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PubMed1997

Solution structure of an RNA x DNA hybrid duplex containing a 3'-thioformacetal linker and an RNA A-tract.

Neutral and achiral backbone linkers are promising replacements for the phosphodiester linkages of antisense oligonucleotides that target mRNA sequences. Results are presented here for the solution structure elucidation by NMR of an RNA x DNA hybrid duplex, r(GCGCAAAACGCG) x d(CGCGTT-SCH2O-TTGCGC) (designated RIII), containing a 3'-thioformacetal (3'-TFMA) backbone substitution in the DNA strand. The 3'-thioformacetal linker can be accommodated in the hybrid duplex in a conformation that is drastically different from its form in a DNA x DNA duplex but close to that of the canonical A-form helix, reflecting the sequence requirement for hybridization. While the global features of RIII are similar to what are described in the literature, the 3'-TFMA modification drives sugar puckers of the adjacent residues to more C3'-endo-like conformations and causes distortions in related twist angles and helical rises. The helical conformation analyses of each of the two strands and the hybrid duplex enable a clear account of the conformational variability of both the DNA and RNA strands. The A-tract in the RNA strand features an overall straight helix and a more prominent bend at the 3'-end CG step. The structure of RIII provides a structural basis for the improved thermal stability of RIII compared to the corresponding DNA x DNA duplex and insights into the factors that are important concerns for the design of new, effective antisense oligonucleotides.

Acetals↗
PubMed1997

Bromocontryphan: post-translational bromination of tryptophan.

We demonstrate that post-translational bromination of a tryptophan residue occurs in the biologically active octapeptide bromocontryphan, purified and characterized from Conus radiatus venom. Clones encoding bromocontryphan were identified from a cDNA library made from C. radiatus venom ducts. The mRNA sequence obtained predicts a prepropeptide which has the mature peptide sequence at the C-terminal end, with the L-6-bromotryptophan residue encoded by UGG, the Trp codon. These data provide the first direct evidence for post-translational bromination of a polypeptide which is translated through the normal cellular machinery. In addition to bromination, the peptide, which induces a "stiff tail" syndrome in mice, has several other modifications as shown by the sequence [Formula: See Text] in which Hyp = hydroxyproline. Asterisks indicate post-translational modifications (left to right): proteolytic cleavage at the N-terminus; hydroxylation of Pro3; epimerization of Trp4; bromination of Trp7, and C-terminal amidation. Bromocontryphan appears to have the highest density of post-translational modifications known among gene-encoded polypeptides. The overall result is a molecule which closely resembles marine natural products produced through specialized biosynthetic pathways comprising many enzyme-catalyzed steps.

Amino Acid Sequence↗
PubMed2006

Cloning and real-time PCR testing of 14 potential biomarkers in Eisenia fetida following cadmium exposure.

Important biological activities could be affected in metal exposed species, and amongthe main physiological functions, immunity may provide one (or more) effector(s) which expression can be directly affected by a metal exposure in various macroinvertebrates. As many proteinic effectors showed a high degree of homology between species, we have developed a PCR approach to characterize partial mRNA sequences of selected effectors in the laboratory model, Eisenia fetida. After cloning, levels of expression of each gene were analyzed following exposures (80 and 800 mg/kg) to cadmium spiked soils using real-time PCR. An implemented approach was allowed to test quickly potential biomarkers in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, trancriptionally controlled tumor protein, protein kinase C, and ubiquitin. Most of the selected effectors did not show variations of expression level after exposure. Others expressed weak changes of expression as heat shock proteins. At lastfor catalase and metallothionein, early suitable variations of expression were observed.

Animals↗
PubMed2001

Mapping of 13 horse genes by fluorescence in-situ hybridization (FISH) and somatic cell hybrid analysis.

We report fluorescence in-situ hybridization (FISH) and somatic cell hybrid mapping data for 13 different horse genes (ANP, CD2, CLU, CRISP3, CYP17, FGG, IL1RN, IL10, MMP13, PRM1, PTGS2, TNFA and TP53). Primers for PCR amplification of intronic or untranslated regions were designed from horse-specific DNA or mRNA sequences in GenBank. Two different horse bacterial artificial chromosome (BAC) libraries were screened with PCR for clones containing these 13 Type I loci, nine of which were found in the libraries. BAC clones were used as probes in dual colour FISH to confirm their precise chromosomal origin. The remaining four genes were mapped in a somatic cell hybrid panel. All chromosomal assignments except one were in agreement with human-horse ZOO-FISH data and revealed new and more detailed information on the equine comparative map. CLU was mapped by synteny to ECA2 while human-horse ZOO-FISH data predicted that CLU would be located on ECA9. The assignment of IL1RN permitted analysis of gene order conservation between HSA2 and ECA15, which identified that an event of inversion had occurred during the evolution of these two homologous chromosomes.

Animals↗
PubMed1980

The influence of codon context on genetic code translation.

A class of mutations that increase the deficiency of a suppressor tRNA in translating a particular amber codon has been characterized. The increased efficiency is due to a mutation resulting in a change in the mRNA that affects the nucleotide adjacent to the 3' side of the UAG triplet. Thus the interaction of tRNA with mRNA is influenced by mRNA sequences outside the triplet codon.

Base Sequence↗
PubMed1980

Post-transcriptional regulation of gene expression in guinea pig tissues.

The formation of individual functional mRNA sequences in higher organisms requires many steps in addition to transcription. These include RNA splicing, polyadenylation, base modification, transport from nucleus to cytoplasm and assembly into polyribosomes. Various control mechanisms must also operate. These will function on a quantitative basis to account for the differing frequency of the various classes of cytoplasmic mRNAs, and also on a qualitative basis, because in higher organisms not all the nuclear poly(A)-containing RNA molecules are found in a cytoplasmic poly(A)-containing RNA population from the same tissue. During our studies on the mechanisms controlling the accumulation of the poly(A)-containing RNA sequences which occur with high and moderately high frequency in the cytoplasm of the lactating guinea pig mammary gland, it became apparent that > 75% of the 20,000 or so poly(A)-containing nuclear RNA sequences were not found in the cytoplasmic poly(A)-containing RNA fraction. Here we demonstrate that many of the poly(A)-containing RNA sequences retained in the nucleus of the lactating guinea pig mammary gland are also present in the nucleus and cytoplasm of the liver of the male guinea pig. These observations provide new evidence for a predominant role of post-transcriptional mechanisms in the regulation of structural gene expression in guinea pig tissue.

Animals↗
PubMedDate not supplied

Somatic mutation and the maturation of immune response to 2-phenyl oxazolone.

Studies on the development of the immune response suggest that the repertoire of expressed antibody specificities is strongly influenced by antigen (reviewed in ref. 1). One way in which this influence is manifested is by a progressive increase in the affinity of antibody for antigen with time after immunization. This phenomenon, termed the 'maturation' of the immune response, must be due to a change in the structure of the antibody being synthesized. However, the precise nature of the changes involved and the genetic mechanisms used to produce them have not been clearly defined. We have now investigated the maturation of the immune response to the hapten 2-phenyloxazolone by mRNA sequencing of specific hybridomas. We conclude that somatic mutation of germ-line encoded genes plays a major role in the generation of antibodies with increased affinity for oxazolone with time after immunization.

Animals↗
PubMedDate not supplied

Molecular cloning of a cDNA encoding human antihaemophilic factor.

A complete copy of the mRNA sequences encoding human coagulation factor VIII:C has been cloned and expressed. The DNA sequence predicts a single chain precursor of 2,351 amino acids with a relative molecular mass (Mr) 267,039. The protein has an obvious domain structure, contains sequence repeats and is structurally related to factor V and ceruloplasmin.

Animals↗
PubMed1983

Messenger RNA regulation during compensatory renal growth.

The normal pattern of mRNA metabolism almost certainly becomes altered after uninephrectomy, especially during the first day when ribosomes accumulate in the proximal tubular cells of the remaining kidney. For example, within the first hour after nephrectomy, the fraction of newly synthesized poly(A)-deficient mRNA increases relative to poly(A)-containing mRNA. Investigation of other growth-specific regulatory changes in renal mRNA has been complicated by its heterogeneity with respect to translational activity, polyadenylate content, membrane association, and cytoplasmic distribution. In general, analysis of kidney mRNA metabolism during growth has not been sufficiently thorough in that few timepoints have been examined after nephrectomy and the techniques used have been suited primarily to the study of only abundant mRNA sequences. Application of recombinant DNA methods should eliminate these difficulties and permit quantitative measurement of growth-specific genetic events during compensatory growth of the kidney.

Animals↗
PubMed1985

Activation of human and rabbit prokallikrein by serine and metalloproteases.

Human and rabbit kidney and urine contain an inactive form of kallikrein. Studies on the mRNA sequence suggested that the active form of the enzyme and the propeptide are linked by a peptide bond between a basic and hydrophobic amino acid. We studied the activation of prokallikrein by serine proteases and a neutral metalloproteinase, thermolysin, because serine proteases cleave the peptide chain after a basic amino acid and thermolysin before a hydrophobic amino acid. The activity of kallikrein was measured by RIA and with a fluorogenic peptide substrate. Trypsin was used as a standard reference activator. We found that human plasmin and plasminogen, activated by urokinase, activate prokallikrein. Pronase coupled to Sepharose also enhanced the activity of the renal kallikrein zymogen. On a molar basis, thermolysin was a more effective activator of prokallikrein than trypsin. The activation by thermolysin was blocked by the inhibitor phosphoramidon, but not by DFP or SBTI. These experiments indicate that, in addition to serine proteases, neutral metalloproteases of tissues may activate prokallikrein.

Animals↗
PubMed2001

Expression of insulin-like growth factor-I (IGF-I) in aneurysmal bone cyst.

The effects of insulin-like growth factor-I on bone tissue and its role in bone development have been extensively investigated, but there is little information on its role in the pathogenesis of aneurysmal bone cyst. Therefore, using the techniques of immunohistochemistry and in situ hybridization, the authors studied the expression of insulin-like growth factor-I in 19 specimens of aneurysmal bone cyst. Insulin-like growth factor-I or specific mRNA sequences encoding for insulin-like growth factor-I were detectable in all specimens tested and were mainly localized in multinucleate giant cells. In contrast, only insignificant levels of insulin-like growth factor-I expression were detectable in normal human bone tissue. Taken together with the previously reported role of insulin-like growth factor-I in the pathogenesis of giant cell tumor, the findings of this study suggest that insulin-like growth factor-I may play a role in the pathogenesis of aneurysmal bone cyst.

Adolescent↗
PubMed1997

Selecting effective antisense reagents on combinatorial oligonucleotide arrays.

An array of 1,938 oligodeoxynucleotides (ONs) ranging in length from monomers to 17-mers was fabricated on the surface of a glass plate and used to measure the potential of oligonucleotide for heteroduplex formation with rabbit beta-globin mRNA. The oligonucleotides were complementary to the first 122 bases of mRNA comprising the 5' UTR and bases 1 to 69 of the first exon. Surprisingly few oligonucleotides gave significant heteroduplex yield. Antisense activity, measured in a RNase H assay and by in vitro translation, correlated well with yield of heteroduplex on the array. These results help to explain the variable success that is commonly experienced in the choice of antisense oligonucleotides. For the optimal ON, the concentration required to inhibit translation by 50% was found to be five times less than for any other ON. We find no obvious features in the mRNA sequence or the predicted secondary structure that can explain the variation in heteroduplex yield. However, the arrays provide a simple empirical method of selecting effective antisense oligonucleotides for any RNA target of known sequence.

Animals↗
PubMed1998

Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.

Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control ribozymes had less effect on the retinal degeneration. Intracellular production of ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.

Animals↗
PubMed2002

Early organization of pre-mRNA during spliceosome assembly.

Intron excision from precursor mRNAs (pre-mRNAs) in eukaryotes requires juxtaposition of reactive functionalities within the substrate at the heart of the spliceosome where the two chemical steps of splicing occur. Although a series of interactions between pre-mRNAs, pre-spliceosomal and spliceosomal factors is well established, the molecular mechanisms of splicing machinery assembly, as well as the temporal basis for organization of the substrate for splicing, remain poorly understood. Here we have used a directed hydroxyl radical probe tethered to pre-mRNA substrates to map the structure of the pre-mRNA substrate during the spliceosome assembly process. These studies indicate an early organization and proximation of conserved pre-mRNA sequences during spliceosome assembly/recruitment and suggest a mechanism for the formation of the final active site of the mature spliceosome.

Adenosine Triphosphate↗
PubMed2004

Control of human potassium channel inactivation by editing of a small mRNA hairpin.

Genomic recoding by A-->I RNA editing plays an important role in diversifying the proteins involved in electrical excitability. Here, we describe editing of an intronless potassium channel gene. A small region of human K(V)1.1 mRNA sequence directs efficient modification of one adenosine by human adenosine deaminase acting on RNA 2 (hADAR2). Mutational analysis shows that this region adopts a hairpin structure. Electrophysiological characterization reveals that the editing event (I/V) profoundly affects channel inactivation conferred by accessory beta subunits. Drosophila melanogaster Shaker channels, mimicking this editing event through mutation, exhibit a similar effect. In addition, we demonstrate that mRNAs for the paralogous D. melanogaster Shab potassium channel are edited at the same position by fly ADAR-a clear example of convergent evolution driven by adenosine deamination. These results suggest an ancient and key regulatory role for this residue in K(V) channels.

Amino Acid Sequence↗
PubMed2004

S100A6 (Calcyclin) is a prostate basal cell marker absent in prostate cancer and its precursors.

S100A6 (Calcyclin) is a calcium-binding protein that has been implicated in a variety of biological functions as well as tumorigenesis. The aim of our study was to investigate the involvement of S100A6 during prostate cancer development and progression. Using immunohistochemistry, the expression of S100A6 was examined in benign (n=66), premalignant (n=10), malignant (n=66) and metastatic prostate (n=5) tissues arranged in a tissue-microarray or whole sections as well as in prostate cancer cell lines. The S100A6 immunostaining pattern in tissues was compared with that of cytokeratin 5 (a basal cell marker) and 18 (a benign luminal cell marker). In all cases of benign epithelium, intense S100A6 expression was seen in the basal cell layer with absent staining in luminal cells. In all cases of prostatic adenocarcinoma (matched), metastatic lesions and 3/10 high-grade prostatic intraepithelial neoplasia lesions, an absence of S100A6 was seen. Western blotting and RT-PCR analysis of cell lines showed S100A6 expression to be absent in LNCaP, LNCaP-LN3 and LNCaP-Pro5 but present in Du145, PC3, PC-3M and PC-3M-LN4. LNCaP cells treated with 5-Azacytidine, caused re-expression of S100A6 mRNA. Sequencing of bisulphite modified DNA showed CpG methylation within the S100A6 promoter region and exon 1 of LNCaP, LNCaP-LN3 and LNCaP-Pro5 cell lines but not in Du145 cells. Our data suggest that loss of S100A6 protein expression is common in prostate cancer development and may occur at an early stage. The mechanism of loss of expression may involve hypermethylation of CpG sites. The finding of intense S100A6 expression in the basal cells of benign glands but loss of expression in cancer could be useful as a novel diagnostic marker for prostate cancer.

Adenocarcinoma↗
PubMed2004

Noncompetitive antagonism of BIBN4096BS on CGRP-induced responses in human subcutaneous arteries.

We investigated the antagonistic effect of 1-piperidinecarboxamide, N-[2-[[5amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS) on the calcitonin gene-related peptide (CGRP)-induced responses by using isometric myograph and FURA-2 technique in human subcutaneous arteries removed in association with abdominal surgery. BIBN4096BS, at the concentration of 1 pm, had no significant effect on the CGRP-induced relaxation in these vessels. At the concentration of 10 pM, BIBN4096BS had a competitive antagonistic-like behaviour characterized by parallel rightward shift in the log CGRP concentration-tension curve with no depression of the E(max). At the higher concentrations (0.1 and 1 nM), BIBN4096BS had a concentration-dependent noncompetitive antagonistic effect on the CGRP-induced responses. The efficacy and potency of CGRP was significantly greater in the smaller (lumen diameter approximately 200 microM) human subcutaneous arteries compared to the larger ones. The apparent agonist equilibrium dissociation constant, K(A), for CGRP(1) receptors in the human subcutaneous arteries was approximately 1 nM. Analysis of the relationship between receptor occupancy and response to CGRP indicates that the receptor reserve is relatively small. Using reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of mRNA sequences encoding the calcitonin receptor-like receptor, receptor activity modifying protein (RAMP1, RAMP2, RAMP3) and receptor component protein were demonstrated in human subcutaneous arteries, indicating the presence of CGRP(1)-like receptor and the necessary component for the receptor activation. In conclusion, the inhibitory action of BIBN4096BS at the low concentration (10 pM) on the CGRP-tension curve (but not intracellular calcium concentration ([Ca(2+)](i)) resembles what is seen with a reversible competitive antagonist. However, at the higher concentrations (0.1 and 1 nM), BIBN4096BS acts as a selective noncompetitive inhibitor at CGRP(1) receptors in human subcutaneous arteries.

Abdominal Wall↗
PubMed2004

Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA.

The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 on the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effects. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron, Thus UK-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.

Alternative Splicing↗