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Human Molecular Genetics Pages 2127-2133  

Evidence for uniparental, paternal expression of the human GABAA receptor subunit genes, using microcell-mediated chromosome transfer
Introduction
Results
   Isolation of monochromosomal hybrids containing a paternal or maternal human chromosome 15
   Maintenance of SNRPN and IPW imprinting in A9 hybrids
   Imprinting analysis of GABAA receptor subunit and E6-AP genes
Discussion
Materials And Methods
   Cell lines
   Construction of mouse A9 clones containing a single human chromosome
   Fluorescence in situ hybridization (FISH)
   Expression analysis
   Methylation analysis
Acknowledgements
References

Evidence for uniparental, paternal expression of the human GABA<sub>A</sub> receptor subunit genes, using microcell-mediated chromosome transfer

Evidence for uniparental, paternal expression of the human GABAA receptor subunit genes, using microcell-mediated chromosome transfer

Makiko Meguro1, Kohzoh Mitsuya1, Hajime Sui1, Katsuyo Shigenami1, Hiroyuki Kugoh1, Mitsuyoshi Nakao2, Mitsuo Oshimura1,3,*

1Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Nishimachi 86, Yonago, Tottori 683, Japan, 2Department of Tumor Genetics and Biology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860, Japan and 3Core Research for Evolutional Science and Technology (CREST), Japan and Science and Technology Corporation (JST), Japan

Received July 9, 1997; Revised and Accepted August 27, 1997

We have constructed mouse A9 hybrids containing a single normal human chromosome 15, via microcell-mediated chromosome transfer. Cytogenetic and DNA-polymorphic analyses identified mouse A9 hybrids that contained either a paternal or maternal human chromosome 15. Paternal specific expression of the known imprinted genes SNRPN (small nuclear ribonucleoprotein-associated polypeptide N gene) and IPW (imprinted gene in the Prader-Willi syndrome region) was maintained in the A9 hybrids. Using this system, we first demonstrated that human GABAA receptor subunit genes, GABRB3, GABRA5 and GABRG3, were expressed exclusively from the paternal allele and that E6-AP (E6-associated protein or UBE3A) was biallelically expressed. Moreover, the 5[prime] portion of the GABRB3 gene was found to be hypermethylated on the paternal allele. Our data imply that GABAA receptor subunit genes are imprinted and are possible candidates for Prader-Willi syndrome, and that this human monochromosomal hybrid system enables the efficient analysis of imprinted loci.

INTRODUCTION

Genomic imprinting is a biological phenomenon in which parental alleles are genetically marked, ensuring functional inequality of paternal and maternal genomes. This phenomenon plays an important role in early development (1), and may provide an explanation for certain genetic disorders which do not exhibit Mendelian inheritance (2). The function and mechanisms involved in genomic imprinting have not been fully clarified, but DNA methylation, chromatin structure and asynchronous replication may play a role in this process (3). At present, 19 imprinted genes have been reported in human and mouse, 12 of them paternally expressed and seven of them maternally expressed (4-6).

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are classical examples of genomic imprinting in humans (7). These two syndromes are commonly caused by a deletion of chromosome 15q11-q13 region, but differ in parental origin. Most PWS patients have a paternally derived deletion of 15q11-q13 or maternal uniparental disomy (UPD), while AS patients have a maternally derived deletion or paternal UPD. These observations indicate that candidate genes for PWS or AS are located on the chromosome 15q11-q13 region and that PWS gene(s) are normally active on the paternal allele, while AS gene(s) are maternally expressed. Five paternally expressed genes from the 15q11-q13 region, SNRPN (8-10), ZNF127, IPW (11), PAR-1 and PAR-5 (12), have been candidate genes for PWS. Although a maternally expressed AS gene has yet to be identified, point mutations in the E6-AP or UBE3A gene have been recently found in AS patients, suggesting that AS is caused by disrupted ubiquitin-mediated protein degradation possibly during brain development (13,14). Three GABAA receptor subunit genes, GABRB3, GABRA5 and GABRG3, have been mapped to the distal region of E6-AP (15). GABAA is a major inhibitory neurotransmitter in the central nervous system and functions through the GABAA receptor. Although the cluster of GABAA receptor subunits localizes to the imprinted 15q11-q13 region, the imprinting status of these genes has not been determined due to relatively low level and tissue-specific expression, and because no polymorphism has been reported in the transcribed regions.

Other examples of monoallelic expression could be found in mammalian X chromosome inactivation, that leads to transcriptional silencing of most genes on one of the two X chromosomes in female somatic cells (16). Previous studies have demonstrated that the inactive human X chromosome remains repressed in mouse/human somatic cell hybrids (17,18), so that the hybrid cells containing the human active or inactive X chromosome have been used to study the X inactivation process and the X-linked gene expression (19-21). To develop an assay system for examination of imprinted gene expression, we have constructed the mouse A9 hybrids, via microcell-mediated chromosome transfer, that contain a single normal human chromosome 15 whose parental origin is determined. Expression and methylation studies revealed that the imprinting of the human 15q11-q13 genes SNRPN and IPW was maintained in mouse A9 hybrids. Using this system, we demonstrated that E6-AP was expressed from both parental alleles, and that GABRB3, GABRA5 and GABRG3 were expressed from only the paternal allele. Potential applications of the human monochromosomal hybrid system for the investigation of genomic imprinting are discussed.

RESULTS

Isolation of monochromosomal hybrids containing a paternal or maternal human chromosome 15

In order to establish an in vitro system for screening human imprinted genes, normal human fibroblasts transfected with pSV2bsr were fused with mouse A9 cells, and microcell-mediated chromosome transfer was performed to construct A9 hybrids containing a single human chromosome tagged with pSV2bsr. Cytogenetic analyses confirmed the presence of a single normal human chromosome 15 in A9 hybrid clones (Fig. 1A, B). Analysis of DNA polymorphisms using CA repeats determined parental origin of transferred human chromosomes (Fig. 1C). Six A9 hybrid clones were identified to contain a single paternal or maternal human chromosome 15, termed A9(15P)-1, -2, -3, or A9(15M)-1, -2, -3, respectively.


Figure 1 Metaphase chromosome spreads of human monochromosomal hybrids isolated via microcell-mediated chromosome transfer. (A) A9(15M)-1 containing a maternal chromosome 15. Metaphase spreads were stained with quinacrine and Hoechst 33258. The arrow indicates the morphologically normal human chromosome 15. (B) A9(15P)-1 containing a paternal chromosome 15. Metaphase chromosomes from mouse A9 hybrids were hybridized in situ with biotin-labeled pSV2bsr DNA probe. The arrow indicates the morphologically normal chromosome 15 with the integrated bsr. (C) Identification of the parental origin of human chromosome 15 in A9 hybrids. Genomic DNAs from chromosome 15 donor fibroblasts, peripheral lymphocytes from the donor's parents, and A9 hybrids were amplified by PCR using primers that span a CA repeat polymorphism (D15S211).

Maintenance of SNRPN and IPW imprinting in A9 hybrids

We first examined the expression of the human imprinted genes SNRPN and IPW on chromosome 15, by RT-PCR analysis of human fibroblasts and A9 hybrids. PCR primers that encompassed a polymorphic BstUI site within exon 4 of SNRPN (22) were used to verify the parental origin of chromosome 15 and analyze SNRPN expression (Fig. 2A). PCR products amplified from genomic DNA were restricted with PstI and BstUI, to generate a 629 bp undigested fragment (allele a, maternal) or a 580 bp digested fragment (allele b, paternal). PCR products amplified from RNA were restricted with BstUI to generate an expected 218 bp undigested product (allele a, maternal) or a 169 bp digested product (allele b, paternal). SNRPN was expressed exclusively from the paternal allele (allele b) in fibroblasts, and was also expressed from the paternal allele in A9(15P) clones. Conversely, SNRPN expression was not detected in A9(15M) clones. Expression of IPW was analyzed using primers designed to amplify a 254 bp product from RNA and a 937 bp product from genomic DNA due to the presence of an intron (Fig. 2B). IPW expression was detected in A9(15P) clones, but not in A9(15M) clones, whereas the control GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was expressed in all lines examined.


Figure 2 Expression studies of human SNRPN and IPW in A9 hybrids containing a paternal or a maternal human chromosome 15 by RT-PCR. (A) Paternal expression of SNRPN in human fibroblasts and A9 hybrids. PCR was performed using primers (arrowheads) that encompassed a BstUI polymorphic site. PCR products from genomic DNA were digested with PstI and BstUI, and RT-PCR products were digested with BstUI. The BstUI-undigested and -digested alleles were designated allele a and b, respectively. Exons 4 and 5 are indicated (59). (B) Paternal expression of IPW in A9 hybrids. Amplification products were visualized by ethidium bromide staining. GAPDH was used as a control. RNA samples were run with (+) and without (-) reverse transcriptase and amplified using the same primers as those used for genomic DNA (G). No PCR product was observed in mouse A9 cells (data not shown).

DNA methylation studies of the SNRPN CpG island were performed by Southern hybridization (12). Digestion of genomic DNA with XbaI and the methylation-sensitive restriction enzyme NotI, and probing with a SNRPN exon 1 DNA fragment, generated 3.0 and 0.9 kb bands for unmethylated DNA and a 4.2 kb band for methylated DNA (Fig. 3A). The methylated 4.2 kb and the unmethylated 3.0 and 0.9 kb bands were observed in human fibroblasts. The 3.0 and 0.9 kb bands were exhibited by A9(15P) clones and A9(15M) clones exhibited only the 4.2 kb band. This indicated that the CpG island of SNRPN was methylated on the maternal but not on the paternal allele. The methylation status of intron 7 of SNRPN was also examined. Genomic DNA was digested with HindIII, or HindIII and MspI, or HindIII and methylation-sensitive restriction enzyme HpaII. Samples were Southern blotted and probed with a SNRPN exon 4-5 DNA fragment. In contrast with maternal methylation in the CpG island, intron 7 of SNRPN was specifically methylated on the paternal allele in A9 hybrids (Fig. 3B), as previously described in normal tissues (8). Any changes in methylation and expression profiles of the human SNRPN and IPW were not observed during at least 8 weeks in the subsequent culture (data not shown). These data indicated that the imprinting status of human SNRPN and IPW are stably maintained in mouse A9 hybrids.

Imprinting analysis of GABAA receptor subunit and E6-AP genes

Expression levels of three GABAA receptor subunit genes (GABRB3, GABRA5 and GABRG3) and E6-AP in mouse A9 hybrids were examined by RT-PCR, using identical cDNA pools as for the SNRPN and IPW analyses. GABRB3, GABRA5 and GABRG3 were expressed in A9(15P) but not in A9(15M) clones (Fig. 4A). These findings were confirmed by concomitant expression in two cell lines, or repression in two cell lines of these three genes and SNRPN in additional A9 hybrids containing a neo-tagged human chromosome 15 whose parental origin could not be determined (data not shown). These findings demonstrated that the three GABAA receptor subunit genes were expressed specifically from the paternal allele. GABRG3 expression was not detected in fibroblasts, whereas its expression was detected in human adult brain (data not shown). This may indicate that expression levels of certain human genes are higher in A9 hybrids than in human fibroblasts. In addition, we examined the amplification of these three GABAA receptor subunit genes from YACs, 49G3 and 335B1 containing GABRB3 and GABRA5 respectively, and LS6 hybrid containing the q13-qter region of human chromosome 15 (9,23). As expected, GABRB3 and GABRA5 were amplified from YAC 49G3 and YAC 335B1, respectively. GABRG3 was not present in these YACs and LS6 hybrid, which suggests this gene is proximal to the q13 breakpoint. On the other hand, E6-AP expression was detected in all the cell lines examined (Fig. 4A). Detailed analysis by RT-PCR revealed that isoforms I, II and III of E6-AP (24) were expressed equivalently from both parental alleles in these hybrids (data not shown).


Figure 3 Methylation studies of the human SNRPN gene in A9 hybrids. (A) Maternal allele-specific methylation of SNRPN CpG island. Genomic DNA was digested with XbaI (X) and NotI (N), Southern blotted and probed with a 4.2 kb XbaI DNA fragment which included exon 1 of SNRPN. (B) Paternal allele-specific methylation in intron 7 of the SNRPN gene. Genomic DNA was digested with HindIII (H) and MspI (M) or HpaII (h), and probed with DNA fragment from exon 4-5 of SNRPN. Black boxes indicate exons (59).


Figure 4 Imprinting studies of human GABRB3, GABRA5, GABRG3 and E6-AP in A9 hybrids. (A) Paternal expression of GABRB3, GABRA5 and GABRG3 and biallelic expression of E6-AP were observed by RT-PCR as described in the legend for Figure 2. Amplification products were not detected in reverse transcriptase (-) controls (data not shown). PCR products were stained with SYBR Green for GABRG3 because of relatively low expression levels. GABRB3 and GABRA5 were amplified from YAC 49G3 and YAC 335B1 respectively, and none of the three GABAA receptor subunit genes were amplified from the LS6 hybrid. (B) Paternal allele-specific methylation of the 5[prime] region of the GABRB3 gene was determined by a HpaII-sensitive PCR assay. Genomic DNA was digested (D) or not digested (N) with HpaII (h), and analyzed by the nested PCR analysis. Arrowheads show the location of primers used for the PCR.

To analyze the methylation status in the 5[prime] region of GABRB3, a HpaII-sensitive PCR assay was performed using primers that were designed to amplify a 460 bp fragment containing four HpaII sites (Fig. 4B). Digestion of genomic DNA with the methylation-sensitive restriction enzyme HpaII followed by PCR amplification revealed that a 460 bp fragment was detected in A9(15P), but not in A9(15M) clones. This indicates that the 5[prime] region of GABRB3 was hypermethylated exclusively on the paternal allele. These findings suggest strongly that GABRB3, GABRA5 and GABRG3 are paternally expressed imprinted genes, while E6-AP exhibits biallelic expression.

DISCUSSION

Analysis of genomic imprinting in normal diploid cells is difficult, as they contain genome copies inherited from each parent that are indistinguishable without sporadic differences as polymorphism. To establish an in vitro assay system for the investigation of human imprinted loci, we have constructed mouse A9 hybrids containing a single human chromosome 15 whose parental origin is determined, via microcell-mediated chromosome transfer. Our expression and methylation studies demonstrated that imprinting status of human SNRPN and IPW, which were located on the imprinted 15q11-q13 region (11,25), was maintained in mouse A9 hybrids. We also confirmed that human H19 and KVLQT1 were expressed exclusively from the maternal allele, and that the paternal allele of human H19 was heavily methylated through the entire gene, including the CpG island upstream of the promoter in mouse A9 hybrids containing a single paternal or maternal chromosome 11 (to be reported elsewhere). These findings indicate that the human monochromosomal hybrids may facilitate the identification of human imprinted loci, by analysis of parental origin-specific expression or differential methylation. In addition, it has been shown that the inactive human X chromosome in a rodent background is maintained to be inactive under appropriate culture conditions, and that the hybrids containing the human active or inactive X chromosome reflect the expression and methylation profiles of the X-linked genes in human cells (26,27), proving the validity of this hybrid system for studies of genomic imprinting.

This study showed clearly that the three GABAA receptor subunit genes, GABRB3, GABRA5 and GABRG3 were expressed exclusively from the paternal allele in mouse A9 hybrids, indicating that they are paternally expressed imprinted genes. Closely linked genes in mice, Gabrb3, Gabra5 and Gabrg3 have previously been reported to be expressed equivalently from maternal and paternal alleles in the brain (28,29). A considerable number of imprinted genes exhibit species- and/or tissue-specific expression profiles. For instance, paternally derived mouse Igf2r is repressed in every tissue type of all strains examined (30), while human IGF2R shows polymorphic expression profiles with maternal expression in only a minority of individuals (31,32). Paternal duplication of the central region of mouse chromosome 7 that is homologous to the human PWS/AS region produced no imprinting effect (29), implying qualitative difference between these imprinted loci in mice and those in humans. Thus, it is feasible that the three GABAA receptor subunit genes represent species- and/or tissue-specific expression profiles, although this was not addressed in this study. In agreement with the previous report (9), E6-AP was expressed from both parental alleles in mouse A9 hybrids. However, this does not exclude that E6-AP could be imprinted in other tissues and cell types, at other stages of development, or that it is expressed at differential levels which can not be detected quantitatively by PCR. Another possibility is that the E6-AP protein could interact with unknown maternal factors during early development. In this case, E6-AP could behave as a maternally expressed gene, even though it is expressed biallelically. The pathogenesis of AS phenotypes due to E6-AP mutations is presently unclear.

In this study, the 5[prime] region of GABRB3 was hypermethylated exclusively on the expressed paternal allele in A9 hybrids. DNA methylation is suggested to play a role in either the negative or positive transcriptional regulation of known imprinted genes (33). It has been reported that hypomethylation activated the silent H19 and Xist alleles, but repressed the active Igf2 and Igf2r alleles in DNA methyltransferase mutant mice (34,35), indicating multiple functions of DNA methylation for gene expression. Thus, the methylation of the 5[prime] region of GABRB3 may correlate to positive regulation of gene expression, as observed in IGF2R (36), SNRPN (8), and ZNF127 (37). DNA replication studies have revealed that the PWS/AS region exhibits reciprocal asynchronous replication (38,39). The loci between ZNF127 and GABRB3 on paternal chromosome 15 replicate earlier than those on maternal chromosome, while GABRA5, which is proximal to GABRG3, replicates earlier on the maternal than paternal homologue (40,41). This replication pattern has only been demonstrated by fluorescence in situ hybridization (FISH) assay, and different replication data have been shown at several loci using FISH and BrdU assays (42,43). Therefore it might be necessary to reexamine precisely by using more direct methods for determination of the allele-specific replication timing at this region. Studies of some familial PWS and AS cases with microdeletions of a 100 kb region containing exon 1 of SNRPN suggests the presence of an imprinting center (IC) which regulates in cis the switching of imprinting status within the PWS/AS region during gametogenesis (44,45). Whether the three GABAA receptor subunit genes are also controlled by the IC is not known. However, it has been reported recently that a temporal and spatial association of homologous chromosomes occurs specifically at imprinted regions between SNRPN and GABRB3/A5 in late S phase, and that PWS and AS patients are deficient in not only asynchronous replication but homologous association (46). Although paternally expressed SNRPN is abundant in maturing neurons (47), it is not necessarily true that lack of SNRPN expression contributes solely to the PWS phenotype (48). Deficiency of Gabrb3 results in cleft palate in mice, implying that Gabrb3 is involved in not only neuronal inhibition but facial development (49,50). Thus, it is possible that a defect in human GABRB3 expression is responsible for the facial dysmorphism in PWS. Our findings indicate that GABRB3, GABRA5 and GABRG3 are paternally expressed imprinted genes. Therefore, disrupted imprinting of these genes could also play a role in the PWS phenotypes, as has been suggested already for other paternally expressed genes (45). We are currently using our in vitro assay for more detailed analyses of the relationships between expression profiles, methylation status and asynchronous replication at these loci.

Finally, to apply this hybrid system to the overall human genome, we are now constructing a mouse A9 hybrid panel containing each human chromosome whose parental origin is determined. A specific human chromosome tagged with a selectable marker could be transferred from mouse A9 hybrids into mammalian cells via microcell-mediated chromosome transfer (51). Introduction of a specific chromosomal region into mouse embryonic stem cells by microcell fusion has been used to generate mice which contain a subchromosomal fragment derived from human fibroblasts (52). Thus, this approach allows the analysis of imprinting effects in mice with parental duplications for specific chromosomal regions. The DT40/human chromosome shuttle system is also available for the targeted modification of the human loci of interest by homologous recombination (53,54). This in vitro assay system will facilitate the analysis of the mechanisms involved in genomic imprinting.

MATERIALS AND METHODS

Cell lines

Fibroblasts were obtained with standard punch biopsies from a normal adult, and established by standard techniques. Fibroblast cultures were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% fetal calf serum. Mouse A9 cells were maintained in DMEM supplemented with 10% bovine calf serum (CS). Mouse A9 hybrids containing a normal human chromosome tagged with the pSV2bsr (55) were maintained in DMEM supplemented with 10% CS and 3 [mu]g/ml of Blasticidin S (BS) and expanded for 4 weeks until sufficient cells were present for nucleic acid extraction.

Construction of mouse A9 clones containing a single human chromosome

Normal human fibroblasts were transfected with pSV2bsr plasmid DNA by the calcium phosphate precipitate method (56). Following fusion of BS-resistant human fibroblasts and mouse A9 cells, the transfer of bsr-tagged human chromosomes in the hybrids to mouse A9 cells was performed via microcell-mediated chromosome transfer (51,57). The resulting BS-resistant clones were isolated, expanded and karyotyped. A9 hybrids containing an intact human paternal or maternal chromosome 15 were identified using CA repeat polymorphism. Genomic DNA was amplified with primers 5[prime]-AAGCAGGTGGAATCCTTG-3[prime] and 5[prime]-AAAAGCCCCAGGTAGGG-3[prime] (D15S211) for 35 cycles at 94/53/72°C for 60/60/60s.

Fluorescence in situ hybridization (FISH)

The pSV2bsr probe was labeled with biotin-16-dUTP by nick translation. After hybridization to denatured chromosomes and incubation with anti-biotin Fab[prime]-alkaline phosphates conjugate, the biotinylated probes were detected with 3[prime]-hydroxy-N-2[prime]- biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP) (58). Slides were counterstained with Hoechst-quinacrine, which allowed detection and simultaneous visualization of Q-bands and HNPP signals.

Expression analysis

Genomic DNA was prepared by standard phenol-chloroform extraction methods. Total RNA was extracted using the acid guanidium thiocyanate procedure. The total RNA samples were digested with RNase-free DNase I (TAKARA) prior to reverse transcription using standard conditions, primed with on oligo (dT)15 primer. To investigate the possibility of DNA contamination in the RNA sample, total RNA was incubated with (+) or without (-) M-MLV reverse transcriptase (Gibco-BRL), including an oligo (dT)15 primer. PCR analysis of SNRPN and IPW were performed as reported previously (10,11). Identical primers and PCR conditions were used for PCR analysis of both cDNA and genomic DNA. PCR primers were as follows: for GABRB3, 5[prime]-TCAGGCGGCATTGGCGATACC-3[prime], 5[prime]-ATAAAAACTTGACAGGCAGAG-3[prime]; for GABRA5, 5[prime]-AATATTGCCTTGATGTTTCTA-3[prime], 5[prime]-GCCTATTCTATTTCTTCGTGT-3[prime]; for GABRG3, 5[prime]-GCGTATTCACATAGACATCTTG-3[prime], 5[prime]-GATTGGTCACTACTGGTCTGG-3[prime]; for E6-AP, 5[prime]-AGCTGCAAAGCATCTAATAG-3[prime], 5[prime]-CTTTAAAATCAATCCTAGCGC-3[prime]; for GAPDH, 5[prime]-CCATCTTCCAGGAGCGAGA-3[prime], 5[prime]-TGTCATACCAGGAAATGAGC-3[prime] (forward and reverse, respectively). Temperature conditions used were: for SNRPN, 33 cycles at 94/62/72°C for 45/90/45 s; for IPW, 35 cycles at 95/56/72°C for 45/30/45 s; for GABRB3, 35 cycles at 94/58/72°C for 45/30/60 s; for GABRA5, 32 cycles at 94/55/72°C for 45/60/60 s; for GABRG3, 36 cycles at 95/62/72°C for 45/30/30 s; for E6-AP, 35 cycles at 94/58/72°C for 45/30/45 s; for GAPDH, 25 cycles of at 94/55/72°C for 45/30/90 s.

Methylation analysis

DNA methylation status of the SNRPN was examined by Southern hybridization. Five [mu]g of genomic DNA was digested with the appropriate restriction enzymes. The 4.2 kb XbaI fragment was used as a probe for the SNRPN CpG island. The exon 4-5 probe generated by PCR in the same way as for expression analysis was used for intron 7 of SNRPN. Radiolabelled DNA probes were hybridized to Southern blots. The DNA methylation status of the GABRB3 was analyzed by a HpaII-sensitive PCR assay. Genomic DNA was digested with HpaII and amplified by nested PCR. PCR primers were as follows: for first round PCR, 5[prime]-GAGGAAGGCTTTTCGGCAT-3[prime], 5[prime]-CATGTTGACTTCGGAAACCA-3[prime]; for nested PCR, 5[prime]-TGGTGTGCTGCGCCCAGA-3[prime], 5[prime]-GATGTTCATCCCCACGCAG-3[prime] (forward and reverse, respectively). Temperature conditions used were 25 cycles at 95/58/72°C for 60/30/60 s.

ACKNOWLEDGMENTS

We would like to thank Dr T. Schulz (CREST; Core Research for Evolutional Science and Technology, Tottori University) for critical reading and comments on this manuscript. This study was supported by the CREST program of the Japan Science and Technology Corporation (JST) and by the Mitsubishi Foundation, Japan.

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*To whom correspondence should be addressed. Tel: +81 859 34 8260; Fax: +81 859 34 8134; Email: oshimura@grape.med.tottori-u.ac.jp

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