Bovine Casein Gene Research Articles

New Combinations of Alleles in the Variants of the Cluster of Bovine Casein Genes and Revision of the Nomenclature of These Genes

New Combinations of Alleles in the Variants of the Cluster of Bovine Casein Genes and Revision of the Nomenclature of These Genes

Identification of Prolyl Endopeptidase (PEP) Inhibitory Peptides from Lactobacillus helveticus Digestion of Four Recombinant Bovine Caseins

Eurygaster integriceps Puton (Sunn pest) is the most damaging insect pest of wheat in areas of North Africa, Eastern Europe and Western and Central Asia. Sunn pest injects an enzyme characterized as a prolyl endylprotease (spPEP) into wheat grains that digests the gluten protein (Darkoh et al., 2010). Previous studies have shown that casein peptides generated by Lactobacillus species have inhibitory effects on mammalian and bacterial prolyl endoproteases (PEP) which makes casein a good natural candidate against spPEP. This study used four purified recombinant bovine casein genes, α1, α2, β and κ to identify which Lactobacillus generated peptides will inhibit the spPEP. Firstly, these four recombinant casein genes were expressed and purified. Then the peptides were generated by incubating each purified casein with Lactobacillus helveticus. These peptides were separated using High Performance Liquid Chromatography (HPLC). The inhibitory peptides were identified by incubating them with recombinant PEP enzyme in the presence the GPpNA substrate. Mass and sequence of the inhibitory peptides were determined using MALDI Mass Spectrophotometry.. Future studies will include the cloning and expression of the identified peptides of different lengths to determine specificity unique to spPEP. Funding: Office of Research and Sponsored Projects, SFASU and Ed and Gwen Cole, Nacogdoches, TX.

Use of Recombinent Casein Peptides as Inhibitors of spPEP

Eurygaster integreceps (Sunn pest) causes major damage in wheat grains by injecting their saliva into the grain degrading the gluten proteins. This saliva protein has been identified and characterized as a prolyl endoprotease (spPEP) in our lab (Darkoh et al, 2010). A natural, biological friendly inhibitor to the spPEP is being sought. Previous studies have shown that casein peptides generated by Lactobacillis species have inhibitory effects on mammalian and bacterial prolyl endoproteases (PEP). The Objective of this study was to clone the four bovine casein genes, α1, α2, β and κ for use in expressing each isomer to identify which peptides inhibit the spPEP. The nucleotide sequences corresponding to bovine casein isomers were identified from the NCBI database to design gene specific primers that would amplify the full length casein proteins. Total RNA was extracted from bovine mammary gland biopsy tissue kindly donated by Dr. Erin Connor, Research Leader, USDA‐ARS, Beltsville, MD and subsequent cDNA has been synthesized. Each casein subunit was cloned into the pGEM T‐Easy vector for sequencing. Verified sequences were cloned into the pET15b E.coli expression vector for protein expression and purification using IMAC chromatography. Purified proteins will be used for subsequent incubation with L. helveticus at varying times. The resulting peptide mixture will be incubated with the spPEP in the presence of the GPpNA substrate to determine which isomer/s contains the spPEP inhibitory sequences.Funding Sources: Office of Research and Sponsored Projects, Stephen F. Austin State University, Nacogdoches, TX and Ed and Gwen Cole, Nacogdoches, TX.

DNA-based identification of novel bovine casein gene variants

In cattle, at least 39 variants of the 4 casein proteins (αS1-, β-, αS2- and κ-casein) have been described to date. Many of these variants are known to affect milk-production traits, cheese-processing properties, and the nutritive value of milk. They also provide valuable information for phylogenetic studies. So far, the majority of studies exploring the genetic variability of bovine caseins considered European taurine cattle breeds and were carried out at the protein level by electrophoretic techniques. This only allows the identification of variants that, due to amino acid exchanges, differ in their electric charge, molecular weight, or isoelectric point. In this study, the open reading frames of the casein genes CSN1S1, CSN2, CSN1S2, and CSN3 of 356 animals belonging to 14 taurine and 3 indicine cattle breeds were sequenced. With this approach, we identified 23 alleles, including 5 new DNA sequence variants, with a predicted effect on the protein sequence. The new variants were only found in indicine breeds and in one local Iranian breed, which has been phenotypically classified as a taurine breed. A multidimensional scaling approach based on available SNP chip data, however, revealed an admixture of taurine and indicine populations in this breed as well as in the local Iranian breed Golpayegani. Specific indicine casein alleles were also identified in a few European taurine breeds, indicating the introgression of indicine breeds into these populations. This study shows the existence of substantial undiscovered genetic variability of bovine casein loci, especially in indicine cattle breeds. The identification of new variants is a valuable tool for phylogenetic studies and investigations into the evolution of the milk protein genes.

Associations between two novel rSNPs in 5′-flanking region of the bovine casein gene cluster and milk performance traits

Ten evolutionary conservative sequences with high identity level to homological sequences in other mammal species were revealed in 5′-flanking region of casein's genes cluster. Five novel SNPs located inside of the evolutionary conservative regions were identified. The binding sites were revealed to be present in one allelic variant of four detected SNPs. So these SNPs were considered as rSNPs. Significant differences of allelic frequencies were revealed between beef cow's group and dairy cow's group in two rSNPs (NCE4, NCE7, p<0.001). Different alleles of those two rSNPs were shown to be associated with some milk performance traits in Black-and-White Holstein dairy cows. Significant difference of protein percentage has been found between cows with G/G and A/A genotypes (P<0.05) and A/G and A/A genotypes (P<0.05) for NCE4 polymorphism. The groups of animals with genotypes G/G and A/G for NCE7 polymorphism were significantly different in milk yield at the first lactation (kg) (P<0.01), milk fat yield (kg) (P<0.05) and milk protein yield (kg) (P<0.01). For the last trait the difference was significant also between cows with genotypes G/G and A/A for rSNP NCE7 (P<0.05).

Functional analysis of the bovine beta- and kappa casein gene promoters using homologous mammary gland derived cell line

Abstract. Bovine casein gene cluster belongs to the best studied regions of the bovine genome. However, molecular basis of the regulation of casein gene expression is still of great interest for the advancement of milk production. Identification of crucial regulatory regions governing casein gene expression would provide valuable information for marker assisted selection in dairy cattle. In our study we performed comparative analysis of the bovine beta- and kappa casein gene promoter sequences with the regulatory sequences from some other species. In addition, we used homologous mammary gland derived cell culture and luciferase reporter gene system to confirm the functionality of the proximal beta and kappa casein promoters. The longer kappa casein promoter (2064 bp) showed the highest expression level, followed by the short kappa casein promoter (925 bp) and beta casein promoter (1692 bp). Here we demonstrate the suitability of the bovine mammary gland derived cell line BME UV1 for transient gene expression under transcriptional control of the bovine casein gene promoters and compare functionality of different fragments of bovine beta- and kappa casein gene promoters using homologous in vitro system.

Association of nucleotide-sequence polymorphism in the 5’‑flanking regions of bovine casein genes with casein content in cow’s milk

The 5'-flanking regions (promoters) of the bovine αs1-, αs2- and β-casein genes were analysed for DNA sequence variants using PCR/RFLP in Polish Red (PR) and Black-and-White (BW) cattle. The polymorphic sites occurred at positions -1084 and -186 in the promoter region of the αs2-casein gene and at -728 and -109 in the αs1- and β-casein genes, respectively. These poly- morphic sites were located within known potential regulatory sequences, suggesting an influence on the binding of transcription factors and expression of the bovine casein genes. Associations were found between various genotypes in the promoter region of the αs2-casein gene and αs1- and β-casein genotypes, thus showing the existence of intergenic haplotypes within the bovine casein locus. Milk proteins derived from cows of varying genotypes in the casein gene promoters were analysed using SDS-PAGE and HPLC techniques. It was shown that nucleotide sequence polymorphism in the pro- moter region of the bovine αs2-casein gene was associated with various contents of αs2- and β-casein in the milk. Bovine casein gene / 5'-flanking region / gene polymorphism / milk protein

Transcription factor binding to variable nucleotide sequences in 5′-flanking regions of bovine casein genes

Different transcription-factor (TF)-binding sites involved in expression of bovine milk protein genes were identified in their 5′-flanking regions (promoters). Previous studies have identified polymorphism within 5′-flanking regions of bovine milk protein genes resulting from the natural variations in nucleotide sequences. Our computer analysis using the TESS program revealed that in the bovine casein genes at least some of the polymorphic sequences are located in putative TF-binding sites, and thus they may influence gene expression. Moreover, in the 5′-flanking region of the bovine αS1-casein gene two putative mammary gland factor/signal transducer and activator of transcription binding sites were identified differing in the G/A nucleotide substitution. Electrophoretic mobility-shift assays showed quantitative differences in the binding of nuclear proteins to variable sequences in bovine αS1- and αS2-casein gene promoters. The differences in protein binding to variable sites of bovine αS2-casein gene at positions –1101/−1100 and −186 were statistically significant (p<0.05). Results of electrophoretic mobility-shift assays competition experiments suggested that TFs such as activator protein 1, cAMP-response element-binding protein, thyroid receptor and Ying Yang 1 might take part in the formation of DNA–protein complexes. Using reverse transcription PCR method, differences in the transcription rates were found for the αS2-casein gene promoter variants, but not in the case of αS1 gene variants.

Transgenic modification of cows milk for value-added processing.

The application of transgenic technologies in dairy cattle has been restricted largely to producing potential pharmaceutical or nutriceutical products in the mammary gland. Broader application of transgenesis in dairy cattle production will require identifying target traits that are both amenable to transgenic modification and economically important to the dairy industry. The casein proteins are the most valuable component of cows milk destined for value-added processing. The four bovine casein genes lie within a single, multi-gene locus of approximately 200 kb in length. The working hypothesis is that this multi-gene locus contains all of the DNA sequences required to regulate the coordinated expression of all four individual casein genes (i.e. a locus control region or LCR). The initial research aim is to clone the entire casein locus into a bacterial artificial chromosome (BAC) vector, thus preserving the extended 5'and 3' regions that flank the locus, as well as maintaining the spatial integrity of the four individual casein genes that comprise the locus. The author's laboratory has prepared a bacterial artificial chromosome (BAC) library of genomic DNA from elite dairy cattle. Partial, non-elite BAC clones of the casein gene locus are being tested in transgenic mice to establish proof of concept. Advances in nuclear transfer of transfected somatic cells should improve the efficiency of producing transgenic calves that possess a BAC casein construct introduced into an elite genetic background.

Restriction map of two yeast artificial chromosomes spanning the murine casein locus.

The caseins are the major mammalian milk proteins (reviewed by Mercier and Vilotte 1993), constituting a dietary supply of amino acids and calcium to the infant. In the presence of calcium, the so-called calcium-sensitive caseins of the a and [3 types form loose, non-crystalline aggregates termed micelles, which are stabilized by calcium-insensitive K casein. In the gut the site-specific cleavage of K casein by rennin causes the milk to clot and remain in the stomach, facilitating digestion. The caseins are encoded by a small gene family, which in cows and sheep consists of four members, ~X~l, [3, oLs2, and K, and in mice and rats five, c~, [3, 7, ~, and K (Yu-Lee and Rosen 1983; Jones et al. 1985; Thompson et al. 1985). The casein genes all map to a single chromosome in rodents, sheep, cows, humans, and pigs (reviewed by Mercier and Villotte, 1993) and are very tightly linked genetically. All four bovine casein genes have been mapped to a single 250-kb locus (Ferretti et al. 1990; Threadgill and Womack 1990). This close linkage might be expected in the case of the evolutionarily closely related calcium-sensitive caseins, but there is no evidence that K casein is evolutionarily related to the other caseins. Both in sequence homology and protein function it appears to be related to -/ fibrinogen (Jolles et al. 1974; Thompson et al. 1985; Alexander et al. 1988), which performs a cleavage-induced clotting function in blood similar to the clotting function of K casein in the stomach. Therefore, the proximity of the bovine K casein gene to the other casein genes is noteworthy. Two yeast artificial chromosome (YAC) clones bearing the five murine caseins were obtained by screening the Imperial Cancer Research Fund mouse genomic YAC library, primarily with two genomic probes for murine [3 casein as described (Cox et al. 1993) and secondarily by screening [3 casein-positive clones with cDNA probes for murine e~, 7, e, and K casein, by colony hybridization and pulsed field gel electrophoresis (PFGE). The two c lones are I C R F y 9 0 2 G 0 7 8 1 , r enam ed MP12, and ICRFy902CI 1116, renamed MPI4. MP14 is approximately 380 kb in size, and MP12 is approximately 435 kb in size, as determined by PFGE and Southern blotting (data not shown). A restriction map of the two YACs was generated by partial digestion with PmeI, SaII, XhoI and ClaI, and probing a single filter with probes specific to the left and right arms of pYAC4 (LA and RA; Fig. 1). The restriction fragments detected by LA and RA are listed in Table 1. When the MP12 and MP14 PmeI band patterns are represented as maps, the two maps can be aligned so that the 121-kb MP14 band is lined up with the 105-kb MP12 band. The alignment is almost perfect over the region of overlap, varying by less than 5 kb. By this analysis, the 26-kb MP14 band should fall some 10 kb from the left telomere of MP12 and therefore give a band in the MP12 lanes, but, given that there are 6 kb

Structure and Function of Milk Protein Genes

Interspecies comparisons of cDNA and mosaic milk protein genes have confirmed their high rate of evolution, but the overall gene organization has been conserved. The three Ca-sensitive casein genes, which share common motifs in the promoter region and contain similar sequences that encode signal peptide and multiple phosphorylation sites, probably derived from a common ancestor. αsl-and αs2-casein genes, divided into many small exons, undergo complex splicing, and the deleted caseins arise from exon skipping. The four bovine casein genes are clustered on 200 kb of chromosome 6. α-Lactalbumin and β-lactoglobulin pseudogenes occur in ruminants.Study of the expression of native and modified milk protein genes in mammary cell lines and transgenic animals and DNA footprinting have shown the occurrence of important regulatory motifs in the proximal 5’ flanking region, including one recognized by a specific mammary nuclear factor. Good stageand tissue-specific expression has been obtained in transgenic animals with milk protein genes having less than a 3-kb 5’ flanking region.Better knowledge of both the structure and function of milk protein genes, which has already allowed the use of powerful techniques for the rapid identification of alleles, offers the potential for the genetic modification of milk composition.

A Simple and Powerful Method for Linkage Analysis by Amplification of DNA from Single Sperm Cells

Haplotypes of three bovine casein loci were analyzed as a way of determining genetic linkage. A total of 330 individual sperm cells from a triply heterozygous bull were selected by means of a new method involving fixing the sperm cells into low-melting-point agarose gels. The method is simple and very accurate with an efficiency close to 100% for picking sperm cells and producing amplifiable DNA templates, circumventing the complicated statistical analysis mandated by automated cell sorting. The DNA was amplified in a two-step polymerase chain reaction (PCR) to achieve the necessary high specificity of amplification. In the first reaction, primers flanking the polymorphic site at each locus were used. The PCR product from the first reaction was then reamplified in a second PCR using primers that create allele-specific restriction sites (ACRS) in the PCR products for two of the three loci, allowing the alleles to be determined by gel electrophoresis. No recombinants were found among the 330 single sperm cells analyzed, giving a lod score higher than 30 and proving a very strong linkage between bovine casein genes.

Analysis of polymorphism in the bovine casein genes by use of the polymerase chain reaction

Methods have been devised for detecting polymorphisms in the bovine beta- and kappa-casein genes using the polymerase chain reaction (PCR) followed either by restriction enzyme digestion (to reveal a restriction fragment length polymorphism (RFLP] or by hybridization of an allele-specific oligonucleotide. These methods, as well as being faster and more sensitive than traditional RFLP methods, are of more general applicability since they can detect any change in DNA sequence. They require only a small sample of blood or semen and are applicable to animals of any age or sex. These methods make possible large-scale screening and thus selection for alleles at these loci. Typing of blood DNA can give erroneous results when the animal concerned is a twin; however, this can be overcome by retesting using milk or semen. Analysis of the kappa-casein genotype of Holstein-Friesian bulls gives frequencies for the A and B alleles of 0.80 and 0.20 respectively. Selection in favour of the B allele, which is superior for cheese production, could thus have a large effect. The A3 and B alleles at the beta-casein locus have been shown to be rare in the Holstein-Friesian population. Linkage disequilibrium exists between beta-casein B and kappa-casein B.

The potential for improving sire selection in dairy cattle by molecular genetic analysis of bovine casein genes

Population studies have shown useful correlations between alleles at the casein loci and important production parameters such as compositional characteristics of milk, cheese yield and lactation yield. Molecular genetic analysis of genotype at the casein loci could therefore enhance sire selection in dairy herds. We are Investigating polymorphisms in K-casein, where the B-variant gives improved cheesemaMng and in β-casein, where the A3 allele is associated with improved lactation yields. We have previously reported a restriction fragment length polymorphism in the K-casein gene, enabling us to identify the alleles at this locus [Perry, B.N., Savva, D., Radley, E., Skldmore, C.J. &amp; Lovell, R.D. (1989) Anim. Prod. 48, 661]. Our current work makes use of the polymerase chain reaction (PCR) to increase dramatically the speed of di agnosis.

Long range restriction analysis of the bovine casein genes

Pulsed field gel electrophoresis (PFGE) was used to analyse the organization of the bovine alpha s1, alpha s2, beta and kappa casein genes. High molecular weight DNA was prepared from fibroblasts and lymphocytes embedded in agarose and was digested with the restriction endonucleases Clal, Sall, Smal, Xhol. The digestion products were separated by PFGE, transfered to nitrocellulose filters and hybridized to probes corresponding to the cDNAs of the four bovine casein genes. The casein genes were demonstrated to be physically linked within a region of 300 kb, represented by two adjacent Xhol fragments in fibroblasts and by a single fragment in lymphocytes. A restriction map of the casein locus was derived and the order of the genes was shown to be kappa, alpha s2, beta, alpha s1.