At Least One Class I Gene in Restriction Fragment Pattern-Y
(Rfp-Y), the Second MHC Gene Cluster in the Chicken, Is
Transcribed, Polymorphic, and Shows Divergent Specialization
in Antigen Binding Region1,2
Marielle Afanassieff,3*† Ronald M. Goto,* Jennifer Ha,* Mark A. Sherman,‡ Lingwen Zhong,*
Charles Auffray,§ Françoise Coudert,† Rima Zoorob,§ and Marcia M. Miller4*
MHC genes in the chicken are arranged into two genetically independent clusters located on the same chromosome. These are the
classical B system and restriction fragment pattern-Y (Rfp-Y), a second cluster of MHC genes identified recently through DNA
hybridization. Because small numbers of MHC class I and class II genes are present in both B and Rfp-Y, the two clusters might
be the result of duplication of an entire chromosomal segment. We subcloned, sequenced, and analyzed the expression of two class
I loci mapping to Rfp-Y to determine whether Rfp-Y should be considered either as a second, classical MHC or as a region
containing specialized MHC-like genes, such as class Ib genes. The Rfp-Y genes are highly similar to each other (93%) and to
classical class Ia genes (73% with chicken B class I; 49% with HLA-A). One locus is disrupted and unexpressed. The other, YFV,
is widely transcribed and polymorphic. Mature YFV protein associated with !2m arrives on the surface of chicken B (RP9)
lymphoma cells expressing YFV as an epitope-tagged transgene. Substitutions in the YFV Ag-binding region (ABR) occur at four
of the eight highly conserved residues that are essential for binding of peptide-Ag in the class Ia molecules. Therefore, it is unlikely
that Ag is bound in the YFV ABR in the manner typical of class Ia molecules. This ABR specialization indicates that even though
YFV is polymorphic and widely transcribed, it is, in fact, a class Ib gene, and Rfp-Y is a region containing MHC genes of specialized
function. The Journal of Immunology, 2001, 166: 3324 –3333.
M
HC class I genes are often described as being either
classical (class Ia) or nonclassical (class Ib). Class Ia
genes are ubiquitously expressed encoding the polymorphic transplantation Ags that are the basis of rapid graft rejection.
They include human HLA-A, -B, and -C; murine H2-K, -D, and -L;
two loci in the chicken B system; and additional loci in a number
of other species. Class Ia molecules present peptide-Ag to CTL. At
the cell surface class Ia molecules are tripartite, composed of the
polymorphic heavy chain, bound peptide, and !2-microglobulin
*Department of Molecular Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010; †Institut National de la Recherche
Agronomique, Station de Pathologie Aviaire et Parasitologie, Nouzilly, France; ‡Department of Biology, Beckman Research Institute, City of Hope National Medical
Center, Duarte, CA 91010; and §Centre National de la Recherche Scientifique, Unité
Propre de Recherche 420, Génétique Moléculaire et Biologie du Développement,
Villejuif, France
Received for publication June 12, 2000. Accepted for publication December 11, 2000.
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
This material is based upon work supported in part by the U.S. Department of
Agriculture/National Research Initiative Competitive Grants Program (92-372048244), the U.S. Department of Agriculture/Foreign Agricultural Service/International
Collaborative Research/Research and Scientific Exchange Division (58-3148-5-023),
and by the National Science Foundation under Grants 9118199 and 9604589.
2
Sequences submitted to the GenBank database are Y-FVw*7.1 (AF218783) and
Y-FVIw*7.1 (AF218784).
3
Current address: Laboratoire de Biologie Moléculaire et Cellulaire, Centre National
de la Recherche Scientifique Unité Mixte de Recherche 5665, Institut National de la
Recherche Agronomique, LA 913, Ecole Normale Supérieure de Lyon, Lyon, France.
4
Address correspondence and reprint requests to Dr. Marcia M. Miller, Department
of Molecular Biology, Beckman Research Institute of the City of Hope National
Medical Center, 1450 East Duarte Road, Duarte, CA 91010-3011. E-mail address:
[email protected]
Copyright © 2001 by The American Association of Immunologists
(!2m).5 By contrast, the MHC class Ib genes, such as human
HLA-E, -F, -G, H, I, and J; murine H2-Q, -T, and -M; mammalian
CD1, Hfe, and Xenopus NCI genes, are generally not polymorphic
in the manner of class Ia; but, there are exceptions, such as Q2 (1).
Nonclassical molecules generally have weak influences in graft
rejection. Some class Ib loci lie within or near the MHC, and a
portion of these are phylogenetically close to class Ia loci. Other,
usually older class Ib loci are located in paralogous regions on
entirely different chromosomes. Many class Ib molecules are restricted in expression to particular tissues. Some class Ib molecules
have critical roles in regulating NK cell activity (2), and trafficking
of these to the cell surface can be dependent upon the binding in
the Ag binding region (ABR) of peptide derived from signal sequences of other class I molecules (3). Still others, such as FcRn
(4) and Hfe (5), function in the delivery of particular molecules
across cellular boundaries via molecular interactions independent
of the ABR. Although divergent to various degrees in primary
sequences, the class Ib and class Ia heavy chain molecules share
many elements of tertiary structure in common. Many, but not all,
class Ib molecules bind !2m.
A characteristic often useful for distinguishing between class Ia
and class Ib molecules is a set of 8 aa present in the "1 and "2
domains of the class I heavy chain (6, 7). These amino acids in the
ABR are effectively invariant in all class Ia molecules and contrast
strikingly with the many polymorphic residues in the region. These
residues are essential in sequence-independent anchoring of peptide-Ag. Substitutions are present at one or more of these eight
5
Abbreviations used in this paper: !2m, !2-microglobulin; Rfp-Y, restriction fragment pattern-Y, ABR, Ag binding region; SSCP, single-stranded conformational
polymorphism.
0022-1767/01/$02.00
The Journal of Immunology
positions in nearly all class Ib molecules, although a few exceptions to this are found among H2-Q and H2-T loci. In some class
Ib molecules, particular substitutions are associated with anchoring
special forms of Ag (8). For example, N-formylated peptides (9)
are bound by the mouse-nonclassical H2-M3 molecules in an ABR
made more hydrophobic by the presence of phenylalanine and
leucine at two of the eight critical positions. Glycolipids and lipoglycans are presented by transporter associated with Ag processing
(TAP)-independent CD1 molecules (10, 11). In the ABR of CD1
there are multiple, generally hydrophobic substitutions occurring
at the eight residues. Other substitutions are present in other class
Ib molecules and are associated with other modifications of the
ABR, such as closing of the groove in FcRn (12).
Restriction fragment pattern-Y (Rfp-Y) and B are two genetically independent clusters of MHC class I and class II! genes in
the chicken that map to a single microchromosome (chromosome
16) (13–16). Also mapping to chromosome 16 is the single nucleolar organizer region found within the chicken genome (17) and a
single nonpolymorphic classical class II" locus (18). A chromosomal region supporting highly frequent meiotic recombination,
perhaps associated with the nucleolar organizer region, separates B
and Rfp-Y such that the two clusters are genetically unlinked even
though they are located on the same microchromosome (15, 16).
This arrangement is quite different from the arrangement of class
I loci in the mouse into H-2K and H-2D where the loci remain
linked despite physical separation. Rfp-Y was detected initially
when two sets of polymorphic restriction fragments revealed by B
system class I and class II probes were found to assort independently of one another in families of fully pedigreed animals (13).
Rfp-Y was later found to correspond to the cosmid clusters II/IV
and III in the molecular map (19) of chicken MHC genes (14, 15).
At least two class I" heavy chain genes (YFV and YFVI), three
class II! genes (YL!III, YL!IV, and YL!V) (20), a c-type lectin
gene (21), and two additional genes (13.1 and 17.8) of unknown
identity map to Rfp-Y. The classical B system is a compact gene
region that determines rapid allograft rejection. A large portion of
the B cluster has been sequenced and found to contain 19 genes
within a 92-kb region, virtually all of which have counterparts in
the human MHC (22). Included among these are two polymorphic
class I heavy chain and two polymorphic class II! loci (19,
23–25).
Because the small number of chicken class I" heavy chain and
class II! chain genes are essentially equally divided between B and
Rfp-Y, the two clusters might originate from duplication of an entire chromosomal segment providing duplicate sets of loci with
similar functions. Alternatively each may perform specialized,
complementary functions as is becoming apparent for mammalian
classical and nonclassical regions (8). For example, the Rfp-Y loci
might in some instances provide molecules supplementing the less
than comprehensive Ag presentation that is so characteristic of the
B system (26). To begin to define the basis of the organization of
chicken MHC genes into two genetically independent clusters, we
subcloned and fully sequenced the YFV and YFVI loci located in
the Rfp-Y cosmid cluster map. We analyzed the sequences of these
loci with respect to those of known class Ia and class Ib loci and
evaluated their polymorphism. We extensively analyzed gene transcription and the capacity of YFV cDNA to produce mature Rfp-Y
class I molecules upon transfection.
Materials and Methods
Animals
Clone c!10 was isolated from a cosmid library made from line CB (B*12,
Yw*7.1) (27). (We use the w* notation with all Rfp-Y haplotypes to indicate
that assignments are subject to further refinement.) For transcription anal-
3325
ysis 12- and 19-day-old line CB embryos were provided by Pierrick Thoraval from stock maintained at Institut National de la Recherche
Agronomique (Nouzilly, France). One-year-old male birds from line C
were provided by Larry Bacon from stock maintained at the US Department of Agriculture Avian Disease and Oncology Laboratory (ADOL)
(East Lansing, MI). Lines C and CB both originate from the Reaseheath
line RH-C. Lines CB and C are inbred and homozygous for the same B and
Rfp-Y haplotypes. Other Rfp-Y haplotypes analyzed include Yw*1.3,
Yw*2.1, Yw*3.1, and Yw*6.1 from Northern Illinois University (13);
Yw*4.2 and Yw*5.3 from University of New Hampshire (R.L. Taylor, Jr.
and M.M., unpublished data); and Yw*7.2, Yw*8.1, and Yw*9.1, provided
by Larry Bacon (28).
Subcloning and sequencing of YFV and YFVI
The 3.55- and 4.8-kb BglII fragments of c!10 (27) containing the
YFVw*7.1 and YFVIw*7.1 genes, respectively, were subcloned into the
BamHI site in Bluescript II KS (Stratagene, La Jolla, CA) to provide pYFVw*7.1 and pYFVIw*7.1. Sequences of the YFVw*7.1 and YFVIw*7.1
genes were obtained through the successive application of two techniques.
First, the sequences of exons 2, 3, 4, and 5, and introns 2, 3, and 4 of each
gene were defined by sequencing of products obtained by PCR with primers designed from the B-FIV*12 gene sequence (29). Two hundred nanograms of cosmid c!10 clone DNA, 10 ng of pYFVw*7.1 and pYFVIw*7.1
plasmid DNA were used as templates. PCR amplifications consisted of 35
cycles of 95°C for 1 min, 60°C for 45 s, and 72°C for 45 s using Taq DNA
polymerase buffer with 400 nM of each primer, 200 #M of each dNTP
(Pharmacia, Piscataway, NJ), and 1 U of Taq DNA polymerase (PE Biosystems, Foster City, CA). A fraction of each reaction product was cloned
using the TA cloning vector (Invitrogen, San Diego, CA), and the nucleotide sequence of the insert was fully determined by dideoxy chain termination with a Prism Ready Reaction Dye Terminator Cycle Sequencing Kit
and a 370A DNA Sequencer (PE Biosystems). To identify any errors due
to misincorporation by Taq polymerase, three to six independent PCR were
conducted for each primer set and 4 –10 clones per PCR were fully sequenced. The sequences upstream of exon 2 and downstream of exon 5
were obtained by direct sequencing of the pYFVw*7.1 and pYFVIw*7.1
plasmids with annealing primers designed from previously determined
sequences.
Isolation of additional YFV clones
A YF-specific clone, 163/164f, was generated by PCR from YFVw*3.2
DNA and corresponds to exons for Cy1, Cy2, Cy3, and portions of surrounding introns of YFV (see Fig. 1). Clone 163/164f was used in turn to
isolate a full-length cDNA clone, c36f, from a cDNA library made from the
small intestine of a UCD line 330 young adult bird. An additional clone,
cos2, was also obtained by 163/164f screening from a SuperCos I cosmid
library (Stratagene) produced from a bird (wb3078) heterozygous for two
additional Rfp-Y haplotypes designated Yw*1.1 and Yw*5.1. Cos2 was determined to originate from Yw*5.1 by restriction fragment pattern. A YFV
subclone, pcr75171–3, was derived from cos2 and sequenced from the
clone margins.
RNA purification
RNA was extracted from frozen tissues with RNAzol B (Tel-Test, Friendswood, TX). For 12-day-old whole embryos and 19-day-old embryo tissues
RNA was purified on cesium chloride gradients.
Southern blot analysis
Samples containing 10 #g of genomic DNA were digested with restriction
endonucleases, electrophoresed in 1% agarose gels, and analyzed by
Southern hybridization (13). Probes included 1) a 32P-labeled oligonucleotide (TGGGGCTGGGGCTGGGGCT) designed from the exon 1 of
YFVIw*7.1; 2) a 0.9-kb SacI fragment of pYFVIw*7.1 corresponding to
exons 1 to 2; and 3) 163/164f.
Transcript analysis and single-stranded conformational
polymorphism (SSCP) assays
Transcription analysis by RT-PCR was performed as follows: 1) firststrand cDNA was synthesized for 15 min at 37°C using 1 #g of total RNA,
40 nM primer, 200 #M dNTPs, 20 U of AMV transcriptase (Life Sciences,
St. Petersburg, FL) in reverse transcriptase buffer, and a single oligonucleotide reverse primer RTBYF"2 (CCTCGAGGATGTCACAGCC) corresponding to a site in exon 3 identical in the BF and YF genes; 2) cDNA
was tested for purity with PCR performed using primer pairs that span the
"1 exon/intron/"2 exon boundary so that any product originating from
3326
genomic DNA can be recognized by product length; RTBYF" with
BFIV"1-5! (GGGCAGCCGTGGTTCGTGACT) and with YF"1-5! (GT
GGACGACAAAATCTTCGGTA). Products of the PCR (35 cycles at
95°C for 1 min, at 60°C for 45 s, and at 72°C for 45 s) were analyzed for
fragment length on agarose gels; 3) cDNA free of genomic DNA was used
as template for PCR performed with primers specific for the two YF loci
consisting of YF"1-5! and YF"1-3! (TTTGTTGTAGCGTTCCG
GCAGCC). For BFI the primer pair was BFI"1-5! (GGGCTGCCGTG
GTTCGTGGAC) and BFI"1-3! (GTGTTCAAGCTCACTTCCACAC).
For BFIV the primer pair was BFIV"1-5! and BFIV"1-3! (ATGCCCAG
GTTCTCGCGGTCAA); and 4) presence and absence of the BF and YF
transcripts were scored on the presence or absence of amplicon in agarose
gels. To distinguish the locus of origin for YF amplicons obtained with
YF"1-5! and YF"1-3! primers were analyzed by SSCP (30). For this, 1–3
#l of the PCR products were denatured in formamide at 80°C for 5 min and
electrophoresed for 105 min at 200 V in 10% polyacrylamide, 0.5% TBE
(44.5 mM Tris-borate, 44.5 mM boric acid, 1 mM EDTA) gels in a Miniprotean II apparatus (Bio-Rad, Richmond, CA). The gels were fixed and
stained with a Silver Stain Plus Kit (Bio-Rad) and dried in gel wrap
(BioDesign, New York, NY). The resulting patterns were scored in comparison with those provided by PCR in which line C DNA, c!10,
pYFVw*7.1, and pYFVIw*7.1 served as template.
Transfection, immunoprecipitation, and immunoblotting
A FLAG epitope tag sequence was incorporated into the YFV cDNA clone
c36f for tagging mature protein at the N-terminal end. The modified clone
was transferred into the replication-competent RCASBP(A) vector (31),
the viral plasmid was transfected into avian DF1 cells (32), and packaged
virus in the DF1 culture supernatant was used to infect avian RP9 cells
(33). Extracts were made of intact RP9 cells expressing FLAG-tagged c36F
YFV molecule and control cells (uninfected RP9 cells, RP9 cells infected
with RCASBP(A) containing FLAGc36f in the nonsense orientation, and
RP9 cells expressing FLAGBFIV21; Ref. 34), electrophoresed, blotted, and
developed using M2 anti-FLAG mAb and ECL reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Proteins were also immunoprecipitated
with M2 anti-FLAG mAb (Sigma, St. Louis, MO) or with anti-chicken
!2m mAb (35), generously provided by Jim Kaufman (Institute for Animal
Health, Compton, U.K.). The immunoprecipitates were electrophoresed
and blotted as above.
Sequence analysis
Sequences were assembled with PC Gene and DNAStar. Similarity indices
were determined with Wilbur-Lipman (DNA) and Lipman-Pearson (protein) algorithms. Deduced amino acid sequences were aligned using a mutation matrix, together with visual inspection of the modeled structures of
YFVw*7.1 and B-FIV*12 and with Megalign (DNAStar). Pileup (GCG)
and PaupSearch (maximum parsimony) were used to construct gene trees
and to assign bootstrap values.
Molecular modeling
A homology model of the YFVw*7.1 structure was built using Insight II
(Molecular Simulations, San Diego, CA) software. File 2clr.ent (HLAA*0201) was chosen from the protein database as the template. HLAA*0201 was one of several class Ia molecules giving essentially equal
scores in FASTA alignments with YFVw*7.1 sequence. None of the class
Ib molecules scored well when aligned with YFV. Amino acid insertions
and deletions were readily placed between secondary structure elements at
positions minimizing disruption of the overall fold by searching a highresolution subset of the Brookhaven database for loops having the required
length and similar context. Wherever more than one loop was present, the
loop of the highest sequence homology was chosen. Once all coordinates
were assigned and several conflicting side chains repositioned, the models
were energy minimized with DISCOVER within Insight II using the consistent valence force field and default parameters. Similar steps were followed in modeling the avian !2m chain on the structure of human !2m.
Results
YFVw*7.1 and YFVIw*7.1 are class I-like loci, but only
YFVw*7.1 is intact
BglII subclones containing YFVw*7.1 and YFVIw*7.1 were prepared from the cosmid c!10 (14, 27) and fully sequenced. The two
loci are oriented with 3! ends opposed and are separated by 11.5
kb. The sequences of both loci are presented in Fig. 1 aligned with
the sequence of the BFIV*12 (29), an allele at the classical class I
CHICKEN Rfp-Y CLASS Ib GENES
locus that is most strongly expressed in the chicken and with which
the Rfp-Y class I genes are highly similar. The exon/intron junctions for the YFVw*7.1 and YFVIw*7.1 were deduced based on the
sequence of BFIV*12 and confirmed by sequencing a YFV cDNA
(c36f) clone. The intron and exon organization in all three chicken
genes is typical of class I genes. Eight exons are present, and their
size is generally conserved with variations in the Rfp-Y genes confined to one or two codon differences from BFIV*12. Introns are
generally small compared with mammalian class I genes with intron length varying between Rfp-Y and B system genes by 1–29
nucleotides. The Rfp-Y class I genes are C"G rich as has been
noted for the B system class I loci (36).
The two YF loci are highly similar (93%) in nucleotide sequence
(Table I) except for a large repeat sequence (48 copies of the hexanucleotide GGGCTG) that disrupts exon 1 of YFVIw*7.1 (Fig. 1).
This insertion and the absence of a polyadenylation signal sequence downstream of the stop codon indicate that YFVI w*7.1 is
most likely unexpressed. As noted below, no transcripts from the
YFVI locus were found in any organs examined in RT-PCR assays.
To determine whether the hexanucleotide repeat is present in other
YFVI alleles in other Rfp-Y haplotypes two probes were prepared
for Southern hybridizations. When an oligonucleotide containing
three hexanucleotide repeats and a subclone of exon 1 from
YFVIw*7.1 were used to probe DNA representing seven additional
Rfp-Y haplotypes, only one was found to hybridize (data not
shown), indicating that the repeat is not commonly present in other
Rfp-Y haplotypes.
There is substantial sequence similarity between the sequence of
YFVw*7.1 and other MHC class I genes in extracellular domains
(Table I). Overall the nucleotide/predicted amino acid sequence
similarities for YFVw*7.1 with BFIV*12 and HLA-A2 are 73%/
60% and 49%/38%, respectively, and the similarity is generally
uniform over the exons corresponding to extracellular domains.
However, YFVw*7.1 and BFIV*12 are relatively less similar to
each other in exon 2/"1 domain sequences (66%/49%) than they
are in other extracellular exon/domain sequences suggesting that
the "1 domains of YFV and BFIV molecules have divergent functional constraints. The exons corresponding to transmembrane and
cytoplasmic domains of YFVw*7.1, BFIV*12, and HLA-A2 are
mostly dissimilar suggesting further specialization associated with
the Rfp-Y locus. As with other class Ib molecules, YFV-encoded
molecules lack the phosphorylation motif found in mouse and human MHC class Ia molecules (37).
The predicted, mature product of the YFVw*7.1 gene is a 332residue protein that has a single potential n-glycosylation site at the
same residue found in many class Ia molecules (noted by F in Fig.
2A). The amino acids critical for folding of class I molecules are
generally conserved in the YFVw*7.1 amino acid sequence. The
four cysteine residues that form the basis of the highly conserved
class I disulfide loops (C98C101-C161C164, C199C203-C255C259)
are present (the superscript denotes the position in HLA-A2). All
18 invariant residues (noted by I in Fig. 2A) known to form various
contacts within and between class I domains that are strictly conserved in the sequences of class I molecules (38) are present in
YFVw*7.1. Molecular modeling of YFVw*7.1 provides a structure highly similar to that of HLA-A2 (Fig. 2B). The structural
integrity of the ! strands and the " helices forming the ABR is
mostly conserved in the YFV protein, even though the YFV ABR
is three residues shorter than that of HLA-A2. The positions of the
“missing” residues are easily assigned to the margins of the !
strand and " helical regions and most likely do not disrupt domain
folding. As reflected in the model, a proline substitution at P51E53
in YFVw*7.1 is likely to disrupt the H1 helical region typical of
The Journal of Immunology
3327
FIGURE 1. Nucleotide sequences of the YFVw*7.1 and YFVIw*7.1 loci are aligned with the sequence of chicken classical class I locus BFIV*12. Exon
sequences are presented as codons, and introns as uninterrupted sequence. Intron boundary sequences and the ATG start site are in bold print and underlined.
The sequence of the hexanucleotide repeat region in YFVIw*7.1, the gene regions in YFVw*7.1 amplified with the YF"1-5! and YF"1-3! primer pair, the
gene region corresponding to the YF gene-specific probe 163/164f, and the polyadenylation signal sequence are labeled and in bold print. Identity is marked
by a dot, and a gap is shown as a dash. The GenBank accession numbers are AF218783 (YFVw*7.1) and AF218784 (YFVIw*7.1).
the "1 domain of classical class I molecules. In addition, the presence of a contiguous pair of flexible glycine residues G67A69
G68H70 in YFVw*7.1 indicate that the long H2 helical region of
the "1 domain may be broken into two shorter helices. Also, the
natural break in the "2 domain " helix is further accentuated in
YFV by the insertion of a glycine between positions 150 and 151
3328
CHICKEN Rfp-Y CLASS Ib GENES
Table I. Sequence similarity indices among YFw*7.1 loci and classical class I genes
YFVw*7.1 vs YFVIw*7.1
YFVw*7.1 vs BFIV*12
YFVw*7.1 vs HLA-A*0201
Entirea
Ex1/SP
Ex2/"1
Ex3/"2
Ex4/"3
Ex5/TM
Ex6-8/Cy
93/–
73/60
49/38
86/–
79/56
47/38
97/–
66/49
55/40
98/–
74/64
56/46
95/–
87/77
44/39
76/–
73/51
NS
94/–
66/54
NS
a
Similarity indices in nucleotide/predicted amino acid sequences as determined by Wilbur-Lipman algorithm and Lipman/Pearson alignments, respectively. Nucleotide
sequence for the disrupted YVIw*7.1 gene is compared with the hexanucleotide repeat excluded.
(HLA-A2 numbering). In summary, it is likely that YFVw*7.1 will
have a structure overall highly similar to HLA-A2 with the ABR
displaying a degree of specialization associated with the locus, a
feature commonly encountered in comparisons between class Ia
and class Ib molecules.
YFVw*7.1 encodes a distinctive class Ib molecule
In class Ia molecules eight highly conserved residues define the
“left” (Y7, Y59, Y159, and Y171) and “right” (Y84, T143, K146,
and W147) pockets of the ABR and secure peptide Ag by bonding
with main chain atoms. In YFV two of the left-pocket tyrosine
residues are replaced by H57Y59 and E156Y159. Further substitutions occur in the right pocket. Position 84 in YFV is polymorphic
occupied by R, Q, and C in different Rfp-Y alleles. A further, albeit
conserved substitution, R143K146, is also present in the right
pocket. These left and right pocket substitutions make it highly
unlikely that YFV class I molecules present Ag in the manner of
class Ia molecules. Hence the YFV locus fails to meet a major
criterion for inclusion in the class Ia category and, therefore,
should be considered a class Ib locus.
The substitutions that are found in the predicted YFV molecules
at four of the eight subclass-defining residues in the ABR are extremely rare among class Ib molecules. The E156Y159 substitution
is unique. Substitution of Y159 is generally rare with substitutions
of phenylalanine (H2-M9, H2-Q5k, H2-Mb-1, DLA79, FcRn), glycine (H2-T10), aspartic acid (H2-T9, H2-T22), tryptophan (Mr1),
alanine (MICA), and leucine (hCD1c and mCD1) occurring in a
limited number of class Ib molecules. The H57Y59 substitution is
shared so far only with Mr1 and some Xenopus class Ib loci. The
Y59 is conserved at most class Ib loci with other substitutions such
as phenylalanine (H2-M9) and leucine (mCD1) only rarely occurring. The substitution of three different amino acids at tyrosine 84
(R/Q/C82Y84) in different alleles at any class I locus is unprecedented. Generally alternatives to tyrosine at this position are very
rare with isoleucine and glutamic acid found at mouse H2-Mb-1
and CD1. Finally, the R143K146 substitution occurs occasionally in
class Ib molecules (H2-M3, FcRn) and is common among chicken
class Ia (BFIV) alleles. Thus no other known class I locus is
closely similar to YFV in the substitutions at these four positions
suggesting that YFV defines a new type of class Ib locus.
YFVw*7.1 is a recently derived class Ib locus
The "3 domain sequences of the Rfp-Y class Ib loci were aligned
using PileUp (Genetics Computer Group, Madison, WI) with the
corresponding sequences from class Ia and class Ib molecules from
several vertebrate species. The alignment was analyzed with phylogenetic analysis using parsimony to generate the gene tree and
bootstrap values presented in Fig. 3 (class Ib are underlined). The
two Rfp-Y class Ib "3 sequences are closely similar to those of
chicken and quail class Ia molecules (see large bracket). This
group forms a clade restricted to gallinaceous birds indicating that
the Rfp-Y loci may be relatively young genes sharing recent ancestors with class Ia genes in gallinaceous birds. Similar relationships occur in other taxa between class Ib loci and class Ia loci as
can be seen in the other bracketed regions of the tree where Xenopus, humans, and mice also form clades in which class Ib and
class Ia share recent ancestry. Most of the class Ib molecules
within these clades are known to require TAP for processing of
specialized Ag indicating that these relatively recently derived
class Ib share Ag processing pathways with class Ia (8). These
molecules contrast with more distantly derived class Ib molecules
known to be TAP independent in the presentation of
specialized Ag.
YF class Ib genes display both haplotypic and allelic variation
Because polymorphism or the lack thereof is another characteristic
that is often used to separate class Ia from class Ib loci, we examined Rfp-Y class I genes for evidence of haplotypic and allelic
polymorphism. We first examined Rfp-Y class I haplotypic variability using a Rfp-Y class I specific probe, 163/164f, in Southern
blots. Nine different TaqI restriction fragment patterns were obtained from nine previously defined Rfp-Y haplotypes (Fig. 4A).
Surprisingly, the number of restriction fragments varies among the
haplotypes from only two in Yw*1.3 and Yw*7.2 to at least 10 in
Yw*4.2 and Yw*6.1. Similar differences were found in PstI and
BglI restriction fragment patterns (data not shown). It is likely that
the number of class I loci varies among Rfp-Y haplotypes. Similar
variation in gene number occurs in the class Ib region in H-2 haplotypes (39).
Predicted amino acid sequences for YFVw*7.1, YFVw*3.2, and
YFVw*5.1 are aligned in Fig. 2A. It is likely that these three sequences all originate from the YFV locus. YFVw*7.1 and
YFVw*3.2 are identical in cytoplasmic domains and differ by only
one amino acid in the transmembrane domain (TM). In contrast,
YFVw*3.2 differs from YFVIw*7.1 by 15 of 34 aa over the same
regions making it highly unlikely that YFVw*3.2 originates from
the YFVI locus. The third clone, YFVw*5.1, was obtained by PCR
using primers designed for YFV specificity and differs from
YFVw*7.1 by only a single amino acid in transmembrane domain
sequence.
The three clones display considerable sequence variability (Fig.
2, A and B). The "1 domain variability among the three sequences
(21%, 18 of 87 aa) is nearly as great as the variability that is
present among BFIV alleles (27%, 24 of 88 aa in 11 alleles) (40).
This variability is almost entirely confined to the helical region of
the "1 domain and to the floor of the ABR with a small remainder
of variation present in loop regions (Fig. 2B). Amino acid replacements are most often nonconservative. For example, charged residues are interchanged with neutral, nonpolar (70R/L, 59D/A,
67D/G, 85K/I, 87K/G) or with neutral, polar residues (37D/N,
55Q/R, 66Q/R, 73D/C, 82R/Q/C). In other instances neutral, polar
residues are interchanged with neutral, nonpolar residues (32N/I,
35T/I, 60T/A, 78W/G, 75N/F/L, 77N/G). Conservative substitutions (47V/A and 71D/E) in these regions are more rare.
The "2 domain is less variable (10%, 9 of 92 aa). Substitutions
are mostly confined to two ! sheet strands in the floor of the ABR,
as they are in the classical BFIV alleles (40). The residues are often
The Journal of Immunology
3329
FIGURE 2. Comparison of the predicted amino acid sequence of YFVw*7.1 with the sequences of human (HLA-A2) and chicken (BFIV*12) classical
class I molecules. A, Structure-based alignment of the deduced amino acid sequences of three YFV alleles with chicken class Ia (BFIV*12) and human class
Ia (HLA-A2) sequences. Polymorphic residues in the YFV sequences are noted in red with the position underlined. B, Stereoview of the model for the three
extracellular domains of YFVw*7.1 and chicken !2m (both in red) modeled after the crystal structures of HLA-A2 and human !2m (both in black).
YFVw*7.1 apparently lacks the H1 helical region (noted by arrow) found in the "1 domain of HLA-A2 and other class Ia molecules.
3330
FIGURE 3. Gene tree showing the relationship of YFVw*7.1 and
YFVIw*7.1 to selected vertebrate class Ia and class Ib (underlined) genes
in the "3 domain. Note that the appearance of YFVw*7.1 and YFVIw*7.1
predates the separation of chicken and Japanese quail (larger bracket).
These genes share a relationship with class Ia genes similar to the relationship among class Ia and subsets of class Ib molecules in Xenopus,
human, and mouse (smaller brackets).
hydrophobic, and substitutions are often conservative. One nonpolar residue often substitutes for another (92L/M, 95 M/I, 96I/F,
120L/I), or charged residues are interchanged (117R/K). In other
instances the substitutions are nonconservative (91T/M, 94 M/R,
119F/H/Y, and 178R/T). Similar to the chicken class Ia molecules,
some variability is also present in the "3 domain (Fig. 2A).
Whether this reflects functional specialization among the YFV alleles is not yet understood.
Nonsynonymous-to-synonymous substitution ratios vary across
" helical and ! sheet regions of the "1 and "2 domains of the three
YFV sequences (data not shown). Briefly, the " helical portion of
the "1 domain has a high ratio compared with the ! sheet region
suggesting that, as in class Ia loci (40, 41), this region may be
under selection for interactions with diverse Ag or variability in a
counterreceptor. In contrast, in the "2 domain the values for the
nonsynonymous-to-synonymous substitution ratios are reversed.
The YFV "2 " helical region has an extremely low ratio indicating
that diversification of this region of the molecule is restricted, as
apparently it is in BFIV alleles (40). So although YFV molecules
are nonclassical and unlikely to bind typical peptide Ag, they are
polymorphic with the distribution of sequence variability among
alleles not unlike the classical class Ia molecules of the chicken.
YFV is transcriptionally active in many organs and can be
expressed as a transduced gene
To determine whether the YFVw*7.1 and YFVIw*7.1 are transcriptionally active and to learn whether transcription is confined to
particular organs as often is found for class Ib genes, we performed
RT-PCR SSCP assays using a YF gene-specific primer set. With
the exception of small regions immediately upstream of the start
site, the regulatory and promoter sequence elements (see GenBank
sequences) of YFVw*7.1 (and YFVIw*7.1) genes are similar to
those of class Ia genes suggesting that YF could be generally active
in many tissues. RNA free of genomic DNA was obtained from
CHICKEN Rfp-Y CLASS Ib GENES
embryos and from a number of organs of young adult line C birds
(Fig. 5 and Table II). The YF-specific primers provided a means of
specifically detecting transcripts from the Rfp-Y class I loci, and
SSCP provided a means of distinguishing between YFVw*7.1 and
YFVIw*7.1 transcripts. RT-PCR assays for BFI and BFIV served
as positive controls. YFVw*7.1 transcripts were detected in all organs tested, except for three (brain, heart, and pancreas). No evidence was found for transcriptional activity of YFVIw*7.1 locus.
Results of the full analysis are summarized in Table II and are
consistent with a limited number of RNase protection assays demonstrating the presence of YFVw*7.1 transcripts in several tissues
(42). We conclude that YFVw*7.1 is constitutively transcriptionally active in many organs much like MHC class Ia genes. It remains to be determined whether YFV transcription is inherent in all
or many of the tissues in these organs or whether transcription is
limited to a particular cellular subset with perhaps only limited
quantities of YFV reaching the surface of these cells.
To determine whether mature protein can be produced from expression of YFV cDNA, the clone c36f was FLAG-epitope tagged,
inserted into the RCASBP(A) retroviral vector, and expressed in
the chicken B cell lymphoma line RP9. FLAG-tagged protein was
found by flow cytometry to be at the surface of cells expressing
FLAGc36f inserted into the vector in the sense orientation but not
in the nonsense orientation (data not shown). In immunoprecipitations with the anti-FLAG mAb M2, RP9 cells expressing
FLAGc36f and the positive control FLAGBFIV*21 were both
found to produce FLAG-tagged protein with masses (#48 kDa)
typical of class I molecules (Fig. 6A). The FLAGc36f encoded
protein could also be immunoprecipitated with an anti-chicken
!2m mAb (Fig. 6B) providing evidence for association between
YFV protein and !2m. Hence protein similar to typical class I
molecules in molecular mass and in !2m association can be obtained by expressing the YFV cDNA c36f as a transgene.
Discussion
In these experiments we have shown that the YFV locus shares
many qualities with MHC class Ia genes. As summarized in the
structural model in Fig. 2B, the YFV molecule is likely to be
structurally similar to class Ia, as are several other class Ib molecules. The unusual substitutions in the putative ABR of the YFV
molecules are the strongest characteristic separating YFV from
class Ia loci. Alleles at the YFV locus differ from one another by
multiple changes in predicted amino acid sequence. Many of the
polymorphic residues surround the ABR. Over 20 aa differences in
the ABR separate the three YFV alleles in this study from each
other. Such variability is on par with the differences between alleles at class Ia loci and between alleles at the one previously
identified highly polymorphic class Ib locus, H2-Q2 (1). In addition, there may be many YFV alleles. SSCP analyses of the Rfp-Y
class I exon 2 sequences in a variety of genetic lines provide indirect evidence for many more YFV alleles than the three presented
here (M.M., unpublished data). As with class Ia loci, polymorphism and the relatively high ratios of nonsynonymous-to-synonymous substitutions over portions of the YFV ABR indicate that
this region may be under selection for diversity. YFVw*7.1 and
YFVIw*7.1 occupy positions in the evolutionary tree in Fig. 3 that
further emphasize their close relationship with class Ia genes in
gallinaceous birds. The gene tree in Fig. 3 indicates that the
YFVw*7.1 and YFVIw*7.1 were derived relatively recently with
their appearance likely predating only the separation of gallinaceous taxa (Fig. 3). Perhaps YFVw*7.1 and YFVIw*7.1 are intermediates in the evolutionary tide postulated by Shawar and colleagues (37) to flow between loci encoding molecules with less
selective (class Ia) and highly selective (class Ib) ABRs.
The Journal of Immunology
3331
FIGURE 4. Evidence for genetic polymorphism associated with Rfp-Y class I genes. A,
Southern blot analysis. Restriction fragment
polymorphism of YF class Ib loci in nine different Rfp-Y haplotypes revealed by a YF-specific
probe, 163/164f, in TaqI-digested DNA. B, Stereoview of "-carbon backbone of the "1 and "2
domains of the YFVw*7.1 model structure. Residues that are polymorphic among the three
Rfp-Y sequences in Fig. 2A are marked by black
circles with the amino acids variants presented in
single letter code.
Given the close relationship the YF loci have with avian class Ia
genes and their sequence polymorphism, it seems likely that YFV
molecules bind peptide Ag. The unique substitutions of glutamic
acid and histidine in the left pocket of the ABR may provide a
means for selecting a particular form of Ag. Perhaps the charged
residues form salt bridges with Ag in the ABR providing a means
for selection of a particular subset of Ags. Or alternatively, perhaps
the left end of the putative ABR of YFV is actually closed by
interactions between these and other residues surrounding this region of the groove. In this instance, shorter forms of peptide might
fill the remaining open portion of the groove through a selective
interaction based on another characteristic of antigenic peptide,
such as hydrophobicity of amino acid side chains. Because YFV
molecules are apparently close relatives of the MHC class Ia molecules, it seems likely that peptide Ag load into the ABR through
a TAP-dependent pathway. It would make sense that the YFV Ag
is peptide, but this remains to be determined. How YFV molecules
bind Ag and what form the Ag has will be the subject of additional
experiments, as will be the consequence of YFV expression in
cellular interactions with cytotoxic T and NK cells.
The YF loci have other features that define them as class Ib.
Most class Ib loci have little or only weak influences in graft rejection. The structural identification of YFV as a class Ib gene is
consistent with conclusions drawn by Pharr and colleagues (28) on
the influence of Rfp-Y incompatibility on transplantation immunity. In experiments conducted with carefully defined genetic
stock, Pharr et al. found skin graft rejections attributable to Rfp-Y
incompatibilities occurred at moderate rates. They were clearly
slower than B incompatibilities but significantly faster than RfpY-compatible grafts. These authors suggested that one reason for
FIGURE 5. Illustration of the RT-PCR SSCP assays used for scoring
YF gene expression in organs from line C birds. The SSCP step was used
to distinguish between transcripts originating from YFVw*7.1 and
YFVIw*7.1 loci. Controls include SSCP patterns for PCR products originating from line C DNA, cosmid c!10, and plasmids separately containing
the YFVw*7.1 and YFVIw*7.1 genes. Results of the full analysis are presented in Table II.
3332
CHICKEN Rfp-Y CLASS Ib GENES
Table II. Distribution of Rfp-Y and B class I transcripts determined
using RT-PCR
Organa
Adult
Blood
Brain
Bursa of Fabricius
Caecum
Eye
Gizzard
Heart
Kidney
Liver
Lung
Ovary
Pancreas
Skeletal muscle
Skin
Small intestine
Spleen
Thymus
19-day-old embryo
Bursa of Fabricius
Spleen
Thymus
Whole 12-day-old embryo
YFV
YFVI
BFIV
BFI
"
$
"
"
"
"
$
"
"
"
"
$
"/$
"
"
"
"
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"/$
"
"
"/$
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"
"/$
$
$
$
$
"
"
"
"
"
"
"
"/$
a
RNA was extracted and assays were performed on organs from four adult chickens, four 12-day-old embryos, and two 19-day-old embryos from the C or CB lines
(B*12 and Yw*7.1).
the observed intermediate rate of graft rejection with Rfp-Y incompatibility might be the presence of class I-like (nonclassical) loci
within the Rfp-Y gene region.
The YFV locus may share another feature with class Ib genes.
There may be little YFV normally on the surface of cells despite
the presence of YFV transcripts. Other investigators have found no
evidence that Y-FV molecules are immunoprecipitated by antichicken !2m (43) and so YFV molecules may be normally less
abundant at the cell surface than, for example, chicken class Ia
molecules derived from the BFIV locus. It will be interesting to
learn whether there are conditions under which surface expression
of YFV becomes abundant. Because the Ag for YFV is likely to be
atypical, it may be that it is not normally abundant and that trafficking of YFV to the cell surface is limited by Ag availability.
This would be particularly interesting to explore given the evidence that in some but not all instances Rfp-Y haplotype has been
found to influence resistance to virally induced tumors in chickens
(44 – 46). If YFV is dependent on TAP molecules encoded in the
B system for Ag processing, it could also be that interaction with
chicken TAP affects YFV surface expression. Because chicken
TAP genes are themselves polymorphic (47) and the YFV and TAP
loci are unlinked, it might be that in particular combinations of
TAP and YFV alleles there is either less or more YFV at the cell
surface even in the presence of ample YFV Ag.
Finally, the organization of chicken class I genes into two genetic units composed of class Ia and class Ib loci is not unique. The
class Ia and class Ib loci in Xenopus are also located in two independent genetic units and, just as in chickens, the two regions map
to the same chromosome (48) separated by a region supporting
highly frequent recombination. Considering the evolutionary relationship that exists between class Ia and class Ib genes in these two
species, as illustrated in Fig. 3A, it is likely that this manner of
organizing class I genes has been arrived at independently in these
two species. Genetic separation of the two class I subclasses could
be a means by which the integrity of class Ia and class Ib loci are
maintained. Perhaps the class Ia loci isolated by this arrangement
evolve in concert with adjacent Ag processing loci as has been
suggested by others (47, 48), whereas the class Ib loci are free to
evolve in a different manner. In isolation the class Ib loci may be
able to change in number, allelic variation, and ABR specificity
through a variety of recombination events in a system for selective
Ag presentation that evolves rapidly in response to disease
challenge.
Acknowledgments
We thank Larry Bacon for providing C line birds and DNA from Cornell
line N and P; Henry Hunt for providing RP9 cells expressing FLAGBFIV*21; Pierrick Thoraval for providing tissue from line CB embryos;
and Jim Kaufman for providing anti-!2m mAb. Elwood Briles and Robert
Taylor, Jr. generously provided blood samples from Rfp-Y-typed birds. We
thank Larry Bacon, Pamela Bjorkman, Louis DuPasquier, Henry Hunt, and
Iwona Stroynowski for helpful discussions.
References
FIGURE 6. Immunoblot and immunoprecipitation of FLAG-tagged
YFV molecules. A, Immunoblot was developed with anti-FLAG M2 mAb.
Lysates are from RP9 cells expressing FLAGBFIV*21 (positive control),
FLAGc36f in anti-sense ($) and sense (") orientations, and uninfected
cells. B, Proteins were immunoprecipitated with anti-FLAG M2 or antichicken !2m mAb from lysates of RP9 cells expressing FLAGc36f in antisense ($) and sense (") orientations. Immunoblot was developed with
anti-FLAG M2 mAb. The Ig H and L chains are derived from the precipitating mAbs. Approximate mass of the FLAG-tagged products is noted
with arrowheads.
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