Molecular cloning and expression of bovine (Bos taurus) leptin receptor isoform mRNAs

We characterized Bos taurus leptin receptor (Ob-R) isoform mRNAs as well as their expression in different tissues, including some adipose depots (perirenal, subcutaneous and intermuscular adipose tissues). Based on the GenBank database sequences of the bovine partial Ob-R, primers were designed to amplify cDNAs of bovine Ob-R isoforms. The full-length cDNAs of bovine the Ob-R isoforms were cloned by combination with 3'-and 5'-RACE. Three bovine Ob-R isoform cDNAs were cloned and the sequence analyses revealed that these cDNAs were bovine Ob-R isoforms, i.e., the long form (Ob-Rb), the middle form (Ob-Ra) and the short form (Ob-Rc). The open reading frames of Ob-Ra, Ob-Rb and Ob-Rc gene were 2688, 3498 and 2673 bp, respectively. The deduced amino acid sequences suggested that the isoforms were single transmembrane proteins, and differed in the C-terminal amino acid sequences. The amino acid sequence of these bovine Ob-R isoforms showed 73-75% identity compared with the corresponding mouse isoforms. The tissue-specific expression of the bovine Ob-R isoforms were measured by semi-quantitative RT-PCR. Expression of Ob-Rb was highest in liver, heart, spleen and kidney, with lower expression in lung and testis, and slight expression in muscle. Ob-Ra was highly expressed in liver and spleen, whereas moderate expression was observed in heart, testis, and muscle, and its expression was the lowest in lung and kidney. Ob-Rc mRNA was expressed in the liver, heart, testis, kidney and muscle, but not in the lung and spleen. In adipose tissues, higher expression of Ob-Ra and Ob-Rb mRNA was observed in intermuscular adipose tissue than in subcutaneous or perirenal adipose tissues. Ob-Ra mRNA level was positively correlated with Ob-Rb mRNA level in the adipose tissues (r=0.81, P<0.05). The results demonstrated that each Ob-R isoform mRNA was differentially expressed in various tissues of cattle, which may be involved in the difference of peripheral actions for leptin.     

Avian adipose lipoprotein lipase: cDNA sequence and reciprocal regulation of mRNA levels in adipose and heart

cDNA clones for chicken adipose lipoprotein lipase were isolated from an expression library in lambda gt11 by antibody screening and characterized by hybridization selection and nucleotide sequencing. Based on the cDNA sequence and on N-terminal sequence analysis of the purified enzyme, chicken adipose lipoprotein lipase is a mature protein of 465 amino acids with a signal peptide of 19 or 25 amino acids, depending on which of two methionine residues is used for translation initiation. The predicted amino-acid sequence was found to be 73-77% identical to the four known mammalian adipose lipoprotein lipase sequences, with conservation of position of cysteine residues and putative functional domains, and number of potential N-glycosylation sites. Chicken lipoprotein lipase differs from mammalian lipoprotein lipases with respect to the position of one N-glycosylation site and the presence of an additional 15-17 C-terminal amino acids. 32P-labeled cDNA clones hybridized to mRNA species of 3.7 and 4.0 kb in Northern blots of heart and adipose, but not of liver RNA. In chickens that were fasted for 48 h and then refed, lipoprotein lipase mRNA levels in adipose increased to a maximal level of 350% that of controls at 10 h, whereas heart lipoprotein lipase mRNA levels fell to 40% of controls at 14 h. Concomitantly, no changes in total RNA were observed. Thus, avian lipoprotein lipase is subject to reciprocal pretranslational regulation in adipose and heart.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

Gene expression and cDNA cloning identified a major basic protein constituent of bovine seminal plasma as bovine monocyte-chemoattractant protein-1 (MCP-1).

P6 is one of the major basic proteins of bovine seminal plasma. Using cell-free translation of poly(A)+RNA from bovine seminal vesicle tissue and monospecific anti-P6-IgGs, we show that P6 is a secretory product of the seminal vesicles. Immunohistochemical experiments supported this finding. Immunoscreening of a lambda gt11 cDNA library derived from seminal vesicle poly(A)+RNA furnished a number of positive cDNA clones, from which clone pH42 was characterized by sequencing. The partial amino acid sequence of a CNBr-fragment of P6 permitted identification of the reading frame of clone pH42 encoding the precursor protein of P6. The P6 precursor contains a signal peptide of 23 amino acids followed by the mature P6 sequence of 76 amino acid residues. The cDNA sequence of pH42 was 80% homologous with that of the human monocyte-chemoattractant protein-1 (hMCP-1). The respective amino acid sequences for the precursor molecules are 72% identical. Northern analysis of seminal vesicle poly(A)+RNA using pH42 as probe probe identified a 0.9-kb P6 mRNA. Stimulation of P6 mRNA expression by phytohemagglutinin in bovine peripheral mononuclear leukocytes suggests that P6 is identical to bovine MCP-1.     

Structural characterization of the calcium binding protein s100 from adipose tissue

A partial amino acid sequence for bovine adipose tissue S100 was elucidated by characterization of peptides generated by cyanogen bromide cleavage. The cyanogen bromide peptides were aligned by homology with the bovine brain S100 beta sequence. The results demonstrate that adipose S100 beta is probably identical to brain S100 beta, and suggest that S100 beta is a conserved protein among tissues of the same species.     

cDNA clones encoding bovine gamma-crystallins

We have determined the nucleotide sequence of two bovine lens gamma-crystallin cDNA clones, pBL gamma II-1 and pBL gamma III-1. The 644 bp cDNA insert of pBL gamma II-1 contains coding information for the entire amino acid sequence of bovine gamma II-crystallin. The 497 bp cDNA insert of pBL gamma III-1 encodes a homologous but different gamma-crystallin polypeptide, and appears to lack the coding information for the C-terminal 17 amino acid residues. While the nucleotide and predicted amino acid sequences of the coding regions of the clones show a high degree of homology, the untranslated leader sequences are relatively dissimilar. The leader sequence of pBL gamma III-1 is strikingly homologous to a portion of a rabbit immunoglobulin alpha-heavy chain mRNA.     

Human liver cDNA clones encoding proteolipid subunit 9 of the mitochondrial ATPase complex

Clones encoding the proteolipid subunit 9 of the mitochondrial ATPase complex have been isolated from a lambda gt10 library of human liver cDNA sequences, using a probe of Neurospora crassa cDNA encoding subunit 9. From nucleotide sequence analysis it is concluded that the amino acid sequence of mature human subunit 9 is identical to that of its bovine counterpart. By comparing the sequence of two cDNA clones (denoted human 1 and 2) to those of two bovine cDNA clones (denoted P1 and P2) recently described by Gay and Walker (EMBO J. 4, 3519-3524, 1985) it is evident that there are close sequence relationships between human 1 and bovine P1, and between human 2 and bovine P2, although both human clones are truncated at their 5'-ends. Thus, as in bovine cells there appears to be at least two human genes encoding subunit 9.     

Characterization of the cytochrome P-450IID subfamily in bovine liver. Nucleotide sequences and microheterogeneity.

To elucidate the molecular mechanisms underlying drug detoxification, the structures of the members of the microsomal cytochrome P-450IID subfamily were analyzed by isolating, mapping and sequencing cytochrome P-450IID (CYP2D) cDNA clones from bovine liver. The screening was performed under nonstringent conditions so that most of the P-450IID subfamily members could be obtained. 114 of the 147 positive clones were classified into four groups on the basis of their restriction-enzyme maps. The maps of the four groups were highly similar, however, the clones of one group contained an insertion of approximately 500 bp in the coding region. Analysis of partial nucleotide sequences of several representative clones from each group showed that the bovine P-450IID subfamily in liver consisted of several, not many, highly similar members, differing by less than 7% in their nucleotide sequences. The location of the insertion found in the minor group corresponded to intron 7 and the GT/AG rule was found at the exon/intron boundary, suggesting that intron 7 was retained in this group. The complete nucleotide sequences of two clones from the major group were examined to determine the structures of the P-450IID subfamily in bovine liver. A full-length cDNA clone (1615 bp) and a partial cDNA clone (1538 bp) contained open reading frames encoding 500 and 487 amino acid residues, respectively. The partial clone lacked the nucleotide sequence corresponding to the first 13 N-terminal amino acid residues. The deduced amino acid sequences of the two clones were 98% similar, and 80% and 68% similar to those from human CYP2D6 and rat CYP2D1, respectively. Comparisons of the amino acid sequences of the P-450IID subfamily members showed the highly conserved C-terminal region of their molecules and the high similarity between the members in one species, especially in cattle and man.     

Structural analysis of mouse S-antigen

Mouse S-antigen clones were isolated from a mouse retinal cDNA library using a bovine S-antigen cDNA probe. The largest clone (MSC-242) comprised 1532 bp and contained the entire coding sequence. The nucleotide sequence homology between the mouse and bovine coding regions was 84%, while non-coding regions appeared to be more divergent. The deduced amino acid sequence indicated that the mouse S-antigen had 403 residues and its molecular ratio was 44,930. An overall amino acid sequence similarity of 84% was observed between the mouse and bovine proteins. This degree of similarity dropped to 60% and 47% at the N and the C termini, respectively. The local homology with alpha-transducin observed in the bovine proteins, including the putative phosphoryl and rhodopsin binding sites, was conserved in the mouse as well. There was no overall sequence similarity with other proteins listed in the National Biomedical Research Foundation (NBRF) protein sequence database. Among the uveitopathogenic sites for experimental autoimmune uveitis (EAU), peptides N and M were identical to their bovine counterparts. Peptides 3 and K, however, were more divergent. The short repeats within these peptides were conserved.     

cDNA cloning and the response to overfeeding in the expression of stearoyl-CoA desaturase 1 gene in Landes goose

Stearoyl-CoA desaturase 1 (SCD1) is a rate limiting enzyme in the biosynthesis of monounsaturated fatty acids. It has been cloned from several species: Rattus norvegicus, Mus musculus, Homo Sapiens and Gallus gallus, but not from Anser anser. This study was conducted to isolate the SCD1 cDNA sequence and investigate the effect of overfeeding on SCD1 gene tissue expression in Landes goose. The complete cDNA is 3294 bp in length, with an ORF of 1.083 bp encoding a predicted polypeptide of 360 amino acids and 5′/3′-UTR of 74 and 2137 bp, respectively. Quantitative real time PCR (qPCR) was used to examine SCD1 expression in heart, liver, spleen, lung, kidney, gizzard, glandular stomach, intestine, crureus, pectoral muscle, hypothalamus and adipose tissue (abdominal fat) in both the overfed and control group. SCD1 mRNA was highly expressed in goose fatty liver, and the expression levels of SCD1 in liver and fat of overfeeding group were more than double that of the control group. During the overfeeding period, SCD1 expression in liver and adipose tissue reached the highest level after 70 days, but declined at 79 days. In the control group, after fasting 24 h, the expression level of SCD1 gene in tissues declined sharply. However, SCD1 gene expression in hypothalamus was unaffected. The results of this study provide a theoretical basis to study the relationship between SCD1 gene expression and the formation of fatty liver of Landes goose in response to overfeeding.     

Stearoyl-CoA desaturase 1 coding sequences and antisense RNA affect lipid secretion in transfected chicken LMH hepatoma cells

Hepatic stearoyl CoA desaturase (SCD) activity in chickens from a fat line is higher than that of chickens from a lean line and correlates with plasma triacylglycerol concentrations. Furthermore, in these lines, the hepatic SCD1 mRNA level is positively correlated with the adipose tissue weight. To analyze the contribution of the SCD1 gene in the regulation of adiposity in the early stages of triacylglycerol secretion, SCD1 coding sequence and antisense RNA expression vectors were transfected in LMH cells. After selection, these cells were analyzed with regard to SCD1 expression and lipid secretion. The amounts of secreted triacylglycerols and phospholipids were shown to be higher in LMH cells transfected with the SCD1 gene, but reduced in those transfected with the SCD1 antisense sequences when compared to cells transfected with the vector alone (without SCD1 sequences). These results provide direct evidence that the expression of the SCD1 gene plays a major role in the triacylglycerol and phospholipid secretion process.     

The sequence of cDNA encoding lipoprotein lipase. A member of a lipase gene family

cDNA clones corresponding to the entire coding region of mature lipoprotein lipase were identified by antibody screening of a mouse macrophage library and sequenced. The predicted amino acid sequence indicates that the mature protein contains 447 amino acids with a molecular weight of 50,314. Comparison of the nucleotide and amino acid sequence with those of rat hepatic lipase and porcine pancreatic lipase reveals extensive homology among the enzymes, indicating that they are members of a gene family of lipases. Most striking is a conservation of five disulfide bridges in all three enzymes, strongly suggesting that the enzymes have similar overall folding patterns. Lipoprotein lipase is also shown to be extraordinarily conserved among mouse, human, and bovine species. The mRNA for lipoprotein lipase is abundant in heart and adipose tissue but is also present in a wide variety of other tissues. There are two major species of mRNA in mouse and human tissues examined, 3.6 and 3.4 kilobases (kb) in size. Rat tissues, on the other hand, contain only the 3.6-kb species while bovine tissues contain an additional 1.7-kb species.     

Isolation and characterisation of a bovine cDNA encoding eukaryotic initiation factor 2 alpha

Two cDNA clones have been isolated, from a bovine lymphosarcoma library, that encode the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha). The predicted 315 amino acid sequence showed more than 99% amino acid identity with rat and human eIF-2 alpha. Galactose-regulated expression of a full length bovine eIF-2 alpha cDNA in yeast resulted in the synthesis of a polypeptide of the predicted molecular mass (36 kDa). Furthermore, the expressed polypeptide cross-reacted with an antibody raised against rabbit eIF-2 alpha confirming the identity of the cDNA.     

Expression of new members of the prolactin growth hormone gene family in bovine placenta. Isolation and characterization of two prolactin-like cDNA clones

Two prolactin-like proteins (bPLP-I and bPLP-II) were deduced from the nucleotide sequence analyses of the cDNA clones derived from a bovine (Bos taurus) term placenta. These proteins resembled bovine prolactin but were different from the reported bovine placental lactogens or prolactin-related proteins. The predicted amino acid sequences of these clones showed 45-51% identity with bovine prolactin and 23-24% with bovine growth hormone. The two new clones show 62 and 39% overall homology with each other at the levels of nucleotide and amino acid sequences, respectively. bPLP-I, bPLP-II, placental lactogens, prolactins (PRLs), and other prolactin-like proteins isolated from cow, mouse, and rat share 7 common amino acid residues. Five of the 7 residues are conserved by other members of the family such as growth hormones, suggesting that they may be essential for the common structural features of the gene family. The other 2 residues are uniquely conserved in bovine, mouse, and rat placental lactogens, PRLs, and PRL-like proteins, predicting their indispensable roles in binding to the specific receptors. bPLP-I and bPLP-II, as well as bPLP-III, are shown to be expressed stage specifically and predominantly in full-term bovine placentas.