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Biomedical subjects

R K Gaur

Publications and source records attributed to R K Gaur.

17 recordsLinked to original sources
PubMed2000

Role of adenine functional groups in the recognition of the 3'-splice-site AG during the second step of pre-mRNA splicing.

The AG dinucleotide at the 3' splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG --> GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG --> CG) or uracil (AG --> UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG --> HG). When adenine was replaced by a purine base (AG --> PG) or by 7-deazaadenine (AG --> c(7)AG), little effect on the second step was observed, suggesting that the 6-NH(2) and N(7) groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG --> 2-APG) had no effect on the second step. Taken together, our results suggest that the N(1) group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.

Adenine↗
PubMed1999

Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65).

U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF(65) and U2AF(35). U2AF(65) binds to the polypyrimidine (Py) tract upstream from the 3' splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF(35) facilitates U2AF(65) binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF(35) remain controversial. Here we show that recombinant U2AF(65) is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF(65), U2AF(35), and the interaction between them are required for splicing of an immunoglobulin micro; pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF(35) occurs without changes in U2AF(65) cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF(35) and a novel function for this factor in pre-mRNA splicing.

Animals↗
PubMed1997

Functional group substitutions of the branchpoint adenosine in a nuclear pre-mRNA and a group II intron.

Splicing of nuclear mRNA precursors (pre-mRNAs) takes place in the spliceosome, a large and complex ribonucleoprotein. Nuclear pre-mRNA splicing and group II intron self-splicing occur by a chemically identical pathway involving recognition of a specific branchpoint adenosine and nucleophilic activation of its 2'-hydroxyl group. The chemical similarity between these two splicing reactions, as well as other considerations, have suggested that the catalytic core of the spliceosome and group II introns may be related. Here we test this hypothesis by analyzing splicing and RNA branch formation of a pre-mRNA and a group II intron in which the branchpoint adenosine was substituted with purine base analogues. We find that replacement of the branchpoint adenosine with either of two modified adenosine analogues or guanosine leads to remarkably similar patterns of splicing and RNA branch formation in the two systems.

Adenosine↗
PubMed1996

Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA [corrected].

The mammalian splicing factor U2AF65 binds to the polypyrimidine tract adjacent to the 3' splice site and promotes assembly of U2 small nuclear ribonucleoprotein on the upstream branch point, an interaction that involves base pairing with U2 small nuclear RNA (snRNA). U2AF65 contains an RNA binding domain, required for interaction with the polypyrimidine tract, and an arginine-serine-rich (RS) region, required for U2 snRNP recruitment and splicing. Here it is reported that binding of U2AF65 to the polypyrimidine tract directed the RS domain to contact the branch point and promoted U2 snRNA-branch point base pairing even in the absence of other splicing factors. Analysis of RS domain mutants indicated that the ability of U2AF65 to contact the branch point, to promote the U2 snRNA-branch point interaction, and to support splicing are related activities, requiring only a few basic amino acids. Thus, the U2AF65 RS domain plays a direct role in modulating spliceosomal RNA-RNA interactions.

Amino Acid Sequence↗
PubMed1996

Differences in the interaction of Escherichia coli RNase P RNA with tRNAs containing a short or a long extra arm.

The phosphorothioate footprinting technique was applied to the investigation of phosphate moieties in tRNA substrates involved in interactions with M1 RNA, the catalytic subunit of Escherichia coli RNase P. In general agreement with previous data, all affected sites were localized in acceptor stem and T arm. But the analyzed examples for class I (Saccharomyces cerevisiae pre-tRNA(Phe) with short variable arm) and class II tRNAs (E. coli pre-tRNA(Tyr) with large variable arm) revealed substantial differences. In the complex with pre-tRNA(Phe), protection was observed at U55, C56, and G57, along the top of the T loop in the tertiary structure, whereas in pre-tRNA(Tyr), the protected positions were G57, A58, and A59, at the bottom of the T loop. These differences suggest that the size of the variable arm affects the spatial arrangement of the T arm, providing a possible explanation for the discrepancy in reports about the D arm requirement in truncated tRNA substrates for eukaryotic RNase P enzymes. Enhanced reactivities were found near the junction of acceptor and T stem (U6, 7, 8 in pre-tRNA(Phe) and G7, U63, U64 in pre-tRNA(Tyr)). This indicates a partial unfolding of the tRNA structure upon complex formation with RNase P RNA.

Base Sequence↗
PubMed1995

Enzymatic synthesis of 2'-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates.

For the first time mosaic nucleic acids composed of 50% RNA and 50% DNA can be obtained as transcripts with T7 RNA polymerase. Two NTPs could be replaced simultaneously in a transcription reaction. This means more than 40 deoxynucleotides were inserted in one transcript. Previously, a maximum of two deoxynucleotides could be incorporated and 2'-O-methyl-NTPs were not substrates at all. We obtained reasonable transcript yields with a maximal level of 99% 2'-O-methyl-NTPs, and the products contained up to 58% 2'-O-methylnucleotides at more than 20 positions. Sequence-specific nucleotide incorporation was monitored by sequence ladders (partial alkali or iodine cleavage). No base misincorporations were detected with 100% dGTP, dCTP and dTTP, and with partial incorporation of dATP alpha S, 2'-O-methyl-GTP alpha S and 2'-O-methyl-CTP alpha S, whereas they were found with dATP, 2'-O-methyl-ATP alpha S and 2'-O-methyl-UTP alpha S. Quantitative data allow predetermined modification levels of partially modified transcripts. Highly modified transcripts can be used for structural and functional studies, in modification interference approaches and for in vitro evolution procedures. Modification interference studies revealed a small number of important phosphate and ribose moieties in RNase P substrates. The conversion of T7 RNA polymerase to a DNA polymerase extends the observation that there is no absolute distinction between RNA and DNA polymerases. Accordingly, an adapted concept of a primordial RNA world is presented.

Base Sequence↗
PubMed1995

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2',5'-phosphodiester bond (RNA branch) with the 5' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction.

Base Sequence↗
PubMed1993

Modification interference approach to detect ribose moieties important for the optimal activity of a ribozyme.

A new approach for modification interference studies is presented. It involves the use of phosphorothioates as a handle to analyze any desired base or sugar modification. This method was applied to identify ribose and phosphate moieties which could be important in the pre-tRNA recognition of E. coli RNase P RNA (M1 RNA). The utility of this technique was confirmed by detecting the inhibitory effect of a deoxyribose in the 5'-flank (position-1). This site was already known to interfere with RNase P cleavage, if modified. We have analyzed pre-tRNA(Tyr) and pre-tRNA(Phe) and found different interference patterns for both tRNAs. Two unpaired regions were involved in both pre-tRNAs. Phosphorothioates interfered at the transition between acceptor- and D-arms. The results with deoxythymidines in the T-loop indicated that deoxyribose moieties or the extra methyl group in thymidine could interfere with RNAse P cleavage. These data suggest that even in complete pre-tRNAs, only a few intact ribonucleotides are important in the substrate recognition by RNase P. We have demonstrated the potential of this new approach which offers many future applications in all fields involving nucleic acids, for example RNA processing, action of ribozymes, tRNA charging and studies related to DNA promoter recognition.

Base Sequence↗
PubMed1993

Enzymatic RNA synthesis with deoxynucleoside 5'-O-(1-thiotriphosphates).

We have investigated the incorporation of 2'-deoxynucleoside-5'-O-(1-thiotriphosphates) into RNA transcripts using T7 RNA polymerase. With the exception of [alpha-S]dGTP, we obtained full-length transcripts of pre-tRNA(Phe) and pre-tRNA(Tyr) using an appropriate mixture of 2'-deoxynucleoside 5'-O-(1-thiotriphosphate) and the corresponding normal nucleoside triphosphate. The yields of the transcripts were comparable to those obtained with unmodified NTPs. Both substrates, [alpha-S]dTTP and [alpha-S]dATP, were inserted specifically. However, [alpha-S]dCTP was excluded at specific sites. We could not obtain transcripts using the deoxyguanosine derivative.

Base Sequence↗
PubMed1989

A spectrophotometric method for the estimation of amino groups on polymer supports.

A simple and sensitive method for the estimation of polymer-supported amino groups is reported. The polymer support is treated either with N-succinimidyl-4-O-(4,4'-dimethoxytrityl)-butyrate or 2,4-dinitrophenyl-4-O-(4,4'-dimethoxytrityl)-butyrate and a catalytic amount of 4-dimethylaminopyridine. After removal of the excess reagent through washing, a weighed quantity of the polymer support is treated with perchloric acid to release the 4,4'-dimethoxytrityl cation from the solid support into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption (epsilon 498 = 70,000/M) at 498 nm, is determined spectrophotometrically. A comparative study of these reagents with N-succinimidyl-3-(2-pyridyldithio)-propionate, 4,4'-dimethoxytrityl chloride, and sodium 2,4,6-trinitrobenzenesulfonate methods is also included.

Acylation↗
PubMed1989

A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups.

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.

Amines↗
PubMed1986

Traditional concepts of mental disorder among Indian psychiatric patients: preliminary report of work in progress.

In a medically pluralistic setting a range of health care providers offer not only different forms of treatment, but different ways of understanding illness. Even within a single tradition, these concepts evolve over time. Chapters in the classical texts of Ayurveda describe varieties of severe mental disorder (unmada) arising from a particular humoral imbalance (dosa) or arising in association with specific demons and deities (bhuta) that produce distinct character changes and symptom patterns. Patients currently presenting for treatment of mental disorder may describe their illness with reference to these concepts, but they also rely on other indigenous traditional concepts such as astrology, karma, the effects of other humoral relationships, such as semen loss and so forth; or they may rely on ideas derived from cosmopolitan medicine or both. Patients presenting to allopathic psychiatric centers in India were studied to determine whether patterns of help seeking could be predicted from the conceptual model by which they understood their illness. We elicited explanatory models from patients and obtained a history of prior consultations to other types of healer. Preliminary findings were notable for the pervasiveness of prior use of folk healers and the prominence of somatic symptoms among patients presenting to these allopathic physicians. Hypotheses regarding the impact of explanatory models on patterns of medical help seeking require further study from a larger and more diverse data base.

Attitude to Health↗