Nonradioisotopic PCR heteroduplex analysis: a rapid, reliable method of detecting minor gene mutations.
Explore the source record for details and available documents.
DNA Mutational Analysis↗
Biomedical subjects
Y Xing
Publications and source records attributed to Y Xing.
Reproducibility of an esophageal biopsy sampling procedure in a high-incidence area for esophageal cancer in northern China.
The objective of this study was to evaluate the reproducibility of the biopsy sampling procedure in research on esophageal lesions. Biopsies were taken from the middle and lower thirds of the esophagus, one from each site, from 25 subjects in a high-incidence area for esophageal cancer in Xinye County of Henan Province, China. The biopsy sampling procedure was repeated on the same subjects 10 days later. When the biopsies were analyzed together and those with worse pathologies were used for diagnosis, 52% of the subjects had the same grade of lesions in the second biopsy examination, 32% had lower-grade lesions, and 16% had higher-grade lesions.
Adult↗
Treatment of 119 cases of verruca vulgaris and verruca plana by external application of pulvis pepper alba.
Explore the source record for details and available documents.
Administration, Topical↗
Molecular-dynamics simulation of fragmentation of C60 colliding with H2.
Explore the source record for details and available documents.
Journal Article↗
Thermodynamics of RNA unfolding: stabilization of a ribosomal RNA tertiary structure by thiostrepton and ammonium ion.
RNAs with interesting secondary and tertiary structures tend to melt in several broad and overlapping transitions over a wide temperature range, and it has been consequently difficult to resolve the thermodynamics of individual unfolding steps. In the case that a ligand selectively binds a single folded state of the RNA, it is possible to obtain reliable thermodynamic parameters for both RNA unfolding and RNA-ligand binding simply from the hyperchromicity of RNA denaturation. The analysis procedure involves fitting a three-dimensional surface to absorbance data collected as a function of both temperature and ligand concentration. Analysis of the unfolding of a fragment of the large subunit ribosomal RNA (Escherichia coli sequence 1051 to 1109) is presented; both an antibiotic (thiostrepton) and ammonium ion specifically stabilize a tertiary structure within this RNA. A consistent set of thermodynamic parameters (delta H and tm) for the first two sequentially linked unfolding transitions is obtained from the experiments, and the binding constants obtained for the two ligands are consistent with other independent measurements. The approach is applicable to a variety of RNAs that specifically bind proteins, antibiotics, ions or other ligands.
Ammonia↗
Stabilization of a ribosomal RNA tertiary structure by ribosomal protein L11.
Interactions between ribosomal protein L11 and a domain of large subunit rRNA have been highly conserved and are essential for efficient protein synthesis. To study the effects of L11 on rRNA folding, a homolog of the Escherichia coli L11 gene has been amplified from Bacillus stearothermophilus DNA and cloned into a phage T7 polymerase-based expression system. The expressed protein is 93% homologous to the L11 homolog from Bacillus subtilis, denatures at temperatures above 72 degrees C, and has nearly identical rRNA binding properties as the Escherichia coli L11 in terms of RNA affinity constants and their dependences on temperature, Mg2+ concentration, monovalent cation, and RNA mutations. Mg2+ and NH4+ are specifically bound by the RNA-protein complex, with apparent ion-RNA affinities of 1.6 mM-1 and 19 M-1, respectively, at 0 degree C. The effect of the thermostable L11 on the unfolding of a 60 nucleotide rRNA fragment containing its binding domain has been examined in melting experiments. The lowest temperature RNA transition, which is attributed to tertiary structure unfolding, is stabilized by approximately 25 degrees C, and the interaction has an intrinsic enthalpy of approximately 13 kcal/mol. The thermal stability of the protein-RNA complex is enhanced by increasing Mg2+ concentration and by NH4+ relative to Na+. Thus L11, NH4+, and Mg2+ all bind and stabilize the same rRNA tertiary interactions, which are conserved and presumably important for ribosome function.
Amino Acid Sequence↗
Cloning of yeast HAP5: a novel subunit of a heterotrimeric complex required for CCAAT binding.
The CCAAT-binding factor is a conserved heteromeric transcription factor that binds to CCAAT box-containing upstream activation sites (UASs) within the promoters of numerous eukaryotic genes. The CCAAT-binding factor of Saccharomyces cerevisiae activates the transcription of these genes in response to growth in a nonfermentable carbon source. Previous studies have demonstrated that the HAP2, HAP3, and HAP4 subunits of the yeast CCAAT-binding factor are required for the transcriptional activation of genes containing a CCAAT box. Using the two-hybrid screening method, we have identified an additional component of the CCAAT-binding factor. We present the identification and characterization of a novel gene, HAP5, that encodes an additional subunit of the CCAAT-binding factor required for the assembly and DNA-binding activity of the complex. In a hap5 mutant, we show that CCAAT-binding activity is abolished in vitro. Furthermore, we demonstrate that purified recombinant HAP2, HAP3, and HAP5 are able to reconstitute CCAAT-binding activity in mobility shift analysis. These data suggest that the HAP2/3/5 heterotrimer represents a unique DNA-binding factor in which all three subunits of the complex are absolutely required for DNA-binding activity.
Amino Acid Sequence↗
Nonrandom gene organization: structural arrangements of specific pre-mRNA transcription and splicing with SC-35 domains.
This work demonstrates a highly nonrandom distribution of specific genes relative to nuclear domains enriched in splicing factors and poly(A)+ RNA, and provides evidence for the direct involvement of these in pre-mRNA metabolism. As investigated in hundreds of diploid fibroblasts, human collagen I alpha 1 and beta-actin DNA/RNA showed a very high degree of spatial association with SC-35 domains, whereas three nontranscribed genes, myosin heavy chain, neurotensin, and albumin, showed no such preferential association. Collagen I alpha 1 RNA accumulates within the more central region of the domain, whereas beta-actin RNA localizes at the periphery. A novel approach revealed that collagen RNA tracks are polarized, with the entire gene at one end, on the edge of the domain, and the RNA extending into the domain. Intron 26 is spliced within the RNA track at the domain periphery. Transcriptional inhibition studies show both the structure of the domain and the gene's relationship to it are not dependent upon the continued presence of accumulated collagen RNA, and that domains remaining after inhibition are not just storage sites. Results support a model reconciling light and electron microscopic observations which proposes that transcription of some specific genes occurs at the border of domains, which may also function in the assembly or distribution of RNA metabolic components. In contrast to the apparently random dispersal of total undefined hnRNA synthesis through interdomain space, transcription and splicing for some genes occurs preferentially at specific sites, and a high degree of individual pre-mRNA metabolism is compartmentalized with discrete SC-35 domains.
Actins↗
Western blot analysis of anti-Escherichia coli serum immunoglobulins in women susceptible to recurrent urinary tract infections.
The basis for increased susceptibility of some women to recurrent urinary tract infections (UTIs) is not clear; increased susceptibility may be due to host factors that promote increased colonization of the vaginal and bladder mucosa with uropathogens or to decreased immune responses to uropathogens. Anti-Escherichia coli antibody specificities in sera from UTI patients and controls were comprehensively assessed to determine whether UTI-susceptible and -nonsusceptible women differed in their capacities to make antibodies to individual E. coli antigens. Sera were analyzed by one-dimensional Western blots using antigens prepared from uropathogenic E. coli. The results showed that sera from subjects without a history of recurrent UTIs contained IgG antibodies to specific E. coli antigens more often than did sera from UTI-susceptible patients. These data suggest that hyporesponsiveness to specific E. coli antigens may be linked to increased UTI susceptibility in some women.
Adult↗
Protein recognition of a ribosomal RNA tertiary structure.
Ribosomal protein L11 recognizes a highly conserved, 58 nucleotide domain of large subunit ribosomal RNA. This domain has a set of tertiary interactions that are specifically stabilized by Mg2+ and NH4+ ions. The protein recognizes this tertiary structure, in the sense that ions stabilizing the tertiary structure also promote L11 binding, and a heat stable form of the protein (from Bacillus stearothermophilus) prevents RNA unfolding. We also find that these RNA-binding properties are confined to a approximately 75 amino acid domain of the protein. The larger issue of L11 function within the ribosome is discussed in light of these findings.
Bacterial Proteins↗
Brain growth retardation due to the expression of human insulin like growth factor binding protein-1 in transgenic mice: an in vivo model for the analysis of igf function in the brain.
Three lines of transgenic (Tg) mice carrying a fusion gene linking the mouse metallothionein-I promoter to a cDNA encoding human insulin-like growth factor binding protein-1 (hIGFBP-1) were found to express the transgene in brain. As judged by comparing Tg brain weights to those of non-transgenic littermates, adult hemizygotic Tg mice of each line exhibited brain growth retardation (16.2%, 14.4% and 8.1% reductions in weight, respectively in each line). In two lines, total brain DNA and protein content were decreased. Further analysis indicated that the brain growth retardation was manifested in the second week of postnatal life. Given that the insulin-like growth factors (IGFs) stimulate cell proliferation and/or survival in neural cultures and that hIGFBP-1, when present in a molar excess, inhibits IGF interactions with their cell surface receptors, the brain growth retardation in hIGFBP-1 Tg mice likely results from hIGFBP-1 inhibition of IGF-stimulated growth-promoting actions. These hIGFBP-1 Tg mice should prove useful in defining IGF actions during postnatal brain maturation.
Aging↗
Subunit interaction in the CCAAT-binding heteromeric complex is mediated by a very short alpha-helix in HAP2.
We dissected the domain of HAP2 that mediates subunit association in the heteromeric CCAAT-binding complex, first by genetic mutational analysis and then by structural studies. The mutational data suggest that a very short region in HAP2 mediates protein-protein association and that the structure of this domain is likely to be an alpha-helix. The CD analyses of a 15-residue synthetic oligopeptide covering this region confirm this surmise. The oligopeptide indeed formed an unusually thermal stable alpha-helix in aqueous solution. Eight amino acids that lie along one face of this helix, including three arginines, are found to be critical for protein-protein association. The partner that interacts with this helical motif is likely to be another subunit in the HAP complex, since the CCAAT-binding factor is shown to contain one molecule of HAP2. Our results suggest that very short regions in proteins can encode precise structures and mediate stable and specific protein-protein recognition and interactions.
Amino Acid Sequence↗
Human insulin-like growth factor-binding protein-1 (hIGFBP-1) in transgenic mice: characterization and insights into the regulation of IGFBP-1 expression.
Three hemizygous transgenic (Tg) mouse lines were generated with a fusion gene composed of the mouse metallothionein promoter (mMT-I) and a full-length human insulin-like growth factor binding protein-1 (hIGFBP-1) complementary DNA that was truncated in its 3'-untranslated region. Despite high serum hIGFBP-1 levels (120-2570 micrograms/liter) before puberty in two of these lines, no significant alterations were observed in somatic growth, nor were marked alterations noted in fasting or random serum glucose or in the response of young adult Tg mice to ip glucose. The transgene was expressed in a number of tissues from each line, but liver was a significant site of transgene expression in only one line. Unexpectedly, liver hIGFBP-1 messenger RNA (mRNA) expression in this line was regulated in fashion similar to the native liver IGFBP-1 mRNA: 1) its abundance waned with advancing postnatal age and became minimal in early adult life, despite continuous zinc supplementation to stimulate its transcription; and 2) fasting increased its abundance 3- to 4.3-fold. The decline in transgene expression with aging was not due to a deletion, rearrangement, or a change in the methylation of liver transgene DNA. Transcriptional mechanisms also were not likely to account for the observed regulation of the transgene mRNA, because liver expression of the mMT-I gene, which shares identical genomic 5'-regulatory elements with the transgene, was not similarly altered by aging or fasting. Because cycloheximide (CHX) treatment of cultured rat H4IIE cells has been shown to prolong IGFBP-1 mRNA half-life while decreasing its transcription, Tg mice were treated with CHX to test the possibility that instability of the liver transgene mRNA influenced its abundance. After CHX and under conditions of chronic zinc supplementation, liver transgene mRNA abundance increased in parallel with that of the native IGFBP-1 mRNA. Although CHX is known to activate mMT-I transcription by mechanisms involving the 5'-regulatory regions contained in the transgene, CHX-induced transcription only in part accounted for the increase in liver transgene mRNA, because CHX induced an earlier and greater increase in liver transgene mRNA than in mMT-I mRNA. Taken together, these data indicate that both transgene and native IGFBP-1 liver mRNA are regulated by factors that alter mRNA stability. The finding that native liver IGFBP-1 mRNA abundance is influenced by transgene expression further supports the concept that both mRNAs share some common mechanisms of regulation.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Higher level organization of individual gene transcription and RNA splicing.
Visualization of fibronectin and neurotensin messenger RNAs within mammalian interphase nuclei was achieved by fluorescence hybridization with genomic, complementary DNA, and intron-specific probes. Unspliced transcripts accumulated in one or two sites per nucleus. Fibronectin RNA frequently accumulated in elongated tracks that overlapped and extended well beyond the site of transcription. Splicing appears to occur directly within this RNA track, as evidenced by an unambiguous spatial separation of intron-containing and spliced transcripts. Excised introns for neurotensin RNA appear free to diffuse. The transcription and processing site of the fibronectin gene localized to the nuclear interior and was associated with larger transcript domains in over 88 percent of the cells. These results support a view of nuclear function closely integrated with structure.
Animals↗
Mutations in yeast HAP2/HAP3 define a hybrid CCAAT box binding domain.
We describe a detailed genetic analysis of the DNA-binding regions in the HAP2/HAP3 CCAAT-binding heteromeric complex. The DNA-binding domain of HAP2 is shown to be a 21 residue region containing three critical histidines and three critical arginines. Mutation of an arginine at position 199 to leucine alters the DNA-binding specificity of the complex to favor CCAAC over CCAAT. Residues in HAP3 that are critical for DNA-binding comprise a short, seven amino acid region. Three different mutations in the HAP2 DNA-binding domain are suppressed by a mutation in the HAP3 DNA-binding domain. This HAP3 mutation also suppresses mutations in a different region of HAP2 which promotes subunit assembly of the complex. These findings suggest that short regions of HAP2 and HAP3 comprise a hybrid DNA-binding domain and that this domain can help hold the two subunits together in the CCAAT-binding complex.
Amino Acid Sequence↗
Nuclear RNA tracks: structural basis for transcription and splicing?
Knowledge of how the biochemical machineries governing metabolism and transport of several distinct classes of RNA may be organized and integrated into the structure of the nucleus remains very limited. Recent observations, including advances in the detection of specific nucleotide sequences directly within the nucleus, have heightened the long-standing interest in the structural organization of pre-mRNA transcription and processing.
Journal Article↗
Transformation-sensitive cells in G2/M-phase are not promoted by TPA following 137Cs gamma-rays.
Mouse C3H 10T1/2 cells are most sensitive to radiation-induced neoplastic transformation in the G2/M-phase of the cell cycle. When synchronized 10T1/2 cells were exposed to phorbol 12-myristate 13-acetate (TPA) after irradiation, transformation of cells not in the transformation-sensitive window was enhanced, but transformation of cells already in the transformation-sensitive window was not. Earlier work showed that (a) TPA enhances the frequency of transformation of both high and low dose-rate gamma-irradiated cells by about the same factor, but that (b) TPA enhances the transformation of cells exposed to low dose-rate fission spectrum neutrons appreciably less than cells exposed to high dose-rate fission spectrum neutrons. The latter observation is consistent with the inability of TPA to promote cells in the transformation-sensitive window and with the role of such cells in enhancing transformation by protracted doses of neutrons. The data provide a cellular basis for studying the biochemical/molecular aspects of TPA promotion in vitro.
Animals↗
Oxygen radicals as second messengers for expression of the monocyte chemoattractant protein, JE/MCP-1, and the monocyte colony-stimulating factor, CSF-1, in response to tumor necrosis factor-alpha and immunoglobulin G. Evidence for involvement of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase.
The potential involvement of reactive oxygen species in the expression of genes involved in immune response was examined in mesangial cells. Tumor necrosis factor (TNF-alpha) and aggregated (aggr.) IgG increased mRNA levels for the monocyte chemoattractant protein, JE/MCP-1, and the colony-stimulating factor, CSF-1. Scavengers for free radicals such as di- and tetra-methylthiourea (DMTU and TMTU) attenuated the increase in mRNA levels in response to TNF-alpha and aggr. IgG. Generation of superoxide anion by xanthine oxidase and hypoxanthine increased mRNA levels of these genes, but exogenous H2O2 did not. Addition of NADPH to activate a membrane-bound NADPH-oxidase generated superoxide and caused a dose-dependent increase in mRNA levels and further enhanced the stimulation by TNF-alpha or aggr. IgG. An inhibitor of NADPH-dependent oxidase 4'-hydroxy-3'-methoxy-acetophenone attenuated the rise in mRNA levels in response to TNF-alpha and aggr. IgG. By nuclear run-on experiments TNF-alpha, aggr. IgG and NADPH increased the transcription rates for JE/MCP-1 and CSF-1, effects inhibited by TMTU. We conclude that generation of reactive oxygen species, possibly by NADPH-dependent oxidase, are involved in the induction of the JE/MCP-1 and CSF-1 genes by TNF-alpha and IgG complexes. The concerted expression of leukocyte-directed cytokines represents a general response to tissue injury.
Animals↗