Polysomal mRNA Research Articles

Mevalonate regulates polysome distribution and blocks translation-dependent suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA: relationship to translational control.

We reported previously that 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis is regulated at the translational level by mevalonate. To determine at what stage mevalonate affects reductase synthesis, we examined the distribution of reductase mRNA in polysomes from cells treated with lovastatin alone; lovastatin and 25-hydroxycholesterol; or lovastatin, 25-hydroxycholesterol, and mevalonate. In lovastatin-treated cells, reductase mRNA was primarily associated with heavy polysome fractions. When 25-hydroxycholesterol was added to lovastatin-treated cells, reductase mRNA levels were reduced approximately fourfold in all polysome fractions, with no accompanying redistribution of reductase mRNA into lighter polysome fractions. However, addition of both 25-hydroxycholesterol and mevalonate to lovastatin-treated cells shifted reductase mRNA from heavier to lighter polysome fractions. No change in the distribution of control beta-actin or ribosomal protein S17 mRNA occurred with any of the treatments. These results suggest that mevalonate suppresses reductase synthesis at the level of initiation. When the translation inhibitor cycloheximide was added to all three regimens, reductase mRNA shifted into heavy polysome fractions. Treatment with either lovastatin alone or lovastatin plus 25-hydroxycholesterol resulted in a 50% greater loss of reductase mRNA from the heavy polysome fractions compared to the same fractions from noncycloheximide-treated cells. No loss of reductase mRNA occurred when cycloheximide was added to cells treated with both 25-hydroxycholesterol and mevalonate. beta-Actin mRNA levels and polysome distribution were not significantly changed by cycloheximide under any of these conditions. Translationally mediated suppression of reductase mRNA did not occur when protein synthesis was inhibited with puromycin. Our results indicate that regulation of reductase mRNA levels is translation-dependent and is linked to the rate of elongation.

Poly (A) binding protein is bound to both stored and polysomal mRNAs in the mammalian testis

RNA-binding proteins that bind to the 3' untranslated region of mRNAs play important roles in regulating gene expression. Here we examine the association between the 70 kDa poly (A) binding protein (PABP) and stored (RNP) and polysomal mRNAs during mammalian male germ cell development. PABP mRNA levels increase as germ cells enter meiosis, reaching a maximum in the early postmeiotic stages, and decreasing to a nearly nondetectable level towards the end of spermatogenesis. Most of the PABP mRNA is found in the nonpolysomal fractions of postmitochondrial extracts, suggesting that PABP mRNA is either inefficiently translated or stored as RNPs during spermatogenesis. Virtually all of the testicular PABP is bound to either polysomal or nonpolysomal mRNAs, with little, if any, free PABP detectable. Analysis of several specific mRNAs reveals PABP is bound to both stored (RNP) and translated forms of the mRNAs. Western blot analysis and immunocytochemistry indicate PABP is widespread in the mammalian testis, with maximal amounts detected in postmeiotic round spermatids. The presence of PABP in elongating spermatids, a cell type in which PABP mRNA is nearly absent, suggests that PABP is a stable protein in the later stages of male germ cell development. The high level of testicular PABP in round spermatids and in mRNPs suggests a role for PABP in the storage as well as in the subsequent translation of developmentally regulated mRNAs in the mammalian testis.

The beneficial effects of incubating bovine embryos in groups are due to platelet-derived growth factor

This report presents an analysis of histone gene expression in the cleaving embryo of the sea urchin, Strongylocentrotus purpuratus, with emphasis on whether the regulatory site(s) in the pathway of gene expression change as development proceeds. The analysis focuses on the equation, dP∗dt = M·f·n·At, where dP∗dt = the absolute rate of histone synthesis; M = the mole quantity of histone messenger RNA; f = the fraction of histone mRNA in polysomes; n = the polysome size; and At = the rate of elongation of nascent histone polypeptide chains. The embryo solves this rate equation differently at different times. Measurements were made (at 15°C) of absolute rates of histone synthesis (dP∗dt). The rate of histone synthesis increases at least 48-fold during the first 6 hr after fertilization from less than 0.5 to 24 pg embryo−1 hr−1; in the period from 6 to 12 hr, this rate rises to 182 pg embryo−1 hr−1, an additional 7.7-fold rise, resulting in an overall increase of 370-fold between the 1-cell and 200-cell stage. The fraction of newly synthesized (zygotic) histone messenger RNA that partitions into polysomes (fzygotic) has also been measured during the first 12 hr of development. This fraction increases from 0.2 in the 2-hr embryo to 0.8 in the 6-hr embryo (16-cell stage), increasing slowly thereafter to near unity by 12 hr. The size of histone-synthesizing polysomes (n) does not change substantially over the 12-hr interval, remaining constant at a weighted mean of 5 ribosomes per polysome (range 3 to 7). Utilizing the data on fzygotic and dP∗dt, the rate of elongation of nascent histone polypeptide chains (At) during the first 6 hr of development was estimated; At remains constant at 1.11 codons per second. This calculated value is in fair agreement with a direct measurement of histone peptide elongation rate in the 12-hr embryo. It is proposed that histone gene expression in cleaving sea urchin embryos be divided into two phases, distinguished on the basis of their pivotal translational parameters: Phase I (0–6 hr), during which f is rate determining, and Phase II (6 hr on), during which M is the rate-determining parameter.

In male mouse germ cells, copper-zinc superoxide dismutase utilizes alternative promoters that produce multiple transcripts with different translation potential.

Copper-zinc superoxide dismutase (SOD-1) is an enzyme that is widely expressed in eukaryotic cells and performs a vital role in protecting cells against free radical damage. In mouse testis, three different sizes of SOD-1 mRNAs of about 0.73, 0.80, and 0.93 kilobases (kb) are detected. The 0.73-kb mRNA is found in early stages of male germ cells and in all somatic tissues. The mRNAs of 0.80 and 0.93 kb are exclusively detected in post-meiotic germ cells. RNase H digestions and Northern blot analyses reveal that the three SOD-1 mRNAs are derived from two transcripts, a ubiquitously expressed transcript and a post-meiotic transcript, which differ by 114-120 nucleotides. RNase protection assays demonstrate that the additional nucleotides present in the post-meiotic mRNA are solely in the 5'-untranslated region. Using a probe derived from the 5'-untranslated region of the 0.93-kb SOD-1 mRNA, we have established that it originates from an alternative upstream promoter contiguous with the somatic SOD-1 promoter. Polysomal gradient analysis of the three mouse testis SOD-1 mRNAs reveals that the 0.93-kb SOD-1 mRNA is primarily non-polysomal, while the 0.80- and 0.73-kb SOD-1 mRNAs are mostly polysome associated. A faster migrating form of the 0.93-kb SOD-1 mRNA is present on polysomes as a result of partial deadenylation. In a cell-free translation system, the 0.73-kb SOD-1 mRNA translates about 2-fold more efficiently than the 0.93-kb SOD-1 mRNA. These data demonstrate that male germ cells transcribe two size classes of SOD-1 mRNAs with different translation potential by utilizing two different promoters, post-meiotic SOD-1 mRNAs undergo adenylation changes, and one of the post-meiotic SOD-1 mRNAs is transcribed during mid-spermiogenesis and translated days later in a partially deadenylated form.

Cytoplasmic localization of transcripts of a complex G+C-rich crab satellite DNA

The primary sequence and higher order structures of a G+C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis have been described previously. The repeat unit of the satellite is approximately 2.1 kb. In exploring a possible function for this satellite, we asked whether it is transcribed. As a probe for transcripts, we used a segment of DNA amplified from a 368 bp EcoRI fragment from the very highly conserved 3' end of the satellite DNA. During polymerase chain reaction (PCR) amplification, the probe was simultaneously either radiolabeled or biotinylated. Tissue- and stage-specific transcripts were observed when blots of poly(A)+ mRNAs recovered from polysomes isolated from crab tissues [including midgut gland (hepatopancreas), limb bud, and claw muscle] were probed with the satellite DNA fragment. The presence of satellite transcripts in polysomal mRNAs is strong evidence that the transcripts had reached the cytoplasm. To corroborate the presence of transcripts in the cytoplasm, we investigated in situ hybridization of satellite probes with RNAs in tissue sections. Biotinylated satellite DNA probes were applied to sections of midgut gland, limb bud papilla, ovary, or testis of anecdysial crabs. Retention of RNAs in tissue sections was improved by UV-irradiation prior to hybridization. Transcripts were abundant in the cytoplasm of all tissues except testis. Sections of crab midgut gland treated with RNase A prior to hybridization and sections of mouse pancreatic tumor served as controls; neither showed any signals with the probe.

Soleus muscle nascent polypeptide chain elongation slows protein synthesis rate during non-weight-bearing activity

Protein synthesis rate of the soleus muscle decreases rapidly during non-weight-bearing activity. We isolated polysomes from 18-h non-weight-bearing soleus muscle to investigate the mechanism of this phenomenon. The distribution of polysomal alpha-actin mRNA and 18S rRNA on sucrose density gradients shows that polysomes shift to larger sizes (more ribosomes per mRNA) during non-weight-bearing activity. Furthermore, RNA is mobilized into the polysome pool of the non-weight-bearing soleus muscle; these data indicate that initiation of protein synthesis is not rate limiting. We explain these results as the slowing of nascent polypeptide chain elongation, such that there is a "traffic jam" of ribosomes on the mRNAs, increasing the number of ribosomes per mRNA while, at the same time, decreasing protein synthesis rate. In support of this hypothesis, myoblasts treated with a low dose of cycloheximide (a specific elongation inhibitor) show a similar shift in polysome size. A numerical model of protein synthesis further shows that elongation is more effective than initiation and termination in affecting protein synthesis and polysome size. We conclude that the non-weight-bearing-induced decrease in postural muscle protein synthesis rate is initially caused by slowing of nascent polypeptide chain elongation.

Vir-115 gene product is required to stabilize D1 translation intermediates in chloroplasts

The nuclear gene mutant of barley, vir-115, shows a developmentally induced loss of D1 synthesis that results in inactivation of Photosystem II. Translation in plastids isolated from 1 h illuminated vir-115 seedlings is similar to wild type. In wild-type barley, illumination of plants for 16 to 72 h results in increased radiolabel incorporation into the D1 translation intermediates of 15-24 kDa. In contrast, these D1 translation intermediates were not observed in vir-115 plastids isolated from plants illuminated for 16-72 h. In addition, after 72 h of illumination, radiolabel incorporation into D1 was undetectable in vir-115 plastids. The level and distribution of psbA mRNA in membrane-associated polysomes was similar in wild-type and vir-115 mutant plastids isolated from plants illuminated for 16-72 h. Toeprint analysis showed similar levels of translation initiation complexes on psbA mRNA in vir-115 and wild-type plastids. These results indicate that translation initiation and elongation of D1 is not significantly altered in the mutant plastids. Ribosome pausing on psbA mRNA was observed in wild-type and vir-115 mutant plastids. Therefore, the absence of D1 translation intermediates in mutant plastids is not due to a lack of ribosome pausing on psbA mRNA. Based on these results, it is proposed that vir-115 lacks or contains a modified nuclear-encoded gene product which normally stabilizes the D1 translation intermediates. In wild-type plastids, ribosome pausing and stabilization of D1 translation intermediates is proposed to facilitate assembly of cofactors such as chlorophyll with D1 allowing continued D1 synthesis and accumulation in mature chloroplasts.

Temporal Expression Patterns of Alpha-amylase Isozymal Genes in Polysomal and Total RNAs of Germinating Barleys

Analyses of alpha-amylase gene expression have focused mainly upon the gibberellin-treated isolated aleurone system. In order to determine how the alpha -amylase isozymes are synthesized in the intact system, high and low pI alpha-amylase transcripts were analyzed in total RNA and polysomal (polyribosomal) messenger RNA from seedlings of the Morex and Himalaya cultivars. Two growing conditions, malting and germination on paper, were compared. Unlike isolated aleurone systems, where high and low pI mRNA levels are roughly equal, the intact seedlings had five- to 10-fold more high pI mRNA than low pI mRNA. This corresponded to the differences in high and low pI enzyme activities observed on isoelectric focusing gels. Both the high and low pI and mRNAs had similar temporal accumulation patterns when analyzed in the total RNA population, but distinctly different patterns were observed in polysomal mRNA. Hihg pI mRNA increased rapidly, reached a peak in 4 to 5 days after the initiation of imbibition, and then declined. Low pI mRNA increased slowly but progressively for at least 6 days. Surprisingly, when gene expression in total RNA reached a peak for either isozymal RNA, only 2·1 to 3·9% of the RNA was bound to polysomes. Gene expression reflected in total RNA from paper-germinated seeds was similar to that in malted seeds, except that peaks in expression occurred 1 day earlier, and the high pI/low pI RNA ratio was lower.

The 5'-end structure of ovalbumin mRNA in isolated nuclei and polysomes.

We used the chemical reagents dimethylsulfate and 4'-aminomethyl-4,5',8-trimethylpsoralen and the enzyme T1 ribonuclease to compare the 5'-end structure of ovalbumin mRNA in situ in purified hen oviduct nuclei and polysomes with that of the isolated mRNA. The qualitative pattern of structure-dependent base modifications and T1 ribonuclease cleavage sites in intranuclear and polysomal ovalbumin mRNAs was found to be nearly identical to those in isolated ovalbumin mRNA. These structural data are consistent with the presence of a trigonal stem-loop structure at the 5'-end of ovalbumin mRNA (hairpin-1) in nuclei and polysomes. Similar results were obtained for a coding region structure (hairpin-3) in intranuclear ovalbumin mRNA. We have recently shown that hairpin-1 positively affects the rate of ovalbumin mRNA translation in vitro and is part of a high affinity binding site for eucaryotic initiation factor-2 (eIF-2). The presence of hairpin-1 in ovalbumin mRNA in both a pretranslation state (nuclei) and active translation state (polysomes) is consistent with its hypothesized biological function as an intracellular initiation signal that facilitates the translation of this mRNA.

Heterogeneity in the 5' untranslated region of mouse cytochrome cT mRNAs leads to altered translational status of the mRNAs.

Previous studies have shown that the differential regulation of mouse somatic cytochrome c (cyt cS) and testicular cytochrome c (cyt cT) during spermatogenesis is accompanied by changes in mRNA length [Hake et al. (1990) Development, 110, 249-257]. When analyzed by polysomal gradient sedimentation, cytochrome cT sediments in two broad size classes: non-polysomal mRNAs are about 0.6 to 0.75 kb and polysomal mRNAs range from 0.7 to 0.9 kb. Both classes of mRNAs shorten to about 0.5 kb following deadenylation. Oligonucleotide-directed cleavage of the cytochrome cT RNAs by RNase H reveals that the size heterogeneity of cytochrome cT mRNAs resides in the 5' untranslated regions (UTRs). Ribonuclease protection assays reveal that multiple cytochrome cT mRNAs are transcribed from six different transcriptional start sites spanning a region of 59 nucleotides in the 5'UTR from +1 to +59. Transcripts derived from the first and second transcriptional initiation sites are not loaded onto polysomes as efficiently as those transcripts initiated from the other start sites. Each of the longer mRNAs has an upstream open reading frame, which starts at +8 and ends at +136 in the 5'UTR of the cytochrome cT transcript. Computer analysis suggests that the lengthened 5'UTR sequences allow additional hairpin structures to be formed. Since the upstream open reading frame and the additional stem loop structure are absent in the 5' UTRs of the cytochrome cT mRNAs initiated from the four downstream start sites, we suggest that these sequences in the two longest cytochrome cT transcripts hinder their loading onto polysomes.

In vivo regulation of low-density lipoprotein receptors by estrogen differs at the post-transcriptional level in rat and mouse.

Rats and mice are frequently used in studies of the regulation of lipoprotein metabolism. Although the species are closely related, they differ dramatically in the responses of their lipoproteins to estrogen administration. In rats, estrogens produce profound decreases in the levels of all plasma lipoproteins and this is attributed largely to estrogen-induced increases of hepatic low-density lipoprotein receptor (LDL-receptor) activity. Estrogens affect mouse plasma lipoproteins to a much lesser extent. Therefore, one of our aims was to compare the regulation of LDL-receptor gene expression in rats and mice at several potential loci of regulation. To assess the specificity of the estrogen effect, we also compared the responses of apolipoprotein AI (apoAI), apolipoprotein B (apoB), and beta-actin to the response of the LDL-receptor. In male Sprague Dawley rats given 17 beta-estradiol or 17 alpha-ethinyl estradiol at supraphysiological doses of 5 micrograms/g body mass/day, plasma total cholesterol and triacylglycerols fell to approximately 5% and approximately 50%, and, plasma apoAI and apoB fell to approximately 12% and approximately 16% of controls, respectively. By contrast, in male C3H/HeJ mice the above parameters dropped only to approximately 65% of controls and apoB concentrations rose to approximately 200% of controls. In rats, relative rates of LDL-receptor mRNA transcription (nuclear 'run-off' assay) and total hepatic, nuclear and polysomal LDL-receptor mRNA levels (RNase protection assay) increased by 1.5-2-fold, while synthesis of LDL-receptor protein on hepatic polysomes (in a wheat-germ translation system) increased 8-fold and LDL-receptor protein mass in hepatic plasma membranes increased 10-fold (by immunoblotting). In mouse liver, too, LDL-receptor mRNA levels increased 1.5-fold and the LDL-receptor mRNA transcription start sites in rat and mouse were found to be the same, but mouse LDL-receptor protein mass did not change, i.e. LDL-receptors of mice were similar to rat with respect to transcriptional regulation, but differed in their post-transcriptional control mechanisms. In rats, estrogen administration increased apoAI mRNA transcription rates 1.6-fold and also apoAI mRNA levels in total liver homogenates, nuclei and polysomes, (2-fold for each) consistent with transcriptional regulation. However, apoAI synthesis on total RNA increased less than apoAI mRNA, indicating that apoAI translational control mechanisms, at least in part, also regulate hepatic rates of apoAI production. ApoB mRNA transcription rates and levels showed small increases following estrogen administration. Hepatic beta-actin mRNA transcription and levels did not change.(ABSTRACT TRUNCATED AT 400 WORDS)

A complex pattern of changes in polysomal mRNA populations is evident in the leaves of Arabidopsis thaliana (L.) Heynh. during photoperiodic induction of flowering

Previous studies have indicated that the photoperiodic induction of flowering results in qualitative changes in gene expression in the leaves of the induced plant. However, we obtained different results when leaf tissue was examined at different sampling times after flower induction, thus pointing to the possibility that alterations in gene expression during photoperiodic induction are only transient. We analyzed the composition of mRNA populations in the leaves of the long-day plant Arabidopsis thaliana (L.) Heynh. at various times during photoperiodic induction. Polysomal mRNA populations were assayed during illumination programs consisting of different photoperiodic cycles. Quantitative changes of distinct leaf mRNAs were observed during the time course. The expression levels of most of these mRNAs oscillated in a diurnal or circadian rhythm with respect to the photoperiod. However, no novel mRNA appeared or decreased to undetectable levels during the changes in photoperiod. After the onset of inducing photoperiods, alterations in the phase and amplitude of these oscillations were seen. Various types of oscillation pattern were observed. These changes occurred either very rapidly, within hours after the additional light period began or appeared after the first altered photoperiod was complete. The majority of the changes seen at the beginning of inductive photoperiods were transient and the mRNA levels reverted to the original pattern when the plant was returned to non-inductive photoperiods. In addition, some of these alterations were found to be transient, irrespective of the length of subsequent light periods.

Molecular basis of the thyroglobulin synthesis defect in Dutch goats

A thyroglobulin (Tg) synthesis defect in Dutch goats causes congenital goiter and hypothyroidism. The disease is inherited in an autosomal recessive way and is linked to restriction fragment length polymorphisms (RFLPs) in the Tg gene. Previous studies showed that Tg mRNA isolated from the goiters was of normal size (8.4 kilobases). Translation of high mol wt polysomal Tg mRNA isolated from goiter in a cell-free rabbit reticulocyte lysate resulted in a single 35,000 mol wt Tg polypeptide. Tg antigens analyzed in T4-arrested goiters were glycosylated and had mol wt of 40,000 and 32,000. The aim of this study was to identify the molecular lesion responsible for this disease. Polysomal Tg mRNA, therefore, was isolated, and cDNA was made using oligonucleotides as primers. This cDNA was multiplied by the polymerase chain reaction and cloned. In comparing the normal and abnormal sequences, we found a C—>G point mutation in exon 8 causing a change from TAC (Tyr)—>TAG (termination signal) at amino acid position 296. This mutation resulted in the appearance of a KpnI restriction site in the goiter DNA. The sequence of Tg mRNA preceding the stop codon was equal for normal and goitrous goats, except for one C—>T mutation in exon 5 which gave a Ser—>Leu transition. The KpnI site introduced by the C—>G point mutation was present in chromosomal DNA of the goitrous goats, making it possible to distinguish goats heterozygous for the defect from normal and goitrous animals. We calculated that the stop codon in exon 8 would result in a Tg polypeptide chain with a mol wt of 39,000, in good agreement with the mol wt of the in vitro and in vivo translation products. In conclusion, the C—>G mutation causing a stop codon in exon 8 is responsible for the Tg synthesis defect in Dutch goats.

Molecular basis of the thyroglobulin synthesis defect in Dutch goats.

A thyroglobulin (Tg) synthesis defect in Dutch goats causes congenital goiter and hypothyroidism. The disease is inherited in an autosomal recessive way and is linked to restriction fragment length polymorphisms (RFLPs) in the Tg gene. Previous studies showed that Tg mRNA isolated from the goiters was of normal size (8.4 kilobases). Translation of high mol wt polysomal Tg mRNA isolated from goiter in a cell-free rabbit reticulocyte lysate resulted in a single 35,000 mol wt Tg polypeptide. Tg antigens analyzed in T4-arrested goiters were glycosylated and had mol wt of 40,000 and 32,000. The aim of this study was to identify the molecular lesion responsible for this disease. Polysomal Tg mRNA, therefore, was isolated, and cDNA was made using oligonucleotides as primers. This cDNA was multiplied by the polymerase chain reaction and cloned. In comparing the normal and abnormal sequences, we found a C-->G point mutation in exon 8 causing a change from TAC (Tyr)-->TAG (termination signal) at amino acid position 296. This mutation resulted in the appearance of a KpnI restriction site in the goiter DNA. The sequence of Tg mRNA preceding the stop codon was equal for normal and goitrous goats, except for one C-->T mutation in exon 5 which gave a Ser-->Leu transition. The KpnI site introduced by the C-->G point mutation was present in chromosomal DNA of the goitrous goats, making it possible to distinguish goats heterozygous for the defect from normal and goitrous animals. We calculated that the stop codon in exon 8 would result in a Tg polypeptide chain with a mol wt of 39,000, in good agreement with the mol wt of the in vitro and in vivo translation products. In conclusion, the C-->G mutation causing a stop codon in exon 8 is responsible for the Tg synthesis defect in Dutch goats.

Localization of small heat shock proteins to the higher plant endomembrane system.

Three related gene families of low-molecular-weight (LMW) heat shock proteins (HSPs) have been characterized in plants. We describe a fourth LMW HSP family, represented by PsHSP22.7 from Pisum sativum and GmHSP22.0 from Glycine max, and demonstrate that this family of proteins is endomembrane localized. PsHSP22.7 and GmHSP22.0 are 76.7% identical at the amino acid level. Both proteins have amino-terminal signal peptides and carboxyl-terminal sequences characteristic of endoplasmic reticulum (ER) retention signals. The two proteins closely resemble class I cytoplasmic LMW HSPs, suggesting that they evolved from the cytoplasmic proteins through the addition of the signal peptide and ER retention motif. The endomembrane localization of these proteins was confirmed by cell fractionation. The polypeptide product of PsHSP22.7 mRNA was processed to a smaller-M(r) form by canine pancreatic microsomes; in vivo, GmHSP22.0 polysomal mRNA was found to be predominantly membrane bound. In vitro-processed PsHSP22.7 corresponded in mass and pI to one of two proteins detected in ER fractions from heat-stressed plants by using anti-PsHSP22.7 antibodies. Like other LMW HSPs, PsHSP22.7 was observed in higher-molecular-weight structures with apparent masses of between 80 and 240 kDa. The results reported here indicate that members of this new class of LMW HSPs are most likely resident ER proteins and may be similar in function to related LMW HSPs in the cytoplasm. Along with the HSP90 and HSP70 classes of HSPs, this is the third category of HSPs localized to the ER.

Some effects of aluminium on rat brain protein synthesis

1. Infant rats and rabbits received intraperitonal aluminium (Al) chloride (5, 10 or 20 mg Al/kg body weight) every third day from one to four weeks of age. 2. When the polysomal fraction was tested in a protein synthesizing system, a significant increase in the incorporation of [ 14C] leucine, [ 14C] phenylalanine, or [ 35S] methionine into proteins in vitro was observed at the higher doses in rats but not rabbits. 3. The incorporation of [ 35S]methionine into brain ferritin was measured using polysomal mRNA or mRNA “stored” in the ribonucleoprotein (RNP) particle fraction. 4. The results suggest that Al exposure causes the mobilization of ferritin mRNA from the latter fraction to the polysomal fraction for increased ferritin synthesis.

Effect of growth hormone on levels of differentially processed insulin- like growth factor I mRNAs in total and polysomal mRNA populations

Effect of growth hormone on levels of differentially processed insulin- like growth factor I mRNAs in total and polysomal mRNA populations

Gene expression of calpains and their specific endogenous inhibitor, calpastatin, in skeletal muscle of fed and fasted rabbits.

To investigate the role of calpains in myofibrillar protein degradation in skeletal muscle and the regulation of their activity in vivo, we studied the effects of fasting on gene expression of calpains and calpastatin in the skeletal muscle of rabbits. In response to fasting, myofibrillar protein degradation increased 2-fold and mRNA levels of calpain I, calpain II and calpastatin were also increased. However, calpain and calpastatin activities remained unchanged. To investigate this discrepancy, we analysed polysomal calpain mRNA. Results indicated that fasting caused a 2-fold increase in the loading of calpain I and II mRNAs on ribosomes. Thus transcription of genes encoding calpain may be increased during fasting to ensure adequate synthesis of the proteinases needed to mobilize muscle protein reserves. The effect of fasting on calpain and calpastatin mRNA expression is shared by cathepsin D and proteasome C2 but not by beta-actin, implying that fasting invokes control of several proteolytic systems in skeletal muscle and underscores the possibility that each proteolytic system plays a role in the adaptation of skeletal muscle to the fasted state.

The mechanism of the antifertility effects of the luteinizing hormone-releasing hormone (LH-RH) and its agonist

Research results demonstrated that repeated dosages of luteinizing hormone-releasing hormone (LH-RH) or its agonists (LH-RH A) terminate pregnancy when given at the periimplantation stage in female rats. Several scientists proposed that the mechanism for this action directly inhibits metabolism and development of the uterus. An endocrinologist at the Institute of Zoology of Academia Sinica in Beijing China experimented with female rats to identify LH-RH receptors in endometrial tissue. Very specific LH-RH binding occurred and binding was especially great on day 3 of pregnancy. Other research revealed that 250 mcg/day of LH-RH A administered to rats and 300 mcg/day of LH-RH A administered to rabbits delayed implantation for 2 days. LH-RH A did not affect blastocyst development. It did inhibit protein synthesis of the rabbit uterus particularly uteroglobin however. 50 mcg/2ml culture medium of LH-RH inhibited secretory protein (50000-60000 MW) synthesis in human trimester trophoblast. 300 mg/kg/day of LH-RH A was administered for 4 8 and 11 days to rabbits to examine the effect LH-RH A has on endometrial polysomal mRNA. The amount of this mRNA in pregnant rabbits was consistently lower than that in the nonpregnant rabbits. Administration of LH-RH A also reduced the translation activity of endometrial polysomal mRNA in the reticulolysate system. The endometrial polysomal mRNA in pregnant rabbits caused 2 important uterine proteins with MWs varying from 22000 to 69000 to almost disappear. These results suggested that LH-RH A inhibits polysomal mRNA and thus plays a role in the regulation of uterine protein synthesis.

Alterations in brain polyribosomal RNA translation and lymphocyte proliferation in prenatal ethanol-exposed rats.

The long-term effects of prenatal ethanol exposure on the properties of brain polysomes and the proliferative responses of lymphocytes to mitogenic stimulation in adult offspring were assessed. Female Sprague-Dawley rats either ingested the control or 6.6% ethanol-containing Lieber-DeCarli liquid diet during the 3rd trimester of pregnancy. Controls were age-matched and pair-fed. At 42 to 72 days of age, ethanol effects were evaluated on the (1) polysomal properties in the cerebral hemispheres, cerebellum, and hippocampal regions of the brain after translation in a messenger RNA (mRNA)-dependent rabbit reticulocyte lysate system and (2) immunologic functions of lymphocytes cultured from spleen cells by measuring their responses to mitogenic stimulation. Results showed long-term adverse effects of in utero ethanol exposure on the polysomal RNA translation in each of the three brain regions tested with free polysomal mRNAs affected more than the bound polysomal mRNAs. Of these, the hippocampal region appeared to sustain the most injurious effects. In addition, a suppression of the mitogen-induced lymphocyte proliferative responses were present under these conditions. The degree of suppression varied with the specific mitogen used. Data suggest that the ethanol effects on the CNS and lymphocyte proliferation are most possibly irreversible, and in the case of the CNS, a post-translational modification by ethanol is indicated. The reduced lymphocyte responses are suggestive of a possible interference by ethanol of the synthesis of interleukin-2 (IL-2) and/or a reduced binding of IL-2 with its receptor (IL-2 receptors).