Mapping cynomolgus monkey MHC class I district on chromosome 6p13 using pooled cDNAs

The cynomolgus monkey (Macaca fascicularis) is a frequently used animal model for studying human diseases, especially immune related ones. For a better understanding of its major histocompatibility complex (MHC) class I district chromosome location, we selected seven cDNA clones as probes for fluorescence in situ hybridization (FISH) from a lymphocyte cell line cDNA library. Expressed sequence tags (ESTs) from these clones were assembled into three clusters and annotated Mafa-A and Mafa-B genes. Further bioinformatics analysis shows that they had multiple duplications spanning approximately 2.8 Mb on the rhesus macaque MHC class I district. Using the FISH technique, we mapped the seven pooled cDNA clones to the short arm of the cynomolgus monkey chromosome 6 on 6p13. To our knowledge, this is the first report of the location of cynomolgus monkey MHC class I district. Using pooled adjacent cDNAs as probes also allows affordable, specific genome region mapping research.     

MHC class I characterization of Indonesian cynomolgus macaques

Cynomolgus macaques (Macaca fascicularis) are quickly becoming a useful model for infectious disease and transplantation research. Even though cynomolgus macaques from different geographic regions are used for these studies, there has been limited characterization of full-length major histocompatibility complex (MHC) class I immunogenetics of distinct geographic populations. Here, we identified 48 MHC class I cDNA nucleotide sequences in eleven Indonesian cynomolgus macaques, including 41 novel Mafa-A and Mafa-B sequences. We found seven MHC class I sequences in Indonesian macaques that were identical to MHC class I sequences identified in Malaysian or Mauritian macaques. Sharing of nucleotide sequences between these geographically distinct populations is also consistent with the hypothesis that Indonesia was a source of the Mauritian macaque population. In addition, we found that the Indonesian cDNA sequence Mafa-B7601 is identical throughout its peptide binding domain to Mamu-B03, an allele that has been associated with control of Simian immunodeficiency virus (SIV) viremia in Indian rhesus macaques. Overall, a better understanding of the MHC class I alleles present in Indonesian cynomolgus macaques improves their value as a model for disease research, and it better defines the biogeography of cynomolgus macaques throughout Southeast Asia.     

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.     

Assignment of 118 novel cDNAs of cynomolgus monkey brain to human chromosomes

In order to isolate genes that may not be represented in current human brain cDNA libraries, we have sequenced about 20,000 sequence tags of cDNA clones derived from cerebellum and parietal lobe of cynomolgus monkeys (Macaca fascicularis). We determined the entire cDNA sequence of approximately 700 clones whose 5'-terminal sequences showed no homology to annotated putative genes or expressed sequence tags in current databases of genetic information. From this, 118 clones with sequences encoding novel open reading frames of more than 100 amino acid residues were selected for further analysis. To localize the genes corresponding to these 118 newly identified cDNA clones on human chromosomes, we performed a homology search using the human genome sequence and fluorescent in situ hybridization. In total, 108 of 118 clones were successfully assigned to specific regions of human chromosomes. This result demonstrates that genes expressed in cynomolgus monkey are highly conserved throughout primate evolution, and that virtually all had human homologs. Furthermore, we will be able to discover novel human genes in the human genome using monkey homologs as probes.     

Chromosome mapping of MHC class I in rainbow trout (Oncorhynchus mykiss)

The major histocompatibility complex (MHC) is well-studied in mammals. Much research has addressed the genomic organisation of MHC genes and it is well established that human MHC class I genes are located on chromosome 6. However, information on the organisation of the MHC complex in rainbow trout is only beginning to become available. In the present study it was determined that rainbow trout MHC class I sequences are located on chromosome 18. This is the first reported use of fluorescence in situ hybridisation (FISH) to identify the chromosomal location of genes involved in the immune system of fish.     

Assignment of the genes encoding human interleukin-8 receptor types 1 and 2 and an interleukin-8 receptor pseudogene to chromosome 2q35.

Two human cDNA clones that encode different interleukin-8 (IL8) receptors have recently been isolated. The interleukin-8 receptor type 1 (IL8R1) binds IL8 only, whereas the interleukin-8 receptor type 2 (IL8R2) (previously designated IL8RA) also binds growth regulated gene (GRO), and neutrophil activating protein-2 (NAP-2) with high affinity. In the process of screening a genomic library with these cDNAs to obtain large clones for use in chromosomal localization studies, we isolated an interleukin-8 receptor pseudogene (IL8RP) that bears greatest similarity to IL8R2. Using Southern hybridization analysis of human x rodent somatic cell hybrid DNAs with cDNA probes for IL8R1 and IL8R2 and probes from the IL8RP locus, we assigned the three loci to chromosome 2; fluorescence in situ hybridization (FISH) to metaphase chromosome preparations using genomic clones from each locus refined this localization to chromosome 2, band q35, for all three. By virtue of their chromosomal location, IL8R1 and IL8R2 may be considered candidate genes for several human disorders in which the involved locus has been mapped to distal 2q or that are associated with structural abnormalities of this segment, including van der Woude syndrome and the neoplastic diseases rhabdomyosarcoma and uterine leiomyomata. In addition, because this region of chromosome 2q is homologous to proximal mouse chromosome 1 in the segment containing the Lsh-Ity-Bcg locus involved in mediating host resistance to infection with intracellular pathogens, examination for abnormalities of the murine homologues of the IL8R genes should be considered in mice affected by mutations of this locus.     

A cytogenetically characterized, genome-anchored 10-Mb BAC set and CGH array for the domestic dog

The generation of a 7.5x dog genome assembly provides exciting new opportunities to interpret tumor-associated chromosome aberrations at the biological level. We present a genomic microarray for array comparative genomic hybridization (aCGH) analysis in the dog, comprising 275 bacterial artificial chromosome (BAC) clones spaced at intervals of approximately 10 Mb. Each clone has been positioned accurately within the genome assembly and assigned to a unique chromosome location by fluorescence in situ hybridization (FISH) analysis, both individually and as chromosome-specific BAC pools. The microarray also contains clones representing the dog orthologues of 31 genes implicated in human cancers. FISH analysis of the 10-Mb BAC clone set indicated excellent coverage of each dog chromosome by the genome assembly. The order of clones was consistent with the assembly, but the cytogenetic intervals between clones were variable. We demonstrate the application of the BAC array for aCGH analysis to identify both whole and partial chromosome imbalances using a canine histiocytic sarcoma case. Using BAC clones selected from the array as probes, multicolor FISH analysis was used to further characterize these imbalances, revealing numerous structural chromosome rearrangements. We outline the value of a combined aCGH/FISH approach, together with a well-annotated dog genome assembly, in canine and comparative cancer studies.     

Detection of 14 alleles derived from the MHC class I<Emphasis Type="Italic"> A</Emphasis> locus in cynomolgus monkeys

A basic understanding of the major histocompatibility complex (MHC) class I, which, together with T-cell receptors, is a key player in antigen recognition by cytotoxic T lymphocytes, is necessary to study the cellular immune response to intracellular pathogens. The MHC has hardly been reported in cynomolgus monkeys (Macaca facicularis), although cynomolgus monkeys have been frequently used as the surrogate animal model. We attempted to determine the nucleotide sequences of the MHC class I A locus of cynomolgus monkeys (Mafa-A) and eventually 34 independent sequences of Mafa-A were obtained from 29 cynomolgus monkeys. These 34 sequences were classified into 14 Mafa-A alleles according to the results of phylogenetic analyses using the neighbor-joining method. One to three Mafa-A alleles were obtained from a single animal. We also tried to establish a multiplex PCR-SSP method for convenient typing of Mafa-A alleles. cDNA from a family of cynomolgus monkeys, which is composed of four sirs and four dams, were examined by multiplex PCR-SSP. The result of multiplex PCR-SSP showed that an individual cynomolgus monkey had two or three Mafa-A alleles, suggesting that the A locus of cynomolgus monkeys might be duplicated.     

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     

Targeting of marker loci to chicken chromosome 16 by representational difference analysis

Representational difference analysis (RDA) was initially used to identify differences between two inbred lines of chickens, line N and line 15I, on which the Compton mapping reference population is based. RDA was subsequently used to identify marker loci targeted specifically to chicken chromosome 16. Chromosome 16 contains the major histocompatibility complex (MHC), nucleolar organiser region (NOR) and Rfp-Y complex. To generate markers specific for this chromosome a bird was selected from the Compton mapping reference population which had inherited N line alleles for the MHC, NOR and Rfp-Y regions on this chromosome. DNA from this bird was compared with pooled DNA from 16 of its siblings, all of which had inherited line 15I alleles for the MHC, NOR and Rfp-Y regions. Initially amplicons were derived from Bam HI digested samples, RDA products were cloned after the first round of hybridisation and 113 clones were investigated: 45 of these identified Bam HI polymorphisms in this population. Of the 45 polymorphic clones, 17 have been mapped in the reference population so far, and these have identified seven new loci on chromosome 16. Interestingly a group of 16 other loci were linked on chromosome 4. The same birds were also compared by RDA following digestion with Taq I. Again large numbers of clones were generated of which 65 were investigated. Of these 17 clones were polymorphic and of five clones mapped so far three lie on chromosome 16. Two of the loci mapped to chromosome 16 have been used to identify yeast artificial chromosome (YAC) clones (GenBank accession numbers: AF057302, AF057303, AF057304, AF063218, AF06347, AF06348, AF06349, AF06350, AF063#51, AF06353, AF06354, AF06355, AF06356).     

A BAC clone-based physical map of ovine major histocompatibility complex

An ovine bacterial artificial chromosome (BAC) library containing 190,000 BAC clones was constructed and subsequently screened to construct a BAC-based physical map for the ovine major histocompatibility complex (MHC). Two hundred thirty-three BAC clones were selected by 84 overgo probes designed on human, mouse, and swine MHC sequence homologies. Ninety-four clones were ordered by DNA fingerprinting to form contigs I, II, and III that correspond to ovine MHC class I-class III, class IIa, and class IIb. The minimum tiling paths of contigs I, II, and III are 15, 4, and 4 BAC clones, spanning approximately 1900, 400, and 300 kb, respectively. The order and orientation of most BAC clones in each contig were confirmed by BAC-end sequencing. An open gap exists between class IIa and class III. This work helps to provide a foundation for detailed study of ovine MHC genes and of evolution of MHCs in mammals.     

Clusters of genes encoding mouse transplantation antigens

We constructed a cosmid library from BALB/c mouse sperm DNA and isolated 64 cosmid clones with cDNA probes for transplantation antigens (class I molecules). Of these clones, 54 mapped into 13 gene clusters containing 36 distinct class I genes and encompassing 837 kilobases of DNA. One gene cluster mapped to the L region and a second cluster with seven genes to the Qa-2,3 region of the major histocompatibility complex. Restriction map and Southern blot analyses suggest that there are subgroups of class I genes. Using a 5' flanking sequence of the L gene as a hybridization probe, we show the L gene to be present in mouse strains expressing this antigen but deleted or mutated in strains failing to express it. Our data suggest that gene duplication and deletion presumably by homologous but unequal crossing-over has altered the size and organization of the class I clusters in different mouse strains and probably is an important mechanism for generating polymorphism in these genes. Analysis of the 36 class I genes with cDNA probes specific for the 5' and 3' ends shows that the exon encoding the third external domain is far more conserved than those encoding the first and second external domains of the transplantation antigen. These differences in variability have interesting functional implications.     

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.     

Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae

Key message

A cytogenetic map of wheat was constructed using FISH with cDNA probes. FISH markers detected homoeology and chromosomal rearrangements of wild relatives, an important source of genes for wheat improvement.

Abstract

To transfer agronomically important genes from wild relatives to bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) by induced homoeologous recombination, it is important to know the chromosomal relationships of the species involved. Fluorescence in situ hybridization (FISH) can be used to study chromosome structure. The genomes of allohexaploid bread wheat and other species from the Triticeae tribe are colinear to some extent, i.e., composed of homoeoloci at similar positions along the chromosomes, and with genic regions being highly conserved. To develop cytogenetic markers specific for genic regions of wheat homoeologs, we selected more than 60 full-length wheat cDNAs using BLAST against mapped expressed sequence tags and used them as FISH probes. Most probes produced signals on all three homoeologous chromosomes at the expected positions. We developed a wheat physical map with several cDNA markers located on each of the 14 homoeologous chromosome arms. The FISH markers confirmed chromosome rearrangements within wheat genomes and were successfully used to study chromosome structure and homoeology in wild Triticeae species. FISH analysis detected 1U-6U chromosome translocation in the genome of Aegilops umbellulata, showed colinearity between chromosome A of Ae. caudata and group-1 wheat chromosomes, and between chromosome arm 7S#3L of Thinopyrum intermedium and the long arm of the group-7 wheat chromosomes.