Immunogenetics (2001) 52 : 186±194 DOI 10.1007/s002510000270
O R I G I N AL P AP E R
Shohei Chida ´ Hirohiko Hohjoh ´ Momoki Hirai Katsushi Tokunaga
Haplotype-specific sequence encoding the protein kinase, interferon-inducible double-stranded RNA-dependent activator in the human leukocyte antigen class II region Received: 10 August 2000 / Revised: 5 October 2000 / Published online: 14 December 2000 Springer-Verlag 2000
Abstract The protein kinase, interferon-inducible double-stranded (ds)RNA-dependent activator (PRKRA) is a dsRNA-binding protein which activates a protein kinase participating in the antiviral activity of interferon. Our previous studies indicated that the nucleotide sequence encoding PRKRA, which appeared to be an intronless gene, was present in PAC HS265J14 containing the human leukocyte antigen (HLA) DR subregion. In this study, we further investigated and characterized the PRKRA gene on the human genome by means of Southern blotting and polymerase chain reaction with homozygous typing cell lines for HLA genes. Results indicated that the presence of PRKRA in the DR subregion was dependent on the DR53 group. Consistently, fluorescence in situ hybridization profiles with PRKRA as a probe showed that the hybridization signal on Chromosome (Chr) 6p21.3 was seen only in the samples carrying the DR haplotypes that belonged to the DR53 group. Interestingly, another hybridization signal, which was mapped on Chr 2q31.2±q32.1, was always detected in the samples examined, i.e., even in the samples negative for the DR53 group. The outcome of a sequence-database homology search further indicated that the PRKRA
S. Chida ´ H. Hohjoh ()) ´ K. Tokunaga Department of Human Genetics, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan E-mail:
[email protected] Phone: +81-3-58413693 Fax: +81-3-58028619 M. Hirai Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan
gene with introns appeared to be present in a recently opened draft-sequence, RP11-65L3 (GenBank accession number AC009948), which is located between D2S335 and D2S2257. Together, the data presented here indicate that the PRKRA gene in the DR subregion is a processed pseudogene (PRKRAC), which could have been generated only on the DR53 common ancestor's genome, and that the master copy of PRKRAC is most probably present on Chr 2q31.2±q32.1. Keywords PRKRA ´ Double-strand RNA-binding protein ´ DR53 group ´ Processed pseudogene
Introduction The human leukocyte antigen (HLA) region is located on the short arm of chromosome (Chr) 6 at 6p21.3 (Campbell and Trowsdale 1993). The region spans approximately 3.6 Mb and consists of three subregions, HLA class I, II, and III. The HLA region is characterized by the highest density of genes in the human genome (Abdulla et al. 1996; Janer and Geraghty 1998; MHC Sequencing Consortium 1999; Yamazaki et al. 1999), the presence of highly polymorphic genes with large numbers of alleles (Bodmer et al. 1999), and the presence of many genes associated with various human diseases and disorders (Hill et al. 1991; Juji et al. 1984; Todd et al. 1987; Winchester et al. 1994). Determination of the nucleotide sequence of the entire HLA region has been recently completed (MHC Sequencing Consortium 1999), and the sequence data are now available. In previous studies, we investigated the reported genomic DNA sequences of the HLA class II region, and found a sequence with potential to encode an RNA-binding protein in PAC HS265J14 (GenBank accession number Z84477) containing the HLA-DR subregion (Chida et al. 1998, 1999). Independently of
187
our studies, Patel and Sen (1998) isolated a cDNA clone encoding the protein kinase, interferon-inducible double-stranded (ds)RNA-dependent activator (PRKRA), which was able to bind to dsRNA molecules, i.e., was a dsRNA-binding protein. The PRKRA cDNA and the RNA-binding protein gene we found in PAC HS265J14 have a nucleotide sequence identity of 98.4%, suggesting that the PRKRA gene is present in the HLA-DR subregion. Regarding the nomenclature of this gene, although it was provisionally named HSPBP in our previous study (Chida et al. 1999), we will henceforth refer to it as PRKRA, according to the suggestion of HUGO, the Human Gene Nomenclature committee. The PRKRA gene has significant sequence homology to the Xenopus laevis dsRNA-binding protein gene, Xlrbp (Eckmann and Jantsch 1997), and to the human gene for the cytoplasmic protein bound to the transactivation response element of human immunodeficiency virus type-1 (HIV-1) RNA (TRBP) (Gatignol et al. 1991, 1993), both of which encode polypeptides with an ability to bind to dsRNA molecules, suggesting that these genes, including PRKRA, belong to a multigene family for dsRNA-binding proteins. Another feature of the PRKRA gene in the DR subregion is that it appears to have no intron sequences. In this study, we further investigated and characterized features of PRKRA in the human genome by means of Southern blotting, polymerase chain reaction (PCR) with homozygous typing cell lines (HTCs), and fluorescence in situ hybridization (FISH) analyses. The data presented here indicate that the PRKRA gene in the HLA-DR subregion is specifically present in the DR53 group, and that the gene is a processed pseudogene, which could have been generated in the common ancestor of the DR53 group. In addition, the results of the FISH analysis and a homology search of the most recent sequence database lead to the conclusion that the master copy of the gene is most probably present on Chr 2q31.2±q32.1.
Materials and methods Materials Peripheral blood samples were obtained from healthy individuals. The isolation of genomic DNA from peripheral lymphocytes was carried out as described previously (Sambrook et al. 1989). The genomic DNA samples prepared from various kinds of HTCs for HLA (Marsh et al. 1997), and from individuals homozygous for DRB1 alleles that belong to the DR53 group were kindly provided by Dr. Y. Ishikawa (Japanese Red Cross Central Blood Center, Tokyo, Japan) and Dr. N. Tsuchiya (University of Tokyo, Tokyo, Japan), respectively. HLA-DRB1 allele typing HLA-DRB1 allele typing was carried out using polymerase chain reaction (PCR)-microtitre plate hybridization (Kawai et al. 1996).
Southern blotting analyses Genomic DNA (~10 g) was digested with restriction enzymes, electrophoretically separated on 1% agarose gels and blotted onto nylon membranes. The membranes were UV cross-linked, hybridized with an alkaline phosphatase-labeled probe in hybridization buffer (Alkphos Direct; Amersham) at 52 C overnight, and washed twice in the primary wash buffer (50 mM Na phosphate, 150 mM NaCl, 10 mM MgCl2, 2 M urea, 0.1% SDS) at 55 C for 10 min, and twice in the secondary wash buffer (50 mM Tris base, 100 mM NaCl, 2 mM MgCl2) at room temperature for 5 min. After washing, the membranes were treated in CDP-star detection reagent (Amersham), and exposed to X-ray films. The probe DNA used in the hybridization was prepared from plasmid pCRL-ExpR (Chida et al. 1999). After digestion of the plasmid DNA with BamHI and HindIII, the inserted DNA fragment containing the entire PRKRA sequence was isolated, heat-denatured and chemically cross-linked with thermostable alkaline phosphatase (AlkPhos Direct). The alkaline phosphatase-labeled DNA fragment was used as the probe. PCR analyses PCR was carried out in 50 l of reaction solution [10 mM TrisHCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 250 M dNTP, 0.5 M of each oligonucleotide primer, ~100 ng genomic DNA, and 1 unit Taq polymerase (PE Applied Biosystems)]. The hr2L, hr3R, and hrExpR oligonucleotide primers previously described (Chida et al. 1999) were used in the PCR, and the oligonucleotide primers newly synthesized were as follows: hr6L; 59-TTGGTTCATTACAGGAATTGG-39, hr7L; 59-CAGGGGGAGAAAATTAAACC-39, hr7R; 59-GTAA ATTTTACTTAGGCCATC-39. The Gene Amp PCR system 9600 (PE Applied Biosystems) was used as the thermal cycler. The thermal cycling profile was as follows: heat denaturation at 96 C for 10 min, 35 cycles of amplification including denaturation at 96 C for 30 s, annealing at 54 C for 30 s, and extension at 72 C for 2.5 min, and a final extension at 72 C for 5 min. Direct sequencing PCR products were purified with a commercial kit (Gene Clean; Bio 101) after agarose gel electrophoresis, and used as templates in direct sequencing. The sequence reaction was carried out with the dRhodamine Terminator Cycle Sequencing FS Ready Reaction Kit (PE Applied Biosystems) according to the directions provided by the manufacturer. The resultant samples were analyzed by an automatic DNA sequencer (ABI PRISM 310 Genetic Analyzer; PE Applied Biosystems). FISH analysis To determine the chromosomal localization of the PRKRA gene, FISH was performed as described previously (Hirai et al. 1994). In brief, plasmid pBSL-3R containing the entire PRKRA sequence (900 bp) was labeled with biotin-14-dATP by nicktranslation and hybridized with R-banded chromosomes prepared from phytohemagglutinin-stimulated cultured lymphocytes. After overnight hybridization in a humidified chamber at 37 C, the slides were washed in 50% formamide/2SSC at 37 C for 10 min, followed by a wash in 1SSC at room temperature for 15 min. Hybridization signals were amplified using rabbit anti-biotin (Enzo) and fluorescein-labeled goat anti-rabbit IgG (Enzo). The chromosomes were counterstained with propidium iodide.
188
Results Southern blot analysis with the PRKRA gene as a probe We examined genomic DNA prepared from several individuals with distinct HLA-DRB1 genotypes by means of Southern blot analysis using the PRKRA gene as a probe. The hybridization profiles showed multiple hybridization signals (Fig. 1), which agreed with our previous results (Chida et al. 1999). The important point to note in this profile is that additional hybridization signals were observed in the lanes of individuals c and d: lanes 3, 4, 7, 8, 11, and 12 (indicated by arrows in Fig. 1). The results of HLA-DRB1 typing showed that individuals c and d were DRB1*0901 (DR9) and DRB1*0405 (DR4) homozygotes, respectively, which appear to belong to the DR53 group in terms of polymorphism of genome organization in the HLA-DR subregion, whereas individuals a and b were a DRB1*0101 (DR1)/DRB1*1302 (DR13) heterozygote and DRB1*1501 (DR15) homozygote, respectively, belonging to DR groups other than DR53 (Fig. 2). It should be noted that the HS265J14 clone, in which the PRKRA gene was found (Chida et al. 1999), contains a part of the DR subregion that belongs to the DR53 group. Therefore, these results suggested that the PRKRA gene could be specifically present in the DR subregion that belongs to the DR53 group.
Fig. 1 Southern blot hybridization profiles of the PRKRA gene. Genomic DNA was prepared from unrelated healthy individuals (a±d), and examined by Southern blotting. Ten micrograms of each genomic DNA was digested with the restriction enzymes indicated (HindIII, BamHI, and NdeI), electrophoretically separated on 1% agarose gels, and transferred onto nylon membranes. The membranes were subjected to hybridization with alkaline phosphatase-labeled PRKRA as a probe. The hybridization, washing, and visualization of the hybridized probe are detailed in Materials and methods. Arrows indicate the additional hybridization bands detected in individuals c and d. Size-marker DNA fragments (lDNA/HindIII fragments) are indicated by bars. The HLADRB1 genotypes of individuals a±d are as follows: a, DRB1*0101/*1302 heterozygote; b, DRB1*1501 homozygote; c, DRB1*0901 homozygote; d, DRB1*0405 homozygote
PCR analysis with HTCs To address the question whether the PRKRA gene is specifically present in the DR53 group, we investigated various HTCs for HLA (Table 1) using PCR with three sets of PCR primers, which were designed on the basis of the HS265J14 sequence: hr7L and hr7R primers, hr2L and hrExpR primers, and hr6L and hr3R primers (Fig. 3A). When BOLETH, BSM, Table 1 HLA-DRB1 alleles and groups of the homozygous typing cell lines (HTCs). The cell lines are homozygous at the DRB1 locus Name
DRB1
DR group
SA JESTOM HOM-2 SCHU TOKUNAGA HOR STEINLIN VAVY BOLETH BSM AWELLES MLF MOU PITOUT BM9 OLGA TAB089
*0101 *0101 *0101 *1501 *1502 *0301 *0301 *0301 *0401 *0401 *0401 *0401 *0701 *0701 *0801 *08022 *0801
DR1 DR1 DR1 DR51 DR51 DR52 DR52 DR52 DR53 DR53 DR53 DR53 DR53 DR53 DR8 DR8 DR8
189
Fig. 2 Schematic drawing of the genome organization in each DR group. Solid and hatched boxes indicate functional genes and pseudogenes, respectively. The arrow indicates the position of the PRKRA sequence in the DR subregion. The DRB types (serological specificities) that belong to each DR group are indicated in parentheses
MOU, PITOUT, AWELLES, and MLF cell lines were examined by PCR followed by agarose gel electrophoresis, PCR-amplified bands, whose sizes were almost the same as those estimated from the HS265J14 sequence, were observed (Fig. 3B). By direct sequencing with the PCR products, the amplified DNA fragments were shown to correctly encode the PRKRA sequence (detailed below). In contrast, no amplified bands were observed when the other cell lines were examined by the same PCR assay with any of the primer sets used (Fig. 3B). The DRB1 types of the HTCs used are as follows: BOLETH, BSM, AWELLES, and MLF are known to be homozygous for DRB1*0401, and MOU and PITOUT are homozygous for DRB1*0701 (Table 1). Thus, the DR subregions containing these DRB1 alleles belong to the DR53 group. The other cell lines, which showed no amplification in PCR assay, are homozygous for DRB1 alleles that belong to DR groups other than the DR53 group (Table 1). Accordingly, these observations strongly suggested that the presence of the PRKRA gene in the DR subregion is dependent on the DR53 group.
FISH analysis with the PRKRA gene as a probe To determine the chromosomal localization of the PRKRA gene, we performed FISH analysis using the PRKRA gene as the probe. Metaphase chromosomes were prepared from several individuals and examined by FISH. When chromosomes prepared from an individual homozygous for DRB1*0405 were examined, the hybridization signals were detected on Chr 6 and 2: at Chr 6p21.3, where the HLA region is located, and at Chr 2q31.2±q32.1 (Fig. 4. Table 2). When chromosomes prepared from the DRB1*1501 homozygous and DRB1*0802/DRB1*0803 heterozygous individuals
Table 2 Summary of FISH analyses (+ presence of the hybridization signals) Sample name
DRB1 genotypea
N28
*1501 (DR15)/*1501 (DR15) *0405 (DR4)/*0405 (DR4) *0802 (DR8)/*0803 (DR8) *0803 (DR8)/*0901 (DR9) *0901 (DR9)/*0901 (DR9)
N48 NH60 N3 N88 a
Signals on 6p21.3
on 2q31.2±q32.1
Undetectable + +
+
Undetectable + +b
+
+
+
Serological specificities are indicated in parentheses The hybridization signal was observed on one of the homologous chromosomes, but not on the other
b
190 Fig. 3A,B PCR analysis with homozygous typing cell lines (HTCs). A Schematic drawing of the PRKRA gene in the DR subregion and PCR primers. Open rectangle represents the open reading frame of the PRKRA gene. The first methionine (Met) and stop (Stop) codons are indicated by bars. PCR primers are shown by arrows together with their names. Broken lines show the PCR-amplified regions, and the predicted size of each region is indicated above the line. B Aganose gel electrophoresis profile. PCR was carried out with hr7L and hr7R primers, and the resultant PCR products were analyzed by 1% agarose gel electrophoresis followed by ethidium bromide staining. The HTCs used in the assay are indicated. The arrow indicates the predicted size (2.2 kb) of the PCR product on the basis of the PAC HS265J14 sequence. Lane M shows a 1-kb DNA ladder marker
Fig. 4 FISH profiles of the PRKRA gene. Metaphase chromosomes were prepared from the peripheral blood sample of an individual homozygous for DRB1*0405, and hybridized with the labeled PRKRA probe. Hybridization signals (indicated by arrows) were detected on Chromosome 2 and 6. The estimated chromosomal localization of each signal is indicated under the panel
were analyzed, the hybridization signal was detected only on Chr 2q31.2±q32.1, but not on Chr 6 (Table 2). When chromosomes prepared from a DRB1*0803/ DRB1*0901 heterozygote were examined, while the
hybridization signal on Chr 2q31.2±q32.1 was detected on both of the homologous chromosomes, the hybridization signal on 6p21.3 was observed on only one of a pair of Chr 6 (Table 2). Since the DR subregion carry-
191
Fig. 5 Variations observed in the PRKRAC gene and its 59 flanking region. Open and hatched boxes represent the predicted amino acid coding region of the PRKRAC gene, the hatched boxes indicating the dsRNA-binding domains previously described by Burd and Dreyfuss (1994). The adenine residue in the first methionine codon (ATG) is given as +1. Arrows indicate the variations observed in this study. Nonsynonymous substitutions at positions +98, +239, and +602, and a synonymous substitution at position +474 show the related amino acid residues in parentheses. Note that the variations from position ±219 to +602 are in the PRKRAC gene, i.e., in the transposed PRKRA cDNA sequence
ing either DRB1*0405 or DRB1*0901 belongs to the DR53 group, these FISH profiles demonstrated that the PRKRA gene in the DR subregion was specific for the DR53 group.
Variations in the PRKRA gene When nucleotide sequences of the PCR products shown in Fig. 3 were examined by direct sequencing, several nucleotide sequence variations were found in the amplified PRKRA region. To further investigate these variations, we selected individuals homozygous for DRB1 alleles that belong to the DR53 group, and examined their PRKRA genes in the DR subregion by PCR direct sequencing. Figure 5 summarizes the variations observed in the PRKRA region. A total of ten variations were found, four in the amino acid coding region. Nonsense mutations were never observed, although synonymous and nonsynonymous substitutions were seen. From alignment of these variations, i.e., haplotypes with the variations, the haplotypes could be classified into three groups (Table 3).
Table 3 Nucleotide sequence variations in the PRKRA gene in the DR subregion. All the samples except for the PAC HS265J14 clone are homozygenous at the DRB1 locus (NA no answer because of the absence of the DRB1 gene in the clone) Sample name
PAC HS265J14 AWELLES BOLETH BSM MLF #56 13465 13708 11980 12802 13224 3427 GN48 G94 G175 13707 13800 9530 13144 G77 GN88 13763 13887 MOU PITOUT
DRB1 type
NA *0401 *0401 *0401 *0401 *0402 *0403 *0403 *0404 *0406 *0406 *0407 *0405 *0405 *0405 *0405 *0405 *0410 *0410 *0901 *0901 *0901 *0901 *0701 *0701
Sequence variations (based on numbering shown in Fig. 5) ±508
±296
±219
±73
±54
del del del del del del del del del del del del
T T T T T T T T T T T T A A A A A A A A A A A T T
G G G G G G G G G G G G C C C C C C C C C C C G G
C C C C C C C C C C C C A A A A A A A A A A A A A
del del del del del del del del del del del del
G G G G G G G G G G G G
GC GC GC GC GC GC GC GC GC GC GC GC
del GC del GC
±44
+98
+239
+474
+602
C C C C C C C C C C C C C C C C C C C C C C C T T
A A A A A A A A A A A A A A A A A A A A A A A G G
C C C C C C C C C C C C T T T T T T T T T T T C C
T T T T T T T T T T T T T T T T T T T T T T T C C
A A A A A A A A A A A A T T T T T T T T T T T T T
192
Discussion Haplotypes of the HLA-DR subregion are mainly classified into five DR groups in terms of its genome configuration with active DRB genes and their pseudogenes: DR1, DR8, DR51, DR52, and DR53 groups (Fig. 2). The data presented here demonstrated that the PRKRA gene in the DR subregion was specifically present in the DR53 group. Together with the previous observation that the PRKRA gene in the DR subregion appeared to have no intron sequences, it was suggested that the gene in the DR subregion could have been generated as a pseudogene, PRKRAC, on the common-ancestral genome of the DR53 group. A question was raised by the above possibility: where is the master copy of the PRKRA gene on the human genome? The results of the present FISH analyses provide us with a reliable answer. When individuals with the DR subregion belonging to the DR53 group were examined by FISH using PRKRAC as the probe, the hybridization signals were detected not only on Chr 6p21.3, to which the HLA region maps, but also on Chr 2q31.2±q32.1 (Fig. 4, Table 2). While detection of the signal on Chr 6p21.3 depended on DR53 group presence, the signal on Chr 2q31.2±q32.1
was always detected (Table 2). These observations, therefore, suggest that the master copy of the PRKRA gene could be present on Chr 2q31.2±q32.1. Given that no PCR amplification using hr7L and hr7R primers was observed in the HTC samples that belonged to DR groups other than DR53 (Fig. 2), the master copy of PRKRA may contain relatively long intron sequences such that the PCR amplification with these primers failed. To find and characterize the master copy, we have attempted to search the GenBank database using the BLAST computer program (Altschul et al. 1990). We have recently found a draft-sequence containing the PRKRA gene with introns (Fig. 6A). The name of the clone is RP11-65L3 (GenBank accession number
Fig. 6A,B Schematic drawing of the PRKRA and PRKRAC genes (A) and exon/intron boundaries (B) in the gene. A Schematic structure of the PRKRA and PRKRAC genes found in the RP11-65L3 and HS265J14 clones, respectively. Numbered open boxes represent exons. Vertical figures indicate the nucleotide positions based on the numbering used in the clone. In HS265J14, the 59 and 39 flanking sequences of PRKRAC are shown. Arrows indicate a direct sequence repeat. B Nucleotide sequences of the exon/intron boundaries in the PRKRA gene. Uppercase letters represent exon sequences. All introns follow the GT-AG role, highlighted by underlining
193
AC009948), and it is located between D2S335 and D2S2257. By alignment of the RP11-65L3 sequences and PRKRAC, the PRKRA gene in RP11-65L3 was shown to consist of eight exons (i.e., seven introns), some of which were >2 kb long, and that the exon/intron boundaries perfectly followed the GT-AG rule (Fig. 6B). The alignment also allowed us to define PRKRAC as a processed pseudogene: the presence of an A stretch at the 39 end of PRKRAC and the presence of direct sequences(59-CCCTTCTC-39) at both 59 and 39 ends were recognized (Fig. 6B). Taken all the data together, we conclude that PRKRAC is a processed pseudogene, specifically present in the DR53 group, and that the master copy of PRKRAC is most probably present on Chr 2q31.2±q32.1. Direct sequencing data with the PCR products (Fig. 5, Table 3) and the information on the boundaries of PRKRAC (Fig. 6A), together indicate that PRKRAC possesses at least eight nucleotide-sequence variations; i.e., the variations from ±219G/C to +602A/T (Fig. 5) (the hr7L primer used in the PCR is located in the 59 flanking region of PRKRAC). Interestingly, a nonsense mutation has never been observed in the amino acid coding region, although a synonymous and three nonsynonymous substitutions have been seen in the region. In our previous study using an in vitro translation system (Chida et al. 1999), the PRKRAC transcript synthesized in vitro was translatable. The PRKRAC transcript, if it were expressed, might encode a functional PRKRA. Accordingly, it would be of interest to see if PRKRAC is an expressed processed pseudogene, in other words, if the 59 flanking region of PRKRAC has promoter activity (in a preliminary experiment using an in vitro transcription system with HeLa nuclear extract, we could not, however, detect any promoter activities in the region; data not shown). From alignment of the variations in PRKRAC, the resultant variation profiles (haplotypes) could be classified into three groups (Table 3). Based on the classification, we attempted to divide the DR53 group further into three subgroups, provisionally named the DR53 subgroups I, II and III: the DR subregions containing DRB1*0401, *0402, *0403, *0404, *0406, and *0407 are grouped in DR53 subgroup I; the DR subregions containing DRB1*0405, *0410, and *0901 are in subgroup II; DRB1*0701 belongs to DR53 subgroup III. Since the frequency of DRB1 alleles that belong to the DR53 group appears to be increased in patients with adult T-cell leukemia (ATL) or human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparasis (HAM/TSP) caused by HTLV-I infection (Sonoda et al. 1996), and since PRKRA has high sequence homology to TRBP that binds to the HIV-I RNA genome (Chida et al. 1999), PRKRAC, if expressed, might be associated with the HTLV-I RNA genome, which could influence susceptibility to ATL and HAM/TSP. Classification of the DR53 subgroups I, II, and III in such patients may
provide us with significant information regarding a detailed relationship of the DR53 group with ATL and HAM/TSP. Finally, based on the seven base substitutions in the PRKRAC gene, i.e., the base substitutions at positions ±219, ±73, ±44, +98, +239, +474, and +602 (Table 3), we attempted to estimate the divergence time of each DR53 subgroup using the formulas described by Jukes and Canter (1969). When the average substitution rate (one substitution/site per year) was set to be 3.810±9 (for a pseudogene), the divergence times of either subgroup II or III from subgroup I and of subgroup II from subgroup III were estimated to be 0.3 and 0.375 million years ago, respectively. When the divergence orders of these subgroups were considered on the basis of the variations including ±296T/A and ±54 (delGC) (Table 3), the order may have been as follows: DR53 subgroup II diverging first from subgroup I and III, followed by the divergence of subgroup III from subgroup I. The divergence orders and times are very similar to those estimated from the variations in the DRB9 pseudogene, located at the telomeric terminus of the DR subregion (Gongora et al. 1996). Therefore, the variations in PRKRAC may be useful markers for studying the molecular evolution of the DR53 group. Acknowledgements We would like to thank Drs. Y. Ishikawa (Japan Red Cross Blood Center) and N. Tsuchiya (University of Tokyo) for kindly providing the genomic DNA samples, and Dr. J. Ohashi for his helpful discussion and suggestions. This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.
References Abdulla S, Alderton RP, Glynne RJ, Gut IG, Hosking LK, Jackson A, Kelly A, Newell WR, Radley E, Sanseau P, Thorpe KL, Trowsdale J. Beck S (1996) DNA sequencing of the MHC class II region and the chromosome 6 sequencing effort at the Sanger Centre. DNA Seq 7:5±7 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403±410 Bodmer JG, Marsh SG, Albert ED, Bodmer WF, Bontrop RE, Dupont B, Erlich HA, Hansen JA, Mach B, Mayr WR, Parham P, Petersdorf EW, Sasazuki T, Schreuder GM, Strominger JL, Svejgaard A, Terasaki PI (1999) Nomenclature for factors of the HLA System, 1998. Hum Immunol 60:361±395 Burd CG, Dreyfuss G (1994) Conserved structures and diversity of functions of RNA-binding proteins. Science 265:615±621 Campbell RD, Trowsdale J (1993) Map of the human MHC. Immunol Today 14:349±352 Chida S, Wakui M, Hohjoh H, Ishikawa Y, Tsuchiya N, Juji T, Tokunaga K (1998) A possible new gene encoding an RNA binding protein in the class II region and a MICA-MICB null haplotype in the class I region. In: Talwar GP and Gangly NK (eds) The10th International Congress of Immunology. Monduzzi, Bologna, pp 39±42 Chida S, Hohjoh H, Tokunaga K (1999) Molecular analyses of the possible RNA-binding protein gene located in the human leukocyte antigen (HLA)-DR subregion. Gene 240:125±132
194 Eckmann CR, Jantsch MF (1997) Xlrbpa, a double-stranded RNA-binding protein associated with ribosomes and heterogeneous nuclear RNPs. J Cell Biol 138:239±253 Gatignol A, Buckler-White A, Berkhout B, Jeang KT (1991) Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science 251:1597±1600 Gatignol A, Buckler C, Jeang KT (1993) Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen. Mol Cell Biol 13:2193±2202 Gongora R, Figueroa F, Klein J (1996) The HLA-DRB9 gene and the origin of HLA-DR haplotypes. Hum Immunol 51:23±31 Hill AV, Allsopp CE, Kwiatkowski D, Anstey NM, Twumasi P, Rowe PA, Bennett S, Brewster D, McMichael AJ, Greenwood BM (1991) Common west African HLA antigens are associated with protection from severe malaria. Nature 352:595±600 Hirai M, Suto Y, Kanoh M (1994) A method for simultaneous detection of fluorescent G-bands and in situ hybridization signals. Cytogenet Cell Genet 66:149±151 Janer M, Geraghty DE (1998) The human major histocompatibility complex: 42,221 bp of genomic sequence, high-density sequence-tagged site map, evolution, and polymorphism for HLA class I. Genomics 51:35±44 Juji T, Satake M, Honda Y, Doi Y (1984) HLA antigens in Japanese patients with narcolepsy: all the patients were DR2 positive. Tissue Antigens 24:316±319 Jukes TH, Cantor CR (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism. Academic Press, New York, pp 21±123
Kawai S, Maekawajiri S, Tokunaga K, Kashiwase K, Miyamoto M, Akaza T, Juji T, Yamane A (1996) Routine low and high resolution typing of the HLA-DRB gene using the PCRMPH (microtiter plate hybridization) method. Eur J Immunogenet 23:471±486 Marsh SGE, Packer R, Heyes JM, Bolton B, Fauchet R, Charron D, Bodmer JG (1997) The 12th international histocompatibility workshop cell line panel: list of cell lines. In: Charron F (ed) HLA. EDK, Paris, pp 633±647 MHC Sequence Consortium (1999) Complete sequence and gene map of a human major histocompatibility complex. Nature 401:921±923 Patel RC, Sen GC (1998). PACT, a protein activator of the interferon-induced protein kinase, PKR. EMBO J 17:4379±4390 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Sonoda S, Fujiyoshi T, Yashiki S (1996) Immunogenetics of HTLV-I/II and associated diseases. J Acquir Immune Defic Syndr Hum Retrovirol 13:S119±S123 Todd JA, Bell JI, McDevitt HO (1987) HLA-DQ beta gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature 329:599±604 Winchester R (1994) The molecular basis of susceptibility to rheumatoid arthritis. Adv Immunol 56:389±466 Yamazaki M, Tateno Y, Inoko H (1999) Genomic organization around the centromeric end of the HLA class I region: largescale sequence analysis. J Mol Evol 48:317±327
