Biomedical subjects

L X Shen

Publications and source records attributed to L X Shen.

11 recordsLinked to original sources

PubMed2001

[Cloning and expression of the genes of glutathione synthetases].

The genes(gsh-I,gsh-II) for gamma-glutamyl-cysteine synthetase(GSH-I) and glutathione synthetase(GSH-II) from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively. The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc-gsh. E. coli BL21 was transformed by pTrc-gsh for expression of the related enzymes. Analysis of SDS-PAGE showed that the expected products were expressed. E. coli BL21(pTrc-gsh) were incubated at 37 degrees C and pH 7.2 to OD550 = 0.5. The conditions were then switched to 34 degrees C and pH6.7 after the addition of 0.1 mmol/L IPTG. The expressed products were up to 25% of the total protein of the bacteria. Acetone-treated cells of the engineered strain could synthesize GSH efficiently.

Cloning, Molecular↗

PubMed2001

[Studies on synthesis of glutathione by E. coli BL21 (pTrc-gsh) coupled with Saccharomyces cerevisiae].

During synthesis of GSH by the engineered strain E. coli BL21(pTrc-gsh) coupled with Saccharomyces cerevisiae producing ATP from adenosin, the inconsistency of two systems in the concentration of phosphate buffer was solved by decreasing concentration to 250 mmol/L. The conditions under 250 mmol/L phosphate buffer were optimized and the yield of GSH was 1.6 g/L, which was higher than that of summation by two systems under the same conditions respectively. Addition of glycine later after glutamate and cysteine weakened the inhibition of GSH to GSHI. It made the yield of GSH reach to 2.13 g/L which was 30.7% higher than the control.

Adenosine↗

PubMed1999

Single-nucleotide polymorphisms can cause different structural folds of mRNA.

Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation in man. Genes containing one or more SNPs can give rise to two or more allelic forms of mRNAs. These mRNA variants may possess different biological functions as a result of differences in primary or higher order structures that interact with other cellular components. Here we report the observation of marked differences in mRNA secondary structure associated with SNPs in the coding regions of two human mRNAs: alanyl tRNA synthetase and replication protein A, 70-kDa subunit (RPA70). Enzymatic probing of SNP-containing allelic fragments of the mRNAs revealed pronounced allelic differences in cleavage pattern at sites 14 or 18 nt away from the SNP, suggesting that a single-nucleotide variation can give rise to different mRNA folds. By using phosphorothioate oligodeoxyribonucleotides complementary to the region of different allelic structures in the RPA70 mRNA, but not extending to the SNP itself, we find that the SNP exerts an allele-specific effect on the accessibility of its flanking site in the endogenous human RPA70 mRNA. This further supports the allele-specific structural features identified by enzymatic probing. These results demonstrate the contribution of common genetic variation to structural diversity of mRNA and suggest a broader role than previously thought for the effects of SNPs on mRNA structure and, ultimately, biological function.

Alanine-tRNA Ligase↗

PubMed1999

Pseudo-cyclic oligonucleotides: in vitro and in vivo properties.

We have designed and studied antisense oligodeoxynucleotides (oligonucleotides; oligos) which we call 'pseudo-cyclic oligonucleotides' (PCOs). PCOs contain two oligonucleotide segments attached through their 3'-3'- or 5'-5'-ends. One of the segments of the PCO is an antisense oligo complementary to a target mRNA, and the other is a short protective oligo that is 5-8 nucleotides long and complementary to the 3'- or 5'-end of the antisense oligo. As a result of complementarity between the antisense and protective oligo segments, PCOs form intramolecular pseudo-cyclic structures in the absence of the target RNA. The antisense oligo segment of PCOs used for the studies described here is complementary to an 18-nucleotide-long site on the mRNA of the protein kinase A regulatory subunit RIalpha (PKA-RIalpha). Thermal melting studies of PCOs in the absence and presence of the complementary RNA suggest that the pseudo-cyclic structures formed in the absence of the target RNA dissociate, bind to the target RNA, and form heteroduplexes. The results of RNase H cleavage assays suggest that PCOs bind to complementary RNA and activate RNase H in a manner similar to that of an 18-mer conventional antisense PS-oligo. In snake venom (a 3'-exonuclease) or spleen (a 5'-exonuclease) phosphodiesterase digestion studies, PCOs are more stable than conventional antisense oligos because of the presence of 3'-3'- or 5'-5'-linkages and the formation of intramolecular pseudo-cyclic structures. PCOs with a phosphorothioate antisense oligo segment inhibited cell growth of MDA-MB-468 and GEO cancer cell lines similar to that of the conventional antisense PS-oligo, suggesting efficient cellular uptake and target binding. The nuclease stability studies in mice suggest that PCOs have higher in vivo stability than antisense PS-oligos. The studies in mice showed similar pharmacokinetic and tissue distribution profiles for PCOs to those of antisense PS-oligos in general, but rapid elimination from selected tissues.

Animals↗

PubMed1998

Impact of mixed-backbone oligonucleotides on target binding affinity and target cleaving specificity and selectivity by Escherichia coli RNase H.

All phosphorothioate mixed-backbone oligonucleotides (MBOs) composed of deoxyribonucleotide and 2'-O-methylribonucleotide segments were studied for their target binding affinity, specificity, and RNase H activation properties. The 2'-O-methylribonucleotide segment, which does not activate RNase H, serves as a high affinity target-binding domain and the deoxyribonucleotide (DNA) segment, which binds to the target with a lower affinity than the former domain, serves as an RNase H-activation or target-cleaving domain. In order to understand the influence of the size and position of the DNA segment of MBOs on RNase H-mediated cleavage of the RNA target, we designed and synthesized a series of 18-mer MBOs with the DNA segment varying from a stretch of two to eight deoxyribonucleotides in the middle, at the 5'-end, or at the 3'-end, of the MBOs. UV absorbance melting experiments of the duplexes of the MBOs with the complementary and singly mismatched RNA targets suggest that the target binding affinity of the MBOs increases as the number of 2'-O-methylribonucleotides increases, and that the binding specificity is influenced by the size and position of the DNA segment. Analysis of RNase H assay results indicates that the minimum substrate cleavage site and cleavage efficiency of RNase H are influenced by the position of the DNA segment in the MBO sequence. RNA cleavage efficiency decreases as the position of the DNA segment of the MBO.RNA heteroduplex is changed from the 3'-end to the middle and to the 5'-end of the target strand. Studies with singly mismatched targets indicate that the RNase H-dependent point mutation selectivity of the MBOs is affected by both the position and size of the DNA segment in the MBO sequence.

Binding Sites↗

PubMed1996

A characteristic bent conformation of RNA pseudoknots promotes -1 frameshifting during translation of retroviral RNA.

The structures of four different RNA pseudoknots that provide one of the signals required for ribosomal frameshifting in mouse mammary tumor virus have been determined by NMR. The RNA pseudoknots have similar sequences and assume similar secondary structures, but show significantly different frameshifting efficiencies. The three-dimensional structures of one frameshifting and one non-frameshifting RNA pseudoknot had been determined previously by our group. Here we determine the structures of two new RNA pseudoknots, and relate the structures of all four pseudoknots to their frameshifting abilities. The two efficient frameshifting pseudoknots adopt characteristic bent conformations with stem 1 bending towards the major groove of stem 2. In contrast, the two poor frameshifting pseudoknots have structures very different from each other and from the efficient frameshifters. One has linear, coaxially stacked stems, the other has stems twisted and bent, but in the opposite direction to the efficient frameshifters. Changes in loop size that favor bending (shorter loops) increase frameshifting efficiency; longer loops that allow linear arrangement of the stems decrease frameshifting. Frameshifting pseudoknots in feline immunodeficiency virus and simian retrovirus have different loop sequences, but the sequences at their stem junctions imply the same bent conformation as in the mouse mammary tumor viral RNA. The requirement for a precise pseudoknot conformation for efficient frameshifting strongly implies that a specific interaction occurs between the viral RNA pseudoknot and the host protein-synthesizing machinery.

Base Sequence↗

PubMed1995

Synthesis and NMR of RNA with selective isotopic enrichment in the bases.

Efficient syntheses of pyrimidine and purine nucleosides and nucleotides with selective 13C enrichment in the base moieties are described. Uridine and cytidine are labeled at position C6 and adenosine and guanosine are labeled at position C8. The selectively labeled nucleosides were converted to nucleoside triphosphates and used with in vitro transcription to synthesize labeled RNA. Isotope-edited 12C and 13C sub-spectra of a omega 1-1/2-X-filtered NOESY experiment are demonstrated to be useful for making resonance assignments and for deriving structural information in large (> 20 nt) RNA molecules. The labeled RNAs also allow heteronuclear J-couplings and relaxation parameters to be measured without complications from 13C-13C J-couplings.

Adenine↗

PubMed1995

The structure of an RNA pseudoknot that causes efficient frameshifting in mouse mammary tumor virus.

The structure of a 34-nucleotide RNA pseudoknot that causes efficient -1 frameshifting in the messenger RNA of mouse mammary tumor virus has been investigated by NMR. Spectral assignment of the pseudoknot was facilitated by comparative NMR studies on the pseudoknot and on two smaller hairpin RNAs, and by using selective 13C labeling and 13C-edited NMR techniques. The three-dimensional structure of the pseudoknot has been determined. The frameshifter pseudoknot possesses structural features not observed in previously reported model pseudoknots. It has a compact structure with a pronounced bend at the junction of its G.C-rich stems. A single adenylate residue is intercalated between the two stems so that direct coaxial staking of the stems is not possible. The lack of an opposing nucleotide for the stacked, intervening adenylate creates a hinge in the pseudoknot. Most of the loop nucleotides are restrained by base staking interactions which keep the loops from adopting extended conformations. The sterically constrained loops direct the bending of the pseudoknot at the stem-stem junction. The roles of the intercalated adenylate and loop lengths in causing bending can explain their requirement for efficient frameshifting. Our NMR data also indicate that there are internal dynamics associated with the pseudoknot. The unique, compact structure and conformational flexibility of the pseudoknot may be required for recognition and favourable interaction with the translating ribosome, or with translation factors associated with the ribosome.

Base Sequence↗

PubMed1995

Structural and functional studies of retroviral RNA pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting.

Ribosomal frameshifting, a translational mechanism used during retroviral replication, involves a directed change in reading frame at a specific site at a defined frequency. Such programmed frameshifting at the mouse mammary tumor virus (MMTV) gag-pro shift site requires two mRNA signals: a heptanucleotide shifty sequence and a pseudoknot structure positioned downstream. Using in vitro translation assays and enzymatic and chemical probes for RNA structure, we have defined features of the pseudoknot that promote efficient frameshifting. Heterologous RNA structures, e.g. a hairpin, a tRNA or a synthetic pseudoknot, substituted downstream of the shifty site fail to promote frameshifting, suggesting that specific features of the MMTV pseudoknot are important for function. Site-directed mutations of the MMTV pseudoknot indicate that the pseudoknot junction, including an unpaired adenine nucleotide between the two stems, provides a specific structural determinant for efficient frameshifting. Pseudoknots derived from other retroviruses (i.e. the feline immunodeficiency virus and the simian retrovirus type 1) also promote frameshifting at the MMTV gag-pro shift site, dependent on the same structure at the junction of the two stems.

Animals↗

PubMed1995

RNA structure at high resolution.

Studies of RNA structural motifs at high resolution by NMR and X-ray crystallographic methods have provided many insights into the fundamental forces that give rise to the unique structural characteristics of RNA. Non-Watson-Crick purine-pyrimidine, purine-purine, and pyrimidine-pyrimidine base pairing, as well as base-phosphate and base-ribose hydrogen bonding, are important forces for folding and stabilizing RNA structures. Base stacking is as important in determining RNA conformations as hydrogen bonding interactions. With the noncanonical interactions, many single-stranded loop regions such as hairpin loops, bulge loops, and internal loops fold into well-defined secondary structures. Loop-loop and loop-helix interactions can produce tertiary structures such as pseudoknots. Also, single strands adjacent to helical regions can form tertiary contacts with base-paired nucleotides of the helices. As we learn more about the structures of the important motifs we can ask more specific questions about the mechanisms of RNA-mediated functions. Conformational flexibility rather than a specific shape of the RNA may be important for some biological reactions. However, knowledge of the structures and the ease of conformational change of the molecules involved in any process are essential for understanding and eventually controlling the process.

Base Sequence↗

PubMedDate not supplied

A mild and efficient solid-support synthesis of novel oligonucleotide conjugates.

Conjugates of oligodeoxyribonucleotide phosphorothioate (ODN-PS) with folic acid, retinoic acid, arachidonic acid, and methoxypoly(ethylene glycol)propionic acid have been synthesized. The procedure involved the initial solid-phase preparation of 5'-amino-functionalized ODN-PS using N-pent-4-enoyl-derived (PNT) nucleoside phosphoramidites followed by conjugation of the oligonucleotide either to the ligand acids, using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide as a coupling reagent, or to their corresponding succinimidyl derivatives. Subsequent exposure of the support to aqueous ammonium hydroxide (28%, 2 h, 55 degrees C) resulted in the release of the fully deprotected ODN conjugates, which were purified by reversed-phase HPLC or by preparative polyacrylamide gel electrophoresis. The identity of the oligonucleotide conjugates was confirmed by MALDI-TOF mass spectral analysis.

Ammonium Hydroxide↗