Characterization of two new alleles at the goat CSN1S2 locus

Two novel alleles at the goat CSN1S2 locus have been identified: CSN1S2(F) and CSN1S2(D). Sequence analyses revealed that the CSN1S2(F) allele is characterized by a G --> A transition at the 13th nucleotide in exon 3 changing the seventh amino acid of the mature protein from Val to Ile. The CSN1S2(D) allele, apparently associated with a decreased synthesis of alpha s2-casein, is characterized by a 106-bp deletion, involving the last 11 bp of the exon 11 and the first 95 bp of the following intron. Methods (PCR-RFLP and PCR) for identification of carriers of these alleles have been developed.     

Molecular cloning and characterization of buffalo alpha(s1)-casein gene.

Buffaloes in Indian subcontinent play an important role as the producer of milk and milk products. The alpha(s1)-casein constitutes 38% of the total milk proteins. The present study was carried out to characterize the gene in Murrah breed of Riverine buffalo. Buffalo alpha(s1)-casein cDNA was synthesized by RT-PCR, then cloned using pDRIVE-cloning vector and sequenced. The sequencing revealed that the size of alpha(s1)-casein cDNA was of 645 bp with GC content of 45.58%. The alpha(s1)-casein gene coded 214 amino acids precursor with a signal peptide of 15 amino acid residues. The similarity of buffalo alpha(s1)-casein mRNA sequence with that of cattle, goat, sheep, pig, camel, equine and human were estimated as 97.2, 93, 92.3, 57.2, 59.5, 55.9 and 46.6%, respectively. A similar trend was observed when compared amino acid sequences of these species. In the phylogenetic trees, constructed from the data of the alpha(s1)-casein mRNA as well as protein sequences, it has been observed that buffalo, cattle, goat and sheep formed a cluster with a closer relationship between cattle and buffalo followed by goat and sheep.     

An allele associated with a non-detectable amount of αs2 casein in goat milk

The goat CSN1S2 locus is characterized by the presence of three alleles, A, B and C, all associated with about 2.5 g/l of protein per allele. The SDS-PAGE analysis of 441 individual milk samples obtained from goats belonging to a population reared in Southern Italy showed that the milk produced by three goats did not apparently contain alpha s2-casein, whereas milk produced by 37 goats showed a less intense electrophoretic band of this casein fraction (about 50%). These results can be explained by hypothesizing the presence of another allele at this locus, CSN1S2o, associated with a 'null' content of alpha s2-casein. Southern blot, PCR and PCR-RFLP analyses of the DNA region containing the CSN1S2 gene of individuals producing milk with and without alpha s2-casein did not show differences between the two groups. As a consequence, goats producing milk without alpha s2-casein carry an apparently intact gene. The first results obtained by sequencing part of the CSN1S2o allele revealed a G-->A transition at nucleotide 80 of the 11th exon which creates a stop codon and could be responsible for the absence of the alpha s2-casein in goat milk. This mutation eliminates a NcoI restriction site. A test based on this polymorphism has been established in order to identify carriers of the CSN1S2o allele.     

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.     

Evolution of the casein multigene family: conserved sequences in the 5'' flanking and exon regions.

The rat alpha- and bovine alpha s1-casein genes have been isolated and their 5' sequences determined. The rat alpha-, beta-, gamma- and bovine alpha s1-casein genes contain similar 5' exon arrangements in which the 5' noncoding, signal peptide and casein kinase phosphorylation sequences are each encoded by separate exons. These findings support the hypothesis that during evolution, the family of casein genes arose by a process involving exon recruitment followed by intragenic and intergenic duplication of a primordial gene. Several highly conserved regions in the first 200 base pairs of the 5' flanking DNA have been identified. Additional sequence homology extending up to 550 base pairs upstream of the CAP site has been found between the rat alpha- and bovine alpha s1-casein sequences. Unexpectedly, the 5' flanking promoter regions are conserved to a greater extent than both the entire mature coding and intron regions of these genes. These conserved 5' flanking sequences may contain potential cis regulatory elements which are responsible for the coordinate expression of the functionally-related casein genes during mammary gland development.     

Cloning and characterization of alpha(s2)-casein gene of Riverine buffalo.

The present study was carried out to characterize the alpha(s2)-casein gene in Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and alpha(s2)-casein cDNA were synthesized by RT-PCR, then cloned using pDRIVE-cloning vector and sequenced. The sequencing revealed that the size of alpha(s2)-casein was 669 bp with GC content of 41.11%. The gene encoded for 222 amino acid precursors and that it possessed 15 amino acids signal peptide. The similarity of buffalo alpha(s2)-casein mRNA sequence with that of cattle, sheep, goat, pig and camel were estimated as 97.9, 93.6, 93.4, 73.5 and 73.0%, respectively. In the phylogenetic trees, constructed from the data of the alpha(s2)-casein mRNA sequences as well as protein sequences, it has been observed that the cattle and buffalo were in the same group whereas sheep and goat formed another group. The camel and swine were placed in two separate groups.     

Structure of the human ornithine transcarbamylase gene

Complementary and genomic DNA clones corresponding to the human ornithine transcarbamylase (OTC) [EC 2.1.3.3]mRNA have been isolated and analyzed. The OTC gene is about 73 kilobase pairs (kb) long and contains 10 exons interrupted by 9 introns of highly variable sizes. The smallest intron is 80 base pairs and the largest, 21.7 kb. The 5'- and 3'-flanking regions, entire exons and all the exon/intron boundaries were sequenced. The nucleotide and deduced amino acid sequences of isolated OTC cDNAs as well as the corresponding regions of the genomic DNA were compared with those of human OTC cDNA (Horwich, A.L., Fenton, W.A., Williams, K.R., Kalousek, F., Kraus, J.P., Doolittle, R.F., Koningsberg, W., & Rosenberg, L.E. (1984) Science 224, 1068-1074). We found 20 nucleotide substitutions among these sequences, of which 6 related to amino acid changes. The nature of these nucleotide substitutions is 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 α_s1-casein and of β-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 β-elimination/thiol derivatization. Water buffalo α_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 α_s1-casein C variant, the water buffalo α_s1-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine βA₂-casein variant, the two water buffalo β-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 β-casein variants seem to be homologous to bovine βA₂-casein.     

Human alpha 3(VI) collagen gene. Characterization of exons coding for the amino-terminal globular domain and alternative splicing in normal and tumor cells

We recently reported the isolation and sequencing of human cDNA clones corresponding to the alpha 3 chain of type VI collagen (Chu, M.-L., Zhang, R.-Z., Pan, T.-c., Stokes, D., Conway, D., Kuo, H.-J., Glanville, R., Mayer, U., Mann, K., Deutzmann, R., and Timpl, R. (1990) EMBO J. 9, 385-393). The study indicates that the amino-terminal globular domain of the alpha 3(VI) chain consists of nine repetitive subdomains of approximately 200 amino acid residues (N1-N9) and the gene appeared to undergo alternative splicing since some clones lacked regions encoding the N9 and part of the N3 subdomains. In the present study, we report the exon structure for the region encoding the amino-terminal globular domain of the human alpha 3(VI) chain. The nine repetitive subdomains are encoded by 10 exons spanning 26 kilobase pairs of genomic DNA. Eight of the repetitive subdomains (N2-N9) were found to be encoded by separate exons of approximately 600 base pairs each. The only exception is the N1 subdomain which is encoded by two exons of 417 and 146 base pairs. Characterization of the exon/intron structure showed that the cDNA variants were the result of splicing out of exon 9 (encoding the N9 subdomain) and part of exon 3 (encoding the N3 subdomain). Nuclease S1 analysis and the polymerase chain reaction demonstrated that exon 7 (N7 subdomain) was also subject to alternative splicing in normal skin fibroblasts. Examination of these splicing events by nuclease S1 analysis in normal fibroblasts, three different human tumor cell lines, and several human tissues showed that splicing out of exon 9 is much more efficient in normal as compared to tumor cells.     

Complete sequence of ovine alpha s2-casein messenger RNA

The primary structure of mRNA coding for ovine alpha s2 casein has been determined by chemical sequencing of three cDNA clones and the primer extension products of the longest one. The mRNA was 1,024 nucleotides long, excluding the poly(A) tail. The length of the 5' noncoding, coding and 3' noncoding regions was 53, 669 and 302 nucleotides, respectively. A comparison of the nucleotide sequence of ovine alpha s2-casein and guinea-pig casein A mRNAs revealed an extensive homology in the 5' and 3' noncoding regions. The deduced amino acid sequence of ovine alpha s2-casein was compared with its bovine and guinea-pig counterparts. Moreover, an heterogeneity was evidenced in the mRNA population of the alpha s2-casein.     

Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms

We report an allele-specific sequencing method using allele-specific long-range polymerase chain reaction (PCR) to determine if multiple (specifically, more than three) single nucleotide polymorphisms (SNPs) are located on the same allele. We sequenced the glucocorticoid receptor (GR) gene as a model and detected four nucleotide changes, including two novel variations, in intron 4 and exons 6, 8, and 9 alpha in four of the investigated cell lines. The terminal SNPs (intron 4 and exon 9 alpha) were separated by 19 kb. Following SNP identification, the first round PCR allele-specific primers are designed at the both distal SNP sites (intron 4 and exon 9 alpha), placing the SNP positions at the primer 3'-end. Using these first round PCR products as template, the second round PCR was performed to separately amplify exons 6 and 8. These second round PCR products were subsequently sequenced. The sequencing results showed that the four SNPs were located on the same allele, i.e., forming a haplotype. This allele-specific long-range PCR/sequencing (ALP/S) method is rapid and applicable to the allelic assignment for more than three SNPs.     

A systematic genetic polymorphism analysis of the CYP2C9 gene in four different geographical Han populations in mainland China

While many studies have been focused on CYP2C9*2 and *3 there was a lack of large full gene sequencing on CYP2C9, and this study was designed to fill this gap. We used direct sequencing to systematically screen genetic polymorphisms of the CYP2C9 gene including the 5' -flanking region (2kb), all exons and their adjoining intron regions and the 3' UTR in 400 unrelated healthy Chinese Han volunteers. A total of 27 different CYP2C9 polymorphisms were identified, 3 of which were novel, including one in intron 6, a synonymous variant (1137T>C, Tyr379Tyr), and a deletion mutation in the 3'UTR (1739-1740ATdel), which potentially influences the stability of CYP2C9 mRNA. We identified CYP2C9*1, *2, *3, *8, *11, and *31, of which alleles *8 was identified for the first time in Chinese population while *11 first in Asian. This is the first systematic screening of genetic polymorphisms of CYP2C9 in the Chinese Han population.     

Proteomic tools to characterize the protein fraction of Equidae milk

The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, with or without tryptic digestion, on a matrix-assisted laser desorption/ionization-time of flight spectrometer. Based on the sequence and molecular mass information obtained, identifications were achieved through a protein database search using homology or pattern research algorithms. This methodological approach was shown to be rapid, efficient and reliable in identifying the principal proteins in pony mare's milk. kappa-, alpha(s1)-, alpha(s2)-, and beta-casein, lysozyme C, alpha-lactalbumin and beta-lactoglobulin I and II were thus identified. alpha(s1) and beta-caseins displayed polymorphic patterns, probably due to alternative splicing processes leading to casual exon skipping events involving exons 7 and 14 in alpha(s1)-casein and exon 5 in beta-casein. Edman N-terminal microsequencing over 35 amino acid residues, for pony alpha(s1)-casein, clearly demonstrated the occurrence, in Equidae, of a splicing pattern similar to that reported in rodents, characterized by the constitutive outsplicing of exon 5. Pony mare's milk SDS-PAGE and RP-HPLC patterns were compared with those obtained for other milks (cow, goat and human), as were the relative levels of caseins and major whey proteins in these milks. Our results provide further evidence to support the notion that Equidae milk is closer to human breast milk than milk from bovine and caprine with respect to the casein and lysozyme C contents and casein/whey proteins ratio.     

Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein.

The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.