Expression of three forms of thyroid hormone receptor in human tissues

At least two thyroid hormone receptor (hTR) genes are present in humans, but the significance of this multiplicity is unknown. These receptors could have differences in tissue distribution or possess different functions. We studied the distribution and abundance of three hTR mRNAs (hTR beta, hTR alpha 1, and hTR alpha 2) by Northern blot analysis. Three mRNAs were expressed in all tissues examined. hTR beta was strongly expressed in brain and prostate predominantly as a 10.0-kilobase (kb) mRNA. This mRNA was also expressed in thyroid and was much less abundant in liver, kidney, placenta, tonsil, and spleen. hTR alpha 1 is represented by two mRNAs with sizes of 6.0 and 3.2 kb. The 6.0-kb mRNA was constantly less abundant than the 3.2-kb mRNA. hTR alpha 2 was detected as a single mRNA with a size of 3.2 kb, using a probe unique for this mRNA. Both hTR alpha 1 and hTR alpha 2 were strongly expressed in brain, prostate, and thyroid and much less in other tissues. The relative amounts of the three hTR mRNAs were roughly parallel in each tissue. It is of interest that none of these hTRs was abundant in liver, which is the major thyroid hormone-responsive organ. Another hTR may be present in liver.     

Variation in insulin receptor messenger ribonucleic acid expression in human and rodent tissues

The expression of insulin receptor mRNA was studied in human and rodent tissues by Northern analysis. Human EBV-transformed lymphocytes contained four receptor mRNA species of sufficient length to encode the entire proreceptor: 9.5, 7.9, 7.1, and 5.7 kb. In human fibroblasts, the same four species were observed; however, the 7.9 and 5.7 kb mRNAs were markedly decreased. In mouse liver, rat hepatoma cells, and normal rat brain, kidney, liver, and muscle only two mRNA species (7.4 and 9.6 kb) were detected. Each of these human and rodent mRNAs hybridized equally well with cDNA sequences encoding the binding and kinase domains of the insulin receptor. Several smaller polyadenylated mRNAs (approximately 1.8 to 3.3 kb) were also identified in human cell lines that appeared to separately encode either alpha- or beta-subunit sequences of the receptor. In rats, liver had the highest content of insulin receptor mRNA, followed by kidney, brain, and muscle. The relative amount of the two mRNA species also varied among the rat tissues. The ratio of the 9.6-7.4 kb species was 2.7 in brain but only 1.0 to 1.6 in the other tissues (P less than 0.025). Dexamethasone treatment increased the content of the two insulin receptor mRNAs in rat liver by 2-fold. The half-life of both mRNA species was 70 min in rat hepatoma cells. These findings indicate that insulin receptor gene expression is complex and regulated with differential expression of insulin receptor mRNA and/or alterations in mRNA processing among various tissues.     

Nucleotide sequence of a cDNA clone for human aldolase: a messenger RNA in the liver

Nearly complete cDNA clones for human aldolase A mRNA were isolated from human liver cDNA library and the nucleotide sequence determined. Using the cDNA clone as a probe the length of human aldolase A mRNAs, isolated from the skeletal muscle, liver and placenta tissues, was measured by RNA blotting and estimated to be 1,600 nucleotides for skeletal muscle mRNA and 1,700 nucleotides for both the liver and placenta mRNAs, indicating that different species of mRNA coding for human aldolase A were expressed in the different tissues.     

Somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues

Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study 32P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyse rat Sm-C/IGF-I and IGF-II mRNAs in poly(A+) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobases (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. The abundance of a 7.5-kb Sm-C/IGF-I mRNA in poly(A+) RNAs from adult rat liver was 10-50-fold higher than in other adult rat tissues which provides further evidence that in the adult rat the liver is a major site of Sm-C/IGF-I synthesis and source of circulating Sm-C/IGF-I. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A+) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. Some samples of adult rat intestine contained the 4.7- and 3.9-kb IGF-II mRNAs and some samples of adult liver and lung contained the 4.7-kb mRNA. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded.     

Fat feeding stimulates only one of the two mRNAs encoding rat intestinal membranous and secreted alkaline phosphatase

We have identified two mRNAs in rat intestinal mucosa by Northern blot analysis, using cloned cDNAs encoding human placental alkaline phosphatase (PLAP). Probes from both the NH2- and COOH-terminal ends of the human PLAP coding region identified, in rat intestine (especially duodenum), an mRNA of nearly identical size (3 kb) to that found in human placenta. A smaller mRNA (2.7 kb), detected only with the COOH-terminal probe, was more prevalent in jejunum. Following feeding of triacylglycerols, the prevalence of the 2.7 kb mRNA increased over 2-fold. The tissue distribution and response of the 2.7 kb mRNA to fat feeding corresponds exactly with the known behavior of the secreted alkaline phosphatase.     

Expression of ceruloplasmin gene in mammalian organs from the data of hybridization analysis with complementary DNA probes

Expression of ceruloplasmin (Cp)-coding gene in rat and human liver and brain tissues was studied by Northern blot hybridization and by in situ hybridization with cloned species-specific cDNA probes. In rat brain structures, different levels of Cp mRNA were detected, the maximal one was found in cerebellum. The steady-state level of Cp mRNA in rat and human brain was several times lower than in parenchymatous liver cells. The size heterogeneity of Cp mRNA was found. Polyadenylated RNA prepared from human liver contains two equally abundant Cp mRNAs differing in their chain length (3.6 and 4.5 kb) while brain polyadenylated RNA contains a single Cp mRNA (4.5 kb).     

Cloning and sequencing of a rat CuZn superoxide dismutase cDNA. Correlation between CuZn superoxide dismutase mRNA level and enzyme activity in rat and mouse tissues

The molecular cloning and nucleotide sequence of a cDNA clone (pR SOD) for rat CuZn superoxide dismutase (CuZnSOD) is reported. Nucleotide sequence homology with human superoxide dismutase is 86% for the coding region and 71% for the 3' untranslated region. The deduced amino acid sequence is given and the homologies with the sequences reported for other species are presented. Northern blot analysis of total RNA from various rat and mouse tissues and from two mouse cell lines show that pR SOD hybridizes with one mRNA species of about 0.7 kb. The amount of CuZnSOD mRNA in each tissue, measured by densitometry of the Northern blot autoradiograms, correlates with the enzymatic activity based on protein content. These results indicate that the control of CuZnSOD activity in mammalian tissues is largely dependent on the regulation of CuZnSOD mRNA levels. In human liver, fibroblasts and FG2 hepatoma cells, two CuZnSOD mRNAs (0.7 kb and 0.9 kb) are observed. The level of CuZnSOD mRNA in FG2 is 25% that of the liver and four times more abundant than in fibroblasts.     

Demonstration of L1-related mRNAs in rat brain using DNA oligonucleotide probes.

Only one copy of the cell adhesion molecule L1 gene is present in the mouse genome, and only one mRNA of 6 kilobases (kb) is expressed in mouse brain [1987, Neurosci. Lett. 82, 89-94]. We have constructed 5 synthetic oligonucleotide probes covering different parts of the published mouse L1 cDNA sequence. Using these probes 3 distinct mRNAs of 9.0, 7.0 and 6.0 kb in rat brain could be demonstrated. Hybridizations performed at different stringency conditions indicated that the 9.0 and 7.0 kb mRNAs were highly related to the L1 mRNA of 6.0 kb expressed in rat brain. The 7.0 kb mRNA is possibly coding for a rat homologue of chicken Nr-CAM, whereas the 9.0 kb mRNA may code for a new member of the L1 family.     

Apolipoprotein gene expression in the rabbit: abundance, size, and distribution of apolipoprotein mRNA species in different tissues

We have used human apolipoprotein cDNAs as hybridization probes to study the relative abundance and distribution of apolipoprotein mRNAs in rabbit tissues by RNA blotting analysis. The tissues surveyed included liver, intestine, lung, pancreas, spleen, stomach, skeletal muscle, testis, heart, kidney, adrenal, aorta, and brain. We found that liver is the sole or major site of synthesis of apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, and apoE, and the intestine is a major site of synthesis of apoA-I, apoA-IV, and apoB. Minor sites of apolipoprotein mRNA synthesis were as follows: apoA-I, liver and skeletal muscle; apoA-IV, spleen and lung; apoB, kidney; apoC-II and apoC-III, intestine. ApoE mRNA was detected in all tissues surveyed with the exception of skeletal muscle. Sites with moderate apoE mRNA (10% of the liver value) were lung, brain, spleen, stomach, and testis. All rabbit mRNAs had forms with sizes comparable to their human counterparts. In addition, hybridization of hepatic and intestinal RNA with human apoA-IV and apoB probes produced a second hybridization band of approximately 2.4 and 8 kb, respectively. Similarly, hybridization of rabbit intestinal RNA with human apoC-II produced a hybridization band of 1.8 kb. The 8 kb apoB mRNA form may correspond to the apoB-48 mRNA, whereas the apoA-IV- and apoC-II-related mRNA species have not been described previously. This study provides a comprehensive survey of the sites of apolipoprotein gene expression and shows numerous differences in both the abundance and the tissue distribution of several apolipoprotein mRNAs between rabbit and human tissues. These findings and the observation of potentially new apolipoprotein mRNA species are important for our understanding of the cis and trans acting factors that confer tissue specificity as well as factors that regulate the expression of apolipoprotein genes in different mammalian species.     

Molecular cloning of a non-isopeptide-selective human endothelin receptor.

We isolated several complementary DNA (cDNA) clones encoding a non-isopeptide-selective human endothelin receptor (ETBR) from a human placenta cDNA library. The clones, different in the length of their 3'-untranslated regions, encoded the same 442-amino acid protein with a transmembrane topology similar to that of other G protein-coupled receptors. The rank order of the binding of ET isopeptides (ET-1, ET-2 and ET-3) to the receptor expressed in COS-7 cells was ET-1 = ET-2 = ET-3. Northern blot analysis identified three mRNA species, 4.3 kb, 2.7 kb and 1.7 kb in size, probably generated by their use of alternative polyadenylation sites. These mRNAs were expressed in a wide variety of human tissues, at the highest level in the brain and at a significant level in cultured endothelial cells.     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.     

The house musk shrew (Suncus murinus): a unique animal with extremely low level of expression of mRNAs for CYP3A and flavin-containing monooxygenase

Expression of drug-metabolizing enzymes including cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) in various tissues of Suncus murinus (Suncus) were examined. Northern blot analysis showed that mRNAs hybridizable with cDNAs for rat CYP1A2, human CYP2A6, rat CYP2B1, human CYP2C8, human CYP2D6, rat CYP2E1, human CYP3A4 and rat CYP4A1 were expressed in various tissues from Suncus. The mRNA level of CYP2A in the Suncus lung was very high. Furthermore, it was found that the level of CYP2A mRNA in the Suncus lung was higher compared to the Suncus liver. The expression level of mRNA hybridizable with cDNA for human CYP3A4 was very low. The presence of CYP3A gene in Suncus was proven by the induction of the CYP with dexamethasone. Very low expression levels of mRNAs hybridizable with cDNAs for rat FMO1, rat FMO2, rat FMO3 and rat FMO5 were also seen in Suncus liver. No apparent hybridization band appeared when human FMO4 cDNA was used as a probe. The hepatic expression of mRNAs hybridizable with cDNAs for UDP-glucuronosyltransferase 1*6, aryl sulfotransferase, glutathione S-transferase 1, carboxyesterase and microsomal epoxide hydrolase in the Suncus were observed. These results indicate that the Suncus is a unique animal species in that mRNAs for CYP3A and FMO are expressed at very low levels.     

Analyses of ornithine decarboxylase antizyme mRNA with a cDNA cloned from rat liver

Ornithine decarboxylase antizyme is a unique inhibitory protein induced by polyamines and involved in the regulation of ornithine decarboxylase. A cDNA was isolated from a rat liver cDNA library by the screening with monoclonal antibodies to rat liver antizyme as probes. The expression products of the cDNA in bacterial systems inhibited rat ornithine decarboxylase activity in a manner characteristic of antizyme and rabbit antisera raised against its direct expression product reacted to rat liver antizyme, confirming the authenticity of the cDNA. On RNA blot analysis with the cDNA probe, an antizyme mRNA band of 1.3 kb was detected in rat tissues. Antizyme mRNA did not increase upon administration of putrescine, an inducer of antizyme, and its half-life after actinomycin D treatment was as long as 12 h in rat liver, suggesting that antizyme mRNA is constitutively expressed and antizyme synthesis is regulated at the translational level. Similar-sized mRNAs hybridizable to the cDNA were also found in various mammalian and non-mammalian vertebrate tissues under physiological conditions. In addition, chicken and frog antizymes showed immunocrossreactivity with rat antizyme. The ubiquitous presence and the evolutionally conserved structure of antizyme in vertebrate tissues suggest that it has an important function.     

Regulation of two c-erbA messenger ribonucleic acids in rat GH3 cells by thyroid hormone

There is recent evidence suggesting that c-erbA is the thyroid hormone nuclear receptor, and that there may be multiple c-erbA genes. We investigated the effect of T3 on two c-erbA mRNAs present in GH3 cells. A partial cDNA was isolated from rat GH3 cells which is nearly identical (99.6% nucleotide identity) to rat c-erbA alpha, except for a unique 3'-region corresponding to the carboxyl terminal region of the predicted protein sequence. This cDNA (c-erbA alpha-2), like rat c-erbA alpha, hybridizes to a 2.6 kilobase (kb) mRNA which is distinct from a 6.2 kb species that hybridizes to c-erbA beta. Since nuclear T3-binding is down-regulated by T3, we hypothesized that one or both c-erbA mRNAs might be regulated by T3. GH3 cells were treated with 10 nM T3 for up to 24 h, a manipulation known to decrease nuclear T3 binding by approximately 2-fold in GH cells. Both the 6.2 kb and 2.6 kb mRNA species decreased to nearly 50% of control values at 24 h. These data indicate that these two c-erbA mRNAs are regulated by T3 and suggest that the T3 effect on T3 binding-activity in GH cells may be mediated, in part, by down-regulation of c-erbA mRNA levels.