Advanced comparative cytogenetic analysis of X chromosomes in river buffalo, cattle, sheep, and human.

Based on a recently generated comprehensive gene map for Ovis aries chromosome X (OARX) with an approximately even locus distribution, we assigned selected bacterial artificial chromosome (BAC) probes corresponding to these OARX loci to Bubalus bubalis (BBU) and Bos taurus (BTA) by comparative fluorescence in-situ hybridization (FISH) to improve cytogenetically the X chromosome maps in these species. Twenty-five added loci in BBUX and BTAX, respectively, contribute to a more detailed description of the cytogenetic organization of these chromosomes. Further seven loci were identified in OARX and two DNA probes were assigned to X and Y chromosomes in river buffalo, cattle, and sheep, respectively, and thus identified loci in the pseudoautosomal region. The additional assignments double the number of cytogenetic loci in BBUX and increase their number in BTAX and OARX. The larger quantity of cytogenetic anchors allows a more precise morphological comparison of bovid X chromosomes among each other and with the Homo sapiens (HSA) X chromosome. The anchor loci confirm and refine syntenic fragments in HSAX and identify several evolutionary breakpoints between the compared chromosomes. The cytogenetic assignments in BBUX, BTAX, and OARX represent useable anchors for the ongoing genome sequence assembly in Bovidae.

Tissue-specific mRNA expression patterns reveal a coordinated metabolic response associated with genetic selection for milk production in cows.

The molecular mechanisms regulating the physiological adaptation of tissues important for nutrient partitioning and metabolism in lactating cows are still not completely understood. The aim of our study was to identify tissue-specific regulatory mechanisms necessary to accommodate metabolic changes associated with different genetic potential for milk performance. For this purpose, we analyzed mRNA expression of genes involved in energy metabolism of segregating F(2) beef type cows with a combined genetic dairy and beef background (Charolais × German Holstein cross, CH×GH) in contrast to purebred German Holstein (GH) dairy cows. Three groups of cows differing in milk performance were examined using quantitative real-time PCR in liver, mammary gland, and skeletal muscle. Our results describe substantial tissue-specific differences in mRNA transcription profiles between cow groups in relation to their genetic potential for milk performance and highlight genes exhibiting specific, partially yet-unknown functions in dairy and beef type cows, e.g., upregulation of PCK2 transcripts in the mammary gland and FBP2 transcripts in skeletal muscle of dairy cows. Noticeably, PCCA and PPARGC1A mRNA abundance varied significantly across experimental groups in all three tissues, pointing to potential key gene functions in the metabolic adaptation relative to divergent milk production performance. Correlations of mRNA expression levels to milk performance traits indicate that gene transcriptional processes may play a regulatory role in liver, mammary gland, and skeletal muscle to enable cows with different genetic potential for milk performance to cope with metabolic lactation-associated challenges.

Extended cytogenetic maps of sheep chromosome 1 and their cattle and river buffalo homoeologues: comparison with the OAR1 RH map and human chromosomes 2, 3, 21 and 1q.

Cytogenetic maps are useful tools for several applications, such as the physical anchoring of linkage and RH maps or genome sequence contigs to specific chromosome regions or the analysis of chromosome rearrangements. Recently, a detailed RH map was reported in OAR1. In the present study, we selected 38 markers equally distributed in this RH map for identification of ovine genomic DNA clones within the ovine BAC library CHORI-243 using the virtual sheep genome browser and performed FISH mapping for both comparison of OAR1 and homoeologous chromosomes BBU1q-BBU6 and BTA1-BTA3 and considerably extending the cytogenetic maps of the involved species-specific chromosomes. Comparison of the resulting maps with human-identified homology with HSA2q, HSA3, HSA21 and HSA1q reveals complex chromosome rearrangements differentiating human and bovid chromosomes. In addition, we identified 2 new small human segments from HSA2q and HSA3q conserved in the telomeric regions of OAR1p and homoeologous chromosome regions of BTA3 and BBU6, and OAR1q, respectively. Evaluation of the present OAR1 cytogenetic map and the OAR1 RH map supports previous RH assignments with 2 main exceptions. The 2 loci BMS4011 and CL638002 occupy inverted positions in these 2 maps.

Evolutionary break point analysis between the proximal half of bovine chromosome 27 and conserved segments of the human genome.

The proximal half of Bos taurus chromosome 27 (BTA27prox) delimited by microsatellite markers BM3507 and CSSM043 reveals complex rearrangements compared to its corresponding Homo sapiens chromosome (HSA) fragments. A comparative mapping approach combining somatic and radiation hybrid cell mapping techniques and related cytogenetic data resulted in an improved physical map for BTA27prox, which provides candidate genes for several important economic traits. The generated comprehensive map includes anchor loci for 103 genes and microsatellite markers. Mapping of genes proximal to BM3507 matching a region from 0.60 to 2.78 megabase pairs (Mb) of HSA8 confirmed recent sequence annotations on BTA27. Assignments of loci predicted to be on BTA27 to BTA1, BTA8, and BTA17 narrowed down evolutionary chromosome break points compared with corresponding chromosome segments in human. New physical anchors obtained in this study confirm in more detail the described evolutionary conservation between the proximal half of BTA27 and homologous segments of HSA4 and HSA8 and will contribute to the completion of the cattle DNA genome sequence.

FISH-mapping comparison between river buffalo chromosome 7 and sheep chromosome 6: assignment of new loci and comparison with HSA4.

Synchronized peripheral blood lymphocytes from both river buffalo (BBU) and sheep (OAR) were treated for late incorporation of both BrdU and H-33258 to obtain R-banded preparations to be used for FISH-mapping. Ovine BAC-clones were hybridized for three days on slides pre-exposed to UV light after H-33258 staining. The following loci were mapped: GPR103 (BBU7q13, OAR6q13), TRAM1L1(OAR6q13dist), PPP3CA (BBU7q21, OAR6q15), SNCA (OAR6q17), PPARGC1A(BBU7q23, OAR6q17), UGDH (BBU7q25prox, OAR6q22prox), KDR (BBU7q27, OAR6q22), CNOT6L (BBU7q32prox, OAR6q32prox), NUP54 (BBU7q32, BBU6q32), DMP1 (BBU7q34dist-q36prox, OAR6q34dist-q36prox), QDPR (BBU7q36, OAR6q36). All loci mapped in homoeologous chromosomes and chromosome bands of the two species and their locations are in agreement with the previous RH-mapping performed on BBU7 with some difference in the distal region of BBU7. However, the present cytogenetic map better anchors the RH-map on specific river buffalo chromosome bands. In addition, eleven loci were assigned for the first time in sheep to OAR6, noticeably extending the cytogenetic map on this important chromosome which encodes caseins. Two loci (TRAM1L1 and SNCA) mapped in sheep were unmapped in river buffalo in three different FISH experiments. Comparisons between integrated cytogenetic maps of BBU7/OAR6 (and BTA6) with human chromosome 4 (HSA4) revealed complex chromosome rearrangements differentiating these chromosomes.

A high-resolution radiation hybrid map of sheep chromosome X and comparison with human and cattle.

A radiation hybrid (RH) map of sheep X chromosome (Ovisaries; OARX) containing 146 physically anchored loci was generated in this study, providing information for comparative X chromosome analysis between the maps of sheep, human, and cattle. Primers typed on the USUoRH5000 ovine whole-genome radiation hybrid panel were designed from sequences predicted to be on the ovine X chromosome, based on comparative mapping within the virtual sheep genome browser (v1.2). The resulting RH map for the ovine X chromosome consists of 4 linkage groups composed of 76 BAC end sequences (BES), 28 gene loci that were confirmed within ovine BAC clones in the CHORI-243 ovine BAC library, 28 additional gene loci from the ovine comparative map and 14 polymorphic sequence tagged sites (STS) from the OARX linkage map. This first-generation RH map of OARX contributes to the expansion of a comprehensive ovine genome map for sheep and provides evidence of rearrangements in loci order compared to the human and cattle orders.

Molecular cytogenetics and gene mapping in sheep (Ovis aries, 2n = 54).

The development of a completely annotated sheep genome sequence is a key need for understanding the phylogenetic relationships and genetic diversity among the many different sheep breeds worldwide and for identifying genes controlling economically and physiologically important traits. The ovine genome sequence assembly will be crucial for developing optimized breeding programs based on highly productive, healthy sheep phenotypes that are adapted to modern breeding and production conditions. Scientists and breeders around the globe have been contributing to this goal by generating genomic and cDNA libraries, performing genome-wide and trait-associated analyses of polymorphism, expression analysis, genome sequencing, and by developing virtual and physical comparative maps. The International Sheep Genomics Consortium (ISGC), an informal network of sheep genomics researchers, is playing a major role in coordinating many of these activities. In addition to serving as an essential tool for monitoring chromosome abnormalities in specific sheep populations, ovine molecular cytogenetics provides physical anchors which link and order genome regions, such as sequence contigs, genes and polymorphic DNA markers to ovine chromosomes. Likewise, molecular cytogenetics can contribute to the process of defining evolutionary breakpoints between related species. The selective expansion of the sheep cytogenetic map, using loci to connect maps and identify chromosome bands, can substantially contribute to improving the quality of the annotated sheep genome sequence and will also accelerate its assembly. Furthermore, identifying major morphological chromosome anomalies and micro-rearrangements, such as gene duplications or deletions, that might occur between different sheep breeds and other Ovis species will also be important to understand the diversity of sheep chromosome structure and its implications for cross-breeding. To date, 566 loci have been assigned to specific chromosome regions in sheep and the new cytogenetic map is presented as part of this review. This review will also summarize the current cytogenomic status of the sheep genome, describe current activities in the sheep cytogenomics research sector, and will discuss the cytogenomics data in context with other major sheep genomics projects.

A radiation hybrid comparative map of ovine chromosome 1 aligned to the virtual sheep genome.

Ovis aries chromosome one (OAR1) is the largest submetacentric chromosome in the sheep genome and is homologous to regions on human chromosomes 1, 2, 3 and 21. Using the USUoRH5000 ovine whole-genome radiation hybrid (RH) panel, we have constructed a RH map of OAR1 comprising 102 framework and 75 placed/binned markers across five linkage groups spanning 3759.43 cR5000, with an average marker density of 21.2 cR5000/marker. The alignment of our OAR1 RH map shows good concordance with the recently developed virtual sheep genome, with fewer than 1.86% discrepancies. A comparative map of OAR1 was constructed by examining the location of RH-mapped orthologues in sheep within the genomes of cow, human, horse and dog. Analysis of the comparative map indicates that conserved syntenies within the five ovine RH linkage groups underwent internal chromosomal rearrangements which, in general, reflect the evolutionary distances between sheep and each of these four species. The ovine RH map presented here integrates all available mapping data and includes new genomic information for ovine chromosome 1.

A comparative radiation hybrid map of sheep chromosome 10.

Comparative radiation hybrid (RH) maps of individual ovine chromosomes are essential to identify genes governing traits of economic importance in sheep, a livestock species for which whole genome sequence data are not yet available. The USUoRH5000 radiation hybrid panel was used to generate a RH map of sheep chromosome 10 (OAR10) with 59 markers that span 1,422 cR over an estimated 92 Mb of the chromosome, thus providing markers every 2 Mb (equivalent to every 24 cR). The markers were derived from 46 BAC end sequences (BESs), a single EST, and 12 microsatellites. Comparative analysis showed that OAR10 shares remarkable conservation of gene order along the entire length of cattle chromosome 12 and that OAR10 contains four major homologous synteny blocks, each related to segments of the homologous human chromosome 13. Extending the comparison to the horse, dog, mouse, and chicken genome showed that these blocks share conserved synteny across species.

A high-resolution comparative radiation hybrid map of ovine chromosomal regions that are homologous to human chromosome 6 (HSA6).

In this study, we constructed high-resolution radiation hybrid (RH) and comparative maps of ovine chromosomes or chromosomal segments that are homologous to human chromosome 6 (HSA6). A total of 251 markers were successfully genotyped across the recently developed USUoRH5000 whole-genome panel; 208 of these markers were assigned to five RH linkage groups distributed on three ovine chromosomes (OAR8, 9 and 20). The RH maps have good correspondence with previous chromosome painting data, although a small centromeric region on OAR9 that is homologous to HSA6 had not been previously detected using human chromosome paints on ovine chromosomal spreads. High percentages of the ovine markers were identified as orthologues in the bovine (86.3%), dog (85.8%), horse (69.3%) and human (88.7%) genomes. These maps contribute to investigations in mammalian chromosome evolution and the search for economic trait loci in sheep.

Forward to a detailed sequence map of bovine chromosome 6.

Numerous QTL for a variety of phenotypic traits in dairy and beef cattle have been mapped on bovine chromosome 6 (BTA6). The complete and validated information on the molecular genome organization is an essential prerequisite for the conclusive identification of the causative sequence variation underlying the QTL. In our study we describe efforts to improve the genomic sequence map assembly of BTA6 by filling-in gaps and by suggesting sequence contig rearrangements. This is achieved by the generation and in silico mapping of BAC-end sequences (BESs) from clones containing sequences placed on our high-resolution radiation hybrid (RH) map of BTA6 onto the genome sequence map. Linking high-resolution RH mapping with in silico mapping of BESs on BTA6 enabled the detection of discrepancies in chromosomal assignments of genome sequence contigs and improved the resolution of non-conclusive assignments on the genome sequence assembly. Furthermore, 37% of BESs enabled chromosomal assignment of contigs previously unassigned. Anchoring of 66% of BESs onto HSA4 confirmed the synteny of the respective region of BTA6 including the known evolutionary breakpoints. The BESs will play an important role in the ongoing efforts to complete the sequence of the bovine genome and will also provide a source for the identification of new polymorphic sites in the genome sequence to resolve QTL-containing intervals.

Comparative FISH-mapping of twelve loci in river buffalo and sheep chromosomes: comparison with HSA8p and HSA4q.

Twelve loci (11 of type I and 1 of type II) previously FISH-mapped in cattle were comparatively FISH-mapped in both river buffalo chromosome 1p (BBU1p) and homologous chromosome 26 of sheep (OAR26), extending the cytogenetic maps in both chromosome species and providing a more precise localization of these loci in single chromosome bands than previous locations on BTA27. Bovine BAC clones containing DCTD, C4orf20, CASP3, TLR3, MSR1, FAT, LONRF1, DLC1, C8orf41, CSSM036, LSM1 and EIF4EBP1 were used for FISH on RBPI-banded chromosomes. All loci were located on the same homologous chromosome bands (R-band positive) of both species further confirming the high degree of banding and gene (order of loci) homologies among bovids. Detailed cytogenetic maps of OAR26 and BBU1p were performed and compared with that of BTA27 as well as with those of both HSA8p and HSA4q, revealing complex chromosome rearrangements differentiating OAR26/BBU1p/BTA27 from human chromosomes.

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 1 (BBU1).

The largest chromosome in the river buffalo karyotype, BBU1, is a submetacentric chromosome with reported homology between BBU1q and bovine chromosome 1 and between BBU1p and BTA27. We present the first radiation hybrid map of this chromosome containing 69 cattle derived markers including 48 coding genes, 17 microsatellites and four ESTs distributed in two linkage groups spanning a total length of 1330.1 cR(5000). The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH(5000)) panel. The retention frequency of individual markers across the panel ranged from 17.8 to 52.2%. With few exceptions, the order of markers within linkage groups is identical to the order established for corresponding cattle RH maps. The BBU1 map provides a starting point for comparison of gene order rearrangements between river buffalo chromosome 1 and its bovine homologs.

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 7 and comparative mapping to the cattle and human genomes.

A preliminary radiation hybrid (RH) map containing 50 loci on chromosome 7 of the domestic river buffalo Bubalus bubalis (BBU; 2n = 50) was constructed based on a comparative mapping approach. The RH map of BBU7 includes thirty-seven gene markers and thirteen microsatellites. All loci have been previously assigned to Bos taurus (BTA) chromosome BTA6, which is known for its association with several economically important milk production traits in cattle. The map consists of two linkage groups spanning a total length of 627.9 cR(5,000). Comparative analysis of the BBU7 RH(5,000) map with BTA6 in cattle gave new evidence for strong similarity between the two chromosomes over their entire length and exposed minor differences in locus order. Comparison of the BBU7 RH(5,000) map with the Homo sapiens (HSA) genome revealed similarity with a large chromosome segment of HSA4. Comparative analysis of loci in both species revealed more variability than previously known in gene order and several chromosome rearrangements including centromere relocation. The data obtained in our study define the evolutionarily conserved segment on BBU7 and HSA4 to be between 3.5 megabases (Mb) and 115.8 Mb in the HSA4 (genome build 36) DNA sequence.

YAC/BAC contig spanning the MHC class III region of cattle.

A contig of the class III region of the bovine major histocompatibility complex (MHC) was established from bacterial and yeast artificial chromosomes using PCR and BAC-end sequencing. The marker content of individual clones was determined by gene and BAC-end specific PCR, and the location of genes and BAC-ends was confirmed analyzing somatic hybrid cells. A comparative analysis indicated that the content and order of MHC class III genes is strongly conserved between cattle and other mammalian species. Fluorescence in situ hybridization localized the bovine class III region to BTA23q21-->q22. The results show that the collection of sequenced BAC-ends is a powerful resource for generating high-resolution comparative chromosome maps.

Identification and gene expression of the bovine C-type lectin Dectin-1.

C-type lectin receptors (CTLR) are cell-surface signalling molecules that recognize a range of highly conserved pathogen molecules and instigate the appropriate immune response. Here, we report the cloning, sequencing, mapping and expression pattern of the bovine C-type lectin domain family 7, member A (CLEC7A; synonyms CLCSF12, Dectin-1). We identified two isoforms, similar to the human system, with a long and short neck. Overall, the organization of the two bovine CLEC7A genes is similar to that of humans and mice. The CLEC7A gene maps on Bos taurus chromosome 5 (BTA5). mRNA transcripts for CLEC7A were detected in bone-marrow cells, monocytes, macrophages and dendritic cells and NK cells, but not in CD4(+) T-cells or CD21(+) B-cells. The increased knowledge of the genomic organization of the bovine CTLR genes may promote our understanding of their evolution and help in the identification of bovine genes underlying disease-resistance traits.

Positional and functional characterisation of apoptosis related genes belonging to the BCL2 family in sheep.

Apoptosis is a process whereby cells die in a controlled manner and it is involved in animal development, tissue homeostasis and a variety of diseases. The B-cell lymphoma 2 family proteins are central regulators of intracellular apoptotic signalling cascades. This work describes the isolation of cDNA and genomic fragments from five sheep BCL2 related genes: BCL2, BCL2L1, BCL2L2, BAX and MCL1. Transcript sequences showed a high homology with BCL2 related genes from other species. Three cattle BAC probes containing the homologous BCL2, BCL2L1 and BCL2L2 genes were identified and used for comparative FISH mapping in sheep. BCL2 was localised in OAR23q27, BCL2L1 in OAR13q22 and BCL2L2 in OAR7q15-->q21. Intron polymorphisms were used for linkage mapping of BAX and MCL1, which were mapped on OAR14 and OAR1 respectively. Moreover, a BCL2L1 pseudogene was also identified and linkage mapped on OAR2. The expression of these genes was analysed in mammary gland, ovary, intestine and brain which are target tissues for sheep pathological processes where apoptosis is involved.

Clarifications on breakpoints in HSAX and BTAX by comparative mapping of F9, HPRT, and XIST in cattle.

The coagulation factor IX gene (F9), the hypoxanthine phosphoribosyl transferase 1 gene (HPRT1), and the X-inactive specific transcript gene (XIST) were physically assigned in cattle to analyze chromosomal breakpoints on BTAX recently identified by radiation hybrid (RH) mapping experiments. Whereas the FISH assignment of XIST indicates a similar location on the q-arm of the human and cattle X chromosomes, the locus of HPRT1 supported the assumption of a chromosome rearrangement between the distal half of the q-arm of HSAX and the p-arm of BTAX identified by RH mapping. F9 previously located on the q-arm of BTAX was assigned to the p-arm of BTAX using RH mapping and FISH. The suggested new position of F9 close to HPRT1 supports the homology between HSAXq and BTAXp. The F9 locus corresponds with the gene order found in the homologous human chromosome segment. XIST was assigned on BTAXq23, HPRT1 and F9 were mapped to BTAXp22, and the verification of the location of F9 in a 5000 rad cattle-hamster whole genome radiation hybrid panel linked the gene to markers URB10 and HPRT1.

Detection and functional characterisation of the transcription factor peroxisome proliferator-activated receptor gamma in lutein cells.

A prominent functional change during differentiation of lutein cells from follicular thecal and granulosa cells is an enhanced production and secretion of progestins. The regulation of this process is not fully understood but may be associated with the expression of transcription factors which activate genes, products of which are involved in pathways of the cholesterol and lipid metabolism. As peroxisome proliferator-activated receptors (PPARs) play a role in both pathways, we were interested in the expression of PPARgamma, a PPAR form which is involved in adipogenic differentiation. First, we were able to show the expression of PPARgamma in bovine lutein cells (day 12 of the ovarian cycle) at the mRNA and protein level by imaging, flow cytometry and blot analysis, and secondly a role of PPARgamma in the secretion of progesterone. The cells (24 h culture) responded dose dependently by increasing progesterone secretion (up to 1.5-fold of the basal level) to an endogenous ligand of PPARgamma, 15-deoxy-delta12,14 prostaglandin J2 (15-dPGJ2) and to the thiazolidinedione ciglitizone. Aurintricarboxylic acid (ATA) was found to reduce the intracellular PPARgamma level and to promote cell cycle progress, indicating that ATA can be used as a tool for experimental changes of PPARgamma proteins in intact cells and for studying the physiological consequences. The ATA-mediated decrease of PPARgamma was accompanied by reduced progesterone production and a progression of the cell cycle, suggesting a function of PPARgamma in both processes. The response to ATA was abrogated by a high dose (>490 nM) of 15-dPGJ2, suggesting that 15-dPGJ2 exerts its effect on steroidogenic activity via PPARgamma and that the 15-dPGJ2-PPARgamma system plays a role in the maintenance of a differentiated quiescent stage in lutein cells.