Assignment of the fibroblast growth factor 10 (FGF10) gene to bovine chromosome 20q16-->q17 by fluorescence in situ hybridization and somatic cell panel analysis.
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Biomedical subjects
Publications and source records attributed to R Weikard.
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The primary aim of this study was to investigate whether previous findings of similar quantitative trait loci (QTL) positions for correlated yield traits are due to a pleiotropic QTL. We applied a multitrait variance component based QTL mapping method to a dataset involving five granddaughter families from the German Holstein dairy cattle population. The marker map contained 16 microsatellite markers, distributed across chromosome BTA6. A chromosomewise significance threshold was used, because BTA6 is known to harbor QTL for several milk traits. To evaluate the results from the multivariate, across-family analysis, we also conducted single-family analyses using the least squares method of QTL estimation. The results provided two significant QTL findings at 49 and 64 cM for milk yield in different families and putative QTL at 68 cM for fat yield and at 71 cM for protein yield in another family. The results for fat and protein yield were confirmed by a univariate, across-family variance components analysis. The multivariate analysis of three bivariate trait combinations resulted in a significant pleiotropic QTL finding at 68 cM for fat yield and protein yield, bracketed by markers TGLA37 and FBN13. The estimates of variance contribution due to this QTL were 23% and 25%, respectively.
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.
Interval mapping was carried out to identify quantitative trait loci (QTL) for milk production traits in five granddaughter design families of the German Holstein population. Fourteen randomly generated markers spanning the whole of BTA6 and six targeted microsatellite markers from BTA6q21-31 were included in the analysis. In one family a QTL with effects on milk fat yield and milk protein yield was mapped to the interval TGLA37-FBN13 (3 CM proximal to FBN13, lodscore 3.22) in the middle part of the chromosome. Although there are several reports about QTL with effects on milk production traits on BTA6 in the literature, a QTL with effects on milk fat and milk protein yield has not been previously described.
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A methodical strategy for the isolation of microsatellite markers specific for targeted regions of bovine chromosomes is presented. The procedure involves directed microdissection of one defined subchromosomal area, its DOP-PCR-amplification and cloning. With this approach, a library specific to the BTA 6q21-31 chromosomal region was constructed. Eleven unique microsatellite-containing sequences were isolated, converted into sequence-tagged microsatellite sites, and characterized concerning their species-specific origin. Seven primer pairs generated bovine-specific PCR products and provided a set of microsatellite markers that generally revealed high informativity in the HF breed. Linkage analysis assigned six of them to their predefined subchromosomal origin on BTA 6 corresponding to the specific rehybridization signal of the DOP-PCR product generated from the microdissected chromosome area 6q21-31. The results underline the usefulness of the BTA 6q21-31 library for targeted isolation of unique sequences that are specific for the dissected chromosomal region as demonstrated here by the isolation of microsatellite markers.
1. Male and female broiler chickens (144 in total) were given diets supplemented with clenbuterol (CB) at 0 (control) and at 1 mg/kg between 28 and 49 d of age to study the effect of CB on growth, carcase and skeletal muscle. 2. CB improved growth in males by increasing daily weight gain and final live weight and by lowering food conversion ratio. In females it changed the carcase composition by reducing abdominal fat pad and by increasing the proportion of protein. Consequently, carcase protein gain was increased in both sexes (11% and 16%, respectively). 3. Skeletal muscle weights were enhanced by between 6% and 22%. Muscle fibre diameters were increased in extensor hallucis longus (EHL) but not in gastrocnemius (GAS) muscle. This increase was more pronounced in females. EHL total muscle fibre number remained unchanged. The proportion of fast-twitch glycolytic fibres was increased at the expense of fast-twitch oxidative fibres in males only. Nuclear/cytoplasm and DNA/protein ratios tended to be decreased by CB. 4. From the elevated EHL muscle RNA/DNA, unchanged protein/RNA and translation activity it is suggested that CB stimulated protein synthesis at the pretranslational level. Reduced protein degradation is deduced from decreased neutral calcium-dependent proteolytic activity. 5. It is concluded that broiler chickens respond to long-term CB treatment as has been shown in various mammals. However, the sex-specific response in growth, carcase composition and skeletal muscle cellularity is more clearly apparent in broiler chickens.
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.
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The effects of clenbuterol (CB) on the weights of 6 hindleg muscles as well as on structural and biochemical characteristics of extensor digitorum longus muscle (EDL) were studied on Wistar rats. Different CB-doses (5 vs. 18 mg/kg diet) were tested and rats of different sex were used. The 12 days treatment accelerated the growth of the 6 hindleg muscles by 13 to 24% without predominant response of fast twitch or slow twitch muscles. The higher muscle weights were achieved by fibre hypertrophy as demonstrated by increased fibre diameters (+13 to +16%), and not by fibre multiplication. There was no evidence for a selective fibre hypertrophy of one of the metabolic fibre types. Fibre type composition shifted to the fast glycolytic fibres (FTG +0.7 to +4.9%-units). There were no significant differences in responses between the sexes of CB-doses. Clenbuterol decreased the nucleus/cytoplasm ratio, measured as nuclei number/mm2 fibre area, between 24 and 28% and the DNA/protein ratio by 17%. RNA content and RNA/DNA ratio were enhanced by 25 and 22%, respectively; the protein/RNA ratio remained unchanged. The results suggest that clenbuterol changes fibre type composition and stimulates fibre growth in a pretranslational stage of protein synthesis without addition of nuclei by satellite cell proliferation.
The administration of porcine somatotropin (pST) to pregnant sows during early gestation (day 10 to 24) induced the formation of significantly more muscle fibres in the semitendinosus muscle of the fetuses, representing a higher growth capacity of skeletal muscle. The pST-treatment during late pregnancy (day 80 to 94) accelerated the development of the fetus resulting in higher body weights and advanced stage of maturity at birth.
In 3 experimental groups 9 female Wistar rats (initial live weight 150 g) were fed either the control diet, the control diet supplemented with 5 mg clenbuterol or the combination of 5 mg clenbuterol and 500 mg propranolol per kg diet over a 12-day period. The N-balance was estimated over days 6 to 10 followed by a 15N-tracer experiment for determining the influence of the feed additives on characteristics of protein metabolism on day 12. All differences in the means were concluded to be significant for P < 0.05. Live weight gain and feed efficiency were improved by clenbuterol. The animals treated with clenbuterol had 18%-24% higher muscle weights whereas the combined treatment increased the muscle weights by 10%-16% only. The good correlation between the increase of muscle weights and the total protein content indicates that clenbuterol does not change the relation between protein- and water accumulation. Histological-histochemical investigations showed that the higher muscle weights were achieved through muscle fibre hypertrophy. The number of muscle fibres remained constant. Concerning the distribution of the fibre types, clenbuterol increased the proportion of FTG-fibres (white, fast-twitch, glycolytic) on the expense of the FTO-fibres (fast-twitch, oxidative). While the number of nuclei per muscle fibre did not change, the nucleus-cytoplasm relation decreased by 24%. Compared to the animals fed the control diet, the N-balance in the clenbuterol-treated group was increased by 41%. Feeding the combination of clenbuterol and propranolol resulted in an increase of 24% only. Clenbuterol increased the N-content of the carcass by 6% and reduced the carcass fat content by 30%. In the group fed clenbuterol and propranolol, the N-content of the carcass only tended to be increased and the influence on carcass fat reduction was only 16%. The stimulated N-deposition in the carcass of clenbuterol-treated rats was obtained, since the calculated protein degradation rates were more reduced than protein synthesis rates. In-vitro investigations of the muscle protein synthesis and -protease activities support these results. The clenbuterol-induced increase in muscle protein was accompanied by an inhibition of the Ca-dependent protease activity and an increase of muscle DNA- and RNA-content. The additional application of propranolol reduced these effects of clenbuterol again. Since propranolol partly prevented the effects of clenbuterol on protein metabolism it is suggested that not only the lipolytic but also protein anabolic effects are caused by the beta-adrenergic action of clenbuterol.
The recent paper deals with the influence of nutritional situation on the ability of cell sap fractions of liver and muscle of rats to stimulate protein biosynthesis in a cell-free system. The incorporation of 3H-leucine into polypeptides from tissues of ad libitum fed rats was 1.25-1.46 fold higher in the liver resp. 1.2-1.7 fold higher in the muscle in comparison to the incorporation data of the starved rats. Significant differences were also shown in the course of translation (incorporation of amino acids in dependence of concentration and time). The amino acid incorporation of starved animals rapidly reached a plateau at a low level of incorporation (liver 40,000 dpm, muscle 9,000 dpm) whereas at higher cell sap concentrations and prolonged times of incubation (120 min) degradation effects (proteolysis) must be taken into account. The incorporation of amino acids in tissues of ad libitum fed animals has still increased under the same conditions (liver 55,000 dpm, muscle 16,000 dpm). These results obtained with in-vitro-methods confirm findings got with in-vivo-techniques and may complete the investigations of protein metabolism sensibly.