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PubMed1994

Analysis of the role of the Shine-Dalgarno sequence and mRNA secondary structure on the efficiency of translational initiation in the Euglena gracilis chloroplast atpH mRNA.

Chloroplast mRNAs in Euglena gracilis fall into two classes. One class has a Shine-Dalgarno sequence 5' to the AUG start codon while the other group of mRNAs does not have any conserved sequence elements near the start codon. The chloroplast mRNA encoding the atpH gene has been selected as an example of a message which has a Shine-Dalgarno sequence (GGAGUU) located in the initiation region. Mutations in the Shine-Dalgarno sequence result in 2-5-fold reductions in the efficiency of the message in initiation complex formation depending on the precise mutation. Secondary structure mapping of the initiation region of the atpH mRNA suggests that a number of mRNA conformations are present in an equilibrium mixture. Mutations in the Shine-Dalgarno sequence had little effect on the overall structure of the mRNA. Directed mutations which place the Shine-Dalgarno sequence into a region of higher secondary structure drastically reduce the activity of the message. The monocistronic form of the atpH mRNA has a 5'-untranslated leader region slightly over 70 nucleotides in length. Deletions in the leader suggest that a minimum length of 10 bases 5' to the start codon is important for activity in initiation. The presence of the full leader increased efficiency in initiation about 2-fold compared to messages with leaders 25-40 residues in length.

Animals↗
PubMed1986

Changes in apolipoprotein A-I mRNA level in the liver of rats with experimental nephrotic syndrome.

In previous studies we had shown that: one of the most specific feature of hyperlipoproteinemia found in rats with experimental nephrotic syndrome is the accumulation of apolipoprotein A-I-rich HDL in plasma and this disorder is associated with an overproduction of apolipoprotein A-I by the liver. The present study was designed to investigate whether the increased hepatic synthesis of apolipoprotein A-I was due to an accumulation of functionally active apolipoprotein A-I mRNA in liver of nephrotic rats. Hepatic mRNA was translated in vitro by rabbit reticulocyte lysate in the presence of [35S]methionine and in vitro synthesized apolipoprotein A-I, albumin and apolipoprotein E were immunoprecipitated by specific rabbit IgG. In nephrotic rats the amount of in vitro synthesized apolipoprotein A-I was almost twice that found in the controls, suggesting that functionally active apolipoprotein A-I mRNA was increased in liver of nephrotic rats. To confirm that this difference in apolipoprotein A-I mRNA activity was due to an actual increase of hepatic apolipoprotein A-I mRNA sequences, we performed nucleic acid hybridization experiments (northern blot) using several cloned cDNA probes (rat and human apolipoprotein A-I, rat apolipoprotein E and apolipoprotein A-II). The results indicate that in nephrotic rats the amount of hybridizable apolipoprotein A-I mRNA sequences was about 3-fold higher than that in controls. In contrast, there was no difference in the amount of hybridizable apolipoprotein A-II and apolipoprotein E mRNA sequences, indicating that the change in apolipoprotein A-I mRNA induced by the nephrotic state was specific for this mRNA.

Animals↗
PubMed1975

alpha-and beta-Globin complementary deoxyribonucleic acids of human and rabbit. Specificity of hybridization.

The specificity of hybridization was compared between the human and rabbit alpha and beta-globin complementary DNAs (cDNAs) and the corresponding alpha and beta-globin messenger RNAs (mRNAs). The globin chain-specific mRNAs of rabbit were prepared from polysomes incubated with O-methylthreonine (alpha and beta) or from postribosomal supernatant (alpha). Enrichment for either the alpha- or beta-globin mRNA was demonstrated by cell-free protein synthesis and by RNA-cDNA hybridization. Human mRNAs, active as templates for RNA-directed DNA polymerase, were prepared from reticulocytes of patients with hemolytic anemia, alpha-thalassemia (hemoglobin H disease), and beta-thalassemia. Because there was partial cross-hybridization between human mRNA and rabbit cDNA, the rabbit alpha- and beta-globin cDNAs could be used to demonstrate that the beta-thalassemia mRNA was enriched in human alpha-globin mRNA sequences and that the alpha-thalassemia mRNA was enriched in human beta-globin mRNA sequences. These results were confirmed by preparation of thalassemia globin cDNAs and subsequent hybridization to their template mRNAs. The amount of cross-hybridization between the human and rabbit alpha-globin mRNA and the two alpha-globin cDNAs was comparable to the cross-hybridization between the two beta-globin mRNAs and the two beta-globin cDNAs, indicating a similar degree of evolutionary divergence in the nucleotide sequences of the two globin genes.

Animals↗
PubMed1977

Distribution of rat liver albumin mRNA membrane-bound and free in polyribosomes as determined by molecular hybridization.

Recently, we purified rat liver albumin mRNA and prepared albumin [(3)H]cDNA. Using albumin [(3)H]cDNA in molecular hybridization experiments, we have now determined the distribution of albumin mRNA sequences in membrane-bound and free liver polyribosomes prepared by techniques in which there is high recovery of polyribosomes without evidence of degradation. By using molecular hybridization to measure specific mRNA sequence content, numerous problems could be avoided in interpretation of results as obtained by cell-free protein synthesis or immunological methods. Under these conditions, 98% of albumin mRNA sequences in polyribosomes are found in the membrane-bound fraction.

Albumins↗
PubMed1980

IgE production in vitro by two rat myeloma tumor lines (IR2 and IR162).

Two IgE-synthesizing rat myeloma tumor lines (IR2 and IR162) were maintained in growing cultures that were viable for prolonged periods of up to three months in the presence of primary fibroblasts. The production of IgE by explanted tumor cells was studied in short-term and long-term cultures. The IR 162 tumor line retained expression of both IgE heavy (H) and light (L) chain synthesis for up to three months under in vitro conditions, while the IR2 tumor line initially lost H chain expression after approximately one month in culture, followed by subsequent loss of L chain about one month later. Total polysomal RNA extracted from IR2 cells, before and after loss of IgE-expression, was separated by sucrose gradient centrifugation, and gradient fractions were analysed for poly(A)-containing mRNA by a [3H]poly(U)binding assay. The results for the long-term IR2 cultures showed a disappearance of mRNA sedimenting at 18-20 S and 15 S, found to represent H and L chain mRNAs, respectively. Furthermore, no IgE L chain mRNA sequences could be detected in long-term IR2 cultures when analysed by hybridization assays, using as a probe a 3H-labelled DNA complementary to IgE L chain mRNA, L chain mRNA sequences were also absent from the nuclear RNA in long-term IR2 cultures. The loss of IgE synthesis in long-term cultures of IR2 cells therefore appears to be correlated with a concomitant loss of IgE mRNA sequences indicating a dedifferentiation in vitro modulated at the transcriptional level.

Animals↗
PubMed1988

Amino acid sequence of the signal peptide of mitochondrial nicotinamide nucleotide transhydrogenase as determined from the sequence of its messenger RNA.

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase was recently deduced from isolated cDNAs and reported [Yamaguchi, M., Hatefi, Y., Trach, K., and Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767]. The cDNAs lacked the N-terminal coding region, however, and the 8 N-terminal residues were determined by protein sequencing. In the present study, the nucleotide sequence of the 5' upstream region was determined by dideoxynucleotide sequencing of the transhydrogenase messenger RNA, and amino acid sequences of the N-terminal region and the signal peptide of the enzyme were deduced from the nucleotide sequence. The N-terminal sequence of the enzyme as deduced from the mRNA sequence is the same as that determined by protein sequencing, with one difference. Protein sequencing showed Ser as the N-terminal residue. The mRNA sequence indicated that Ser is the second N-terminal residue, and the first is Cys. That preparations of the enzyme are mixtures of two polypeptides, one polypeptide being one residue shorter at the N terminus than the other, has been pointed out in the above reference. The signal peptide consists of 43 residues, is rich in basic (4 Lys, 2 Arg) and hydroxylated (4 Thr, 3 Ser) amino acids, and lacks acidic residues.

Amino Acid Sequence↗
PubMed1994

Potential secondary structure at the translational start domain of eukaryotic and prokaryotic mRNAs.

In order to identify conserved potential secondary structures within translational start sites, mRNA sequences derived from different species were studied with programs able to depict such features. The potential secondary structure of 71 bases around the initiator AUG or AUGs in the coding sequences of 290 eukaryotic mRNAs was first examined and compared to 290 similarly analyzed regions derived from prokaryotic mRNA sequences (Nucleic Acids Res (1987) 15, 345-360). In both sets of sequences the initiator codon was often found to be in an open potential structure whereas a denser region characterized by nearly-periodic spacings defined the coding regions. Randomization of the sequences obliterated the observed patterns suggesting that the structure of the mRNA may determine these differences. Three sets of eukaryotic and prokaryotic mRNAs of approximately equal length were analyzed and found to preserve an open unpaired non-coding region 5' to the start codon. The start codon was found free of potential secondary structure in over 80% of all the sequences analyzed. These data, and study of mutants that restrict the accessibility of the start codon to the ribosomal initiation complex, suggest that both the prokaryotic and eukaryotic mRNA start sites must occur free of potential secondary structure for efficient initiation. A striking difference of the eukaryotic mRNA sequences analyzed was the high propensity of the coding region vicinal to the start codon to form secondary structures. Certain translation-defective mutants exhibit impaired formation of these secondary structures suggesting that the structure of the coding regions adjacent to the start codons of eukaryotic mRNAs may be an important, thus far unexamined, determinant of initiation. We propose that, for all genes studied, the transition in secondary structure between the coding and non-coding regions may be an important determinant of initiation.

Animals↗
PubMed1989

Homologous rat hepatic protease inhibitor genes show divergent functional responses to inflammation.

The genes encoding three distinct serine protease inhibitors (Spi) have been cloned from rat liver. These inhibitors are highly homologous with each other and are similar to alpha 1-antitrypsin at the nucleic and amino acid sequence level. Although previous investigators have examined the regulation of the Spi 2 locus by inflammation, the use of various techniques and the complexity of this genetic locus have led to incomplete and somewhat confusing results. Oligonucleotide probes specific for Spi 2.1, Spi 2.2, Spi 2.3, and a 3' mouse cDNA probe for alpha 1-antitrypsin mRNA were used to measure these mRNA after induction of inflammation with subcutaneous turpentine in Fischer rats. alpha 1-Antitrypsin mRNA increased 1.8-fold, and Spi 2.2 increased 7-fold. In contrast, Spi 2.1 and 2.3 mRNA sequences decreased fourfold. The maximal changes occurred between 24 and 48 h after inflammation, with a gradual return toward normal over the next 4 days. Since Spi 2.1, Spi 2.3, and alpha 1-antitrypsin mRNA sequences are responsive to growth hormone, two other growth hormone-responsive mRNA sequences, alpha 2u-globulin and insulin-like growth factor I, were measured, and they also decreased after induction of inflammation. The results of this study show that, despite a marked similarity of nucleotide sequence, Spi 2.1 and 2.3 genes respond very differently from Spi 2.2 and alpha 1-antitrypsin to both growth hormone and inflammation. We speculate that the functions of Spi 2.1 and 2.3 products are different from those of Spi 2.2 and alpha 1-antitrypsin and may involve the regulation of growth.

Animals↗
PubMed1983

Regulation of protein synthesis in mitogen-activated bovine lymphocytes. Analysis of actin-specific and total mRNA accumulation and utilization.

In response to the mitogenic lectin concanavalin A, the rate of protein synthesis in bovine small lymphocytes increased about 3-fold in 10 h and about 6-fold in 24 h. The rate of synthesis of the cytoskeletal protein actin, which comprises about 10% of total protein synthesis, increased in parallel with total protein synthesis. The cellular levels of actin mRNA were determined at various times after mitogen addition using a cDNA probe derived from a recombinant plasmid carrying a fragment coding for bovine actin. Actin mRNA sequences were found to increase significantly within 3 h and to increase 3.6 +/- 1.2-fold within 10 h after mitogen addition. Therefore, the increase in actin synthesis after mitogenic activation seems to be accounted for by accumulation of actin mRNA sequences. Total translatable mRNA and poly(A) sequences also accumulated in parallel with total protein synthesis, suggesting that regulation of actin synthesis was not unique. Two direct studies of mRNA utilization indicated that there is little, if any, translational regulation of actin synthesis. A detailed examination of actin mRNA-containing polysomes and total polysomes revealed no significant change in the average size of polysomes after cell activation. Therefore, given that the rates of polypeptide elongation and termination are constant after mitogen addition (Kay, J.E., Ahern, T., Lindsay V.J., and Sampson, J. (1975) Biochim. Biophys. Acta 378, 241-250), the rate of translational initiation per active mRNA must be unchanged. In addition, no significant amount of unutilized, nonpolysomal actin mRNA was detected in total cell extracts from either unstimulated or activated cells. Together these data suggest that elevated protein synthesis in activated lymphocytes is regulated by mRNA level.

Actins↗
PubMed2003

Antisense transcripts with rice full-length cDNAs.

BACKGROUND: Natural antisense transcripts control gene expression through post-transcriptional gene silencing by annealing to the complementary sequence of the sense transcript. Because many genome and mRNA sequences have become available recently, genome-wide searches for sense-antisense transcripts have been reported, but few plant sense-antisense transcript pairs have been studied. The Rice Full-Length cDNA Sequencing Project has enabled computational searching of a large number of plant sense-antisense transcript pairs. RESULTS: We identified sense-antisense transcript pairs from 32,127 full-length rice cDNA sequences produced by this project and public rice mRNA sequences by aligning the cDNA sequences with rice genome sequences. We discovered 687 bidirectional transcript pairs in rice, including sense-antisense transcript pairs. Both sense and antisense strands of 342 pairs (50%) showed homology to at least one expressed sequence tag other than that of the pair. Microarray analysis showed 82 pairs (32%) out of 258 pairs on the microarray were more highly expressed than the median expression intensity of 21,938 rice transcriptional units. Both sense and antisense strands of 594 pairs (86%) had coding potential. CONCLUSIONS: The large number of plant sense-antisense transcript pairs suggests that gene regulation by antisense transcripts occurs in plants and not only in animals. On the basis of our results, experiments should be carried out to analyze the function of plant antisense transcripts.

DNA, Antisense↗
PubMed1986

Measurement of human prolactin messenger RNA in decidual tissues using complementary DNA probe cloned in M13mp9 bacteriophage.

A human prolactin (PRL) cDNA clone was digested with restriction enzyme Pst I and the resultant fragments were cloned into bacteriophage M13mp9. Single stranded recombinant DNAs having a coding strand of the PRL cDNA were selected by hybridization with 125I-labeled PRL mRNA obtained from human prolactinoma tissue. One of the single stranded recombinant DNAs was purified by agarose gel electrophoresis and labeled with 125I to a specific activity of 1.4 X 10(8) cpm per microgram of DNA. The probe could be successfully used in RNA dot hybridization. Analysis of poly (A) RNAs from prolactinoma, liver and placental tissues revealed that this probe was specific to PRL mRNA sequence. Hybridization of poly (A) RNA from decidual tissue to this probe revealed the presence of PRL mRNA sequence. However, PRL mRNA in decidua was at least 20,000 times less than that in pituitary prolactinoma.

Cloning, Molecular↗
PubMed1978

Regulation of translation of ovalbumin messenger RNA by estrogens and progesterone in oviduct of withdrawn chicks.

In the oviduct of chicks withdrawn from previous treatment with estrogens, no ovalbumin synthesis can be detected, although there are a limited number of ovalbumin mRNA sequences. These sequences are predominately associated with membrane-bound ribosomes. However, the size of the polysomes is small compared to those from the laying hen, suggesting that the inability to detect ovalbumin synthesis is the result of inefficient initiation of ovalbumin synthesis. When the rate of peptide chain elongation is reduced by treatment of chicks with cycloheximide, there is an increase in the average size of polysomes and a shift of ovalbumin mRNA sequences from small to large-sized polysomes. Readministration of estrogen to withdrawn chicks results in a time-dependent shift of monosomes to polysomes and a proportional shift of ovalbumin mRNA sequences between the two fractions, indicating that estrogen stimulates the rate of initiation of all mRNA species in the oviduct to essentially the same extent. In contrast, progesterone administration results in a preferential shift of ovalbumin mRNA relative to total RNA, suggesting a preferential effect of progesterone on initiation of protein synthesis with ovalbumin mRNA.

Animals↗
PubMed2005

Identification and phylogenetic analysis of 15 MHC class II DRB1 beta1 expressed alleles in a ewe-lamb flock.

Ovar-DRB1 is part of the major histocompatibility complex (MHC) class II of sheep and functions by presenting extracellular-derived peptides to the immune system. Although there are over 100 different Ovar-DRB1 DNA sequences reported in GenBank, only two Ovar-DRB1 mRNA sequences have been reported. As a first step in understanding MHC Class II function as it relates to disease progression in sheep, Ovar-DRB1 transcripts encoding the peptide-binding site or the first domain (beta1) of Ovar-DRB1 in a 32-ewe-lamb flock were identified and characterized by using reverse transcriptase-polymerase chain reaction, cloning, sequencing, and phylogenetic analysis. Fourteen new Ovar-DRB1 beta1 cDNA sequences out of a total of 15 Ovar-DRB1 beta1 cDNA sequences in a ewe-lamb flock of 32 sheep were identified. One Ovar-DRB1 beta1 cDNA sequence was 100% identical to M93432, one of the two Ovar-DRB1 mRNA sequences reported in GenBank. Twelve out of 15 Ovar-DRB1 beta1 cDNA sequences were 100% identical to the corresponding previously reported Ovar-DRB1 genomic DNA sequences, indicating that these Ovar-DRB1 genomic DNA sequences are also transcribed. One of three of the remaining Ovar-DRB1 beta1 cDNA sequences, DRB1*07012, had a synonymous substitution resulting in an identical deduced amino acid sequence to DRB1*0701. Two of the remaining three Ovar-DRB1 beta1 cDNA sequences had nucleotide differences and subsequent deduced amino acid sequence differences when compared to known Ovar-DRB1 beta1 genomic DNA sequences, and therefore, DRB1*0206 and DRB1*0353 represent new Ovar-DRB1 beta1 expressed alleles. Phylogenetic analysis of the 15 Ovar-DRB1 beta1 cDNA sequences revealed that DRB1*0206 had a strong phylogenetic relationship to DRB1*0203, and DRB1*0353 had a strong phylogenetic relationship to DRB1*0303.

Alleles↗
PubMed1979

Vitellogenin in Xenopus laevis is encoded in a small family of genes.

Vitellogenin, the yolk protein precursor, is produced in X. laevis liver from a 6.3 kilobase (kb) mRNA. Sequences of this mRNA have been transcribed into cDNA and cloned in E. coli. Some properties of 21 of these cloned DNAs, ranging in size from 1 to 3.7 kb, have been reported by Wahli et al. (1978b). This paper reports restriction endonuclease mapping, cross hybridization, heteroduplex mapping in the electron microscope and heteroduplex melting experiments with these DNAs. We conclude that the cloned DNAs fall into two main groups of sequences which differ from each other in approximately 20% of their nucleotides. Each main group contains two subgroups which differ from each other by about 5% sequence divergence. By hybridizing cloned DNAs with restricted genomic DNA, we showed that sequences corresponding to all four sequence groups are present in a single animal. Furthermore, we have obtained tentative evidence for the presence of large intervening sequences in genomic vitellogenin DNA. Analysis of R loop molecules demonstrated that all four sequences are present in the vitellogenin mRNA population purified from individual animals. While some alternate explanations are not entirely excluded, we suggest that vitellogenin is encoded by a small family of related genes in Xenopus.

Animals↗
PubMed1986

Murine myb protooncogene mRNA: cDNA sequence and evidence for 5' heterogeneity.

We have sequenced two overlapping cDNA clones from a murine pro-B cell library to generate a composite sequence that includes 3413 bases of the murine c-myb mRNA. There is a single long open reading frame, beginning at the first base of this sequence, and continuing from the first methionine codon at nucleotide 265 to a TGA termination codon at nucleotide 2173. The predicted murine translation product contains 636 amino acid residues and is about 71 kDa long, which is in good agreement with the 75-kDa molecular size determined for the avian c-myb protein. The murine c-myb protein shows a striking 82% amino acid homology in the region (amino acids 71-444) where it can be compared to the published avian c-myb gene sequence. S1 nuclease protection analysis indicates extreme heterogeneity at the 5' end of steady-state murine c-myb mRNA.

Animals↗
PubMed2000

Determination of the cDNA sequence and mRNA expression of interleukin-1 receptor antagonist in horses.

OBJECTIVE: To determine the complementary DNA (cDNA) sequence of interleukin-1 receptor antagonist (IL-1ra) in horses and compare messenger RNA (mRNA) expression of IL-1ra among horses of various breeds. SAMPLE POPULATION: Blood samples from neonatal and adult horses examined for a variety of diseases. PROCEDURE: A polymerase chain reaction procedure was used to amplify a 220 base pair (bp) portion of the genomic DNA. The upstream and downstream regions of the cDNA sequence were determined by means of 5' and 3' rapid amplification of cDNA ends (RACE) procedures. Northern blot hybridization was used to examine steady-state mRNA expression of IL-1ra. RESULTS: The consensus sequence of the cDNA obtained with the 5'-RACE procedure and the sequence for the 220 bp portion of the genomic DNA represented the putative sequence for secreted IL-1ra. The predicted secreted IL-1ra amino acid sequence contained 176 residues with an in-frame stop codon; the N-terminal 25 amino acid residues resembled the signal peptide reported for human secreted IL-1ra. An approximately 1.3 kilobase pair (kb) band that represented a portion of the 3' end of the coding region and the 3' untranslated region was obtained by use of the 3' -RACE procedure. Northern blot hybridization detected a 1.6 kb transcript in blood RNA from adult Arabian, Belgian, Thoroughbred, and Standardbred horses. CONCLUSIONS: Results suggest that the DNA for equine secreted IL-1ra has a short (29 bp) 5' untranslated region, a 534 bp coding region, and a long (approximately 1,080 bp) untranslated region.

Amino Acid Sequence↗
PubMed2002

Protein similarity search under mRNA structural constraints: application to targeted selenocysteine insertion.

Selenocysteine is the 21th amino acid, which occurs in all kingdoms of life. Selenocysteine is encoded by the STOP-codon UGA. For its insertion, it requires a specific mRNA sequence downstream the UGA-codon that forms a hairpin like structure (called Sec insertion sequence (SECIS)). We consider the computational problem of generating new amino acid sequences containing selenocysteine. This requires to find an mRNA sequence that is similar to the SECIS-consensus, is able to form the secondary structure required for selenocysteine insertion, and whose translation is maximally similar to the original amino acid sequence. We show that the problem can be solved in linear time when considering the hairpin-like SECIS-structure (and, more generally, when considering a structure that does not contain pseudoknots).

Amino Acid Sequence↗
PubMed2004

Presence of a poly(A) binding protein and two proteins with cell cycle-dependent phosphorylation in Crithidia fasciculata mRNA cycling sequence binding protein II.

Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.

Amino Acid Sequence↗