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The Jackson Laboratory, Bar Harbor, ME 04609
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several strategies have been used to attempt to characterize mHA and to clone the corresponding genes. This includes purification and sequencing of natural mHA peptides (1, 2, 3, 4, 5), screening of random peptide libraries (6, 7), and expression cloning (8, 9, 10). Positional cloning approaches have been limited to situations in which the genome from which the Ag is derived is smaller, as in the case of mitochondrial-encoded Ags (11), or when the region of the chromosome containing the mHA gene is well studied, as in the case of Y chromosome-encoded H-Y Ag genes (2, 12, 13). However, the systematic molecular characterization of the more numerous autosomal mHA genes remains elusive.
Of the autosomal mHA loci, the H3 locus on mouse Chromosome 2 is of historical significance (14). This locus is complex, in that it contains at least three genes that can elicit an immune response (15, 16, 17, 18). H3 spans 10 to 12 cM, and additional mHA genes have been reported to map to the complex (19, 20, 21). The H3a gene within H3 is of particular interest, as genetic differences in this gene can elicit cytotoxic immune responses after transplantation or immunization between H3-congenic mouse strains. These CTLs are directed against a peptide, encoded by H3a, presented on the H2-Db class I MHC molecule.
We have taken a positional cloning approach to identify H3a. High resolution genetic linkage studies have mapped H3a between D2Mit444 and B2m (17, 18), an estimated genetic distance of 1 to 2 cM. We report the construction of a physical map of YAC clones spanning this region. The use of YACs was advantageous for two reasons. One was to minimize the number of clones required to cover the relatively large region, and the second was to utilize established molecular genetics methods for modifying YACs in yeast to incorporate an expression screen for H3a. This screen exploits the fact that the H3a Ag can be assayed in mouse cell lines. Transfer of retrofitted YACs into mammalian cells can be used to identify a YAC clone that contains H3a.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The primary YAC library screened was the B6-derived
Whitehead/MIT library (22), purchased from Research Genetics
(Huntsville, AL). Other libraries were screened with specific markers
to fill gaps or to provide additional coverage. One was the B6-derived
library constructed at Princeton University (Princeton, NJ) (23). The
other library is a composite of a (C3H x B6)F1
library (24), and a B10 library (25). These latter two libraries have
been pooled and are distributed by the mouse YAC screening service at
Baylor College of Medicine (Houston, TX). YACs M3H6, M3H10, and M5G7
have been described previously (26). Libraries were screened by
successive rounds of PCR to identify YACs that carry desired
D2Mit loci and known genes. D2Mit primer pairs
were purchased from Research Genetics. Sequence tagged sites (STS) were
generated to genes known from high resolution mapping studies (18) to
map within the H3a region. B2m- and
Ltk-specific PCR primers have been described before (18).
Primers designed to other genes screened in this study are listed in
Table I
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Intact chromosomes were prepared from yeast strains containing YACs using an Imbed kit (New England Biolabs, Beverly, MA). Chromosomes were resolved by electrophoresis in a Bio-Rad (Richmond, CA) CHEF-DR III unit using the manufacturer’s recommended parameters.
Cloning and sequencing of YAC ends
Total DNA was prepared from yeast clones containing YACs (27).
YAC ends were cloned by inverse PCR following published procedures and
pYAC4 vector-specific oligonucleotide primers (28, 29). DNA sequencing
of gel-purified inverse PCR products was performed manually, after
[
-32P]ATP labeling of appropriate sequencing
primers, using the AmpliCycle sequencing kit (Perkin-Elmer, Norwalk,
CT), or by The Jackson Laboratory Microchemistry Service (Bar Harbor,
ME) using an Applied Biosystems (Foster City, CA) Model 373 Stretch
automated fluorescent DNA sequencer.
STS primer design and determination of chimerism
Genomic DNA sequences derived from YAC ends were compared with DNA sequences within GenBank by BLAST homology searches. Oligonucleotide primers, with predicted annealing temperatures of 55°C or greater, were designed to nonrepetitive regions. Normally, if the primers could amplify a DNA fragment of the expected size from B6 or B10 genomic DNA, and also from a hamster-mouse somatic cell monochromosomal hybrid that contains only mouse Chromosome 2 (EAS5-17c; 30 , this was sufficient to initially establish nonchimerism. No PCR product, or one the same size as the mouse genomic DNA-derived product, should be amplified from yeast and hamster genomic DNAs. In the case of any STS that mapped to Chromosome 2, but could only amplify DNA from the YAC from which it was derived, the STS was additionally mapped either on The Jackson Laboratory BSS backcross panel (31), or on a panel of (B10 x 129)F1 variants that demonstrate loss of heterozygosity (LOH) around H3a (18). If the STS mapped to the H3a region, then the YAC from which the STS was derived was considered nonchimeric at that end.
Simple sequence-length polymorphism, single-stranded conformational polymorphism, and Southern blotting
These techniques were performed as described previously (18).
YAC transduction
The method of Spencer et al. (32) was used to transfer YACs from the host strain AB1380 into the strain YPH925, to allow His+ selection in subsequent retrofitting experiments (see below). Individual YAC transductants were screened for all markers previously determined to be present on the YAC to ensure that YACs had been transferred intact.
YAC retrofitting
Selected YACs, containing either B6- or B10-derived DNAs, were
modified by retrofitting to introduce the mammalian selectable
NeoR gene into one of the two YAC arms, thus
permitting selection for YAC transfer into mammalian cells for
functional studies. In this context, retrofitting refers to process
whereby existing YAC clones are modified, by using the efficient
homologous recombination mechanisms of yeast cells, to insert the
Neo gene into the YAC arm. One of two plasmids was used to
retrofit YACs. Plasmid pRV1 (33) contains homology to the
ura3 gene present on the YAC arm, and carries the yeast
lys2 gene and NeoR gene. Retrofitting
of YACs with pRV1 was performed as described (33). YAC-containing
AB1380 yeast Lys+ transformants were screened for a
Ura- phenotype, as these were likely to have arisen as a
result of specific integration of the retrofitting plasmid into the YAC
arm. Because the lys2 mutation present within AB1380 reverts
high frequency, some YAC-bearing AB1380 strains were already
Lys+. In these cases, a second retrofitting plasmid
(obtained from Bruce Lamb, Case Western University, Cleveland, OH) was
used. Plasmid phis3PYF101neobpA (a derivation of pPol2sneobpA; 34
was used to transform YAC-containing YPH925 clones to His+.
YPH925 contains a chromosomal deletion in the his3 gene, and
because the plasmid carries the intact his3 gene, as well as
homology to the ß-lactamase gene present in the trp arm of
the YAC, all transformants arose through integration of the
ScaI-linearized plasmid into the YAC arm. Plasmids were
introduced into yeast cells by electroporation using a Bio-Rad Gene
Pulser and Pulse Controller. The manufacturer’s suggested procedures
were followed. The presence of the retrofitting plasmid-derived
Neo gene in yeast transformants was confirmed by PCR with
gene-specific primers (Table I
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Transfer of YACs into mammalian cells by spheroplast fusion
Yeast strains containing retrofitted YACs were grown in media selective for both the YAC-encoded ura and trp genes (in the case of YACs retrofitted with phis3PYF101neobpA), or for the trp gene only (in the case of pRV1-retrofitted YACs). Cells were spheroplasted and fused to Db-L cells (obtained from Larry Pease, Rochester, MN), as previously described (35). YAC-containing L cells were selected in DMEM media containing 500 µg/ml G418 (Life Technologies, Gaithersburg, MD). Typically, 10 to 100 clones were generated after 2 wk in culture. Usually three independent clones of each transfection were screened for expression of the YAC-encoded H3aa Ag.
CTL and CML assay
G418R Db-L cells or Con A-stimulated splenocytes were used as target cells in a standard CML assay (36). Effector cells were the H3aa-specific CTL clone C1 (15), and either the H47a-specific clone CTL3 (37) or a H3ab-specific B/L line derived from immunization of B10 mice with B10.LP-H3b splenocytes (Ref. 17; this manuscript). L cells express the H47a and H3ab Ags; therefore, cytolysis by either CTL3 or B/L provided a positive control. Percent specific lysis of CML assays was calculated as (51Cr release in the presence of effector - spontaneous chromium release) divided by (maximal chromium release caused by SDS addition - spontaneous release).
Flow cytometry
Ab staining of cells was performed as described (38). Relative immunofluorescence was analyzed on a Becton Dickinson flow cytometer FACScan (Sunnyvale, CA). Monoallelic Abs specific for ß2mb (S19.8; 39 and H-2Db (28-13-3; 40 were supplied from Flow Cytometry Service of The Jackson Laboratory (Bar Harbor, ME).
Database accession numbers
The nucleotide sequences of STS and YAC ends reported in this manuscript have been deposited with GenBank under the accession numbers AF037327, AF037453, and G36380–G36415.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1 shows the relative
positions of YAC clones that span the interval between
D2Mit444 and D2Mit17. Yeast clones containing
desired YACs were identified after screening of YAC libraries with
gene-specific primers (Table I) and D2Mit loci shown to map
within this region (18). Thirty-three YAC clones were recovered (Table II). An additional three YACs (M3H6,
M5G7, and M2H10), previously reported by Richard and Beckmann (26) to
contain the Capn3 gene (encodes the calpain 3 protease
subunit) and to be derived from this region of the genome, were
included in this study.
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). In
some cases, the libraries were rescreened to identify overlapping
clones positive for a specific STS.
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; Fig. 1). The nucleotide sequence of 14 of these
ends demonstrated no homology to any known gene or EST. For one end,
rescued from the left arm of YAC 12, we observed a 39-bp region of
sequence identity with the mouse Nnt NADP transhydrogenase
gene (EMBL accession number Z49204). The sequence of STS 12L
most likely identifies an exon-intron boundary within this gene.
Nnt maps to mouse Chromosome 13 (41), and as YAC 12 also
contains B2m, this chimeric YAC contains DNA derived from
Chromosomes 2 and 13. YAC 6 is also chimeric. The 471-bp cloned DNA fragment from the right arm of YAC 6 (GenBank accession No. AF037453) was mapped by Southern hybridization using a BglII restriction fragment-length polymorphism, on a panel of (B10 x 129)F1 somatic cell variants that demonstrate LOH on Chromosome 2 (18). All variants remain heterozygous at the STS 6HR locus, signifying that it probably maps to a chromosome other than Chromosome 2 (data not shown). The other end of YAC 6 was derived from the B2m gene. The sequence of the cloned 120-bp DNA fragment was identical to nucleotides 268 to 388 of intron 1 of B2m (GenBank accession No. M15535). YAC 6 was identified because it contains exon 3 of B2m, and subsequently the YAC was found to contain Trnah, a gene that is distal to B2m on the physical map. These data suggest that the genomic orientation of B2m is the same as the orientation of the chromosome, with transcription proceeding from centromere to telomere.
Of the 36 YACs recovered, only five nonchimeric YACs (1, 23, 33, 40, 45) have been positively identified. This number is likely an
underrepresentation of the actual number of nonchimeric YACs. Seven YAC
ends contained repetitive sequences that precluded the determination of
chimerism because they could not be mapped, and some YAC ends could not
be isolated. Therefore, for some YACs shown in Figure 1, only one end
has been characterized. If this end was deduced to be chimeric, then
the orientation of this YAC with respect to the other YACs in the
contig is unknown, and their alignment is thus ambiguous. In total, 23
new STS markers from the H3a region have been generated
(Table III). These new markers identify unique loci in that they do not
amplify DNA from a known gene or EST. These STS loci increase the
genetic resolution surrounding H3a.
The entire region from D2Dcr20 to D2Mit17 has been cloned, save for one putative gap between D2Dcr16 and D2Dcr22. D2Dcr16 is present on YAC 33 (from whence it was derived) and YAC 34, but not on YACs 45 and 47. D2Dcr22 (derived from YAC 45) is present on YACs 45 and 47, but not on YAC 34. As no informative markers could be generated from the ends of YAC 47, and because YAC 34 contains repeat sequences at both ends, it is not known whether these two YACs overlap.
Four YACs showed evidence of internal deletion. Three of these YACs (YAC 23, 45, and M3H6) did not contain one predicted STS locus. YAC 17 did not contain two predicted STS loci, D2Dcr10 and D2Dcr3. The extents of the deletions have not been further characterized.
Initial YAC characterization had involved the determination of the size
of the cloned DNA fragment within YACs. Whereas most yeast colonies
carried only a single YAC, two yeast clones, 131E11 and 183G8, harbored
two different sized YACs within the same cell (Table II). As 131E11 was
identified after screening for B2m-positive YACs, a Southern
hybridization was sufficient to establish that the smaller 150-kb YAC
(designated YAC 2) contained the B2m gene (data not shown).
Yeast 183G8 was isolated after screening for YACs carrying
D2Mit190. Two YACs of 450 and 570 kb were found within a
single yeast clone. These YACs were segregated from each other after
yeast 183G8 was mated with YPH925. D2Mit190-positive
transductants contained only the smaller 450-kb YAC (designated YAC
16). The fact that many of the YAC clones were either chimeric or
contained internal deletions, limits estimates of physical distances
between STS markers. Based on the sizes of nonchimeric YACs 1 and 23,
we can only effectively state that D2Dcr11 must be at least
685 kb from D2Dcr3.
YAC retrofitting and expression screening for the H3aa Ag
Transfer of YACs into mammalian cells allows one to rapidly screen for genes that confer a recognizable phenotype (42, 43, 44, 45). To identify which YAC carried H3a, we reasoned that transferring selected YACs into an H3aa Ag-negative mammalian cell line, and screening for the presence of the B6- or B10-encoded H3aa Ag would be a successful strategy.
L cells are H3ab-expressing C3H-derived fibroblasts. H3aa Ag expression has been observed in a large variety of cell types, including fibroblasts, and so L cells would be likely to express this Ag when fused to YACs containing the genomic H3aa gene. However, since CTL recognition of the H3aa Ag requires that the cells coexpress the H2-Db class I MHC molecule, a H2Db-transfected L cell line was used as our host cell for YAC transfer.
As some of the YACs in the contig could be C3H derived, and these would not be appropriate for an H3aa Ag expression screen in L cells, YACs derived from the Baylor library, and the three isolated by Richard and Beckmann (26), were typed with DNA markers that would distinguish between B6 (or B10) and C3H. YAC M3H6 is B6 (or B10) derived. The other YACs, M5G7, M2H10, 44, 45, and 47, are derived from the C3H mouse (data not shown).
To ensure YAC transfer and expression of a YAC-encoded gene by L cells,
we took advantage of the allele-specific Ab directed against the
ß2mb protein expressed from B6-derived DNA, but not from
the host L cell line (genotype: B2ma). YAC 1 carries
B2mb. It was retrofitted and transferred into
Db-L cells. Three independent clones were screened by Ab
staining for levels of ß2mb expression (Fig. 2). All three clones expressed
ß2mb on the cell surface ranging from 2.5- to 6-fold
times the parental Db-L cell control. The normal
distribution pattern of fluorescence in all three clones suggests that,
once integrated, the expression of YAC-encoded B2mb
is stable. Thus, we established that YAC transfer and expression of
YAC-encoded genes occurred efficiently.
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identifies other
B6- or B10-derived YACs that were retrofitted, transferred into
Db-L cells, and assayed for H3aa
expression. Only YAC M3H6 conferred antigenic activity. Two independent
clones arose as a result of M3H6 transfer into the Db-L
cells, and both were lysed by the H3aa-specific CTL clone
C1 at levels approaching mitogen-stimulated lymphoblast target cells
(Fig. 4). The parental Db-L
cells are not lysed by C1, but are lysed by the
H3ab-specific CTL line B/L, as expected.
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). Because M3H6 does not contain
D2Mit190, D2Dcr24 most likely maps between
Epb4.2 and D2Mit190. Based on this data, it was
expected that M3H6 should also contain Epb4.2. However, no
amplification was detected with Epb4.2-specific PCR primers.
Thus, M3H6 also contains an internal deletion. The extent of this
deletion is not known. Nevertheless, the expression data from a
retrofitted M3H6 clone suggest that this YAC does carry an intact
H3a gene, or at least enough of the gene to express the
peptide Ag. The internal deletion would also most likely eliminate
Epb4.2 as a candidate for H3a. Additional support
for this comes from the observation that retrofitted YACs 14 and 28 do
not express the H3aa Ag, even though they contain
Epb4.2. Therefore, H3a is most likely to map
proximal to this gene. M3H6 overlaps with YAC 45, M5G7, and M2H10
because they all contain exon 1 of Capn3. These three YACs
could not be screened for expression of H3aa, because they
are C3H derived. The 3' end of Capn3 is present on M3H6 and
M5G7, but not on YAC 45 or M2H10 (Fig. 5). This defines one of the ends
of YAC 45 (D2Dcr21) and M2H10 as being between
exon 1 and exon 19 of this gene. D2Dcr21 is not homologous
to the mouse Capn3 cDNA sequence and, therefore, is likely
to map within an intron of this gene.
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). Thus, Tyro3 can be
eliminated as a candidate for H3a. YAC 50, M2H10, and YAC 45
contain the 5' and 3' ends of Tyro3, and are likely to carry
the whole gene. M5G7 carries only the 3' end. This YAC also carries
Capn3. These data, and those deduced from the STS content of
YAC 45 and M2H10, suggest that Tyro3 and Capn3
are orientated similarly on the chromosome with the direction of
transcription of both genes proceeding from centromere to telomere. YAC
45 also contains Ltk and D2Mit395 (Fig. 1). It
does not, however, contain D2Dcr23 (Fig. 5). Thus, YAC 45,
despite being a nonchimeric clone, also contains an internal deletion
of unknown extent. Genetic confirmation of the location of H3a
H3aa Ag-loss variants have been generated after in vitro immunoselection with H3aa-Ag-specific CTL (17). The parental line from which these variants were derived was a (B10 x 129)F1 pre-B cell line that expresses the H3aa Ag heterozygously. Many of these variants demonstrate LOH at loci surrounding H3a, and this has provided useful information on the fine mapping of H3a with respect to flanking DNA markers (17, 18). Variant 2A12, however, did not demonstrate LOH for any marker tested. We reasoned that perhaps 2A12 contained a much smaller region of LOH, and the genetic resolution with the markers tested was not sufficient to identify this region. Therefore, we analyzed this variant in greater detail.
The analysis of YAC M3H6 suggested H3a maps in close
proximity to Capn3 (Figs. 4 and 5). To determine whether
2A12 carried a LOH in this region, we typed 2A12 at Epb4.2,
Capn3, and Ltk. No polymorphism was observed
between B10 and 129 for Tyro3 and, hence, this locus could
not be typed. Variant 2A12 demonstrated a LOH at Capn3, but
not at any other marker (Fig. 6). Because
this LOH is causally related to the lack of expression of the
H3aa Ag (17), the LOH provides genetic proof that
H3a also maps in close proximity to Capn3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We developed a novel assay whereby retrofitted YACs were transferred into mouse Db-L cells, and YAC-containing mouse cell lines were then screened for lysis after coincubation with H3aa-specific CTLs. The technique was valuable in that it allowed the rapid mapping of H3a from a large genomic region, without the necessity of generating and analyzing higher resolution genetic linkage maps. By systematically retrofitting and expression screening 14 YACs that covered the majority of the H3a region, we identified a single YAC clone, M3H6, that carries H3a. Others have reported the use of an expression screen in refining the location of desired genes within larger YAC contigs (43, 44, 45). We show that this approach can be extended to include the screening of genes that encode Ags recognized by T cells, and that considerable physical distances can be covered. The localization of H3a to the region of DNA covered by YAC M3H6 has been confirmed genetically, because of the discrete LOH observed at Capn3 in the H3aa Ag loss immunoselected variant 2A12.
The gene order determined from this region of mouse Chromosome 2 is in perfect agreement with that determined from a physical map of the syntenic region on human Chromosome 15q (46, 47), and the placement of the H3a-containing YAC M3H6 within the physical map eliminates many genes as H3a candidates. H3a maps in close proximity to Capn3, a gene that encodes a member of the calpain protease family. The close proximity of these genes suggests that they could be the same. However, published reports on the expression pattern of Capn3 suggest that it is muscle specific (48, 49, 26). In contrast, expression of H3a is observed in a wide variety of tissues, including skin, lymphoid-derived T and B cells, myoblasts, and fibroblasts. These data suggest that Capn3 is unlikely to encode the H3aa Ag, and that H3a is an unknown gene contained within the Tyro3 to Epb4.2 region.
Additional genes from the human region have been identified, primarily as ESTs expressed in human muscle, and subsequent coexpression of a subset of these genes in other tissues (47). Some of these ESTs map in close proximity to Capn3. Examination of the human transcript map for 15q21 (http://www.ncbi.nlm.nih .gov) indicates that, to date, 185 cDNA transcripts have been mapped in close vicinity to CAPN3. The majority of these are "unidentified" in that they are not homologous to any known gene. Although some of these cDNA fragments are likely to be derived from the same gene, the abundance of ESTs mapped to this small region of the chromosome suggests a moderate to high gene density surrounding H3a.
The ability to retrofit and routinely transfer YAC clones, identified as a result of completion of the human and mouse physical maps, into mammalian cells should allow one to efficiently bridge the gap from physical map to gene, and could be applied to the positional cloning of any gene whose expression can be assayed in cell lines. YACs have been transferred into a number of different human and mouse cell types in addition to murine L cells. These include embryonic stem cells, embryonic carcinoma cells, renal carcinoma cells, and Chinese hamster ovary cell lines (35, 45, 50, 51, 52). There are conflicting reports on the effects of YAC size on transfer into mammalian cells. Gobin et al. (53) suggest that YAC size is not limiting for transfers involving spheroplast fusion. However, Blunt et al. (43) find that smaller YACs undergo fewer rearrangements as a result of YAC transfer than larger ones. Nevertheless, we found the fusion approach to be reliable in that YACs carrying B2m or H3a reproducibly conferred strong and stable Ag expression in mouse L cells. We never observed Ag expression from YACs that were derived from other regions of the contig. In light of the real possibility of rearrangements and internal YAC deletions, a prudent approach in expression screening of this type would require testing multiple independent and overlapping YACs if at all possible. The use of CTLs specific for mHA in expression screening of retrofitted YAC clones toward the positional cloning of clinically significant human and mouse mHA genes is just one application for this technology. T cell clones can be generated to autoantigens that are responsible for, or contribute to, an array of autoimmune diseases, including autoimmmune diabetes and multiple sclerosis, thus making the genes encoding these Ags amenable to a positional cloning approach.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. R. Zuberi. The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609. E-mail address:
3 Abbreviations used in this paper: mHA, minor histocompatibility antigen; CML, cell-mediated lympholysis; EST, expressed sequence tag; LOH, loss of heterozygosity; STS, sequence tagged site; YAC, yeast artificial chromosome.
Received for publication January 20, 1998. Accepted for publication March 20, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
. Open circles at the ends of some YACs
signify that the DNA is repetitive and, hence, uninformative. Box
symbols at YAC ends identify chimerism. Boxed areas within YACs signify
the absence of STS markers expected to be present, and identify an
internal deletion within the YAC. Ends of YACs without symbols indicate
that their origin was not determined.
) and the
H3ab Ag-specific CTL clone B/L (symbol: o). The effect on
target ratio is shown on the x axis. Target cells in A and
B are two independent clones of H2-Db-expressing
L cells containing YAC M3H6. In C, the target cell is the
parental H2-Db-expressing L cell line. Target cells in
D and E are Con A-treated splenocytes of B10
(genotype: H3aa) and B10.LP-H3b
(genotype: H3ab) mice, respectively.
