Rat beta casein cDNA: sequence analysis and evolutionary comparisons.

The complete sequence of a 1072 nucleotide rat beta-casein cDNA insertion in the hybrid plasmid pC beta 23 has been determined. Primer extension was employed to determine the sequence of an additional 82 5'-terminal nucleotides in beta-casein mRNA. Rat beta-casein mRNA consists of a 696 nucleotide coding region, flanked by 52 nucleotide 5' and 406 nucleotide 3' noncoding regions, including a 40 nucleotide poly(A) tail. The derived 216 amino acid sequence of rat beta-casein was compared to the previously determined sequences of beta-caseins from several other species. Approximately 38% of the amino acids have been conserved among the rat, ovine, bovine and human sequences and these conserved amino acids occurred in clusters throughout the protein. One such cluster containing the majority of the potential casein phosphorylation sites was located near the amino terminus. Contrary to the considerable divergence observed for the processed beta-casein, 14 of 15 amino acids in the signal peptide sequence of the precasein were identical between the rat and ovine caseins.     

Separation and partial characterization of guinea-pig caseins.

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.     

The structure and expression of the guinea pig Fc receptor for IgG1 and IgG2 (Fc gamma 1/gamma 2R).

A cDNA clone encoding the receptor for guinea pig immunoglobulin G was isolated from a guinea pig peritoneal macrophage cDNA library. The cloned cDNA encoded 271 amino acids containing an N-terminal signal sequence. The deduced amino acid sequence is most homologous to murine Fc gamma RII beta 2. The receptor protein could be expressed in COS-7 and L cells transfected with the cDNA, suggesting that the expression of this receptor does not require the co-expression of a second chain such as gamma chain of Fc epsilon RI or CD3 zeta chain. The transformant L cells showed the binding to both the guinea pig IgG1 and IgG2 antibodies complexed with antigen, indicating that the cDNA we cloned was the one for guinea pig Fc gamma 1/gamma 2R.     

Identification of two amino acid residues in the epsilon subunit that promote mammalian muscle acetylcholine receptor assembly in COS cells

We have used a species difference in epsilon subunits of the acetylcholine receptor (AChR) to investigate regions of the subunit protein that are important in receptor assembly. Upon transient transfection of COS cells, mouse epsilon subunit cDNA is approximately 10 times more effective than that of the rat in supporting expression of surface AChRs when the other subunits are from either mouse or rat. In cells transfected with only alpha and epsilon subunit cDNAs, the formation of an alpha epsilon heterodimer, a presumed assembly intermediate, is also less efficient with rat than with mouse epsilon subunit. By site-directed mutagenesis, we have found that these differences can be accounted for by 2 amino acid differences in the N-terminal domain at positions 106 and 115 of the rat and mouse epsilon subunits, suggesting that the region near these 2 amino acid residues is important for AChR assembly.     

Cloning and expression of the cDNA coding for a human lymphocyte IgE receptor.

Low-affinity receptors (Fc epsilon R) and secreted factors (IgE-BF) which bind to immunoglobulins of the IgE isotype play a key role in the regulation of human IgE synthesis. We report here the cloning of a cDNA coding for the Fc epsilon R of the human B-lymphoblast cell line RPMI 8866. The nucleotide sequence of this cDNA predicts a polypeptide with 321 amino acids and a mol. wt of 36,281 daltons. A functional Fc epsilon R capable of binding IgE was expressed in Chinese hamster ovary cells after stable transformation with the cDNA which had been cloned into a mammalian expression vector. Amino acid sequence analysis of IgE-BF purified from RPMI 8866 cells revealed an amino-terminal sequence of 19 residues which coincides with the predicted amino acid sequence of the Fc epsilon R, starting at residues 148 and 150. A computer search with the translated amino acid sequence of the Fc epsilon R revealed a domain of 120 amino acids having striking homology to the human asialoglycoprotein receptors.     

Cloning of a human collagen-binding protein, and its homology with rat gp46, chick hsp47 and mouse J6 proteins.

Several cDNA clones encoding a collagen-binding protein were isolated from human fibroblasts. The cDNA encoded a 417 amino acid protein, containing two potential N-linked oligosaccharide binding sites and a C-terminal RDEL sequence, which has been shown to act as an endoplasmic retention signal in other systems. The derived amino acid sequence of the protein shows close homology with gp46 from rat skeletal myoblasts, J6 protein from mouse F9 embryonal carcinoma cells and hsp47 from chick embryo fibroblasts. It also shows sequence similarity with members of the serpin family.     

Structural comparisons between mouse and human prealbumin

In an attempt to construct model systems for familial amyloidotic polyneuropathy, prealbumin cDNA was cloned from a mouse liver cDNA library, using previously cloned human prealbumin cDNA as a hybridization probe. The primary structure of mouse prealbumin deduced from the cDNA sequence shows that it consists of 147 amino acids, including a whole prealbumin sequence (127 amino acids) and a putative signal sequence (20 amino acids). These numbers are in complete agreement with those determined for the human prealbumin. Among the 127 amino acid residues of the mature human prealbumin, 25 are replaced by different amino acids in the mouse prealbumin. Interestingly, 24 out of the 25 substituted amino acids are located at the outer surface of the protein, and the regions corresponding to the core and central channel of the protein are almost completely conserved. The cloned cDNA provided essential information for manipulating amyloidosis in mice.     

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.     

Nucleotide sequence determination of guinea-pig casein B mRNA reveals homology with bovine and rat alpha s1 caseins and conservation of the non-coding regions of the mRNA.

Nucleotide sequence analysis of cloned guinea-pig casein B cDNA sequences has identified two casein B variants related to the bovine and rat alpha s1 caseins. Amino acid homology was largely confined to the known bovine or predicted rat phosphorylation sites and within the 'signal' precursor sequence. Comparison of the deduced nucleotide sequence of the guinea-pig and rat alpha s1 casein mRNA species showed greater sequence conservation in the non-coding than in the coding regions, suggesting a functional and possibly regulatory role for the non-coding regions of casein mRNA. The results provide insight into the evolution of the casein genes, and raise questions as to the role of conserved nucleotide sequences within the non-coding regions of mRNA species.     

Sequence of rat alpha- and gamma-casein mRNAs: evolutionary comparison of the calcium-dependent rat casein multigene family.

The complete sequences of rat alpha- and gamma-casein mRNAs have been determined. The 1402-nucleotide alpha- and 864-nucleotide gamma-casein mRNAs both encode 15 amino acid signal peptides and mature proteins of 269 and 164 residues, respectively. Considerable homology between the 5' non-coding regions, and the regions encoding the signal peptides and the phosphorylation sites, in these mRNAs as compared to several other rodent casein mRNAs, was observed. Significant homology was also detected between rat alpha- and bovine alpha s1-casein. Comparison of the rodent and bovine sequences suggests that the caseins evolved at about the time of the appearance of the primitive mammals. This may have occurred by intragenic duplication of a nucleotide sequence encoding a primitive phosphorylation site, -(Ser)n-Glu-Glu-, and intergenic duplication resulting in the small casein multigene family. A unique feature of the rat alpha-casein sequence is an insertion in the coding region containing 10 repeated elements of 18 nucleotides each. This insertion appears to have occurred 7-12 million years ago, just prior to the divergence of rat and mouse.     

Messenger RNA expressed in mouse teratocarcinoma stem cells and down-regulated by a tumor-promoting phorbol ester codes for a novel transmembrane protein

Cloning and sequence analysis of a DNA complementary to the mRNA expressed in undifferentiated mouse F9 teratocarcinoma stem cells but disappearing rapidly after treatment with a tumor-promoting phorbol ester revealed it to be a 1.9 kilobase pairs-long cDNA encoding a protein of 323 amino acid residues. Computer-assisted analyses of the deduced amino acid sequence indicated that this protein contains a typical hydrophobic signal peptide consisting of 33 amino acid residues and six putative membrane-spanning segments. The deduced amino acid sequence, as a whole, bears no significant sequence homology to any previously described protein.     

Identification of a novel FGF, FGF-21, preferentially expressed in the liver

We isolated cDNA encoding a novel FGF (210 amino acids) from mouse embryos. As this is the 21st documented FGF, we tentatively term it FGF-21. FGF-21 has a hydrophobic amino terminus ( approximately 30 amino acids), which is a typical signal sequence, and appears to be a secreted protein. The expression of FGF-21 mRNA in mouse adult tissues was examined by Northern blotting analysis. FGF-21 mRNA was most abundantly expressed in the liver, and also expressed in the thymus at lower levels. We also isolated human cDNA encoding FGF-21 (209 amino acids). Human FGF-21 is highly identical ( approximately 75% amino acid identity) to mouse FGF-21. Among human FGF family members, FGF-21 is most similar ( approximately 35% amino acid identity) to FGF-19.     

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.     

Cloning, recombinant expression and biochemical characterization of the murine CD83 molecule which is specifically upregulated during dendritic cell maturation

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC) and represents the best marker for mature DC. Here, we report the cloning of the cDNA encoding mouse CD83 (mCD83) from a murine bone marrow-derived DC (BM-DC) cDNA library. DNA sequence analysis revealed a 196 amino acid protein including a signal peptide of 21 amino acids which shares 63% amino acid identity with hCD83. Using Northern blot analyses, mCD83 mRNA was found to be strongly expressed in mouse BM-DC and its expression was upregulated following stimulation with LPS or TNF-alpha. Transfection experiments using COS-7 cells revealed that mCD83 is glycosylated. Furthermore, the extracellular CD83 domain was recombinantly expressed in Escherichia coli and one-dimensional NMR data strongly support that the protein is structurally folded.     

Characterization of a camel milk protein rich in proline identifies a new beta-casein fragment

A camel milk whey protein has been isolated by reverse-phase high performance liquid chromatography. The protein is, like caseins, rich in proline (25% of the whole protein). The N-terminal amino acid sequence shows that the protein is homologous with a C-terminal region of beta-caseins analyzed from other species. The protein is concluded to be a fragment of beta-casein, derived from a non-tryptic type of cleavage of the parent molecule, and increasing the multiplicity of known casein products.