Search

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.

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., (Copyright 2009 S. Karger AG, Basel.)

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., (Copyright 2009 S. Karger AG, Basel.)

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., (Copyright (c) 2008 S. Karger AG, Basel.)

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., (Copyright 2003 S. Karger AG, Basel)

A PCR-based method for sex determination of bovine DNA samples and embryo biopsies is presented. Using only one primer pair both the male-specific sequence FBNY (127 bp) and a sex-independent control PCR-fragment, the microsatellite marker FBN17 (136-140 bp) are generated in the same PCR reaction. Synteny mapping assigned the male-specific sequence to bovine chromosome Y (BTA Y), whereas FBN17 was mapped to bovine chromosome 2. Localisation of FBNY on BTA Y was confirmed by fluorescence in hybridisation of two BAC clones containing the male-specific sequence. There was no amplification of the male-specific target sequence FBNY in sheep, pig, goat, mice, man, and several wild species of the tribe Bovini. The bovine male-specific fragment was detected in dilutions containing as little as 10 pg genomic DNA and in blastomeres from embryo biopsies. The PCR assay presented here does require neither restriction endonuclease digestion of the PCR product nor additional nested PCR steps. Owing to the advantage of parallel amplification of the autosomal locus FBN17 no additional control fragment is necessary to detect PCR failure. The results of sex determination in embryo biopsies using FBNY were in agreement with the outcome from a reference assay used in commercial breeding programs., (Copyright 2001 Wiley-Liss, Inc.)

mRNA differential display was applied to identify hepatic and intestinal expressed sequence tags (ESTs) in lactating cows of different metabolic types (milk type, meat/milk type, meat type) that are potentially associated with energy turnover and involved in the regulation of these processes. Altogether, 277 ESTs (liver: 161, intestine: 116) were identified. For 150 transcripts (liver: 99, intestine: 51), the sequences showed similarity to previously described genes and ESTs. Many of these homologous sequences are reported to be involved in hepatic metabolism. Ninety-four ESTs (liver: 43, intestine: 51) did not match with any database entries. Semi-quantitative RT-PCR revealed quantitative differences in transcript represented by randomly chosen ESTs in liver samples of animals of the Holstein and Charolais breeds. One hundred twenty-two ESTs were mapped physically by using a bovine-hamster somatic cell hybrid panel (SCP) and a 5000-rad bovine whole genome radiation hybrid panel (WGRH). These ESTs were assigned to the bovine syntenic groups and positioned in the recently established RH-based ordered comparative map of the cattle and human genomes. The mapped, differentially expressed sequence tags are a useful prerequisite for cloning of genetic variation underlying economic traits.

An alternative approach for the direct analysis of chromosome regions corresponding to economical traits on the basis of chromosome microdissection is described. Large fragment clones isolated with primer pairs designed from chromosome fragment-specific DNA sequences were localized by FISH to the scraped chromosome region of interest. The chromosome fragment-specific clones are a useful tool for the generation of region specific high density marker and gene maps and represent the source material for the development of a DNA contig including the economical trait.

Results of fluorescence in situ hybridization (FISH) of Bos taurus and B. indicus Y chromosomes using the bovine locus-specific Y probes BC1.2 and lambda ES6.0 and region-specific probes of B. indicus and B. taurus Y chromosomes, which were generated by microdissection and DOP-PCR, indicate that the Y chromosomes of B. indicus (BIN Y) and B. taurus (BTA Y) differ by a pericentric inversion. Parts of the short and long arms of the Y chromosome in B. taurus and the distal half of the Y chromosome in B. indicus were microdissected, amplified by DOP-PCR, biotinylated, and rehybridized in situ to the corresponding metaphase chromosomes to test the chromosome fragment specificity of the DNA probes. The region-specific painting probes were used for hybridization to metaphase chromosomes of the other species. The DNA painting probes BTA Yp12 and BTA Yq12.1-ter derived from BTA Y hybridized to the distal and proximal halves of BIN Y, respectively. Complex hybridization signals on BTA Yq12.1-->qter were generated with the DNA probe BIN Yqcen-centr (centromere-central) after FISH. The results demonstrate that BTA Yp is homologous to the distal half of BIN Y and that BTA Yq corresponds to the proximal part of BIN Yq. Hybridization of the Y chromosome-specific DNA probes lambda ES6.0 to BTA Yp12-->p11 and near to the telomere of BIN Y and BC1.2 to BTA Yq12-->q13 and to the telomere of BIN Y indicate an opposite orientation of the homologous chromosome fragments BTA Yp and of the distal half of BIN Yq.

A rapid procedure for the defined isolation and characterization of single bovine chromosome fragment specific probes is described. This has been developed as a technical prerequisite for the directed generation of bovine DNA sequences. The specific regions 1q13-24, 5q21-24, 6q31-32, 7q21-22, 12q24-ter, and 20q12-ter of bovine GTG-banded metaphase chromosomes were microdissected and amplified by PCR with a degenerate oligonucleotide primer and subsequently cloned into pBluescript II SK. The DNA probes generated were characterized by gel electrophoresis, dot blot analysis and rehybridization in situ to GTG-banded metaphase spreads. The position and size of the hybridization sites on the chromosomes correspond exactly to the dissected chromosome areas and indicate the complexity and specificity of the microdissected and amplified chromosome material.

A cosmid clone containing the complete sheep inhibin subunit beta B gene (INHBB) was assigned to sheep and cattle homologous chromosome bands 2q31-q33 by fluorescence in situ hybridization. The assignment of INHBB in sheep excludes another candidate gene as the site of the FecB mutation.

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 cR5,000. Comparative analysis of the BBU7 RH5,000 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 RH5,000 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. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract In this study the mRNA differential display method was applied to identify mastitis-associated expressed DNA sequences based on different expression patterns in mammary gland samples of non-infected and infected udder quarters of a cow. In total, 704 different cDNA bands were displayed in both udder samples. Five hundred-and-thirty two bands, (75.6%) were differentially displayed. Ninety prominent cDNA bands were isolated, re-amplified, cloned and sequenced resulting in 87 different sequences. Amongst the 19 expressed sequence tags showing a similarity with previously described genes, the majority of these sequences exhibited homology to protein kinase encoding genes (26.3%), to genes involved in the regulation of gene expression (26.3%), to growth and differentiation factor encoding genes (21.0%) and to immune response or inflammation marker encoding genes (21.0%). These sequences were shown to have mastitis-associated expression in the udder samples of animals with and without clinical mastitis by quantitative RT-PCR. They were mapped physically using a bovine-hamster somatic cell hybrid panel and a 5000 rad bovine whole genome radiation hybrid panel. According to their localization in QTL regions based on an established integrated marker/gene-map and their disease-associated expression, four genes (AHCY, PRKDC, HNRPU, OSTF1) were suggested as potentially involved in mastitis defense.

The article focuses on the isolation of bovine genomic partial BCL2 and BCL2L1 gene sequences. Through the use of fluorescence in situ hybridization, the cytogenetic loci of the cattle chromosome bands is identified. A fragment representative of bovine BCL2 has been determined through DNA sequencing. Similarity of the bovine sequences for BCL2 and BCL2 connects the DNA contigs with the corresponding gene-specific chromosome bands.