Polyribosomal RNA Research Articles

Deep brain stimulation of the subthalamic nucleus preferentially alters the translational profile of striatopallidal neurons in an animal model of Parkinson's disease.

Deep brain stimulation targeting the subthalamic nucleus (STN-DBS) is an effective surgical treatment for the motor symptoms of Parkinson's disease (PD), the precise neuronal mechanisms of which both at molecular and network levels remain a topic of debate. Here we employ two transgenic mouse lines, combining translating ribosomal affinity purification (TRAP) with bacterial artificial chromosome expression (Bac), to selectively identify changes in translational gene expression in either Drd1a-expressing striatonigral or Drd2-expressing striatopallidal medium spiny neurons (MSNs) of the striatum following STN-DBS. 6-hydroxydopamine lesioned mice received either 5 days stimulation via a DBS electrode implanted in the ipsilateral STN or 5 days sham treatment (no stimulation). Striatal polyribosomal RNA was selectively purified from either Drd2 or Drd1a MSNs using the TRAP method and gene expression profiling performed. We identified eight significantly altered genes in Drd2 MSNs (Vps33b, Ppp1r3c, Mapk4, Sorcs2, Neto1, Abca1, Penk1, and Gapdh) and two overlapping genes in Drd1a MSNs (Penk1 and Ppp1r3c) implicated in the molecular mechanisms of STN-DBS. A detailed functional analysis, using a further 728 probes implicated in STN-DBS, suggested an increased ability to receive excitation (mediated by increased dendritic spines, increased calcium influx and enhanced excitatory post synaptic potentials) accompanied by processes that would hamper the initiation of action potentials, transport of neurotransmitters from soma to axon terminals and vesicular release in Drd2-expressing MSNs. Finally, changes in expression of several genes involved in apoptosis as well as cholesterol and fatty acid metabolism were also identified. This increased understanding of the molecular mechanisms induced by STN-DBS may reveal novel targets for future non-surgical therapies for PD.

Precocious germination and its regulation in embryos of triticale caryopses

Triticale var. Lasko embryos, isolated from grain gathered at milk ripeness, the beginning of wax ripeness and at full ripeness, were allowed to germinate for 48 h on agar with glucose. The highest incorporation of tritiated adenosine into polyribosomal RNA during germination was found in the ribosome fractions from embryos of grain gathered at full ripeness, lower incorporation was in preparations from embryos of milk ripe grain and the lowest in preparations from embryos of wax ripe grain. Different tendencies were observed in respect to the synthesis of ribosomal proteins. The highest incorporation of <sup>14</sup>C-amino acids into ribosomal proteins was found in preparations of ribosome fractions from embryos of milk ripe grain, lower in preparations of embryos from fully ripe grain, the lowest in preparations of embryos from wax ripe grain. ABA (10<sup>-4</sup> M) completely inhibited the external symptoms of germination of immature embryos, while its inhibition of the synthesis of polyribosomal RNA and ribosomal proteins was greater the more mature the embryos that were germinated. The greatest stimulation of precocious germination by exogenous BA and GA<sub>3</sub> was demonstrated in the least mature embryos isolated from milk ripe grain. Under the influence of both stimulators, an increase of the proportion of polyribosomes in the total ribosome fraction occurred in this sample, as did a rise in the intensity of ribosomal protein synthesis. The incorporation of <sup>3</sup>H-adenosine into polyribosomal RNA, however, was lower than in the control sample. The results obtained suggest that the regulation of precocious germination of triticale embryos by phyto-hormones is not directly related to transcription.

The use of transcription inhibitors in the study of the mechanism of abscisic acid action in germinating triticale caryopses

The study was conducted on germinating triticale (var. Grado) caryopses. The purpose of the experiments was to compare the effect of selected inhibitors of transcription with the action of abscisic acid during germination of caryopses. The following inhibitors were used: α-amanitin, cordycepin, cycloheximide and 5-fluorouracil. Studied were the synthesis of total and polyribosomal RNA, the process of polyribosome formation and the synthesis of ribosomal proteins. The effect of exogenous ABA, especially in the early stages of germination, was not similar to any of the four above inhibitors of transcription. After 12 h of imbibition at a lowered temperature and 3 h of germination, ABA caused a relatively low level of inhibition of RNA synthesis, whereas all of the inhibitors used halted RNA synthesis in embryos by about 50-60%. After 6 h of germination, the same proportion of polyribosomes in the total ribosome fraction (46%) was found in both the embryos from the control sample and treated with ABA. The use of inhibitors brought this figure down to below 40%. The conclusion is drawn that in the early stages of germination, regulation of protein synthesis by ABA in triticale caryopses must occur on a level other than transcription.

The role of the embryoless parts of triticale caryopses in inhibiting precocious germination and transcription in the embryo during development and maturation of caryopses

The experiments were conducted on developing and ripening triticale cv. Dagro caryopses. Increasing capability for precocious germination of the caryopses was seen as development and maturation progressed. A significant role of the embryoless parts of the caryopses (testa, pericarp and endosperm) in preventing germination processes was found. Isolated embryos (after 8 days of incubation) germinated by 100% from the 32nd day after flowering, while only 10% of whole caryopses from this sample germinated. Removal of the outer pericarp strongly stimulated germination of unripe caryopses. However, incising the caryopses near the embryo only slightly stimulated this process, which indicates that hypoxia of the embryo is not the cause of triticale embryo dormancy. Another very sensitive indicator of release of dormancy in the caryopses was the increased synthesis of embryo polyribosomal RNA induced by germination. The results of investigations on RNA synthesis in embryos - which undergoes extreme intensification when germination processes are initiated in the caryopses - were in agreement with those of biological studies based on observation of the elongation of the radicle. The lowest inhibition of transcription in the embryo was found when it was completely separated from the testa, pericarp and endosperm. A smaller effect was seen upon removal of the outer pericarp from developing and ripening caryopses, and decidedly the smallest effect still of incising the caryopses near the embryo.

The role of zeatin and gibberellic acid in breaking of the abscisic acid-induced dormancy in Triticale caryopses

The investigations were conducted on the germinating embryos and the whole caryopses of <em>Triticale</em>. During preimbibition and 24 hours germination caryopses were treated with abscisic acid (ABA), which produced 63% inhibition of embryo growth. Gibberellin-A<sub>3</sub> (GA<sub>3</sub>) reversed the ABA effect in 18%, while zeatin in 22%. The clear synergic reaction was observed (36%) when both stimulators acted together. There was no significant effect of ABA, ABA and GA<sub>3</sub>, as well as ABA and zeatin on the synthesis of polyribosomal RNA in the initial period of germination of excised embryos. However, during 24 hours germination of whole caryopses ABA caused a twofold decrease in <sup>3</sup>H-uridine incorporation into the total fraction of embryonic ribosomes. While the incorporation of <sup>14</sup>C-aminoacid mixture into ribosomal proteins was even three-fold lower. Effect of GA<sub>3</sub> and zeatin on breaking of the ABA-induced "dormancy" was studied. It was confirmed that the higher polyribosome contribution to the sum total of ribosomes the more intensive synthesis of ribosomal proteins. No higher <sup>3</sup>H-uridine incorporation into polyribosomal fraction was observed. From the results it may be inferred that in the initial period of germination of <em>Triticale</em> caryopses regulation of protein biosynthesis occurs rather at the translation than transcription level.

Discovery of the breast cancer gene BASE using a molecular approach to enrich for genes encoding membrane and secreted proteins.

To identify unknown membrane proteins that could be used as targets for breast and prostate cancer immunotherapies and secreted proteins to be used as diagnostic markers, a cDNA library was generated from membrane-associated polyribosomal RNA derived from four breast cancer cell lines, one normal breast cell line, and a prostate cancer cell line. The membrane-associated polyribosomal cDNA library was subtracted with RNA from normal brain, liver, lung, kidney, and muscle. Of the 15,581 clones sequenced from the subtracted cDNA library, sequences from 10,506 clones map to known genes, but 5,075 sequences, representing 3,181 unique transcripts, are not associated with known genes. As one example, we experimentally investigated expression of a previously uncharacterized breast cancer gene that encodes a secreted protein designated BASE (breast cancer and salivary gland expression). BASE is expressed in many breast cancers but not in essential normal tissues including the five organs used for subtraction. Further analysis of this library should yield additional gene products of use in the diagnosis or treatment of breast or prostate cancer.

Identification of transcripts translated on free or membrane-bound polyribosomes by differential display

Differential display has been widely and successfully used to identify differentially expressed genes based on physiological treatments or genetic variation. We used differential display to identify genes based on their site of translation. Identification was made based on the fractionation of RNA from soybean leaves into total RNA, free polyribosomal RNA, and membrane-bound (MB) polyribosomal RNA. Sequences were identified representing RNAs uniquely translated on free or MB polyribosomes. The compartmentalization was confirmed on RNA blots. Differential display of free and MB polyribosomal RNA from genetic mutants or physiological studies has 2 potential advantages. First, the sensitivity of the method is increased. Second, localization of mRNAs to the free or MB compartments may identify genes that are controlled at the level of translation or that switch compartments in response to a treatment.

The abundant latency-associated transcripts of herpes simplex virus type 1 are bound to polyribosomes in cultured neuronal cells and during latent infection in mouse trigeminal ganglia.

During herpes simplex virus type 1 (HSV-1) latency, limited viral transcription takes place. This transcription has been linked to the ability of the HSV-1 genome to reactivate and consists of abundant 2.0- and 1.5-kb collinear latency-associated transcripts (LATs), spanned by minor hybridizing RNA (mLAT). The 1.5-kb LAT is derived from the 2.0-kb LAT by splicing, and both transcripts contain two large overlapping open reading frames. The molecular action mechanisms of the latency-associated gene expression are unknown, and no HSV-1 latency-encoded proteins have been convincingly demonstrated. We have cloned the entire latency-associated transcriptionally active HSV-1 DNA fragment (10.4 kb) under control of a constitutive promoter and generated a neuronal cell line (NA4) stably transfected with the viral LAT's region. NA4 cells produced the 2.0- and the 1.5-kb LATs. Northern blotting and reverse transcription-PCR analysis of RNA from NA4 cells and from trigeminal ganglia of mice latently infected with HSV-1 revealed that the two abundant LAT species were present in the polyribosomal RNA fractions. After addition of EDTA, which causes dissociation of mRNA-ribosome complexes, both LATs could be detected only in subpolyribosomal, but not in polyribosomal fractions. These results show that (i) HSV-1 LATs are bound to polyribosomes during latency in vivo, as well as in neuronal cells in vitro, and therefore might be translated, and that (ii) the NA4 cell line is a suitable tool with which to look for HSV-1 latency-encoded gene products.

Evidence for differences in the post-transcriptional regulation of rat metallothionein isoforms.

The expression of metallothionein (MT)-1 and -2 mRNAs in rat liver following administration of Cd or Cu was investigated using specific oligonucleotides. The specificity was confirmed using a competitive prehybridization assay. Cd injection caused a biphasic induction of both isoform mRNAs, whereas Cu induced a sustained, monophasic response. Analysis of polyribosomal RNA showed that, after both Cd and Cu treatments, the recruitment of MT-1 mRNA into polyribosomes paralleled the increase in transcription, but the increase of polyribosomal MT-2 mRNA was less than that of total MT-2 mRNA. This indicates that not all the MT-2 mRNA induced was translated, suggesting that there is translational control of MT-2 mRNA expression, but not of MT-1 mRNA. This hypothesis was supported by the observation that, after Cu treatment, the induction of MT-1 protein was induced to the same extent as MT-1 mRNA, whereas the total MT protein (MT-1 + MT-2) was increased far less (7-fold) than MT-2 mRNA (30-fold).

Comparative studies on tryptophan binding to hepatic nuclear envelopes in Sprague-Dawley and Lewis rats

Since Lewis rats are susceptible to many inflammatory diseases and have been used in an experimental model of the eosinophilia-myalgia syndrome, we investigated whether Lewis rats would respond to L-tryptophan as have Sprague-Dawley rats reported earlier. In this comparative study using females of both strains, we observed a decrease in the affinity of in vitro L-tryptophan binding to hepatic nuclei and nuclear envelopes of Lewis rats compared with Sprague-Dawley rats. However, in vivo stimulatory effects of administering L-tryptophan on hepatic polyribosomal aggregation, protein synthesis, and nuclear RNA release were similar in both strains. In vitro [3H]tryptophan binding to hepatic nuclear envelopes, using L-tryptophan implicated in cases of the eosinophilia-myalgia syndrome, revealed less specific binding than when using nonimplicated L-tryptophan in both strains. The possible significance of the quantitative difference in the binding affinity of L-tryptophan to hepatic nuclei of Lewis rats compared with those of Sprague-Dawley rats is as yet undetermined.

Expression of Mitochondrial Genes in the Germinating Conidia of Neurospora crassa

The germinating asexual spores (conidia) of Neurospora crassa were employed to study steps in the accumulation of transcripts of groups of mitochondrial genes, including those for peptide subunits of cytochrome c oxidase (CO), ATPase (ATP), and apocytochrome b (COB). Physically clustered groups of genes were expressed as cohorts: transcripts of the ATP8-ATP6-mtATP9-CO2 genes were almost undetectable in the dormant spores, and they accumulated rapidly as a group immediately after spore activation. Transcripts of COB and the adjacent CO1 were abundant in the dormant spores, and the dormant and germinating spores contained size forms of the COB transcripts that were not evident in vegetative cells. Polyribosomes were prepared from mitochondrial lysates, and the polyribosomal RNA was probed to identify the mRNAs of specific genes; in several instances polycistronic mRNAs were present in the polyribosomes as were the smaller end products of the inferred transcript processing pathways. The expression of the physically dispersed genes for subunit peptides of cytochrome c oxidase appears to be regulated to the level of translation; these transcripts are accumulated in the total mitochondrial RNA with sharply different kinetics, but they appeared in the polyribosomes uniformly, their appearance correlating with the uniform synthesis of the subunit peptides. Transcripts for a previously reported non-functional mitochondrial gene, homologous to the functional nuclear gene for ATPase subunit 9, were found in the germinating spores, but were not detected in vegetative cells. These mtATP9 transcripts were also present in the polyribosomes and were apparently translated into a protein in vivo whose synthesis was insensitive to cycloheximide and detectable with an anti-ATP9 subunit antibody. Transcripts for two nuclear genes for mitochondrially localized proteins, ATP9 and CO5, were accumulated in unison and especially rapidly during spore germination

Inhibition of protein synthesis by an efficiently expressed mutation in the yeast 5.8S ribosomal RNA.

Recent studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides strongly suggested that this RNA plays an important role in eukaryotic ribosome function. To evaluate this possibility further, a ribosomal DNA transcription unit from Schizosaccharomyces pombe was cloned into yeast shuttle vectors with copy numbers ranging from 2 to approximately 90 per cell; to allow direct detection of expressed RNA and to disrupt the function of the 5.8S rRNA molecule, a five base insertion was made in a universally conserved GAAC sequence. The altered mobility of the mutant RNA was readily detected by gel electrophoresis and analyses indicated that mutant RNA transcription reflected the ratio of plasmid to endogenous rDNA. The highest copy number plasmid resulted in about 40-50% mutant RNA. This mutant RNA was readily integrated into the ribosome structure resulting in an in vivo ribosome population which was also about 40-50% mutant; the rates of growth and protein synthesis were equally reduced by approximately 40%. A comparable level of inhibition in protein synthesis was demonstrated in vitro and polyribosomal profiles revealed a consistent increase in size. Subsequent RNA analyses indicated a normal distribution of mutant RNA in both monoribosomes and polyribosomes, but elevated tRNA levels in mutant polyribosomes. Additional mutations in alternate GAAC sequences revealed similar but cumulative effects on both protein synthesis and polyribosome profiles. Taken together, these results suggest little or no effect on initiation but provide in vivo evidence of a functional role for the 5.8S rRNA in protein elongation.

Characterization of Defective Interfering RNA Components That Increase Symptom Severity of Broad Bean Mottle Virus Infections

Several strains of the broad bean mottle virus (BBMV), an icosahedral tripartite plant RNA virus, which show distinct reactions on certain plant hosts have been described (K. M. Makkouk et el., Neth. J. Plant Pathol. 94, 195-212, 1988). Here we report defective interfering (DI) RNAs encapsidated in two BBMV strains from Morocco and Tunisia. While not effective in some plants, these DI RNAs exacerbated the severity of symptoms in others. The most dramatic, lethal effect of DI RNAs has been found on pea ( Pisum sativum, cv. Rondo) seedlings. Sequence analysis has revealed that the DI RNAs were derived by single in-frame central deletions of 448 to 537 nt in the corresponding genomic RNA2 components. A comparison of the intensities of full-length RNA2 bands from DI molecule-containing and DI molecule deficient virion RNA preparations revealed that the DI RNAs decreased the level of RNA2 components in total RNA preparations. The differences between corresponding virion RNAs were much smaller. This suggests an interference with RNA replication. In vitro assays and an analysis of the polyribosomal RNA fractions confirmed the translational activity of DI RNAs. This paper reports the first description of natural DI RNAs in tripartite isometric plant RNA viruses.

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).

Polyubiquitin RNA characteristics and conditional induction in sea urchin embryos

A cDNA of the sea urchin Strongylocentrotus purpuratus was identified as encoding polyubiquitin and used to detect a single gene with transcripts containing multiple ubiquitin coding units. Polyubiquitin transcripts exist as a 3.2-kb RNA in polyribosomes and as three higher molecular weight RNAs in purified nuclei. The amount of polyubiquitin RNA is essentially constant at 10 4–10 5 transcripts per embryo during the egg-to-blastula period and then declines during further development. Heat shock elicits a transient increase in the level of polyubiquitin RNA, while Zn(II) ions induce a sustained accumulation, that is influenced by developmental parameters: One round of Zn(II) induction elicits the accumulation of the nuclear 7.6- and 5.6-kb RNAs, as well as the 3.2-kb polysomal RNA; however, a second round of induction yields only the 5.6- and 3.2-kb RNAs, suggestive of a change in pre-mRNA size or processing. Polyubiquitin RNA is expressed equally in ectodermal and mesoendodermal tissues and is induced in both tissue fractions by treatment of pluteus larvae with Zn(II). However, in isolated and cultured tissue fractions, polyubiquitin RNA is not inducible by Zn(II), in contrast to the full inducibility of metallothionein mRNAs. Polyubiquitin RNA induction thus appears to be conditioned by the integrity of the embryo, as well as by previous exposure to inducer.

Post-Transcriptional Control of Sucrose Synthase Expression in Anaerobic Seedlings of Maize

We have examined post-transcriptional control of expression of the anaerobically induced sucrose synthase 1 (SS1) isozyme mRNA encoded by the shrunken (Sh) gene of maize (Zea mays L.). The SS1 transcript level is increased in maize seedling roots during anaerobiosis without a concomitant increase in the SS1 protein level. We show that the anaerobic SS1 RNA was loaded onto polyribosomes and that SS1 proteins produced by in vitro translation of polyribosomal RNA from anaerobic roots and immature kernels were indistinguishable based on abundance and apparent molecular weight. [(35)S]Methionine uptake in control and anaerobically stressed seedling roots indicated a detectable, but only slight, increase in radiolabel in the SS1 polypeptide as compared to the sucrose synthase 2 isozyme, SS2. However, this slight increase in [(35)S]methionine uptake did not contribute to a detectable increase in the steady state level of SS1 protein relative to SS2 protein. Chase experiments with unlabeled methionine indicated that SS1 protein was relatively stable in the anaerobic environment. From these results we conclude that SS1 protein was not rapidly turned over in the anaerobic environment and that expression of anaerobically induced SS1 transcripts was blocked at some step beyond polyribosomal loading.

Translational regulation of ferritin synthesis in rat spleen: Effects of iron and inflammation

Translational control of ferritin synthesis was studied in rat spleen, and compared with that for liver, heart and brain, in response to iron and inflammation. Spleen concentrations of total RNA in the ribonucleoprotein (mRNP) fraction was comparable to that for liver, while polyribosomal RNA was less. Both fractions were ten-fold lower in heart and brain. In untreated animals, the mRNP fraction of all tissues had the largest portion of the ferritin mRNA, as determined by slot blot hybridization with 32P-labeled cDNA for the L subunit. Acute treatment with ferric ammonium citrate shifted the spleen ferritin mRNA to the polyribosome fraction. This was also so in liver but not in the heart and brain which took up much less iron. The findings were confirmed by hybridization studies of mRNPs and polyribosomes separated in sucrose gradients. Turpentine-induced inflammation also caused a shift in ferritin mRNA from the mRNP to the polyribosome fraction of spleen and liver, over 12 h. We conclude that as in liver, spleen ferritin synthesis is under translational control by iron, and that both tissues also respond to inflammation by shifting of ferritin mRNA to the polyribosomes.

Polyribosome fractions and protein synthesis in stem rust infected wheat

The effects of infection by stem-rust fungus on polyribosomal RNA fractions and protein synthesis in vitro and in vivo in near-isogenic resistant (Sr6) and susceptible (sr6) lines of wheat were determined. In infected resistant leaves the proportion of ribosomes present as polyribosomes was greater than that in healthy (uninfected) leaves at 1, 3, and 6 days and that in susceptible leaves at 1 and 3 days after inoculation. In the latter there were large increases in the pelletable RNA content (ribosomes, ribosomal subunits, and polyribosomes) and proportion of ribosomes present as polyribosomes from day 6. In vitro translation failed to detect any marked differences in polyribosomal translation products in resistant and susceptible leaves in response to infection. Labelling of polypeptides in vivo and separation by one- and two-dimensional electrophoresis showed that at 1 day after inoculation, two groups of high molecular mass polypeptides (80–96 and 100–110 kDa) were more heavily labelled and two novel polypeptides were present in resistant and susceptible leaves in response to infection. Synthesis of the high molecular weight and two novel polypeptides was maintained in infected resistant leaves up to 6 days after inoculation. In susceptible leaves the amount of radiolabel incorporated into these polypeptides and several proteins prominently labelled in uninfected controls declined rapidly from 3 days after inoculation.

Synthesis of 3-ketoacyl-CoA thiolase of rat liver peroxisomes on free polyribosomes as a larger precursor. Induction of thiolase mRNA activity by clofibrate.

The site of synthesis and induction by clofibrate of peroxisomal 3-ketoacyl-CoA thiolase (acetyl-CoA acyltransferase; EC 2.3.1.16) was investigated. Free and membrane-bound polyribosomal RNA species from the livers of normal rats and rats treated with clofibrate, a hypolipidaemic drug that causes marked proliferation of peroxisomes, were translated in a nuclease-treated rabbit reticulocyte-lysate cell-free protein-synthesizing system with [35S]methionine as label. The cell-free translation products were immunoprecipitated with monospecific X rabbit anti-thiolase serum and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. Thiolase mRNA was found predominantly in free polyribosomes, in both normal and clofibrate-treated rats. Clofibrate treatment increased mRNA activity for thiolase approx. 20-fold. The translation product of clofibrate-induced thiolase mRNA migrated slightly faster in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than did the translation product of normal thiolase mRNA. Both the normal and the clofibrate-induced translation products were approx. 6000 Da larger than the 41000-Da subunit of the purified enzyme. Immunoblot analysis of liver homogenates, isolated peroxisomes and the purified enzyme indicated that the thiolase subunit was approx. 41000 Da in all samples, ruling out proteolysis during the purification of thiolase. Thiolase biogenesis thus differs from that of rat liver peroxisomal proteins studied previously in that it is synthesized as a larger precursor, implying post-translational import of thiolase into peroxisomes with proteolytic processing. Clofibrate apparently alters the size as well as the amount of the translation product.

Cell-free translation of adenovirus 2 E1a- and E1b-specific mRNAs and evidence that E1a-related polypeptides are produced from E1a-E1b overlapping mRNA.

We have characterized the polypeptides translated in vitro by mRNAs of early region 1 (E1) of human adenovirus (Ad) type 2. Poly (A+) polyribosomal RNA was isolated from early Ad2-infected cells, the viral specific mRNAs were selected by hybridization to Ad2 E1a and E1b DNA, and the mRNAs were translated in vitro using [35S]methionine as a labeled precursor with a rabbit reticulocyte lysate. E1a-selected mRNA was translated to the 45-58-kDa cluster of polypeptides. We show here that E1b-selected mRNA can also be translated to the 45-58-kDa cluster of polypeptides in addition to the major 19-kDa polypeptide. The E1b 58-kDa polypeptide was produced only at a low level unless E1b mRNA is fractionated before translation to enrich for the 58-kDa mRNA. Translation of E1b region-selected mRNAs that have been fractionated by size shows that the 22 S mRNA fraction is translated to at least the 53-58-kDa E1a-related polypeptides as well as to E1b 58- and 19-kDa polypeptides. Our experiments suggest that the 22 S mRNA fraction includes E1a-E1b overlapping mRNA which was translated to E1a-related polypeptides as well as E1b 22 S mRNA. When compared by two-dimensional gel electrophoresis and by tryptic peptide mapping, the cluster of polypeptides translated from E1a-selected mRNA and the cluster translated from E1b-selected mRNA were distinguishable. A possible explanation for this is discussed, based upon splicing sites of the E1a-E1b overlapping mRNA which would result in an amino acid sequence with a COOH-terminal end slightly different from that of E1a polypeptides.