Changes In Steady-state mRNA Levels Research Articles

Global analysis of posttranscriptional gene expression in response to sodium arsenite.

Background:Inorganic arsenic species are potent environmental toxins and causes of numerous health problems. Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription.Objectives:We evaluated the prevalence of changes in mRNA stability in response to sodium arsenite in human fibroblasts.Methods:We used microarray analyses to determine changes in steady-state mRNA levels and mRNA decay rates following 24-hr exposure to noncytotoxic concentrations of sodium arsenite, and we confirmed some of these changes using real-time reverse-transcription polymerase chain reaction (RT-PCR).Results:In arsenite-exposed cells, 186 probe set–identified transcripts were significantly increased and 167 were significantly decreased. When decay rates were analyzed after actinomycin D treatment, only 4,992 (9.1%) of probe set–identified transcripts decayed by > 25% after 4 hr. Of these, 70 were among the 353 whose steady-state levels were altered by arsenite, and of these, only 4 exhibited significantly different decay rates between arsenite and control treatment. Real-time RT-PCR confirmed a major, significant arsenite-induced stabilization of the mRNA encoding δ aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. This change presumably accounted for at least part of the 2.7-fold increase in steady-state ALAS1 mRNA levels seen after arsenite treatment. This could reflect decreases in cellular heme caused by the massive induction by arsenite of heme oxygenase mRNA (HMOX1; 68-fold increase), the rate-limiting enzyme in heme catabolism.Conclusions:We conclude that arsenite modification of mRNA stability is relatively uncommon, but in some instances can result in significant changes in gene expression.Citation:Qiu LQ, Abey S, Harris S, Shah R, Gerrish KE, Blackshear PJ. 2015. Global analysis of posttranscriptional gene expression in response to sodium arsenite. Environ Health Perspect 123:324–330; http://dx.doi.org/10.1289/ehp.1408626

Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels

Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme. However, recent investigations have attributed unique roles to PARP-2 in metabolic regulation in the liver. We assessed changes in hepatic lipid homeostasis upon the deletion of PARP-2 and found that cholesterol levels were higher in PARP-2−/− mice as compared to wild-type littermates. To uncover the molecular background, we analyzed changes in steady-state mRNA levels upon the knockdown of PARP-2 in HepG2 cells and in murine liver that revealed higher expression of sterol-regulatory element binding protein (SREBP)-1 dependent genes. We demonstrated that PARP-2 is a suppressor of the SREBP1 promoter, and the suppression of the SREBP1 gene depends on the enzymatic activation of PARP-2. Consequently, the knockdown of PARP-2 enhances SREBP1 expression that in turn induces the genes driven by SREBP1 culminating in higher hepatic cholesterol content. We did not detect hypercholesterolemia, higher fecal cholesterol content or increase in serum LDL, although serum HDL levels decreased in the PARP-2−/− mice. In cells and mice where PARP-2 was deleted we observed decreased ABCA1 mRNA and protein expression that is probably linked to lower HDL levels. In our current study we show that PARP-2 impacts on hepatic and systemic cholesterol homeostasis. Furthermore, the depletion of PARP-2 leads to lower HDL levels which represent a risk factor to cardiovascular diseases.

Distinct Translational Control in CD4+ T Cell Subsets

Regulatory T cells expressing the transcription factor Foxp3 play indispensable roles for the induction and maintenance of immunological self-tolerance and immune homeostasis. Genome-wide mRNA expression studies have defined canonical signatures of T cell subsets. Changes in steady-state mRNA levels, however, often do not reflect those of corresponding proteins due to post-transcriptional mechanisms including mRNA translation. Here, we unveil a unique translational signature, contrasting CD4+Foxp3+ regulatory T (TFoxp3+) and CD4+Foxp3− non-regulatory T (TFoxp3−) cells, which imprints subset-specific protein expression. We further show that translation of eukaryotic translation initiation factor 4E (eIF4E) is induced during T cell activation and, in turn, regulates translation of cell cycle related mRNAs and proliferation in both TFoxp3− and TFoxp3+ cells. Unexpectedly, eIF4E also affects Foxp3 expression and thereby lineage identity. Thus, mRNA–specific translational control directs both common and distinct cellular processes in CD4+ T cell subsets.

Lipin 2 Is a Liver-enriched Phosphatidate Phosphohydrolase Enzyme That Is Dynamically Regulated by Fasting and Obesity in Mice

Lipin 1 is a bifunctional intracellular protein that regulates fatty acid metabolism in the nucleus via interactions with DNA-bound transcription factors and at the endoplasmic reticulum as a phosphatidic acid phosphohydrolase enzyme (PAP-1) to catalyze the penultimate step in triglyceride synthesis. However, livers of 8-day-old mice lacking lipin 1 (fld mice) exhibited normal PAP-1 activity and a 20-fold increase in triglyceride levels. We sought to further analyze the hepatic lipid profile of these mice by electrospray ionization mass spectrometry. Surprisingly, hepatic content of phosphatidate, the substrate of PAP-1 enzymes, was markedly diminished in fld mice. Similarly, other phospholipids derived from phosphatidate, phosphatidylglycerol and cardiolipin, were also depleted. Another member of the lipin family (lipin 2) is enriched in liver, and hepatic lipin 2 protein content was markedly increased by lipin 1 deficiency, food deprivation, and obesity, often independent of changes in steady-state mRNA levels. Importantly, RNAi against lipin 2 markedly reduced PAP-1 activity in hepatocytes from both wild type and fld mice and suppressed triglyceride synthesis under conditions of high fatty acid availability. Collectively, these data suggest that lipin 2 plays an important role as a hepatic PAP-1 enzyme.

Gap Junctions and Osteoblast-like Cell Gene Expression in Response to Fluid Flow

Bone formation occurs in vivo in response to mechanical stimuli, but the signaling pathways involved remain unclear. The ability of bone cells to communicate with each other in the presence of an applied load may influence the overall osteogenic response. The goal of this research was to determine whether inhibiting cell-to-cell gap junctional communication between bone-forming cells would affect the ensemble cell response to an applied mechanical stimulus in vitro. In this study, we investigated the effects of controlled oscillatory fluid flow (OFF) on osteoblastic cells in the presence and the absence of a gap-junction blocker. MC3T3-E1 Clone 14 cells in a monolayer were exposed to 2 h of OFF at a rate sufficient to create a shear stress of 20 dyne/cm(2) at the cell surface, and changes in steady-state mRNA levels for a number of key proteins known to be involved in osteogenesis were measured. Of the five proteins investigated, mRNA levels for osteopontin (OPN) and osteocalcin were found to be significantly increased 24 h postflow. These experiments were repeated in the presence of 18 beta-glycyrrhetinic acid (BGA), a known gap-junction blocker, to determine whether gap-junction intercellular communication is necessary for this response. We found that the increase in OPN mRNA levels is not observed in the presence of BGA, suggesting that gap junctions are involved in the signaling process. Interestingly, enzyme linked immunosorbent assay data showed that levels of secreted OPN protein increased 48 h postflow and that this increase was unaffected by the presence of intact gap junctions.

Co-activation of synovial fibroblasts by laminin-111 and transforming growth factor-β induces expression of matrix metalloproteinases 3 and 10 independently of nuclear factor-κB

We showed previously that the attachment of synovial fibroblasts to laminin (LM)-111 in the presence of transforming growth factor-β induces significant expression of the matrix metalloproteinase (MMP)-3. Here we go...

Activation of synovial fibroblasts by laminin-1 and transforming growth factor beta induces expression of stromelysins independently of TNFα, IL-1β or NF-κB

Recently it was shown that attachment of synovial fibroblasts (SF) from rheumatoid arthritis patients to laminin-111 (LM-111) induced an elevated expression of stromelysin-1 (MMP-3). We therefore investigated the regulation of additional matrix metalloproteinases (MMPs) and their specific tissue inhibitors of matrix metalloproteinases (TIMPs) by attachment to LM-111 in the presence of transforming growth factor beta (TGFβ). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative RT-PCR. Production of MMPs and cytokines was monitored by a multiplexed immunoarray or by ELISA. Signal transduction pathways were studied by immunoblotting. Attachment of SF to LM-111 in the presence of TGFβ induced significant increases in stromelysin-1 mRNA (12.35-fold, P < 0.001) and protein (mean 62 ng/ml, sixfold, P < 0.008). Expression of stromelysin-2 (MMP-10) mRNA (11.68-fold, P < 0.05) and protein (54 ng/ml, 20-fold, P ≥ 0.02) was significantly activated as well. All other TIMPs and MMPs investigated failed to show this LM-111-facilitated TGFβ response. Induction of stromelysin-1 and stromelysin-2 was associated with the activation of transcription factors c-fos and Egr-1, but phosphorylation of NF-κB was not observed. Further, LM-111-activated and TGFβ-activated SF failed to produce remarkable amounts of IL-1β or TNFα. We conclude that costimulation of synovial fibroblasts by LM-111 together with TGFβ suffices to induce significant expression of MMP-3 and MMP-10 by SF and that this induction is independent of phosphorylation of NF-κB.

Evaluation of gene expression endpoints in the context of a Xenopus laevis metamorphosis-based bioassay to detect thyroid hormone disruptors

Thyroid hormones (TH) are important in growth, development and the maintenance of proper cellular metabolism in vertebrates. Amphibian metamorphosis is completely dependent on TH and forms the basis of a screen for thyroid axis disrupting chemicals that currently relies on external morphological endpoints and changes in thyroid gland histology. The requirement for TH-dependent gene expression makes it possible to augment this screen through the addition of molecular endpoints. In order to do this, gene selection, choice of sampling time, tissue sensitivity, and their relationship to morphological change must all be considered. We exposed stage 54 Xenopus laevis tadpoles to a concentration series of the THs, thyroxine (T 4) and 3,5,3′-triiodothyronine (T 3), and three known TH antagonists, methimazole, propylthiouracil (PTU), and perchlorate. The agonists significantly accelerated metamorphosis as defined by developmental stage attained after 14 days. In contrast, the TH antagonists significantly delayed metamorphosis at 14 days and caused an increase in thyroid gland size at day 8. We assessed the changes in steady-state mRNA levels of thyroid hormone receptor α- and β-isoforms and the basic transcription element binding (BTEB) protein by quantitative real-time polymerase chain reaction. Three tissues (brain, tail and hindlimb) were analyzed at 24, 48 and 96 h and we found that TH receptor, TRβ, and BTEB were the most sensitive gene transcripts for the TH agonists, whereas only TRα displayed significant changes upon antagonist exposure. We detected differences in tissue-specific responses between the two agonists. We matched the concentrations of T 3 and T 4 that elicited similar biological responses at 14 days and compared the induction of gene expression. At 96 h, the TRβ and BTEB expression response to T 3 and T 4 was similar in the tail. In contrast, T 3 elicited no concentration-dependent changes in TRβ and BTEB expression in the brain, whereas T 4 elevated their expression. The tail showed the highest correlation between TH concentration and morphological outcome whereas the brain was the most sensitive to antagonist treatment. Only methimazole and perchlorate showed significant changes in TRα gene expression in the brain whereas PTU did not suggesting differences in cellular mechanisms of action. The greatest effect on gene expression occurred within 48 h with many of the hormone-dependent changes disappearing by 96 h. This study accentuates the need to examine multiple tissues and provides critical information required for optimization of exposure regimens and endpoint assessments that focus on the detection of disruption in TH-regulatory systems.

Unanticipated changes in steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase in rat tibiae.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels are commonly used as an internal control to normalize gene expression data based on the belief that this gene is constitutively expressed. However, GAPDH mRNA levels increased by more than 2.5-fold in tibiae of hindlimb unloaded female rats compared to L32 mRNA levels. Similarly, GAPDH mRNA levels increased compared to 18S ribosomal RNA. Treatment with growth hormone and alcohol show no disparity in GAPDH mRNA levels whereas in some experiments, parathyroid hormone and 17beta-estradiol increased GAPDH mRNA levels. Taken together, these findings indicate that it is essential to demonstrate that GAPDH expression is not altered prior to using the gene as an internal control.

Mechanisms of transcriptional dysregulation in Huntington's disease

Recent studies have provided strong evidence for transcription-related deficits in Huntington's disease (HD). These discoveries include consistent changes in steady-state mRNA levels, direct interactions between huntingtin and known transcription factor proteins, sequestration of transcription-related factors into polyglutamine aggregates, and inhibition of enzymes involved in chromatin remodeling. Also, there is increasing evidence that huntingtin itself may be a transcriptional regulator. This review discusses the cumulative body of evidence for transcriptional dysregulation as a mechanism of HD pathogenesis and possible implications for disease progression and treatment.

Reduced IL-2 and IL-4 mRNA Expression in CD4 + T Cells from Bovine Leukemia Virus-Infected Cows with Persistent Lymphocytosis

The role of T-helper (Th) responses in the subclinical progression of bovine leukemia virus (BLV) infection was explored by determining the contribution of CD4 + T cells to the expression of mRNAs encoding interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) in BLV-infected cattle. Relative levels of mRNA encoding IFN-γ, IL-2, IL-4, and IL-10 were measured in fresh and concanavalin A (Con A) activated peripheral blood mononuclear cells (PBMCs) and purified CD4 + T cells from cows seronegative to BLV (BLV −), seropositive without persistent lymphocytosis (BLV +PL −), and seropositive with PL (BLV +PL +) using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The expressions of IFN-γ, IL-2, and IL-4 mRNAs were significantly reduced in the PBMCs from BLV +PL + cows as compared to BLV − cows. Reduced levels of IL-2 and IL-4 mRNAs were detected in fresh CD4 + T cells from BLV +PL + cows. In contrast, Con A stimulated PBMCs and CD4 + T cells did not differ significantly in expression of IFN-γ, IL-2, IL-10, or IL-4 mRNAs among the BLV infection groups. Using flow-sorted CD4 + T cells and semiquantitative RT-PCR the frequencies of CD4 + T cells transcribing IFN-γ, IL-2, IL-4, and IL-10 mRNAs in the peripheral blood of BLV −, BLV +PL −, and BLV +PL + cows were determined. There were no significant differences in the frequencies of CD4 + T cells expressing these cytokine mRNAs among animals in the different BLV infection categories. Thus, the observed differences in IL-2 and IL-4 mRNAs in CD4 + T cells were due to changes in steady-state mRNA levels expressed by individual cells and not to changes in the frequency of cells transcribing IL-2 and IL-4 mRNAs. These results demonstrate that the progression of BLV infection to PL is associated with reduced expression of classical Th1 and Th2 cytokines by CD4 + T cells, thus suggesting aberrant Th regulation in subclinically infected animals.

Decreased expression of the NADH:ubiquinone oxidoreductase (complex I) subunit 4 in 1-methyl-4-phenylpyridinium -treated human neuroblastoma SH-SY5Y cells

Oxidative stress and mitochondrial dysfunction have been implicated in Parkinson's disease (PD) pathology. NADH:ubiquinone oxidoreductase (complex I) (EC 1.6.99.3) enzyme activity is aberrant in both PD and 1-methyl-4-phenylpyridinium (MPP +) models of PD. Reverse transcription polymerase chain reaction of RNA isolated from MPP +-treated human neuroblastoma SH-SY5Y cells identified changes in steady-state mRNA levels of the mitochondrial transcript for subunit 4 of complex I (ND4). Expression of ND4 decreased to nearly 50% after 72 h of MPP + (1 mM) exposure. The expression of other mitochondrial transcripts did not change significantly under the same conditions. Pre-incubation of cells with the free-radical spin-trap, N-tert-butyl-α-(2-sulfophenyl)-nitrone prior to MPP + exposure, prevented decreases in cell viability and ND4 expression. This suggests that functional defects in complex I enzyme activity in PD and MPP + toxicity may result from changes in steady-state mRNA levels and that free radicals may be important in this process.

Effects of spaceflight and simulated weightlessness on longitudinal bone growth

Indirect measurements have suggested that spaceflight impairs bone elongation in rats. To test this possibility, our laboratory measured, by the fluorochrome labeling technique, bone elongation that occurred during a spaceflight experiment. The longitudinal growth rate (LGR) in the tibia of rats in spaceflight experiments (Physiological Space Experiments 1, 3, and 4 and Physiological-Anatomical Rodent Experiment 3) and in two models of skeletal unloading (hind-limb elevation and unilateral sciatic neurotomy) were calculated. The effects of an 11 day spaceflight on gene expression of cartilage matrix proteins in rat growth plates were also determined by northern analysis and are reported for the first time in this study. Measurements of longitudinal growth indicate that skeletal unloading generally did not affect LGR, regardless of age, strain, gender, duration of unloading, or method of unloading. There was, however, one exception with 34% suppression in LGR detected in slow-growing, ovariectomized rats skeletally unloaded for 8 days by hind-limb elevation. This detection of reduced LGR by hind-limb elevation is consistent with changes in steady-state mRNA levels for type II collagen (−33%) and for aggrecan (−53%) that were detected in rats unloaded by an 11 day spaceflight. The changes detected in gene expression raise concern that spaceflight may result in changes in the composition of extracellular matrix, which could have a negative impact on conversion of growth-plate cartilage into normal cancellous bone by endochondral ossification.

Analysis of cytokine mRNA expression following repeated exposure of mice to chemical contact and respiratory allergens.

It has been shown previously that cytokine secretion patterns characteristic of the activation of discrete responses by functional subsets of T cells of type 1 and type 2, respectively, are elicited following topical exposure of BALB/c strain mice to chemical contact and respiratory allergens. In order to investigate if the differences in protein profiles are paralleled by changes in steady-state mRNA levels we have now investigated cultured draining lymph node cell (LNC) cytokine mRNA expression profiles by reverse transcriptase-polymerase chain reaction (RT-PCR) under conditions where divergent cytokine secretion is observed. Mice were exposed topically by repeated application of the respiratory allergen trimellitic anhydride (TMA) or of the contact allergen 2,4-dinitrochlorobenzene (DNCB). An elevation in the expression of mRNA for interleukin 4 (IL-4) and interleukin 10 (IL-10) by LNC from both TMA- and DNCB-treated animals was observed within 6 h of culture, reaching maximal levels after 72 h. Relative mRNA levels for both of these type 2 cytokines were considerably higher in cultured cells derived from TMA-exposed mice, compared with those from DNCB-treated animals. Transient low levels of the type 1 cytokine interferon y (IFN-gamma) were observed in response to treatment with TMA, whereas a substantial upregulation of IFN-gamma gene expression was seen from 24 h onwards in cultured LNC derived from DNCB-exposed mice. Changes in cytokine mRNA in allergen-activated LNC preceded protein production, although the kinetic profiles were similar. These data suggest that the divergent cytokine secretion profiles exhibited by mice treated by repeated topical exposure to contact and respiratory allergens are controlled primarily at the level of transcription. The RT-PCR methodology described herein may be more sensitive for the detection of cytokines expressed in low copy number, such as IL-4, where previously it has been found necessary to stimulate LNC with mitogen to elicit measurable levels of protein secretion. However, this technique was not found to offer major practical advantages when compared with protein detection methods (enzyme-linked immunosorbent assays) for the routine predictive characterization of chemicals as a function of cytokine 'fingerprinting'.

Hyperosmotic Stress as a Stimulant for Proinflammatory Cytokine Production

The synthesis of proinflammatory cytokines involves members of the mitogen-activated protein (MAP) kinase stress pathway, particularly p38 MAP kinase and c-jun NH2-terminal kinase. In this report we used hyperosmotic stress to study changes in steady-state mRNA levels and synthesis of proinflammatory cytokines in freshly obtained human peripheral blood mononuclear cells (PBMC)in vitro.There was no evidence of interleukin (IL)-8 gene expression in freshly obtained human blood despite 30 cycles of amplification of reverse-transcribed mRNA using the polymerase chain reaction. In contrast, exposure of PBMC to hyperosmotic conditions (330–410 mOsM) by the addition of NaCl to tissue culture medium induced gene expression for IL-1α, IL-1β, and IL-8. Routine tissue culture medium is hyperosmotic (305 mOsM) compared to human plasma (280–295 mOsM), but decreasing the osmolarity to the physiological range resulted in a 50% reduction in baseline IL-8 synthesis (P< 0.001). Although hyperosmotically induced accumulation of steady-state mRNA levels for IL-1α and IL-1β increased 50- and 7-fold over control, respectively, these were poorly translated into each respective cytokine. However, in PBMC stimulated by hyperosmotic stress, the addition of femtomolar concentrations of bacterial lipopolysaccharide, IL-1, or 1% normal human serum resulted in a synergistic synthesis (at least twice that expected) of IL-1α, IL-1β, TNF-α, and IL-8.

Alterations in mRNA stability during rat liver regeneration.

We examined the in vivo transcriptional and posttranscriptional regulation of various genes involved in hepatocyte growth and replication that exhibited changes in steady-state mRNA levels after 70% partial hepatectomy (PH). Of the 19 genes examined by nuclear run-on assay, 17 demonstrated no change in transcriptional activity through the first 96 h of regeneration. However, results from in vivo half-life determinations indicated that changes in mRNA stability played a critical role in regulating transcript levels during liver regeneration. For many of the genes, alterations in transcript abundance correlated with similar changes in mRNA half-lives. Inhibition of protein synthesis by cycloheximide was generally associated with increased levels of mRNA expression, but no detectable changes in transcriptional rates in both control and regenerating rat liver. Finally, genomic methylation status was investigated by Southern analysis for several genes that displayed changes in mRNA stability. Interestingly, increases in mRNA half-lives for the genes p53, c-myc, H-ras, and ornithine decarboxylase were associated with decreased genomic methylation. In conclusion, regulation of gene expression beyond the immediate early phase of te cell cycle during rat liver regeneration after PH occurs predominantly at the posttranscriptional level. mRNA stability appears to be a significant factor in this control, and may itself be modulated by the methylation status of the corresponding genomic DNA.

Posttranscriptional regulation of mRNA levels in rat liver associated with deoxycholic acid feeding.

We investigated the effects of bile acid feeding on the mRNA levels and transcriptional activity of genes involved in various facets of hepatic cell function. Rats were maintained for 10 days on standard diet supplemented with combinations of 1 and 0.4% deoxycholic acid and ursodeoxycholic acid. Significant reductions in mRNA levels for liver fatty acid binding protein, albumin, the asialoglycoprotein receptor, connexins 32 and 26, and cytochromes P-450IIB1 and P-450IIE1 were associated with 1% deoxycholic acid feeding. Conversely, the 1% deoxycholic acid-fed animals exhibited increased mRNA levels for cholesterol 7 alpha-hydroxylase, 3-hydroxy-3-methylglutaryl-CoA reductase, multidrug resistance, procollagens, extracellular matrix, protooncogenes, tumor suppressors, and cyclins. The 0.4% deoxycholic acid-fed animals exhibited increased mRNA levels for c-jun, H-ras, p53, cyclins D1 and D3, fibronectin, and procollagens alpha 1(I) and alpha 1(III). Transcriptional rate changes could not account for the observed changes in steady-state mRNA levels. Ursodeoxycholic acid feeding had no significant effect on gene expression and almost completely inhibited the changes associated with 1% deoxycholic acid when coadministered. The results indicate that dietary ingestion of deoxycholic acid profoundly affects hepatic gene expression in the rat, and regulation occurs primarily at the posttranscriptional level.

Parathyroid gland calcium receptor mRNA levels are unaffected by chronic renal insufficiency or low dietary calcium in rats

Extracellular ionized calcium (Ca(2+)) is the primary physiological regulator of parathyroid hormone (PTH) secretion and the G protein-coupled receptor (CaR) that mediates this response has been cloned from bovine and human parathyroid glands. The Ca(2+) set-point for the regulation of PTH secretion is right-shifted in primary hyperparathyroidism (1°HPT), but whether there is a similar shift in 2°HPT is unclear. Additionally, the molecular defects associated with such changes in the set-point remain uncharacterized. These experiments were designed to determine (1) if changes in set-point occur in rats with 2°HPT induced by chronic renal insufficiency (CRI) or dietary Ca deficiency, and (2) whether any changes in set-point are mirrored by changes in steady-state mRNA levels for the parathyroid CaR. CaR mRNA levels were quantified in pairs of glands from individual rats using a solution hybridization assay. Blood urea nitrogen and PTH levels were ∼ 4-fold higher in rats with CRI induced by 5/6 nephrectomy 7 weeks earlier. Rats with CRI were also significantly hypocalcemic and hyperphosphatemic. The setpoint was unchanged in CRI rats and CaR mRNA levels were also unaffected. Normal rats fed a 0.02% Ca diet for 6 weeks were markedly hypocalcemic, and had 10- and 15-fold increases in plasma PTH and 1,25-dihydroxyvitamin D(3) levels, respectively. Technical problems prevented assessment of the set-point in these animals, but parathyroid gland CaR mRNA levels were identical in both dietary groups. Thus, neither alterations in mRNA levels for the CaR nor changes in the set-point play demonstrable roles in the pathogenesis of 2°HPT in these models.

Transcriptional and post-transcriptional mechanisms regulating neurofilament and tubulin gene expression during normal development of the rat brain

The transcription of the beta II-, beta IV- and alpha 1-tubulin genes as well as that of the three neurofilament genes, NF-L, NF-M and NF-H, was examined during the course of postnatal brain development. Changes in the transcriptional activity of these genes were studied using run-off transcription assays with nuclei isolated from the rat cerebral cortex at postnatal days P2, P5, P10 and adult stages. Northern blotting of total RNA isolated from the cerebral cortex was used to compare changes in steady-state mRNA levels with transcriptional changes that occurred in the cerebral cortex during the postnatal interval. Nuclear run-off assays showed that beta II- and alpha 1-tubulin gene transcription rates were maximal from P2-P5 and declined at later times. Changes in the steady-state mRNA levels for these two genes followed the same general pattern as transcription, but in the case of beta II-tubulin mRNA, were more dramatic. beta IV-tubulin gene transcription dropped between P2 and P5 and then increased progressively to the adult stage, coordinate with an increase in beta IV-tubulin steady-state mRNA levels. NF-L and NF-H genes showed similar patterns of transcriptional change during the postnatal interval, with maximal rates of transcription at P5 followed by a decline at later times. The steady-state levels of NF-L and NF-H mRNAs changed in a manner opposite to that of transcription and increased progressively during the postnatal interval. This suggests that mRNA stabilization is the main factor regulating the steady-state levels of NF-L and NF-H mRNAs in postnatal brain. For the NF-M gene, the developmental transcription pattern was also dissociated from steady-state mRNA level changes, but differed from the transcription patterns of the NF-L and NF-H genes. This suggests the importance of post-transcriptional mechanisms in regulating NF-M mRNA levels in brain and also indicates that some differences exist in the regulatory mechanisms which control NF-M compared to NF-L and NF-H mRNA levels.

Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.).

The effects of leaf developmental age on the expression of three nuclear gene families in pea (Pisum sativum L.) coding for enzymes of chlorophyll and heme biosynthesis have been examined. The steady-state levels of mRNAs encoding aminolevulinic acid (ALA) dehydratase, porphobilinogen (PBG) deaminase, and NADPH:protochlorophyllide reductase were measured by RNA gel blot and quantitative slot-blot analyses in the foliar leaves of embryos that had imbibed for 12 to 18 h and leaves of developing seedlings grown either in total darkness or under continuous white light for up to 14 d after imbibition. Both ALA dehydratase and PBG deaminase mRNAs were detectable in embryonic leaves, whereas mRNA encoding the NADPH:protochlorophyllide reductase was not observed at this early developmental stage. All three gene products were found to increase to approximately the same extent in the primary leaves of pea seedlings during the first 6 to 8 d after imbibition (postgermination) regardless of whether the plants were grown in darkness or under continuous white-light illumination. In the leaves of dark-grown seedlings, the highest levels of message accumulation were observed at approximately 8 to 10 d postgermination, and, thereafter, a steady decline in mRNA levels was observed. In the leaves of light-grown seedlings, steady-state levels of mRNA encoding the three chlorophyll biosynthetic enzymes were inversely correlated with leaf age, with youngest, rapidly expanding leaves containing the highest message levels. A corresponding increase in the three enzyme protein levels was also found during the early stages of development in the light or darkness; however, maximal accumulation of protein was delayed relative to peak levels of mRNA accumulation. We also found that although protochlorophyllide was detectable in the leaves immediately after imbibition, the time course of accumulation of the phototransformable form of the molecule coincided with NADPH:protochlorophyllide reductase expression. In studies in which dark-grown seedlings of various ages were subsequently transferred to light for 24 and 48 h, the effect of light on changes in steady-state mRNA levels was found to be more pronounced at later developmental stages. These results suggest that the expression of these three genes and likely those genes encoding other chlorophyll biosynthetic pathway enzymes are under the control of a common regulatory mechanism. Furthermore, it appears that not light, but rather as yet unidentified endogenous factors, are the primary regulatory factors controlling gene expression early in leaf development.