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.     

Multiple mRNA species code for the catalytic subunit of the cAMP-dependent protein kinase from LLC-PK1 cells. Evidence for two forms of the catalytic subunit

We present evidence for the existence of two forms of the catalytic (C) subunit of the cAMP-dependent protein kinase. A lambda gt-11 cDNA library constructed from poly(A)-rich RNA from the porcine kidney cell line, LLC-PK1, was screened using a 1.5-kb EcoRI fragment from a bovine cDNA for the C subunit. Two independent classes of cDNAs were identified on the basis of partial restriction map and sequence data. These two cDNAs, lambda CAT4 and lambda CAT3, apparently encode two forms of C subunit designated C alpha and C beta, respectively. The nucleotide sequence of the C alpha and C beta cDNAs revealed differences in the coding region and particularly in the 3' untranslated region. However, the deducted amino acid sequences of C alpha and C beta subunits were 96% homologous to the sequences so far determined. Specific probes from the 3' coding region of the two cDNA species were used to investigate C subunit mRNA expression in LLC-PK1 cells. Northern analysis showed a major mRNA species of 2.8 kb with the C alpha probe while the C beta probe detected two mRNA species of 5.0 kb and 3.8 kb. These data were supported by genomic blot analysis which showed distinct hybridization patterns with either the C alpha or C beta probes. All the available evidence suggests that at least two distinct genes encode the C subunit which are expressed in LLC-PK1 cells.     

Multiple octamer binding sites in the promoter region of the bovine alpha s2-casein gene.

Using a set of overlapping oligonucleotides from the promoter region of the bovine alpha s2-casein gene we have identified two nuclear factors which probably are involved in expression of this gene and the related calcium sensitive alpha s1- and beta-casein genes. One of these factors which was present in extracts of all tissues that have been tested including Hela cells turned out to be the octamer binding protein OCT-1. Oct-1 binds with different affinity to 4 sites at positions centred around -480, -260, -210 and -50. The strongest of these 4 binding sites, the one around position -50, is highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle. The other nuclear factor (MGF, mammary gland factor) which is specifically expressed in the mammary gland, binds to a site around position -90. This binding site is also highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle.     

Identification of two distinct antibacterial domains within the sequence of bovine alpha(s2)-casein.

Two distinct domains with antibacterial activity were isolated from a peptic hydrolysate of bovine alpha(s2)-casein. The digested alpha(s2)-casein was fractionated by cation-exchange chromatography, after which the peptides in the two active fractions obtained were separated by high-performance liquid chromatography and sequenced by electrospray-ionization tandem mass spectrometry. The major component in each active fraction, f(183-207) and f(164-179), was further purified and the antibacterial activity of these components was tested against several microorganisms. Depending on the target bacterial strain, these peptides exhibited minimum inhibitory concentrations between 8 and 99 microM. Peptide f(183-207) exhibited a consistently higher antibacterial activity than f(164-179), although both peptides showed a comparable hemolytic effect. A method of in situ enzymatic hydrolysis on a cation-exchange membrane to obtain a fraction enriched in the most active antibacterial domain is presented. The antibacterial and hemolytic activities are discussed in relation to the structure and hydrophobicity of the peptides.     

Amyloid fibril formation by bovine milk alpha s2-casein occurs under physiological conditions yet is prevented by its natural counterpart, alpha s1-casein

The calcified proteinaceous deposits, or corpora amylacea, of bovine mammary tissue often comprise a network of amyloid fibrils, the origins of which have not been fully elucidated. Here, we demonstrate by transmission electron microscopy, dye binding assays, and X-ray fiber diffraction that bovine milk alpha s2-casein, a protein synthesized and secreted by mammary epithelial cells, readily forms fibrils in vitro. As a component of whole alpha s-casein, alpha s2-casein was separated from alpha s1-casein under nonreducing conditions via cation-exchange chromatography. Upon incubation at neutral pH and 37 degrees C, the spherical particles typical of alpha s2-casein rapidly converted to twisted, ribbon-like fibrils approximately 12 nm in diameter, which occasionally formed loop structures. Despite their irregular morphology, these fibrils possessed a beta-sheet core structure and the ability to bind amyloidophilic dyes such as thioflavin T. Fibril formation was optimal at pH 6.5-6.7 and was promoted by higher incubation temperatures. Interestingly, the protein appeared to be less prone to fibril formation upon disulfide bond reduction with dithiothreitol. Thus, alpha s2-casein is particularly susceptible to fibril formation under physiological conditions. However, our findings indicate that alpha s2-casein fibril formation is potently inhibited by its natural counterpart, alpha s1-casein, while is only partially inhibited by beta-casein. These findings highlight the inherent propensity of casein proteins to form amyloid fibrils and the importance of casein-casein interactions in preventing such fibril formation in vivo.     

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.     

Structure of the gene encoding rabbit alpha s1-casein.

The complete nucleotide sequence of the entire rabbit alpha s1-casein-encoding gene Aslca and its flanking regions was determined. These data represent the first complete primary sequence of an Aslca gene. The gene consists of 19 exons spread over 16 kb. Highly conserved sequences were found between this gene and other casein-encoding genes mainly upstream from the gene from position -180 to -10. Several repeated interspersed elements of unknown function were also identified within introns.     

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.     

Electro-membrane filtration for the selective isolation of bioactive peptides from an alpha(s2)-casein hydrolysate

For the isolation of the ingredients required for functional foods and nutraceuticals generally membrane filtration has too low a selectivity and chromatography is (too) expensive. Electro-membrane filtration (EMF) seems to be a breakthrough technology for the isolation of charged nutraceutical ingredients from natural sources. EMF combines the separation mechanisms of membrane filtration and electrophoresis. In this study, positively charged peptides with antimicrobial activity were isolated from an alpha(s2)-casein hydrolysate using batch-wise EMF. alpha(s2)-Casein f(183-207), a peptide with strong antimicrobial activity, predominated in the isolated product and was enriched from 7.5% of the total protein components in the feed to 25% in the permeate product. With conventional membrane diafiltration using the same membrane (GR60PP), isolation of this and other charged bioactive peptides could not be achieved. The economics of EMF are mainly governed by the energy costs and the capital investment, which is affected by the flux of the desired peptide. A maximum average transport rate of alpha(s2)-casein f(183-207) during batch-wise EMF of 1.2 g/m2. h was achieved. Results indicate that an increase in the hydrolysate (feed) concentration, the applied potential difference and the conductivity of the permeate and electrode solutions, and a reduction in the conductivity of the feed result in a higher transport rate of alpha(s2)-casein f(183-207). This is in line with the expectation that the transport rate is improved when the concentration, the electrical field strength, or the electrophoretic mobility is increased, provided that the electrophoretic transport predominates. The expected energy consumption of the EMF process per gram of peptide transported was reduced by approximately 50% by applying a low overall potential difference and by processing desalinated hydrolysate. Considerable improvements in transport rate, energy efficiency, and process economics seem to be attainable by additional optimization of the process parameters and the EMF module design.     

Multiple calmodulin mRNA species are derived from two distinct genes.

We have observed three calmodulin mRNA species in rat tissues. In order to know from how many expressed genes they are derived, we have investigated the genomic organization of calmodulin genes in the rat genome. From a rat brain cDNA library, we obtained two kinds of cDNAs (pRCM1 and pRCM3) encoding authentic calmodulin. DNA sequence analysis of these cDNA clones revealed substitutions of nucleotides at 73 positions of 450 nucleotides in the coding region, although the amino acid sequences of these calmodulins are exactly the same. DNA sequences in the 5' and 3' noncoding regions are quite different between these two cDNAs. From these results, we conclude that they are derived from two distinct bona fide calmodulin genes, CaMI (pRCM1) and CaMII (pRCM3). Total genomic Southern hybridization suggested four distinct calmodulin-related genes in the rat genome. By cloning and sequencing the calmodulin-related genes from rat genomic libraries, we demonstrated that the other two genes are processed pseudogenes generated from the CaMI (lambda SC9) and CaMII (lambda SC8) genes, respectively, through an mRNA-mediated process of insertions. Northern blotting showed that the CaMI gene is transcribed in liver, muscle, and brain in similar amounts, whereas the CaMII gene is transcribed mainly in brain. S1 nuclease mapping indicated that the CaMI gene produced two mRNA species (1.7 and 4 kilobases), whereas the CaMII gene expressed a single mRNA species (1.4 kilobases).     

Multiple mRNA species for adenovirus type 2 polypeptides III and pVII.

Fractionation of messenger activities isolated from the cytoplasm of HeLa cells late in infection with adenovirus type 2 reveals that viral polypeptides III and pVII are each synthesized from two different-sized mRNA's. the major messenger activity for each protein has the same sedimentation rate as that previously reported by Anderson et al. (Proc. Natl. Acad. Sci. U.S.A. 71:2756-2760, 1974). The minor messenger activities for III and pVII sediment more rapidly and are not aggregates of the major mRNA's for these proteins. The two minor messenger activities cosediment with two polyadenylated RNA species which are labeled late in infection with 32P and whose molecular weights are estimated to be 2.9 x 10(6) and 2.4 x 10(6). Both of these species hybridize to adenovirus type 2 DNA specific for the mRNA family that is 3' coterminal at adenovirus type 2 map position 49.5 and the mRNA family that is 3' coterminal at 62.0. This is consistent with the possibility that these RNAs have 5'-terminal sequences identical to those of the normal mRNA's for III and pVII but are 3' coterminal at map position 62, the normal 3' terminus of the mRNA's for polypeptides II and pVI. These species are not found in polyadenylated RNA isolated from the nucleus, suggesting that the minor mRNA species are cytoplasmic RNAs.     

Localization of two interchain disulfide bridges in dimers of bovine alpha s2-casein. Parallel and antiparallel alignments of the polypeptide chains.

Carboxymethylation of bovine skimmed milk with 14C-labelled iodoacetic acid followed by purification of the alpha s2-casein dimer showed that all four cysteine residues in the protein are engaged in disulfide linkages. Mass spectrometry and sequence analysis of cystine-containing tryptic peptides revealed the presence of two interchain disulfide bridges in the protein. Sequence analysis of disulfide-linked peptides resulting from an enzymatic cleavage between the bridges demonstrated that the individual chains in the dimers are either aligned in an antiparallel or a parallel orientation. The identity of some of the disulfide-linked peptides was further verified by performic acid oxidation followed by sequence analysis of the resulting peptides.     

Multiple classes of prostaglandin F2 alpha binding sites in subpopulations of ovine luteal cells

A cryostorage procedure was developed to provide ovine luteal cells throughout the period of seasonal anestrus. Corpora lutea obtained from midluteal phase, superovulated ewes were dispersed enzymatically. Some dispersed cells were fractionated into subpopulations by elutriation. Dimethylsulfoxide (7.5% final concentration) in Hanks' buffered saline was added to cells at 4 degrees C, and dispersed cell preparations were frozen in a programmable cell freezer and stored at -196 degrees C. After recovery from cryopreservation, cell viability and prostaglandin F2 alpha (PGF2 alpha) binding characteristics of thawed cells were not different from those of corresponding fresh cells. Additionally, thawed cells retained the capacity to attach to culture dishes and retained responsiveness of progesterone secretion to prostaglandin E2 (PGE2) and ovine luteinizing hormone (LH), although rates of progesterone secretion were attenuated in thawed compared with fresh cells. The cryopreservation procedure will prove useful to relieve constraints in utilization of ovine luteal cells arising from reproductive seasonality in sheep. Cells retrieved from cryostorage were evaluated by studying PGF2 alpha binding characteristics. From saturation analyses (increasing amounts of radiolabeled PGF2 alpha) of PGF2 alpha binding to unfractionated cells, we detected a single class of high affinity binding sites (Kd = 17.4 +/- 2.3 nM) in addition to the nonspecific binding component. Using displacement analyses (constant radiolabeled PGF2 alpha and increasing amounts of unlabeled PGF2 alpha) and unfractionated cells, we detected additional binding sites of lower affinity (Kd = 409 +/- 166 nM) as well as the nonspecific binding component. Small luteal cells obtained by elutriation, which were essentially devoid of large cell contamination, had only low affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Molecular chaperone-like properties of an unfolded protein, alpha(s)-casein.

All molecular chaperones known to date are well organized, folded protein molecules whose three-dimensional structure are believed to play a key role in the mechanism of substrate recognition and subsequent assistance to folding. A common feature of all protein and nonprotein molecular chaperones is the propensity to form aggregates very similar to the micellar aggregates. In this paper we show that alpha(s)-casein, abundant in mammalian milk, which has no well defined secondary and tertiary structure but exits in nature as a micellar aggregate, can prevent a variety of unrelated proteins/enzymes against thermal-, chemical-, or light-induced aggregation. It also prevents aggregation of its natural substrates, the whey proteins. alpha(s)-Casein interacts with partially unfolded proteins through its solvent-exposed hydrophobic surfaces. The absence of disulfide bridge or free thiol groups in its sequence plays important role in preventing thermal aggregation of whey proteins caused by thiol-disulfide interchange reactions. Our results indicate that alpha(s)-casein not only prevents the formation of huge insoluble aggregates but it can also inhibit accumulation of soluble aggregates of appreciable size. Unlike other molecular chaperones, this protein can solubilize hydrophobically aggregated proteins. This protein seems to have some characteristics of cold shock protein, and its chaperone-like activity increases with decrease of temperature.     

Interallelic recombination is probably responsible for the occurrence of a new alpha(s1)-casein variant found in the goat species.

The alphas1-casein (alphas1-Cas) locus in the goat is characterized by a polymorphism, the main feature of which is to be qualitative as well as quantitative. A systematic analysis performed in an autochthon southern Italy breed identified a new rare allele (M), which was characterized at both the protein and genomic level. The M protein displays the slowest electrophoretic mobility of the alphas1-Cas variants described so far. MS and automated Edman degradation experiments showed that this behavior was due to the loss of two phosphate residues in the multiple phosphorylation site (64SP-SP-SP-SP-SP-E-70E) consecutively to a Ser-->Leu substitution at position 66 of the peptide chain (64S-SP-L-SP-SP-E-70E). This was confirmed by sequencing a genomic DNA fragment encompassing exon 9 where the 8th codon (TCG) was shown to be mutated to TTG. Sequencing of amplified genomic DNA segments spanning the 5' and 3' flanking regions of each exon allowed us to identify 23 single nucleotide polymorphisms and two insertion/deletion events in the coding as well as the noncoding regions. A comparison of specific haplotypes defined for each of the alphas1-CasF, A and M alleles indicates that the M allele probably arises from interallelic recombination between alleles A and B2, followed by a C-->T transition at nucleotide 23 of the ninth exon. The region encompassing the recombination break point was putatively located between nucleotide 86 upstream and nucleotide 40 downstream of exon 8. Interallelic recombination therefore appears to be a possible means of generating allelic diversity at the alphas1-Cas locus, at least in the goat. The previously proposed molecular phylogeny must now be revised, possibly starting from two ancestral allelic lineages.     

Rapid screening of antigenically reactive fragments of alpha s1-casein using HPLC and ELISA

Screening of antigenically reactive fragments of alpha S1-casein (alpha S1-CN), the major casein in bovine milk, was done by using HPLC and enzyme-linked immunosorbent assay (ELISA). BALB/c mice (6-week-old) were injected intraperitoneally with alpha S1-CN and complete Freund's adjuvant, and 14 days later, all the mice were boosted with alpha S1-CN and incomplete Freund's adjuvant. Twenty-one days after the 1st immunization, the mice were bled and antiserum was separated. Anti alpha S1-CN antibody fraction was obtained by precipitation from the antiserum with 50% saturated ammonium sulfate. alpha S1-CN was digested with trypsin and chymotrypsin, and 35 peptides were purified from the digests by reversed-phase HPLC with ODS (octadecylsilica) columns. Reactivity of peptides with the antibody were examined by ELISA. The solid phase in the wells of the polystyrene microtiter plate was coated with peptides, and the plate was successively incubated with anti alpha S1-CN antibody, conjugate of anti mouse immunoglobulin with alkaline phosphatase (ALP) and substrate of ALP. Two tryptic fragments (the residues 104-119 and 133-151) and three chymotryptic fragments (33-54, 105-121, and 174-199) were positive in an ELISA test. These five fragments would correspond to four antigenic sites. We could thus find antigenically reactive fragments of alpha S1-CN by the direct and simple detection of specific antigen-antibody interaction.