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Analysis of a 26-kb Region Linked to the Mhc
in Zebrafish: Genomic Organization of the
Proteasome Component β/Transporter
Associated with Antigen Processing-2 Gene
Cluster and Identification of Five New
Proteasome β Subunit Genes
Brent W. Murray, Holger Sültmann and Jan Klein
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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 © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 1999; 163:2657-2666; ;
http://www.jimmunol.org/content/163/5/2657
Analysis of a 26-kb Region Linked to the Mhc in Zebrafish:
Genomic Organization of the Proteasome Component
b/Transporter Associated with Antigen Processing-2 Gene
Cluster and Identification of Five New Proteasome b Subunit
Genes
Brent W. Murray, Holger Sültmann, and Jan Klein1
P
roteasomes are self-compartmentalizing multicatalytic
protease complexes that play a key role in protein degradation throughout the cell (1, 2). A feature common to all
proteasomes is the central core, the 20S unit, in which the proteolytic activity of the complex resides (1). The 20S proteasome is a
barrel-shaped molecule, made up of four stacked rings arranged in
an abba orientation. In eukaryotes, both the a and the b rings are
comprised of seven unique proteasome component (PSM)2 subunits (3). Of these, only three of the b subunits appear to be catalytically active, and these interact with the other b subunits to
form a proteolytic pocket in the center of the ring structure (3, 4).
In mammals, a second 20S proteasome, the immunoproteasome,
has been identified (5). It differs from the “general housekeeping”
20S proteasome at the three catalytically active b subunits. Upon
induction by IFN-g, three additional proteasome component b
(PSMB) subunits are expressed, PSMB8 (5 low molecular mass
protein 7 (LMP7)), PSMB9 (5LMP2), and PSMB10 (5MECL1).
These subunits replace the constitutively expressed housekeeping
subunits PSMB5 (5X, MB1), PSMB6 (5Y, d), and PSMB7 (5Z,
MC14), respectively, during formation of the newly synthesized
proteasome. The resulting immunoproteasome has been shown to
Max-Planck-Institut für Biologie, Abt. Immungenetik, Tübingen, Germany
Received for publication March 24, 1999. Accepted for publication June 11, 1999.
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
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Jan Klein, Max-Planck-Institut
für Biologie, Abt. Immungenetik, Corrensstrasse 42, D-72076, Tübingen, Germany.
E-mail address: [email protected]
2
Abbreviations used in this paper: PSM, proteasome component; AA, amino acid;
ABC, ATP-binding cassette; BAC, bacterial artificial chromosome; LMP, low molecular mass protein; PAC, P1 artificial chromosome; PSMB, proteasome component
b; UTR, untranslated region; C/EBP, CCAAT/enhancer-binding protein; IRF, IFN
regulatory factor.
Copyright © 1999 by The American Association of Immunologists
exhibit an altered proteolytic activity, producing peptides that are similar to those presented by the MHC (Mhc) class I molecules (6, 7).
The TAP molecule is part of a large superfamily of ATP-binding cassette (ABC) membrane bound transporters (8). In mammals, TAP molecules transport antigenic peptides produced in the
cytosol, preferentially those destined for binding to class I molecules, into the lumen of the rough endoplasmic reticulum (9). The
TAP molecule is comprised of two noncovalently associated subunits, TAP1 and TAP2. The genes coding for both of these subunits are located in the mammalian Mhc, where they are tightly
linked with the PSMB8 and PSMB9 genes (10). The PSMB8,
TAP1, PSMB9, and TAP2 genes of the cluster occur in the following orientation: 4 3 3 3. Evidence suggests that the PSMB8
and TAP1 genes are coregulated from a shared bidirectional
promoter (11, 12).
Proteasome and ABC transporter genes have been used to provide evidence in support of the hypothesis that two rounds of chromosome duplication, presumably preceding the emergence of
jawed vertebrates (13–17), were critical for the appearance of the
adaptive immune system (15). PSMB and ABC transporter genes
are part of paralogous genomic regions that are central to this
hypothesis. In humans, the PSMB/TAP gene cluster is linked to the
HLA complex located in the 6p21.3 region. Genes paralogous to
several HLA complex genes, including PSMB7, which codes for a
subunit that is replaced by the PSMB10 subunit in the immunoproteasome, and the ABC2 gene, are present in the q33–34 region
on chromosome 9 (15, 16). Kasahara and coworkers (15) hypothesized that the preduplication ancestor of the 6p21.3 region contained three tandemly arranged PSMB housekeeping genes, prePSMB5, pre-PSMB6, and pre-PSMB7. Upon block duplication
(presumably chromosomal), the three genes associated with the
region destined to become the Mhc evolved into the PSMB8,
PSMB9, and PSMB10 genes. The PSMB10 gene was subsequently
translocated to another chromosome. In the 9q33–34 paralogous
0022-1767/99/$02.00
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Sequencing of zebrafish (Danio rerio) bacterial artificial chromosome and P1 artificial chromosome genomic clone fragments and
of cDNA clones has led to the identification of five new loci coding for b subunits of proteasomes (PSMB). Together with the four
genes identified previously, nine PSMB genes have now been defined in the zebrafish. Six of the nine genes reside in the zebrafish
MHC (Mhc) class I region, four of them reside in a single cluster closely associated with TAP2 on a 26-kb long genomic fragment,
and two reside at some distance from the fragment. In addition to homologues of the human genes PSMB5 through PSMB9, two
new genes, PSMB11 and PSMB12, have been found for which there are no known corresponding genes in humans. The new genes
reside in the PSMB cluster in the Mhc. Homology and promoter region analysis suggest that the Mhc-associated genes might be
inducible by IFN-g. The zebrafish class I region contains representatives of three phylogenetically distinguishable groups of PSMB
genes, X, Y, and Z. It is proposed that these genes were present in the ancestral PSMB region before Mhc class I genes became
associated with it. The Journal of Immunology, 1999, 163: 2657–2666.
2658
PROTEASOME GENES IN ZEBRAFISH
Table I. PCR primers used for cDNA analysis
Primer
a
b
ACC ATT CTT gCA gTC AAg TTC
gTC ATC ATT ggg TCT gAC TC
C AgA CAT CAT CCA TAC TCT CAA g
g TCT gAC TCC AgA gCT TCC ATg
gC ATC AgC CAg TgA TCC AgC
AA TAT TCg ATC gTg CAC CTg
gT gCC AAg ATT CAC TAC ATT gCA
g gAg AAg ACA ACA gAT ATg CTg TC
T gTA CAA gTg ACT gCC AgT gCA g
TTg gAT CgC ATC ACT TAC CA g Ag
TC CAT gAC AAg ATC TA C TgT gC
gAC gCT CAA ACT ATT gCT gAg
CAg AgTgAg AgC ATT CAC TAC
gT CAg CAA ACC ACT CAA TgT Tg
CAg AgA CAg ACT gTT gAT CAC
C ATC TTT gTC AgT AgT ATC CAC
T CTC gCT gTC AAT ggT AAC gAg
CAT CgC TgC Cag AgA TCC AgA C
gTA TgC ggA gAC CTg TTC gCT A
gAC TTg ACC ATA gAT gAg gCT Tg
AgCAA gTTCA gCCTg gTTAAg
CTTAT gAgTA TTTCT TCCAg ggTA
Gene
Orientationa
Relative Locationb
PSMB11
PSMB11
PSMB11
PSMB11
PSMB11
PSMB11
PSMB12
PSMB12
PSMB12
PSMB12
PSMB9A
PSMB9A
PSMB9A
PSMB9A
PSMB9B
PSMB9A
PSMB9B
PSMB7
PSMB5
PSMB5
l Vector
l Vector
F
F
F
F
R
R
F
F
R
R
F
F
R
R
R
R
R
R
F
F
Not applicable
Not applicable
2–8
12–18
215 to 27
15–22
53–47
43–37
32–39
53–61
115–106
163–156
38–45
52–58
164–158
123–116
164–158
185–178
185–178
138–131
123–130
148–155
Not applicable
Not applicable
An F or R indicates the 39 end of the primer faces forward or reverse relative to the reading frame, respectively.
The position of the primer, 59–39, is given relative to the amino acid positions given in Fig. 1.
region, the PSMB7 is postulated to be the sole remainder of the
alternate duplicated PSMB three-gene cluster.
To test the hypothesis of block duplication of the proposed
paralogous regions, Hughes (18) conducted a phylogenetic analysis of the genes involved. He found the estimated times of the
duplication events to vary considerably among the pairs of paralogous genes. This observation is inconsistent with the block duplication hypothesis. In particular, the divergence of the TAP1/2 and
ABC2 genes appears to have occurred before the divergence of
eukaryotes from bacteria. As an alternative explanation, he proposed that there may be a selective advantage to the clustering of
broadly expressed genes in regions likely to have a wide range of
transcriptional activity. According to this hypothesis, the association of the PSMB and ABC transporter genes with the two proposed paralogous regions has occurred purely by chance, but was
subsequently maintained due to selective advantage.
The comparative analysis of nonmammalian jawed vertebrate
genomes will undoubtedly lead to a greater understanding of the
evolution of gene associations. Unfortunately, little is known about
the genomic organization of any nonmammalian vertebrate. Most
advanced in this regard is the study of the zebrafish, Danio rerio
(17). The zebrafish, a representative of the bony fishes (Osteichthyes), is one of the model organisms in a variety of studies, but
particularly in developmental biology (19). In this species, four
proteasome genes have been identified (20): two from complete
cDNA sequences, Dare-PSMB8 and -PSMB6 (formerly DareLMP7 and -Y, respectively), and from two partial cDNA sequences, Dare-PSMB9 and -PSMB5 (formerly Dare-LMP2 and -X,
respectively). Linkage studies (20) and analysis of zebrafish
genomic bacterial artificial chromosome (BAC) clones containing
class I genes (21) have shown that the Dare-PSMB8 and -PSMB9
are linked to Mhc class I genes. However, in zebrafish, Mhc class
I and class II are not linked (22). Genes representing four families
of the human 6p21.3 and 9q33–34 paralogous groups (PSMB,
TAP, RING3, and RXRB-like genes) have been found in zebrafish
to be linked to Mhc class I genes (20, 23). All of these and the Mhc
class I genes are present on a 500-kb contig of BAC and P1 artificial chromosome (PAC) clones, with the PSMB, TAP, RING3,
and a class I gene present on a single BAC clone (B. Murray, V.
Michalová, H. Sültmann, and J. Klein, manuscript in preparation).
The aim of the present study was to determine the composition
and organization of the Mhc-linked PSMB and TAP2 genes in the
zebrafish.
Materials and Methods
Sequencing of BAC 7 and 716 subclones
Genomic regions flanking the previously identified Dare-TAP2 and
-PSMB8 genes (20) were targeted for nucleotide sequencing. Various restriction enzymes were used to construct subclone libraries of BAC clones
7 and 716 (two overlapping clones previously shown to contain Mhc class
I genes; Ref. 21). Restriction fragments were excised from 1% agarose gels
(Carl Roth, Karlsruhe, Germany), extracted via the QIAEX II kit (Qiagen,
Hilden, Germany), and cloned into the pGEM-7Zf(1) plasmid vector (Promega, Mannheim, Germany). Subcloned fragments spanning the region of
the known Dare-TAP2 and PSMB8 genes were sequenced using the
Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech,
Braunschweig, Germany), the LI-COR DNA sequencer 4200 (MWG-Biotech, Ebersberg, Germany), and fluorescently labeled primers. Sequences
were compared with those in the GenBank through both FASTA nucleotide
and BLASTx searches.
Analysis of zebrafish cDNA library
Two rounds of PCR amplification were used to obtain full-length cDNA
sequences of clones in a zebrafish cDNA library constructed from 20 adult
individuals (24). In the first round, a vector-specific and a gene-specific
primer were used; in the second round, the same vector primer and a second gene-specific primer located just downstream of the first were applied
(Table I). PCR amplifications were conducted using the PTC-100 programmable thermal controller (MJ Research, Watertown, MA). In each case, 1
ml of the phage suspension was used as a template and the concentrations
of reagents in a 25-ml volume were as follows: 5 pmol of each primer, 1
U Taq DNA polymerase (Amersham Pharmacia Biotech), 100 mM NaCl,
10 mM Tris, pH 7.8, and 1.5 mM MgCl2. The thermal profile consisted of
4 min at 94°C followed by 35 cycles of 94°C for 15 s, 50°C or 55°C for
30 s, 72°C for 2 min, and completion at 72°C for 8 min. The PCR fragments were cloned into the pGEM-T vector (Promega) .
Sequence analysis
Sequence alignments were made with the aid of the computer program
CLUSTAL W (25) with minor improvements made by eye. Phylogenetic
trees were constructed by the neighbor-joining method (26) from pairwise
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BM-a37-7F1
BM-a37-7F2
BMpr5-F3
BMpr5-F4
BM-b18-3R1
BM-b18-3R2
Dare*Z1-E3F1
Dare*Z1-E4F2
Dare*Z1-E5R2
Dare*Z1-E7R1
Dare*LMP2.F1
Dare*LMP2.F2
Dare*LMP2.R1
Dare*LMP2.R2
Dare*LMP2.R3
Dare*LMP2.R4
Dare*LMP2.R5
Dare*PSMB7.R1
Dare*X.F1
Dare*X.F2
lGT10-F1
lGT10-R1
Sequence
The Journal of Immunology
distances estimated from amino acid (AA) alignments using both the programs “neighbor” and “protdist” contained in the PHYLIP, version 3.5c,
computer package (27) and the MEGA, version 1.02, computer program
(28). In each case, any positions containing indels were excluded from the
analysis. The trees were bootstrapped 500 times (29).
Screening of PAC library
The PAC zebrafish genomic library no. 706 was obtained from the Resource Center/Primary Database of the German Human Genome Project,
Max-Planck Institute for Molecular Genetics (Berlin-Charlottenburg, Germany; http://www.rzpd.de). It was screened with PCR-amplified fragments
of the Dare-PSMB9A and Dare-PSMB9B cDNA pGEM clones. The PCR
products were isolated from a low-melting-point agarose gel, labeled with
50 mCi [a-32P]dCTP by the random priming method using the ReadyTo-Go kit (Amersham Pharmacia Biotech) and hybridized to the PAC filters following the suggested protocol (Resource Center/Primary Database).
Genomic organization
Analysis of promoter regions
DNA regions extending 700 bp upstream of the initiation codon of each
gene were analyzed for possible regulatory motifs. Sequences were compared with the TRANSFAC database (30) with the computer program
TFSEARCH, version 1.3 (Y. Akiyama, TFSEARCH: Searching Transcription Factor Binding Sites, http://www.rwcp.or.jp/papia/).
Nomenclature
This paper follows the convention for naming Mhc genes (31). Genes with
homology to previously named genes in other organisms are given the
same name in zebrafish, (e.g., Psmb in mouse, PSMB in human and zebrafish). Alleles are designated by an asterisk and a numerical code.
Results
Identification of new genes
To identify new genes in the Mhc class I region of zebrafish, subclone libraries of the overlapping zebrafish BAC clones 7 and 716
(21) were constructed. Through direct sequencing of targeted subcloned fragments, genomic sequences were found that contained
exons of new proteasome genes. From a TaqI subclone library, we
recovered genomic fragments that contained possible exons with
sequence similarity to the mammalian proteasome genes PSMB6
or PSMB9 (LMP2; Refs. 32–34). In the second subclone library, an
XhoI fragment (3.3 kb) was found to contain exons related to the
proteasome genes PSMB7 and PSMB10 (35).
Based on the exons detected, primers were designed (Table I) to
screen the zebrafish cDNA library. In addition, primers were designed based on the partial zebrafish PSMB5 and PSMB9 gene
sequences described previously (20) and on EST sequences with
similarity to PSMB7. Through the screening of the cDNA library,
six full-length and one partial cDNA sequences were found (Fig.
1). These included sequences of two new genes, Dare-PSMB11
and -PSMB12, the originally described Dare-PSMB5 and DarePSMB9 (now called PSMB9A) genes (20), a second related PSMB9
gene, named Dare-PSMB9B, and the Dare-PSMB7 gene.
Dare-PSMB11
The cDNA sequence of Dare-PSMB11 (Fig. 1) is based on two
overlapping amplified fragments. The first fragment extends 238
bp from the position of the BM-b18-3R2 primer to the beginning
of the 59 untranslated region (UTR), while the second fragment
extends 720 bp from the position of the BM-a37-7F1 primer to the
beginning of the polyA tail. The sequences are identical in the
84-bp region of overlap. The entire cDNA sequence is 855 bp
long, extends up to the beginning of the polyA tail, and includes 74
bp of the 59 UTR and 127 bp of the 39 UTR. A possible polyadenylation signal is found at sites 103–108 of the 39 UTR. The AA
sequence of the entire polypeptide is 217 AA residues long. It is
comprised of a 16-AA propeptide and a 201-AA mature protein
deduced from similarity to other PSMB subunits (Fig. 2) and from
the presence of the correct glycine (G)-threonine (T) motif, which
is the site of the cleavage of the N-terminal propeptide (1).
Dare-PSMB12
The Dare-PSMB12 cDNA sequence (Fig. 1) is also based on two
overlapping PCR-amplified fragments. The first fragment extends
462 bp from the Dare*Z1-E5R2 primer to the transcription initiation site. The second fragment extends 723 bp from the position of
the Dare*Z1-E4F2 primer to the beginning of the polyA tail. No
nucleotide differences are found in the 132-bp overlap. No 59 UTR
is detected in the 1002-bp sequence, while a 161-bp 39 UTR is
found that contains a polyadenylation signal at sites 148 –153 and
a polyA tail after site 161. However, analysis of the genomic sequence reveals the presence of a probable initiation codon (shown
in lower case and italics in Fig. 1) that is lacking in the cDNA
sequence. Based on this initiation codon the deduced mature protein is 237-AA residues long after the removal of a 44-AA residue
long propeptide (assuming cleavage at the GT motif).
Dare-PSMB9
Based on a fragment of the Dare-PSMB9 gene reported previously
(20), primers were designed to characterize the full-length cDNA
sequence. Three sequences were detected: two alleles of the original locus renamed Dare-PSMB9A and a second locus identified on
the basis of a divergent sequence and denoted Dare-PSMB9B.
The full-length Dare-PSMB9A*01 cDNA sequence (Fig. 1) was
derived from two overlapping PCR fragments. The sequence of the
first fragment is 594 bp long, extending from the 59 UTR to the
position of the Dare*LMP2.R1 primer. The second fragment,
which is identical in the 291-bp overlap to the first one, extends
from the position of the Dare*LMP2.F2 primer to the end of the
shown 39 UTR. The Dare-PSMB9A*01 sequence is identical
throughout its length to the previously reported fragment (20) and
is most likely the full-length cDNA clone of this gene. The sequence is 885 bp long and contains 54 bp of the 59 UTR and 174
bp of the 39 UTR. Although a polyA tail has not been found, a
potential polyadenylation signal is present at sites 167–172. The
sequence codes for a deduced propeptide of 19-AA residues and a
mature protein of 199-AA residues (assuming cleavage at the GT
motif).
An additional PCR fragment was found with high sequence similarity to the PSMB9A*01 gene. The similarity extends from the
position of the Dare*LMP2.R1 primer along the entire length of
the 594 bp PSMB9A*01 fragment, but the sequence extends by 263
bp into the 59 UTR. Thirteen substitutions differentiate these two
sequences, six in the 59 UTR and seven synonymous substitutions
in the coding region. The 39 UTR sequence of this cDNA sequence
was not determined. Because of its high similarity to the DarePSMB9A*01 gene, the sequence is interpreted as being an allele of
this gene and as such it is designated Dare-PSMB9A*02 (not
shown).
Another full-length cDNA sequence with similarity to the DarePSMB9A genes was found based on two overlapping PCR fragments (Fig. 1). The 59 part of the sequence extends 643 bp from the
position of the Dare*LMP2.R3 primer; the 39 part of the sequence
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Overlapping DNA sequence fragments generated from the analysis of the
subclone libraries were identified and organized with the computer program AssemblyLIGN (Eastman Kodak, Rochester, NY). For each contig,
the exon/intron organization was deduced from the known cDNA sequences. Intron-specific PCRs were conducted to join the existing contigs,
confirm exon/intron boundaries, and estimate the sizes of introns. The
PCR, cloning of amplified fragments, and DNA sequencing were performed as described above (primer sequences available upon request). In
all cases, BAC 716 DNA was used as a PCR template.
2659
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PROTEASOME GENES IN ZEBRAFISH
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FIGURE 1. Nucleotide sequences of five cDNAs derived from zebrafish (Dare) PSMB7, PSMB9A, PSMB9B, PSMB11, and PSMB12 genes. Sequence
identity with the uppermost sequence is indicated by a dash and an indel by an asterisk. Missing coding sequence information was deduced from the
genomic sequence and is indicated by lower case italicized letters. The possible polyadenylation sites in the 39 UTR are underlined.
The Journal of Immunology
2661
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FIGURE 2. Amino acid alignment of proteasome subunits: (A) propeptide sequence and (B) mature protein sequences (used for phylogenetic analysis).
Sequences can be identified from their GenBank accession numbers and the references contained therein. Proteasome subunits are from Saccharomyces
cerevisiae, Sace PRE2 (M96667), PRE3 (X78991), PUP1 (X61189); Lampetra japonica, Laja PSMB6 (D87690); Danio rerio, Dare-PSMB5, -PSMB7,
-PSMB9A, -PSMB9B, -PSMB11, -PSMB12 (AF155576 – 81), -PSMB6 (AF0323392); Xenopus laevis, Xela-PSMB6 (D87689), -PSMB8A (D44540),
PSMB8B (D44549), -PSMB9 (D87687); and Homo sapiens, Hosa-PSMB5 (D29011), -PSMB6 (D29012), -PSMB7 (D38048), -PSMB8 (Z14982), -PSMB9
(U01025), -PSMB10 (X71874). The zebrafish subunits linked to Mhc class I genes are underlined. Positions relative to the N terminus of the mature protein
(11) are shown. At each position, identity to the consensus sequence is shown by a dash while an indel is indicated by an asterisk.
2662
PROTEASOME GENES IN ZEBRAFISH
FIGURE 3. Midpoint rooted neighbor-joining
dendrogram based on protein sequence distances
among proteasome subunits (mature protein) estimated by the p-distance method (28). Values placed
to the left of each node indicate the percentage of
times the subunits joined by the node were found in
a monophyletic clade in the consensus tree of the
bootstrap analysis. The zebrafish subunits linked to
Mhc class I genes are underlined. Other subunits include Saccharomyces cerevisiae, Sace-PRE2
(M96667), -PRE3 (X78991), -PUP1 (X61189);
Lampetra japonica, Laja-PSMB6 (D87690); Danio
rerio,
Dare-PSMB5
(AF155578),
-PSMB6
(AF0323392), -PSMB7 (AF155581); Xenopus laevis, Xela-PSMB6 (D87689), -PSMB8A (D44540),
-PSMB8B (D44549), -PSMB9 (D87687); and Homo
sapiens,
Hosa-PSMB5
(D29011),
-PSMB6
(D29012), -PSMB7 (D38048), -PSMB8 (Z14982),
-PSMB9 (U01025), -PSMB10 (X71874).
Dare-PSMB7
Five partial zebrafish cDNA sequences with similarity to the human PSMB7 gene were recovered from the GenBank EST library
(AA605681, AA606112, AI331717, AI332003, and AI332014). A
consensus sequence was generated which extends 704 bp 59 of the
end of the polyA tail. The primer Dare*PSMB7.R1 (Table I) was
designed to amplify the remaining 59 end of the cDNA sequences.
A 528-bp fragment was recovered that was identical with the initial consensus in the 248-bp overlap. No 59 UTR or initiation
codon is present in the resulting 962-bp transcript (Fig. 1). The
deduced 275-AA polypeptide is comprised of a 41-AA propeptide
and a 234-AA mature protein (assuming cleavage at the GT motif).
The 39 UTR is 100 bp long up to the start of the polyA tail and
contains a possible polyadenylation signal at sites 90 –95.
Dare-PSMB5
The partial sequence of the previously reported Dare-PSMB5 gene
(20) was extended in the 39 direction to conduct a phylogenetic
analysis that included the complete mature protein of all known
zebrafish PSMB subunits. We amplified a 706-bp fragment that
extended from the primer Dare*X.F2 (Table I) to the end of a
polyA tail (including a possible polyadenylation signal). The sequence is identical with the previous (AF032391) in the 43-bp
overlap and extends the deduced polypeptide 37 AA up to the stop
codon (Fig. 2). The complete Dare-PSMB5 cDNA sequence is
1296 bp long (not shown; GenBank accession no. AF155578).
Phylogenetic analysis
Phylogenetic trees were constructed based on distance estimates
from an AA alignment (Fig. 2). Three distance estimates were
used, the first based on simple proportional (p) distances (28), the
second based on the Dayhoff PAM matrix, and the third based on
the categories method (27). All three distance estimates resulted in
the same topology (except for the PSMB6 clade of jawed vertebrates). For this reason, only the tree based on the p-distances is
shown (Fig. 3). Bootstrap values show strong support (99 –100) for
the grouping of each of the three types of b subunits with one of
the ancestral yeast subunits, Y (PRE3, PSMB6, PSMB9), Z
(PUP1, PSMB7, PSMB10), and X (PRE2, PSMB5, PSMB8), and
for each of the PSMB clades (PSMB5–9). The positions of the
Dare-PSMB9A and -PSMB9B subunits are consistent with previous phylogenies (20) and support the introduced nomenclature.
The new Dare-PSMB11 and -PSMB12 subunits are clearly members of the Y (PRE3) and Z (PUP1) clades, respectively. In both
cases, they are only distantly related to the other subunits and their
phylogenetic positions within the clades are unclear. In the Y
clade, PSMB11 is a sister subunit to all other subunits, while PRE3
is a sister subunit to the PSMB6 clade. In the Z clade, PSMB12 is
a sister subunit to the PSMB7 clade. However, in no case are these
positions supported by the bootstrap analysis (Fig. 3).
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extends 530 bp from the position of the Dare*LMP2.F2 primer. No
differences are found in the 291-bp long overlap. This sequence
has a 119-bp long 59 UTR, a 57-bp long 39 UTR, and a potential
polyadenylation signal at sites 33–38, but no polyA tail. A 227-AA
polypeptide is deduced from the coding region, and a 17-AA
propeptide and a 210-AA residue long mature protein are postulated based on the conserved GT motif. A comparison with the
PSMB9A*01 sequence shows low similarity in the 59 and 39 UTR
and a slightly longer coding region containing 66 nucleotide differences. The sequence divergence suggests that this sequence represents a second PSMB9 locus, which we designate DarePSMB9B, and this suggestion is borne out by mapping studies.
Screening of a zebrafish genomic PAC library with fragments of
the PSMB9A and PSMB9B cDNA clones has shown the PSMB9A
and PSMB9B genes to reside on different Mhc class I gene-containing PAC clones: the PSMB9A gene resides on BAC clones 7
and 716 and the PSMB9B gene on the PAC clone
BUSMP706A2470Q3 (B. Murray, V. Michalovà, H. Sültmann,
and J. Klein, unpublished observations).
Finally, the screening of the PAC clones revealed the presence
of a third PSMB9-like locus on a clone (BUSMP706P02172Q3)
that does not contain either the PSMB9A or the PSMB9B loci. PCR
amplification of this clone with PSMB9-specific primers yielded a
product that contained parts of intron 5 and exon 6. The sequence
is divergent from both PSMB9A and -B but more closely related to
PSMB9B (data not shown). On the basis of its position on the
zebrafish Mhc map and its sequence divergence, we interpret the
sequence as being derived from a third PSMB9 locus that we designate Dare-PSMB9C. A transcript of PSMB9C was not found in
our cDNA library.
The Journal of Immunology
2663
Genomic organization
Analysis of promoter regions
Six regions of contiguous DNA sequence (contigs), spanning a
segment of about 26 kb (Fig. 4), were assembled based on the
sequence information derived from the analysis of subclone libraries and intron-specific PCRs. All contigs are joined by clones for
which the complete sequence was not determined. For each gene,
a detailed map of the intron/exon organization was deduced. In
every case, correct splice signals were found at the intron/exon
boundaries. The Dare-TAP2 gene organization was deduced from
the partial zebrafish cDNA sequence (exons 8 –11; Ref. 20) and
from the salmon (Salmo salar) TAP2 gene (36). Eleven exons of
similar sizes and splice site locations to salmon (36) and human
(32) TAP genes were identified. The exact size of exon 1 is not
known and the given estimate is based on the position of a methionine codon most similar to that identified as the start of translation in salmon. Two other possible initiation codons exist. Six
exons of the Dare-PSMB8 were deduced from the existing cDNA
sequence (20). Only a partial sequence of exon 6 was available for
analysis; however, based on the length of the Dare-PSMB8 cDNA
and the similarity of the organization to other PSMB8 genes, this
is most likely the last exon of the gene. The intron/exon organization of the Dare-PSMB9A, -PSMB11, and -PSMB12 genes was
deduced from the cDNA sequences reported herein. The DarePSMB9A and -PSMB11 genes have a very similar organization,
each possessing six exons of similar size and splice site locations.
The Dare-PSMB12 gene contains eight exons. Analysis of the first
exon reveals a probable initiation codon one codon upstream of the
end of the reported cDNA sequence.
The analysis of the promoter regions for transcription factor binding motifs showed many possible transcription factor binding sites
(data not shown). Of interest here is that no SP1 sites, found in the
mammalian PSMB8 and PSMB9 genes (33, 37, 38), were found in
any of the proteasome gene promoter regions searched. Further, in
each promoter, a possible CCAAT/enhancer-binding protein (C/
EBP)-b (NF-IL-6) site is present (Figs. 4 and 5). In each case, the
position of the transcription factor motif is given relative to the
initiation codon (Fig. 5). The C/EBP-b nuclear factor is an activator of various acute-phase proteins (39) and is present in the
mammalian PSMB10 promoters (35). Of particular interest is the
presence of the IFN regulatory factor (IRF, also known as ISRE)
motif in the TAP2, PSMB11, and PSMB12 promoters (Figs. 4 and
5). This motif binds both the activator IRF-1 and repressor IRF-2
transcription factors (40) and is present in the mammalian TAP2,
PSMB8, and PSMB10 promoters (32, 35, 41, 42), as well as the
bidirectional promoter of the TAP1 and PSMB9 genes (11, 12).
The zebrafish PSMB9A and PSMB8 genes lack this element. The
initiation codons of the PSMB11 and PSMB12 genes are 159 bp
apart. The IRF element of the PSMB11 gene is located in the first
intron of the PSMB12 gene and vice versa.
Discussion
In this and an earlier publication (20), we have identified nine
PSMB loci in the zebrafish genome. Phylogenetically, the loci fall
into three groups, which, for convenience, we designate X, Y, and
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FIGURE 4. Genomic organization of the TAP2, PSMB9A, PSMB11, PSMB12, and PSMB8 genes. Positions of HindIII (H), XhoI (X), SalI (S), and MluI
(M) restriction sites are listed on the full map. Exons are indicated by filled boxes. The positions and relative sizes of the six contiguous (CTG) regions
of DNA sequence are shown below the full map. Detailed maps of the TAP2, PSMB9A, PSMB11, PSMB12, and PSMB8 genes are given underneath the
full map. Sizes of introns and exons are given in base pairs. The locations of the C/EBP-b and IRF transcription binding sites are indicated by asterisks
and circumflexes, respectively.
2664
Z, the symbols used originally for some of the human homologues
(Fig. 3). The groups are identified not only by shared substitutions,
the length of propeptide sequences (Fig. 2), and insertions/deletions (indels; group X-specific indel at site 95, group Y-specific
indels at sites 109, 218, 219, and group Z-specific indels at sites
184 –209; Fig. 2), but also by a characteristic exon-intron organization (six exons in genes of groups Y and X, eight exons in group
Z genes, whereby the exons in genes of groups Y and X are distinguished by their different lengths and splice site locations; Fig.
4). In each of these groups, the human counterparts are distinguishable into two types on the basis of their expression—the constitutively expressed genes and genes inducible by IFN-g (5). For
brevity, we refer to these as the c and i types, respectively. The
zebrafish loci are distributed among these groups as follows: group
X (PSMB5, PSMB8), group Y (PSMB6, PSMB9A, PSMB9B,
PSMB9C, PSMB11), and group Z (PSMB7, PSMB12; Fig. 3). The
human c types in these three groups are PSMB5 (X), PSMB6 (Y),
and PSMB7 (Z); the human i types are PSMB8 (LMP7), PSMB9
(LMP2), and PSMB10 (MECL1). The constitutiveness vs inducibility of the zebrafish PSMB genes has not been tested (if for no
other reason than the zebrafish IFN-g has not been identified as
yet), but a tentative assignment of the types can be made by two
criteria—sequence homology and the presence or absence of relevant sequence elements (transcription factor binding sites) in the
genes’ promoter regions (Fig. 5). Specifically, we interpret the
presence of the NF-IL-6 and IRF binding sites as an indication that
the associated gene might be of the i type. By these criteria, we
classify the zebrafish PSMB8, PSMB9A, and PSMB9B loci as being most probably of the i type, the PSMB11 and PSMB12 loci as
being probably of the i type (all these loci are in the zebrafish Mhc
class I region), and the PSMB5, PSMB6 (two loci that are not in the
Mhc region), and PSMB7 loci as being of the c type.
The location of the IRF-binding sites in the zebrafish PSMB
clusters may not be fortuitous. In humans, an IRF site is positioned
in the region between the TAP1 and the PSMB9 (LMP2) genes (32,
43) and is believed to regulate the bidirectional expression of both
these genes, which are arranged in a head-to-head orientation (11,
12). A similar head-to-head arrangement exists in zebrafish, except
in this case both the PSMB11 and PSMB12 promoters possess a
separate IRF-binding site. The central position of these promoters
may influence the expression of four or five genes, PSMB11,
PSMB9 (LMP2), and TAP2 in one direction, as well as PSMB12
and PSMB8 (LMP7) in the other direction (Fig. 4). However, the
TAP2 gene also has an additional IRF-binding site in its own promoter region.
In humans (and other mammals tested thus far), only two PSMB
loci are present in the Mhc (PSMB8 and PSMB9) within the class
II region (32, 43). Both loci are of the i type; the third i-type locus
(PSMB10), as well as all the other PSMB loci, are found outside of
the Mhc (5). In the zebrafish, the situation is more complicated.
Here, there are at least six PSMB loci in the Mhc, but not in the
class II region; instead they are all in the class I region. Because
the i-type PSMB genes are functionally tied to the class I and not
to the class II Mhc genes (5), it can be argued that the zebrafish
arrangement makes more sense, particularly because, in this species, the class I and class II loci are on different chromosomes (22).
Furthermore, in the zebrafish, the Mhc-associated loci are presumably all of the i type and they represent all three PSMB groups (in
mammals, the two loci in the Mhc represent the X and Y groups;
the i-type locus of the Z group is on a different chromosome). Four
of the six Mhc-associated zebrafish PSMB loci are in a single main
cluster; the other two are at a distance of ;60 kb (PSMB9C) and
;120 kb (PSMB9B) from the cluster (B. Murray, V. Michalovà, H.
Sültmann, and J. Klein, manuscript in preparation). The association of i-type PSMB genes with Mhc class I in zebrafish is in
agreement with Hughes’ (18) hypothesis of a selective advantage
to the clustering of genes that have similar broad range expression
patterns.
In both humans and zebrafish, the PSMB clusters are associated
closely with the TAP loci (TAP1 and TAP2 loci in humans and
TAP2 locus in the zebrafish; the TAP1 locus could not be identified
in this species thus far) and loosely with the RING3 locus (32). The
conservation of a close linkage of the TAP2 gene and the PSMB
cluster between bony fish and mammals, despite genomic rearrangement, again suggests that it might have a selective advantage (18).
The degree of sequence divergence between the Dare-PSMB9A
and Dare-PSMB9B genes is similar to that reported for Xenopus
laevis, Xela-PSMB8A (LMP7A) and Xela-PSMB8B (LMP7B)
genes (44). Both sets of genes have highly diverged 59 and 39
UTRs and a similar degree of AA identity in the mature protein
sequence (85% for Dare and 90% for Xela). However, segregation
studies in Xenopus indicate that the two Xela genes may be allelic
(44). In contrast, the Dare genes reside at different loci.
The main PSMB cluster, which extends over ;18 kb, consists of
the PSMB9A, PSMB11, PSMB12, and PSMB8 loci, arranged in
this order in the following orientation 4 4 3 3 (Fig. 4). The
two PSMB loci outside the main cluster are apparently the result of
a duplication of the PSMB9 locus. Because phylogenetically the
PSMB9B and PSMB9C loci appear to be more closely related to
each other than either of them is to PSMB9A, they are presumably
derived from a common ancestor that had a common ancestor with
the PSMB9A locus. Whether the B and C loci are functional is
unclear at this time; in a cDNA library only the transcript of the B
locus has been found. However, the PSMB9B locus is apparently
present in some haplotypes and absent in others (B. Murray, V.
Michalovà, H. Sültmann, and J. Klein, manuscript in preparation).
The two extra loci in the main zebrafish PSMB cluster, PSMB11
and PSMB12, are of special interest because of their location and
their phylogenetic relationships. The PSMB11 locus is a member
of the Y group, which can be divided into two subgroups represented in humans by the PSMB6 and PSMB9 loci. Because the
zebrafish genome contains close relatives of these two loci (DarePSMB6 and Dare-PSMB9, respectively) and on the phylogenetic
tree the branch leading to Dare-PSMB11 splits off before the
PSMB6 and PSMB9 branches split from each other (Fig. 3),
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FIGURE 5. Consensus sequences of two transcription factor binding
motifs, C/EBP-b and IRF, and sequences of the PSMB9A, PSMB11,
PSMB12, and PSMB8 promoter regions with similarity to these motifs.
Nucleotide identity is shown in bold, nonidentity in lower case. For positions allowing any base pair (i.e., n), the presence of a base pair is shown
by a dash. The relative position of the motif upstream from the translation
initiation codon is indicated.
PROTEASOME GENES IN ZEBRAFISH
The Journal of Immunology
combination with differential peptide binding by different families
of Mhc class I molecules. The various immunoproteasomes could
have coevolved with separate Mhc molecules, and the apparent
presence of the PSMB9B gene in some haplotypes but not in others
may be a reflection of this evolution. In humans, immunoproteasomes have been shown to produce peptides with hydrophobic or
basic carboxyl termini (6), which are well suited for binding in the
C-terminal anchor pocket of the HLA class I molecules (7). Speculatively, each immunoproteasome may produce a set of peptides
with a specific type of C termini. This hypothesis predicts the
existence of corresponding class I molecules specialized for binding the products of different immunoproteasomes. In an attempt to
test the binding specificity of fish class I molecules, Okamura et al.
(48) compared the AA variation at the C-terminal anchor pocket of
carp class I genes with the four conserved residues (Tyr84, Thr143,
Lys146, and Trp147) of the mammalian classic class I molecules.
The comparison revealed conservation of between one and three of
these residues in carp molecules, indicating a possible expansion
of peptide binding specificity (48). In the zebrafish, three class I
loci have been described (49), all of which possess the same three
conserved residues (Thr143, Lys146, and Trp147), as found in the
most conserved carp gene. However, the full range of class I genes
in zebrafish has yet to be described. Additional zebrafish class I
genes have been identified (H. Sültmann, B. Murray, and J. Klein,
unpublished observations), and the degree of allelic diversity is
being investigated at these loci. The above hypothesis predicts the
presence of class I molecules with diverse C-terminal anchor
pocket motifs in the zebrafish.
Acknowledgments
We thank Dr. Colm O’hUigin for critical reading of the manuscript, as well
as Ms. Jane Kraushaar for editorial and Ms. Sabine Jantschek for technical
assistance. We also thank Ms. Vera Michalovà for sharing the PAC clones,
and Dr. Masanori Kasahara for providing access to the unpublished nurse
shark and hagfish PSMB7-like subunit sequences.
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the PSMB11 gene appears to have arisen before the divergence
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sense, the zebrafish arrangement of PSMB genes resembles the
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or translocation) of one or more genes from the ancient PSMB
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deletions within the PSMB cluster on the different chromosomes.
If we assume functionality of all five Y group genes in the zebrafish (PSMB6, PSMB9A, PSMB9B, PSMB9C, and PSMB11) and
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