Specific mRNA Species Research Articles

Quantitation of mRNA by the polymerase chain reaction.

A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.

Quantitative measurement of mRNAs by polymerase chain reaction

Although polymerase chain reaction (PCR) has been used to detect the presence of specific mRNA species, there are no reports indicating that PCR can be used as a reliable, reproducible assay to quantify the relative level of an mRNA. In this study we examined the enzymatic steps (reverse transcription and PCR) required to analyze RNA by PCR and determined the conditions under which the product obtained reproducibly reflects the relative amounts of amplified species in the starting material. Aliquots of total RNA from rat ovaries and GH 3 pituitary cells were used to prepare cDNAs for PCR amplification of ß-actin and prolactin (PRL) sequences, respectively. Assay of equivalent dilutions of ovarian cDNAs made from 10, 2, and 0.4 μg of RNA demonstrated that the amount of PCR product obtained was proportional to both the amount of cDNA amplified and the amount of RNA transcribed, with a relatively small variability for both reactions. cDNAs were also made against RNA prepared from GH 3 cells cultured in the presence or absence of Ca 2+, which induces PRL gene expression. Measurement of PRL mRNA by PCR gave results comparable to those obtained by Northern blot (4.7-fold induction vs. 5.9-fold), and again was highly reproducible. Additionally, PCR analysis of cDNA against GH 3 nuclear RNA allowed us to detect an apparent splice variant of the PRL nuclear RNA that is also Ca 2+ regulated. These results indicate the sensitivity and reliability of PCR as a quantitative assay for specific mRNAs, and demonstrate the possibilities for obtaining data not readily available by other means.

The isolation and characterization of a cDNA clone encoding Lupinus angustifolius root nodule glutamine synthetase.

Glutamine synthetase, purified from Lupinus angustifolius legume nodules, was carboxymethylated and succinylated prior to chemical or enzymatic cleavage. Peptides were purified and sequenced. An oligonucleotide probe was constructed for the sequence MPGQW. This probe was used to identify a glutamine synthetase cDNA clone, pGS5, from a lupin nodule cDNA library constructed in pBR322. pGS5 was sequenced (1043 bp) and computer-assisted homology searching revealed a high degree of conservation between this lupin partial cDNA clone and other plant glutamine synthetases at both the amino acid (greater than 90%) and nucleotide (greater than 80%) level. Northern and Southern analyses using pGS5 supported the conclusion that a multigene glutamine synthetase family exists in lupin which is differentially expressed in both an organ-specific and temporal manner. Western and Northern blot analyses indicated the accumulation of a glutamine synthetase specific mRNA species during nodule development corresponded to the appearance of a novel glutamine synthetase polypeptide between 8 and 10 days after rhizobial inoculation.

Lipopolysaccharide-inducible Macrophage Early Genes Are Induced in Balb/c 3T3 Cells by Platelet-derived Growth Factor

We have previously described the isolation and characterization of a set of cDNA clones encoding lipopolysaccharide (LPS)-induced early genes in murine peritoneal macrophages. The treatment of macrophages with LPS also stimulates the expression of four early or competence genes (c-fos, c-myc, JE, and KC) described in platelet-derived growth factor-stimulated Balb/c 3T3 cells. These latter findings led to the hypothesis that long term, adaptive responses such as DNA synthesis in fibroblasts and functional activation of macrophages may share multiple mechanistic pathways. To test this possibility, we have examined the expression of four LPS-inducible macrophage genes in platelet-derived growth factor-stimulated Balb/c 3T3 fibroblasts. The results demonstrate that three of these four genes are expressed in 3T3 cells in a fashion reminiscent of other growth factor-stimulated competence genes. All three mRNAs are expressed even in the presence of cycloheximide and two of the three exhibit superinducibility. The accumulation of these specific mRNA species was dependent upon the stimulation of transcription as determined by nuclear "run-off" studies. The platelet-derived growth factor dose dependence is comparable both for stimulation of DNA synthesis and expression of the three early genes. Furthermore, expression of all three genes preceded the entry of the cells into S phase, suggesting an association with cell cycle entry. Stimulation of 3T3 cells with epidermal growth factor resulted in DNA synthesis but not early gene expression. This latter result indicates that these early gene products are not necessary for 3T3 cell mitogenesis. Nevertheless, the expression of these genes in two different cell types in association with two distinct functional responses suggests that they contribute common functions either in terms of the physiologic response in which these cells participate (e.g. inflammation) or in the regulatory mechanisms which govern such responses.

Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.

A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed.

Quantitative in-situ hybridization histochemistry of anterior pituitary hormone mRNA species in human pituitary adenomas.

We have examined the anterior pituitary hormone messenger (m) RNA species contained in biopsies of 41 pituitary tumours obtained at hypophysectomy using in-situ hybridization histochemistry. The adenoma were grouped clinically into 12 prolactinomas, 8 somatotrope adenomas, 16 non-functioning, 4 Nelson's syndrome, and 1 thyrotrope adenoma. Of these, 10 contained no detectable anterior pituitary hormone mRNA species and 11 appeared to be expressing the gene responsible for the patients' clinical state in isolation. In a number of cases the accumulation of specific mRNA species was not accompanied by an increase in the circulating levels of the corresponding hormone or subunit. Evidence of activation of more than 1 anterior pituitary hormone gene was present in 16 adenomas of which only 7 showed a pattern of activation or amplification of gene expression which would suggest deregulation of either the inositol phospholipid or cAMP second messenger pathway. It was therefore not possible from these data to postulate that isolated deregulation of a single second messenger transduction pathway is a common etiological factor in pituitary tumour formation.

Time course of response of individual messenger RNAs in the rat heart to T3

The time course of response of specific mRNAs following administration of triiodothyronine (T3) to hypothyroid rats was examined. We were particularly interested in identifying mRNAs showing a rapid response. Hypothyroid rats were injected with 0.2 mg of T3/100 g body wt and total cardiac RNA was prepared 0.5, 1, 2, 3, 5, 12 and 24 h later. RNA was translated in vitro in the presence of [35S]-methionine, the labeled peptides separated by two-dimensional electrophoresis and quantitated by digital matrix photometry. Of a total of 427 translational products 13 were identified to be selectively responsive to thyroid hormone. A specific mRNA coding for a protein designated as spot 72b (Mr 81,600, pI 5.34) was observed to show the most rapid response to T3. Administration of T3 to the hypothyroid animal resulted in an increase in the level of spot 72b by 2.6-fold within 1 h. The lag time between injection of T3 and response of other specific mRNA species varied between 5 to 24 h. These results demonstrate the diversity of response of individual cardiac mRNAs. The specific T3 responsive mRNA species described in the heart have not been demonstrated in other tissues indicating that induction of distinctive mRNA species is highly tissue specific. Relatively late responses may represent indirect effects of T3 mediated by interaction with other hormonal or metabolic signals. The rapid induction of spot 72b suggests it may result from the interaction of T3 with the nuclear receptor leading to a direct effect on the expression of this gene in the heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Disruption of intracellular processing of epidermal growth factor by methylamine inhibits epidermal growth factor-induced DNA synthesis but not early morphological or transcriptional events.

Upon internalization, epidermal growth factor (EGF) is proteolytically processed from its COOH terminus as it traverses intracellular vesicles and lysosomes. This report describes experiments which were conducted to determine whether lysosomotropic amines such as methylamine, which are known to inhibit degradation of EGF, are able to significantly inhibit the COOH-terminal processing of EGF, and whether disruption of EGF processing would negatively affect EGF-stimulated events such as DNA synthesis and induction of specific mRNA species. The results of these experiments indicated that, whereas methylamine treatment had no effect on EGF binding or internalization, vesicular translocation from endocytic vesicles to lysosomes was halted and processing of EGF was severely inhibited. The stimulation of DNA synthesis beginning 12 h after EGF exposure was also markedly inhibited by methylamine treatment. However, addition of methylamine alone produced a non-specific inhibition of DNA synthesis. The ability of EGF to induce specific transcription of the rat transin gene within 6 h of treatment was also not inhibited by methylamine treatment, but was actually increased in the presence of methylamine. These results suggest that at least some early transcriptionally regulated events induced by EGF do not require vesicular processing of EGF (or its receptor) and that the signal transduced by the binding of EGF to its receptor occurs in, or proximal to, the endocytic vesicles.

Molecular cloning of hepatic mRNAs in Rana catesbeiana responsive to thyroid hormone during induced and spontaneous metamorphosis.

Amphibian metamorphosis affords a useful experimental system in which to study thyroid hormone regulation of gene expression during postembryonic vertebrate development. In order to isolate gene-specific cDNA probes which correspond to thyroid hormone-responsive mRNAs, we employed differential colony hybridization of a cDNA library constructed from poly(A)+ RNA of thyroxine-treated premetamorphic tadpole liver. From an initial screening of about 6000 transformants, 32 "potentially positive" colonies were obtained. The recombinant cDNA-plasmids from 13 of these colonies plus two "potentially negative" colonies were purified for further study. Southern blot analysis of the plasmid DNA was employed to determine whether different cDNAs encoded for the same mRNA. The effect of thyroid hormone on the relative levels of specific mRNA species was examined by Northern analysis of liver RNA from premetamorphic tadpoles, thyroxine-treated tadpoles, and adult bullfrogs. Three independent cDNA clones were obtained which encoded thyroid hormone-enhanced mRNAs. We also obtained two independent cDNA clones encoding thyroid hormone-inhibited mRNAs and three independent clones encoding thyroid hormone-unresponsive mRNAs. The levels of two thyroid hormone-enhanced mRNAs and one thyroid hormone-inhibited mRNA were essentially the same in the thyroid hormone-treated tadpole liver and adult liver, suggesting that thyroid hormone induces stable changes in liver gene expression during spontaneous metamorphosis. Using selected cDNAs, RNA dot blot analysis of liver mRNA from tadpoles at different stages of metamorphosis showed that the level of one thyroid hormone-enhanced mRNA increased during late prometamorphosis and metamorphic climax. Similarly, a mRNA which was strongly inhibited by thyroid hormone treatment was observed to decline during prometamorphosis and reach undetectable levels during metamorphic climax. One mRNA was detected which was reproducibly inhibited by thyroid hormone treatment but which remained essentially unchanged during spontaneous metamorphosis. These results provide the first direct evidence for the coordinate and selective pretranslational regulation by thyroid hormone of several liver genes during the developmental process of metamorphosis.

Some aspects of the regulation of pyruvate kinase levels in Neurospora crassa.

Pyruvate kinase levels were monitored in Neurospora crassa mycelium (grown on different carbon sources for varying time intervals) by immunoprecipitation using polyclonal antibodies raised against a purified enzyme preparation. Pyruvate kinase specific mRNA was demonstrated by hybridization of Northern and dot blots of total RNA with a N. crassa pyruvate kinase gene fragment. Two pyruvate kinase specific mRNA species were detected in mycelia of all ages examined. An age-dependent and carbon source dependent variation in the pyruvate kinase protein and mRNA levels was encountered: both registered an increase for up to about 20 h and a subsequent decline; growth on acetate and sucrose resulted in significantly higher yields of both, relative to that on medium containing ethanol and alanine. Stress caused by heat shock depressed the pyruvate kinase mRNA levels.

PC12 variants deficient in catecholamine transport.

We have isolated PC12 cell variants deficient in transporter-mediated uptake of 3,4-dihydroxyphenylethylamine (dopamine). The variants either were obtained nonselectively, or they were selected by resistance to guanethidine or N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Dopamine uptake into guanethidine-resistant cells occurred with a decreased Vmax; the Km for dopamine and inhibition by guanethidine were normal. MPTP-resistant cells lacked the capacity to take up dopamine. Most of the variants resembled wild-type PC12 in their response to nerve growth factor and the storage and secretion of dopamine. MPTP-resistant cells exhibited several deficiencies in addition to dopamine transport, i.e., no measurable storage of dopamine or acetylcholine and no observable response to nerve growth factor. Wild-type and variant cells were compared with respect to the labeling of cell proteins with [3H]xylamine, which binds covalently to certain proteins apparently only after entering PC12 via the catecholamine transporter. When intact variant cells were used, there was markedly reduced labeling of the proteins by [3H]xylamine. Almost all of these proteins were readily labeled when cell homogenates were exposed to [3H]xylamine. However, MPTP-resistant cells were missing three of these proteins. Northern blot analysis with cDNA clones revealed that the MPTP-resistant cells had markedly reduced levels of several specific mRNA species.

Rapid effects of insulin on in vitro translational activity of specific mRNA in diabetic rat heart.

We studied the time course of response of specific cardiac mRNA after administration of insulin to diabetic rats. The primary aim was to identify specific cardiac mRNA, which show a rapid response to insulin administration. Diabetic rats were injected with 2 U of regular insulin intravenously, and total cardiac RNA was prepared 0.5, 1.5, 3, 5, 12, and 24 h later. RNA was translated in vitro in the presence of [35S]methionine and the translational products separated by two-dimensional electrophoresis and quantitated by digital matrix photometry. A rapid change in the translational activity of five specific mRNA species was observed within 0.5 h after administration of insulin to the diabetic animal. One translational product exhibits a more delayed response at 1.5 h. The predominance of three of these products was increased, while that of three was decreased. Two specific mRNA coding for translation products designated as spots 97 and 106 show the most significant change, with a dramatic decrease of 15-fold and 6.5-fold, respectively, within 0.5 h after insulin administration. The change in levels of these specific mRNA species could result from effects of insulin at various sites of mRNA synthesis or degradation. However, the rapidity of the response is compatible with a direct effect of insulin on gene expression. The very quick response of these specific mRNA species to insulin could thus serve as a useful model system to examine the molecular mechanisms of insulin action in the heart.

Synthesis of specific brain proteins is influenced by testosterone at mRNA level in the neonatal rat

Sex differences in brain protein synthesis were investigated by analyzing the in vitro translation products of poly (A) + mRNA isolated from the hypothalamus-preoptic area of male, female and androgenized female (injected with testosterone propionate at day 4) rats at different stages of development. Poly (A) +-enriched mRNA, prepared by oligo (dT)-chromatography of total RNA extracted by guanidine thiocyanate, was translated in either a rabbit reticulocyte lysate or cell-free wheat germ system supplemented with either [ 35S]methionine or a [ 3H]amino acid mix. The translation products were analyzed by one- and two-dimensional SDS-PAGE. Several developmental and androgen-induced changes were found in the translation products of specific mRNA species, the most important of which was a sex difference (higher in the male) in the apparent amount of mRNA coding for proteins with the electrophoretic characteristics of actin and tubulin. These developmental and sex differences in the apparent amounts of specific mRNA species in brain may be the cause or result of functional and structural changes such as neurite growth and synapse formation.

Influence of starvation and total protein deprivation on cardiac mRNA levels.

The effect of starvation and of protein-deprivation on the extractable amount of cardiac mRNA was investigated in male rats. Cardiac mRNA was determined by either (a) isolation of cardiac mRNA by SDS-Phenol/oligo-dT-cellulose, or by (b) hybridization of cardiac mRNA to 3H-Poly(U). During starvation (1-6 days) the extractable amount of cardiac microsomal RNA decreased from 870 micrograms/g heart (controls) to 606 micrograms/g (3 days) and to 547 micrograms/g (6 days), the extractable amount of mRNA fell from 28.6 micrograms/g heart (controls) to 18.7 micrograms/g (3 days) and to 14.5 micrograms/g (6 days). When a normocaloric but protein-deficient diet was fed, the decreases in cardiac microsomal RNA and mRNA were qualitatively similar, but slightly less severe. An analysis of the intracellular distribution of cardiac microsomal RNA and mRNA in the hearts of normal animals and of animals starved or fed a protein-deficient diet indicates that during starvation cardiac mRNA does not accumulate in the cell sap, but gets rapidly degraded. In the refeeding period, mRNA is transported from the nucleus to the cytoplasm and engages in polyribosome formation. The specific mRNA species coding for the major myofibrillar cardiac proteins are affected to a similar extent by these changes during starvation/protein-deprivation and refeeding.

Mapping of the structural gene of pseudorabies virus glycoprotein A and identification of two non-glycosylated precursor polypeptides.

Cell-free translation of pseudorabies virus RNA isolated during the late phase of the infectious cycle yielded a variety of polypeptides. A monoclonal antibody directed against one of the major viral glycoproteins, gA, immunoprecipitated two polypeptides ranging in molecular weight from 78K to 83K. To localize the structural gene for gA, we used cloned BamHI fragments of the viral DNA to select specific mRNA species and immunoprecipitated their in vitro translation products with the anti-gA monoclonal antibody. This allowed us to map the genomic region encoding the mRNA for the gA within the short unique region of the viral genome on BamHI fragments 7 and 12. Additional polypeptides encoded by this region were characterized by their electrophoretic mobility. In three virus strains tested a similar, but strain-specific, pattern of the two gA precursors was found which was not dependent on the host cell or the state of infection after reaching the late phase.

Gene expression in cardiac hypertrophy in rat and human heart muscle.

Changes in the cardiac contents of microsomal RNA and poly(A)-containing mRNA have been examined during induction and regression of heart muscle hypertrophy in rat hearts, as well as possible changes in the subcellular distribution and protein-synthetic activity of cardiac mRNA. In addition, cardiac biopsies from patients with mitral valve diseases were evaluated for their mRNA contents. Induction of heart muscle hypertrophy was accompanied by substantial increases in cardiac microsomal RNA (30-60%) and cardiac mRNA (20-80%). During regression of hypertrophy increased levels of cardiac microsomal RNA and mRNA returned to normal values within 10-16 days. In general, cardiac mRNA levels were lower in human heart muscle than in rat heart muscle. Since the subcellular distribution of the microsomal RNA and of the mRNA as well as the protein-synthetic activity of the mRNA were not changed in the hypertrophied animals as compared with normal animals, and since the cardiac contents of most specific cardiac mRNA species (mRNAs for MHC, MLC1 and MLC2, actin, tropomyosin, troponin-T, myoglobin) increased proportionately, it is concluded that during induction of hypertrophy activation of gene expression occurs and affects the genes coding for the major cardiac proteins to a similar extent. This does, however, not exclude the possibility of more specific shifts in isoprotein patterns and in the levels of their corresponding mRNAs. It is proposed that changes in cardiac mRNA levels are the major regulatory factor in causing changes in cardiac protein synthesis rates leading to the induction and regression of cardiac hypertrophy.

Programmed expression of beta-tubulin genes during development and differentiation of the chicken.

We have previously demonstrated that the chicken genome contains at least four different, functional beta-tubulin genes. By using gene specific probes we have now analyzed the relative levels of expression of the four encoded messenger RNA (mRNA) transcripts as a function of chicken development and differentiation. We have found that the RNA transcript from the beta 2 gene is present in large amounts in embryonic chick brain and is also preferentially expressed in spinal cord neurons, indicating that this transcript encodes the dominant neuronal beta-tubulin polypeptide. The beta 3 mRNA is present in overwhelming amounts in RNA from chicken testis suggesting that this gene encodes a flagellar or meiotic spindle tubulin. However, both of these genes are transcribed to varying, but lesser, degrees in a number of additional cell and tissue types indicating that they are not neuronal or testis specific, respectively. Beta 4' transcripts are present at moderate levels in all cell and tissue types examined, suggesting that this mRNA encodes a constitutive beta-tubulin polypeptide that is involved in an essential or housekeeping microtubule function. Transcripts from the beta 1 gene are a minor component of the beta-tubulin mRNA populations in all cells and tissues tested. Overall, we conclude that specific beta-tubulin mRNA species are expressed in markedly different ratios in different tissues in the chicken. Such developmental regulation may reflect the function(s) of the individual beta-tubulin polypeptides or, alternatively, may be required for precise control of tubulin gene expression in cells that utilize microtubules for divergent purposes.

Alterations in the relative amounts of specific mRNA species in the developing human brain in Down's syndrome.

Total cellular polyadenylated RNA [poly(A)+ RNA] was prepared after guanidinium thiocyanate extraction of frozen brain tissue from age-matched normal and Down's-syndrome (trisomy 21) human foetuses. Poly(A)+ RNA populations were analysed by translation in vitro, followed by two-dimensional gel analysis by using both isoelectric focusing (ISODALT system) and non-equilibrium pH-gradient electrophoresis (BASODALT system) as the first-dimension separation. The relative concentrations of poly(A)+ RNA species coding for seven translation products were significantly altered in Down's syndrome, as determined by both visual comparisons of translation-product fluorograms from normal and Down's-syndrome samples and by quantitative radioactivity determination of individual translation products. The relative concentrations of mRNA species coding for two proteins (68 kDa and 49 kDa) were increased in Down's syndrome and may represent genes located on chromosome 21. The relative concentrations of mRNA species coding for five proteins (37 kDa, 35 kDa, 25.5 kDa, 24.5 kDa, 23 kDa) were decreased in Down's syndrome, these probably representing secondary effects of the trisomy. Six Down's-syndrome-linked translation products (49 kDa, 37 kDa, 33 kDa, 25.5 kDa, 24.5 kDa, 23 kDa) did not migrate with appreciable amounts of cellular proteins on two-dimensional gels and hence may represent either proteins of high turnover rates or those that are post-translationally modified in vivo. One translation product (68 kDa) comigrated with a major cellular protein species, which was identified as a 68 kDa microtubule-associated protein by limited peptide mapping. The significance of these changes is discussed in relation to the mechanisms whereby the Down's-syndrome phenotype is expressed in the human brain.

Influence of thyroid hormone on the in vitro translational activity of specific mRNAs in the rat heart.

The influence of thyroid hormone on the translational activity of specific cardiac mRNA was determined by in vitro translation of RNA isolated from the heart of normal, hypothyroid, and 3,3',5-triiodo-L-thyronine-injected hypothyroid rats. Proteins synthesized in vitro in the presence of [35S]methionine were separated by two-dimensional gel electrophoresis and quantitated by a novel scanning procedure using digital matrix photometry. A total of 421 translational products were detected by fluorography and changes in the predominance of 12 of these were influenced by the thyroid state of the animals. The relative predominance of 8 species was increased in euthyroid animals, whereas 4 translational products were increased in hypothyroid animals. The majority of these thyroid hormone-related alterations occurred in spot pairs of similar molecular weights, but slightly different isoelectric points. In contrast, the relative predominance of mRNAs coding for the major contractile proteins, light chain 1, light chain 2, tropomyosin, actin, and myosin heavy chain was not altered by the thyroid status of the animals. The relative levels of these abundant mRNA species remained unaltered in spite of a thyroid hormone-related increase in total RNA levels. In vivo effects of thyroid hormone on cardiac RNA levels are complex. In addition to a general increase in total RNA and mRNA levels, increases or attenuations in the predominance of a small number of specific mRNA species are observed when euthyroid and hypothyroid animals are compared.

A genetic locus regulates the expression of tissue-specific mRNAs from multiple transcription units.

129 GIX- mice, unlike animals of the congeneic partner strain GIX+, do not express significant amounts of the retroviral antigens gp70 and p30. Evidence is presented indicating that the GIX phenotype is specified by a distinct regulatory gene acting on multiple transcription units to control the levels of accumulation of specific mRNA species. The steady-state levels of retroviral-homologous mRNA from the tissues of GIX+ and GIX- mice were examined by blot hybridization using as probes DNA fragments from cloned murine leukemia viruses. RNA potentially encoding viral antigens was reduced or absent in GIX- mice, even though no differences in integrated viral genomes were detected between these congeneic strains by DNA blotting. Tissue-specific patterns of accumulation of these RNA species were detected in brain, epididymis, liver, spleen, and thymus, and several distinct RNA species were found to be coordinately regulated with the GIX phenotype. Measurements of RNA synthesis suggest a major role for transcriptional control in the regulation of some retroviral messages.