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At least 865 records · Page 48Linked to original sources
PubMed2002

Translation initiation AUG context varies with codon usage bias and gene length in Drosophila melanogaster.

The relationship between the codon usage bias and the sequence context surrounding the AUG translation initiation codon was examined in 1100 Drosophila melanogaster mRNA sequences. The codon usage bias measured by the "codon adaptation index" (CAI), and the effectiveness of the AUG context for translation initiation assessed by the "AUG context adaptation index" (AUGCAI), showed a significant positive relationship (correlation coefficient: r = 0.34, p <0.0001), indicating that these two factors are evolutionally under a similar natural selection constraint at the translational level. The importance of each position of the AUG context in relation to codon usage bias was examined, and the preference for the nucleotide at the -13, -12, -11, -10, -7, -6, -5, -4, -3, -2, and -1 positions showed a significant positive correlation to the codon usage bias, suggesting the action of natural selection on these very specific positions of the Drosophila genome. The relationship between AUGCAI value and gene length was also examined, and a significant negative relationship was found (r = -0.15, p <0.0001), suggesting a general tendency of higher expressivity of shorter genes, and of lower expressivity of longer genes in D. melanogaster.

Animals↗
PubMed2004

Isolation and characterization of genomic and transcribed retrotransposon sequences from sorghum.

Reverse transcriptase sequences from both major classes of retrotransposons were amplified from sorghum genomic DNA, leaf mRNA and callus protoplast mRNA. Sequence analysis of clones derived from genomic DNA demonstrated the presence of a wide variety of copia-like and gypsy-like elements. Twenty-four families of copia-like elements were found, of which at least thirteen were expressed in callus protoplasts. Two families (containing forty-eight subfamilies) of gypsy-like elements were discovered, both closely related to Huck of maize. At least twenty-seven of these subfamilies were expressed in callus protoplasts. Most of these elements were expressed at high levels in protoplasts derived from embryogenic callus, but expression of only a few was detected (at low levels) in leaves. Sequence divergence within individual families was quite high, and all relatedness profiles were consistent with vertical transmission of these elements. These data indicate that sorghum contains a large number and diversity of retrotransposons, and that some may be useful as transposon tagging systems in callus protoplasts.

Base Sequence↗
PubMed1977

Number and distribution of polyadenylated RNA sequences in yeast.

The poly(A)-containing RNA, isolated from the budding yeast Saccharomyces cerevisiae, has been characterized with regard to the number and distribution of sequences by a kinetic analysis of RNA-cDNA hybridization. In agreement with results previously obtained on metazoan eucaryotes (Bishop et al., 1974), discrete complexity classes were observed. There exist low, medium, and high complexity classes which contain approximately 20, 400, and 2400 sequences, respectively. This measurements of the number of sequences has been verified by hybridization with single copy DNA. 20% of the single copy fraction of the yeast genome is rendered double-stranded by poly(A)-containing RNA. Assuming asymmetric transcription, this is equivalent to approximately 4000 poly(A)-containing sequences, verifying the results obtained with RNA-cDNA hybridization. In addition, the first-order kinetics of the hybridization with single copy DNA verified the notion that most of the sequence complexity is present at the same intracellular concentration. The same number and distribution of sequences were found in poly(A)-containing polysomal RNA and in total RNA, suggesting that most or all of the sequence complexity is on polysomes and is adenylated. The results indicate that RNA-cDNA hybridization is an accurate method for determining sequence complexity values and that yeast, grown under vegetative conditions, has 3000-4000 different mRNA sequences.

Base Sequence↗
PubMed1984

Characterisation and translation studies of messenger RNA from the human malaria parasite Plasmodium falciparum and construction of a cDNA library.

RNA was isolated from trophozoites, schizonts and mixed populations of Plasmodium falciparum. 5% of the total was poly(A+) message, of average length 1.2 kb (10-12 kb maximum) and a poly(A) content of 10%. The mRNA fractions could be translated in vitro by reticulocyte lysates supplemented either with exogenous or P. falciparum tRNA. The patterns from two independent isolates, one cloned (T9-94) and one uncloned (K1) were virtually identical. Major translation products from 16-230 kDa have been measured. The most abundant is lactate dehydrogenase (34.8 kDa). Trophozoite mRNA codes principally for proteins of less than or equal to 93 kDa, while schizont mRNA codes for additional proteins of higher molecular mass. There are marked similarities between the in vitro translation products and proteins synthesised in vivo in synchronous cultures. A number of schizont mRNA translation products (principally those of 230, 203, 185, 170, 115, 101 and 71 kDa) are specifically precipitated without post-translational modification by sera from humans exposed to malaria. A cDNA library has been constructed in phage lambda from total poly(A+) RNA and partially characterised. About 10% of the clones derive from abundant mRNA sequences. Putative actin clones have been isolated from this library and the parasite actin mRNA sized at approx. 2.8 kb.

Actins↗
PubMed1988

Mechanisms of mRNA decay in bacteria: a perspective.

Messenger RNA decay plays an important role in prokaryotic gene expression. The disparate stabilities of bacterial messages in vivo are a consequence of their differential susceptibility to degradation by cellular endoribonucleases and 3' -exoribonucleases, which in turn results from differences in mRNA sequence and structure. RNase II and polynucleotide phosphorylase, the major bacterial exonucleases involved in mRNA turnover, rapidly degrade single-stranded RNA from the 3' end, but are impeded by 3' stem-loop structures. At present, the identify and substrate specificity of the endonucleases that control mRNA decay rates are relatively poorly defined. Ribosomes and antisense RNA also can influence the stability of transcripts with which they associate. Differences in mRNA stability can contribute to differential expression of genes within polycistronic operons and to modulation of gene expression in response to changes in bacterial growth conditions.

Bacteria↗
PubMed1999

Cytoplasmic localization of the trypanothione peroxidase system in Crithidia fasciculata.

Tryparedoxin I (TXNI) and tryparedoxin peroxidase (TXNPx), novel proteins isolated from Crithidia fasciculata, have been reported to reconstitute a trypanothione peroxidase activity in vitro (Nogoceke, E.; Gommel, D. U.; Kiess, M.; Kalisz, H. M.; Flohé, L. Biol. Chem. 378:827-836; 1997). Combined with trypanothione reductase, they may form an NADPH-fueled trypanothione-mediated defense system against hydroperoxides in the trypanosomatids. In situ confocal microscopy of antibody-stained TXNI and TXNPx and electron microscopy of the immunogold labeled proteins revealed their colocalization in the cytosol. Insignificant amounts of the enzymes were detected in the nucleus and vesicular structures, whereas the kinetoplast and the mitochondrion are virtually free of any label. Comparison of the PCR product sequences obtained with genomic and cDNA templates rules out any editing typical of kinetoplast mRNA. Sequence similarities with any of the established maxicircle genes of trypanosomatids were not detectable. It is concluded that both, TXNI as well as TXNPx are encoded by nuclear DNA and predominantly, if not exclusively localized in the cytosol. Working in concert with trypanothione reductase, they can function as an enzymatic system that reduces hydroperoxides at the expense of NADPH without any impairment of the flux of reduction equivalents by cellular compartmentation.

Animals↗
PubMed1986

cDNA sequences for human von Willebrand factor reveal five types of repeated domains and five possible protein sequence polymorphisms.

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened with two previously described cDNA inserts for human von Willebrand factor. Among 16 positive isolates, two that hybridized with a probe corresponding to the amino terminus of von Willebrand factor were sequenced. Together, these four cDNA inserts span 6.5 kilobases of the von Willebrand factor mRNA sequence, completely specifying the 2050 amino acids of the subunit of mature, secreted von Willebrand factor and 24 residues of a precursor peptide. Approximately 77% of the sequence is contained in five types of repeated domains. Domain A consists of 193-220 amino acids and is present in three tandem copies between residues 497 and 1111. Domain B contains 25-35 amino acids and is present in three copies between residues 1533 and 1636. Domain C consists of 116-119 amino acids and is duplicated between residues 1637 and 1899. In contrast to the essentially contiguous repetition of domains A-C, the two copies of domains D and E are each separated by 804 and 1383 amino acids, respectively. Domain D1 contains 289 amino acids between residues 79 and 367, while domain D2 consists of 270 amino acids between residues 1171 and 1440. Domain E1 consists of 46 amino acids between residues 25 and 70, and domain E2 consists of 46 amino acids between residues 1453 and 1498. The triplicated A domains are notably poor in Cys content, while the remaining domains are Cys-rich. The A domains appear to be homologous to a 225-residue segment of complement factor B.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1982

Human somatostatin I: sequence of the cDNA.

RNA has been isolated from a human pancreatic somatostatinoma and used to prepare a cDNA library. After prescreening, clones containing somatostatin I sequences were identified by hybridization with an anglerfish somatostatin I-cloned cDNA probe. From the nucleotide sequence of two of these clones, we have deduced an essentially full-length mRNA sequence, including the preprosomatostatin coding region, 105 nucleotides from the 5' untranslated region and the complete 150-nucleotide 3' untranslated region. The coding region predicts a 116-amino acid precursor protein (Mr, 12.727) that contains somatostatin-14 and -28 at its COOH terminus. The predicted amino acid sequence of human somatostatin-28 is identical to that of somatostatin-28 isolated from the porcine and ovine species. A comparison of the amino acid sequences of human and anglerfish preprosomatostatin I indicated that the COOH-terminal region encoding somatostatin-14 and the adjacent 6 amino acids are highly conserved, whereas the remainder of the molecule, including the signal peptide region, is more divergent. However, many of the amino acid differences found in the pro region of the human and anglerfish proteins are conservative changes. This suggests that the propeptides have a similar secondary structure, which in turn may imply a biological function for this region of the molecule.

Amino Acid Sequence↗
PubMed1984

Locus unlinked to alpha-fetoprotein under the control of the murine raf and Rif genes.

The levels of alpha-fetoprotein mRNA in mice are determined by at least two trans-acting, unlinked genes, raf and Rif. raf determines the basal levels of alpha-fetoprotein mRNA in adult mice, while Rif determines its degree of inducibility during liver regeneration. To determine whether these regulatory loci affect other structural genes, we screened a murine fetal liver cDNA library for clones containing mRNA sequences that decrease after birth. One such clone, termed pH19, was identified, and its mRNA was shown to be under the control of both raf and Rif. The single-copy gene for H19 mRNA was localized to chromosome 7, and genetic crosses established that it was unlinked to either raf or Rif. It encodes a 2.5-kilobase mRNA that was identified in those tissues that produce alpha-fetoprotein: visceral endoderm, liver, and fetal gut. The repression of H19 mRNA in neonatal liver occurs several days after the decrease in alpha-fetoprotein mRNA, whereas inductions of both mRNAs during the differentiation of F9 teratocarcinoma cells into visceral endoderm were identical. The tissue-specific expression of H19 mRNA is different from that of alpha-fetoprotein in that H19 mRNA was detected also in both cardiac and skeletal muscle where no alpha-fetoprotein mRNA is produced. Despite the fact that the levels of H19 mRNA decline to 1/10th to 1/20th in cardiac muscle after birth, the adult basal levels are not under the influence of raf. This observation argues that the raf gene is a tissue-specific regulator of mRNA levels.

Animals↗
PubMed1990

Differences in the modulation of P450IA1 and epoxide hydratase expression by benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse embryo versus mouse hepatoma-derived cell lines.

Polycyclic aromatic hydrocarbon (PAH)-induced C3H/10-T1/2/CL8 mouse embryo fibroblasts (10T1/2) and mouse hepatoma-derived Hepa 1c1c7 cells (Hepa-1), exhibit comparable total cytochrome P450 levels and total PAH-metabolizing activities but very different distributions of PAH metabolites. Based on anti-P450IA1-IgG inhibition data, P450IA1 contributes essentially all PAH metabolism in Hepa-1 microsomes but is not involved in PAH metabolism by 10T1/2 cells. In addition, the microsomal epoxide hydratase (EHm) in Hepa-1 cells is far less effective in dihydrodiol (diol) formation compared to that in 10T1/2 microsomes [Pottenger, L.H. and Jefcoate, C.R. Carcinogenesis, 11, 321-327 (1990)]. In the present study, the levels of expression of P450IA1 and EHm proteins and the corresponding mRNAs, both prior to and following exposure to benz[a]anthracene (BA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been correlated with microsomal PAH metabolism by each cell type. In 10T1/2 cells, P450IA1 protein (56 kd) and mRNA (2.6 kb) were detectable at extremely low levels in only two of five cell preparations and then only after maximum induction by TCDD and BA. Thus although 10T1/2 cells contain functional Ah receptors, their capacity to induce P450IA1 is highly suppressed, representing at most 2% of the total P450. TCDD (10 nM) was 4-fold more effective than BA (10 microM) in inducing P450IA1 mRNA, while the levels of immunodetectable protein were comparable. An even greater discrepancy between P450IA1 mRNA and protein levels was seen in BA-induced Hepa-1 cells, where a 4-fold increase in mRNA was paralleled by a 20-fold increase in protein. This difference is probably due to the greater effect of BA depletion on mRNA compared to protein levels. In 10T1/2 cells, BA and TCDD were equally effective at increasing expression of an unidentified 1.9 kb mRNA sequence that blotted very weakly with the P450IA1 cDNA probe. The expression of this mRNA was independent from that of P450IA1. A similar band was visible in Hepa-1 cells less than 1% of the P450IA1 mRNA. EHm mRNA was almost 3-fold higher in 10T1/2 compared to Hepa-1 cells and was unaffected by cell treatments. In Hepa-1 cells, BA and TCDD elevated EHm protein and hydrating activity to levels comparable to those expressed in 10T1/2 cells. It is, therefore, suggested that the relative ineffectiveness of Hepa-1, compared to 10T1/2 EHm, to hydrate low levels of PAH-epoxides is due to differences between the two proteins or their disposition in the microsomal membrane.

Animals↗
PubMed2004

Genomic structure of the human gene for protein kinase A regulatory subunit R1-beta (PRKAR1B) on 7p22: no evidence for mutations in familial hyperaldosteronism type II in a large affected kindred.

OBJECTIVE: Familial hyperaldosteronism type II (FH-II) is characterized by inheritance of primary aldosteronism (PAL) but, unlike FH-I, is not glucocorticoid remediable and not associated with the hybrid CYP11B1/CYP11B2 gene mutation. Analysis of two pedigrees previously demonstrated linkage of FH-II with a locus at chromosome 7p22. We sought to determine whether mutations in the exons or intron/exon boundaries in PRKAR1B (encoding protein kinase A regulatory subunit R1-beta), which resides within the linked locus, are associated with FH-II. METHODS: Primers enabling sequencing of all exons and intron/exon boundaries were designed by BLAT search using known mRNA sequence, and comparison with an orthologous mouse gene. Sequences from four affected and two unaffected subjects from an Australian family with FH-II demonstrating linkage at 7p22 were compared with published sequences. RESULTS: A probable two-nucleotide GenBank sequence error, resulting in an amino acid change, was detected. Two of seven single nucleotide polymorphisms (SNPs) identified were in exons and five in introns. Neither exon-localized SNP resulted in an amino acid change. All intron-localized SNPs were at least 16 nucleotides from the closest intron/exon boundary and therefore unlikely to interfere with gene splicing. Importantly, none of the identified SNPs was exclusively associated with affectation status. CONCLUSIONS: Mutations in the exons or intron/exon boundaries of PRKAR1B do not appear to be responsible for FH-II in this family, but a mutation in the promoter or remaining intronic or 5' or 3' untranslated regions could be. Alternatively, a mutation within another gene residing at the 7p22 locus may be responsible.

Adult↗
PubMed1985

Suppressible and nonsuppressible +1 G-C base pair insertions induced by ICR-170 at the his4 locus in Saccharomyces cerevisiae.

Fifteen independent ICR-170-induced his4 mutations in Saccharomyces cerevisiae were examined by DNA sequence analysis. All of the mutations contained a +1 G-C base pair addition in the HIS4 coding region. Eleven different sites of insertion were identified. Combined with previous DNA sequence data, 21 ICR-170-induced his4 mutations distributed at 16 different sites were analyzed. The insertions were always located in a consecutive run of two or more G-C base pairs, with all base pairs in each run having identical orientation. Long consecutive G-C runs were preferred target sites over short runs. Although some consecutive G-C runs appeared to be preferred target sites over others of identical length, such preference was not due to any particular type of nucleotide pair immediately adjacent to a given target site. In addition, DNA sequence analyses of the his4 mutations provided a basis for examining the mechanism of mRNA sequence recognition by extragenic suppressors of ICR-170-induced mutations. The implications of these results for mechanisms of frameshift suppression are discussed.

Aminoacridines↗
PubMed2002

RNA helicase p54 (DDX6) is a shuttling protein involved in nuclear assembly of stored mRNP particles.

Previously, we showed that an integral component of stored mRNP particles in Xenopus oocytes, Xp54, is a DEAD-box RNA helicase with ATP-dependent RNA-unwinding activity. Xp54 belongs to small family of helicases (DDX6) that associate with mRNA molecules encoding proteins required for progress through meiosis. Here we describe the nucleocytoplasmic translocation of recombinant Xp54 in microinjected oocytes and in transfected culture cells. We demonstrate that Xp54 is present in oocyte nuclei, its occurrence in both soluble and particle-bound forms and its ability to shuttle between nucleus and cytoplasm. Translocation of Xp54 from the nucleus to the cytoplasm appears to be dependent on the presence of a leucine-rich nuclear export signal (NES) and is blocked by leptomycin B, a specific inhibitor of the CRM1 receptor pathway. However, the C-terminal region of Xp54 can act to retain the protein in the cytoplasm of full-grown oocytes and culture cells. Cytoplasmic retention of Xp54 is overcome by activation of transcription. That Xp54 interacts directly with nascent transcripts is shown by immunostaining of the RNP matrix of lampbrush chromosome loops and co-immunoprecipitation with de novo-synthesized RNA. However, we are unable to show that nuclear export of this RNA is affected by either treatment with leptomycin B or mutation of the NES. We propose that newly synthesized Xp54 is regulated in its nucleocytoplasmic distribution: in transcriptionally quiescent oocytes it is largely restricted to the cytoplasm and, if imported into the nucleus, it is rapidly exported again by the CRM1 pathway. In transcriptionally active oocytes, it binds to a major set of nascent transcripts, accompanies mRNA sequences to the cytoplasm by an alternative export pathway and remains associated with masked mRNA until the time of translation activation at meiotic maturation and early embryonic cell division.

Active Transport, Cell Nucleus↗
PubMedDate not supplied

Amino acid sequence determination of three monoclonal antibodies to angiotensin II.

To relate the immunochemical specificity of three murine monoclonal antibodies directed against the octapeptide hormone angiotensin II (AII) to their primary structure, we have sequenced mRNA for the variable regions of their heavy and light chains. Comparison of the sequences indicates that each of the antibodies utilize very closely related V genes in both heavy and light chains. Two of these antibodies, ICH2 and ICA10, display an epitope preference for the carboxy terminus of AII. A third monoclonal antibody, KAA8, displays a lesser specificity for the carboxy terminus of AII than either ICH2 or ICA10. This antibody differs in sequence from the others mainly in the light chain complementarity determining regions (CDR) 1 and 3 and in CDR 3 (D region) on the heavy chain. We interpret these sequence differences as the basis for the observed specificity preferences.

Amino Acid Sequence↗
PubMed1987

Chicken proto-oncogene c-ros cDNA clones: identification of a c-ros RNA transcript and deduction of the amino acid sequence of the carboxyl terminus of the c-ros product.

Three overlapping cDNA clones that hybridized to a v-ros probe were isolated from a cDNA library constructed from chicken kidney mRNA. Sequence analysis of these clones showed that they were all derived from c-ros mRNA. Using hybridization probes synthesized from the cDNA clones, a c-ros mRNA transcript of approximately 8.3 kb was detected in chicken kidney RNA, but not in chick embryo fibroblast RNA. The amino acid sequence predicted from the cDNA sequence indicates that the carboxyl terminus of the chicken c-ros protein contains 58 amino acids which are not present in v-ros. The predicted amino acid sequence of the chicken c-ros protein differs by 25% from that of its closest known human counterparts within the tyrosine protein kinase catalytic domain, and by 66% downstream of this domain. Despite these differences, both the chicken and human amino acid sequences share a potential site of tyrosine phosphorylation near their carboxyl termini that is absent from v-ros.

Amino Acid Sequence↗
PubMed1991

In the Escherichia coli lacZ gene the spacing between the translating ribosomes is insensitive to the efficiency of translation initiation.

We have constructed a series of 44 Escherichia coli strains in which the chromosomal region corresponding to the Ribosome Binding Site (RBS) of the lacZ gene, has been replaced by small DNA fragments harboring either RBSs from other genes, or artificial RBSs. The beta-galactosidase expression from these strains ranges from 1 to 130 per cent of that of the parental strain. Using this collection, we demonstrate here that strain-to-strain variations in expression are paralleled by nearly equivalent variations in lacZ mRNA content. We propose that, in this system, polarity and mRNA stability are tightly coupled to translation initiation, so that changes in RBS efficiency are detected mainly as changes in mRNA concentration rather than in the spacing between translating ribosomes. In addition, we show that the mRNA sequence immediately downstream from the initiator codon influences per se the lifetime of the lacZ mRNA. We discuss the mechanism of the interdependence between translation, transcription and degradation in this gene, and speculate about the general role of this interdependence in determining the expression of bacterial genes.

Acetylglucosaminidase↗
PubMedDate not supplied

Identification of scrapie prion protein-specific mRNA in scrapie-infected and uninfected brain.

To date no nucleic acid has been found in the purified infectious agent which causes the spongiform encephalopathy known as scrapie. In an attempt to identify a unique scrapie virus-associated messenger RNA in tissues of infected animals, we have synthesized an oligonucleotide probe complementary to the mRNA sequence corresponding to the amino-acid sequence of the prion protein, PrP27-30 (ref. 1). We report here that, with this probe, a complementary DNA clone representing PrP27-30 was obtained from scrapie-infected mouse brain; the DNA sequence of this clone could be translated into a protein that matches exactly the published sequence of PrP27-30. The cDNA clone hybridized to a single 2.4-2.5-kilobase (kb) mRNA from both normal and scrapie-infected brain. Thus, the PrP27-30 mRNA is not uniquely associated with scrapie infectivity, suggesting that PrP27-30 may be a normal component of mouse and hamster brain.

Amino Acid Sequence↗
PubMed1982

Somatic cell hybridization of growth hormone-producing rat pituitary cells and mouse fibroblasts results in extinction of growth hormone expression via a defect in growth hormone RNA production.

In the GH3 line of pituitary cells, growth hormone (GH) production is stimulated by thyroid and glucocorticoid hormones, yet non-pituitary cells, for example mouse fibroblast cells, which also contain receptors for these hormones do not produce GH. We have chosen to study both types of control over GH production in GH3 rat pituitary x GH nonexpressing LB82 mouse fibroblast hybrid cells. Most hybrid cells fail to produce GH in either the absence or presence of the inducers triiodothyronine and dexamethasone although these hormone receptors are present. Rat and mouse GH genes were detected in the hybrid cells' DNA by Southern hybridization following restriction enzyme digestion and gel electrophoresis. The probe was a cloned, full length rat GH cDNA which cross-hybridizes with the mouse GH gene. Analyses with multiple restriction enzymes that cut inside and outside the rat and mouse GH coding sequences were performed. We conclude that 1) both rat and mouse GH genes are present in the hybrid cells in copy number equivalent to those in the parental cell lines, and 2) the rat GH structural gene and flanking sequences are identical in the GH3 and hybrid cell DNA at the level of sensitivity afforded by restriction enzyme analyses. GH mRNA was not detected in total cell or poly(A+) RNA isolated from control or hormone-treated hybrid cells assayed by translation in 2 in vitro systems or by Northern hybridization analyzes. The latter experiments showed that no hybridizable GH mRNA sequences were present in the hybrids as either incomplete transcripts, mature mRNA, or unprocessed high molecular weight precursors. We conclude that extinction of GH in these hybrid cells results from a transcriptional block and/or rapid transcript degradation.

Animals↗