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Biomedical subjects

W Xiao

Publications and source records attributed to W Xiao.

At least 109 records · Page 6Linked to original sources
PubMed1997

A mutation in the MSH5 gene results in alkylation tolerance.

DNA methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are potent carcinogens; their carcinogenic effect is mainly due to the effect of production of O6-methylguanine (O6 MeG) on DNA. O6 MeG is not only mutagenic but also toxic to the cell because Mer-/Mex- cells unable to remove O6 MeG are very sensitive to killing by MNNG. It has been proposed that repeated futile mismatch correction of O6 MeG-containing bp is responsible for the genotoxicity of the O6 MeG lesion and that loss of mismatch repair activity results in cellular tolerance to O6 MeG, but the hypothesis has not been proved. We used yeast as a model to test this hypothesis and found that chromosome deletion of any known nuclear mitotic mismatch repair genes, including MLH1, MSH2, MSH3, MSH6, and PMS1, did not rescue mgt1delta O6 MeG DNA repair methyltransferase-deficient cells from killing by MNNG. A large number of mgt1delta, MNNG-tolerant revertants were isolated, among which one cell line, XS-14, has been found to carry a mutated allele of the MSH5 gene. The mutation also affected spore survival but did not increase the spontaneous mutation rate. We further demonstrated that a mutated form of the MSH5 gene, msh5-14, not the msh5delta-null mutation, is responsible for the cellular tolerance to MNNG in XS-14 cells. This observation offers an alternative model that may reconcile seemingly contradictory observations of yeast and mammalian cells.

Alkylation↗
PubMed1997

Correlation of selective modifications to a 2',5'-oligoadenylate-3',5'-deoxyribonucleotide antisense chimera with affinity for the target nucleic acid and with ability to activate RNase L.

The use of an antisense oligonucleotide to address a specific targeted RNA sequence and subsequent localized activation of the 2-5A-dependent RNase (RNase L) to effect selective RNA degradation is a new approach to the control of gene expression called 2-5A-antisense. The previously reported biological activity of the 2-5A:AS chimeric oligonucleotide [p5'(A2'p)3A-antiPKR1], directed against nucleotides 55-73 of the coding sequence of the PKR mRNA, has been used as a point of reference to examine the effect of introducing mismatches into the chimeric oligonucleotide, altering the chain length of the antisense domain of the chimeras, removal of the 5'-monophosphate moiety, shortening the 2',5'-oligoadenylate domain, and substitution of 3',5'-linked 2'-deoxyadenosine nucleotides for the 2-5A domain. The general formula for the novel chimeric oligonucleotides is p5'(A2'p)3A2'p(CH2)4p(CH2)4p(5'N3'p)mN, where N is any nucleoside and m is any integer. When the biological activity of these new chimeric oligonucleotides was compared to that of the parent chimera, 2-5A-aPKR, for their ability to effect target PKR RNA cleavage in a cell-free and in an intact cell assay, it was determined that there was a close correlation between the activity of 2-5A-antisense chimeras and their affinity (Tm) for a targeted nucleic acid. In addition, there was also a close correlation between activity of the 2-5A-antisense chimeras and their ability to activate the 2-5A-dependent RNase L.

Adenine Nucleotides↗
PubMed1997

Targeting RNA decay with 2',5' oligoadenylate-antisense in respiratory syncytial virus-infected cells.

Treatment of human cells with 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) results in the selective cleavage of targeted RNA species by 2-5A-dependent RNase L. Here we show that 2-5A-antisense containing stabilizing modifications at both termini are effective in suppressing the replication of respiratory syncytial virus (RSV) in human tracheal epithelial cells. The affinity of 2-5A-antisense for different regions in the RSV M2 and L mRNAs was predicted from a computer-generated model of the RNA secondary structure. The most potent 2-5A-antisense molecule caused a highly effective, dose-dependent suppression of RSV yields when added to previously infected cells. In contrast, control oligonucleotides, including an inactive dimeric form of 2-5A linked to antisense, 2-5A linked to a randomized sequence of nucleotides, and antisense molecules lacking 2-5A, had minimal effects on virus replication. The specificity of this approach was shown by reverse transcriptase-coupled PCR analysis of RSV M2, P, and N mRNA and of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The RSV M2 mRNA amounts were depleted after treating RSV-infected cells with 2-5A-antisense targeted to this mRNA, whereas the amounts of the other RNA species were unchanged. These studies demonstrate that 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) can effectively suppress RSV replication by directing the cellular RNase L to selectively degrade an essential viral mRNA.

Adenine Nucleotides↗
PubMed1997

UAS(MAG1), a yeast cis-acting element that regulates the expression of MAG1, is located within the protein coding region of DDI1.

MAG1 and DDI1 are two divergently transcribed DNA damage-inducible genes from Saccharomyces cerevisiae. Previous studies have shown that MAG1 induction requires an upstream activating site (UAS) located between nucleotides -376 and -330. Here we show that a 24-bp oligonucleotide from within the UAS(MAG1) region forms a sequence-specific DNA-protein complex with partially purified proteins from S. cerevisiae. Point mutations introduced into the 24-bp oligonucleotide inhibited the formation of the DNA-protein complex and decreased the level of induction of MAG1-lacZ. By determining the transcription and translation start points of both MAG1 and DDI1, an interesting, indeed unprecedented feature of genome organization in eukaryotes was revealed: UAS(MAG1) actually lies within the protein-coding region of DDI1. Although tightly linked to each other, and co-induced upon treatment with DNA-damaging agents, DDI1 does not share the UAS(MAG1) required for DNA damage induction of MAG1. Furthermore, MAG1 and DDI1 respond differently in the presence of the protein synthesis inhibitor cycloheximide, suggesting that these two genes are regulated by different mechanisms in the absence of de novo protein synthesis.

Amino Acid Sequence↗
PubMed1997

Bidirectional regulation of two DNA-damage-inducible genes, MAG1 and DDI1, from Saccharomyces cerevisiae.

MAG1 encodes a Saccharomyces cerevisiae 3-methyladenine DNA glycosylase that initiates a base-excision-repair pathway and protects yeast cells from killing by methylating agents such as methyl methanesulphonate (MMS). In the promoter region of the MAG1 gene, there is an 8 bp GC-rich direct repeat (DR). Here we report that the DR sequence functions as an upstream activating site (UAS) that upregulates the expression of MAG1 as well as another DNA-damage-inducible gene, DDI1, which is transcribed divergently from MAG1. Deletions, or point mutations, within this repeat completely abolished DNA-damage induction and resulted in a reduced basal-level expression for both MAG1 and DDI1 genes. Furthermore, yeast cells carrying the MAG1 gene with the DR deletion displayed an increased sensitivity to MMS compared with wildtype cells. The DR sequence alone can activate transcription of a CYC1 minimal promoter and confer a partial DNA-damage responsiveness. Electrophoretic mobility-shift assays indicate that the DR function is not due to interaction with the yeast RPA. Like MAG1, the DDI1 gene is also controlled by an upstream repressing site (URS) located 5' to the direct repeat. Based on this and previous studies, a model is proposed whereby the constitutive expression of MAG1 and DDI1 is controlled by two functionally opposite regulatory elements, UAS and URS, probably through an antagonistic mechanism, whereas the damage-induced expression appears to be regulated by mechanisms of derepression at the URS as well as activation at the UAS.

Base Sequence↗
PubMed1997

Robustness of bioequivalence procedures under Box-Cox alternatives.

This study investigates the robustness of Schuirmann's two one-sided tests procedure under a set of Box-Cox alternatives. A simulation is conducted to study the level of significance and the power of the procedure. Empirical results show that Schuirmann's procedure is robust under Box-Cox alternatives when the mean of the underlying distribution of the reference formulation is known, which suggests that the assumptions of normality or lognormality are sufficient but may not be necessary. It appears that Schuirmann's procedure cannot maintain the proper level of significance when the mean of the reference formulation is unknown, even under the normality or lognormality assumptions.

Chemistry, Pharmaceutical↗
PubMed1997

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.

Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (pDD-2) containing only 165 bp of AAV sequence: two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. pDD-2 replication intermediates were characteristic of the AAV replication scheme in which linear monomer, dimer, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV DNA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both rAAV vector production and synthetic plasmid vector delivery.

DNA, Viral↗
PubMed1997

Identification of a promoter-specific transactivation domain in the herpes simplex virus regulatory protein ICP4.

ICP4 is expressed during the immediate-early phase of infection by herpes simplex virus (HSV) and activates transcription of viral genes during subsequent phases of productive infection. Several members of the alpha-herpesvirus family encode regulatory proteins that have extensive homology with ICP4 and exhibit a transactivation domain (TAD) at the N terminus. The portions of ICP4 required for nuclear localization, DNA binding, and dimerization have been defined, but a domain that is specifically required for transactivation has not been identified. We have defined a promoter-specific ICP4 TAD by analysis of the activity of GAL4-ICP4 fusion proteins cotransfected into HeLa cells with a luciferase reporter gene linked to a promoter with five GAL4 binding sites. The transactivation activity of GAL4-ICP4 hybrids is located entirely within the first 139 residues of ICP4 and is significantly less potent than the activity of GAL4-TAD hybrids derived from ICP4 homologs. ICP4 residues 97 to 109 are a critical component of this N-terminal TAD. Transient transfection assays performed with nonfusion forms of ICP4 and luciferase genes linked to the HSV glycoprotein D (gD) or thymidine kinase (tk) promoter revealed that ICP4 residues 97 to 109 are required for induction of the gD promoter but are not required for induction of the tk promoter. Comparative experiments with ICP4 homologs revealed that the pseudorabies virus TAD is a potent activator of the gD promoter and a weak activator of the tk promoter. Complementation assays revealed that loss of ICP4 residues 97 to 109 reduced the yield of virus from infected cells nearly 500-fold compared to wild-type ICP4. We conclude that ICP4 residues 97 to 109 are a core component of a promoter-specific transactivation domain that is required for efficient replication of herpes simplex virus.

Animals↗
PubMed1996

High efficiency, long-term clinical expression of cottontail rabbit papillomavirus (CRPV) DNA in rabbit skin following particle-mediated DNA transfer.

The ability of skin to support long lasting expression of genes delivered with a particle-mediated system was evaluated in rabbits inoculated with cottontail rabbit papillomavirus (CRPV) DNA. The optimal delivery force for maximal gene expression in rabbit skin was determined in transient beta-galactosidase assays. Forty-five sites in four rabbits were then inoculated at 350-400 p.s.i. with CRPV DNA. All sites (100%) formed papillomas with multiple papillomas at most sites. These results support the feasibility of using a particle-mediated delivery system for gene therapy and suggest that some papillomavirus features, such an origin of replication, may be well suited for use in vectors to target long term expression to skin.

Animals↗
PubMed1996

The 2 micron plasmid of laboratory yeast strains is a type-1/type-2 hybrid.

Industrial yeast strains carry one of two homeologous 2 microns plasmids designated as type-1 or type-2. The 2 microns plasmid, Scp1, found in common laboratory strains of Saccharomyces cerevisiae is considered a type-2 plasmid, since the ori, STB, RAF and REP1 loci and intergenic sequences of the right-unique region of Scp1 are homologous to the corresponding loci in industrial strain type-2 plasmids. However, within both its 599 bp inverted repeats Scp1 has 142-bp sequences homologous to the bakers' yeast type-1 plasmid. DNA sequence analyses and oligonucleotide hybridizations indicate that the 142-bp insertion in Scp1 was probably due to homeologous recombination between type-1 and type-2 plasmids. These results suggest that some of the plasmid and chromosomal sequence polymorphisms seen in laboratory yeast strains result from homeologous recombination in their ancestral breeding stock.

Base Sequence↗
PubMed1996

Modulation of interferon (IFN)-inducible gene expression by retinoic acid. Up-regulation of STAT1 protein in IFN-unresponsive cells.

Interferons (IFN) and retinoids failed to inhibit the growth of a number of breast tumor cell lines. However, a combination of these two biological response modifiers significantly suppressed the cell growth at pharmacologically achievable doses. The molecular basis for such enhancement was investigated in MCF-7, a breast tumor cell line resistant to growth inhibition by IFN-beta. Pretreatment of cells with retinoic acid (RA) for 16 h followed by IFN-beta, but not the converse, induced cytotoxic effects in the cells. Continuous presence of RA was not necessary, although it enhanced the degree of cell death when present. Further analyses revealed that IFN-beta failed to activate IFN-stimulated gene transcription. However, IFN-beta strongly up-regulated the gene expression in RA-pretreated cells. Both IFN-beta- and IFN-gamma-inducible gene expression were enhanced via a modulation of the transcriptional factor IFN-stimulated gene factors-3 and GAF binding to respective cognate regulatory elements. STAT1 was undetectable in these cells prior to RA treatment. RA increased the levels of this crucial regulator, thereby restoring IFN responses. Thus, RA augmentation of STAT1 may be an early step in the cooperative anti-tumor effects of IFN and RA.

Animals↗
PubMed1996

Particle-mediated delivery of recombinant expression vectors to rabbit skin induces high-titered polyclonal antisera (and circumvents purification of a protein immunogen).

Polyclonal antibodies were generated in rabbits by delivery to skin of gold particles coated with mammalian expression vectors encoding a cytoplasmic (beta-galactosidase) or a nuclear (L1 capsid of cottontail rabbit papillomavirus) protein. One primary and one booster immunization of 30 micrograms DNA per rabbit yielded specific antisera with titers from 1:24 000 to 1:120 000 in each of eight rabbits, as detected by ELISA and Western blot analysis. Genetic immunization requires relatively small amounts of DNA, eliminates the need to purify the protein immunogen, and does not require irritating adjuvants.

Animals↗
PubMed1996

Asymmetry requirements in the photosynthetic reaction center of Rhodobacter capsulatus.

Nine large-scale symmetry reaction center mutants were constructed in Rhodobacter capsulatus by replacing segments of the M subunit gene with the homologous region of the L subunit gene. Between them, the mutations resulted in symmetrization of essentially the entire region from the carboxy terminal portion of the C helix through most of the E helix. The amino acids in this region define about 80% of the environment of the reaction center cofactors. These studies show that roughly 80% of the amino acids that come in close contact with the cofactors involved in initial electron transfer can be made symmetric in a piecewise manner without loss of the ability to grow photoheterotrophically. However, the amino acid regions near the quinones and iron atom are much more sensitive to symmetrization and most of the large-scale changes in this region resulted in the loss of photosynthetic viability, probably due to loss of stable reaction centers from the photosynthetic membrane. More detailed analysis of the isolated photosynthetic membranes from these mutants showed that in all cases but one, there was some amount of charge separation occurring in the mutant reaction centers. This bank of mutants serves as a useful starting point for more detailed studies of the differential molecular interactions which occur between the two reaction center subunits and their associated cofactors.

Amino Acid Sequence↗
PubMed1996

Low-temperature femtosecond-resolution transient absorption spectroscopy of large-scale symmetry mutants of bacterial reaction centers.

Reaction centers isolated from three large-scale symmetry mutants sym0, sym2-1, and sym5-2 described in the previous article of this issue [Taguchi, A. K. W., Eastman, J. E., Gallo, D. M., Jr., Sheagley, E.. Xiao, W., & Woodbury, N. W. (1996) Biochemistry 35, 3175-3186] have been investigated by low-temperature ground state and ferntosecond-resolution transient absorption spectroscopy. All three of these large-scale symmetry mutants undergo electron transfer at 20 K. The mutants sym0 and sym5-2 have yields and dominant rates of charge separation comparable to wild type. However. the sym2-mutant shows a roughly 35%, quantum yield at this temperature, and the major kinetic component of the initial electron transfer is slower than wild type by nearly a factor of 100. The sym0 mutant showed substantial changes in the monomer bacteriochiorophyll ground state and transient spectra, and both sym0 sym2-1 showed changes in the bacteriopheophyll ground state and transient spectra. In particular, sym2-1 shows a small absorbance decrease in the region of the Qx band of the B side bacteriopheophytin which could be attributed to 10%-20% electron transfer along the B pathway.

Cold Temperature↗
PubMed1996

The repair of DNA methylation damage in Saccharomyces cerevisiae.

The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion. An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the damaged base, followed by an Apn1 apurinic/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent repair. MMS is also regarded as a radiomimetic agent, since a number of DNA radiation-repair mutants are also sensitive to MMS. To understand how these radiation-repair genes are involved in DNA methylation repair, we performed an epistatic analysis by combining yeast mag1 and apn1 mutations with mutations involved in each of the RAD3, RAD6 and RAD52 groups. We found that cells carrying rad6, rad18, rad50 and rad52 single mutations are far more sensitive to killing by MMS than the mag1 mutant, that double mutants were much more sensitive than either of the corresponding single mutants, and that the effects of the double mutants were either additive or synergistic, suggesting that post-replication and recombination-repair pathways recognize either the same lesions as MAG1 and APN1, or else some differ- ent lesions produced by MMS treatment. Lesions handled by recombination and post replication repair are not simply 3MeA, since over-expression of the MAG1 gene does not offset the loss of these pathways. Based on the above analyses, we discuss possible mechanisms for the repair of methylation damage by various pathways.

Adenosine Triphosphatases↗
PubMed1996

Synthesis and characterization of composite nucleic acids containing 2', 5'-oligoriboadenylate linked to antisense DNA.

Composite nucleic acids, known as 2-5A antisense chimeras, cause the 2-5A-dependent ribonuclease (RNase L) to catalyze the specific cleavage of RNA in cell free systems and in intact cells. Such 2-5A antisense chimeras are 5'-monophosphorylated, 2,'5'-linked oligoadenylates covalently attached to antisense 3',5'-oligodeoxyribonucleotides by means of a linker containing two residues of 1,4-butanediol phosphate. Here we report a fully automated synthesis of 2-5A antisense chimeras on a solid support using phosphoramidite methodology with specific coupling time modifications and their subsequent purification by reverse-phase ion-pair and anion exchange HPLC. Purified 2-5A antisense chimeras were characterized by [1H]NMR and [31P]NMR, MALDIMS, and capillary gel electrophoresis. The synthetic 2',5'-linked oligoadenylate showed no phosphodiester isomerization to 3',5' during or after synthesis. In addition, we have developed facile methodologies to characterize the chimeras using digestion with various hydrolytic enzymes including snake venom phosphodiesterase I and nuclease P1. Finally, Maxam-Gilbert chemical sequencing protocols have been developed to confirm the entire sequence of these chimeric oligonucleotides.

2',5'-Oligoadenylate Synthetase↗