Genotyping bovine milk proteins using allele discrimination by primer length and automated DNA sizing technology

A method for genotyping K-casein ( A, B, E ), β-casein ( A ¹, A ², A ³, A⁵, B ) and β-lactoglobulin ( A, B ) simultaneously by the use of allele discrimination by primer length combined with automated detection of fragments with a sequencing instrument is described. Seven different mutations within the milk protein genes were analysed in order to distinguish between the alleles examined. The samples were amplified in two separate multiplex polymerase chain reactions (PCRs), which were then pooled and separated according to size in a single lane on the gel. By using stringent PCR conditions, we have been able to achieve allele-specific amplifications and minimize amplification of mismatched primer for all seven mutations.     

An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of β -casein expression in milk. Alleles CSN2 ^A , CSN2 ^{A 1}, CSN2 ^B , CSN2 ^C , CSN2 ^D and CSN2 ^E have been associated with normal content (allele effects of about 5 g of β -casein per litre), whereas the CSN2 ⁰ and CSN2 ⁰¹ alleles have been associated with non-detectable levels of β -casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region ( AJ011018 :g.1311T>C), which is associated with the absence of β -casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.     

Whole genome scan to detect quantitative trait loci for bovine milk protein composition

The objective of this study was to perform a whole genome scan to detect quantitative trait loci (QTL) for milk protein composition in 849 Holstein–Friesian cows originating from seven sires. One morning milk sample was analysed for the major milk proteins using capillary zone electrophoresis. A genetic map was constructed with 1341 single nucleotide polymorphisms, covering 2829 centimorgans (cM) and 95% of the cattle genome. The chromosomal regions most significantly related to milk protein composition ( P _genome < 0.05) were found on Bos taurus autosomes (BTA) 6, 11 and 14. The QTL on BTA6 was found at about 80 cM, and affected α_S1-casein, α_S2-casein, β-casein and κ-casein. The QTL on BTA11 was found at 124 cM, and affected β-lactoglobulin, and the QTL on BTA14 was found at 0 cM, and affected protein percentage. The proportion of phenotypic variance explained by the QTL was 3.6% for β-casein and 7.9% for κ-casein on BTA6, 28.3% for β-lactoglobulin on BTA11, and 8.6% for protein percentage on BTA14. The QTL affecting α_S2-casein on BTA6 and 17 showed a significant interaction. We investigated the extent to which the detected QTL affecting milk protein composition could be explained by known polymorphisms in β-casein , κ -casein , β-lactoglobulin and DGAT1 genes. Correction for these polymorphisms decreased the proportion of phenotypic variance explained by the QTL previously found on BTA6, 11 and 14. Thus, several significant QTL affecting milk protein composition were found, of which some QTL could partially be explained by polymorphisms in milk protein genes.     

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

D. FIRA, M. KOJIC, A. BANINA, I. SPASOJEVIC, I. STRAHINIC AND L. TOPISIROVIC. 2001 . The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6·5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both α_S1-casein and β-casein, showing very low activity towards κ-casein. The BGPF1 proteinase completely hydrolysed only β-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA–DNA hybridization and PCR analysis showed that BGPF1 contains the prtB -like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.     

Allele frequencies of the major milk proteins in the Finnish Ayrshire and detection of a new K-casein variant

A total of 20990 Finnish Ayrshire cows were phenotyped for the major milk proteins by isoelectric focusing in polyacrylamide gels. The predominant alleles in the Finnish Ayrshire were α_S1-casein B (0.999), α_S2-casein A (0.991), β-casein A₁ (0.509) and α₂ (0.490), α₂₂-casein A (0.612) and β-lactoglobulin B (0.716). The K-casein E allele (0.307) was also rather common in the Finnish Ayrshire. A new K-casein variant (K-casein F) was demonstrated in two Finnish Ayrshire cows, a dam and a daughter.     

Genomic organization and chromosomal localization of the human casein gene family

Five yeast artificial chromosome (YAC) clones containing the human casein gene family were isolated and characterized to study the control mechanisms for the expression of these genes. Partial restriction analysis in conjunction with the chromosomal fragmentation method and fluorescence in situ hybridization (FISH) analysis were performed to construct a detailed physical map of the casein gene family and to determine the chromosomal localization of these genes. The isolated YAC clones 748F3, 750D11, 882G11, 886B3 and 960D2 were 1.2 Mb, 860 kb, 800 kb 1.5 Mb and 1.5 Mb in size, respectively. The clones 748F3, 882G11, 886B3 and 960D2 contained the entire casein gene family, while the κ-casein gene was absent in 750D11. The human αS1-, β- and κ-casein genes were found to be closely linked and arranged in the order αS1-β-κ. The distance between αS1 and β, and between αS1 and κ was approximately 10 and 300 kb, respectively. The β-casein gene was oriented in the opposite direction to the αS1- and κ-casein genes. The casein gene family was localized to chromosome 4q21.1 by FISH analysis. Received: 7 July 1996 / Revised: 29 October 1996     

Oleate and linoleate stimulate degradation of β-casein in prolactin-treated HC11 mouse mammary epithelial cells

Although virtually all cells store neutral lipids as cytoplasmic lipid droplets, mammary epithelial cells have developed a specialized function to secrete them as milk fat globules. We have used the mammary epithelial cell line HC11 to evaluate the potential connections between the lipid and protein synthetic pathways. We show that unsaturated fatty acids induce a pronounced proliferation of cytoplasmic lipid droplets and stimulate the synthesis of adipose differentiation-related protein. Unexpectedly, the cellular level of β-casein, accumulated under lactogenic hormone treatment, decreases following treatment of the cells with unsaturated fatty acids. In contrast, saturated fatty acids have no significant effect on either cytoplasmic lipid droplet proliferation or cellular β-casein levels. We demonstrate that the action of unsaturated fatty acids on the level of β-casein is post-translational and requires protein synthesis. We have also observed that proteasome inhibitors potentiate β-casein degradation, indicating that proteasomal activity can destroy some cytosolic protein(s) involved in the process that negatively controls β-casein levels. Finally, lysosome inhibitors block the effect of unsaturated fatty acids on the cellular level of β-casein. Our data thus suggest that the degradation of β-casein occurs via the microautophagic pathway.     

Implication of C-Terminal Deletion on the Structure and Stability of Bovine β-casein

Bovine β-casein (β-CN) with its C-terminal truncated by chymosin digestion, β-CN-(f1-192), was examined and characterized using circular dichroism (CD) under various temperature conditions. CONTIN/LL analysis of the CD data revealed significant secondary structure disruption in β-CN-(f1-192) relative to its parent protein,β-CN, in the temperature range (5° to 70°C) studied. Near-UV CD spectra indicated significant temperature dependent structural changes. Analytical ultracentrifugation results showed significant reduction but not complete abolishment of self-association in β-CN-(f1-192) compared to whole β-casein at 2°–37°C. Furthermore, binding experiments with the common hydrophobic probe – 8-anilino-1- naphthalene sulfonic acid (ANS) illustrated that β-CN-(f1-192) is nearly incapable of binding to ANS relative to whole β-CN, suggesting a nearly complete open overall tertiary structure brought about by the C-terminal truncation. It has been demonstrated clearly that the tail peptide β-CN-(f193-209) is important in maintaining the hydrophobic core of β-CN but the residual association observed argues for a minor role for other sites as well.     

Comparison of the Chaperoning Action of Glycerol and β-Casein on Aggregation of Proteins in the Presence of Crowding Agent

β-Casein is one of the major components of the milk micelles of most mammals and has been shown to exhibit in vitro chaperone-like activity. Glycerol is a chemical chaperone belonging to the polyol family, which increases protein stability and inhibits protein aggregation. These prompted us to compare the chaperone-like activity of β-casein and glycerol. In this study, the effect of β-casein and glycerol on folding of the target proteins (ovotransferrin, insulin and α-lactalbumin) in the presence of dextran, as a macromolecular crowding agent, is examined using visible absorption spectroscopy, intrinsic fluorescence spectroscopy, 1-anilino-8-naphthalene sulfonic acid fluorescence binding and near CD spectroscopy. In the presence of dextran, the rate and extent of aggregation of target proteins was enhanced and β-casein was less effective in preventing the aggregation and precipitation of target proteins. These data support the hypothesis that β-casein interacts more effectively with slowly aggregating rather than rapidly aggregating target proteins. It is proposed that dextran-induced changes to protein conformation and the rate of intermolecular association are in a kinetic competition with the chaperoning action of β-casein; however our results demonstrated the higher activity of glycerol, as a chemical chaperone, than β-casein on the folding of target proteins, especially in the presence of dextran. This is likely due to the stabilizing effect of glycerol on protein structure and environment. The implications for the in vivo functions of β-casein and glycerol, based on their exhibiting such in vitro chaperone-like activities, are discussed.     

The Primary Structure of Water Buffalo αs1- and β-Casein: Identification of Phosphorylation Sites and Characterization of a Novel β-Casein Variant

The primary structure of water buffalo _s1-casein and of -casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a -elimination/thiol derivatization. Water buffalo _s1-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine _s1-casein C variant, the water buffalo _s1-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine A₂-casein variant, the two water buffalo -casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo -casein variants seem to be homologous to bovine A₂-casein.     

Characterization and genetic analysis of bovine αs1-casein I variant

The aim of this study was to identify the molecular genetic origin underlying the I variant of α_s1-casein and to develop a DNA-based test for this polymorphism as a tool for genetic analyses independent of milk sample testing. All coding exons and flanking regions of the α _{s 1} -casein gene were sequenced in DNA samples from cattle of known α _{s 1} -casein genotypes ( BI , CI , II , CC ), determined by isoelectric focusing of milk samples. A nucleotide substitution (A>T) in exon 11 (g.19836A>T) leads to the exchange of Glu with Asp at amino acid position 84 of the mature protein (p.Glu84Asp) and perfectly co-segregated with the presence of the α_s1-casein I variant in the milk of the analysed animals. Genotyping of a total of 680 DNA samples from 31 Bos taurus and Bos indicus cattle breeds and from Bos grunniens , Bison bison and Bison bonasus by restriction fragment length polymorphism analysis revealed the occurrence of Asp at position 84 at low frequencies in Bos taurus and Bos indicus breeds and established its origin from the α _s1-casein C variant (p.Glu192Gly). Ten different intragenic haplotypes in the gene region from intron 8 to intron 12 were observed by sequencing, of which two occurred in Bison bison and one in Bison bonasus only. Using available casein gene complex information, an association of Asp at position 84 to β-casein A ² and κ-casein B was shown in the Bos indicus breed Banyo Gudali . Taken together, we can postulate that the α _s1-casein variant I is caused by a non-synonymous nucleotide substitution in exon 11 of the gene and that it originated within Bos indicus and spread to Bos taurus subsequently.     

Expression of the human milk protein β-casein in transgenic potato plants

A 1177 bp cDNA fragment encoding the human milk protein -casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase mas12) promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human -casein cDNA. The presence of human -casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human -casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human - casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human -casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as -casein. The above experiments demonstrate the expression of human milk - casein as part of an edible food plant. These findings open the way for reconstitution of human milk inedible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children     

Detection of bovine αs1-casein genomic variants using the allele-specific polymerase chain reaction

A method was developed to distinguish between genotypic variants B and C of bovine alpha s1-casein, using the allele-specific polymerase chain reaction (ASPCR). The alpha s1-casein genotype determined for 17 Jersey cows by the ASPCR method was confirmed by typing the alpha s1-casein milk proteins on isoelectric focusing gels. Using the ASPCR method described, rapid analysis of the alpha s1-casein genotype of bulls is now possible. In addition, kappa-casein genotypes can be determined from the same PCR reaction.     

The use of flow cytometry and fluorescein-labeled antibodies to measure specific milk proteins in bovine mammary epithelial cells

Summary A flow cytometric technique was developed to measure the relative concentration of whey protein and β-casein in individual fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells were incubated with rabbit anti-bovine whey protein (α-lactalbumin + β-lactoglobulin) or β-casein primary antibodies followed by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T cells. Explant epithelial cells contained 2.9 and 5.1 times more β-casein than primary or MAC-T cells. The higher concentrations of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types.     

Chaperone-like activity of β-casein and thermal stability of alcohol dehydrogenase

Correlation between structural peculiarities of beta-casein and its chaperon-like activity was investigated using the recombinant forms of the protein containing the cysteine residues in the polypeptide chain. Aggregation of native and modified forms of β-casein was studied, as well as their chaperon-like activity towards alcohol dehydrogenase thermal aggregation. It has been shown that dimeric and oligomeric forms of β-casein, which are formed due to intermolecular disulfide bonds, significantly differ in their physicochemical and chaperon-like properties from monomeric forms. The thermal stability of alcohol dehydrogenase has been found to depend on the β-casein concentration.     

Expression and regulation of hFIX minigene and cDNA driven by β-casein gene in mouse mammary gland

Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β-casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and Lac2 gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ-carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β-casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β-casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector. Project supported by the State High Technology Development Program and Shanghai Science and Technology Commission.     

The influence of extracellular matrix and prolactin on global gene expression profiles of primary bovine mammary epithelial cells in vitro

An in vitro bovine mammosphere model was characterized for use in lactational biology studies using a functional genomics approach. Primary bovine mammary epithelial cells cultured on a basement membrane, Matrigel, formed three-dimensional alveoli-like structures or mammospheres. Gene expression profiling during mammosphere formation by high-density microarray analysis indicated that mammospheres underwent similar molecular and cellular processes to developing alveoli in the mammary gland. Gene expression profiles indicated that genes involved in milk protein and fat biosynthesis were expressed, however, lactose biosynthesis may have been compromised. Investigation of factors influencing mammosphere formation revealed that extracellular matrix (ECM) was responsible for the initiation of this process and that prolactin (Prl) was necessary for high levels of milk protein expression. CSN3 (encoding κ-casein) was the most highly expressed casein gene, followed by CSN1S1 (encoding αS1-casein) and CSN2 (encoding β-casein). Eighteen Prl-responsive genes were identified, including CSN1S1 , SOCS2 and CSN2, however, expression of CSN3 was not significantly increased by Prl and CSN1S2 was not expressed at detectable levels in mammospheres. A number of novel Prl responsive genes were identified, including ECM components and genes involved in differentiation and apoptosis. This mammosphere model is a useful model system for functional genomics studies of certain aspects of dairy cattle lactation.     

Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution

We examined whether crude DNA extracts prepared from gene-engineered mouse tissues are suitable as a template for zygosity determination by SYBR Green real-time genomic PCR. A crude DNA solution was prepared by brief incubation with lysis buffer containing ear, tail, or fetus of ROSA26 mouse, a gene-trapped strain carrying the β-galactosidase (β-gal) gene. Five serially diluted crude DNA samples (original, 2-, 4-, 8-, 16-diluted) were next prepared and then subjected to three-step (95°C, 60°C and 72°C) reactions of real-time PCR to detect the β-gal gene and the receptor-activity-modifying protein 3 (ramp3) gene (as an internal reference gene). The slopes of standard curves obtained from the real-time PCR indicated that amplification efficiency was approximately 99%, and the efficiencies of target and reference were almost equal. With this system, we next determined the zygosity of mice derived from mating heterozygous ROSA26 females and males, and found a sharp distinction in zygosity, wild-type, heterozygous and homozygous. Assessment of crude DNA samples from other gene-engineered mice including B6ZP3Cre-Tg, B6rAM-Tg, and Ramp2-gene-targeted strains revealed that our method was effective for determination of zygosity. The present method is more convenient and rapid than formerly published methods employing purified genomic DNA as a template. Our method will be particularly useful for experiments requiring rapid and accurate genotyping of gene-modified animals/fetuses.