correspondences between bovine chromosome 1, human chromosome 21 and
3.4. Chromosomal assignment For the chromosomal localization of BAC clone RP42564N14, the BAC DNA was used as probe in a FISH experiment on bovine metaphase chromosomes. The assignment localized the GART gene to BTA 1q12.1–q12.2 (Fig. 4). The assignment of the bovine GART gene is in good agreement with established syntenies among human chromosome 21q, murine chromosome 16 and bovine chromosome 1q (McAvin et al., 1988; Rexroad and Womack, 1999; Band et al., 2000). 3.5. Analysis of flanking sequences Apart from the GART gene, the sequenced BAC clone also contained the complete SON DNA binding protein (SON), downstream neighbor of SON (DONSON) and crystalline zeta (quinone reductase)-like 1 (CRYZL1) genes as well as parts of the intersectin 1 (ITSN1) gene thus anchoring these genes as well to BTA 1q12.1–q12.2. In fact, the gene order of all four genes located on the sequenced BAC clone corresponds exactly to the human gene order (NCBI map viewer, build 34.3). The overall GC content of the reported BAC sequence is 40.4%. In the investigated sequence, five CpG islands are located. Repetitive elements constitute 23.2% of the analyzed DNA sequence. 3.6. Conclusions In conclusion, our results provide the complete annotated genomic sequence of the bovine GART gene. Expression studies showed that two GART transcripts are expressed in different bovine tissues. We identified 11 SNPs within the
Fig. 4. Chromosomal assignment of the bovine GART gene by FISH analysis. The digoxigenin labeled BAC clone RP42-564N14 containing the bovine GART gene was hybridized to GTG-banded metaphase chromosomes of a normal cattle. The chromosomes were counterstained with propidium iodide and subsequently identified by DAPI staining. (A) GTGbanding of the bovine metaphase spread. (B) Double signals are visible on both chromosomes 1q12.1–q12.2.
A. Wo¨hlke et al. / Gene 348 (2005) 73–81
murine chromosome 16, respectively. The presented study provides detailed information towards comparative mapping of the bovine genome.
Acknowledgments The authors would like to thank Heike Klippert-Hasberg and Stefan Neander for expert technical assistance. We gratefully acknowledge the Dr. h.c. Karl Eibl Foundation, Neustadt/Aisch, Germany and the Deutsche Forschungsgemeinschaft DFG (DI 333/8-1) for their financial support of this study.
References Aimi, J., Hong, Q., Williams, J., Zalkin, H., Dixon, J.E., 1990. De novo purine nucleotide biosynthesis: cloning of human and avian cDNAs encoding the trifunctional glycinamide ribonucleotide synthetase– aminoimidazole ribonucleotide synthetase–glycinamide ribonucleotide transformylase by functional complementation in E. coli. Nucleic Acids Res. 18, 6665 – 6672. Band, M.R., et al., 2000. An ordered comparative map of the cattle and human genomes. Genome Res. 10, 1359 – 1368. Brodsky, G., Barnes, T., Bleskan, J., Becker, L., Cox, M., Patterson, D., 1997. The human GARS-AIRS-GART gene encodes two proteins which are differentially expressed during human brain development and temporally over expressed in cerebellum of individuals with Down syndrome. Hum. Mol. Genet. 6, 2043 – 2050. Chadefaux, B., et al., 1984. Assignment of human phosphoribosylglycinamide synthetase locus to region 21q22.1. Hum. Genet. 66, 190 – 192.
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Daubner, S.C., et al., 1985. A multifunctional protein possessing glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase activities in de novo purine biosynthesis. Biochemistry 24, 7059 – 7062. Gardiner-Garden, M., Frommer, M., 1987. CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261 – 282. ISCNDB, 2000. International System for Chromosome Nomenclature of Domestic Bovids, Di Berardino, D., Di Meo, G.P., Gallagher, D.S., Hayes, H., Iannuzzi, L., (coordinator) (eds) 2001. Cytogenet. Cell Genet. 92, 283–299. Kan, J.L., Moran, R.G., 1995. Analysis of a mouse gene encoding three steps of purine synthesis reveals use of an intronic polyadenylation signal without alternative exon usage. J. Biol. Chem. 270, 1823 – 1832. Kan, J.L., Moran, R.G., 1997. Intronic polyadenylation in the human glycinamide ribonucleotide formyltransferase gene. Nucleic Acids Res. 25, 3118 – 3123. Kan, J.L., Jannatipour, M., Taylor, S.M., Moran, R.G., 1993. Mouse cDNAs encoding a trifunctional protein of de novo purine synthesis and a related single-domain glycinamide ribonucleotide synthetase. Gene 137, 195 – 202. Marino, S., Hoogervoorst, D., Brandner, S., Berns, A., 2003. Rb and p107 are required for normal cerebellar development and granule cell survival but not for Purkinje cell persistence. Development 130, 3359 – 3368. McAvin, J.C., Patterson, D., Womack, J.E., 1988. Mapping of bovine PGRS and PAIS genes in hybrid somatic cells: syntenic conservation with human chromosome 21. Biochem. Genet. 26, 334 – 398. Neuman, E., Sellers, W.R., McNeil, J.A., Lawrence, J.B., Kaelin Jr., W.G., 1996. Structure and partial genomic sequence of the human E2F1 gene. Gene 173, 163 – 169. Rexroad, C.E., Womack, J.E., 1999. Parallel RH mapping of BTA1 with HSA3 and HSA21. Mamm. Genome 10, 1095 – 1097. Warren, W., et al., 2000. Construction and characterization of a new bovine bacterial artificial chromosome library with 10 genome-equivalent coverage. Mamm. Genome 11, 662 – 663.
Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1–q12.2 Anne Wfhlke*, Cord Drfgemqller, Heidi Kuiper, Tosso Leeb, Ottmar Distl Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bu¨nteweg 17 p, 30559 Hannover, Germany Received 30 August 2004; received in revised form 19 November 2004; accepted 22 December 2004 Received by M. D’Urso
Abstract The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report. D 2004 Elsevier B.V. All rights reserved. Keywords: GART; Cattle; Gene structure; BAC clone; FISH; SNP
1. Introduction The glycinamide ribonucleotide formyltransferase (GART) gene is part of a gene family that encodes multifunctional enzymes (Aimi et al., 1990). These enzymes have been found in several metabolic pathways in eukaryotes. In mammals and birds, the GART gene encodes Abbreviations: BAC, bacterial artificial chromosome; bp, base pair; BLASTN, basic local alignment search tool nucleotide; BTA, bovine chromosome; E2F, E2 transcription factor; EST, expressed sequence tag; FISH, fluorescence in situ hybridization; GART, glycinamide ribonucleotide formyltransferase; HSA, human chromosome; kb, kilobase; kDa, kilodalton; MMU, mouse chromosome; nt, nucleotide; Rb, retinoblastoma; RH, radiation hybrid; RT-PCR, reverse transcriptase polymerase chain reaction; SNP, single nucleotide polymorphism; Sp1, stimulating protein 1. * Corresponding author. Tel.: +49 511 9538872; fax: +49 511 9538582. E-mail address: [email protected] (A. Wfhlke). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.12.038
enzymes of purine synthesis, which catalyze three steps of this pathway (glycinamide ribonucleotide synthetase (GARS), glycinamide ribonucleotide formyltransferase (GART) and aminoimidazole ribonucleotide synthetase (AIRS)) (Kan et al., 1993). The enzymes GARS, GART and AIRS catalyze the second, third and fifth step of the de novo purine biosynthetic pathway, respectively (Daubner et al., 1985). The human gene encodes not only the trifunctional protein of 110 kDa, but also a monofunctional GARS protein of 50 kDa (Brodsky et al., 1997). Previous studies on the human and mouse GART locus demonstrated that the mRNA for monofunctional GARS is produced by the use of polyadenylation signals present in intron 11 (Kan and Moran, 1995, 1997). The expression of the monofunctional and trifunctional proteins is regulated during development of the human cerebellum (Brodsky et al., 1997). All three proteins are expressed at a high level in the cerebellum during normal prenatal development and become undetect-
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A. Wo¨hlke et al. / Gene 348 (2005) 73–81
able in the cerebellum shortly after birth. In individuals with Down syndrome the expression of these proteins continues in the postnatal development of the cerebellum (Brodsky et al., 1997). The human GART locus maps to human chromosome (HSA) 21q22.1 (Chadefaux et al., 1984). The human GART gene consists of 22 exons and spans 38.1 kb (NCBI map viewer, human genome build 34.3). The murine Gart gene also consists of 22 exons spanning about 25.5 kb on chromosome MMU 16C3-C4 (NCBI map viewer, mouse genome build 32.1). Because the bovine genome represents an evolutionary clade distinct from primate or rodent genomes we selected the bovine GART gene for sequencing. Until now no sequence information of the orthologous bovine gene has been reported. In this report, we provide the cloning, chromosomal assignment, genomic organization and the complete sequence of the bovine GART gene, respectively. Additionally, we present data on new single nucleotide polymorphisms (SNPs) in this gene.
2. Materials and methods
Table 1 Sequences for annotation of bovine GART exons Exon no.
EST accession nos.
1 2–4 5 6 7 8–11 12–14 15–17 18–20 21 22 23
BE487216 BE487216 BI681173 CN789820 CN789820 BM967887 CK837096 CB457481 CB457481
Experimental bovine cDNAs
Human mRNA isoform 1
AJ783707 AJ783707
NM_000819 NM_000819 NM_000819 NM_000819 NM_000819 NM_000819 NM_000819 NM_000819 NM_000819 NM_000819
AJ783709 AJ783709
genomic alignment program Spidey (http://www.ncbi. nlm.nih.gov/IEB/Research/Ostell/Spidey/index.html) was used. The putative promoter sequence was analyzed in silico with the program MatInspector from Genomatix (http:// www.genomatix.de/cgi-bin/matinspector_prof/). GC content was calculated with the EBI toolbox CpG Plot/CpGreport (http://www.ebi.ac.uk/Tools/sequence.html).
2.1. Cloning and sequencing of the bovine GART gene 2.2. cDNA synthesis, RT-PCR For the isolation of a bovine BAC clone with the GART gene the bovine BAC library RPCI-42 (Warren et al., 2000) was initially screened with a 32P-labeled insert of a human IMAGE cDNA clone (IMAGp998J037162Q2) provided by the German Human Genome Resource Center/Primary Database (http://www.rzpd.de/) from the orthologous human gene. DNA from the clone RP42564N14 was isolated using the Qiagen Large Construct kit (Qiagen, Hilden, Germany). BAC DNA was mechanically sheared to obtain fragments of approximately 2 kb. Sheared BAC DNA was used to construct a shotgun plasmid library. Plasmid subclones were sequenced with the ThermoSequenase kit (Amersham Biosciences, Freiburg, Germany) and a LICOR 4200 automated sequencer (MWG Biotech, Ebersberg, Germany). Sequence data were analyzed with Sequencher 4.1.4 (GeneCodes, Ann Arbor, MI). Remaining gaps were closed by a primer walking strategy until both strands were completely sequenced. Repetitive elements were detected with Repeatmasker 2 (http://www.repeatmasker.genome. washington.edu/). The genomic structure of the bovine GART gene was determined by using the genomic DNA sequence as query in BLASTN (basic local alignment search tool nucleotide) analyses of the bovine expressed sequence tag (EST) databases (http://www.ncbi.nlm.nih.gov/BLAST/). The ESTs which were used to determine the exon organization of the bovine GART gene are given in Table 1. Only when no corresponding bovine EST could be detected the human GART mRNA (Table 1) was used to annotate the GART exons on the genomic sequence. For the exact localization of the exon/intron boundaries the mRNA-to-
RNeasyk 96 Universal Tissue kit (Qiagen) was used to extract total RNA from bovine liver, heart muscle, spleen, kidney, brain, small intestine, pituitary, lung and skin, respectively, from a normal German Holstein calf according to the manufacturer’s protocol. Aliquots of 1 Ag total RNA were reverse transcribed into cDNA using 20 pmol (T)24V primer and Omniscriptk Reverse Transcriptase (Qiagen) in 20 Al reactions. One microliter of the cDNA was used as template in a reverse transcriptase polymerase chain reaction (RT-PCR) reaction. The reaction was performed in a total of 25 Al containing 100 AM dNTPs, 25 pmol of each primer, the reaction buffer supplied by the manufacturer (Qiagen) and 1 U Taq polymerase. After a 5 min initial denaturation at 94 8C, 35 cycles of 30 sec at 94 8C, 1 min at 58 8C and 45 sec at 72 8C were performed in a MJ Research thermocycler (Biozym, Hess. Oldendorf, Germany). To detect the two possible splice variants, we created a single forward primer overlapping the exon 10/11 junction (Ex10-11_F 5V-AGA TAA CAG GGT TTC CTG AG-3V). The GART isoform 1 was detected by a combination of this forward primer with a reverse primer situated at the junction of exon 13/14 (Ex13-14_R 5V-ACT GCT GGG CAA TCT TCA GT-3V) and the GART isoform 2 by a combination with a reverse primer located in intron 11 (GEx11_R 5V-CCT TTG TTC AGG TTC AGT GG-3V). For detection of the 3V end of GART isoform 1, we used a forward primer situated at the junction of exon 21/22 (Ex21-22_F ACT TTG TAG CTG AAG ATG TAG ATG C) and a reverse primer (poly A_R ACC ATC TGT GTG GGT TTT CC) near the predicted polyadenylation signal.
A. Wo¨hlke et al. / Gene 348 (2005) 73–81
75
lation using a Nick-Translation-Mix (Boehringer Mannheim, Mannheim, Germany). Fluorescence in situ hybridization (FISH) on GTG-banded bovine chromosomes (ISCNDB, 2000) was performed using 750 ng of digoxigenin-labelled BAC DNA. One Ag sheared total bovine DNA and 10 Ag salmon sperm were used as competitors in this experiment. After hybridization over night, signal detection was performed using a Digoxigenin-FITC Detection Kit (Quantum Appligene, Heidelberg, Germany). The chromosomes were counterstained with 4,6-diamino-2phenylidole (DAPI) and propidium iodide and embedded in antifade. Thirty metaphases that were previously photographed were re-examined after hybridization with a Zeiss Axioplan 2 microscope equipped for fluorescence.
The obtained RT-PCR products were directly sequenced using the PCR primers as described below. 2.3. Mutation analysis To identify variations within the bovine GART sequence, exons with flanking regions were PCR amplified and sequenced from 24 unrelated animals from the German Fleckvieh (17), German Holstein (3) and Pinzgauer (4) breed. PCR primers and conditions for the amplification of GART exons with flanking sequences are given in Table 2. PCR primers were developed with the Primer 3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). PCR assays were performed as described above. The obtained PCR products were directly sequenced with the DYEnamic ET Terminator kit (Amersham Biosciences) and a MegaBACE 500 capillary sequencer (Amersham Biosciences), using the PCR primers as sequencing primers.
3. Results and discussion
2.4. Fluorescence in situ hybridization
3.1. Analysis of the genomic organization of the bovine GART gene
The 120 kb bovine BAC clone containing the bovine GART gene was labelled with digoxigenin by nick trans-
A human GART cDNA clone was used to screen a genomic bovine BAC library and five positive clones were
Table 2 PCR primers for the amplification of GART exons Exon
Primer
Sequence (5V–3V)
Exon 1
GEx1_F GEx1_R GEx2_F GEx2_R GEx3_F GEx3_R GEx4_F GEx4_R GEx5_F GEx5_R GEx6-7_F GEx6-7_R GEx8_F GEx8_R GEx9-10_F GEx9-10_R GEx11_F GEx11_R GEx12-14_F GEx12-14_R GEx15-16_F GEx15-16_R GEx17_F GEx17_R GEx18_F GEx18_R GEx19-20_F GEx19-20_R GEx21-22_F GEx21-22_R GEx23_F GEx23_R
TGA CAA GCC TGC ATG AAC TCT CAA TGG GCA GAA CCA TGA ACT ATT GAT TCA CCT GGT CTT TGT CCA CAG TAG AGT AAA GCT ATC GAC AAA CCC TGA
Exon 2 Exon 3 Exon 4 Exon 5 Exons 6–7 Exon 8 Exons 9–10 Exon 11 Exons 12–14 Exons 15–16 Exon 17 Exon 18 Exons 19–20 Exons 21–22 Exon 23
GAG CAC ACA TAT GTG TTT GCC ATG TAT GTG CCT AAG TAA CAA CAT GGC CCC TTG ACT ATA TTG CAA ATG ACC TGA GTG TGT ACT AAT CCT AGA ATG
Product length (bp) TAA ACA TTT AAC AAA TCC ATC TAT GCT AGA GGA TGA CAT GGT GGA CTC TAG TTC CTT CGC CAG GAA GAT CTG AGT GAA TCC GGG GAA GAA TTT ACT
ATC AGC GTC CTG CTC CAA TGC CCT CTC CCC AAG GGC GGT TTT GGA ACA AGC AGG TCC AGG AAT GTC CCC AGG TGG GGA TCA TCA AAC AGA CAG TGG
CGC AGC TCT AAC TCC GGT CTT TTC AGG AGC GAG CAT GCC GGA AGT CCC AGA TTC TTC GCT ACA CAA CAG CAG CAG CTG TCC TTC ACC CCA CTT TCA
CAA TGA TGT AAC TGC AAT ATC CCC GTA ACA GCT AAA CTT ATG TTA TTA ACA AGT CCA GAA GCT ATG AAA AAA TGT AGG TTC TCT CAA CTG CAC GAG
AC AC CC AGG A AA GTT G TC CTA GG AC AG TC ACC CAG C C GG GG TAC C AC TCC ATG G TG CC ATG AG GG CC GAG AG GAC
Annealing temperature (8C)
451
58
872
58
373
58
513
58
659
58
1092
58
500
58
976
58
518
58
1422
58
956
58
533
58
506
58
934
55
623
55
888
58
76
A. Wo¨hlke et al. / Gene 348 (2005) 73–81
Table 3 Exon/intron boundaries of the bovine GART gene
3'-Splice site
5'-Splice site
Exon
Intron phase
Intron size
... (exon 1,
>38 bp) ...
-42 CGCCGGgtgagtttcctatgc
-41 gttttctttctgcagCTTTCA ... (exon 2,
186 bp) ...
+145 ATACTGgtaatgacttttttt
1
1082 bp
+146 tttctcttttcacagACATCT ... (exon 3,
96 bp) ...
+241 CTGCTGgtaaagttcatttct
1
477 bp
+242 tattttcatcactagGAATTG ... (exon 4,
175 bp) ...
+416 TATGAGgtagatagaagacag
2
3244 bp
+417 cctctgtcttcacagTGCAGA ... (exon 5,
112 bp) ...
+528 ATGCAGgtaagttgatccttt
0
969 bp
+529 tttatctccaataagGGTAAA ... (exon 6,
69 bp) ...
+597 GTGTCTgtatgtatatccatc
0
502 bp
+598 ttgttttttctgcagTGTCTG ... (exon 7,
126 bp) ...
+723 CCTCAGgtagccatcatttgg
0
1077 bp
+724 ctctgtattccccagGTTTCT ... (exon 8,
88 bp) ...
+811 ACACAGgtaagcacatggatg
1
489 bp
+812 tcttttttaatgaagGTGTCC ... (exon 9,
86 bp) ...
+897 TGCCAAgtgagtaaaaaaaga
0
164 bp
+898 ttgtgtgttttccagGTGATC ... (exon 10, 169 bp) ...
+1066 TAACAGgtgaacatcagggaa
1
2972 bp
2686 bp
Isoform 2 only +1067 +1302 tgctttctcttacagGGTTTC ... (exon 11, 236 bp) ... GCCCAGGTAAACTT ... TTCCAATAAAAAA Isoform 1 only +1067 tgctttctcttacagGGTTTC ... (exon 11, 232 bp) ...
+1298 GCCCAGgtaaacttgtgaccc
2
1879 bp
+1299 ttttgaactttttagGGGCCT ... (exon 12,
+1393 GACCAGgtaagtcagatcctg
1
294 bp
+1394 ttttctctccaacagGCTGTG ... (exon 13, 110 bp) ...
+1503 GCTGAAGgtaatcagactta
0
342 bp
+1504 tatacccttcggtagATTGCC ... (exon 14, 199 bp) ...
+1702 TCCTCGgtatgcacttccatt
1
1042 bp
+1703 ttctcttcctgccagGAGGTG ... (exon 15, 252 bp) ...
+1954 CCTTAGgtacacacgctcacg
1
292 bp
+1955 cttcccaatatacagGTGACT ... (exon 16, 153 bp) ...
+2107 ATTTAGgtaagatcactaaaa
1
2003 bp
+2108 cttgcctgtttttagATGCCC ... (exon 17, 207 bp) ...
+2314 CCAAGGgtaacctcttccttc
1
1085 bp
+2315 acatttgcaccaaagGTTCTC ... (exon 18, 138 bp) ...
+2452 GGACAGgtgagacatggctcc
1
993 bp
+2453 tttgccttttttcagGATCAA ... (exon 19, 131 bp) ...
+2583 ACGAGAgtaagttactttgaa
0
241 bp
+2584 ccatttctcttctagGTAATT ... (exon 20, 142 bp) ...
+2725 GGAATGgtaagtaaaaaactg
1
657 bp
+2726 ttaattaatttccagGGAAAA ... (exon 21, 116 bp) ...
+2841 GTAGCTgtgagtatggctcgt
0
82 bp
+2842 tgctttcttttccagGAAGAT ... (exon 22, 230 bp) ...
+3071 AGCATTgtaggtgctgcaggg
+3072 tctttgtgtttgaagGCCTCC ... (exon 23, 378 bp) ...
+3449 GATGAATAAAAGACTTCCTAA
95 bp) ...
765 bp
A. Wo¨hlke et al. / Gene 348 (2005) 73–81
77
GART (A) AATAAA
AATAAA
] 1
2
3 4
5
6 7
8 9 10
11
12 13 14
15 16
17
18
19 20 21 22 23
(B)
1kb SINE LINE
(C)
ATG
isoform 2
(D)
2
3 4
5
6 7
8 9 10
11
1
ATG 2 3 4
5
6 7
8 9 10
TAA 11
]
12 13 14
1516
17
18
19 20 21 22 23
] CpG Island
isoform 1
TGA
1
GC content
60%
40%
20%
Fig. 1. Genomic structure of the bovine GART gene. (A) Translated exons are shown as solid boxes. Untranslated regions of exons are shown as open boxes. Polyadenylation sites are indicated. (B) Repetitive sequences are indicated beneath the genomic structure. (C) In this part of the figure, the splicing of transcript isoforms 1 and 2 is illustrated. (D) GC content and CpG islands are shown in the lower part of the figure. For the calculation of the GC content, a 300 bp window was used.
isolated. Comparative BLAST analysis of the bovine BAC clone end sequences with respect to the human genome suggested that the clone RP42-564N14 contained the entire open reading frame of the GART gene. This BAC clone was selected for sequencing and the complete 120,377 bp DNA sequence was determined. As the last untranslated exon of the bovine GART gene was not contained on the BAC clone, the genomic sequence was extended for another 1581 bp using cattle whole genome shotgun sequences from the trace archive (Trace Archive identifier TI171578883, TI168408439, TI75419957, TI80262915). Thus, a contiguous sequence of 121,958 bp harbouring the complete GART gene was submitted to the EMBL nucleotide database (accession no. AJ780930). Using EST sequences and
RT-PCR analyses for cDNA-genomic sequence comparisons we detected that the bovine GART gene consists of 23 exons with exon/intron boundaries that conform perfectly to the GT/AG rule (Table 3). During cDNA sequence analyses it was determined that the bovine GART gene consists of 23 exons contrary to the human and murine GART genes, which only consist of 22 exons. This is caused by an additional intron in the 3VUTR of the bovine GART gene. The exon sizes range from 69 to 252 bp, the introns between these exons span between 82 and 3244 bp, the total size of the bovine GART gene is approximately 27 kb. However, the sequence homology between the human, murine and bovine GART gene is limited to the coding sequence of exons 2 to 22. In the coding sequence of isoform 1, the
Notes to Table 3: Exon sequences are shown in uppercase letters and intron sequences in lowercase letters. Untranslated regions are shown in italics. The conserved GT/AG exon/intron junctions are shown in boldface type. For exons 11 and 23, the polyadenylation signals are shown underlined instead of an exon/intron junction. Position +1 corresponds to the adenine of the translation initiation codon ATG.
78
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1 10
11 12 13 14
10
11
2
ntc liv mus spl 1 2 1 2 1 2 1 2
TAA
kid brn int pit lng 1 2 1 2 1 2 1 2 1 2
skn 1 2
M 459 bp 328 bp
Fig. 2. RT-PCR amplification of two GART mRNA isoforms. RNA of nine different bovine tissues (liv: liver, mus: heart muscle, spl: spleen, kid: kidney, brn: brain, int: small intestine, pit: pituitary, lng: lung, skn: skin) was reverse transcribed and amplified using a forward primer located at the exon 10/11 junction in combination with two alternative reverse primers located at the exon 13/14 junction (1) or in intron 11 (2), respectively. (M: 100 bp ladder, ntc: no template control).
bovine GART gene displays 88.0% similarity to the human GART gene and 81.9% similarity to the murine Gart gene, respectively. The coding sequence of isoform 2 displays 89.0% and 82.7% similarity to the human and murine GART gene, respectively. In the untranslated regions including the entire first exon the sequence similarity between human, mouse and cattle is rather low. The repeat content in the 27 kb region of the bovine GART gene is 27.1% (Fig. 1). The fraction of the SINE (11.7%) and LINE (13.8%) elements are nearly balanced. Other repetitive elements constitute 1.7%. The entire bovine GART gene has an overall GCcontent of 40.3% which is in the range of the mammalian average of 41% (Fig. 1). The bovine GART gene contains one CpG island (Gardiner-Garden and Frommer, 1987) in the region of the first exon (Fig. 1) and the putative promoter (GC content of 58.6% over 483 bp). Sequence analysis of the genomic region upstream of the putative transcription start site indicated the absence of TATA and CCAAT boxes, but the presence of one Sp1 and one E2F binding site. E2F is involved in cell cycle regulation such as DNA synthesis and plays a critical role in the expression of genes involved in differentiation and development (Neuman et al., 1996). E2F interacts with the retinoblastoma (Rb) family member p107 protein. Previous studies described that the p107 protein is required for normal cerebellar development (Marino et al., 2003), which seems consistent with the postulated role of GART in cerebellar development. A possible regulation of the GART gene by E2F seems plausible as an upregulation of GART during S phase of the cell cycle could be required for sufficient purine biosynthesis during DNA replication. 3.2. Analysis of the bovine GART cDNA The bovine mRNA to genomic alignment indicated the existence of two transcript isoforms produced by the bovine
GART gene. In order to confirm the existence of the two transcripts in cattle, cDNA was synthesized. Expression studies of the gene in nine different organs by RT-PCR amplification showed that both GART transcripts are widely expressed in bovine tissues (Fig. 2). The three obtained RTPCR products were sequenced and the generated sequence data were submitted to the EMBL nucleotide database (accession nos. AJ783707, AJ783708, AJ783709). The bovine GART gene showed an additional non coding exon no. 23 at the 3V end. The transcript of isoform 1 is encoded by the exons 1–23 and the shorter transcript of isoform 2 by the exons 1–11, respectively (Fig. 1; accession no. AJ783708). The cDNA of bovine GART isoform 1 contains an open reading frame of 3033 nt coding for a protein of 1010 amino acids and the GART isoform 2 transcript contains an open reading frame of 1302 nt coding for a protein of 433 amino acids, respectively. The translation start codon was assigned based on the homology to the human ortholog (Fig. 1). The polyadenylation signal AAUAAA of the longer isoform 1 is located approximately 1.2 kb downstream of the stop codon (Fig. 1). The second shorter GART transcript is terminated by a polyadenylation signal AAUAAA 504 bp downstream of the stop codon in intron 11 (Fig. 1). The bovine GART isoform 1 protein displays 88.3% and 84.6% similarity to the human and murine GART proteins (Fig. 3). The bovine GART isoform 2 protein displays 89.8% similarity to the human GART protein and 84.8% to the murine Gart protein, respectively (Fig. 3). 3.3. Polymorphisms within the bovine GART gene The search for sequence variations within the GART gene revealed a total of 11 SNPs shown in Table 4. One out of these 11 SNPs located in exon 17 affect the amino acid sequence at position 771 of bovine GART protein. This SNP causes an amino acid change from lysine to glutamic
Fig. 3. Alignment of the bovine GART isoform 1 protein (1010 amino acids) with known orthologous GART isoform 1 protein sequences. The sequences were derived from GenBank entries with the accessions NP_000810 (human GART) and NP_034386 (mouse Gart). The bold amino acid indicates the last position of the shorter GART isoform 2 protein at position 433. Identical residues are indicated by asterisks beneath the alignment, while colons and dots represent very similar and similar amino acids, respectively.
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79
cattle human mouse
MAARVLVIGNGGREHTLAWKLAQSTHVKQVLVTPGNAGTACSEKISNTDISISDHTALAQ MAARVLIIGSGGREHTLAWKLAQSHHVKQVLVAPGNAGTACSEKISNTAISISDHTALAQ MAARVLVIGSGGREHTLAWKLAQSPQVKQVLVAPGNAGTAGAGKISNAAVSVNDHSALAQ ****** **:**************::****** *******: :**** : * :** ****
60 60 60
cattle human mouse
FCKDEKIEFVVVGPEAPLAAGIVGNLNSVGVRCFGPTAQAAQLESSKRFAKEFMDRHGIS FCKEKKIEFVVVGPEAPLAAGIVGNLRSAGVQCFGPTAEAAQLESSKRFAKEFMDRHGIP FCKDEKIELVVVGPEAPLAAGIVGDLTSAGVRCFGPTAQAAQLESSKKFAKEFMDRHEIP *** :***:***************:*:*:**:******:******** *********:*:
120 120 120
cattle human mouse
TARWRAFTKPKEACDFIMSADFPALVVKASGLAAGKGVIVAKSKEEACEAVREIMQGKAF TAQWKAFTKPEEACSFILSADFPALVVKASGLAAGKGVIVAKSKEEACKAVQEIMQEKAF TAQWRAFTNPEDACSFITSANFPALVVKASGLAAGKGVIVAKSQAEACRAVQEIMQEKSF **:* ***:*: **:** **:**********************::***:**:****:* *
180 180 180
cattle human mouse
GEAGETVVIEELLEGEEVSCLCFTDGRTVAPMPPAQDHKRLLEGDEGPNTGGMGAYCPAP GAAGETIVIEELLDGEEVSCLCFTDGKTVAPMPPAQDHKRLLEGDGGPNTGGMGAYCPAP GAAGETVVVEEFLEGEEVSCLCFTDGKTVAEMPPAQDHKRLLDGDEGPNTGGMGAYCPAP *:**** * **:* ************ ***:*********** **:**************
240 240 240
cattle human mouse
QVSKDLLLKIKNNILQRTVDGMQEEGMPYTGVLYAGIMLTKNGPKVLEFNCRFGDPECQV QVSNDLLLKIKDTVLQRTVDGMQQEGTPYTGILYAGIMLTKNGPKVLEFNCRFGDPECQV QVSKDLLVKIKNTILQRAVDGMQQEGAPYTGILYAGIMLTKDGPKVLEFNCRFGDPECQV ***:*** ***:: *** *****:**:**** *********:******************
300 300 300
cattle human mouse
ILPLLKSDLYEVIQSILDGLLCTSLPVWLDNCAAVTVVMASKGYPGDYTKGVEITGFPEA ILPLLKSDLYEVIQSTLDGLLCTSLPVWLENHTALTVVMASKGYPGDYTKGVEITGFPEA ILPLLKSDLYEVMQSTLGGLLSASLPVWLENHSAVTVVMASKGYPGAYTKGVEITGFPEA ************ **:*:***: ****** *: * ***********:*************
360 360 360
cattle human mouse
QALGLEVFQAGTALKDGKVVTNGGRVLTVTAIRENLISALEEARKGLAAIKFEGAVYRKD QALGLEVFHAGTALKNGKVVTHGGRVLAVTAIRENLISALEEAKKGLAAIKFEGAIYRKD QALGLQVFHAGTALKDGKVVTSGGRVLTVTAVQENLMSALAEARKGLAALKFEGAIYRKD *****:**:******:*****:***** *** :*** ***:** ***** ***** ****
420 420 420
cattle human mouse
IGFRAIAFLQQPRGLTYKESGVDIAAGNMLVQKIKPLAKATSRPGCDVDLGGFAGLFDLK VGFRAIAFLQQPRSLTYKESGVDIAAGNMLVKKIQPLAKATSRSGCKVDLGGFAGLFDLK IGFRAVAFLQRPRGLTYKDSGVDIAAGNMLVKKIQPLAKATSRPGCSVDLGGFAGLFDLK **** ****:**:**** ************:**:********:**:*************
480 480 480
cattle human mouse
AAGFTDPLLACGTDGVGTKLKIAQQCSKHDTIGQDLVAMCVNDILAQGAEPLFFLDYFSC AAGFKDPLLASGTDGVGTKLKIAQLCNKHDTIGQDLVAMCVNDILAQGAEPLFFLDYFSC AAGFKDPLLASGTDGVGTKLKIAQLCNKHDSIGQDLVAMCVNDILAQGAEPLFFLDYFSC ****:*****:*************:*:*** *****************************
540 540 540
cattle human mouse
GKLDLRTTEAVITGIAKACKKAGCALLGGETAEMPDMYPPGEYDLAGFAVGAMERDQKLP GKLDLSVTEAVVAGIAKACGKAGCALLGGETAEMPDMYPPGEYDLAGFAVGAMERDQKLP GKLDLSTTEAVIAGIAAACQQAACALLGGETAEMPNMYPPGEYDLAGFAVGAMERHQKLP *****::**** ***:**::*:************:*******************:****
600 600 600
cattle human mouse
QLERITEGDAVIGIASSGLHSNGFSLVRKIVAKSSLEYSSPAPGGCGDQTLGDLLLTPTK HLERITEGDVVVGIASSGLHSNGFSLVRKIVAKSSLQYSSPAPDGCGDQTLGDLLLTPTR QLERITEGDAVIGVASSGLHSNGFSLVRKIVERSSLQYSSPAPGGCGDQTLGDLLLTPTR :********:* * *****************: ***:******:***************
660 660 660
cattle human mouse
IYSRSLLPVLRSGRVKAVAHITGGGLLENIPRVLPQKLGVNLDAQTWRVPRIFSWLQQEG IYSHSLLPVLRSGHVKAFAHITGGGLLENIPRVLPEKLGVDLDAQTWRIPRVFSWLQQEG IYSHSLLPIIRSGRVKAFAHITGGGLLENIPRVLPQKFGVDLDASTWRVPKVFSWLQQEG ***:**** ***:***:*****************:*:**:***:*** * ********
720 720 720
cattle human mouse
HLSEEEMARTFNCGIGAALVVSEDLVKQTLQDIEQHQEEACVIGRVVACPKGSPRVKVEH HLSEEEMARTFNCGVGAVLVVSKEQTEQILRDIQQHKEEAWVIGSVVARAEGSPRVKVKN ELSEEEMARTFNCGIGAALVVSKDQAEQVLHDVRRRQEEAWVIGSVVACPEDSPRVRVKN :************* **:****: :::*:*:* ::::***:***:***::::**** *::
780 780 780
cattle human mouse
LIETMQINGSVLENGTLRNHFSVQPKKARVAVLISGTGSNLQALIDSTREPSSLAHIVIV LIESMQINGSVLKNGSLTNHFSFEKKKARVAVLISGTGSNLQALIDSTREPNSSAQIDIV LIETIQTNGSLVANGFLKSNFPVQQKKARVAVLISGTGSNLQALIDSTRDPKSSSHIVLV *** *:*** :** *:::*::::************************ *:*: :*: *
840 840 840
cattle human mouse
ISNKAAVAGLDKAEKAGIPTRVINHKLYKNRAAFDTAIDEVLEEFSTDIVCLAGFMRILS ISNKAAVAGLDKAERAGIPTRVINHKLYKNRVEFDSAIDLVLEEFSIDIVCLAGFMRILS ISNKAAVAGLDRAERAGIPTRVINHKLSKNRVEFDNAVDHVLEEFSVDIVCLAGFMRILS *********** ** ************:***::** * *:******:*************
900 900 900
cattle human mouse
GPFVRKWNGKMLNIHPSLLPSFKGSNAHEQVLDAGVTVTGCTVHFVAEDVDAGQIILQEA GPFVQKWNGKMLNIHPSLLPSFKGSNAHEQALETGVTVTGCTVHFVAEDVDAGQIILQEA GPFVRKWDGKMLNIHPSLLPSFKGSNAHEQVLEAGVTITGCTVHFVAEDVDAGQIILQEA ****:**:**********************:* *** *********************
960 960 960
cattle human mouse
VPVKRGDTVETLSERVKLAEHKIFPSALQLVASGAVRLGENGRICWVTED VPVKRGDTVATLSERVKLAEHKIFPAALQLVASGTVQLGENGKICWVKEE VPVRRGDTVATLSERVKVAEHKIFPAALQLVASGAVQLREDGKIHWAKEQ **** *****:******* ******* ******* *:*:*:* *:*::*
1010 1010 1010
80
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Table 4 Single nucleotide polymorphisms within the bovine GART gene Location of polymorphic site
Positiona
Intron 4 Intron 5 Intron 5 Intron 5 Intron 11 Intron 15c Exon 17 Intron 18c Exon 19c Exon 19c Intron 20c
3054 245 253 837 1689 9 204 898 5 95 84
a b c
Bovine cDNA position
+2311 +2457 +2547
Nucleotide polymorphism TYA TYC TYC CYT CYG GYA AYG insA AYG GYC CYT
Amino acid substitution
771
LysY771Glu
silent silent
Allele frequencies
Genotype frequenciesb
0.90/0.10 0.79/0.21 0.92/0.08 0.85/0.15 0.42/0.58 0.98/0.02 0.67/0.33 0.83/0.17 0.94/0.06 0.94/0.06 0.96/0.04
19/5/0 19/0/5 20/4/0 17/7/0 6/8/10 23/1/0 12/8/4 20/0/4 21/3/0 21/3/0 22/2/0
Numbering refers to the position of the polymorphic nucleotide within the given exon or intron, respectively. Genotypes are given as number of animals [homozygous for allele 1/heterozygous/homozygous for allele 2]. These SNPs showed only a polymorphism in the German Fleckvieh animals.
acid. In the human and murine GART proteins the corresponding amino acid is glutamic acid. Six out of the observed 11 SNPs were polymorphic in all three examined breeds (Table 4).
bovine GART gene. Consistent with the human–cattle comparative map the localization of four genes on the sequenced BAC clone confirmed syntenic correspondences between bovine chromosome 1, human chromosome 21 and
3.4. Chromosomal assignment For the chromosomal localization of BAC clone RP42564N14, the BAC DNA was used as probe in a FISH experiment on bovine metaphase chromosomes. The assignment localized the GART gene to BTA 1q12.1–q12.2 (Fig. 4). The assignment of the bovine GART gene is in good agreement with established syntenies among human chromosome 21q, murine chromosome 16 and bovine chromosome 1q (McAvin et al., 1988; Rexroad and Womack, 1999; Band et al., 2000). 3.5. Analysis of flanking sequences Apart from the GART gene, the sequenced BAC clone also contained the complete SON DNA binding protein (SON), downstream neighbor of SON (DONSON) and crystalline zeta (quinone reductase)-like 1 (CRYZL1) genes as well as parts of the intersectin 1 (ITSN1) gene thus anchoring these genes as well to BTA 1q12.1–q12.2. In fact, the gene order of all four genes located on the sequenced BAC clone corresponds exactly to the human gene order (NCBI map viewer, build 34.3). The overall GC content of the reported BAC sequence is 40.4%. In the investigated sequence, five CpG islands are located. Repetitive elements constitute 23.2% of the analyzed DNA sequence. 3.6. Conclusions In conclusion, our results provide the complete annotated genomic sequence of the bovine GART gene. Expression studies showed that two GART transcripts are expressed in different bovine tissues. We identified 11 SNPs within the
Fig. 4. Chromosomal assignment of the bovine GART gene by FISH analysis. The digoxigenin labeled BAC clone RP42-564N14 containing the bovine GART gene was hybridized to GTG-banded metaphase chromosomes of a normal cattle. The chromosomes were counterstained with propidium iodide and subsequently identified by DAPI staining. (A) GTGbanding of the bovine metaphase spread. (B) Double signals are visible on both chromosomes 1q12.1–q12.2.
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murine chromosome 16, respectively. The presented study provides detailed information towards comparative mapping of the bovine genome.
Acknowledgments The authors would like to thank Heike Klippert-Hasberg and Stefan Neander for expert technical assistance. We gratefully acknowledge the Dr. h.c. Karl Eibl Foundation, Neustadt/Aisch, Germany and the Deutsche Forschungsgemeinschaft DFG (DI 333/8-1) for their financial support of this study.
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