Alpha-tubulin RNA Research Articles

Molecular cloning and characterization of a Leishmania donovani alpha-tubulin gene.

We have isolated a cDNA for an alpha-tubulin mRNA from L. donovani promastigotes and determined its complete nucleotide sequence. Both nucleotide and deduced amino acid sequence analysis of this cDNA showed significant similarity with a previously reported, partial sequence of an L. enriettii alpha-tubulin and the complete sequence of human alpha-tubulin. Further, the in vitro translated L. donovani alpha-tubulin gene product was specifically immunoprecipitated with a monoclonal antibody against human alpha-tubulin. Northern blot analysis revealed that there was little change in the expression of the L. donovani alpha-tubulin RNA during parasite differentiation from promastigote to the in vitro grown "amastigote" form. Southern blot analysis revealed a simple genomic organization for the L. donovani alpha-tubulin gene with more than one copy of the alpha-tubulin gene in the parasite genome. To our knowledge, this is the first complete sequence of an alpha-tubulin for Leishmania to be reported in the literature.

Trans splicing in trypanosomes requires methylation of the 5' end of the spliced leader RNA.

Trypanosoma brucei spliced leader (SL) RNA contains an unusual cap 4 structure consisting of 7-methylguanosine linked to four modified nucleosides. During RNA maturation, trans splicing transfers the first 39 nucleotides of the SL RNA including the cap structure to the 5' end of all mRNAs. Here we show that exposure of permeable trypanosome cells to S-adenosyl-L-homocysteine inhibits methylation of the nucleosides adjacent to 7-methylguanosine of newly synthesized SL RNA and prevents utilization of the SL RNA in trans splicing. However, trans splicing of the SL RNA preexisting in the cells is not inhibited by S-adenosyl-L-homocysteine as shown by the observation that newly synthesized alpha-tubulin RNA is trans spliced at the same level as in control cells. Therefore, it appears that the newly synthesized SL RNA is the only known component of the trans-splicing machinery that is impaired in its function by inhibition of methylation. Undermethylation does not alter either the stability of the SL RNA or the electrophoretic mobility and chromatographic behavior of the core SL ribonucleoprotein particle. Taken together, our data suggest that the cap 4 structure of the SL RNA plays an essential role in the trans-splicing process.

Permeable trypanosome cells as a model system for transceiption and trans-splicing

We have established conditions for Trypanosoma brucei permeable cells to study transcription and trans-splicing. We found that the concentration of monovalent and, to a lesser extent, divalent ions plays a critical role for the expression of a number of different genes. Most remarkably, the synthesis of the spliced leader (SL) RNA was optimal at 20 mM KCl, whereas higher potassium concentrations were inhibitory. In addition, MgCl2 concentrations above 3 mM led to the accumulation of a 3' end shortened SL RNA species, which has been previously reported not to participate in trans-splicing. Using conditions optimal for the synthesis of the SL RNA, we observed accurate trans-splicing of newly-synthesized alpha-tubulin RNA. Moreover, we detected the SL intron both joined to high molecular weight RNAs in the form of branched Y-structures and as a free linear molecule, which rapidly turned over. Furthermore, ionic concentrations that inhibit the synthesis of the SL RNA produced exclusively unspliced alpha-tubulin RNA, thus demonstrating that transcription and trans-splicing can be uncoupled.

Expression of RNAs for calmodulin, actins, and tubulins in rat testis cells.

Messenger RNAs encoding calmodulin, alpha- and beta-tubulins, and actins were analyzed in nucleic acid isolated from purified rat testis cell populations. Cell-specific patterns were discovered that suggest coordinate regulation of members of different gene families. Genes that are candidates for coordinate regulation include those encoding 1.4 kb calmodulin RNA and 1.8 kb alpha-tubulin RNAs; 1.6 kb calmodulin RNA and a 1.8 kb beta-tubulin RNA; and 1.6 kb actin and 2.1 kb alpha-tubulin RNAs. The steady state level of the 2.2 kb actin RNA(s) varies less than any other RNA analyzed. Actin RNAs of approximately 1.6 kb are observed in fractions enriched for A spermatogonia or round spermatids or cytoplasts shed from elongating spermatids. The level of 1.8 kb alpha-tubulin RNA increases in a fraction enriched for leptotene-zygotene spermatocytes and peaks in pachytene spermatocytes. As the level of this alpha-tubulin RNA species decreases in maturing spermatids a new 2.1 kb alpha-tubulin RNA increases. beta-Tubulin RNAs of 2.7 and 1.8 kb are present in somatic and premeiotic cell fractions. beta-Tubulin RNA transcripts of 1.8 kb that increase in pachytene spermatocytes are derived from a different gene than that expressed in premeiotic cells. The major RNA that hybridizes to the chicken calmodulin cDNA is 1.4 kb; it increases during the development of spermatocytes, reaching a maximum level in pachytene spermatocytes before it declines during spermiogenesis. Calmodulin RNAs of 1.6 and 1.0 kb are clearly evident in the round spermatid fraction. These data reveal that mechanisms exist for cell-selective elevation of mRNAs encoding cytoskeletal proteins during rat spermatogenesis.

MTrasT24, a metallothionein-ras fusion gene, modulates expression in cultured rat liver cells of two genes associated with in vivo liver cancer.

We studied the effects of a zinc-inducible metallothionein-ras fusion gene (MTrasT24) in cultured rat liver epithelial (RLE) cells on expression of two genes induced during liver carcinogenesis in vivo: gamma-glutamyltransferase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] and glutathione S-transferase-P (RX:glutathione R-transferase, EC 2.5.1.18). Expression of MTrasT24 increased steady-state RNA levels of gamma-glutamyltransferase and glutathione transferase-P 6- to 100-fold and 1.6- to 6-fold, respectively; in contrast, levels of alpha-tubulin RNA fell slightly or were unchanged. RNA gel blots verified that gamma-glutamyltransferase and glutathione transferase-P RNAs were of the appropriate size, and results from immunocytochemistry on transfected cells demonstrated that RLE cells carrying MTrasT24 synthesized immunoreactive, appropriately localized gamma-glutamyltransferase and glutathione transferase-P. Zinc induction studies indicated that gamma-glutamyltransferase and glutathione transferase-P RNA levels were directly dependent on MTrasT24 RNA levels. These data suggest that expression of gamma-glutamyltransferase and glutathione transferase-P expression are part of a reorientation of cellular gene expression during carcinogenesis and that activated ras expression, like chemical carcinogens, can bring about this change.

Developmental Regulation of Calmodulin, Actin, and Tubulin Rnas during Rat Testis Differentiation1

Postnatal testis differentiation involves transition through neonatal, pre-meiotic, meiotic, haploid, and mature stages. We have examined the qualitative and quantitative changes in rat testis RNAs that specifically hybridize to cDNAs encoding the cytoskeletal proteins, calmodulin, beta-actin, alpha- and beta-tubulin at ages corresponding to each of these developmental periods. We compared the species and relative levels of specific RNAs from testes of animals engaged in normal spermatogenesis with RNA from germ cell-depleted, Sertoli cell-enriched (SCE) testis. Distinct developmental patterns of expression of the specific RNAs were found with each of the cDNAs in the two animal models. A 2.2 kb (kilobase) actin RNA and a 2.7 kb beta-tubulin RNA are maximal at 5-10 days of age, suggesting these RNAs are required by somatic and germ cells in the postnatal phase prior to puberty. Between 19 and 29 days, when pachytene spermatocytes appear in significant numbers, there is a slight increase in the 2.2-kb actin RNA, but a 4- to 10-fold increase in RNAs hybridizing to cDNAs for calmodulin, alpha- and beta-tubulin. These changes are much less pronounced in the SCE testis than in the normal testis, indicating increases in these RNAs are related to germinal cell maturation. The germ cell-related increase in 1.8-kb beta-tubulin RNA appears to reflect a developmental "switch" in the gene from which the RNA is derived. This hypothesis is based on the observation that the ratio of hybridization of a chicken brain beta-tubulin cDNA versus a rat spleen beta-tubulin cDNA to the 1.8-kb RNA band increases more than 40-fold between 5 and 29 days of age in normal testis, but is constant in SCE testis. These data suggest that a specific beta-tubulin gene is activated in maturing germ cells. Analogously, a 2.1-kb alpha-tubulin RNA is found only in maturing normal testis and increases as spermatids are produced. A 2.0-kb beta-tubulin RNA, not found in normal testes, is maximal in maturing SCE testes, suggesting this RNA is of somatic cell origin. All of the RNA species studied, except the 2.0-kb beta-tubulin RNA, decrease between 5 and 19 days in SCE testes, as Sertoli cell mitotic activity wanes, indicating that their levels may be regulated by the developmental signals that influence mitosis.

Inhibition of steroid-inducible tyrosine aminotransferase gene expression by N-methyl-N'-nitro-N-nitrosoguanidine in a rat hepatoma cell line.

Treatment of rat hepatoma cells (the Fao clone of Reuber H35 cells) with a 500 ng/ml concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a marked inhibition of steroid-inducible tyrosine aminotransferase (TAT) gene expression. Following a 1-h treatment period with MNNG, addition of 1 microM dexamethasone (DEX) for 6 and 24 h resulted in, respectively, 78 and 51% less induction of TAT enzyme activity in MNNG-treated than in solvent-treated cells. MNNG had little effect on the basal level of TAT activity 6 h after treatment with the carcinogen, but the uninduced level of TAT activity declined to 36% of the control value 24 h following MNNG treatment. MNNG-treated cells concurrently exhibited 75% less total steroid-induced TAT RNA following addition of 1 microM of DEX for 4 h, but showed little or no change in the basal level of total TAT RNA or in the level of total alpha-tubulin RNA. In addition, MNNG treatment resulted in 45% less induction of TAT enzyme activity following a 4-h treatment period with 0.5 mM N6,O2'-dibutyryl-adenosine 3',5'-cyclic monophosphate. The concentration of MNNG used in these experiments caused only a 14% decline in cell number 24 h following carcinogen treatment and had no effect on cell viability as determined by trypan blue exclusion. The data indicate that the inhibitory effects of MNNG appear to be mediated at a pre-translational step. MNNG inhibits the accumulation of total DEX-inducible TAT RNA either by preventing the steroid-mediated increase of the rate of TAT RNA transcription or decreasing total TAT RNA stability, or both.

Multiple polymorphic alpha- and beta-tubulin mRNAs are present in sea urchin eggs.

Multiple alpha- and beta-tubulin RNAs were found in the mature unfertilized eggs of the sea urchin Lytechinus pictus. The alpha-tubulin RNAs were polymorphic in number, size, and relative amounts in the eggs of different females. Five to seven different size classes [1.75-4.2 kilobases (kb)] were detected on RNA gel blots. All egg preparations contained variable amounts of 1.8- and 2.25-kb beta-tubulin RNAs, and a few of them contained an additional 2.9-kb beta-tubulin RNA. The total amount of alpha-tubulin RNA did not always parallel that of beta-tubulin RNA. A portion of all of the various alpha- and beta-tubulin RNAs were polyadenylylated. RNase H digestions ruled out the possibility that some of these RNAs represented a single transcript bearing different lengths of 3' poly(A). One class of alpha-tubulin RNAs (2.4-2.65 kb) was reduced to 2 kb by RNase H, suggesting the presence of internal oligo(A) regions. All of the egg beta-tubulin RNAs sedimented as free ribonucleoprotein particles. Only a small portion of the 1.75- to 3.6-kb alpha-tubulin RNAs, but most of the 4.2-kb alpha-tubulin RNA, were found on polysomes before fertilization. In the 30-min embryo, small amounts of each of the various alpha- and beta-tubulin RNAs were recruited onto polysomes. Thus, each of the multiple polymorphic alpha- and beta-tubulin RNAs in the egg represent translationally competent mRNA.

Cell cycle regulation of tubulin RNA level, tubulin protein synthesis, and assembly of microtubules in Physarum.

The temporal relationship between tubulin expression and the assembly of the mitotic spindle microtubules has been investigated during the naturally synchronous cell cycle of the Physarum plasmodium. The cell cycle behavior of the tubulin isoforms was examined by two-dimensional gel electrophoresis of proteins labeled in vivo and by translation of RNA in vitro. alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin synthesis increases coordinately until metaphase, and then falls, with beta 2 falling more rapidly than beta 1. Nucleic acid hybridization demonstrated that alpha- and beta-tubulin RNAs accumulate coordinately during G2, peaking at metaphase. Quantitative analysis demonstrated that alpha-tubulin RNA increases with apparent exponential kinetics, peaking with an increase over the basal level of greater than 40-fold. After metaphase, tubulin RNA levels fall exponentially, with a short half-life (19 min). Electron microscopic analysis of the plasmodium showed that the accumulation of tubulin RNA begins long before the polymerization of mitotic spindle microtubules. By contrast, the decay of tubulin RNA after metaphase coincides with the depolymerization of the spindle microtubules.

Differential mRNA accumulation and translation during Spisula development.

The patterns of proteins synthesized in developing Spisula embryos and larvae were compared with in vitro translation products by one-dimensional gel electrophoresis. Major changes in the in vivo pattern occur at fertilization; these are regulated at the translational level (Rosenthal, Hunt, and Ruderman, 1980, Cell 20, 487-494). The pattern is further altered by midcleavage, and subsequent development is accompanied by frequent changes in the kinds of proteins made. By midcleavage many of the in vivo changes are paralleled by alterations in mRNA levels. Three cDNA clones containing developmentally regulated, nonmitochondrial sequences were isolated from a library constructed from veliger larval RNA. Clone 3v4 encodes alpha-tubulin. Clone 12v4 encodes a 35,000-D protein of unknown function. The protein product of clone 10v8 has not been identified. The concentration of alpha-tubulin RNA is relatively low through midcleavage, increases by the swimming gastrula stage, and is maintained at a moderately high level throughout larval development. 10v8 and 12v4 RNAs first appear in trochophore larvae; their concentrations peak 10-12 hr later, and then decline. The proportions of alpha-tubulin and 10v8 RNA that are translated vary with developmental stage. During early cleavage very little alpha-tubulin RNA is on polysomes; in swimming gastrulae 64% of this mRNA is polysomal. Seventy percent of 10v8 RNA is translated in the trochophore larva, while only approximately 40% is polysomal in the 21-hr veliger. These results show that translational regulation may be superimposed on changes in cytoplasmic mRNA concentrations to determine the level of gene expression during embryogenesis.