Characterization of the Mouse PA28 Activator Complex Family: Complete Organizations of the Three Member and a Physical Map of the ∼150-kb Region Containing the α- and β This information is current as -Subunit Genes of September 28, 2021. Keiko Kohda, Teruo Ishibashi, Naoki Shimbara, Keiji Tanaka, Yoichi Matsuda and Masanori Kasahara J Immunol 1998; 160:4923-4935; ; http://www.jimmunol.org/content/160/10/4923 Downloaded from

References This article cites 66 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/160/10/4923.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Characterization of the Mouse PA28 Activator Complex Gene Family: Complete Organizations of the Three Member Genes and a Physical Map of the ϳ150-kb Region Containing the ␣- and ␤-Subunit Genes1,2

Keiko Kohda,* Teruo Ishibashi,* Naoki Shimbara,†‡ Keiji Tanaka,‡§ Yoichi Matsuda,¶ and Masanori Kasahara3*‡

The is a multisubunit protease responsible for the generation of peptides loaded onto MHC class I molecules. Recent evidence indicates that binding of an IFN-␥-inducible PA28 activator complex to the 20S proteasome enhances the generation of

␣ ␤ ␣␤ Downloaded from class I binding peptides. The - and -subunits, which constitute the PA28 activator complex in the form of an ( )3 hetero- hexamer, show significant amino acid sequence similarity to a , designated Ki or the ␥-subunit, that is capable of binding to the 20S proteasome. In this study, we describe the complete nucleotide sequences of the mouse genes, Psme1, Psme2, and Psme3, coding for the ␣-, ␤-, and ␥-subunits, respectively. The overall exon-intron organizations of the three Psme genes are virtually identical, thus providing evidence that they are descended from a single ancestral gene. The promoter regions of the Psme1 and Psme2 genes contain sequence motifs that qualify as IFN-stimulated response elements, consistent with the observation that their /expression is induced strongly by IFN-␥. The Psme1 and Psme2 genes are located ϳ6 kb apart with their 3؅-ends pointing toward http://www.jimmunol.org each other on bands C2 to D1 of mouse 14, supporting the idea that they emerged by tandem duplication. The Journal of Immunology, 1998, 160: 4923–4935.

HC class I molecules bind peptides produced by pro- rings are composed of ␤-type subunits that carry catalytic sites. teolysis of cytosolic and display them on the The 20S proteasome is an ancient enzyme found in organisms M cell surface (1, 2). The peptides are translocated from ranging from archaebacteria to humans (7). Thus, the vertebrate the cytosol into the endoplasmic reticulum by the TAP, where they immune system appears to have recruited preexisting machinery

bind to nascent class I molecules. Class I molecules bearing for- for peptide generation and have modified it to accommodate its by guest on September 28, 2021 eign peptides are recognized by the Ag receptor of cytotoxic T specific needs. One of such modifications seems to be the inven- cells, thus enabling the immune system to destroy abnormal cells tion of three IFN-␥-inducible ␤-type subunits known as low mo- that synthesize viral or other foreign proteins. Accumulated evi- lecular mass polypeptide 2 (LMP2)4, LMP7, and PSMB10 (orig- dence indicates that the proteasome is responsible for the genera- inally described as MECL1) (8). On stimulation with IFN-␥, these tion of cytosolic peptides presented by class I molecules (3–5). subunits are incorporated into the 20S proteasome by displacing The 20S proteasome, which constitutes the proteolytic core of the homologous housekeeping ␤-type subunits (9–15). Such pro- the proteasomal complex, is a cylinder-shaped particle made up of teasomes appear to produce class I binding peptides more effi- four layers of rings, each composed of seven subunits (6). The ciently (16–19). Consistent with the idea that the IFN-␥-inducible outer two rings are made up of ␣-type subunits, while the inner two ␤-type subunits are the specialized subunits dedicated to class I- mediated Ag presentation, phylogenetic analysis (20, 21) showed that LMP2 and LMP7 become detectable first in the cartilaginous *Department of Biochemistry, Hokkaido University School of Medicine, Sapporo fish, the most primitive class of vertebrates in which the MHC has † 060, Japan; Biomedical R&D Department, Sumitomo Electric Industries, Yokohama been identified (22). Recently, the hypothesis was put forward that 244, Japan; ‡CREST (Core Research for Evolutional Science and Technology), Japan Science and Technology Corporation, Japan; §Tokyo Metropolitan Institute of Med- the three IFN-␥-inducible ␤-type subunits emerged simultaneously ical Science, Tokyo 113, Japan; and ¶Laboratory of Animal Genetics, Nagoya Uni- in a common ancestor of jawed vertebrates as a result of chromo- versity School of Agricultural Sciences, Nagoya 464-01, Japan somal duplication involving the MHC region (23, 24). Received for publication October 16, 1997. Accepted for publication January Other proteasome subunits that might have a role specialized for 16, 1998. class I-mediated Ag presentation are those constituting the PA28 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance activator complex, also known as the 11S regulator of the 20S with 18 U.S.C. Section 1734 solely to indicate this fact. proteasome (4, 6). The PA28 activator complex (25, 26) is a ring- 1 This work was supported in part by Grants-in-Aid for Scientific Research from The shaped hexameric structure of ϳ200 kDa, made up of alternating Ministry of Education, Science, Sports and Culture of Japan (to M.K., K.T., and ϳ28-kDa ␣- and ϳ28-kDa ␤-subunits with a stoichiometry of Y.M.) and by a grant for Group Research Project “Biosystems Science” from The ␣␤ ␣ Graduate University for Advanced Studies, Hayama, Japan (to M.K.). ( )3; this complex binds to the outer -rings of the 20S protea- 2 Sequence data reported in this paper have been submitted to the DDBJ, EMBL, and some and stimulates its peptidase activity in an ATP-independent GenBank Nucleotide Sequence Databases under accession numbers AB007136 (mouse Psme1 genomic), AB007137 (human PSME1 genomic), AB007138 (mouse Psme2 genomic), and AB007139 (mouse Psme3 genomic). 4 Abbreviations used in this paper: LMP, low molecular mass polypeptide; BAC, 3 Address correspondence and reprint requests to Dr. Masanori Kasahara at his cur- bacterial artificial chromosome; FISH, fluorescence in situ hybridization; GAS, ␥-ac- rent address: Department of Biosystems Science, The Graduate University for Ad- tivated site; ISRE, IFN-stimulated response element; PFG, pulsed field gel; RACE, vanced Studies, Hayama 240-0193, Japan. E-mail address: [email protected] rapid amplification of cDNA ends.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 4924 MOUSE PA28 ACTIVATOR COMPLEX manner (27–29). Recent evidence (30, 31) indicates that the PA28 GAAGACATCCACCTG-3Ј for Psme2, and 5Ј-ACTTGTGATCCGCTCT CTG-3Ј for Psme3. The amplified DNA fragments were cloned into the activator complex enhances the generation of class I binding pep- ϩ tides by altering the cleavage pattern of the proteasome. Also, pBluescript SKII vector and transformed into bacteria. At least six clones were sequenced for each gene. enhanced expression of the PA28 ␣-subunit in virus-infected fi- broblasts results in more efficient presentation of viral peptides to Cloning of the human gene (PSME1) coding for the PA28 cytotoxic T cells (32). Like other key molecules of the class I Ag ␣-subunit ␣ ␤ presentation machinery, expression of the PA28 - and -subunits The genomic DNA segment encompassing putative exons 3 to 6 of the is induced strongly by IFN-␥ (33–37). PSME1 gene was isolated by PCR using human genomic DNA as a tem- Recently, we showed that the PA28 ␣- and ␤-subunits are struc- plate. The PCR reaction mixture (50 ␮l) contained 0.25 ␮g of human turally related to a Ki antigen (35), a protein originally identified genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, ␮ ␮ Ј with autoantibodies found in sera of patients with systemic lupus 200 M deoxynucleotide triphosphates, 2 M sense primer (5 -CAAGAA GATTTCTGAGCTGGATG-3Ј), 2 ␮M antisense primer (5Ј-TGACATC erythematosus (38). Interestingly, the Ki antigen forms a homo- CTTGATCTCAGG-3Ј), 1 ␮l of Perfect Match enhancer (Stratagene), and hexamer and binds to the 20S proteasome (39), suggesting that it 2.5 units of Taq DNA polymerase. The sense and antisense primers were might also modulate the proteasome activity. On the basis of its designed based on the previously published human PSME1 cDNA se- structural similarity to the ␣- and ␤-subunits and its ability to bind quence (33, 34). The conditions of amplification were 35 cycles of 40 s at 94°C, 1 min at 54°C, and 1 min at 72°C. The DNA fragment of ϳ600 bp to the 20S proteasome, Tanahashi et al. (39) proposed that the Ki thus obtained was cloned into the pBluescript SKIIϩ vector and sequenced. antigen should be renamed the PA28 ␥-subunit. To eliminate potential PCR artifacts such as base misincorporations, PCR As an initial step toward understanding the biologic functions of was conducted three times, and two clones were sequenced for each am- Downloaded from the PA28 activator complex gene family, we present here detailed plification. An identical sequence was obtained from all clones. structural analysis of the mouse genes, Psme1, Psme2, and Psme3, Isolation of bacterial artificial clones (BAC) coding for the ␣-, ␤-, and ␥-subunits, respectively.5 We also show that the Psme1 and Psme2 genes are located ϳ6 kb apart with their The mouse pBeloBAC11 library (45) constructed from the embryonic stem Ј cell line, CJ7, derived from 129/Sv mice (Research Genetics, Huntsville, 3 -ends pointing toward each other on bands C2 to D1 of mouse AL) was screened using a mouse Psme1 genomic DNA fragment (nucle- . otides 2725–4099 in Fig. 2) as a probe. Screening of high density BAC filters was conducted at Research Genetics. The positive clones were sent http://www.jimmunol.org/ Materials and Methods to us in the form of bacterial colonies. BAC DNA was isolated using the standard alkaline lysis method (41). Isolation of ␭ phage clones containing the mouse Psme1, Psme2, and Psme3 genes Pulsed field gel (PFG) analysis of the BAC clone A ␭ FIX II genomic library of 129/SvJ mice (catalog no. 946309; Strat- Restriction mapping of BAC DNA was performed as described previously agene, La Jolla, CA) was screened sequentially using the full-length mouse (46), with minor modifications. Briefly, BAC DNA was completely di- Psme1, Psme2, and Psme3 cDNA clones (40) as probes. Plaque hybrid- gested with NotI and then partially digested with MluI, NruI, or SalI. After ization was conducted according to the standard protocol (41) at 42°C for PFG electrophoresis and transfer to a nitrocellulose membrane, the restric- 24 h in a solution containing 50% formamide, 1 M NaCl, 10ϫ Denhardt’s tion fragments were hybridized with a fluorescein-3Ј-end-labeled T7 or ␮ by guest on September 28, 2021 solution, 50 mM Tris-HCl (pH 7.5), 1% Na4P2O7, 1% SDS, and 150 g/ml SP6 oligonucleotide primer in a solution containing 6ϫ SET (1ϫ SET is sheared and denatured salmon sperm DNA. After hybridization, the filters 150 mM NaCl, 15 mM Tris-HCl (pH 8.3), 1 mM EDTA), 0.1% SDS, 5ϫ were washed twice with 2ϫ SSC (1 ϫSSC is 150 mM NaCl, 15 mM Denhardt’s solution, and 100 ␮g/ml sheared and denatured salmon sperm sodium citrate, pH 7.0), 0.1% SDS at 60°C and once with 0.2ϫ SSC, 0.1% DNA at 40°C for 12 h. After hybridization, the membrane was washed four SDS at 60°C. Restriction mapping of the positive ␭ clones was conducted times for 5 min each in 6ϫ SSC, 0.1% SDS at room temperature. Hybrid- as previously described (42). The accuracy of the maps was verified by ization signals were detected with the CDP-star nucleic acid chemilumi- double digestion. The restriction fragments containing the genes were nescence reagent (NEN Life Science Products, Boston, MA). PFG elec- ϩ cloned into the pBluescript SKII vector (Stratagene) for further trophoresis was performed using the biased sinusoidal field gel characterization. electrophoresis system (47) following the instructions of the manufacturer (ATTO, Tokyo, Japan). A low range PFG marker (New England Biolabs, Determination of transcription initiation sites Beverly, MA) was included as a molecular mass size marker. Transcrip- Transcription initiation sites of the mouse Psme genes were determined by tional orientation of the Psme1 and Psme2 genes and their distance were the rapid amplification of cDNA ends (RACE) essentially as described by determined by long range PCR (ELONGASE amplification system; Life ␮ Technologies, Gaithersburg, MD) using the BAC DNA as a template. The Frohman (43) and Hirzmann et al. (44). Briefly, total liver RNA (7 g) Ј isolated from a 129/SvJ mouse was reverse transcribed using a gene-spe- primers used to measure the intergenic distance were: 5 -CTGCAAT GAGAAGATTGTGGT-3Ј (nucleotides 3479–3499 in Fig. 2) and 5Ј-CT cific primer as described previously (42). The gene-specific primers were Ј 5Ј-GTGACATCCTTGATCTCAGG-3Ј for Psme1,5Ј-CCTCGTTCTGAG GTAGCCAAGGCTTCCAAG-3 (nucleotides 3714–3733 in Fig. 4) for Ј Ј Ј primer pair A; and 5Ј-ATCCTGAAGAACTTTGAGAAGC-3Ј (nucleo- AAGTACTT-3 for Psme2, and 5 -AGGTTCATGTCTGAGTGG-3 for Ј Ј Psme3. The cDNA thus obtained was tailed with dATP (for Psme1)or tides 4446–4467 in Fig. 2) and 5 -CTGTAGCCAAGGCTTCCAAG-3 dGTP (for Psme2 and Psme3). We tailed the Psme1 cDNA with dATP (nucleotides 3714–3733 in Fig. 4) for primer pair B. Ј because tailing with dGTP almost exclusively produced truncated 5 - DNA sequencing RACE products. The tailed cDNA was amplified by PCR (30 cycles of 40 s at 94°C, 1 min at 52°C, and 1 min at 72°C) using a second, more proximal The nucleotide sequence was determined by the chain termination method gene-specific primer (5Ј-AGAACGCATCCAACTCTGAG-3Ј for Psme1, (48) using an automated DNA sequencer (model 4000L, LI-COR, Lincoln, 5Ј-CCGTGGCAAGAAAGTGCAG-3Ј for Psme2, and 5Ј-TGCCACCAA NE) and the SequiTherm Long-Read cycle sequencing kit (Epicentre Tech- Ј GTCTTCTGCCTCAC-3 for Psme3) and the (dT)17- or (dC)12-adapter nologies, Madison, WI). Both strands of the DNA were sequenced at Ј primer. The sequences of the (dT)17- and (dC)12-adapter primers were 5 - least once. Ј Ј GACTCGAGTCGACATCGAT17-3 and 5 -TTCTAGAATTCGGATC12- 3Ј, respectively. The second round of PCR (30 cycles of 40 s at 94°C, 1 min Fluorescence in situ hybridization (FISH) at 54°C, and 1 min at 72°C, and a final extension of 10 min at 72°C) was performed with 1/25th of the material from the first round of PCR using Chromosomal localization of the mouse BAC clone containing the Psme1 another more proximal gene-specific primer and the (dT) - or (dC) - and Psme2 genes was determined using the direct R-banding FISH method 17 12 ϳ adapter primer. The gene-specific primers used for the second round of as described previously (49, 50). Briefly, the BAC DNA of 150 kb was PCR were 5Ј-CACAGGTCTTCACGGAACAC-3Ј for Psme1,5Ј-GTCT labeled by nick translation with biotin-16-dUTP (Boehringer Mannheim, Mannheim, Germany) following the manufacturer’s protocol. Following denaturation and preannealing with the mouse Cot-1 DNA (Life Technol- 5 These gene symbols were approved officially by the Nomenclature Committee of ogies), the biotinylated probes were hybridized overnight at 37°C to the The Mouse Genome Database, The Jackson Laboratory, Bar Harbor, ME. R-banded mouse . Hybridization signals were detected with The Journal of Immunology 4925 Downloaded from

FIGURE 1. Organization and restriction map of the mouse Psme gene family. Exons are shown as solid boxes and numbered. Two overlapping ␭ clones, http://www.jimmunol.org/ ␭B2-1 and ␭B6-1, containing the Psme2 gene are indicated by horizontal bars. The restriction maps of the Psme1 and Psme3 genes were constructed using ␭ clones ␭A6-1 and ␭K6-1, respectively. The exon encoding the 3Ј-untranslated region of the Psme3 gene is drawn in length corresponding to the longer transcript.

fluoresceinated avidin (Vector Laboratories, Burlingame, CA). After wash- (57). 2) The sequence identical with nucleotides 3455–3475 is ing, the slides were stained with propidium iodide (0.75 ␮g/ml). R- and found at nucleotides 3407–3427 (indicated by brackets). This du- G-bands (51, 52) were detected with Nikon B-2A (excitation wavelength, plication creates a potential miniexon capable of encoding five 450–490 nm) and UV-2A (excitation wavelength, ϳ365 nm) filter sets, additional amino acids (PPCGP), although there is no evidence respectively. Kodak Ektachrome ASA100 films (Eastman Kodak, Roches- by guest on September 28, 2021 ter, NY) were used for microphotography. that it is actually used by alternative splicing. To examine whether these unusual features of the mouse Psme1 gene are shared in the Sequence analysis human counterpart (designated PSME1), we isolated the corre- Putative regulatory elements in the promoter regions were identified by sponding region of PSME1. Figure 3 shows that the PSME1 gene searching the TFMATRIX transcription factor binding site profile database also has a variant 5Ј-splice site in putative intron 4 but contains no (release 2.4) with the computer programs TFSEARCH (version 1.3) (53) duplicated sequence in the corresponding intron. and MatInspector (version 2.1) (54). Repetitive sequences in the mouse Psme genes were identified using the RepeatMasker 2 program (http:// The transcription initiation sites of the Psme1 gene were deter- ftp.genome.washington.edu/RM/). Putative sorting signals were analyzed mined by 5Ј-RACE. Of 13 clones subjected to sequence analysis, with the computer program PSORT (http://www.nibb.ac.jp). 2 had inserts starting at nucleotide 1595 and another 2 had inserts at nucleotide 1602. The remaining 9 clones started at nucleotides Results 1592, 1600, 1604, 1631, 1635, 1636, 1648, 1655, and 1661. Thus, Genomic organization of the mouse Psme1 gene like many other genes that lack a TATA box, the Psme1 gene To clone the mouse Psme1 gene, we screened the ␭ FIX II appears to have multiple transcription initiation sites. genomic library of 129/SvJ mice using the previously isolated A computer search showed that the putative promoter region of mouse cDNA probe. Approximately 30 positive clones were iden- this gene has several transcription factor-binding motifs (Fig. 2). tified from ϳ1 ϫ 106 plaques. Preliminary analysis of the ␭ clones Of interest is the existence of a putative IFN-stimulated response revealed that they fall into at least two distinct groups, one con- element (ISRE) at nucleotides 1628–1641. The observed sequence taining the Psme1 gene; and the other containing a Psme1-like (GCTTTCGCTTTCCC) contains the core sequence (TTCNNTTT) sequence without introns. The latter was found to contain a pro- that binds IFN-regulatory factors 1 and 2 and shows 85.7% identity cessed pseudogene thought to be derived from the Psme1 gene to the consensus ISRE motif (AGTTTCNNTTTCNY; Y ϭ CT, (data not shown). We chose one clone, designated ␭A6-1, con- N ϭ GACT) that functions in both orientations (58, 59). This is taining the Psme1 gene and subjected it to detailed analysis. Figure consistent with the observation that expression of Psme1 is in- 1 shows the restriction map of this ␭ clone. Figure 2 shows the duced strongly by IFN-␥ (33–36). No sequence that qualifies as a complete nucleotide sequence of the Psme1 gene. The exonic se- ␥-activated site (GAS; consensus sequence, TTCNNNGAA) (60) quence of the genomic clone was identical with the cDNA se- was found in the putative promoter region of the Psme1 gene. quence obtained from the C57BL/6NHsd mouse (40). Two fea- tures of the Psme1 gene are noteworthy (Fig. 2). 1) The 5Ј-splice Genomic organization of the mouse Psme2 gene site of intron 4 has GC instead of GT, thus deviating from the To clone the mouse Psme2 gene, we screened the genomic library canonical GT/AG rule (55). This type of rare nonconsensus splice using the mouse cDNA probe. Approximately 35 positive clones junction sequence (56) has been shown to allow the 5Ј-site to be were identified from ϳ1 ϫ 106 plaques. Here again, preliminary accurately cleaved, albeit more slowly than the usual GT sequence analysis of the clones revealed the existence of at least two distinct 4926 MOUSE PA28 ACTIVATOR COMPLEX Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 The Journal of Immunology 4927 types of clones, one containing the Psme2 gene and the other con- taining a Psme2-like gene without introns. Two overlapping ␭ clones, ␭B2–1 and ␭B6-1, containing the Psme2 gene were chosen and subjected to restriction enzyme mapping (Fig. 1). Figure 4 shows the complete nucleotide sequence of the Psme2 gene. The exonic sequence of the genomic clone differed from the previously reported cDNA sequence of the C57BL/6NHsd mouse (40) by 5 bp. These nucleotide substitutions, which most likely reflect an allelic variation, are all synonymous and do not change the amino acid residues. The exon-intron boundaries of the Psme2 gene con- formed to the canonical GT/AG rule without exception. The loca- tions and the splicing phases of the exon-intron boundaries in the Psme2 gene are essentially identical with those of the Psme1 gene (Fig. 5). The transcription initiation sites of the mouse Psme2 gene were determined by 5Ј-RACE. Sequence analysis of seven randomly chosen clones showed that they all have inserts with different lengths. The 3Ј-termini of two clones, starting at nucleotides 866 Downloaded from and 872, contained a C residue thought to have resulted from the reverse transcription of the 5Ј-cap of mRNA. The remaining five clones started at nucleotides 850, 860, 864, 868, and 873. Thus, the FIGURE 3. Partial nucleotide and deduced amino acid sequences of the Psme2 gene, which does not have a TATA or CAAT box, appears human PSME1 gene. Exonic and intronic sequences are written in capital to have multiple transcription initiation sites. and lowercase letters, respectively. The conserved dinucleotides (GT/AG and GC/AG) at the 5Ј- and 3Ј-splice sites are doubly underlined. Amino Like the Psme1 gene, expression of the Psme2 gene is induced ␥ acids are shown with a standard single-letter code under the nucleotide http://www.jimmunol.org/ strongly by IFN- (35, 36). Consistent with this observation, the sequence. The amino acid sequence shown here is identical with that de- putative promoter region of the Psme2 gene contains a consensus duced from the previously described cDNA sequence (34). ISRE sequence at nucleotides 837–850. The inversely comple- mentary sequence of nucleotides 837–850 (GCTTTCGCTT nucleotides 2725–4099 of the Psme1 gene (Fig. 2) was amplified TCAC) contains the core sequence of ISRE (TTCNNTTT), and by PCR from this BAC clone; 2) FISH analysis showed that clone shows 85.7% identity with the consensus ISRE motif (AGTTTC 236C3 maps to bands C2 to D1, most likely to the proximal region NNTTTCNY; Y ϭ CT, N ϭ GACT). Another sequence motif that of band D1 of mouse chromosome 14 (Fig. 6, A and B). This might be functionally important is the potential NF-␬B-binding cytogenetic localization is in good accord with the map position of

site (consensus sequence, GGGRNNYYCC) located at nucleotides Psme1 obtained by interspecific backcross mapping (62). Figure by guest on September 28, 2021 335–344 (61). Besides the consensus ISRE sequence and potential 6C shows the restriction map of clone 236C3. Hybridization anal- NF-␬B-binding site, the putative promoter region of the Psme2 ysis with the cDNA probes indicated that the ϳ32-kb SalI frag- gene contains two potential SP1-binding sites at nucleotides 230– ment contains both Psme1 and Psme2 genes. To establish that the 238 and 800–808 (consensus sequence, KRGGCKRRK; K ϭ GT, gene hybridizing with the Psme2 cDNA probe is the functional R ϭ AG; thus, a 1-bp mismatch in both sites). No sequence that Psme2 gene, we amplified from the BAC clone the 540-bp DNA qualifies as GAS was found. fragment and confirmed that its sequence is identical with the cor- responding region (nucleotides 3716–4255 in Fig. 4) of the Psme2 Physical map of the ϳ150-kb region containing the mouse gene. Finally, we determined the distance and relative orientation Psme1 and Psme2 genes of the two Psme genes using the primer pairs described in Mate- We showed previously, using an interspecific backcross panel rials and Methods. Primer pairs A and B produced a band of ϳ8 (40), that the Psme1 and Psme2 genes are tightly linked and map and ϳ7 kb, respectively. Therefore, taken together, Psme1 and ϩ close to the Atp5g1 locus (a gene coding for the ATP synthase, H Psme2 are located ϳ6 kb apart with their 3Ј-ends pointing toward transporting, mitochondrial Fo complex, subunit c, isoform 1) on each other on bands C2 to D1 of mouse chromosome 14, indicating mouse chromosome 14. The distance between Psme1 and Psme2 that they emerged by tandem duplication. Subsequent analysis of was predicted to be Ͻ3.8 cM at the 95% confidence level (40). To the ␭ clones, ␭B6-1 containing the Psme2 gene and ␭A6-1 con- examine the possibility that the two Psme genes are located adja- taining the Psme1 gene (Fig. 1), revealed that their 3Ј-ends have an cent to each other, we isolated BAC clones using the Psme1 overlap of ϳ2.5 kb, thus confirming the analysis of the BAC clone genomic fragment as a probe. Two distinct types of BAC clones, (Fig. 6, C and D). one containing the Psme1 gene (clone 236C3) and the other con- taining a processed pseudogene related to Psme1 (clone 7N2), Genomic organization of the mouse Psme3 gene were identified. Two lines of evidence indicated that clone 236C3 We also isolated the Psme3 gene and determined its restriction contains the functional Psme1 gene: 1) the sequence identical with map (Fig. 1) and complete nucleotide sequence (Fig. 7). Consistent

FIGURE 2. Nucleotide and deduced amino acid sequences of the mouse Psme1 gene. Exonic and intronic sequences are written in capital and lowercase letters, respectively. The 5Ј-end of the previously published cDNA clone (40) is shown as the upstream boundary of the 5Ј-untranslated region. It does not indicate major transcription initiation sites. The conserved dinucleotides at the 5Ј- and 3Ј-splice sites (GC/AG in intron 4 and GT/AG in all the other introns) are doubly underlined. Also doubly underlined is the putative ISRE. An in-frame stop codon (TAG) preceding the ATG translation initiation codon is indicated by asterisks. The duplicated sequence in intron 5 that could function as a miniexon (nucleotides 3407–3427) is indicated by brackets. The putative polyadenylation signal AATAAA (nucleotides 4633–4638) is doubly underlined. Potential transcription factor-binding sites are also indicated. Amino acids are shown with a standard single-letter code under the nucleotide sequence. The KEKE motif located at amino acid positions 70–97 is boxed. 4928 MOUSE PA28 ACTIVATOR COMPLEX Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 The Journal of Immunology 4929 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Locations of the exon-intron boundaries in the mouse PA28 ␣-, ␤-, and ␥-subunits. Ϫ and * indicate identity with the top sequence and absence of residues, respectively. Œ indicate exon-intron boundaries. Exon-intron classes were defined as follows: class 0, splice site between codons; class 1, splice site after codon position 1; and class 2, splice site after codon position 2. The mouse sequences were taken from Figures 2, 4, and 7. The sources of the human sequences were PA28 ␣-subunit (34), ␤-subunit (35), and ␥-subunit (38). Nuclear localization signals predicted with the PSORT program are boxed. The KEKE motif in the ␣-subunit is underlined. with the previous observation that Psme3 is a single-copy gene gives rise to an alternatively spliced transcript in fetal brain tissues. (36), only one type of genomic clone was identified (data not This transcript encodes a larger ␥-subunit, in which 13 additional shown). Comparison of the exonic sequence in Figure 7 and the amino acids (PSGKGPHICFDLQ) are inserted between amino cDNA sequence obtained from C57BL/6NHsd mice (40) revealed acid residues 135 and 136 in Figure 5. However, this appears to a total of 4 bp substitutions and an insertion of 7 bp in the genomic be unique to the human gene because the corresponding region sequence. These substitutions (located at nucleotides 6970, 7467, of the mouse Psme3 gene (intron 6) does not contain an open 7468, and 7868 in Fig. 7) and the 7-bp insertion (at nucleotides reading frame capable of encoding similar amino acids (Fig. 7). 8312–8318 in Fig. 7) are located in the 3Ј-untranslated region. The size of the Psme3 gene is larger than that of the other Psme Thus, the PA28 ␥-subunits of 129/SvJ and C57BL/6NHsd mice genes, mainly because the former has two large introns (introns have an identical amino acid sequence. The exon-intron bound- 4 and 10). However, the overall exon-intron organization of the aries of the Psme3 gene obeyed the GT/AG rule. Albertson et al. Psme3 gene is essentially identical with that of the Psme1 and (63) showed that the human gene coding for the PA28 ␥-subunit Psme2 genes, with the exon-intron boundaries located at nearly

FIGURE 4. Nucleotide and deduced amino acid sequences of the mouse Psme2 gene. Exonic and intronic sequences are written in capital and lowercase letters, respectively. The 5Ј-end of the previously published cDNA clone (40) is shown as the upstream boundary of the 5Ј-untranslated region. It does not indicate major transcription initiation sites. The conserved dinucleotides at the 5Ј- and 3Ј-splice sites (GT/AG) are doubly underlined. Also doubly underlined are the putative ISRE and the potential NF-␬B-binding site. An in-frame stop codon (TAG) preceding the ATG translation initiation codon is indicated by asterisks. The putative polyadenylation signal AATAAA (nucleotides 4319–4324) is doubly underlined. Potential transcription factor-binding sites are also indicated. Amino acids are shown with a standard single-letter code under the nucleotide sequence. 4930 MOUSE PA28 ACTIVATOR COMPLEX

Table I. Distribution of repetitive sequences in the mouse Psme genes

Positionb Length % Name Classa From To Location (bp) Orientationc Similarityd

Psme 1 B1 SINE/Alu 293 333 5Ј-Flanking 41 ϩ 85.4 B1 SINE/Alu 426 571 5Ј-Flanking 146 Ϫ 91.8 Ј ϩ (CATA)n Simple repeat 648 745 5 -Flanking 98 76.5 B1 SINE/Alu 2081 2229 Intron 1 149 Ϫ 79.9 MT2B LTR/MaLR 2144 2252 Intron 1 109 Ϫ 72.5 ϩ (TA)n Simple repeat 2276 2321 Intron 1 46 78.3 B1 SINE/Alu 2332 2476 Intron 1 145 Ϫ 93.8

Psme2 B1 SINE/Alu 1843 1988 Intron 3 146 ϩ 95.9 ϩ (TA)n Simple repeat 2604 2660 Intron 6 57 73.7 B1 SINE/Alu 2722 2826 Intron 6 105 Ϫ 95.2 B2 SINE/B2 2829 3006 Intron 6 178 Ϫ 93.3 B3 SINE/B2 3007 3192 Intron 6 186 ϩ 72.9 Ј ϩ B3 SINE/B2 4426 4479 3 -Flanking 54 79.6 Downloaded from

Psme3 MER2 DNA/MER2 143 211 5Ј-Flanking 69 ϩ 75.4 B1 SINE/Alu 245 383 5Ј-Flanking 139 ϩ 93.5 Poly(A) Simple repeat 400 468 5Ј-Flanking 69 ϩ 85.5 Ј ϩ (CGGG)n Simple repeat 1255 1339 5 -Flanking 85 71.8 ϩ (GGGA)n Simple repeat 1948 2001 Intron 1 54 81.5 Ϫ http://www.jimmunol.org/ (GA)n Simple repeat 2548 2576 Intron 3 29 100 B1 SINE/Alu 3106 3230 Intron 4 125 Ϫ 80.0 Ϫ (CAAAA)n Simple repeate 3302 3370 Intron 4 69 73.9 ID3 SINE/ID 3371 3427 Intron 4 57 Ϫ 66.7 B2 SINE/B2 3428 3638 Intron 4 211 ϩ 88.6 B1 SINE/Alu 3743 3829 Intron 4 87 ϩ 67.8 RSINE1 SINE/B4 4083 4175 Intron 4 93 Ϫ 67.7 Ϫ (CA)n Simple repeat 4272 4299 Intron 4 28 92.9 B1 SINE/Alu 6165 6281 Intron 10 117 Ϫ 76.1 B3 SINE/B2 6350 6494 Intron 10 145 Ϫ 66.9 Ј Ϫ

RSINE2 SINE/B4 7975 8020 3 -Untranslated 46 82.6 by guest on September 28, 2021

a SINE, short interspersed nucleotide elements; LTR, long terminal repeats; MaLR, mammalian apparent LTR-retrotransposons. b Starting and ending position of match (nucleotide numbers correspond to those in Figs. 2, 4, and 7). c Ϫ Match is with the complement of the consensus sequence in the database. d Percent substitutions in matching region compared with the consensus.

equivalent positions and having identical splicing phases ments are thought to have emerged in the rodent lineage after its (Fig. 5). separation from the primate lineage (64), virtual absence of shared We determined the transcription initiation sites of the mouse Psme3 B1 or B2 sequences indicates that the duplication events that gave gene by 5Ј-RACE. Sequence analysis of six randomly chosen clones rise to the three Psme genes took place before mammalian radia- showed that they all have inserts with different lengths. The clones tion. This is consistent with the fact that all three members of the started at nucleotides 1356, 1371, 1395, 1398, 1432, and 1452. Thus, Psme gene family have been identified in both humans and mice. the Psme3 gene appears to have multiple transcription initiation sites. Previous studies showed that IFN-␥ treatment induces a tran- sient, modest increase in the Psme3 mRNA level (35, 36). How- Discussion ever, the putative promoter region of the mouse Psme3 gene does In the present study, we determined the complete nucleotide se- not contain any sequence motifs that qualify as ISRE or GAS (Fig. quences of the three known members of the mouse PA28 activator 7). There is one potential NF-␬B-binding site at position 840–849 complex gene family. The 3 Psme genes show striking similarity (1 bp mismatch from the consensus). The Psme3 gene contains no in exon numbers (they all have 11 exons), locations of exon-intron obvious TATA or CAAT box in its putative promoter. boundaries, and phases of splicing sites (Figs. 1 and 5). This ob- servation provides further evidence that they are descended from a Repetitive sequences in the mouse Psme gene family single ancestral gene. The ␣- and ␤-subunits are more closely re- Table I summarizes the distribution of repetitive sequences in the lated to each other (ϳ50% amino acid sequence identity) than they mouse Psme gene family. The repetitive sequences occupy 14.5, are to the ␥-subunit (ϳ40% and ϳ32% amino acid sequence iden- 16.6, and 16.8% of the total sequences in the Psme1, Psme2, and tity). Thus, the Psme1 and Psme2 genes appear to have emerged by Psme3 genes, respectively. Thus, the three Psme genes contain duplication after its common ancestor had diverged from the almost the same proportion of repetitive sequences. With the ex- Psme3 gene. Our observation that the Psme1 and Psme2 genes are ception of the fact that the 5Ј-flanking regions of the Psme1 and located ϳ6 kb apart with their 3Ј-ends pointing toward each other Psme3 genes contain the B1 repeats, none of the short interspersed on a single BAC clone (Fig. 6) provides convincing evidence that nucleotide elements is shared at the corresponding positions they arose by tandem duplication. Invertebrates such as the brown among the members of the Psme gene family. Because these ele- ear tick (65) and Caenorhabditis elegans (40) have a Psme3-like The Journal of Immunology 4931 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Localization of the BAC clone to bands C2-D1 of mouse chromosome 14 and a physical map of the ϳ150-kb region containing the Psme1 and Psme2 genes. The metaphase spreads were photographed with Nikon UV-2A (A) and B-2A (B) filters. A and B, G-band and R-band patterns, respectively. Specific hybridization signals are indicated by white arrows (B). C, Restriction map of the BAC clone (clone 236C3). The orientations of the Psme1 and Psme2 genes are indicated by arrows. Individual exons of the genes are not shown. The regions of the BAC clone covered by the ␭ clones ␭A6-1, ␭B2–1, and ␭B6-1 (Fig. 1) are shown at the bottom of the figure. The BAC clone contains a ϳ2.5-kb MluI fragment ϳ70 to 90 kb away from the left end, the precise location of which could not be determined. D, Restriction map of the region between the Psme1 and Psme2 genes. Exons are shown as solid boxes and numbered. gene. In contrast, the ␣- and ␤-subunits appear to have a role in which the MHC has been identified (N. Yamamoto and M. dedicated to class I-mediated Ag presentation (30, 32), suggesting Kasahara, unpublished observations). Interestingly, attempts to that they are of more recent origin and perhaps unique to the ver- identify the Psme2 gene in the cartilaginous fish have been unsuc- tebrate. Consistent with this prediction, our preliminary phyloge- cessful. Thus, the tandem duplication that gave rise to the ␤-sub- netic analysis indicates that the Psme1 gene becomes detectable unit might have taken place at a later stage in vertebrate evolution. first in the cartilaginous fish, the most primitive class of vertebrates Available evidence indicates that the ␣-subunit alone is capable of 4932 MOUSE PA28 ACTIVATOR COMPLEX Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 The Journal of Immunology 4933 Downloaded from http://www.jimmunol.org/

FIGURE 7. Nucleotide and deduced amino acid sequences of the mouse Psme3 gene. Exonic and intronic sequences are shown in capital and lowercase letters, respectively. The 5Ј-end of the previously published cDNA clone (40) is shown as the upstream boundary of the 5Ј-untranslated region. It does not indicate major transcription initiation sites. The conserved dinucleotides at the 5Ј- and 3Ј-splice sites (GT/AG) are doubly underlined. An in-frame stop codon (TGA) preceding the ATG translation initiation codon is indicated by asterisks. The Psme3 cDNA occurs in two distinct forms differing in the length of the 3Ј-untranslated region (36, 38, 39, 40). Œ, Poly(A) addition sites observed previously in the shorter cDNA clones (40). The putative polyadenylation by guest on September 28, 2021 signals utilized in the shorter form (nucleotides 6983–6994 and 7020–7025) and in the longer form (nucleotides 8511–8516 and 8519–8530) are doubly underlined. Amino acids are shown with a standard single-letter code under the nucleotide sequence. Potential transcription factor-binding sites in the putative promoter region are also indicated.

facilitating the production of class I binding peptides in vitro (30, that both Psme1 and Psme2 have putative ISRE, but no GAS (Figs. 31, 39). However, optimal peptidase activity is achieved when 2 and 4). With the possible exception of the LMP2 gene, which both ␣- and ␤-subunits are present (28, 39). It appears, therefore, shares a bidirectional promoter with the TAP1 gene (71, 68) and that the tandem duplication event that created the ␤-subunit made hence might be induced rapidly by IFN-␥, all known IFN-␥-in- the PA28 activator complex more efficient in producing class I ducible subunits of the proteasome appear to be activated with binding peptides. similar kinetics, thus presumably contributing to their coordinated The promoter regions of IFN-␥-inducible genes often contain expression. ISRE, GAS, or both (59, 66). Genes with a GAS element are Some IFN-␥-inducible genes, most notably the TAP1 and MHC activated rapidly by IFN-␥, without a need for new protein syn- class I genes, are also activated by TNF-␣ (67). Such genes usually thesis. Typically, expression of these genes is induced within 1 h contain both ISRE and an NF-␬B-binding site in their promoters and maximal mRNA induction is achieved by 6 h. Among the (66). Recent evidence indicates that TNF-␣ activates NF-␬Bby genes involved in class I-mediated Ag presentation, TAP1 appears degrading its cytoplasmic inhibitor I␬B (61). The activated NF- to fall into this category (67, 68). In contrast, genes with ISRE, but ␬B, which translocates to the nucleus and binds to the NF-␬B- without a GAS element, are activated by IFN-␥ more slowly, be- binding site of the target genes, then interacts with the IFN-␥- cause their induction requires the synthesis of transcription factors induced transcription factors that bind to the ISRE (66). This that bind to the ISRE. In these genes, the mRNA levels usually interaction results in synergistic induction of transcription by the increase only after6hormore and reach the maximum between 24 two cytokines. In this regard, it is notable that the putative pro- and 48 h. MHC class I genes (69), and the genes coding for the moter of the mouse Psme2 gene contains the potential NF-␬B- ␤-type proteasome subunits LMP7 (70) and PSMB10 (13) appear binding site (nucleotides 335–344 in Fig. 4) besides the ISRE. to be of this type. Previous studies showed that the Psme1 and Thus, expression of this gene might be induced synergistically by Psme2 mRNAs increase gradually, attaining the maximum level in TNF-␣ and IFN-␥. 24 to 36 h after IFN-␥-stimulation (35, 36). Although absence of IFN-␥ treatment also induces a transient, modest increase in the information on the half-life of the mRNA precludes us from draw- Psme3 mRNA level (35, 36). The magnitude of induction was, ing definitive conclusions, the kinetics of the response appears to however, ϳ10% of that observed for the Psme1 or Psme2 gene suggest that Psme1 and Psme2 also fall into the latter category of (36), and the mRNA levels returned to control levels in 48 h after the IFN-␥-inducible genes. This is consistent with our observation exposure to IFN-␥ (35). The putative promoter region of the mouse 4934 MOUSE PA28 ACTIVATOR COMPLEX

Psme3 gene does not contain sequence motifs that qualify as ISRE 8. Larsen, F., J. Solheim, T. Kristensen, A.-B. Kolstø, and H. Prydz. 1993. A tight or GAS (Fig. 7). Thus, it appears that the Psme3 mRNA is induced cluster of five unrelated human genes on chromosome 16q22.1. Hum. Mol. Genet. 2:1589. by IFN-␥ through a pathway distinct from that mediated by ISRE 9. Akiyama, K., K. Yokota, S. Kagawa, N. Shimbara, T. Tamura, H. Akioka, or GAS. The Psme3 mRNA occurs in two forms that differ in the H. G. Nothwang, C. Noda, K. Tanaka, and A. Ichihara. 1994. cDNA cloning and Ј interferon ␥ down-regulation of proteasomal subunits X and Y. Science 265: length of the 3 -untranslated region (Fig. 7). These two forms, 1231. which encode the same polypeptide, appear to be produced by 10. Belich, M., R. J. Glynne, G. Senger, D. Sheer, and J. Trowsdale. 1994. Protea- differential transcription termination. Interestingly, the ␥-subunit some components with reciprocal expression to that of the MHC-encoded LMP proteins. Curr. Biol. 4:769. genes of humans and cows also have two mRNA forms that differ 11. Fru¨h, K., M. Gossen, K. Wang, H. Bujard, P. A. Peterson, and Y. Yang. 1994. only in the length of the 3Ј-untranslated region and hence are pre- Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a sumably produced by differential transcription termination (38, newly discovered mechanism for modulating the multicatalytic proteinase com- plex. EMBO J. 13:3236. 39). This observation suggests that the existence of two mRNA 12. Groettrup, M., R. Kraft, S. Kostka, S. Standera, R. Stohwasser, and P.-M. Kloet- forms might have some functional significance. zel. 1996. A third interferon-␥-induced subunit exchange in the 20S proteasome. The PA28 ␣- and ␤-subunits reside both in the cytoplasm and Eur. J. Immunol. 26:863. 13. Hisamatsu, H., N. Shimbara, Y. Saito, P. Kristensen, K. B. Hendil, T. Fujiwara, the nucleus, whereas the ␥-subunit exists almost exclusively in the E. Takahashi, N. Tanahashi, T. Tamura, A. Ichihara, and K. Tanaka. 1996. Newly nucleus (37). A computer search of the deduced amino acid se- identified pair of proteasomal subunits regulated reciprocally by interferon-␥. ␣ ␤ ␥ J. Exp. Med. 183:1807. quences of the -, -, and -subunits for sorting signals indicates 14. Nandi, D., H. Jiang, and J. J. Monaco. 1996. Identification of MECL-1 (LMP-10) that the ␣- and ␥-subunits have two putative nuclear localization as the third IFN-␥-inducible proteasome subunit. J. Immunol. 156:2361. signals, respectively (boxed in Fig. 5). One of the predicted nuclear 15. Hayashi, M., T. Ishibashi, K. Tanaka, and M. Kasahara. 1997. The mouse genes ␣ encoding the third pair of ␤-type proteasome subunits regulated reciprocally by Downloaded from localization signals in the -subunit is embedded in the KEKE IFN-␥: structural comparison, chromosomal localization, and analysis of the pro- motif (72) proposed to be involved in protein-protein interac- moter. J. Immunol. 159:2760. tions. Therefore, this motif might also serve as a nuclear local- 16. Driscoll, J., M. G. Brown, D. Finley, and J. J. Monaco. 1993. MHC-linked LMP gene products specifically alter peptidase activities of the proteasome. Nature ization signal. No nuclear localization signal was predicted for 365:262. ␤ ␣␤ the -subunit. Thus, the assembly of the ( )3 heterohexamer 17. Fehling, H. J., W. Swat, C. Laplace, R. Ku¨hn, K. Rajewsky, U. Mu¨ller, and might take place in the cytoplasm and this complex might be H. von Boehmer. 1994. MHC class I expression in mice lacking the proteasome

subunit LMP-7. Science 265:1234. http://www.jimmunol.org/ translocated to the nucleus by virtue of the sorting signal carried 18. Gaczynska, M., K. L. Rock, T. Spies, and A. L. Goldberg. 1994. Peptidase ac- by the ␣-subunit. tivities of are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7. Proc. Natl. Acad. Sci. USA 91: The structural organizations of the mouse Psme gene family 9213. described in this study provide the basic information required to 19. Van Kaer, L., P. G. Ashton-Rickardt, M. Eichelberger, M. Gaczynska, create knockout mice. Given the growing evidence implicating the K. Nagashima, K. L. Rock, A. L. Goldberg, P. C. Doherty, and S. Tonegawa. ␣ 1994. Altered peptidase and viral specific T cell response in LMP2 mutant mice. -subunit in the generation of class I binding peptides (30, 32), it Immunity 1:533. seems reasonable to assume that disruption of the Psme1 gene will 20. Kandil, E., C. Namikawa, M. Nonaka, A. S. Greenberg, M. F. Flajnik, impair class I-mediated Ag presentation. In contrast, the ␤-subunit T. Ishibashi, and M. Kasahara. 1996. Isolation of low molecular mass polypeptide complementary DNA clones from primitive vertebrates: Implications for the or-

alone does not stimulate peptidase activity of the 20S proteasome. igin of MHC class I-restricted antigen presentation. J. Immunol. 156:4245. by guest on September 28, 2021 However, it enhances the peptidase activity in the presence of the 21. Nonaka, M., C. Namikawa-Yamada, M. Sasaki, L. Salter-Cid, and M. F. Flajnik. ␦ ␣-subunit (28, 39), suggesting an auxiliary role for this subunit. 1997. Evolution of proteasome subunits and LMP2: complementary DNA clon- ing and linkage analysis with MHC in lower vertebrates. J. Immunol. 159:734. Thus, inactivation of the Psme2 gene might impair class I-medi- 22. Kasahara, M., M. F. Flajnik, T. Ishibashi, and T. Natori. 1995. Evolution of the ated Ag presentation to a lesser extent than that of the Psme1 gene. major histocompatibility complex: a current overview. Transplant Immunol. 3:1. 23. Kasahara, M., M. Hayashi, K. Tanaka, H. Inoko, K. Sugaya, T. Ikemura, and Less predictable is the outcome of the disruption of the Psme3 T. Ishibashi. 1996. Chromosomal localization of the proteasome Z subunit gene gene. The existence of a Psme3-like gene in the tick (65) and C. reveals an ancient chromosomal duplication involving the major histocompati- elegans (40), the organisms with no adaptive immune system, sug- bility complex. Proc. Natl. Acad. Sci. USA 93:9096. 24. Kasahara, M., J. Nakaya, Y. Satta, and N. Takahata. 1997. Chromosomal dupli- gests that Psme3 presumably has (a) nonimmune function(s). On cation and the emergence of the adaptive immune system. Trends Genet. 13:90. the other hand, the observation that expression of the ␥-subunit is 25. Dubiel, W., G. Pratt, K. Ferrell, and M. Rechsteiner. 1992. Purification of an 11S down-regulated by IFN-␥ at the protein level (39) suggests that regulator of the multicatalytic protease. J. Biol. Chem. 267:22369. 26. Ma, C.-P., C. A. Slaughter, and G. N. DeMartino. 1992. Identification, purifica- this subunit might also have an immunomodulatory function. At- tion, and characterization of a protein activator (PA28) of the 20S proteasome tempts to create Psme3-deficient mice are currently in progress in (macropain). J. Biol. Chem. 267:10515. our laboratories. 27. Gray, C. W., C. A. Slaughter, and G. N. DeMartino. 1994. PA28 activator protein forms regulatory caps on proteasome stacked rings. J. Mol. Biol. 236:7. 28. Kuehn, L., and B. Dahlmann. 1996. Reconstruction of proteasome activator PA28 from isolated subunits: optimal activity is associated with an ␣,␤-heterodimer. Acknowledgments FEBS Lett. 394:183. 29. Song, X., J. D. Mott, J. von Kampen, B. Pramanik, K. Tanaka, C. A. Slaughter, We thank Ms. Noriko Namerikawa for her secretarial help. and G. N. DeMartino. 1996. A model for the quaternary structure of the protea- some activator PA28. J. Biol. Chem. 271:26410. 30. Dick, T. P., T. Ruppert, M. Groettrup, P. M. Kloetzel, L. Kuehn, References U. H. Koszinowski, S. Stevanovic´, H. Schild, and H.-G. Rammensee. 1996. Co- ordinated dual cleavages induced by the proteasome regulator PA28 lead to dom- 1. Heemels, M.-T., and H. Ploegh. 1995. Generation, translocation, and presentation inant MHC ligands. Cell 86:253. of MHC class I-restricted peptides. Annu. Rev. Biochem. 64:463. 31. Niedermann, G., R. Grimm, E. Geier, M. Maurer, C. Realini, C. Gartmann, 2. York, I. A., and K. L. Rock. 1996. Antigen processing and presentation by the J. Soll, S. Omura, M. C. Recksteiner, W. Baumeister, and K. Eichmann. 1997. class I major histocompatibility complex. Annu. Rev. Immunol. 14:369. Potential immunocompetence of proteolytic fragments produced by proteasomes 3. Monaco, J. J., and D. Nandi. 1995. The genetics of proteasomes and antigen before evolution of the vertebrate immune system. J. Exp. Med. 185:209. processing. Annu. Rev. Genet. 29:729. 32. Groettrup, M., A. Soza, M. Eggers, L. Kuehn, T. P. Dick, H. Schild, H.-G. 4. Groettrup, M., A. Soza, U. Kuckelkorn, and P.-M. Kloetzel. 1996. Peptide anti- Rammensee, U. H. Koszinowski, and P.-M. Kloetzel. 1996. A role for the pro- gen production by the proteasome: complexity provides efficiency. Immunol. To- teasome regulator PA28␣ in antigen presentation. Nature 381:166. day 17:429. 33. Honore´, B., H. Leffers, P. Madsen, and J. E. Celis. 1993. Interferon-␥ up-regu- 5. Tanaka, K., N. Tanahashi, C. Tsurumi, K. Yokota, and N. Shimbara. 1997. Pro- lates a unique set of proteins in human keratinocytes. Molecular cloning and teasomes and antigen processing. Adv. Immunol. 64:1. expression of the cDNA encoding the RGD-sequence-containing protein IGUP 6. Coux, O., K. Tanaka, and A. L. Goldberg. 1996. Structure and functions of the I-5111. Eur. J. Biochem. 218:421. 20S and 26S proteasomes. Annu. Rev. Biochem. 65:801. 34. Realini, C., W. Dubiel, G. Pratt, K. Ferrell, and M. Rechsteiner. 1994. Molecular 7. Goldberg, A. L. 1995. Functions of the proteasome: the lysis at the end of the cloning and expression of a ␥-interferon-inducible activator of the multicatalytic tunnel. Science 268:522. protease. J. Biol. Chem. 269:20727. The Journal of Immunology 4935

35. Ahn, J. Y., N. Tanahashi, K. Akiyama, H. Hisamatsu, C. Noda, K. Tanaka, 54. Quandt, K., K. Frech, K. Karas, H. Wingender, and T. Werner. 1995. MatInd and C. H. Chung, N. Shimbara, P. J. Willy, J. D. Mott, C. A. Slaughter, and MatInspector: new fast and versatile tools for detection of consensus matches in G. N. DeMartino. 1995. Primary structures of two homologous subunits of PA28, nucleotide sequence data. Nucleic Acids Res. 23:4878. a ␥-interferon-inducible protein activator of the 20S proteasome. FEBS Lett. 366: 55. Mount, S. M. 1982. A catalogue of splice junction sequences. Nucleic Acids Res. 37. 10:459. 36. Jiang, H., and J. J. Monaco. 1997. Sequence and expression of mouse proteasome 56. Jackson, I. J. 1991. A reappraisal of non-consensus mRNA splice sites. Nucleic activator PA28 and the related autoantigen Ki. Immunogenetics 46:93. Acids Res. 19:3795. 37. Soza, A., C. Knuehl, M. Groettrup, P. Henklein, K. Tanaka, and P.-M. Kloetzel. 57. Aebi, M., H. Hornig, and C. Weissmann. 1987. 5Ј cleavage site in eukaryotic 1997. Expression and subcellular localization of mouse 20S proteasome activator pre-mRNA splicing is determined by the overall 5Ј splice region, not by the complex PA28. FEBS Lett. 413:27. conserved 5Ј GU. Cell 50:237. 38. Nikaido, T., K. Shimada, M. Shibata, M. Hata, M. Sakamoto, Y. Takasaki, 58. Friedman, R. L., and G. R. Stark. 1985. ␣-Interferon-induced transcription of C. Sato, T. Takahashi, and Y. Nishida. 1990. Cloning and nucleotide sequence of HLA and metallothionein genes containing homologous upstream sequences. Na- cDNA for Ki antigen, a highly conserved nuclear protein detected with sera from ture 314:637. patients with systemic lupus erythematosus. Clin. Exp. Immunol. 79:209. 59. Darnell J. E., Jr., I. M. Kerr, and G. R. Stark. 1994. Jak-STAT pathways and 39. Tanahashi, N., K. Yokota, J. Y. Ahn, C. H. Chung, T. Fujiwara, E. Takahashi, transcriptional activation in response to IFNs and other extracellular signaling G. N. DeMartino, C. A. Slaughter, T. Toyonaga, K. Yamamura, N. Shimbara, and proteins. Science 264:1415. K. Tanaka. 1997. Molecular properties of the proteasome activator PA28 family proteins and ␥-interferon regulation. Genes Cells 2:195. 60. Schindler, C., and J. E. Darnell, Jr. 1995. Transcriptional responses to polypep- 40. Kandil, E., K. Kohda, T. Ishibashi, K. Tanaka, and M. Kasahara. 1997. PA28 tide ligands: the JAK-STAT pathway. Annu. Rev. Biochem. 64:621. subunits of the mouse proteasome: primary structures and chromosomal local- 61. Baldwin, J. A. B. 1996. The NF-␬B and I␬B proteins: new discoveries and in- ization of the genes. Immunogenetics 46:337. sights. Annu. Rev. Immunol. 14:649. 41. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Lab- 62. Abbadi, N., and J. H. Nadeau. 1997. Mouse chromosome 14. Mamm. Genome oratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 7:S238. 42. Kandil, E., M. Noguchi, T. Ishibashi, and M. Kasahara. 1995. Structural and 63. Albertsen, H. M., S. A. Smith, S. Mazoyer, E. Fujimoto, J. Stevens, B. Williams,

phylogenetic analysis of the MHC class I-like Fc receptor gene. J. Immunol. P. Rodriguez, C. S. Cropp, P. Slijepcevic, M. Carlson, M. Robertson, P. Bradley, Downloaded from 154:5907. E. Lawrence, T. Harrington, Z. Mei Sheng, R. Hoopes, N. Sternberg, 43. Frohman, M. A. 1990. RACE: rapid amplification of cDNA ends. In PCR Pro- A. Brothman, R. Callahan, B. A. J. Ponder, and R. White. 1994. A physical map tocols: A Guide to Methods and Applications. M. A. Innis, D. H. Gelfand, and candidate genes in the BRCA1 region on chromosome 17q12–21. Nat. Genet. J. J. Sninsky, and T. J. White, eds. Academic Press, San Diego, p. 28. 7:472. 44. Hirzmann, J., D. Luo, J. Hahnen, and G. Hobom. 1993. Determination of mes- 64. Schmid, C. W. 1996. Alu: structure, origin, evolution, significance, and functions senger RNA 5Ј-ends by reverse transcription of the cap structure. Nucleic Acids of one-tenth of human DNA. Prog. Nucleic Acids Res. Mol. Biol. 53:283. Res. 21:3597. 65. Paesen, G. C., and P. A. Nuttall. 1996. A tick homologue of the human Ki nuclear 45. Swiatek, P. J., and T. Gridley. 1993. Perinatal lethality and defects in hindbrain autoantigen. Biochim. Biophys. Acta 1309:9. http://www.jimmunol.org/ development in mice homozygous for a targeted mutation of the zinc finger gene 66. Boehm, U., T. Klamp, M. Groot, and J. C. Howard. 1997. Cellular responses to Krox20. Genes Dev. 7:2071. interferon-␥. Annu. Rev. Immunol. 15:749. 46. Tartof, K. D., and C. A. Hobbs. 1988. New cloning vectors and techniques for 67. Epperson, D. E., D. Arnold, T. Spies, P. Cresswell, J. S. Pober, and easy and rapid restriction mapping. Gene 67:169. D. R. Johnson. 1992. Cytokines increase transporter in antigen processing-1 ex- 47. Shikata, T., and T. Kotaka. 1991. Biased sinusoidal field gel electrophoresis for pression more rapidly than HLA class I expression in endothelial cells. J. Im- size-dependent DNA separation. Biopolymers 31:253. munol. 149:3297. 48. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain- 68. Min, W., J. S. Pober, and D. R. Johnson. 1996. Kinetically coordinated induction terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463. of TAP1 and HLA class I by IFN-␥: the rapid induction of TAP1 by IFN-␥ is 49. Matsuda, Y., Y. N. Harada, S. Natsuume-Sakai, K. Lee, T. Shiomi, and mediated by Stat1␣. J. Immunol. 156:3174. V. M. Chapman. 1992. Location of the mouse complement factor H gene (cfh)by FISH analysis and replication R-banding. Cytogenet. Cell Genet. 61:282. 69. Ting, J. P.-Y., and A. S. Baldwin. 1993. Regulation of MHC gene expression. 50. Matsuda, Y., and V. M. Chapman. 1995. Application of fluorescence in situ Curr. Opin. Immunol. 5:8. by guest on September 28, 2021 hybridization in genome analysis of the mouse. Electrophoresis 16:261. 70. Glynne, R., S. H. Powis, S. Beck, A. Kelly, L.-A. Kerr, and J. Trowsdale. 1991. 51. Evans, E. P. 1996. Standard G-banded karyotype. In Genetic Variants and Strains A proteasome-related gene between the two ABC transporter loci in the class II of the . M. F. Lyon, and A. G. Searle, eds. Oxford University region of the human MHC. Nature 353:357. Press, Oxford, p. 1446. 71. Wright, K. L., L. C. White, A. Kelly, S. Beck, J. Trowsdale, and J. P.-Y. Ting. 52. Somssich, I. E., and H. Hameister. 1996. Standard karyotype of early replicating 1995. Coordinate regulation of the human TAP1 and LMP2 genes from a shared bands (RBG-banding). In Genetic Variants and Strains of the Laboratory Mouse. bidirectional promoter. J. Exp. Med. 181:1459. M. F. Lyon, and A. G. Searle, eds. Oxford University Press, Oxford, p. 1450. 72. Realini, C., S. W. Rogers, and M. Rechsteiner. 1994. KEKE motifs: Proposed 53. Wingender, E. 1994. Recognition of regulatory regions in genomic sequences. roles in protein-protein association and presentation of peptides by MHC class I J. Biotechnol. 35:273. receptors. FEBS Lett. 348:109.