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I
i
MAJOR
LIKE
HISTOCOMPATIBILITY
GENES
IN CHICKENS:
COMPLEX
A LESSON
(MHC)
FROM
I
Department
Marlene G.ofEmara
Animal
andand
N. Food
Lakshmanan
Sciences
University of Delaware
I
Newark,
DE 19717-1303
emara@udel.edu
302-831-1344
I
AND MHC-
BROILERS
INTRODUCTION
I
The major histocompatibility complex (MHC) is one of the most intensively studied
genetic regions of the vertebrate genome. Its involvement in organ transplantation, antigen
I
processing
and presentation
T lymphocytes
during
the immune
and
other disease
associationsto has
made the MHC
an active
area ofresponse,
research. autoimmunity
Three classes
of molecules are encoded in what is commonly known as the classical MHC or MHC
I
proper.
Classes
I and
present antigenic
to CD8
CD4 . T are
lymphocytes,
respectively
during
theIIimmune
response. peptides
The Class
I and+IIand
molecules
expressed at
high levels; they are genetically diverse (one to three functional loci per haplotype, with
I
numerous
alleles at each
theyTrowsdale,
share common
their
function (Salter-Cid
and locus);
Flajnik, and
1995;
1995;structural
Kasahara motifs
et al., related
1996). to
The
human HLA-A, -B, -C and the mouse H-2-K, -D, -L loci encode the Class I MHC molecules,
I
whereas the HLA-D and H-2-I loci encode Class II molecules. In contrast, the Class III
genes encode a range of molecules, including complement factors C2, C4 and Bf, as well as
tumor necrosis factor and the heat shock 70 protein (Salter-Cid and Flajnik, 1995). Other
genes,
immune-related
including Lmp (low molecular weight protein) and TAP (transporter
associated with antigen processing), as well as genes with no immune function, such as G7a
(valyl-tRNA synthetase), G1 (calmodulin-like) and RING3 (serine-threonine kinase) are also
proper
found in the MHC
(Arnaiz-Villena, 1993; Salter-Cid and Flajnik, 1995). Several
genes with unknown function are also scattered throughout the mouse and human MHCs.
I
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To
or
matters,
further complicate
novel families of MHC-like
non-classical MHC
genes have also been described in humans and mice, as well as bony fishes and amphibia
(Shawar et al., 1994; Salter-Cid and Flajnik, 1995). The majority of the non-classical genes
II-related
have also been
are related to the Class I MHC genes; however, Class
genes
described. In the mouse, there are over 58 non-classical Class I genes, pseudogenes and
gene fragments that have been described (Powis and Geraghty, 1995). Some of the Class l-
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like genes and their products include MIC (MHC Class I chain-related genes), the CDi
family, Zn-ct2-glycoprotein and the neonatal Fc receptor (Bahrain et al., 1994; Hashimoto
i
generally
monomorphic,
with1996).
limitedThese
tissuenon-classical
expression, or
as "Class
compared
the classical
et al., 1995;
Kasahara et al.,
Ib" to
molecules
are or
"Class ia'" molecules (Nei et al., 1997). The non-classical MHC genes are structurally
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similar to their classical MHC homologs: however, the homology between the classical and
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1
non-classical genes is very low. For example, there is only 23-34% amino acid homology
between the mouse Fc receptor and the Class Ia MHC proteins of several vertebrate species
(Ahouse et al., 1993) and an average of 25% amino acid homology between the MICA
protein and the Class Ia proteins of humans (Bahram et al., 1994). In several instances, the
non-classical Class Ib gene products are also associated with 132-microglobulin, suggesting
that this binding region is conserved between classical and non-classical Class I genes
(Salter-Cid and Flajnik, 1995). Many of the Class Ib genes (mouse H-2Q, T and M loci;
human HLA-E, F, G, H, I and d loci) are located adjacent to classical MHC regions;
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however, some of them, i.e., CD1, Zn-_2-glycoprotein and the IgG Fc receptor are located
outside the MHC-bearing chromosome (Salter-Cid and Flajnik, 1995; Kasahara et al.,
1996). Like the classical MHC molecules, the non-classical MHC products have been
1
reported to participate in immune functions and they are recognized by T lymphocytes
(reviewed by Shawar et al., 1994; Salter-Cid and Flajnik, 1995). The Class Ib molecules
exhibit unique peptide-binding activities, especially for bacterial antigens (Kurlander and
Nataraj, 1997); they affect maternal immune tolerance (Rouas-Freiss et al., 1997); and they
either inhibit or activate NK cell activity (Rolstad et al., 1997). For example, the mouse
H2-M3 molecule binds N-formylated peptides and it has been suggested to play an
important role in bacterial and mitochondrial peptide presentation to cytotoxic T cells
(Shawar et al., 1991). Similarly, the CD 1 molecules present non-peptide, lipid-containing
epitopes to T cells and they are also suggested to have an important role in bacterial antigen
presentation (Porcelli et al., 1998). Although the majority of the n0n-elassical genes are
Class I-related, Class II-like genes have also been described in humans. The non-classical
Class II genes include HLA-DM and -DO, and they are reported to have chaperone-like
activity during antigen processing (vogt et al., 1997; Kropshofer et al., 1998).
II
Therefore, in summary, the term "MHC" has expanded to include the classical
MHC or MHC proper, in addition to the novel MHC-like genes, oiten referred to as nonclassical MHC genes (summarized in Table 1). As the genetic organization, structure,
diversity and function of the MHC and its related genes is realized, it is becoming apparent
that the MHC is one of the most complex and intriguing regions of the vertebrate genome.
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THE CHICKEN MHC (B) AND RFP-Y SYSTEM
n
The chicken MHC was originally identified by Briles and coworkers as the B blood
group locus (Briles et al., 1950). A decade later, it was observed that B blood group
differences were responsible for skin graft rejections and therefore, it was concluded that
the "B locus" was the chicken MHC (Schierman and Nordskog, 1961). Soon after this
discovery, other immunological phenomena such as the mixed lymphocyte reaction and the
graft-versus-host reaction were also associated with the chicken B blood group locus (Jaffe
and McDermid, 1962; Miggiano et al., 1974). These findings suggested that the red blood
cell antigens of the B system were similar to the histocompatibility antigens of the skin and
to the membrane proteins on lymphocytes. It was the classical experiments of Zeigler and
Pink (1976), who used B-specific antisera to purify the B blood group antigens and
discovered that more than one 13antigen existed on white blood cell membranes. These
moleculcs were designated B-F tbr the histocompatibility antigen (Class I) and B-L for the
l
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immune-associated antigen (Class II). Evidence for a third type of MHC molecule soon
followed. Recombinational events had occurred in two birds which caused the red blood
G].,
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cells from each of these birds to react with 2 different B-typing reagents (Hala et
1976).
One of the proteins was expressed on both red and white blood cells (B-F), and the other
i
was
concluded only
that the
chicken
locusand
wasit actually
a complex
genes IV).
that encoded
was expressed
on red
bloodBcells
was designated
B-Gof(Class
Therefore,at it
least three distinct groups of MHC proteins; Classes I (B-F), II (B-L) and IV (B-G) (Pink et
i
molecular
tissueMHC
specificity
andcan
biological
function (reviewed
by Guillemot
al.,
1977). structure,
Each of these
proteins
be differentiated
from the others
by their and
Auffray, 1989; Plachy et al., 1992). The Class I or B-F proteins are composed of a single
I
on the surface
polypeptide
chain
of all
(orcell
chain),
typesininassociation
the body, including
with 132-microglobulin
erythrocytes (Crone
and theyet are
al., expressed
1985). In
contrast, the Class II or B-L proteins are heterodimeric (ct and 13chains) and their
I
expression
is restricted
to cells (Crone
(B cells,etspecialized
cells,B-L
macrophages,
activated
T
cells) of the
immune system
al., 1981). epithelial
The B-F and
molecules function
in antigen presentation to T lymphocytes, similar to their mammalian Class I and II
I
homologs,
respectively.
The
Class
IV or B-G proteins
are unique to forms
chickens
andred
they
were originally
reported to
exist
in homodimeric
and heterodimeric
on the
blood
cell membrane (Kline et al., 1988). However, the Class IV proteins are also expressed on
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thrombocytes,
bursal
cells, peripheral
B and
T cells,
stromal
the
thymus, bursa thymocytes,
and cecal tonsil
and Bepithelial
cells of the
small
intestine,
cecacells
and of
liver
(reviewed by Kaufman et al., 1991). Although the exact function of the B-G molecule
I
remains
been found
exert an adjuvant
during The
antibody
responsesis
to other unknown,
blood groupit has
antigens,
ex. B-Fto molecules
(Hala eteffect
al., 1981).
B-G molecule
also speculated to have either an antigen presentation function similar to that of Class I and
I
II molecules; to bind antigen similar to that of B cells; or to participate in the development
of the B cell repertoire (Kaufman and Salomonsen, 1992).
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It has been well over two decades since Pink and colleagues (1977) described the
three locus model of the chicken B complex. With the advent of molecular genetic
techniques, rapid progress has been made in characterizing the molecular structure and
genetic
diversity of the chicken MHC. The genomic organization of the chicken MHC is
somewhat different from that of mammals (Guillemot and Auffray, 1989; Trowsdale,
1995; reviewed by Kaufman et al., 1995). In general, the chicken MHC is compact (due to
shorter
introns within the chicken MHC genes); it does not contain discrete Class I, II and
Ill subregions; and there is a low rate of recombination between the MHC genes. An
extensive genomic map of the chicken MHC was originally developed by Guillemot and
were mapped
coworkers (1988). In total,
20 genes
within 4 cosmid clusters, and at least I l
of them were MHC genes. There were 5 B-Lf3 genes (B-L_I to B-L_v) and 6 B-F genes
(B-FI to B-Fvl) and they were found to be intermingled among each other within 250 Kb of
DNA. Interestingly, the B-LI3 genes could be separated into two isotypic families based on
their differential hybridization to a 3' untranslated specific oligonucleotide probe. This
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mapped has
finding
to aproven
second toMHC
be ofsystem
significance
in chickens,
becausenamely
one ofthe
the Rfp-Y
isotypic
system
families
(Miller
has et
since
al., been
1994). In addition, Guillemot and coworkers (1988) identified one B-G gene that was 12 kb
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upstream of the B-F and B-L genes, as well as several non-MHC genes whose functions
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were not characterized.
Since the original finding, at least one of these genes was reported
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to encode a polypeptide which shares 22% amino acid homology with the 13subunit of the
human guanine nucleotide-binding proteins and bovine transducin (Guillemot et al., 1989),
and a second gene (17. 5) encodes a product that is 16-23% homologous to the C-type
animal lectin proteins (Bernot et al., 1994). Although it has been demonstrated that
complement (Class III) levels are associated with the chicken MHC (Chanh et al., 1976), the
specific complement genes that are involved were not determined. In fact, for a long time, it
was thought that the complement genes (C4, C2, etc.) were located elsewhere in the genome
due to the small size of the chicken MHC. However, direct evidence for Class III genes in
the B region was provided by Spike and Lamont (1995) and Kaufman et al., 1999.
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More recently, Milne and coworkers (Genbank Accession # AL023516; submitted
May, 1998) have provided B-region DNA sequence for cosmid clones cB12, c4.5 and
cBF23 that were originally isolated by Guillemot et al. (1988). This data is a landmark in
chicken MHC research. Not only have these authors identified Class I (B-F) and II (B-LI3)
genes in these cosmids, but also several other MHC-linked genes including TAP, RING3,
tapasin (TAP binding protein) and complement C4. In addition, some non-MHC genes
include a histone H3-1ike gene, a Leu-tRNA gene, a lectin-like natttral killer cell surface
receptor gene, a C-type animal lectin gene and a G (zipper protein)-like gene. The tapasin
gene (involved in assembly of the Class I molecules with 132-microglobulin in the
endoplasmic reticulum) has only recently been published (Frangoulis et al., 1999).
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The B-G region is comprised of numerous loci (at least 18) and it is in linkage
disequilibrium with the classical B region genes (reviewed by Kaufman et al., 1991; 1995).
At least four of the B-G loci are transcribed; the tranlated product is a single
immunoglobulin-like polypeptide with cytoplasmic tails that vary in length (Miller et al.,
1991). The length variation is due to differences in the number ofheptad repeat units
associated with the cytoplasmic tails (Kaufman et al., 1990). Interestingly, the myelinoligodendrocyte glycoprotein (MOG) gene which is suspected to influence susceptibility to
multiple sclerosis and the butyrophilin gene which is expressed in the bovine mammary
gland are both related to the chicken B-G genes (Trowsdale, 1995).
•
As indicated earlier, the chicken MHC genes are grouped into two separate systems,
the B region and the Rfp-Y system. Over the past few years, restriction fragment length
polymorphism (RFLP) analyses of B-F and B-Lfl genes in pedigreed families have revealed
diverse MHC-like restriction fragments which segregated independently of the
serologically-defined MHC (B-region) haplotypes (Chauss6 et al., 1989; Tilanus et al.,
1989; Juul-Madsen et al., 1993). These unexplained restriction fragments led Briles and
coworkers (1993) to identify a second group of MHC genes in chickens, termed Rfp-Y.
Interestingly, the Rfp-Y system maps to the same microchromosome (no. 16) as the classical
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MHC (B region); however, the genes within each system appear to segregate independently
(Miller et al., 1996). Lack of genetic linkage of the B and Rfp-Y systems is hypothesized to
be due to a high frequency of recombination within the nucleolar organizing region (NOR)
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which may separate the two systems. The Rfp-Y system is composed of two Class I and two
Class Ii loci; cosmid clusters 11and IV from Guillemot and coworkers (1988) have since
been proven to originate from the R[P-Y system (Miller el al., 1994). Non-MHC genes have
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also been identified in the Rfp-Y system; for example, the 17. 5 gene described earlier
(Bemot el al., 1994). The physiological functions of the Rfp- Y gene products are not
known; however, a report indicates that Rfp-Y (B-LI3III) transcripts are present in the spleen
(Zoorob et al., 1993). Controversy exists in the role of the Rfp-Y genes in Marek's disease
(MD) resistance or susceptibility (Bacon et al., 1996; Wakenell et al., 1996). The Rfp-Y
genes had no significant effect on MD tumors or viremia in Cornell lines N (MD-resistant)
and P (MD-susceptible) and their backcross; nor in Lines 6 (MD-resistant) and 7 (MDthe y3 haplotype Was
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In
susceptible) and their F2 progeny (Bacon et al., 996).
contrast,
associated with a higher incidence of Marek's disease tumors in an MHC-congenic (Ancona
background; Briles et al., 1993) chicken line (Wakenell et al., 1996). A relationship of the
Rfp-Y gene products to minor histocompatibility antigens has also been demonstrated by
Pharr and coworkers (1996). In this case, Rj'_o-Yincompatibility caused rejection of skin
i
effects
less frequent
than thatand
observed
withthan
classical
grafts atwere
a more
faster rate
Rfp-YB-incompatibility.
compatibility. However, the Rfp-Y
i
each group
consiststwo
of both
classes
I andgenes
II (Miller
et al., on
1994;
FIGURE 1).
The
Therefore,
groups
of MHC
are located
chromosome
16 in
theclassical
chicken Band
system contains B-FI, B-FIV, B-L[5I and B-L[3II loci whereas the Rfp-Y system consists of
I
Y-FV, and
Y-FVI,
Y-Lf3IV
Y-L_Vare
loci.
The classical
B system by
is the
linked
to theWhether
Class IV
genes,
bothY-L_III,
the B and
Rfp-Yand
systems
suggested
to be separated
NOR.
the Rfp-Y genes are classical or non-classical MHC genes is not clear. Due to the polymorphic
I
nature
of the
the B-Lf_
Rfp-Yand
genes
and their minor
differences
from the
B region
homology
between
Y-L[5families;
Zoorob
et al., 1993),
Rj_-Y genes
genes (82%
probably
represent a
distinct isotypic family that is part of the classical chicken MHC repertoire. Interestingly,
I
Zoorob and coworkers (1993) identified a third isotypic family of B-L[3 genes, B-LfSVI. Whether
the B-L_VI genes are expressed and from which system (B or Rfp-Y) they are derived are not
known.
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Lastly, there is a strong linkage disequilibrium of the chicken MHC genes
(Simonsen et al., 1980). That is, certain alleles within the B region tend to segregate as a
unit,
independently.
rather than
This phenomenon is due to the low frequency of
recombination among the loci at the MHC. For instance, there have been no identifiable
recombination events between the B-F and B-L subregions, whereas the frequency of
recombination between
the B-G and B-F/B-L subregions is estimated to be less than .05%
(map distance is less than .56 centimorgans) (Koch et al., 1983; Hala et al., 1988).
Because there have been very few recombination events between MHC subregions, the
of alleles
all loci within the B region
term "haplotype" is used to refer to the combination
at
on a single chromosome. In the chicken, there are over 30 B-haplotypes, thus indicating
the polymorphic nature of this complex (Briles et al., 1982; Heller et al., 1991). Similarly,
at
there
Rfp-Y haplotypes are based on their Class I and II composition. Currently,
are
least 11 R/))-Y haplotypes that have been reported in chickens (Briles et al., 1993;
Wakeneil et al., 1996: JuuI-Madsen el al., 1997).
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BROILER MHC GENES
Although analysis of chicken MHC haplotypes in White Leghorns and experimental
chicken lines has progressed steadily, little research emphasis has been placed on the MHC
haplotypes which segregate in meat-type or broiler chickens. Because broiler chickens are
mostly derived from the White Plymouth Rock and White Cornish breeds, it is expected that
broiler chickens would have additional unique MHC haplotypes. In Denmark, Simonsen
(1987) attempted to identify MHC haplotypes in a White Cornish flock and other broiler
populations using hemagglutinating reagents which were developed for standard White
Leghorn haplotypes. However, these attempts were reported as futile, with many of the
broiler chickens reacting non-specifically with one or more of the reagents. The only
13
.
13.
exception was some B typing reagents, where the B
haplotype was found segregating at
low frequency in the flock of White Cornish chickens. Simonsen (1987) also identified
21
130
133
130
B -like haplotypes which were provisionally designated as B - B . Both the B and
B 131haplotypes were indistinguishable from B 21 based on the graft-versus-host
reaction
21
(GVHR); however, each of these haplotypes only reacted with the B-F reagent, but not the
B-G21reagent. In addition, the Bs30and BI31haplotypes are serologically distinct from each
other. In contrast, B 132was serologically similar to B 13°,but yet a GVHR could be induced
between these two haplotypes. Whether the B 13°and B 131haplotypes confer Marek's disease
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1
resistance similar to that of their B2_homologue in White Leghorns was not determined.
In another study, Heller and coworkers (1991) examined the MHC haplotypes in
broiler chickens that were selected for either early high or early low antibody responses to
Escherichia coli and Newcastle disease virus (NDV). Using a panel of standard blood
typing reagents developed in White Leghorns, they identified 12 antisera out of 27 that
displayed differential hemagglutination reactions in the broiler chickens. Once again,
"provisional" haplotypes were designated for the MHC specificities because it was not
known whether the typing reagents reacted with antigenic epitopes fully identical to those
in White Leghorns. However, based on the hemagglutination patterns, at least five MHC
specificities (B5, B 13, B15,B 19and B°) were identified in this broiler population.
Interestingly, the B° haplotype was identified by its non-reactivitY with the White Leghorn
typing reagents. Of importance, the B5 haplotype was associated with early high antibody
response to E. coli and NDV, whereas the B 15and B 19 haplotypes were markers for early
low antibody responsiveness. At least 40% of the early high antibody responders were of
the B° haplotype. Because the B° haplotype did not react with any of the White Leghorn
typing reagents, it may represent one or more undescribed broiler chicken MHC
haplotypes that are associated with high antibody response (Heller et al., 1991).
Molecular analysis of two broiler MHC haplotypes, designated B A4 and B A4variant
indicated that some of the MHC genes are shared between broilers and Leghoms (Li et al.,
1997). In this study, the B A4 haplotype had similar B-F and B-LfllI gene sequences to that
of the B2zhaplotype; however, its B-G genotype was distinct. Although the BA4"ariant
hapiotype had the same B-G molecular genotype as BA4,the two haplotypes could be
distinguished serologically. This may be due in part to different B-F genes in these
haplotypes. Both the B-F and B-L_ sequences were distinct between these haplotypes and
the B A4variant sequences were unique to broilers. More recent research from the same group
_)()
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focused on additional MHC haplotypes in a broiler chicken line (Li et. al., 1999). In this
!
study,
they Not
compared
B-F
andidentify
B-Lf5 sequences
from alleles,
broilers but
thatthey
were
homozygous
at the
B system.
only did
they
unique broiler
also
observed alleles
that were common to both broilers and White Leghorns. Interestingly, some of the B-F and
I
B-LI3
sequenceswith
thatdifferent
are common
broilers
andbroiler
Whiteline.
Leghorns were found in linkage
disequilibrium
B-G to
alleles
in the
I
In characterized
a separate study,
the MHC haplotypes
in a commercial
meat-type
line were
by restriction
fragment length
polymorphism
(RFLP)grandparent
analysis
using a Class IV DNA probe (Uni et al., 1992). At least 23 polymorphic PvulI
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restriction fragments, ranging in size from 1.1 to 5.2 kb were identified in this
population. These fragments identified different RFLP haplotypes and each haplotype
consisted of between 4 and 16 restriction fragments. Discrimination between
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homozygous and heterozygous RFLP patterns was not performed in this study.
However, in a separate study, this research group further investigated two chicken lines,
originally derived from the commercial line and divergently selected for early or late
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antibody response to E. coli (Landesman et al., 1993; Uni et al., 1993). Fourteen
homozygous RFLP haplotypes were identified in these birds, and five of these
haplotypes were found either in the low antibody response line or in the high antibody
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response line. This research provides the first direct evidence that broiler MHC
haplotypes also influence the immune response to disease organisms. In addition, a
relationship of broiler MHC haplotype and egg production has also been reported
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(Tarleton et al., 1994). Therefore, characterization of MHC haplotypes will be of value
to broiler breeding programs especially where selection for immune response and disease
resistance are of importance. Association of certain broiler MHC haplotypes with
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production traits may also provide a selective advantage.
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MOLECULAR GENOTYPES IN A COMMERCIAL BROILER POPULATION
We are currently characterizing the MHC haplotypes in a commercial broiler
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chicken population. Our laboratory has obtained three DNA probes which identify chicken
MHC Classes I (Dr. Henry Hunt, Avian Disease and Oncology Laboratory, East Lansing,
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II andIIIV
MI),
(Xu
probes
et al.,to1989)
determine
and IVthe(Goto
restriction
et al., fragment
1988), respectively.
length polymorphism
We have used
(RFLP)
the Class
patterns in pedigreed families of Lines A and B. In this study, DNA samples were digested
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22
The enzyme,
PvulIPvulI
was and
chosen
for RFLP analysis
because
it gave
withhours.
the restriction
enzyme,
electrophoresed
in a 0.8%
agarose
gel distinct
at 30 volts for
polymorphisms without loss of resolution in Southern blot analysis. The DNA was
I
transferred
nylon membranes
(Hybond
N+, blot
Amersham
Life(Sambrook
Sciences, et
Inc.,
Heights, IL)tofollowing
the standard
Southern
procedure
al.,Arlington
1989).
Prehybridization, hybridization and stringency washes were described previously (Emara et
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al.,
RFLP data
have
been(1-7)
collected
a total of 3)
293
birds. At least
four
(a-d)1992).
Class The
11(TABLE
2) and
seven
Class from
IV (FIGURE
homozygous
molecular
genotypes (RFLP patterns) are segregating in Lines A and B. The frequencies of the
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molecular genotypes in the two broiler lines are presented in FIGURE 2 (Class II) and
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FIGURE 4 (Class IV). Genetic variation at the MHC between these two lines is apparent.
The Class II "a" allele is in highest frequency in both lines; however, the frequencies of the
other Class II alleles are different between the two lines and in fact, the Class II allele "b"
was not observed in Line B. In contrast, the Class IV alleles are unique to each broiler line.
The Class IV alleles, "3-7" were only observed in Line A, whereas alleles "1-3" were only
m
iI
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observed in Line B. Co-segregation of the Class II and Class IV molecular genotypes have
resulted in the identification of eight molecular MHC haplotypes in this broiler population.
SEROLOGICAL
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ANALYSIS OF BROILER MHC HAPLOTYPES
To facilitate rapid identification of broiler MHC haplotypes, a study was initiated to
produce broiler chicken alloantisera within this population. Reciprocal full- and halfsibling alloimmunizations (n = 36) were established for the production of alloantisera.
Sibling immunizations were used in order to minimize differences at minor blood group
antigens. Briefly, 1.5 ml of donor blood was collected in 1.5 ml sodium citrate solution
and then, samples were injected into the brachial vein of recipients. Chickens received two
injections (once per week for 2 weeks) and blood was collected 5 days after the second
immunization for harvesting of serum. Of the 36 recipients, 22 birds responded to
alloimmunizations with titers of four or greater. Fifteen of these 22 antisera gave
differential hemagglutination reaction patterns among the broiler chickens that were tested.
We have identified five of these antisera, in addition to five antisera kindly supplied by Dr.
W. Elwood Briles that appear to react with certain Class IV molecular genotypes. Analysis
of Class I (B-F) genotypes is in progress and will facilitate further characterization of these
alloantisera. Due to the cross-reactivity of the alloantisera with public MHC epitopes, it is
apparent that a panel of antisera will be necessary to differentiate the MHC haplotypes in
broilers. And, as demonstrated by Li et al. (1999), the presence of B-F and B-G alleles
common to broilers and White Leghorns should make it possible to identify pre-existing
standard "B" reagents that will be useful in typing at the MHC in broiler chickens.
Alternatively, alloantisera produced within the broiler population will also be useful
reagents and this research is ongoing.
i
•
1
•
1
•
1
1
•
1
!
MAPPING OF AN MHC-LIKE GENETIC SYSTEM
TO CHICKEN CHROMOSOME
1
During our molecular genotyping studies in pedigreed broiler families, we identified an
MHC Class II-like system that segregates independently of the classical MHC (B region) and
Rfp-Y system. Hybridization of Pvull-digested genomic DNA with a B-LI3II (B region; Class
II) probe revealed two groups of DNA bands, which segregate independently of each other.
Group 1 was identified as the classical MHC (B) region genes due to their intense hybridization
with the B-LI3II probe, whereas group 2 consisted of weakly-hybridizing DNA bands. A BLI3III probe (Rfp-Y) hybridized to the group 1 (B-region) DNA bands; however, it did not
hybridize to the group 2 DNA bands. Therefore, the group 2 DNA bands were considered to be
part of a separate system, which we have temporarily named DELO001. Segregation data for
the three Class II regions (/3, RJp-Y and DELO001) in a pedigreed broiler family are presented in
,,2
i
i
I
1
1
iIIE
i
!
I
!
!
TABLE 3. It was obvious in the segregation data from pedigreed families that we were
I
molecular genotypes
identifying
an independent
that aresystem.
based on
To digestion
date, we have
of chicken
identified
genomic
three DNA
homozygous
with the DELO001
restriction
enzyme, PvulI (FIGURE 5).
I
Based on the conclusion that DELO001 was independent of the classical B and
R_o-Y systems, we conducted two separate mapping studies to localize this MHC-like
I
system
in the
poultry
genome. To(no.
determine
whether
DELO001
located
the MHCand Rfp-Y
bearing
chromosome
16), genomic
DNA
samplesisfrom
the on
three
genotypes segregating in the UCD-Trisomic line were analyzed. Disomic, trisomic and
I
tetrasomic
andMHC4 copies,
of chromosome in
16.chickens
This
line is idealindividuals
for mappingcontain
genes 2,to 3the
and respectively
Rj_o-Y-bearing
via the detection of a proportional increase in the hybridization intensity of DNA bands.
I
Therefore,
and tetrasomic
was digested chicken
with PvulI
analyzed ingenomic
SouthernDNA
blots from
usingdi-,
thetriB-LI3II
probe. Thebirds
UCD-Trisomic
line and
was
found to be monotypic for allele 1 (2.2 kb PvulI fragment). As expected, there was a
I
proportional
16-related
the triintensity
of classical
B-region
DNA
bands (Groupchromosome
1 DNA bands;
2.8 and increase
4.3 kb) inindi-,
and tetrasomic
birds,
respectively.
In contrast, there was no observable difference in the intensity of the DELO001 DNA
I
band
(2.2 kb)
among di-,
tri- intensity
and tetrasomic
respectively.
Similarly,
there wasamong
no
observable
difference
in the
of a 3.2birds,
kb HindllI
DELO001
DNA fragment
di-, tri- and tetrasomic individuals. Therefore, it was concluded that DELO001 was not
I
located on chromosome 16.
Next, we analyzed DNA samples from the East Lansing reference population in
I
order to map DELO001 to a particular chromosome or linkage group in the chicken genome.
The East Lansing reference population was derived from a Jungle Fowl (JF) X White
Leghorn (WL) cross, and it is described elsewhere (Crittenden et al., 1993). Genomic DNA
I
samples from JF, F1 males, four WL females and 52 backcross (BC1) progeny were used in
the linkage mapping studies. Initial RFLP studies with the restriction enzyme, PvulI
indicated that the JF and WL chickens were monotypic for allele 1. However, subsequent
I
studies revealed that the 3.2 kb HindlII fragment of allele 1 was present in the WL females
and the 1.9 kb HindlII fragment of allele 2 was present in the JF. Therefore, HindlII was
used in Southern hybridizations with the B-L[3II probe to genotype the BC1 progeny in the
I
East Lansing reference population. Segregation data that were based on the inheritance of
the JF (1.9 kb) and WL (3.2 kb) DELO001 alleles in the fifty-two BC1 progeny indicate that
i
DELO001 is located on the p-arm of chromosome 1 (FIGURE 6).
This mapping data has proven to be of significance for two reasons. First, a
I
indicates
thatmapping
other immune-related
genes such
Ly49 (natural
killerLDHB
cell receptor),
NKI
comparative
to the mouse genome
withasclosely
linked loci,
and GAPD,
(natural killer cell-associated antigen), CD69 (very early activation antigen) and CMVI
I
Database (MGD), resistance
(cytomegalovirus
Mouse Genome
! ) are located
lnformatics,
in this1999]
syntenic
(FIGURE
group 6).
[Mouse
And Genome
secondly, in a
separate finding, Bumstead and coworkers (1998) mapped a quantitative trait locus (MDV-
I
1) for Marek's disease resistance to the same region of chicken chromosome I. The
•
identity of DELO001 remains unknown; future research will focus on cloning and
characterization of the DELO001 genes.
Thus far, the chicken MHC loci are reported to reside in two regions on
chromosome 16; the classical B and Rfp-Y regions, both of which contain Classes I and II
loci. Our data demonstrate that a third MHC or MHC-like system exists in chickens and it
contains at least a Class II-like gene. Fine mapping of the region of homology between the
Class II (ccli-7-1; Xu et al., 1989) probe and DELO001 has revealed that the homology lies
in the 5' promoter region of the Class II probe. Thus, the portion of the probe that encodes
the Class II protein does not hybridize to DELO001 and this may explain why the B-LI3III
eDNA probe did not identify this system. Interestingly, we have also identified some Class
i restriction fragments that segregate with DELO001. However, in this case, the Class I
probe is a eDNA probe and our current hypothesis is that the DELO001 system may be a
Class I-like gene with a promoter region that is conserved with the Class II (B-LI3II) gene.
Much like that of the mammalian non-classical MHC genes, the chicken DELO001 system
exhibits limited polymorphisms, with only three molecular genotypes that were identified
in 10 chicken lines studied. Whether this MHC-like system is functional and whether it
contains classical or non-classical MHC genes, remains to be determined. As members of
the immunoglobulin supergene family, these MHC-like genes may prove to be important
genes in NK cell activity, or perhaps be related to the MDV1 locus (responsible for Marek's
disease resistance) mapped by Bumstead and eoworkers (1998).
REFERENCES
!
!
m
n
•
m
ll
m
m
l
•
m
n
m
•
I
I
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•
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_)_
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xu,
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I
I
TABLE 1. CHARACTERISTICS
OF CLASSICAL
HISTOCOMPATIBILITY
COMPLEX GENES.
AND NON-CLASSICAL
MAJOR
•
CLASSICAL MHC GENES
NON-CLASSICAL
MI-IC GENES
l
-') Classes I and II
--) Primarily Class I-like; a few Class II-iike
-) Several (1-3) functional loci
-)
Exhibit all or none of the classical MHC traits
•-) High levels of expression
-)
Low levels of expression
-) Broad tissue distribution
•.) Conserved structural features
-') Limited polymorphisms
-) Found within and external to the MHC
1
•
-) Peptide binding/presentation function
") Highly polymorphic (many alleles/locus)
•
l
From Kasahara et al., 1996.
•
l
I
I
B system
B-G gene clusters
Cluster 1
s
s
s
Rfp-Y system
s
s
NOR
Cluster HI
X
_
_
s S
_
_
s s
'
B-FI & B-FII
B-LI3I & B-LI31I
_
_
III (C4)
=> Class
non-MHC
(TAP 1, TAP2, tapasin, C-Type
lectin, iectin-like natural killer cell
Cluster II/IV
I
I
I
I
I
I
I
I
I
I
I
I
I
=_ Y-FV & Y-FVI
_ Y-LI31II to Y-LI3V
_ non-MHC
gene:lectin)
C-Type (17.5
animal
receptor,
histone H3-1ike
RING3, Leu-tRNA
gene) gene,
FIGURE
model
the Genbank
chicken Accession
B and RFP-Y
systems.
(Adapted
from Miller!. elHypothesized
al., 1994; Miller,
1995;ofand
number
AL023516).
I
I
I
I
I
I
I
I
I
I O0
I
!
!
I
TABLE
2. RESTRICTION
FRAGMENT
LENGTH
POLYMORPHISMS
WITH CLASS
II MOLECULAR
GENOTYPES
IN BROILER
CHICKENS.ASSOCIATED
I
Restriction Fragment
(k_b_)
i
a
Class II Molecular Genotvpes
b
c
6.1
+
5.8
l
+
4.3
+
+
3.1
I
d
+
+
2.8
+
+
!
I
I
0.8
0.7
Z
0.6
xxx
_xx
0.5 0.4
xxx
xxx
_xx
I
_
I
__o_ ...
I
I
<
0.1
0
a
LINE A
b
c
LINEB
d
a
b
c
d
CLASS II MOLECULAR GENOTYPES
i
Southern
blotFour
analysis
commercial
broilerwere
lines.
The Class
FIGURE 2.
ClassinIItwo
molecular
genotypes
identified
by II
homozygous genotypes (alleles) are designated a, b, c and d, as
I
indicated
in Table
alleles
lines, except
allele 2.
"b"All
which
waswere
not observed in the
Linetwo
B. broiler
I
I
I_Jl
I
HOMOZYGOUS
1
2
3
I
4
5
6
4/7
I
k__bb
4.2
- 3.44
I
2.6
I
I
- 2.4
I
|
FIGURE 3. Class IV molecular genotypes in a broiler
population. Southern blot analysis was conducted on 35
pedigreed dam families (n=293). At least six homozygous
genotypes (alleles) were identified and a seventh genotype was
only observed in heterozygous condition,
I
II
m
m
I
!
LINE A
LINE B
0.S
0.5
I
0.3
O.2
0.3
0.2
I
0.I
0.4
0
I
o.1
0.4o
_
3
4
5
6
7
1
2
3
CLASS IV ALLELES
I
i
FIGURE 4. Seven Class IV molecular genotypes were identified by Southern
m
blot analysis in two commercial broiler lines. The Class IV homozygous
genotypes (alleles) are designated 1 to 7. Unique Class IV genotypes were
observed for each broiler line.
1
I
I
I02
I
I
i
TABLE
I
3. SEGREGATION
IN A PEDIGREED
BROILER
OF B (B-LI31I), RFP-Y
AND DEL0001
ALLELES
FAMILY.
GENOTYPE
I
I
SIRE
MHC (B-LI31I) I
ab
RFP-Y 2
215
DELO001 _
2/2
DAM
ab
2/2
1/1
Progeny
1
aa
215
1/2
I
Progeny
2
aa
2/2
1/2
i
Progeny
Progeny
34
ab
ab
2/5
2/2
1/2
1/2
I
Progeny
Progeny
5
6
bb
bb
212
215
1/2
1/2
I
tClass II (B-L_H) genotypes are the same as those described in Table 2.
2Rfp-Y genotypes were kindly assigned by Dr. Marcia M. Miller, City of Hope,
Beckman Research Institute, Duarte, CA.
3DELO001
genotypes
are the same as those described
in Figure 5.
I
I
ASSOCIATED
RESTRICTION
I
G EN O T Y PE
l
DEL0001
i
(allele
PvuIl
WITH
DELO001
FRAGMENTS
H indIII
2.2 kb
3.2
kb
2.0 kb
1.9 kb
3.0 kb
Not
determined
1)
DELO001
I
(allele
2)
I
DELO001
(allele 3)
I
FIGURE 5. Molecular genotypes associated with the chromosome
1
MHC-like genetic system, DELO001. At least three DELO001 alleles
I
have been identified
I
in chickens.
103
I
I
Mouse Ch. 6
.,-_.,o,..0,,
; _
14)
_
HA
157
LGAI_
'"
,- :_
"' :_
_
_,--
IA°_I_T
'T_om
lt.ml,4.1
I
133
•
I
T
_
.,
u..,.,_tDEL0001
I'_'_u]"-,o_,,o
I'_m'°"7
I * MDV-1
,..c__I i
_
I_l
.....
,._-,.,°I=
i_
I I
ii
I
_,,2 ±_,_ Mc_,,,-,_,m_
CMV-I
}
Ly49
• ............................................................................................
• ,///// LDHB
•
l
I
,////J
145U0517
-,
_
259
[J_OlOI
I
l.lo?T
I_-,1 I,--_1=
• ............................................................................................
•
_///_ G_°D
FIGURE 6. Partial genetic map of chicken chromosome 1. The MHC-like system
(DELO001) was mapped to the p-arm of chromosome 1 with closely-linked loci,
glyceraldehyde 3-phosphate dehydrogenase (GAPD) and lactate dehydrogenase
(LDHB). Mouse chromosome 6 contains the syntenic or homologous group of genes
(GAPD and LDHB). Also located on mouse chromosome 6 include genes involved in
NK cell activity (Ly49 complex) andcytomegalovirus resistance (CMV-1). Mapped to
chicken chromosome 1 in the same area as DEL0001 is MDV-1, a QTL that confers
resistance to Marek's disease (Bumstead et al., 1998).
|
I
•
1
•
•
I
I
I
I
I
I
I
I()4
I
I
1
I
DISCUSSION
Larry Bacon:
I
QUESTIONS
AND RESPONSES
i believe there is further verifcation
of B-LI3II types being identified
between broilers and White Leghorns that you may want to verify in publication.
Yes. You are probably referring to the paper in immunogenetics that was just released and
I
includes
research
on broiler
MHC haplotypes
fromB-LfllI
Sandragenes
Ewald's
laboratory
(Li et
1999). These
researchers
observed
both B-F and
that were
common
to al.,
broilers and White Leghorns. They also noted that some of the common B-F and B-LflI1
I
genes were in linkage disequilibrium with different broiler B-G genes, when compared to
the White Leghorn B-G genes.
I
Elwood Briles: Your progress is an excellent example of the new discoveries that can
I
result from collaboration between researchers.
I
Yes, this has been a large group effort with expertise and resources from UC Davis
J.
East
D.
Iowa State
(M. Delany);
University (S. Lamont); USDA,
Lansing (L.
Bacon,
H. Cheng); City of Hope, Beckman Research Institute, Duarte, CA (M. M. Miller); and last,
but not least, yourself (Dr. Briles) for contributing antisera for testing of the broiler MHC
I
haplotypes and your endless time and suggestions. This collaboration originated from a
couple of the regional projects (NE-60 and NC-168) and demonstrates the importance of
i
these federally-funded projects.
I
Dominic Eifick: Have you looked for homology with the DELO001 gene in other
species.
I
We have narrowed the region of homology between the Class II (ccli-7-1) probe and
DELO001 to a 300 base pair region in the promoter region of the Class II gene. We have
I
(except with
submitted
thisitssequence
own sequence,
to Genbank
ccli-7-1).
(Blast)We
and
didthere
see 20-35
really base
wasn'tpair
anymatches
strong with
homology
some
odd genes including the mouse cellular retinoic acid binding protein and fibroblast growth
I
factor. If we consider
mouse
chromosome
6 that found
contains
the syntenic
group (the1), then we
chromosomal
region that
contains
similar genes
on chicken
chromosome
may want to consider such genes as Ly49, CD69, NK1 or CMV-I (FIGURE 6). There are
I
some interesting alignments of the 300 base pair fragment with these genes.
I
I
I
11_5