Regulation of specific rat liver messenger ribonucleic acids by triiodothyronine

A plasmid cDNA library was constructed using poly(A+) RNA isolated from the livers of rats treated with 3,5,3'-triiodothyronine (T3) and fed a high carbohydrate diet. This library was screened by differential colony hybridization with [32P]cDNA probes made from hypothyroid and hyperthyroid rat liver poly(A+) RNA to obtain clones representing T3-inducible mRNAs. Using plasmid cDNAs to 4 different T3-inducible mRNAs, we have studied by hybridization assay the responses of these mRNAs to different thyroidal steady states and to a high carbohydrate diet. The fold of induction (hypothyroid to hyperthyroid) varied from about 4.0 (mRNA 5-8D) to 13.2 (mRNA 4-12B). The linearity of response with regard to nuclear receptor occupancy was estimated by assessing the relative mRNA levels in a euthyroid state. Three of the mRNAs demonstrated nonlinear responses with the largest portion of the induction occurring in the euthyroid to hyperthyroid transition. An induction by the high carbohydrate diet was clearly seen for only one mRNA (5-8D) suggesting that these two pathways of induction are independent. In a study of the response kinetics of each mRNA to a nuclear receptor saturating dose of T3 in hypothyroid animals, an increase was seen within 4 h (the earliest time point examined) for one of the mRNAs. The other 3 mRNAs did not increase significantly until 8 h after the T3 dose. Northern analysis showed a single mRNA corresponding to each of these 4 clones with sizes ranging from about 1375 to 7600 bases. Two mRNAs (5-9E and 4-12B) were shown by hybrid-selected translation to code for proteins of molecular mass of about 27 and 46 kDa, respectively. The availability of several different cDNA probes to T3 responsive liver mRNAs should facilitate future studies on the mechanism of action of this hormone.     

The full length coding sequence of rat liver androsterone UDP-glucuronyltransferase cDNA and comparison with other members of this gene family.

Cloned cDNAs coding for hepatic UDP-glucuronyltransferase (UDPGT) have been isolated from a rat liver cDNA library in the expression vector bacteriophage lambda gt11 using anti-UDPGT antibodies. Four different mRNAs have been identified by sequencing of 15 UDPGT cDNA clones. The sequences of the four classes of cDNA were determined to be 85-95% homologous. Restriction fragments were isolated from the cDNA in each class and used as class specific probes. Hybridisation of these probes to northern blots of total RNA prepared from the livers of normal and genetically deficient Wistar rats identified the cDNA in class 4 with androsterone UDPGT. Translation of the cDNA sequence of clone rlug 23, the longest member of class 4, allowed determination of the complete amino acid sequence of androsterone UDPGT.     

Androgen regulation of prostatic binding protein messenger RNA studied by using cloned complementary DNA

The construction of a double-stranded cDNA library using rat prostatic poly(A)RNA and pBR322/kappa 1776 system and the isolation of three prostatic binding protein (PBP) cDNA clones are described. These cDNA clones were characterized and identified by in situ hybridization, mRNA selection-translation and immuno-precipitation as coding for the three subunit components, C1, C2, and C3, of PBP. These clones were used in hybridization experiments with prostatic poly(A)RNA to determine the effect of testosterone on the levels of PBP-mRNA. The results showed that synthesis of these mRNAs varied in response to either androgen withdrawal or replacement. Accumulation of PBP-mRNAs coding for C2 and C3 components occurred 1 hr after androgen administration to castrated rat, whereas the mRNA coding for the C1 component did not appear until 4 hr after androgen replacement. Quantitation of PBP-mRNA sequences in nuclear and polysomal poly(A)RNAs showed that they did not vary coordinately in response to androgen withdrawal. These results indicate differential regulation of PBP genes and suggest possible multiple levels of androgen control of PBP synthesis.     

Differential accumulation of potato tuber mRNAs during the hypersensitive response induced by arachidonic acid elicitor

Accumulation of messenger RNAs in potato tuber discs was analysed during the hypersensitive response induced by treatment with the biotic elicitor arachidonic acid. In vitro translation of polysomal poly(A)⁺ RNAs indicated that the accumulation of some sixteen mRNAs varied following treatment with arachidonic acid, and that the level of thirteen of these was increased. Two cDNA closes (pSTH-1 and-2) were isolated from a library of elicitor-treated tissue cDNAs. Northern blot analysis using these clones as molecular probes indicated that the levels of at least two mRNAs were markedly increased after elicitor treatment. In hybrid-released translation experiments, each of the cDNA clones selected more than one mRNA. Translation of these mRNAs yielded two polypeptides of M_r 45 000 (for the pSTH-1 clone), and three polypeptides of M_e 17 000 (for the pSTH-2 clone). The low molecular weight polypeptides may correspond to potato pathogenesis-related (PR) proteins.     

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

Changes induced in rat liver polysomal polyadenylated RNAs by depletion of circulating glucocorticoids.

The effects of adrenalectomy on the complexity and the relative abundances of rat liver polyadenylated mRNAs have been investigated. The qualitative and quantitative changes induced by adrenalectomy have been measured by hybridisation of polysomal polyadenylated RNAs from the livers of normal and adrenalectomised rats with total cDNAs, fractionated cDNAs, cDNA representing RNAs specific to normal liver, total unique-sequence DNA and unique-sequence DNA complementary to normal liver polysomal RNA. These analyses indicated that, by 14 days after adrenalectomy, the equivalent of about 7000 sequences of average length 2000 nucleotides can no longer be detected in liver polysomes. Many other sequences are decreased in abundance as compared to normal liver, but some abundant sequences become more abundant. Administration of a glucocorticoid hormone (dexamethasone) very rapidly reverses these changes.     

Isolation and characterization of cDNA clones for castration-induced mRNAs in the rat ventral prostate.

To identify gene products involved in castration-induced involution of the rat ventral prostate, we constructed a subtraction cDNA library of the ventral prostate from rats castrated for 48 h. The library was screened with subtracted cDNA probes enriched for sequences with a low copy number expressed in intact or castrated rats. As a result of differential screening, 48 cDNA clones representing 10 different induced mRNAs were isolated. The time course of these mRNA inductions after castration was examined. Within the first 24 h after castration, the level of mRNAs for these cDNA clones was significantly increased and it reached its peak by 48-72 h after castration. Although mRNAs for these cDNA clones were expressed in various tissues from intact rats, an increase in mRNA as a response to castration was observed only in the ventral prostate. Partial sequence analyses of the 10 cDNA clones indicate that three cDNA clones represent rat glutathione S-transferase Yb-1, Yb-2 and Yb-3 subunit mRNA sequences, but for others respective homologues could not be found in a search of the GenBank database (release 67).     

Identification of two cDNA clones coding for androgen-dependent polypeptides in rat ventral prostate

cDNA clones were obtained by transformation of $\text{E. coli}$ x1776 with pBR322 containing insert of ds cDNA synthesized from total rat prostate poly(A) RNA. Two prostate-specific cDNA clones were isolated by colony hybridization and identified by message selection/translation as encoding polypeptides of M_r: 13,500 and 9,300. Hybridization of poly(A) RNA from normal and castrated rat prostates to the cloned cDNAs indicated that the levels of mRNAs coding for M_r: 13,500 and 9,300 polypeptides are regulated by testosterone.     

Rat tropoelastin is synthesized from a 3.5-kilobase mRNA

A lambda gt11 cDNA library was constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats and screened with a human tropoelastin cDNA clone. DNA sequence analysis of several overlapping rat clones confirmed the presence of DNA sequences coding for murine tropoelastin and DNA sequences coding for the 3'-untranslated region of the rat tropoelastin mRNA. Northern blot analysis of total RNA from aortic tissue of neonatal rats using oligonucleotide probes derived from these rat tropoelastin cDNAs demonstrated the presence of a 3.5-kilobase tropoelastin mRNA. The size of this rat tropoelastin mRNA agrees with previous reports for the size of the mRNA coding for tropoelastin in tissue from several vertebrate species but contrasts with several reports suggesting the presence of a higher molecular weight mRNA species responsible for the synthesis of tropoelastin in rodent tissue.     

Bleomycin treatment of chick fibroblasts causes an increase of polysomal type I procollagen mRNAs. Reversal of the bleomycin effect by dexamethasone

Bleomycin treatment of primary chick skin fibroblasts and chick lung fibroblasts resulted in a selective dose-dependent increase of cell layer procollagen synthesis. Solid support hybridization of total cellular RNA to 32P-labeled pro-alpha 1(I) and pro-alpha 2(I) cDNAs did not indicate an increase of total cellular procollagen type I mRNAs in bleomycin-treated cells. However, bleomycin treatment of chick skin fibroblasts causes a redistribution of procollagen type I mRNAs within the nuclear, cytoplasmic, and polysomal subcellular fractions. Both the nuclear and cytoplasmic procollagen type I mRNAs are significantly decreased in concentration after bleomycin administration. In contrast, the polysomal procollagen type I mRNAs are significantly increased in both chick skin and lung fibroblasts treated with bleomycin. Administration of dexamethasone to bleomycin-treated fibroblasts resulted in a reversal of the bleomycin-induced increase in cell layer procollagen synthesis. The increased amounts of polysomal procollagen type I mRNAs in bleomycin-treated cells were also reduced by subsequent administration of dexamethasone. These data indicate that bleomycin treatment of chick skin and chick lung fibroblasts results in a specific increase in procollagen synthesis in the cell layer which is mediated by elevated levels of polysomal type I procollagen mRNAs via a repartitioning of these mRNAs within the fibroblast. Furthermore, dexamethasone reverses the bleomycin-induced elevations of both cell layer procollagen synthesis and polysomal type I procollagen mRNAs.     

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.     

A generally applicable improved method for preparation of single stranded cDNA probes from clones constructed in M13 vectors

A generally applicable simplified procedure for the preparation of radiolabeled cDNA hybridization probes from cDNA clones in M13 (M13mp8) bacteriophage vectors is described. A cDNA copy of the insert DNA is synthesized by controlled reaction with the Klenow fragment of E. coli DNA polymerase I, primed with oligo-dT or sequencing primer. The cDNA is separated from the recombinant phage DNA template by alkaline gel electrophoresis. Sensitivity of the cDNAs was tested by quantitative measurement of specific mRNAs in solution hybridization under RNA (R0t analysis) or cDNA (RNA titration) excess conditions. The procedure permits measurement of mRNA levels as small as 0.00001-0.00006% in total RNA preparation. Cellular accumulation of hormone-induced mRNAs for the milk proteins, whey acidic protein and epsilon-casein was also measured using the cDNAs.     

Multiple mRNAs encode peripherin, a neuronal intermediate filament protein.

Three cDNA clones of 1.6 (3u), 1.2 (5g) and 0.6 (5b) kbp, specific for peripherin, a neuronal intermediate filament protein (IFP), have been isolated from a murine neuroblastoma cell lambda gt11 library by immunoscreening using peripherin antiserum. Antibodies eluted from the fusion proteins produced by clones 3u and 5g recognize the peripherin spots on immunoblots. Where they overlap the three cDNAs have identical sequences. cDNA 5g exhibits the closest homology to type III IFP cDNAs. cDNA 3u is identical to the corresponding region of cDNA 5g, except for the insertion of a 96 bp fragment at a position corresponding to the junction of exons 4 and 5 in type III IFP cDNAs. cDNA 5b is also identical to the corresponding region of cDNA 5g, except for the deletion of a 62 bp fragment at the junction of exons 8 and 9 in type III IFP cDNAs. S1 mapping experiments performed with probes covering the 3' end of the two unexpected regions show that three distinct mRNAs correspond to the three cDNAs. Moreover, three peripherin products, two minor 61 and 56 kd products in addition to the major 58 kd peripherin, are observed when poly(A)+ RNA is in vitro translated, the 61 kd peripherin being translated from the 3u-selected RNA. The three RNAs originate from alternative splicing of a unique peripherin gene, thus generating polymorphism of peripherin.     

Molecular cloning of rat insulin-like growth factor I complementary deoxyribonucleic acids: differential messenger ribonucleic acid processing and regulation by growth hormone in extrahepatic tissues

Two classes of insulin-like growth factor I (IGF-I) cDNAs were isolated from an adult rat liver library using a human IGF-I cDNA probe. The two types of rat IGF-I cDNA differed by the presence or absence of a 52-base pair insert which altered the derived C-terminal amino acid sequence of the E peptide, but not the 3'-untranslated region or the sequence coding for the mature IGF-I protein. When probes derived from these cDNA clones were hybridized to Northern blots of rat mRNA, specific bands of 8.6, 2.1, and 1.0-1.4 kilobases were seen. Hybridization to poly(A)+ RNA from various tissues from GH-treated and control rats demonstrated an increase in IGF-I mRNA due to GH treatment in all tissues examined.