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Query: UNIPROT:P06889 (Mol)
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In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3' end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.
Mol Biotechnol 2002 Nov
PMID:Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis. 1244 77

RNA editing processes kinetoplastid mitochondrial transcripts post-transcriptionally by inserting and deleting uridylates (Us) to produce functional mRNAs. The activities of the RNA ligases in the multienzyme complex (the editosome) that catalyzes editing and of the recombinant proteins were characterized and found to be similar. Ligation of two RNA fragments was enhanced when bridged by a complementary RNA or DNA, which left no gaps or overhangs. An acceptor nucleotide preference of G>U>C>A was observed in the absence of exogenous ATP but U was preferred upon addition of ATP and ligase activity was increased. The substrate specificity and catalytic characteristics indicate that RNA ligase activity contributes to the accuracy of RNA editing.
Mol Biochem Parasitol 2003 Apr 03
PMID:Kinetoplastid RNA editing ligases: complex association, characterization, and substrate requirements. 1267 25

Due to trans-splicing and polycistronic transcription, the 5' end structure of precursor RNAs of protein coding genes in Trypanosoma brucei has not yet been characterized. In eukaryotes, in general, the 5' ends of transcripts generated by RNA polymerase (pol) I and pol II are different. Pol I derived precursor RNAs contain an unmodified tri- or diphosphate group at their 5' ends. In contrast, pol II primary transcripts, the 5' triphosphate (initially also part of the pre-mRNA) is rapidly modified by the addition of methylated guanosine triphosphate, immediately after transcription initiation. We determined the 5' end structure of precursor RNAs of the rRNA gene and the RNA pol I transcribed protein coding gene by the differential display of RNA ligase mediated amplification of cDNA ends (DDRLACE) method. Comparing the ability of the 5' end of RNA transcripts to ligate with an RNA primer following different pre-treatments, the structure of the 5' end of RNA transcripts was characterized. We found that: (1). the 5' end of putative precursor RNAs from a pol I transcribed protein coding gene and the rRNA gene was uncapped; (2). approximately 20% of the putative rRNA precursor contained a 5' tri- or diphosphate group, representing the primary transcript and approximately 80% of the putative rRNA precursor were dephosphorylated and contained a 5' hydroxyl group; (3). the majority of putative neomycin resistance gene precursor RNAs, driven by the procyclin gene promoter (a pol I promoter), contained a 5' hydroxyl group. The procyclin-neo primary transcript, as being those containing a 5' tri- or diphosphate, was below a detectable level in the steady state RNA; and (4). we did not detect pol I transcribed precursor RNAs that contained a 5' monophosphate group. The observation that the putative pre-RNAs derived from the procyclin gene promoter, similar to those of rRNA do not have a 5' capped structure, is consistent with the notion that transcription of pol I transcribed protein coding genes is crucially dependent on trans-splicing for the cap addition.
Mol Biochem Parasitol 2003 Jun
PMID:The 5' end structure of transcripts derived from the rRNA gene and the RNA polymerase I transcribed protein coding genes in Trypanosoma brucei. 1279 8

The 2'-5' RNA ligase family members are bacterial and archaeal RNA ligases that ligate 5' and 3' half-tRNA molecules with 2',3'-cyclic phosphate and 5'-hydroxyl termini, respectively, to the product containing the 2'-5' phosphodiester linkage. Here, the crystal structure of the 2'-5' RNA ligase protein from an extreme thermophile, Thermus thermophilus HB8, was solved at 2.5A resolution. The structure of the 2'-5' RNA ligase superimposes well on that of the Arabidopsis thaliana cyclic phosphodiesterase (CPDase), which hydrolyzes ADP-ribose 1",2"-cyclic phosphate (a product of the tRNA splicing reaction) to the monoester ADP-ribose 1"-phosphate. Although the sequence identity between the two proteins is remarkably low (9.3%), the 2'-5' RNA ligase and CPDase structures have two HX(T/S)X motifs in their corresponding positions. The HX(T/S)X motifs play important roles in the CPDase activity, and are conserved in both the CPDases and 2'-5' RNA ligases. Therefore, the catalytic mechanism of the 2'-5' RNA ligase may be similar to that of the CPDase. On the other hand, the electrostatic potential of the cavity of the 2'-5' RNA ligase is positive, but that of the CPDase is negative. Furthermore, in the CPDase, two loops with low B-factors cover the cavity. In contrast, in the 2'-5' RNA ligase, the corresponding loops form an open conformation and are flexible. These characteristics may be due to the differences in the substrates, tRNA and ADP-ribose 1",2"-cyclic phosphate.
J Mol Biol 2003 Jun 20
PMID:Crystal structure of the 2'-5' RNA ligase from Thermus thermophilus HB8. 1279 81

In vitro evolution was previously used to identify a small deoxyribozyme, 7Q10, that ligates RNA with formation of a 2'-5' phosphodiester linkage from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. Ligation occurs in a convenient "binding arms" format analogous to that of the well-known 10-23 and 8-17 RNA-cleaving deoxyribozymes. Here, we report the optimization and generality of 7Q10 as a 2'-5' RNA ligase. By comprehensive mutagenesis of its 16-nucleotide enzyme region, the parent 7Q10 sequence is shown to be optimal for RNA ligation yield, although several mutations are capable of increasing the ligation rate approximately fivefold at the expense of yield. The 7Q10 deoxyribozyme ligates any RNA substrates that form the sequence motif UA GR (arrowhead=ligation site and R=purine), providing at least 30% yield of ligated RNA in approximately 1-2 hours at 37 degrees C and pH 9.0. Comparable yields are obtained in approximately 12-24 hours at pH 7.5, which may be more suitable for larger RNAs that are more sensitive to non-specific degradation. For RNA substrates that form the related ligation junction UA GY (Y=pyrimidine), somewhat lower yields are obtained, but significant ligation activity is still observed. These data establish that 7Q10 is a generally applicable RNA ligase. A plot of log(k(obs)) versus pH from pH 6.9 to 9.0 has a slope of just under 1, suggesting that a single deprotonation occurs during the rate-determining reaction step. The compact 7Q10 deoxyribozyme has both practical utility and the potential for increasing our structural and mechanistic understanding of how nucleic acids can mediate chemical reactions.
J Mol Biol 2003 Jul 25
PMID:Optimization and generality of a small deoxyribozyme that ligates RNA. 1286 Jan 24

Continuous in vitro evolution methods were used to study the behavior of an evolving population of RNA ligase ribozymes in response to selection pressures involving conditions of extreme pH. The starting population consisted of randomized variants of a ribozyme that had been optimized for activity at pH 8.5. The ribozymes were subjected to repeated rounds of selective amplification under progressively more acidic or more alkaline conditions. The two final evolved populations of ribozymes were able to operate at either pH 5.8 or pH 9.8, respectively. Representative individuals from the two final populations were isolated and characterized. The low-pH ribozyme exhibited a 10-fold increase in catalytic rate at pH 5.8 compared to the starting molecule. The high-pH ribozyme retained its structural integrity and activity at pH 9.8, whereas the starting molecule was denatured under this condition. These findings demonstrate that a population of functional macromolecules can adapt to stringent environmental conditions through the acquisition of relatively few mutations. The results establish continuous in vitro evolution as a useful model system for exploring the evolution of enzymatic function in extreme environments.
J Mol Evol 2003 Sep
PMID:Continuous in vitro evolution of ribozymes that operate under conditions of extreme pH. 1462 39

In the current issue of Structure, Ho and coworkers report the crystal structure and mechanism of a T4 RNA ligase. These studies provide valuable insights on the mechanism and origin of RNA and DNA ligases, and RNA capping enzymes.
Mol Cell 2004 Feb 27
PMID:RNA ligase; picking up the pieces. 1499 15

MicroRNAs (miRNAs) and other small RNAs can be identified by cloning and sequencing cDNAs prepared from the approximately 22-nt fraction of total RNA. Methods are described for the construction of cDNA libraries from small noncoding RNAs through the use of T4 RNA ligase, reverse transcriptase, and polymerase chain reaction. cDNAs are cloned in lambda or plasmid vectors, and the sequences are compared to annotated genomic sequence databases, and analyzed by RNA folding programs to distinguish miRNA sequences from other small RNAs of similar size. Northern blot hybridization is used to confirm the expression of small RNAs in vivo.
Methods Mol Biol 2004
PMID:Identification of microRNAs and other tiny noncoding RNAs by cDNA cloning. 1510 73

We present here methodology for assaying 5'-terminal editing of mitochondrial tRNAs in the amoeboid protist Acanthamoeba castellanii. This type of editing involves replacement of one or more nucleotides within the first three positions at the 5' end of a tRNA substrate. The assay procedure involves RNA ligase-mediated joining of the 5' and 3' ends of a tRNA, use of the resulting circularized tRNA as template for cDNA synthesis primed by tRNA-specific primers over a region that encompasses the ligated 5' and 3' halves of the acceptor stem, amplification of cDNA via polymerase chain reaction, and cloning and sequencing of double-stranded cDNA product. This approach has the advantage that it simultaneously reveals potential editing events on the 5' and 3' side of an acceptor stem, as well as serves to identify mature tRNAs (characterized by having a 3'-CCAOH motif) and partially processed intermediates.
Methods Mol Biol 2004
PMID:Methods for analysis of mitochondrial tRNA editing in Acanthamoeba castellanii. 1510 81

Neonatal mouse skin is useful for studying changes in gene expression during development of hair follicles, as the mitotic activity of skin cells changes shortly after birth. Using ribonucleic acid (RNA) differential display, a 261-nt message has been identified in the skin, specifically on d 3-5 but not on d 2 after birth. Confirmation of its expression by ribonuclease protection assay showed that stronger expression is seen on d 3-5 compared with d 1-2. Using RNA ligase-mediated rapid amplification of 5' complementary deoxyribonucleic acid ends, we have successfully isolated a 3046-bp gene, which has 93% sequence homology to a mouse teashirt1 gene. Amino acid analysis showed that it has 74% identity to the mouse teashirt1 protein and possesses zinc-finger motifs 1, 2, and 3. In situ hybridization data revealed that it is mainly expressed in the follicle bulb, including dermal papilla and matrix cells. As the proliferation of bulb cells is important to follicle development during this period, the finding of its strong expression on d 3-5 suggests that the identified gene is a potential candidate for follicle growth.
Mol Biotechnol 2004 Sep
PMID:A novel gene homologous to teashirt is differentially expressed in neonatal mouse skin during development of hair follicles. 1545 59


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