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.     

Expression of Tropomyosin Genes During the Development of the Rat Cerebellum

The expression of the tropomyosin genes in the rat nervous system was examined during the postnatal development of the cerebellum, using human-specific alpha-, beta-, gamma-, and delta-tropomyosin cDNA probes and rat-specific alpha-, beta-, and delta-tropomyosin oligonucleotide probes. The beta- and gamma-genes do not seem to be expressed in the rat brain. The delta-tropomyosin gene produces two mRNAs: a major one of 2.4 kb, which is highly concentrated during the first postnatal week and then decreases fourfold in level until the age of 35 days, and a minor one of 2 kb, with the same developmental profile as the 2.4-kb mRNA. A 3-kb mRNA is expressed by the alpha-tropomyosin gene and is characteristic of the mature rat. The expression of the tropomyosin genes during the development of the rat cerebellum does not seem to be regulated through alternative splicing but rather implies the differential expression of two different isogenes. The multiple isoforms of tropomyosin produced during neuronal differentiation may be intimately involved in the regulation of the organization and function of actin microfilaments.     

The size heterogeneity of rat insulin-like growth factor-I mRNAs is due primarily to differences in the length of 3'-untranslated sequence

Previous studies have revealed multiple size classes of rat insulin-like growth factor-I (IGF-I) of estimated size 7.5-7.0, 1.9-1.5, and 1.2-0.9 kilobases (kb). Available sequence information accounts for only 2.1 kb of the 7.5-7.0 kb IGF-I mRNAs. We used oligomer directed ribonuclease H (RNase H) mapping to define the extent to which the unknown sequence in the large molecular weight mRNAs lies 5' or 3' to known sequence. Rat liver polyadenylated RNAs were incubated with oligomer probes complementary to internal rat IGF-I precursor (E domain) coding sequences. RNase H was used to hydrolyze IGF-I mRNAs at the point of annealment with the oligomers. Resultant 5' and 3'-IGF-I mRNA fragments were analyzed on Northern blots. A probe specific for type 1 (class C) 5'-sequences (the most predominant of multiple 5'-sequence types found on rat IGF-I mRNAs) identifies intact IGF-I mRNAs of 7.5-7.0, 1.9-1.5 and 1.2-0.9 kb but, after oligomer directed RNase cleavage of these mRNAs, identified only a single IGF-I mRNA 5'-fragment. Major differences in the length of sequence 5' to the IGF-I coding sequence therefore, do not account for the multiple size classes of type 1 (class C) IGF-I mRNAs. The size of the 5'-fragment suggests that the extent of sequence 5' to the IGF-I coding sequence is 0.4-0.7 kb in type 1 (class C) IGF-I mRNAs. Identification of multiple 3'-fragments of IGF-I mRNAs demonstrated heterogeneity in the 3'-ends of rat IGF-I mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

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).     

Triiodothyronine nuclear binding capacity in rat tissues correlates with a 6.0 kilobase (kb) and not a 2.6 kb messenger ribonucleic acid hybridization signal generated by a human c-erbA probe

Recent studies have raised the possibility of multiple structurally distinctive tissue-specific nuclear T3 receptors, all exhibiting homology with the v-erbA oncogene and represented by mRNAs of various sizes. We have assayed the level of mRNAs recognized by a 32P-labeled cRNA derived from human plancetal c-erbA-A beta cDNA by solution hybridization and by Northern transfer in different rat tissues, as well as human liver and placenta. Two related mRNAs were demonstrated in the rat tissues analyzed, one measuring 6.0 and the other 2.6 kilobases (kb). In human liver and placenta a 6.0 kb mRNA was seen, but not a 2.6 kb mRNA. Only the 6.0 kb sequence correlated with the receptor concentration determined by 125I-T3 displacement analysis.     

Tissue and species distribution of mRNA encoding two ADP-ribosylation factors, 20-kDa guanine nucleotide binding proteins

Cholera toxin catalyzed ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide binding protein of the adenylyl cyclase system, is enhanced by approximately 20-kDa guanine nucleotide binding proteins, termed ADP-ribosylation factors or ARFs. ARF is an allosteric activator of the A1 catalytic protein of the toxin. Bovine ARF cDNA clones, ARF-1 isolated from adrenal (Sewell & Kahn, 1988) and ARF-2B from retina (Price et al., 1988), exhibit nucleotide and deduced amino acid sequences that are 80% and 96% identical, respectively, in the coding region. To determine tissue and species distribution of ARF-like mRNAs, bovine ARF-2B and human ARF-1 cDNAs and 30- or 48-base oligonucleotide probes that distinguish between ARF-1 and ARF-2B cDNAs in coding and 3'-untranslated regions were used for Northern analysis of poly(A+) RNA from different tissues and species. On the basis of hybridization with specific oligonucleotide probes, all bovine tissues contained mRNAs of 1.7 and 2.1 kb that were related to ARF-1 and ARF-2B, respectively. Northern analysis of brain poly(A+) RNA from different species with ARF-2B and ARF-1 cDNAs at low stringency demonstrated several bands varying in size from 0.9 to 3.7 kb. A 1.7-kb band consistently hybridized with an ARF-1 30-base coding-region probe but not with a probe for the 3'-untranslated region. Similar ARF-2B oligonucleotide probes did not hybridize with rat, mouse, rabbit, or human brain mRNA. Cleavage of ARF-2B cDNA with PvuII generated two fragments, one containing coding and the other 3'-noncoding region.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.     

Characterization of N-cadherin mRNA in chicken brain and heart by means of oligonucleotide probes

It has previously been reported, that there is only one mRNA of 4.3 kb for chicken N-cadherin in brain and heart and one gene [(1988) J. Cell Biol. 106, 873-881]. Using three synthetic oligonucleotide probes derived from the published chicken N-cadherin cDNA sequence we found hybridization to at least four mRNAs in chicken brain and heart. The measured sizes were 8.0, 4.7, 3.8, and 3.3 kb. The 8.0 kb mRNA is only seen in brain, and only when a cytoplasmic probe is employed. The 3.8 kb mRNA is only observed in heart.     

Tissue-specific expression of the rat pancreatic elastase I gene in transgenic mice

The gene for rat pancreatic elastase I is selectively expressed to high levels in the rat exocrine pancreas. When the cloned rat elastase I gene with 7 kb upstream and 5 kb downstream flanking sequences was introduced into mice by microinjection into fertilized eggs, the gene was expressed in a pancreas-specific manner. In four of five transgenic mice, the level of rat elastase I mRNA in the pancreas was equal to or greater than the normal rat level (10,000 mRNAs per cell) and correlated with the number of integrated gene copies. In nonpancreatic tissues the levels were at least 10³-fold lower, except for expression in the liver of one mouse. Thus transfer of a 23 kb genomic DNA segment containing the rat elastase I gene to a foreign chromosomal location in the mouse can give rise to qualitatively and quantitatively normal expression.     

Differential expression of two neural cell-specific beta-tubulin mRNAs during rat brain development.

We recently demonstrated that beta-tubulin mRNA expression is regulated during rat brain development. This is manifested by a dramatic decrease in both 1.8- and 2.9-kilobase (kb) mRNAs when extensive neurite elongation is occurring. Coincident with these decreases is the increased production of a 2.5-kb mRNA. (J.F. Bond and S.R. Farmer, Mol. Cell. Biol. 3:1333-1342, 1983). In the present study, we have isolated and characterized three different cDNAs corresponding to beta-tubulin mRNAs (R beta T.1, R beta T.2, and R beta T.3). Hybridization of 3' untranslated region subclones of R beta T.1 and R beta T.2 cDNAs to RNA from a variety of rat tissues and cells revealed that these two cDNAs are neural cell specific. R beta T.1 corresponds to an abundant 1.8-kb mRNA expressed only at early stages of rat brain development. R beta T.2 corresponds to the 2.5-kb mRNA expressed at later stages. These data strongly suggest that there is differential expression of the beta-tubulin multigene family during rat brain development.     

Developmental regulation of calmodulin gene expression in rat brain and skeletal muscle.

Three different calmodulin genes that encode the identical protein have been identified in the rat (Nojima, 1989); however, calmodulin gene expression at the various stages of tissue differentiation and maturation has not been previously determined. We have quantitated the content of mRNAs encoding calmodulin in the developing brain and skeletal muscle using RNA blot analysis with three specific cDNA probes. Our results show that five species of calmodulin mRNAs: 4.0 and 1.7 kb for CaM I, 1.4 kb for CaM II, and 2.3 and 0.8 kb for CaM III are detectable at all ages in the brain as well as in skeletal muscle but exhibit a tissue-specific developmental pattern of expression. The comparison of the temporal pattern of calmodulin gene expression with both mitotic activity, as demonstrated by cyclin A mRNA levels, and differentiation and maturation of specific brain or muscle regions is consistent with calmodulin involvement in development.     

Regulation of expression of the alternative mRNAs of the rat alpha-thyroid hormone receptor gene

The rat alpha-thyroid hormone receptor gene encodes through alternative splicing at least three protein isoforms with different functions, and three mRNA species (2.6, 5.4, 6.8 kilobase (kb) in size) are detected using alpha gene-specific probes (Mitsuhashi, T., Tennyson, G. E., Nikodem, V. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5804-5808). In the present study, the identities of these mRNAs were analyzed by Northern analysis, and it was demonstrated that in rat brain the receptor protein is encoded by the minor 5.4- and 6.8-kb mRNAs and the variant proteins are encoded by the major 2.6-kb mRNA. Relative quantities of these mRNAs were determined by RNase protection assay, and the ratio of the receptor mRNAs to the variant mRNAs was estimated to be 1:6 in adult brain. The ratio between the mRNAs was regulated in both a tissue-specific and developmental stage-specific manner. The receptor mRNA levels were also regulated by the thyroid state of the animal showing an increased level in hypothyroid rat liver while those in brain were not affected. Analysis of the alpha-thyroid hormone receptor gene suggested that the choice between two poly-adenylation sites and subsequent RNA processing appear to generate the 3' heterogeneity of these alternative mRNAs.     

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.