An ordered BAC contig map of the equine major histocompatibility complex

A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.     

A physical map of a BAC clone contig covering the entire autosome insertion between ovine MHC Class IIa and IIb

ABSTRACT: BACKGROUND: The ovine Major Histocompatibility Complex (MHC) harbors genes involved in overall resistance/susceptibility of the host to infectious diseases. Compared to human and mouse, the ovine MHC is interrupted by a large piece of autosome insertion via a hypothetical chromosome inversion that constitutes ~25% of the ovine chromosome 20. The evolutionary consequence of such an inversion and an insertion (inversion/insertion) in relation to MHC function remains unknown. We previously constructed a BAC clone physical map for the ovine MHC exclusive of the insertion region. Here we report the construction of a high-density physical map covering the autosome insertion in order to address the question of what the inversion/insertion had to do with ruminants during the MHC evolution. RESULTS: A total of 119 pairs of comparative bovine oligo primers were utilized to screen an ovine BAC library for positive clones and the orders and overlapping relationships of the identified clones were determined by the DNA fingerprinting, BAC-end sequencing, and the sequence-specific PCR. A total of 368 positive BAC clones were identified and 108 of the effective clones were ordered into an overlapping BAC contig to cover the consensus region between ovine MHC class IIa and IIb. Therefore, a continuous physical map covering the entire ovine autosome inversion/insertion region was successfully constructed. The map confirmed the bovine sequence assembly for the same homologous region. The DNA sequences of 185 BAC-ends have been deposited into NCBI database with the access numbers HR309252 through HR309068, corresponding to dbGSS ID 30164010 through 30163826. CONCLUSIONS: We have constructed a high-density BAC clone physical map for the ovine autosome inversion/insertion between the MHC class IIa and IIb. The entire ovine MHC region is now fully covered by a continuous BAC clone contig. The physical map we generated will facilitate MHC functional studies in the ovine, as well as the comparative MHC evolution in ruminants.     

Physical mapping of the major histocompatibility complex class II and class III regions of the rat

A contig of overlapping bacterial and P1-derived artificial chromosome (BAC, PAC) clones derived from the inbred rat strain BN was constructed that encompasses the class II and the class III regions of the rat MHC (RT1 complex). The genomic structure of the rat, human, and mouse class II and class III regions is highly similar. However, different from human and mouse, a copy of the C4, Cyp21, and Stk19 genes is found that maps to the class II region in the rat. Gene trees constructed from human, rat, and mouse C4, Cyp21, and Stk19 sequences show species-specific clustering of the duplicated genes. The class II/III contig reported here links two previously published PAC contigs of the BN rat that contain the centromeric and the telomeric class I regions, RT1-A and RT1-C/E/M, respectively. Thus, the MHC of the rat is now completely mapped in a single contig of BAC/PAC clones derived from a single RT1 haplotype and encompasses about 3.7 Mb.     

Closing a gap in the physical map of the ovine major histocompatibility complex

A 184 kb gap in an ovine MHC physical map was successfully closed by identification of two overlapping clones (304C7 and 222G18) from a Chinese fine wool merino sheep BAC library. The location and tiling path of the two clones were confirmed by BAC‐end sequencing and PCR amplification of loci in overlapping regions. Full‐length sequencing of the clones identified 13 novel ovine genes in the gap between loci Notch4 and Btnl2, and eight of them belonging to the Butyrophilin‐like (Btn‐like or Btnl) gene family. The scattered distribution of the Btnl gene cluster at the gap provided a clue to explain the difficulties previously experienced in closing the gap. Completed BAC contigs of the ovine MHC will facilitate sequencing of the entire ovine leukocyte antigen (OLA) region, providing detailed information for comparative studies of MHC evolution.     

Physical and genetic mapping of the rainbow trout major histocompatibility regions: evidence for duplication of the class I region

One of the most unexpected discoveries in MHC genetics came from studies dealing with the teleost MHC. Initially discovered in zebrafish, the MHC class I and II regions of all bony fish are not linked. Previous segregation analysis in trout suggested that the class I and II regions reside on completely different chromosomes. To learn more about MHC genomics in trout, we have isolated BAC clones harboring class Ia and Ib loci, a single BAC clone containing an MH class II gene ( DAB), as well as BAC clones containing the ABCB2 gene. Upon PCR and sequence confirmation, BAC clones were labeled and used as probes for in situ hybridization on rainbow trout metaphase chromosomes for determination of the physical locations of the trout MH regions. Finally, SNPs, RFLPs, and microsatellites found within the BAC clones allowed for these regions to be assigned to specific linkage groups on the OSU x Hotcreek (HC) and OSU x Arlee (ARL) genetic linkage maps. Our data demonstrate that the trout MH regions are located on at least four different chromosomes and the corresponding linkage groups, while also providing direct evidence for the partial duplication of the MH class I region in trout.     

A high‐resolution radiation hybrid map of the river buffalo major histocompatibility complex and comparison with BoLA

The major histocompatibility complex (MHC) in mammals codes for antigen‐presenting proteins. For this reason, the MHC is of great importance for immune function and animal health. Previous studies revealed this gene‐dense and polymorphic region in river buffalo to be on the short arm of chromosome 2, which is homologous to cattle chromosome 23. Using cattle‐derived STS markers and a river buffalo radiation hybrid (RH) panel (BBURH₅₀₀₀), we generated a high‐resolution RH map of the river buffalo MHC region. The buffalo MHC RH map (cR₅₀₀₀) was aligned with the cattle MHC RH map (cR₁₂₀₀₀) to compare gene order. The buffalo MHC had similar organization to the cattle MHC, with class II genes distributed in two segments, class IIa and class IIb. Class IIa was closely associated with the class I and class III regions, and class IIb was a separate cluster. A total of 53 markers were distributed into two linkage groups based on a two‐point LOD score threshold of ≥8. The first linkage group included 32 markers from class IIa, class I and class III. The second linkage group included 21 markers from class IIb. Bacterial artificial chromosome clones for seven loci were mapped by fluorescence in situ hybridization on metaphase chromosomes using single‐ and double‐color hybridizations. The order of cytogenetically mapped markers in the region corroborated the physical order of markers obtained from the RH map and served as anchor points to align and orient the linkage groups.     

Conserved haplotype blocks within the sheep MHC and low SNP heterozygosity in the Class IIa subregion

This report describes single-nucleotide polymorphisms (SNPs) in the sheep major histocompatibility complex (MHC) class II and class III regions and provides insights into the internal structure of this important genomic complex. MHC haplotypes were deduced from sheep family trios based on genotypes from 20 novel SNPs representative of the class II region and 10 previously described SNPs spanning the class III region. All 30 SNPs exhibited Hardy-Weinberg proportions in the sheep population studied. Recombination within an extended sire haplotype was observed within the class II region for 4 of 20 sheep chromosomes, thereby supporting the presence of separated IIa and IIb subregions similar to those present in cattle. SNP heterozygosity varied across the class II and III regions. One segment of the class IIa subregion manifested very low heterozygosity for several SNPs spanning approximately 120 Kbp. This feature corresponds to a subregion within the human MHC class II region previously described as a 'SNP desert' because of its paucity of SNPs. Linkage disequilibrium (LD) was reduced at the junction separating the putative class IIb and IIa subregions and also between the class IIa and the class III subregions. The latter observation is consistent with either an unmapped physical separation at this location or more likely a boundary characterized by more frequent recombination between two conserved subregions, each manifesting high within-block LD. These results identify internal blocks of loci in the sheep MHC, within which recombination is relatively rare.     

Molecular organization of the canine major histocompatibility complex

The major histocompatibility complex (MHC) is composed of a tightly linked cluster of genes; in dogs, this is referred to as the dog leukocyte antigen (DLA) region. The canine MHC is located on chromosome 12, and several genes within the DLA region have been identified that have significant sequence similarity to their human counterparts. However, in order to characterize other loci in the DLA region, DNA sequencing has begun using a canine bacterial artificial chromosome (BAC) library. Initially 135 BAC clones were isolated from a BAC library using a mixture of human and canine probes. These BAC clones were screened with locus-specific primers in polymerase chain reactions (PCRs). Fifty-six BAC clones were subjected to FingerPrinted Contig (FPC) analysis and several overlapping clones were identified. One BAC clone RP81-231-G24 has been sequenced. Preliminary sequence analysis of this 150 kb clone indicates that it contains the region where the class I and class III regions are joined and encompasses DLA-12a, DLA-53, DLA-12, DLA-64, TNF-alpha, and a canine gene that appears to resemble the HLA class III gene HSPA1A (HSP70-1).     

A physical map of large segments of pig Chromosome 7q11–q14: comparative analysis with human Chromosome 6p21

The aim of this study was to establish a porcine physical map along the chromosome SSC7q by construction of BAC contigs between microsatellites Sw1409 and S0102. The SLA class II contig, located on SSC7q, was lengthened. Four major BAC contigs and 10 short contigs span a region equivalent to 800 cR measured by IMpRH7000 mapping. The BAC contigs were initiated by PCR screening with primers derived from human orthologous segments, extended by chromosome walking, and controlled and oriented by RH mapping with the two available panels, IMpRH7000Rad and IMNpRH12000Rad. The location of 43 genes was revealed by sequenced segments, either from BAC ends or PCR products from BAC clones. The 220 BAC end sequences (BES) were also used to analyze the different marks of evolution. Comparative mapping analysis between pigs and humans demonstrated that the gene organization on HSA6p21 and on SSC7p11 and q11-q14 segments was conserved during evolution, with the exception of long fragments of HSA6p12 which shuffled and spliced the SLA extended class II region. Additional punctual variations (unique gene insertion/deletion) were observed, even within conserved segments, revealing the evolutionary complexity of this region. In addition, 18 new polymorphic microsatellites have been selected in order to cover the entire SSC7p11-q14 region.     

The feline major histocompatibility complex is rearranged by an inversion with a breakpoint in the distal class I region

In order to determine the genomic organization of the major histocompatibility complex (MHC) of the domestic cat (Felis catus), DNA probes for 61 markers were designed from human MHC reference sequences and used to construct feline MHC BAC contig map spanning ARE1 in the class II region to the olfactory receptor complex in the extended class I region. Selected BAC clones were then used to identify feline-specific probes for the three regions of the mammalian MHC (class II–class III–class I) for radiation hybrid mapping and fluorescent in situ hybridization to refine the organization of the domestic cat MHC. The results not only confirmed that the p-arm of domestic cat B2 is inverted relative to human Chromosome 6, but also demonstrated that one inversion breakpoint localized to the distal segment of the MHC class I between TRIM39 and TRIM26. The inversion thus disjoined the ~2.85 Mb of MHC containing class II–class III–class I (proximal region) from the ~0.50 Mb of MHC class I/extended class I region, such that TRIM39 is adjacent to the Chromosome B2 centromere and TRIM26 is adjacent to the B2 telomere in the domestic cat.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

SPC-P1: a pathogenicity-associated prophage of Salmonella paratyphi C

Background

The presence of closely related genomes in polyploid species makes the assembly of total genomic sequence from shotgun sequence reads produced by the current sequencing platforms exceedingly difficult, if not impossible. Genomes of polyploid species could be sequenced following the ordered-clone sequencing approach employing contigs of bacterial artificial chromosome (BAC) clones and BAC-based physical maps. Although BAC contigs can currently be constructed for virtually any diploid organism with the SNaPshot high-information-content-fingerprinting (HICF) technology, it is currently unknown if this is also true for polyploid species. It is possible that BAC clones from orthologous regions of homoeologous chromosomes would share numerous restriction fragments and be therefore included into common contigs. Because of this and other concerns, physical mapping utilizing the SNaPshot HICF of BAC libraries of polyploid species has not been pursued and the possibility of doing so has not been assessed. The sole exception has been in common wheat, an allohexaploid in which it is possible to construct single-chromosome or single-chromosome-arm BAC libraries from DNA of flow-sorted chromosomes and bypass the obstacles created by polyploidy.

Results

The potential of the SNaPshot HICF technology for physical mapping of polyploid plants utilizing global BAC libraries was evaluated by assembling contigs of fingerprinted clones in an in silico merged BAC library composed of single-chromosome libraries of two wheat homoeologous chromosome arms, 3AS and 3DS, and complete chromosome 3B. Because the chromosome arm origin of each clone was known, it was possible to estimate the fidelity of contig assembly. On average 97.78% or more clones, depending on the library, were from a single chromosome arm. A large portion of the remaining clones was shown to be library contamination from other chromosomes, a feature that is unavoidable during the construction of single-chromosome BAC libraries.

Conclusions

The negligibly low level of incorporation of clones from homoeologous chromosome arms into a contig during contig assembly suggested that it is feasible to construct contigs and physical maps using global BAC libraries of wheat and almost certainly also of other plant polyploid species with genome sizes comparable to that of wheat. Because of the high purity of the resulting assembled contigs, they can be directly used for genome sequencing. It is currently unknown but possible that equally good BAC contigs can be also constructed for polyploid species containing smaller, more gene-rich genomes.     

Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant

The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.     

Mapping of the SLA complex of miniature swine: mapping of the SLA gene complex by pulsed field gel electrophoresis.

The overall order of the regions of the swine major histocompatibility complex (MHC), the SLA complex, was determined by pulsed field gel electrophoresis (PFGE). It was found that the order of the regions is class II-class III-class I. A class I probe hybridized to a 420 kb Mlu I and a 420 kb Not I fragment as did a class III probe for C2. None of the class II probes hybridized to these fragments. Thus, linkage of class I to class III was shown. The class III C2, Bf, and C4 genes were found to residue in a 190 kb Not I fragment. Linkage of class III and class II genes was shown when both the class III C4 and the class II DR probes hybridized to the same 195 kb Sac II and 340 kb Not I fragments. The class I probe did not hybridize to these fragments. The order of the regions, class II-class III-class I, is similar to that of human MHC genes and may have been conserved in evolution so that coordinated expression of MHC genes could be achieved.     

Spatial arrangement of pig MHC class I sequences

Bacterial artificial chromosome (BAC) clones were assigned within the pig major histocompatibility complex (Mhc) by polymerase chain reaction-screening and Southern blot hybridization using sequence-tagged site (STS) markers and BAC end-rescued sequences. In all, 35 BAC clones were discovered containing 12 anchor genes of the SLA class I region and two genes of the SLA class III region. Twenty of these 35 clones comprised two distinct class I gene clusters, each spanning about 100 kilobases. One cluster enclosed three class I related genes (SLA-6 to -8) and two genes (MIC-1 and MIC-2) more distantly related to class I. The other cluster enclosed typical class I genes, of which three (SLA-1, -2, and -3) were transcribed by fibroblasts homozygous for the H01 haplotype which we used to construct a pig BAC library. Ordered clones are certainly helpful in isolating agronomically, biologically, and medically important genes. They would also be useful for inducing genetic modifications in pig cell lines.     

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.     

A bacterial artificial chromosome based physical map of the Ustilago maydis genome.

Ustilago maydis, a basidiomycete, is a model organism among phytopathogenic fungi. A physical map of U. maydis strain 521 was developed from bacterial artificial chromosome (BAC) clones. BAC fingerprints used polyacrylamide gel electrophoresis to separate restriction fragments. Fragments were labeled at the HindIII site and co-digested with HaeIII to reduce fragments to 50-750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P < or = 1 x 10(-6) to 1 x 10(-24)). Each assembly nucleated contigs with different percentages of bands overlapping between clones (from 20% to 97%). The number of clones per contig decreased linearly from 41 to 12 from P < or = 1 x 10(-7) to 1 x 10 (-12). The number of separate contigs increased from 56 to 150 over the same range. A hybridization-based physical map of the same BAC clones was compared with the fingerprint contigs built at P < or = 1 x 10(-7). The two methods provided consistent physical maps that were largely validated by genome sequence. The combined hybridization and fingerprint physical map provided a minimum tile path composed of 258 BAC clones (18-20 Mbp) distributed among 28 merged contigs. The genome of U. maydis was estimated to be 20.5 Mbp by pulsed-field gel electrophoresis and 24 Mbp by BAC fingerprints. There were 23 separate chromosomes inferred by both pulsed-field gel electrophoresis and fingerprint contigs. Only 11 of the tile path BAC clones contained recognizable centromere, telomere, and subtelomere repeats (high-copy DNA), suggesting that repeats caused some false merges. There were 247 tile path BAC clones that encompassed about 17.5 Mbp of low-copy DNA sequence. BAC clones are available for repeat and unique gene cluster analysis including tDNA-mediated transformation. Program FingerPrint Contigs maps aligned with each chromosome can be viewed at http://www.siu.edu/~meksem/ustilago_maydis/.     

Organization of Mhc class II A and B genes in the tilapiine fish Oreochromis

Perch-like fishes of the family Cichlidae are models for the study of speciation. An important tool in these studies is the major histocompatibility complex (Mhc) and its organization. The present study takes the first step toward the elucidation of the Mhc class II gene organization in the tilapiine fish Oreochromis niloticus (Orni). Using class II A- and class II B-specific probes, Mhc-bearing clones were identified and isolated from a bacterial artificial chromosome (BAC) library. The analysis of these clones by a combination of molecular, genetic-mapping, and phylogenetic methods led to the identification of nine class II A and 15 class II B loci. Genes at these loci constitute two families, which we designate as class IIa and class IIb families. Each of the families contains A and B loci. Some genes in both families are expressed and functional. The two families differ in their chromosomal location (they are unlinked) and their mode of evolution. The class IIa family genes are conserved across different teleost taxonomical orders, whereas the class IIb family genes are apparently products of multiple, more recent, rounds of gene duplications. The rounds established at least five monophyletic groups of genes. The founding unit of each monophyletic group might have been a pair of class II A and B loci.     

Analysis of the sheep MHC using HLA class I,II, and C4 cDNA probes

Four cDNA probes for the human major histocompatibility complex (MHC) were used to investigate the sheep MHC, in conjunction with serological typing for ovine lymphocyte antigen (OLA). Lymphocytes from a family (two parents and five offspring) of Romanov sheep were subjected to genomic DNA digestion by the restriction endonuclease Eco RI, followed by gel electrophoresis. A single Southern blot representing all seven individuals was then consecutively hybridized with the class I, alpha-DC, beta-DR, and C4 probes, which were originally designed to identify HLA class I, class II (DC and DR), and C4 products, respectively. Using each of the three class I/class II probes, several bands showing DNA polymorphism were detected. The segregation of these bands in the five offspring exactly paralleled the OLA haplotype segregation established by serological typing. A further eight individuals carrying haplotypes which were phenotypically identical to those in the above-mentioned family showed bands in the corresponding positions when tested with the same three probes. Using the C4 probe, no polymorphism was detected in these fifteen individuals.Abbreviations used in this paper MHC major histocompatibility complex - OLA ovine lymphocyte antigen - kbp kilobase pair(s) - MLR mixed lymphocyte reaction - RFLP restriction fragment length polymorphism