The Mac-T cell system was used to investigate the role of the prolactin (PRL) receptor in cell proliferation and the regulation of milk protein synthesis. This study was designed to investigate whether overexpressing the PRLR in the Mac-T cell line resulted in a change in its growth rate and an enhancement of its ability to produce milk proteins. To accomplish these goals, Mac-T cells were stably transfected with the rabbit prolactin receptor gene. Fifteen clones and a pool of transfectants were obtained. Of these, one clone and the pool were positive for the PRL receptor expression. The clone (S15) and pool (SP) cells were sorted into high (H), medium (M), and low (L) expressors, of the PRLR. The high expressors were used for all subsequent experiments. The presence of high levels of the PRLR on the surface of S15 and SP cells was further confirmed by receptor binding assay and Western Blot. Following the establishment of these cell lines, the cells were used to investigate the effect of increased levels of PRLR on cell proliferation and milk protein synthesis. / It was found that the growth rate of parental cells was depressed in the presence of 5 mug/ml of PRL. In contrast, the growth rate of the transfectants was enhanced by the addition of 5 mug/ml PRL to the culture medium. In addition, both "SP" and "S15" cells produced higher levels of STAT5 upon long-term (48 h) PRL stimulation. No effect on the synthesis of alpha S1- and beta-caseins was noted. It is likely that no differences in protein synthesis were observed because the cells have lost the ability to differentiate, even when cultured on collagen gels in the presence of lactogenic hormones.

A genetic approach to lowering protease (plasmin) levels in milk, requires the presence of polymorphism of bovine plasminogen. This study was conducted to determine to what extent genetic polymorphism exists in dairy cattle. Bovine plasminogen was first purified from Holstein cow plasma by affinity chromatography on Lysine-Sepharose and antibodies to bovine plasminogen were raised by monthly intramuscular injection of the isolated bovine plasminogen into rabbits. For plasminogen phenotyping, blood samples were collected at random from 50 Holstein and Ayrshire cattle, and plasminogen was isolated from the plasma using lysine-Sepharose and then treated with neuraminidase. After separation by isoelectric focusing (pH 3.5-9.5) in polyacrylamide gels, Plasminogen polymorphs were detected immunologically using rabbit anti-bovine plasminogen antibodies. Additionally, the plasminogen isoforms were evaluated with a functional assay (caseinolytic overlay technique) after activation of the plasminogen with urokinase. Six plasminogen phenotypes were identified which represent products of 5 variant alleles. The 5 plasminogen variants were characterized based on their isoelectric points and designated PLG A$ sb2$ (pI 6.5 and 7.0), B$ sb2$ (pI 7.6 and 7.8), C$ sb1$ (pI 6.8), D$ sb2$ (pI 7.8 and 8.0), and E$ sb2$ (pI 6.8 and 7.0). PLG A$ sb2$ and PLG B$ sb2$ were the most common variants in these cattle. The 6 phenotypes were $ rm A sb2A sb2, B sb2B sb2, A sb2B sb2, B sb2C sb1, A sb2D sb2 and D sb2E sb2$. The phenotypic frequencies in Holstein and Ayrshire were very different, $ rm A sb2A sb2 and B sb2B sb2$ being respectively the most frequent phenotype. In addition, DNA polymorphism at bovine plasminogen gene was detected when genomic DNA was digested with the restriction enzyme Msp I and hybridized with mouse plasminogen cDNA. This is the first description of plasminogen polymorphism reported in dairy cattle. If different variants have altered activity, the detrimental effect

No description available.

No description available.

No description available.

It is well known that DNA methylation in gene promoter regions inhibits gene transcription and that tissue-specific gene expression is partially under the control of this transcription regulatory mechanism. In this study, bovine mammary gland tissues were collected from individual animals in lactating and non-lactating stages to investigate the DNA methylation patterns in the kappa-casein gene and alpha-lactalbumin gene core promoter regions using the bisulphite treatment in combination with polymerase chain reaction (PCR) sequencing. Different methylation status of each sample was classified into three categories, namely methylation at known transcription factor binding domains, methylation at core promoter non-binding domains and the absence of cytosine methylation. Real-time quantitative PCR was used to quantify the transcription levels of the kappa-casein and alpha-lactalbumin genes from the collected samples. A comparative method was used and fold-change values were calculated based on the comparison of the normalized threshold values of samples from different physiological stages as well as on various methylation patterns observed in their core promoter regions. Statistical analyses showed that the expressions of the kappa-casein and alpha-lactalbumin genes were significantly different in lactating and non-lactating mammary gland tissues. The methylation observed in the core promoter region of bovine alpha-lactalbumin gene was found to be associated with its gene expression. On the other hand, the methylation found in the core promoter region of bovine kappa-casein gene did not have any effect on its gene transcript levels.

Laboratory scale Cheddar type cheese were made from 411 milk samples originating from Ayrshire, Brown Swiss, Canadienne and Jersey with different phenotypes of kappa-casein (kappa-CN) and beta-lactoglobulin (beta-LG). From the milk input, cheese and whey outputs, the cheese yield, cheese composition and cheesemaking efficiency and other parameters were determined. The overall 37% moisture adjusted cheese yield and cheese yield efficiency were 11.19g, 10.19g, 11.97g, 11.46g and 98.91%, 90.10%, 90.76% and 90.92% for Ayrshire, Brown Swiss, Canadienne and Jersey respectively. The milk of the four respective breeds contained 12.91, 12.69, 13.50, 14.57% total solids; 3.97, 3.58, 4.40, 4.61% fat; 3.61, 3.73, 3.81, 4.00% protein. In Ayrshire, the combination BB/BB and BB/AA (kappa-CN/beta-LG) were associated with higher 37% moisture adjusted cheese yield with the values of 12.51 and 12.83 g/100 g milk respectively. The cheese composition for these two types of milk were 62.28 and 63.96% total solids, 24.22 and 20.40% protein; 32.75 and 37.92% fat. For Brown Swiss, type AA/BB was associated with higher cheese yield (11.18g) with composition of 62.15% total solids, 22.54% protein, 33.23% fat. The phenotype with the highest cheese yield for Canadienne is BB/AB (12.45g) with cheese composition of 63.61% total solids, 23.27% protein and 63.61% fat. In Jersey, the phenotype combination with higher cheese yield (14.59g) is BB/BB. The cheese composition corresponding to this phenotype was 58.64% total solids, 22.96% protein and 29.59% fat. Phenotypes associated with better coagulating properties for Ayrshire, Brown Swiss, Canadienne and Jersey were BB/AB, BB/BB, BB/BB and BB/BB for kappa-CN/beta-LG respectively.

Neutrophil potential phagocytic capacity (NPPC), measured on 25 AI Canadian Holstein bulls, was investigated for evidence of association with production and type traits, SCC, and survival in dairy cows. Bulls were ranked based on different degrees of NPPC (Uptakes of 0, 1, 2, and 3 or more latex beads), using the solutions coming from an animal model. A total of 42,103 first lactation records, collected from 1985 through 1993 in 2,919 Quebec dairy herds, were used to obtain EBV's for SCC and for log SCC (LogSCC) for 697 sires. Correlations between NPPC measurements and somatic cell EBV's were null. Canadian official ETA's for type traits related with mammary system had a tendency of being positively correlated with higher NPPC and negatively with Uptake-0. Canadian official ETA's for production traits were negatively correlated with higher NPPC and positively with Uptake-0. A total of 17,202 first lactation records of daughters of the 25 AI bulls were used to study the effect of NPPC and log SCC on survival in dairy cows. Survival after first lactation was more closely related to sires' NPPC-EBV's than to LogSCC-EBV.

DNA methylation has been shown to be involved in switching a number of genes on or off in particular cells. The relationship between DNA methylation and $ beta$-casein gene expression in the mammary tissue of lactating cows and mammary epithelial cells was examined. A positive correlation existed between hypomethylation of two MspI/HpaII sites in the body and one MspI/HpaII site in the 3$ sp prime$ end of the $ beta$-casein gene and its expression. In addition to these sites, hypomethylation of a distal MspI/HpaII site and HindIII sensitivity at a HindIII site also correlated with gene expression. Five DNase I hypersensitive sites were located within a 8 kb fragment. These sites designated as H1 to H5 were mapped approximately $-5, -1.3, -0.2,$ 1.7 and 2.5 kb with respect to the start site of transcription, respectively. The H2 and H3 sites were within a 1790 bp sequence that has been reported to contain a responsive element for prolactin and extracellular matrix dependent regulation and the binding site for mammary gland specific factor. / To study the dynamic changes in hypomenthylation at the MspI/HpaII sites and HindIII sensitivity, mammary tissues from pregnant heifers were evaluated. Site specific demethylation was observed depending on the stage of gestation. Demethylation of two MspI/HpaII sites (denoted M2 and M4) occurred during the early gestation, progressed slowly until mid-pregnancy, and rapidly during the last part of pregnancy. During the early stages of gestation, changes in the HindIII sensitivity in the coding domain of the $ beta$-casein gene also took place. Despite changes in HindIII sensitivity, the second HindIII site remained resistant to HindIII. By the fifth stage of gestation, the third MspI/HpaII site (M3) became less methylated and during this time the H2 site became more sensitive to HindIII. Northern analysis confirmed that demethylation of the M3 site and the acquisition of HindIII sensitivity at the H2 site was correlated with $ beta$-casein transcription. / Although $ alpha$-lactalbumin and $ beta$-casein genes are structurally and evolutionarily unrelated, they likely share common regulatory features, since both are expressed in the mammary gland during lactation. To investigate this possibility, methylation of the $ alpha$-lactalbumin gene was examined. In vivo studies revealed hypomethylation of the bovine $ alpha$-lactalbumin gene at two MspI sites and a cluster of two HhaI sites during the first and second stage of gestation, respectively. Furthermore, hypomethylation events occured only in the functional gene and not in pseudogenes, and the hypomethylation pattern was established prior to gene expression. / Taken together, the present finding suggest that DNA hypomethylation is necessary for the expression of two mammary-specific milk protein genes, $ beta$-casein and $ alpha$-lactalbumin. Hypermethylation within the body of these genes may silence these genes in non-expressing tissues and in non-epithelial cells within the mammary gland during lactation.

No description available.