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PubMed1996

Logitlinear models for the prediction of splice sites in plant pre-mRNA sequences.

Pre-mRNA splicing in plants, while generally similar to the processes in vertebrates and yeast, is thought to involve plant specific cis-acting elements. Both monocot and dicot introns are typically strongly enriched in U nucleotides, and AU- or U-rich segments are thought to be involved in intron recognition, splice site selection, and splicing efficiency. We have applied logitlinear models to find optimal combinations of splice site variables for the purpose of separating true splice sites from a large excess of potential sites. It is shown that plant splice site prediction from sequence inspection is greatly improved when compositional contrast between exons and introns is considered in addition to degree of matching to the splice site consensus (signal quality). The best model involves subclassification of splice sites according to the identity of the base immediately upstream of the GU and AG signals and gives substantial performance gains compared with conventional profile methods.

Algorithms↗
PubMed1980

Quantitation of proinsulin mRNA sequences in hamster insulinoma cells in culture by molecular hybridization.

The content of proinsulin mRNA sequences was measured in a cultured cell line established from a transplantable hamster islet cell tumor, by hybridization with proinsulin cDNA. The cells cultured in vitro were found to contain a significant amount of proinsulin mRNA sequences, compared with non-insulin-producing hamster tissues, and seem to be a useful system for the study of insulin gene expression.

Adenoma, Islet Cell↗
PubMed2005

Type III secretion of the Salmonella effector protein SopE is mediated via an N-terminal amino acid signal and not an mRNA sequence.

Type III secretion systems (TTSS) are virulence-associated components of many gram-negative bacteria that translocate bacterial proteins directly from the bacterial cytoplasm into the host cell. The Salmonella translocated effector protein SopE has no consensus cleavable amino-terminal secretion sequence, and the mechanism leading to its secretion through the Salmonella pathogenicity island 1 (SPI-1) TTSS is still not fully understood. There is evidence from other bacteria which suggests that the TTSS signal may reside within the 5' untranslated region (UTR) of the mRNA of secreted effectors. We investigated the role of the 5' UTR in the SPI-1 TTSS-mediated secretion of SopE using promoter fusions and obtained data indicating that the mRNA sequence is not involved in the secretion process. To clarify the proteinaceous versus RNA nature of the signal, we constructed frameshift mutations in the amino-terminal region of SopE of Salmonella enterica serovar Typhimurium SL1344. Only constructs with the native amino acid sequence were secreted, highlighting the importance of the amino acid sequence versus the mRNA sequence for secretion. Additionally, we obtained frameshift mutation data suggesting that the first 15 amino acids are important for secretion of SopE independent of the presence of the chaperone binding site. These data shed light on the nature of the signal for SopE secretion and highlight the importance of the amino-terminal amino acids for correct targeting and secretion of SopE via the SPI-1-encoded TTSS during host cell invasion.

Amino Acid Motifs↗
PubMed1983

The phytochrome-controlled accumulation of mRNA sequences encoding the light-harvesting chlorophyll a/b protein of barley (Hordeum vulgare L.).

Double-stranded cDNA was synthesized from polysomal poly(A)-containing RNA of illuminated barley plants and was inserted into the PstI site of the bacterial plasmid pBR322. Two different strategies were used for the screening of the bacterial colonies. Light-regulated sequences were detected by differential hybridization with cDNA of polyadenylated RNA from dark-grown and illuminated barley plants. For a second screening step partially purified mRNAs encoding the light-harvesting chlorophyll a/b protein were used for the synthesis of another cDNA probe. By using these procedures a cDNA clone was isolated which encodes a constitutive polypeptide of the light-harvesting chlorophyll a/b protein. This cloned cDNA has been used to assess the effect of phytochrome on the steady-state level of mRNA sequences encoding the light-harvesting chlorophyll a/b protein. The mRNA is almost undetectable in dark-grown plants. Following treatment with red light, the concentration of this mRNA sequence increases rapidly during the subsequent dark period. This red light effect can be reversed substantially by irradiation with far-red light. These results indicate that not only the amount of mRNA activity as shown previously, but also the steady-state level of mRNA sequences encoding the light-harvesting chlorophyll a/b is controlled by phytochrome.

Base Sequence↗
PubMed1983

Determination of the primary sequence of the duck alpha D globin mRNA and comparison of all adult duck and chick globin mRNA sequences.

The nucleotide sequence of the duck alpha D globin mRNA was determined. Its main feature is an exceptionally short 3' non-coding segment of only 46 nucleotides, placed after the coding sequence of 141 codons. The last of the 6 adult globin mRNA of duck and chicken being thus sequenced, a comparison of all their features has become possible. Comparing the duck alpha D mRNA to the related sequence in the chicken, we found greater homology than comparing it to the linked alpha A globin sequence in the same species. Extensive homology can be found for a same globin chain alpha A, alpha D or beta in between different avian species including also the goose and the ostrich; the avian alpha globin chains show a lower degree of sequence conservation in between species than the beta chains. In contrast, within one species the three globin sequences have further diverged. The divergence between the alpha A and alpha D globin within a same species point to individual functional specificity and hence independent evolution and suggest that a mechanism of 'gene conversion' did not operate in between the avian alpha globin genes. Two segments of the amino acid sequence which we named 'A alpha' and 'B alpha' remain homologous in all avian alpha globins; two other regions 'A beta' and 'B beta' are identical in between the beta globins. Segment A is placed at the 5' end of exon II, and segment B at the 3' end of the same exon; some amino acids in those segments are involved in the Heme binding site. Being almost identical in all know mammalian and avian globins of the alpha respectively the beta type, regions A and B seem to represent the best conserved sequences in adult globin mRNA maintained during the divergence of species.

Animals↗
PubMed1980

Polyadenylate-deficient analogues of poly(A)-containing mRNA sequences in cultured AKR mouse embryo cells.

Five to six percent (by mass) of AKR-2B mouse embryo cell polysomal RNA consists of messenger RNA sequences which may exist in polyadenylated form. In the steady state, however, only 30--40% of these molecules are retained by extensive passage over oligo(dT)-cellulose, the remainder being present in the form of poly(A)-deficient analogues. Within experimental limits, these poly(A)-deficient analogues contain representatives of all poly(A)-containing mRNA sequences in these cells. An analysis of the kinetics of hybridization of cDNA probes enriched for either abundant or rare poly(A)-containing mRNA sequences suggests that the frequency distributions of poly(A)-containing and poly(A)-deficient analogues are dissimilar, and that a relationship exists between the intracellular frequency of a given mRNA sequence and the number of poly(A)-deficient analogues of that sequence. High frequency sequences appear to be enriched in the poly(A)-containing fraction, while low frequency sequences are predominately associated with the poly(A)-deficient fraction, thus, poly(A) may play a role in the regulation of mRNA frequency in the cytoplasm.

Animals↗
PubMed1978

Cleavage of pre-mRNA sequences by ribonucleases bound to nuclear RNP particles of rat liver.

The 30S nuclear RNP particles from rat liver have been shown to split the double-stranded- (ds) and single-stranded (ss) sequences of nuclear pre-mRNA. Experiments performed in vitro have demonstrated that 1) a 5'-exonuclease and an endonuclease specific for double-stranded pre-mRNA sequences exist in the 30S pre-mRNP particles; 2) in dsRNA monophosphorylated 5'-termini arose in the course of incubation with 30S RNP and most of the products remained double-stranded. The analysis of terminal pNp nucleotides revealed a relatively high ratio of pPyp in the cleaved dsRNA, whereas the nucleosides in 5'-terminal pNp of ssRNA showed nearly random distribution. Our results provide a possible explanation for the appearance of pNp termini during the processing of nuclear pre-mRNA of mammalian cells.

Animals↗
PubMed1983

Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA.

Animals↗
PubMed1982

On the localization and transport of specific adenoviral mRNA-sequences in the late infected HeLa cell.

In the nucleus of HeLa cells late after infection with adenovirus type 2 mRNA-sequences which are processed via RNA splicing are attached to the nuclear matrix (Mariman et al., 1982). Although the mRNA, which codes for polypeptide IX, is not formed via splicing, about 70% of the non-polyadenylated pre-mRNA and the polyadenylated pIX mRNA are bound to the matrix structure, indicating that polyadenylation is performed while the RNA is associated with the matrix. Binding to the nuclear matrix seems to be a common property of all mRNA-sequences in the nucleus. At the late stage of infection most of the newly synthesized mRNAs which appear in the cytoplasm are viral specific (Beltz & Flint, 1979). Kinetic analysis of the newly synthesized poly(A)-containing mRNA on sucrose gradients reveals that 7-12 S messengers appear more rapidly in the cytoplasm than messengers larger than 13 S. More specifically, the nuclear exit time of the pIX-mRNA, which is the major 9 S adenoviral messenger late after infection, was determined to be about 4 min, while messengers transcribed from the late region 3 need more than 16 min to arrive in the cytoplasm. In the cytoplasm about 70% of the mRNA is bound to the cytoskeletal framework, while 30% remains as free mRNP. Analysis of the mRNA in both fractions reveals that L3-, E1B- and pIX-specific polyadenylated mRNA preferably exist as cytoskeleton-bound mRNA. However, significant differences occur in the partition of specific messengers over free and cytoskeletal RNA fractions.

Adenoviruses, Human↗
PubMed1986

A low-molecular-weight RNA from mouse ascites cells that hybridizes to both 18S rRNA and mRNA sequences.

A low-molecular-weight RNA species from mouse ascites cells has been selected and purified by its intermolecular RNA X RNA hybridization capabilities. This 4.5S RNA is able to base pair with poly(A)+ mRNA sequences and with 18S rRNA. Melting experiments have shown that the intermolecular hybrids formed with this complementary low-molecular-weight RNA are of comparable stability to other RNA X RNA interactions. Analysis has shown that this hybridizing RNA is 87 nucleotides long and has an unusual sequence structure. Located near the 3' terminus is an alternating pyrimidine dinucleotide region of UUCCUUCCUU. This region along with the 3'-adjacent nucleotides form a 14-nucleotide sequence that exhibits perfect complementarity with 18S rRNA. An additional region of 10 nucleotides at the 3' terminus is perfectly homologous to a similarly located sequence in 5.8S rRNA. An obvious RNA polymerase III binding site is not found internally in this low-molecular-weight RNA sequence. The complementary and homologous character of hybridizing RNA with respect to rRNA and mRNA sequences suggests a potential regulatory role for this RNA in the coupling of ribosome and mRNA functions.

Animals↗
PubMed1979

Declining procollagen mRNA sequences in chick embryo fibroblasts infected with rous sarcoma virus. Correlation with procollagen synthesis.

Chick cells infected with Rous sarcoma virus are characterized by a wide variety of changes known collectively as transformation. Among these are decreases in the level of procollagen biosynthesis and in the level of procollagen mRNA. In this communication, we examine the time course of the decrease in procollagen biosynthesis, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and collagenase assay, and compare it with the decrease in procollagen mRNA sequences measured by hybridization to a complementary DNA. Procollagen biosynthesis and procollagen mRNA sequences decrease simultaneously after infection. Even the initial decrease in procollagen biosynthesis, therefore, is due to a decline in the level of procollagen mRNA.

Animals↗
PubMed1976

Detection of mRNA sequences in nuclear 30S ribonucleoprotein subcomplexes.

RNA from nuclear 30S ribonucleoprotein (RNP) complexes of mouse ascites cells has been shows to contain sequences homologous to poly(A) + mRNA by its ability to hybridize with complementary DNA prepared from poly(A) + mRNA template. Analysis of the hybridization kinetics of poly(A) + mRNA with its own complementary DNA revealed several abundancy classes. The total complexity of poly(A) + mRNA from ascites cells was estimated to be approximately 30,000 sequences of average molecular weight (6 X 10(5)). When the hybridization reaction of 30S RNP-RNA with mRNA-specific cDNA was compared to the homologous reaction the majority, and most probably all, of the poly(A) + mRNA sequences were found to be present in the RNA. The kinetics of hybridization suggest that 10-15% of the RNA in this RNP complex is homologous to poly(A) + mRNA. The 30S RNP subcomplexes therefore contain nuclear poly(A) + mRNA sequences as well as the bulk of heterogeneous RNA.

Base Sequence↗
PubMed1985

mRNA sequence predictions from homologous protein sequences.

A model has been developed that permits the prediction of mRNA nucleic acid sequence from the sequences of the translated proteins. The model relies on the information obtained from the comparison of protein sequences in related species to reduce the number of possible codons for those amino acids where mutations are observed. The predictions so obtained have been tested by applying the model to proteins whose mRNA sequences are known. The model's predictions have been found to be 100% accurate if three or more different amino acids are known at a given position and if the protein sequences are restricted to relatively closely related species (within the same class). The use of this model may permit a reduction of the mRNA sequence degeneracy and therefore be helpful in the synthesis of cDNA probes or for the prediction of restriction endonuclease sites. Computer programs have been developed to ease the use of the model.

Amino Acid Sequence↗
PubMed2006

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus.

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.

Animals↗
PubMed1981

Representation of mRNA sequences in normal and SV40 transformed human diploid fibroblasts.

The representation of mRNA sequences in normal and SV40-transformed WI-38 human diploid fibroblasts was examined. Variations in the hybridization of polysomal RNAs to 3H-labeled DNAs complementary to poly A+ mRNA of normal and SV40-transformed cells suggest differences in the mRNA populations. a comparison of the representation of histone RNA sequences of normal and SV40-transformed human diploid cells did not reveal significant differences.

Base Sequence↗
PubMed1986

Multihormonal regulation and kinetics of induction of a hepatic mRNA sequence which is slowly responsive to triiodothyronine.

In contrast to the rapid response of mRNA-S14 which occurs within 20 min after L-triiodothyronine (T3) administration, the induction of other rat hepatic mRNA sequences exhibits a lag time of several hours. We have studied the induction of mRNA-S11 which codes for a protein of pI 6.1 and an Mr of 22,500 as a model of such a slowly responsive gene. In addition to T3, both glucocorticoids and growth hormone regulate the expression of this gene. For each of these stimuli, the response exhibits a lag time of approximately 6 h. Following this lag time, there is a linear increase in the level of mRNA-S11 induced by a single maximal dose of T3, dexamethasone, and growth hormone to levels of 15-, 6-, and 3-fold, respectively, in excess of the hypothyroid base-line levels. The similarity in the lag time and the differences in maximal responses effectively argues against the possibility that the effect of one hormone is mediated exclusively by a change in the secretion or metabolism of another. Support for a direct action of T3 and glucocorticoids on the hepatic cell comes from the observation that these hormones stimulate mRNA-S11 in rat hepatocytes under primary culture. The increases in in vitro nuclear transcription measured following T3 and dexamethasone administration were clearly insufficient to account for the observed increases in mRNA. Furthermore, the hormonal induction of mRNA-S11 was promptly abrogated by cycloheximide (10 mg/kg) injected 6 h after the administration of T3 or dexamethasone at the time of the expected mRNA increase. The post-transcriptional control of the mRNA-S11 and its sensitivity to cycloheximide are similar to previously documented responses of the rapidly induced mRNA-S14 and suggest for both sequences a requirement for ongoing synthesis of rapidly turning over proteins. We speculate that the lag time of response of a given gene to T3 or glucocorticoid may be an intrinsic characteristic of the gene and may represent a common set of molecular events involved in the activation of that gene by diverse stimuli.

Animals↗
PubMed1985

Translational control of the circadian rhythm of liver sterol carrier protein. Analysis of mRNA sequences with a specific cDNA probe.

The striking changes in amount of rat liver SCP (sterol carrier protein) during a 24-h dark-light cycle are due to alterations in the relative synthetic rate of SCP. However, functional SCP mRNA, measured by a cell-free translational assay, does not fluctuate in the dark-light cycle. Since cell-free translational assays do not always reflect the actual abundance of an mRNA molecule, a specific cDNA hybridization assay was used to directly quantitate SCP mRNA sequences. The cDNA probe was selected from a rat liver library by hybridization to a mixture of synthetic oligonucleotides containing a portion of the sequence of SCP mRNA. The relative amount and size distribution of the SCP mRNA species (approximately 700-800 nucleotides) does not change during the diurnal cycle. To explore possible mechanisms of this translational control, the polysomal distribution of SCP mRNA was compared at the maximum and minimum points of SCP synthesis. No significant amounts of SCP mRNA were present in nonpolysomal ribonucleoprotein particles. Furthermore, no alteration in the relative level of SCP mRNA associated with polysomes or in polysome size occurs at the maximum and minimum points of SCP synthesis. Thus, changes in total SCP mRNA levels or its polysomal distribution cannot account for the diurnal variation in SCP synthesis.

Animals↗
PubMed1982

Paradoxical effects of cycloheximide on the ultra-rapid induction of two hepatic mRNA sequences by triiodothyronine (T3).

Triiodothyronine (T3) rapidly induces the accumulation of two hepatic mRNA sequences (spot 14 and spot CyT) in thyroidectomized rats as revealed by two-dimensional gel electrophoresis of in vitro translated products of isolated poly(A) containing RNA. T3 acting alone induced a 29-fold increase of spot 14 within 4 h, an increase which was completely inhibited by the concomitant administration of cycloheximide. On the other hand, CyT could be detected only after the administration of cycloheximide, and the combined action of cycloheximide and T3 resulted in a 4-fold increase in CyT 4 h after administration of both agents. These observations suggested that the early cellular action of T3 is contingent on the participation of rapidly turning over protein and mRNA.

Animals↗