Saturated fatty acids stimulate and insulin suppresses CIDE-A expression in bovine mammary epithelial cells

Cell death-inducing DNA fragmentation factor-alpha-like effector A (CIDE-A) was first identified by its sequence homology with the N-terminal domain of DNA fragmentation factor (DFF). CIDE-A negatively regulates the activity of uncoupling protein 1 (UCP1) in brown adipose tissue. CIDE-A and UCP1 mRNA were detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and lactating bovine mammary glands. Physiological concentrations of saturated fatty acids (stearate and palmitate), but not unsaturated fatty acids (oleate and linoleate) induced up-regulation of CIDE-A mRNA in bMEC. Treatment with insulin (5-10 ng/ml) induced down-regulation of CIDE-A and UCP1. The expression levels of CIDE-A and UCP1 mRNA in bovine mammary glands at various stages of the lactation cycle were determined by quantitative RT-PCR analysis. CIDE-A mRNA expression at peak lactation (2 months after parturition) was significantly higher than at dry off and non-pregnancy but not late lactation. These results suggest that CIDE-A and UCP1 are regulated by insulin and/or fatty acids in mammary epithelial cells and lactating mammary glands, and thereby play an important role in lipid and energy metabolism.     

Expression of kappa-casein in normal and neoplastic rat mammary gland is under the control of prolactin

An 820-nucleotide-long cDNA clone for the kappa-casein (the casein micelle-stabilizing protein) from rat mammary gland was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence from the nucleotide sequence revealed a signal peptide, 21 amino acids long, and a mature protein of 157 amino acids. The signal peptide of rat kappa-casein was highly homologous to that of the precursor to ovine kappa-casein. However, little homology was apparent when the mature kappa-casein protein sequences from ovine or bovine sources were compared with rat kappa-casein. The kappa-casein mRNA content of the mammary tissue was found to increase during its functional differentiation. Prolactin appears to modulate the production of kappa-casein mRNA. Mammary glands of virgin females had no detectable kappa-casein mRNA; however, a marked induction of kappa-casein mRNA was obtained by intravenous infusion of prolactin. Mammary carcinomas did not follow the same pattern. 7,12-Dimethylbenz[a]anthracene-induced mammary carcinomas had normally low levels of kappa-casein mRNA, but intravenous prolactin infusion increased the levels by 2-fold. The MTW9 mammary carcinoma that grows only in the presence of high levels of mammotropic hormones had kappa-casein mRNA content equivalent to that in 10-day lactating rat mammary gland. Continuous venous infusion of prolactin to MTW9 mammary carcinoma did not modify the kappa-casein mRNA levels. Nitrosomethylurea-induced mammary carcinomas had no detectable kappa-casein mRNA, and intravenous prolactin infusion was unable to induce it.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase.

Aspartyl (asparaginyl) beta-hydroxylase which specifically hydroxylates 1 Asp or Asn residue in certain epidermal growth factor-like domains of a number of proteins, has been previously purified to apparent homogeneity from detergent-solubilized bovine liver microsomes (Wang, Q., VanDusen, W. J., Petroski, C. J., Garsky, V. M., Stern, A. M., and Friedman, P. A. (1991) J. Biol. Chem. 266, 14004-14010). Three oligonucleotides, corresponding to three amino acid sequences of the purified hydroxylase, were used to screen bovine cDNA libraries. Several overlapping positive cDNA clones containing a full length open reading frame of 754 amino acids encoding a 85-kDa protein were isolated, and a cDNA, containing the full length open reading frame, was constructed from two of these clones. The resulting clone was then transcribed and translated in vitro to produce recombinant protein which possessed Asp beta-hydroxylase activity. These results constitute proof that the protein purified from bovine liver is an Asp beta-hydroxylase. Comparisons of deduced amino acid sequences of two other alpha-ketoglutarate-dependent dioxygenases, prolyl-4-hydroxylase and lysyl hydroxylase, with that of Asp beta-hydroxylase showed no significant homologies. Indeed, Asp beta-hydroxylase appears to be unique as no striking homology was found with known protein sequences. Furthermore, structural predictions derived from the deduced amino acid sequence are in accord with earlier Stokes' radius and sedimentation coefficient determinations of the enzyme, suggesting that the enzyme contains a relatively compact carboxyl-terminal catalytic domain and an extended amino terminus. This amino-terminal region has a potential transmembrane type II signal-anchor domain that could direct the catalytic domain into the lumen of the endoplasmic reticulum.     

Bovine lactoferrin mRNA: sequence, analysis, and expression in the mammary gland.

The mRNA sequence for bovine lactoferrin expressed in the mammary gland was determined by sequencing three over lapping cDNA clones and by direct sequencing of the mRNA. The mRNA (2351 bases) codes for a 708 amino acid protein with a 19 amino acid signal peptide immediately preceding a sequence identical to the N-terminal 40 amino acids reported for bovine lactoferrin. A putative destabilizing sequence (AUUUA) was identified in the 3'-untranslated region. The nucleic acid sequence and deduced amino acid sequence are highly homologous with other transferrin family members. Lactoferrin mRNA concentrations in bovine mammary tissue were quite low two days before parturition and during lactation but were high three days after the cessation of milking, a sharp contrast from the pattern of regulation of the other milk proteins.     

Nucleotide and deduced amino acid sequences of a GTP-binding protein family with molecular weights of 25,000 from bovine brain

We have purified a novel GTP-binding protein (G protein) with a Mr of about 24,000 to homogeneity from bovine brain membranes (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904). In the present studies, we have isolated and sequenced the cDNA of this G protein from a bovine brain cDNA library using oligonucleotide probes designed from the partial amino acid sequences. The cDNA of the G protein has an open reading frame encoding a protein of 220 amino acids with a calculated Mr of 24,954. This G protein is designated as the smg-25A protein (smg p25A). The amino acid sequence deduced from the smg-25A cDNA contains the consensus sequences of GTP-binding and GTPase domains. smg p25A shares about 28 and 44% amino acid homology with the ras and ypt1 proteins, respectively. In addition to this cDNA, we have isolated two other homologous cDNAs encoding G proteins of 219 and 227 amino acids with calculated Mr values of 24,766 and 25,975, respectively. These G proteins are designated as the smg-25B and smg-25C proteins (smg p25B and smg p25C), respectively. The amino acid sequences deduced from the three smg-25 cDNAs are highly homologous with one another in the overall sequences except for C-terminal 32 amino acids. Moreover, three smg p25s have a consensus C-terminal sequence, Cys-X-Cys, which is different from the known C-terminal consensus sequences of the ras and ypt1 proteins, Cys-X-X-X and Cys-Cys, respectively. These results together with the biochemical properties of smg p25A described previously indicate that three smg p25s constitute a novel G protein family.     

Cloning of the cDNAs encoding the cellular retinaldehyde-binding protein from bovine and human retina and comparison of the protein structures

A 1173-base pair cDNA encoding bovine cellular retinaldehyde-binding protein (CRALBP) was cloned from a bovine retinal cDNA expression library using as probes both anti-CRALBP polyclonal and monoclonal antibodies. The amino acid sequence deduced from the cDNA corresponds exactly to that determined by direct analysis of NH2-terminally acetylated bovine CRALBP (Crabb, J. W., Johnson, C. M., Carr, S. A., Armes, L. G., and Saari, J. C. (1988) J. Biol. Chem. 263, 18678-18687). Nick-translated bovine CRALBP cDNA probes were then used to clone from a human retinal cDNA library a 1317-base pair cDNA encoding human CRALBP. Bovine and human CRALBP are 92% identical in amino acid sequence and not related to any other known protein sequence. Both the bovine and human proteins contain 316 residues and have calculated molecular weights of 36,378 and 36,347, respectively, exclusive of the NH2-terminal blocking groups. The CRALBP cDNA clones should prove valuable as tools for studying the physiological role of the protein in vision and visual disorders.     

Glutathione utilization by lactating bovine mammary secretory tissue in vitro.

gamma-Glutamyltransferase (D-glutamyl transpeptidase, EC 2.3.2.2) activity has been shown to be located predominantly on the extracellular surface of the plasma membrane of lactating bovine mammary cells. Radioactive label from both oxidized ([14C]-gamma-glutamyl) and reduced ([35S]cysteinyl) glutathione was taken up and incorporated into acid-precipitable proteins of mammary tissue. Uptake was shown to involve the transport of free amino acids, and incorporation was shown to involve the action of gamma-=glutamyltransferase. These results indicate that lactating mammary tissue utilizes the constituent amino acids of glutathione for milk-protein synthesis.     

Molecular cloning of the myelin specific enzyme 2',3'-cyclic-nucleotide 3'-phosphohydrolase

We describe the isolation of cDNA clones for bovine brain 2',3'-cyclic-nucleotide 3'-phosphohydrolase (CNPase, EC 3.1.4.37), the third most abundant protein in central nervous system myelin. The cDNA encodes the complete protein (400 amino acids) and hybridizes to a major size species of mRNA in bovine brain tissue, approx. 2.7 kb in size. CNPase mRNA levels do not appear to be affected in quaking dysmyelinating mutant mice. The sequence reveals probable sites for CNPase phosphorylation by cAMP-dependent protein kinase and a region of homology with haemocyanin.     

Cloning and expression of bovine imc-415 cDNA from mammary gland

Bovine imc-415 cDNA was cloned from mammary gland using RACE PCR; it coded for 245 amino acids. The deduced amino acid sequence of mouse and human showed about 94% identity. Expression of bovine imc-415 increased about 40% in involuted mammary tissues compared with lactating tissues.     

Cloning and Expression of Bovine imc-415 cDNA from Mammary Gland

Bovine imc-415 cDNA was cloned from mammary gland using RACE PCR; it coded for 245 amino acids. The deduced amino acid sequence of mouse and human showed about 94% identity. Expression of bovine imc-415 increased about 40% in involuted mammary tissues compared with lactating tissues.     

Isolation of a full-length complementary DNA coding for human E1 alpha subunit of the pyruvate dehydrogenase complex

A 1.5-kilobase cDNA clone for human pyruvate dehydrogenase E1 was isolated from a lambda gt11 expression library by screening with polyclonal antiserum to the E1 alpha subunit of the porcine pyruvate dehydrogenase complex, a polyclonal antibody against bovine pyruvate dehydrogenase complex and a synthetic oligonucleotide based on the known amino acid sequence of the amino-terminal of the bovine pyruvate dehydrogenase-E1 alpha subunit. Nucleotide sequence analysis of the cDNA revealed a 5'-untranslated sequence of 72 nucleotides, a translated sequence of 1170 nucleotides, and a 3'-untranslated sequence of 223 nucleotides with a poly(A) tail. The cDNA structure predicts a leader sequence of 29 amino acids and a mature protein of 362 amino acids comprising an amino-terminal peptide identical to that of the bovine E1 alpha subunit and three serine phosphorylation sites whose sequence was also identical to those in the bovine E1 alpha subunit. The translated sequence for the mature protein differs substantially from that described by Dahl et al. (Dahl, H. H., Hunt, S. M., Hutchison, W. M., and Brown, G. K. (1987) J. Biol. Chem. 262, 7398-7403) by virtue of a frameslip between bases 390 and 594. This amended sequence is confirmed by the presence of additional restriction sites for the enzymes NaeI and HaeII at the beginning and end, respectively, of this section. The leader sequence is typical for mitochondrial enzymes being composed of a combination of neutral and basic residues. The amino acid composition is strikingly similar to that of the bovine protein. This cDNA clone hybridizes with a 1.8-kilobase mRNA on a Northern blot analysis of human fibroblasts, and a second minor band of 4.4 kilobases is also detected.     

The sequence of porcine αs1-casein cDNA: evidence for protein variants generated by altered RNA splicing

A cDNA library was constructed from mRNA isolated from lactating porcine mammary gland and screened with a bovine alpha s1-casein cDNA clone. Three classes of cDNA isolated varied in the number of bases within the coding region. The full length porcine alpha s1-casein cDNA is 1124bp and codes a preprotein of 206 amino acids. The other two classes of alpha s1-casein cDNA lacked 18bp and 60bp respectively when compared to the 1124-bp cDNA sequence. PCR amplification confirmed the presence of these sequences in total RNA. These differences appear to be due to altered RNA splicing.     

A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs. Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase.

Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues. To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains. Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes. One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J. K., Kadlecek, A., Sherbert, C. H., Seger, D., Sonnenburg, W. K., Charbonneau, H., Novack, J. P., and Beavo, J. A. (1992) J. Biol. Chem. 267, 18676-18682). This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone. The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE. Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin. Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line. These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes. Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides.     

Molecular genetic characterization of a developmentally regulated human perinatal myosin heavy chain

We have isolated a human cDNA which corresponds to a developmentally regulated sarcomeric myosin heavy chain. RNA hybridization and DNA sequence analysis indicate that this cDNA, called SMHCP, encodes a perinatal myosin heavy chain isoform. The nucleotide and deduced amino acid sequences of the 3.4-kb cDNA insert show strong homology with other sarcomeric myosin heavy chains. The strongest homology is to a previously described 970-bp cDNA encoding a rat perinatal isoform (Periasamy, M., D. F. Wieczorek, and B. Nadal-Ginard. 1984. J. Biol. Chem. 259:13573-13578). The homology between the analogous human and rat perinatal myosin heavy chain cDNAs is maintained through the highly isoform-specific final 20 carboxyl-terminal amino acids, as well as the 3' untranslated region. Ribonuclease protection studies show that the mRNA encoding this isoform is expressed at high levels in 21-wk fetal skeletal tissue and not in fetal cardiac muscle. In contrast to the rat perinatal isoform, which was not found to be expressed in adult hind-leg tissue, the gene encoding SMHCP continues to be expressed in adult human skeletal tissue, but at lower levels relative to fetal skeletal tissue.