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Decreased mitochondrial RNA levels without accumulation of mitochondrial DNA deletions in aging Drosophila melanogaster.

Declines in electron transport system (ETS) activity have been reported to occur with advancing age in Drosophila melanogaster and many other animals. It has been proposed that these changes are importantly involved in the aging process. ETS decline has been attributed to mitochondrial nucleic acid damage. We analyzed various ages of D. melanogaster (embryos to 60-day-old adults) for the presence of mutated mitochondrial DNA (mtDNA) genomes. Although mtDNA genomes with large DNA deletions (up to 5 kb) were identified, abundance was low and remained constant throughout adult life. Therefore, these mtDNA deletions do not appear to be sufficiently abundant to cause large declines in ETS activity. Next, we analyzed various ages of D. melanogaster for the abundance of four mitochondrial-encoded and two nuclear-encoded ETS transcripts. The abundance of the mitochondrial transcripts declined 5-10-fold, while the nuclear-encoded transcripts declined 2-5-fold with advancing age. Separation of flies on the basis of flight loss was used to distinguish physiologic age from chronological age. Insects capable of flight at 30 days of age were found to have a 4-fold higher abundance of cox I mitochondrial-encoded RNA compared to flightless insects. No difference, however, was apparent in the nuclear-encoded beta-ATPase RNA level, suggesting only mitochondrial RNA (mtRNA) declines are associated with life expectancy.

Aging↗

Changes in the relative abundance of type I and type II lck mRNA transcripts suggest differential promoter usage during T-cell development.

The lck gene, which encodes the lymphoid cell-specific tyrosine protein kinase p56lck, is expressed from two widely separated promoters. The proximal promoter gives rise to a type I lck transcript, and the distal promoter gives rise to a type II transcript. We found that the ratio of the two transcripts changed during T-cell maturation. Type I lck mRNA was twofold more abundant than the type II transcript in early fetal thymocytes. In the adult, the type I and type II lck mRNAs were present in approximately equal amounts in immature thymocytes expressing the heat-stable antigen. In contrast, there was five- to ninefold more type II lck than type I lck mRNA in more mature thymocytes that did not express the heat-stable antigen and in splenic T cells. This change in relative transcript abundance probably reflects activation of the distal promoter and inactivation of the proximal promoter during T-cell maturation in the thymus. It is possible that the two promoters are regulated by different trans-acting factors whose expression is regulated during T-cell maturation.

Animals↗

Analysis of mRNA expression and protein abundance data: an approach for the comparison of the enrichment of features in the cellular population of proteins and transcripts.

MOTIVATION: Protein abundance is related to mRNA expression through many different cellular processes. Up to now, there have been conflicting results on how correlated the levels of these two quantities are. Given that expression and abundance data are significantly more complex and noisy than the underlying genomic sequence information, it is reasonable to simplify and average them in terms of broad proteomic categories and features (e.g. functions or secondary structures), for understanding their relationship. Furthermore, it will be essential to integrate, within a common framework, the results of many varied experiments by different investigators. This will allow one to survey the characteristics of highly expressed genes and proteins. RESULTS: To this end, we outline a formalism for merging and scaling many different gene expression and protein abundance data sets into a comprehensive reference set, and we develop an approach for analyzing this in terms of broad categories, such as composition, function, structure and localization. As the various experiments are not always done using the same set of genes, sampling bias becomes a central issue, and our formalism is designed to explicitly show this and correct for it. We apply our formalism to the currently available gene expression and protein abundance data for yeast. Overall, we found substantial agreement between gene expression and protein abundance, in terms of the enrichment of structural and functional categories. This agreement, which was considerably greater than the simple correlation between these quantities for individual genes, reflects the way broad categories collect many individual measurements into simple, robust averages. In particular, we found that in comparison to the population of genes in the yeast genome, the cellular populations of transcripts and proteins (weighted by their respective abundances, the transcriptome and what we dub the translatome) were both enriched in: (i) the small amino acids Val, Gly, and Ala; (ii) low molecular weight proteins; (iii) helices and sheets relative to coils; (iv) cytoplasmic proteins relative to nuclear ones; and (v) proteins involved in 'protein synthesis,' 'cell structure,' and 'energy production.' SUPPLEMENTARY INFORMATION: http://genecensus.org/expression/translatome

Algorithms↗

Differences in the abundance of variably spliced transcripts for the second asialoglycoprotein receptor polypeptide, H2, in normal and transformed human liver.

The human hepatic asialoglycoprotein receptor comprises two homologous polypeptides designated H1 and H2. Two distinct complementary DNA clones encoding these receptor subunits have been previously isolated from the human hepatoblastoma cell line HepG2. We discovered that multiple variants of H2 transcripts exist both in HepG2 cells and in the normal human liver that, at least in part, appear to be the result of alternative splicing events. We have found that (a) the complementary DNA clone for H2 previously isolated from HepG2 cells, characterized by a 57-nucleotide insertion within the 5' end of the complementary DNA that is absent from H1, represented only one third of H2-related sequences in an unamplified normal human liver complementary DNA library and less than 10% of H2 clones in HepG2 cells; (b) the predominant message for H2 expressed in the liver and HepG2 cells, designated L-H2, appeared to represent the fully processed product of the gene encoding both L-H2 and H2; and (c) a variant H2 transcript existed in HepG2 cells, designated H2', that contained a novel, 5' 88-bp nucleotide insertion. Poly(A+) RNA analysis of the normal liver and HepG2 cells by complementary RNA hybridization and ribonuclease protection corroborated the observations made during the screening of complementary DNA libraries regarding the abundance of the various messages. A striking incongruity was found between the levels of messenger RNA containing the H2-specific 57-nucleotide sequence and the levels of polypeptide expressed in the liver and HepG2 cells as recognized by antiserum specifically raised against this sequence.

Asialoglycoprotein Receptor↗

Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes.

We have characterized the rat prostanoid EP1, EP2, EP3alpha and EP4 receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The KD values obtained with prostaglandin E2 for the prostanoid receptor subtypes EP1, EP2, EP3alpha and EP4 were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP2, EP3alpha and EP4 receptor subtypes was prostaglandin E2 = prostaglandin E1 > iloprost > prostaglandin F2alpha > prostaglandin D2 > U46619. The rank order at the prostanoid EP1 receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP2, M&B28767 and sulprostone were selective for EP3alpha and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP1 and EP3alpha. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP1 mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP3 transcripts were most abundant in liver and kidney whereas prostanoid EP2 receptor mRNA was expressed in spleen, lung and testis and prostanoid EP1 receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP1 probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP1 gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP1 prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227. 1329-1333].

Aequorin↗

Phtf1 is an integral membrane protein localized in an endoplasmic reticulum domain in maturing male germ cells.

Phtf1 is a gene evolutionarily conserved from Drosophila to human that is abundantly expressed in testis. In adult rat, transcripts were abundant in germinal meiotic and postmeiotic cells. Phtf1-specific antibodies revealed weak activity in a juxtanuclear region of early pachytene spermatocytes. Labeling progressively extended to the entire cytoplasm of step 2-3 spermatids, became intense from step 4, and persisted until the end of spermiogenesis, when it was eliminated in the residual bodies. Phtf1 displayed the properties of an integral membrane protein. In transfected cells and haploid cells of rat seminiferous epithelium, it colocalized with ER markers (calnexin and calmegin, respectively). By using both ER and Golgi markers (TGN-38, p58), we were able to show that, in pachytene spermatocytes and in Golgi phase spermatids, phtf1 labeled a region neighboring the cis-Golgi that probably corresponded to the peripheral Golgi region. Phtf1 staining was not related to beta-COP, AP1, or AP2 aptamers, indicating that it was not transported between Golgi saccules or between the Golgi complex and plasma membrane. However, aptamer labeling showed that chlatrin vesicles could be engaged in a new traffic route, raising the possibility of a meiotic proacrosomal vesicle origin. Colocalization between phtf1 and calmegin decreased during the acrosomal phase. During the maturation phase, phtf1 was able to identify different ER domains, as described previously for the peripheral Golgi region. Phtf1 provides a potential new marker for Golgi modifications as well as for many of the obscure transformations undergone by the endoplasmic reticulum. It could help to elucidate the morphogenic events connected with the transformation of spermatogenic cells.

3T3 Cells↗

The wheat mitochondrial rps13 gene: RNA editing and co-transcription with the atp6 gene.

Northern analyses and reverse transcription-polymerase chain-reaction (RT-PCR) experiments, followed by PCR amplification product sequencing, were performed on total mitochondrial (mt) RNAs from wheat seedlings and tissue cultures. It was shown that the rps13 gene, which encodes ribosomal protein S13, and the atp6 gene, which encodes subunit 6 of the ATP synthase complex, were co-transcribed. However, rps13 transcripts were virtually undetectable in seedlings under conditions where atp6 transcripts appeared abundant. In addition, markedly higher steady state transcript levels were observed in tissue culture. Expression of the mitochondrial rps13 gene was confirmed by showing that its transcripts were edited. Slight differences between editing patterns of tissue-culture and whole-plant transcripts were found. Taken together, these results suggest that in vitro culture could disturb the post-transcriptional regulation of gene expression.

Base Sequence↗

CaAlaAT1 catalyzes the alanine: 2-oxoglutarate aminotransferase reaction during the resistance response against Tobacco mosaic virus in hot pepper.

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P0. To elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P0 and genes specifically up-regulated during the HR were isolated by differential screening. One of the clones, CaAlaAT1 encoding a putative alanine aminotransferase (EC 2.6.1.2) exhibited organ-specific expression pattern and the transcript accumulated abundantly in red (ripe) fruit tissues. CaAlaAT1 transcript was also induced in older leaves during senescence. The expression of CaAlaAT1 gene was increased in the incompatible interaction with TMV-P0 but was not in the compatible interaction with TMV-P1.2. When a strain of Xanthomonas campestris pv. vesicatoria (Xcv) carrying an AvrBs2 gene was infiltrated into the leaves of a pepper cv. ECW 20R carrying Bs2 resistance gene, a marked induction and maintenance of CaAlaAT1 gene expression was observed. The expression of CaAlaAT1 gene was triggered by salicylic acid (SA) and ethylene but not by methyl jasmonate (MeJA). CaAlaAT1 seemed to be localized mostly at the cytosol from the polyethylene glycol (PEG)-mediated transformation experiment. CaAlaAT1 seemed to catalyze alanine: 2-oxoglutarate aminotransferase (AKT) reaction, which was a main activity among the four activities in vitro, during the resistance response against TMV in hot pepper. These results suggest that CaAlaAT1, a protein known to be involved in metabolic reactions, might be one of the components in the plant's defense signal pathway against pathogens.

Alanine Transaminase↗

The sugarless mutation affects the expression of the white eye color gene in Drosophila melanogaster.

Investigation of a modifier locus displaying a darker eye phenotype in white-apricot flies led to the isolation of the gene encoding UDP-glucose dehydrogenase (UDPGDH). The P-element insertion l(3)05007 occurs upstream of the transcription start site of the sugarless (sgl) gene and greatly reduces its transcription at various developmental stages. A single abundant sgl transcript shows a ubiquitous distribution and encodes a 53-kDa protein which is 64% identical in sequence to bovine UDP-glucose dehydrogenase. Overexpression of sgl in E. coli resulted in synthesis of a protein with high levels of UDPGDH activity. Expression of three genes that participate in pigment deposition, white, scarlet and brown, was significantly affected in populations segregating for sgl, suggesting that it is the decrease in UDPGDH level that produces the modifying effect observed. In addition, genetic effects on white-apricot were observed in sgl-wingless and sgl-hedgehog double mutants. Recent data have indicated an effect of UDPGDH on cell surface glycosaminoglycans (GAGs) which modulate the activity of growth factors, and in particular wingless signaling. Our results suggest that the levels of GAGs are rate limiting for cell-cell signaling pathways which mediate changes in gene expression.

ATP-Binding Cassette Transporters↗

Transcription of the Anabaena sp. strain PCC 7120 ntcA gene: multiple transcripts and NtcA binding.

The Anabaena sp. strain PCC 7120 ntcA gene showed multiple transcripts with different 5' ends. The relative abundance of transcripts varied in response to nitrogen availability. The ntcA product, NtcA, showed binding to the promoter region of its own gene. The binding site mapped to a region between the transcription start site used under nitrogen-replete conditions and the start sites used under nitrogen-limiting conditions, suggesting that NtcA regulates its own expression.

Amino Acid Sequence↗

Acetic acid induces expression of the Staphylococcus aureus cidABC and lrgAB murein hydrolase regulator operons.

The Staphylococcus aureus lrg and cid operons encode homologous proteins that regulate extracellular murein hydrolase activity and penicillin tolerance in a diametrically opposing manner. Although their specific regulatory functions remain unknown, it has been postulated that the functions of CidA and LrgA are analogous to those of bacteriophage holins and antiholins, respectively, and that these proteins serve as molecular control elements of bacterial programmed cell death. Although these studies demonstrated that cidBC transcription is abundant in sigmaB-proficient strains, cidABC transcription was only minimally expressed under standard growth conditions. In this study, we demonstrate that cidABC and lrgAB transcription in the clinical isolate UAMS-1 is induced by growth in the presence of 35 mM glucose and that this enhances murein hydrolase activity and decreases tolerance to vancomycin and rifampin. The effect of glucose on murein hydrolase activity was not observed in the cidA mutant, indicating that the induction of this activity was dependent on enhanced cidABC expression. Furthermore, we demonstrate that the effects of glucose on cidABC and lrgAB transcription are mediated by the generation of acetic acid produced by the metabolism of this and other carbon sources. These results shed new light on the control of the S. aureus cidABC and lrgAB genes and demonstrate that these operons, as well as murein hydrolase activity and antibiotic tolerance, are responsive to carbohydrate metabolism.

Acetic Acid↗

Sequences within the R region of the long terminal repeat activate basal transcription from the HIV-1 promoter.

The importance of the R region in basal human immunodeficiency virus type 1 (HIV-1) transcription was addressed by comparing a panel of HIV-1 R region mutants using in vitro and in vivo assays. Using deletion, base substitution mutants, and compensatory mutants, the precise R region sequences essential for basal HIV-1 promoter activity in vitro were mapped to sequences between +17 to +21. Within this regulatory domain, nucleotides +19 and +21 appear to be critical. The effect of these mutations on steady state RNA levels in transfected cells has been analyzed by S1 nuclease protection assay using uniformly labeled probes. Two main conclusions may be drawn from these studies. First, HIV-1 basal transcription is abundant, with the majority of correctly initiated transcripts truncated between sequences +57 to +70. Second, analysis of the compensatory mutants indicates the secondary structure of the nascent R region RNA is not an obligate requirement for the production of the truncated transcripts. Mutations in R region primary sequence that selectively abolish the production of the truncated transcripts in vivo also exhibit reduced promoter activity in vitro. The appearance of high levels of truncated transcripts raise the interesting possibility that-similar to c-myc, c-myb, and c-fos--basal HIV-1 expression is regulated by transcription elongation.

Gene Expression Regulation, Viral↗

Isolation of genes identified in mouse renal proximal tubule by comparing different gene expression profiles.

An expression profile is a list based on a large scale sequencing of 1000 cDNA clones, showing the expressed genes and the abundance of their transcripts in a given cell or tissue (Okubo K et al: Nature Genet 2:173, 1992). We constructed an expression profile of mouse renal proximal tubules (PT) carefully isolated by microdissection in order to characterize its gene expression. Altogether 1000 clones were analyzed; there were 646 types of transcripts in PT, among which 196 were identical or homologous to the previously reported genes. The most abundant transcript was kidney-androgen regulated protein. By comparing the expression profile of PT with those obtained from other sources, several genes were identified only in PT. They included known transcripts and transcripts that were not homologous to the known genes. Three (GS4001, 3991, and 4059) of the non-homologous genes were analyzed by Northern blotting and in situ hybridization, and GS4001 and 4059 were predominantly expressed in the kidney, whereas GS3991 was detected in the liver as well as in the kidney. The sequence analysis of the full-size cDNAs demonstrated that GS4001 was a new member of aspartic proteinases and GS4059 was a novel gene. It also revealed that GS3991 was a mouse homologue of SA gene known to be expressed in PT. The expression profile of mouse PT and its comparison with those of other tissues and cells provide an alternate way of isolating genes predominantly expressed in PT, and also provides probes to study the molecular mechanisms of gene expression in the kidney.

3T3 Cells↗

Abundant expression of erythroid transcription factor P45 NF-E2 mRNA in human peripheral granurocytes.

Transcription factor NF-E2 is crucial for regulation of erythroid-specific gene expression. p45 subunit of NF-E2 contains a basic-leucine zipper domain and dimerizes with the small Maf family protein to form functional NF-E2 complex. While p45 expression was shown to be restricted to erythroid cells, megakaryocytes and mast cells in hematopoietic lineage, we found in this study that p45 mRNA is abundantly transcribed in the granulocyte fraction of human peripheral blood cells. As neutrophils occupy approximately 92% of the cells in granulocyte fraction of human peripheral blood cells. As neutrophils occupy approximately 92% of the cells in this fraction, the cells expressing p45 is most likely to be neutrophils. p45 mRNA is also expressed in HL-60 promyelocytes, albeit the expression level is much lower than that of the granulocyte fraction. HL-60 cells were found to express mafK mRNA, indicating the presence of genuine NF-E2 complex in the cells. Although p45 mRNA is transcribed from two different promoters, aNF-E2 promoter and fNF-E2 promoter, in erythroid and megakaryocytic lineage cells, p45 mRNA is transcribed only from aNF-E2 promoter. The expression of p45 megakaryocytic lineage cells, p45 mRNA is transcribed only from aNF-E2 promoter. The expression of p45 mRNA in the neutrophils declined rapidly after transfer of the cells to in vitro culture and G-CSF could not sustain the expression from the down-regulation, suggesting the E2 may also participate in the regulation of neutrophil-specific gene expression.

Alternative Splicing↗

CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus.

The cell cycle of the budding yeast Saccharomyces cerevisiae has been investigated through the study of conditional cdc mutations that specifically affect cell cycle performance. Cells bearing the cdc68-1 mutation (J. A. Prendergast, L. E. Murray, A. Rowley, D. R. Carruthers, R. A. Singer, and G. C. Johnston, Genetics 124:81-90, 1990) are temperature sensitive for the performance of the G1 regulatory event, START. Here we describe the CDC68 gene and present evidence that the CDC68 gene product functions in transcription. CDC68 encodes a 1,035-amino-acid protein with a highly acidic and serine-rich carboxyl terminus. The abundance of transcripts from several unrelated genes is decreased in cdc68-1 mutant cells after transfer to the restrictive temperature, while at least one transcript, from the HSP82 gene, persists in an aberrant fashion. Thus, the cdc68-1 mutation has both positive and negative effects on gene expression. Our findings complement those of Malone et al. (E. A. Malone, C. D. Clark, A. Chiang, and F. Winston, Mol. Cell. Biol. 11:5710-5717, 1991), who have independently identified the CDC68 gene (as SPT16) as a transcriptional suppressor of delta-insertion mutations. Among transcripts that rapidly become depleted in cdc68-1 mutant cells are those of the G1 cyclin genes CLN1, CLN2, and CLN3/WHI1/DAF1, whose activity has been previously shown to be required for the performance of START. The decreased abundance of cyclin transcripts in cdc68-1 mutant cells, coupled with the suppression of cdc68-1-mediated START arrest by the CLN2-1 hyperactive allele of CLN2, shows that the CDC68 gene affects START through cyclin gene expression.

Amino Acid Sequence↗

Gene overlap and site-specific attenuation of transcription of the viral polymerase L gene of human respiratory syncytial virus.

Sequence analysis of the gene encoding polymerase L protein of respiratory syncytial virus showed that L-gene transcription initiates within its upstream neighbor, the gene encoding the 22-kDa protein 22K. This is an exception to the canonical mode of sequential transcription of nonoverlapping genes described for other nonsegmented negative-strand RNA viruses. As a consequence of the gene overlap, the termination/polyadenylylation signal for the 22K gene is located at nucleotides 56-68 within the L gene. L-gene transcription yielded two RNAs: an abundant, truncated, polyadenylylated transcript resulting from termination at the internal signal and a markedly less abundant large polyadenylylated transcript representing the complete L gene. This result showed that the internal termination/polyadenylylation signal is an attenuator of L-gene transcription.

Amino Acid Sequence↗

Spatially heterogeneous expression of aromatase P450 through promoter II is closely correlated with the level of steroidogenic factor-1 transcript in endometrioma tissues.

Endometriosis is an estrogen-dependent disease of women of reproductive age. Recent studies demonstrate that endometriosis per se express high levels of estrogen synthetase (aromatase P450). The resulting estrogen synthesized in situ may play a role in the development and exacerbation of the disease. For ovarian endometrioma, previous studies have been conducted ex vivo using cells obtained from endometrioma and have demonstrated that steroidogenic factor-1 is involved in the expression of aromatase. The aim of the present study was to provide in vivo evidence that steroidogenic factor-1 plays an important role in the regulation and overexpression of aromatase P450 in situ. First, promoter use of aromatase P450 in endometrioma tissue was determined using quantitative methods. Ovarian endometrioma tissue was chopped into small pieces, and two exon 1-specific transcripts of aromatase P450 (PII-specific and I.4-specific transcripts) were quantified using competitive RT-PCR. PII-specific transcript was more abundant than the I.4-specific transcript in 13 of the 15 endometriomas and less abundant in the remaining two. Spatial distribution of aromatase P450 transcripts in these endometrioma tissues revealed heterogeneous expression in the cyst wall, demonstrating wide variability even in the same endometrioma. Two possible regulators of aromatase expression (steroidogenic factor-1 and IL-1 beta) were then measured in all endometrioma samples and the correlation between aromatase P450 transcripts and these possible regulators in the endometrioma samples were tested using Spearman's rank order correlation test. Levels of steroidogenic factor-1 transcript were found to correlate closely with levels of PII-specific transcript in eight of nine endometriomas examined. On the other hand, the level of IL-1 beta weakly correlated with I.4-specific transcripts in three of the nine endometriomas. We next histologically examined samples of four endometriomas in which complete sets of tissue samples corresponded to the RNA samples. We could not identify any specific pathology to explain the heterogeneous expression of PII-specific transcripts of aromatase P450, although the number of CD-68 positive macrophages in the tissue sections weakly correlated with the level of I.4-specific transcript in two of four endometriomas. These results provide strong evidence that promoter II is the predominant promoter of aromatase P450 in endometrioma tissues in vivo and that steroidogenic factor-1 in situ is a major determinant of aromatase P450 overexpression in endometrioma tissues in vivo.

Adult↗

Post-transcriptional regulation of the abundance of mRNAs encoding alpha-tubulin and a 94,000-dalton protein in teratocarcinoma-derived stem cells versus differentiated cells.

Changes in the expression of the genes encoding alpha-tubulin and a 94,000-dalton protein (p94) specified by a cDNA clone, p4-30, were examined in a differentiated teratocarcinoma-derived parietal endoderm cell line, PYS-2, and an undifferentiated teratocarcinoma stem cell line, F9. Relative to other proteins or mRNA species, the synthesis rate of the alpha-tubulins and of p94, as well as the levels of their corresponding cytoplasmic mRNAs, were lower in PYS-2 than in F9 cells. The decrease was greater for the relative abundance of cytoplasmic alpha-tubulin mRNA than for p94 mRNA. Similarly, induction of differentiation of F9 cells by simultaneous exposure to retinoic acid (RA) and dibutyryl cyclic AMP resulted in reduced relative levels of the cytoplasmic mRNAs for these proteins. The reduction in abundance of the two RNA species was not due to a decrease in growth rate since the differentiated cells, PYS-2, RA-treated F9, and RA plus dibutyryl cyclic AMP-treated F9 cells, grew at a rate similar to that of undifferentiated F9 cells. However, induction of differentiation of F9 cells by treatment with RA alone did not cause down-regulation of the two RNA species. The relative levels of total cellular RNA encoding alpha-tubulin and p94 in PYS-2 cells were also lower than those in F9 cells to an extent comparable to the decrease in the cytoplasmic RNAs. Since the apparent relative rates of RNA transcription were similar in both cell types, we conclude that the reduction in relative levels of the alpha-tubulin and p94 RNAs in the cell depends largely on the relative stability of the two RNAs and not on the relative rates of transcription. The faster disappearance of the two RNA species relative to other cellular RNAs from actinomycin D-treated PYS-2 compared with F9 cells is consistent with this interpretation.

Animals↗