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Biomedical subjects

X Mao

Publications and source records attributed to X Mao.

At least 109 records · Page 6Linked to original sources
PubMed1995

CCAAT-box contributions to human thymidine kinase mRNA expression.

In order to examine the role of two inverted CCAAT boxes near the start of transcription of the human thymidine kinase (TK) gene, a series of constructs were prepared in which one or both CCAAT boxes were deleted or mutated. These altered promoters (1.2 kb of 5'-flanking sequence) were used to express a TK minigene containing the first two exons and introns followed by the remainder of the cDNA. RNA blots were prepared from stable cell lines of ts13 cells containing these constructs under three conditions: 1) serum deprived cells, 2) serum stimulated cells, and 3) cells that had been stimulated with serum, but were arrested in the G1 phase of the cell cycle by the temperature sensitive mutation carried by these cells. TK mRNA expression from each construct was suppressed by the temperature sensitive block to cell cycle progression. Measurement of protein expression from the various altered TK promoters indicated that both CCAAT boxes contribute to promoter strength. These experiments also suggested that the two CCAAT boxes were not equivalent and that the distal CCAAT could substitute for the proximal CCAAT, but the converse was not true.

Animals↗
PubMed1995

Protein that binds to the distal, but not to the proximal, CCAAT of the human thymidine kinase gene promoter.

Mobility shift assays were used to examine protein binding to the human TK gene CCAAT boxes. Similar protein binding patterns were observed with probes containing either the proximal or distal CCAAT. However, probes containing both CCAAT boxes in which one of the CCAAT boxes was inactivated by mutation did not demonstrate identical binding patterns. One of the complexes formed with the longer probes was only observed when the distal CCAAT was intact. This species was not formed with probes that only contained an intact proximal CCAAT, and its formation could only be competed by oligonucleotides containing the distal CCAAT motif. This observation reveals the existence of a protein that can bind to the distal, but not to the proximal, CCAAT of the human TK promoter. This protein may account for the previous observation that the two CCAAT motifs are not functionally equivalent. The protein that binds to the distal, but not to the proximal, CCAAT (DTK-CBP) was also present in two human cell lines. Significantly more DTK-CBP was present in nuclear extracts of HepG2 and WI38 cells than in TK-ts13 cells. However, this protein was not observed in three different murine cell lines and one primary culture. Its abundance in some human cell lines suggests it might modulate the expression of human TK mRNA in cells that express this protein.

3T3 Cells↗
PubMed1995

Structural and functional analyses of the transcription-translation proteins NusB and NusE.

The NusB and NusE (ribosomal protein S10) proteins function in transcription and translation. The two proteins form a complex that binds to the boxA sequence found in the leader RNA of rrn operons; boxA is required for transcription antitermination in rrn operons. Although binding of these two proteins to the boxA RNA of the bacteriophage lambda nut site has not been observed, both NusB and NusE as well as the RNA boxA sequence are required for lambda N-mediated antitermination. Studies identifying the amino acid changes caused by mutations in nusB and nusE and relating these changes to altered function are reported. It is concluded that boxA is essential for an effective NusB contribution to N-mediated antitermination and that by mutation NusB may be changed to allow more-effective binding to boxA variants.

Alleles↗
PubMed1995

The D1 and D12 subunits are both essential for the transcription termination factor activity of vaccinia virus capping enzyme.

Transcription termination by vaccinia virus RNA polymerase during synthesis of early mRNAs requires a virus-encoded termination factor (VTF). VTF is but one of many activities associated with the vaccinia virus mRNA capping enzyme, a heterodimer of 95- and 33-kDa subunits encoded by the D1 and D12 genes, respectively. Although the three catalytic domains involved in cap formation have been assigned to individual subunits or portions thereof, the structural requirements for VTF activity are unknown. We now report that both full-length subunits are required for transcription termination. The 844-amino acid D1 subunit by itself, which is fully active in triphosphatase and guanylyltransferase functions, has no demonstrable VTF activity in vitro. Neither does the D12 subunit by itself. The heterodimeric methyltransferase domain of D1 (residues 498 to 844) and D12 subunits also has no VTF activity. VTF is not affected by a K-to-M mutation of the guanylyltransferase active site at position 260 (K260M) that abolishes enzyme-GMP complex formation or by a H682A/Y683A double mutation of the D1 subunit, which abrogates methyltransferase activity. Thus, the structural requirements for termination are distinct from those for nucleotidyl transfer and methyl transfer.

Base Sequence↗
PubMed1995

Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene.

RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.

Amino Acid Sequence↗
PubMed1994

The secondary structure of the ets domain of human Fli-1 resembles that of the helix-turn-helix DNA-binding motif of the Escherichia coli catabolite gene activator protein.

The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known. By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site. The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet. This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor. Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins. These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins.

Amino Acid Sequence↗
PubMed1994

Intrinsic RNA (guanine-7) methyltransferase activity of the vaccinia virus capping enzyme D1 subunit is stimulated by the D12 subunit. Identification of amino acid residues in the D1 protein required for subunit association and methyl group transfer.

Vaccinia virus mRNA capping enzyme, a heterodimer of virus-encoded D1 and D12 subunits, catalyzes three steps in the synthesis of the m7GpppN cap. By expressing portions of the subunits in bacteria, singly and together, we have localized the RNA (guanine-7) methyltransferase domain to a 305-amino acid carboxyl-terminal segment of the D1 polypeptide (residues 540-844) complexed with the D12 protein. We find that the purified carboxyl D1 protein has a weak intrinsic methyltransferase activity, indicating that the catalytic center resides within this subunit. The basal level of activity can be stimulated 100-fold by addition of purified D12 protein, which is itself catalytically inert. The carboxyl region of D1 forms a heterodimer with the D12 subunit in vivo and in vitro. Analysis of alanine substitution mutants of the D1 protein identifies amino acid residues important for subunit interaction. Our results suggest that subunit heterodimerization is necessary, but not sufficient, for full methyltransferase activity. A mutation of vicinal positions His-682-Tyr-683 that specifically affects catalytic activity but not subunit interaction implicates these residues as constituents of the active site.

Amino Acid Sequence↗
PubMed1994

The FLI-1 and chimeric EWS-FLI-1 oncoproteins display similar DNA binding specificities.

Although recent data have demonstrated that the chimeric EWS-FLI-1 cDNA isolated from cases of Ewing's sarcoma can transform NIH 3T3 cells, little is known about the basis for this transformation. Since FLI-1 and EWS-FLI-1 contain an Ets domain, both proteins may act as sequence-specific transcription factors. Here the DNA binding properties of FLI-1 and EWS-FLI-1 have been examined. An epitope-tagging strategy was developed to determine the optimum DNA-binding sequence of FLI-1. The alignment of cloned binding sequences showed a consensus DNA-binding site of ACCGGAAG/aT/c. This consensus sequence shows greater specificity for sequence 5' of the GGAA core site than those of other Ets proteins. Using several truncated forms of FLI-1, we show that the Ets domain is necessary and sufficient for the DNA binding specificity of FLI-1. The EWS-FLI-1 protein displayed the same DNA binding specificity and affinity as FLI-1 did. Despite their DNA binding similarities, the EWS-FLI-1 translocation product is likely to have a distinct pattern of expression from that of FLI-1 since the translocation results in the replacement of the 5' regulatory region of Fli-1 with that of EWS. Consistent with this we found that Fli-1 mRNA expression in lymphocytes was high in quiescent cells and disappeared upon activation while EWS mRNA expression was low in resting cells and increased in activated T cells. In summary, our data suggest that EWS-FLI-1 might act through the same target genes normally regulated by FLI-1, and EWS-FLI-1-induced transformation may result from dysregulation of FLI-1 target genes during cell proliferation and differentiation.

3T3 Cells↗
PubMed1994

Characterization of the three genotypes of low Km aldehyde dehydrogenase in a Japanese population.

A deficiency in low Km aldehyde dehydrogenase (ALDH2) is regarded as the main factor responsible for "Oriental flushing" and other symptoms due to alcohol sensitivity. In this study, the relationship of the ALDH2 genotype to alcohol-associated symptoms and drinking behavior was investigated in 524 Japanese workers, using a new, rapid, and nonisotopic polymerase chain reaction (PCR) method. Differences in the frequency of alcohol-associated manifestations between the normal homozygote and the other deficient types were apparent. In addition, among the ALDH2-deficient individuals, the atypical homozygote was obviously more hypersensitive to alcohol than the heterozygote, judging from the frequency of flushing or other drinking-associated manifestations with a small dose of alcohol. Drinking frequency also apparently decreased in the following order: typical homozygote, heterozygote, atypical homozygote. Similarly, mean amounts of alcohol consumption also decreased in the same order, although considerable variation existed within the typical homozygote and the heterozygote group. In contrast, neither the manifestations nor the drinking behavior were, in general, influenced by polymorphism of the alcohol dehydrogenase beta-subunit (ADH2) gene in males. These findings further indicate the important contribution of the ALDH2 genotype to alcohol sensitivity in Orientals.

Adult↗
PubMed1994

Heterogeneity of Kupffer cells and splenic, alveolar, and peritoneal macrophages for the production of TNF, IL-1, and IL-6.

Kupffer cells and alveolar, splenic, and peritoneal macrophages from normal rats were incubated for various periods of time in the presence of LPS, and the culture supernatants were analyzed for IL-6, IL-1, and TNF. There was very little difference in the amounts of the cytokines produced by the macrophages when stimulated with 0.01-10 micrograms/ml of LPS. The shapes of the time course curves for the production of the cytokines by the different types of macrophages were generally similar, although only Kupffer cells continued to produce IL-6 throughout the entire incubation period and splenic macrophages showed a lag period in the production of IL-1. Kupffer cells produced more IL-6 than that produced by the other populations of macrophages, and alveolar macrophages produced more IL-1 compared to that produced by splenic cells. Kupffer cells and peritoneal macrophages produced more IL-6 in 24 h than in 6 h of culture, and splenic macrophages produced more IL-1 in 24 compared to 6 h of culture. Alveolar macrophages produced more TNF than that produced by the other populations of cells but only when integrated over the entire incubation period. These results confirm and extend the observed functional heterogeneity of macrophages obtained from different tissues of the same animal. This study and future studies will lead to a better understanding of the role of cytokines in the inflammatory response.

Animals↗
PubMed1994

Solution structure of the ets domain of Fli-1 when bound to DNA.

Members of the ets family of transcription factors share a conserved DNA-binding domain, the ets domain. By using multidimensional NMR, we have determined the structure of the ets domain of human Fli-1 in the DNA-bound form. It consists of three alpha-helices and a four-stranded beta-sheet, similar to structures of the class of helix-turn-helix DNA binding proteins first found in the catabolite activator protein of Escherichia coli. NMR and mutagenesis experiments suggest that in comparison to structurally related proteins, the ets domain uses a new variation of the helix-turn-helix motif for binding to DNA.

Amino Acid Sequence↗
PubMed1994

Mutational analysis defines a C-terminal tail domain of RAP1 essential for Telomeric silencing in Saccharomyces cerevisiae.

Alleles specifically defective in telomeric silencing were generated by in vitro mutagenesis of the yeast RAP1 gene. The most severe phenotypes occur with three mutations in the C-terminal 28 amino acids. Two of the alleles are nonsense mutations resulting in truncated repressor/activator protein 1 (RAP1) species lacking the C-terminal 25-28 amino acids; the third allele is a missense mutation within this region. These alleles define a novel 28-amino acid region, termed the C-terminal tail domain, that is essential for telomeric and HML silencing. Using site-directed mutagenesis, an 8-amino acid region (amino acids 818-825) that is essential for telomeric silencing has been localized within this domain. Further characterization of these alleles has indicated that the C-terminal tail domain also plays a role in telomere size control. The function of the C-terminal tail in telomere maintenance is not mediated through the RAP1 interacting factor RIF1: rap1 alleles defective in both the C-terminal tail and RIF1 interaction domains have additive effects on telomere length. Overproduction of SIR3, a dose-dependent enhancer of telomeric silencing, suppresses the telomeric silencing, but not length, phenotypes of a subset of C-terminal tail alleles. In contrast, an allele that truncates the terminal 28 amino acids of RAP1 is refractory to SIR3 overproduction. These results indicate that the C-terminal tail domain is required for SIR3-dependent enhancement of telomeric silencing. These data also suggest a distinct set of C-terminal requirements for telomere size control and telomeric silencing.

Alleles↗
PubMed1994

Cardiorespiratory responses to low-intensity heart rate-controlled exercise in female subjects.

This study compares selected cardiorespiratory responses of six healthy females during exercise on rowing (R), cycle (C) and arm (A) ergometers. Their mean +/- sd age, mass and height were 22.3 +/- 3.0 years, 55.3 +/- 6.3 kg and 161.5 +/- 7.4 cm, respectively. Subjects reported to the laboratory on three separate days without engaging in intense physical activity the day prior to testing. Subjects sat quietly for five minutes on an ergometer prior to a 15 minute, steady-state exercise bout. Target heart rate during the exercise period was set at 115 bpm, and was monitored continually with workload adjustments applied to the ergometers to ensure that the target rate was maintained. CO2 rebreathing using the Sensormedics Horizon MMC was performed twice at steady-state exercise and averaged to examine changes in Q and SV. A one-way repeated measures ANOVA revealed that at similar levels of heart rate (R = 114.0 +/- 2.8; C = 116.0 +/- 2.8; A = 114.8 +/- 6.9 bpm) there were significantly lower Q and SV during arm ergometry (8.43 +/- 0.93 l/min and 72.61 +/- 7.82 ml) than rowing (13.01 +/- 1.61 l/min and 111.57 +/- 13.52 ml) and cycle (11.10 +/- 2.19 l/min and 88.23 +/- 21.94 ml) ergometry, and there were no significant differences in Q and SV between rowing and cycling. These results suggest that arm work during low intensity steady-state aerobic exercise is associated with a reduced stroke volume which is probably related to a reduced venous return during arm work in comparison to other types of exercise.

Adult↗
PubMed1994

[Molecular cytogenetic studies of soft tissue tumors].

Molecular cytogenetic studies of 25 cases of soft tissue tumors, including 8 malignant fibrous histiocytomas (MFH), 13 rhabdomyosarcomas (RMS), 3 Ewing's sarcomas (ES) and 1 synovial sarcoma (SS), were performed. Chromosomal analysis showed that of 17 analyzable cases, 15 had chromosomal numerical and structural abnormalities. In RMS, ES and SS consistent chromosomal abnormalities, t(2;13) (q37;q14), t(11;22) (q24;q12) and t(X;18) (p11;q11), respectively, were found, and the remaining 2 had normal karyotypes. Southern blot analysis demonstrated that in 14 cases with matched normal (N) and tumor tissue (T) DNA (5 MFH, 6 RMS, 2 ES, 1 SS), RFLPs changes including loss of allele, partial gene deletion, rearrangement and amplication had occurred at 8 loci (D1S57, D2S44, MYL1-3, D2S3, D13S1, D13S30, ESD and D17S5, respectively). Among these loci, gene changes on D1S57 and D2S44 were found to be shared by MFH, RMS, ES and SS, and D17S5, D2S3 and ESD abnormalities were shared by MFH, ES and SS, as well as by MFH and RMS. These results suggest that a multilocus and multistep process may be involved in the carcinogenesis of MFH, RMS, ES and SS.

Adolescent↗
PubMed1993

bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death.

We report the isolation of bcl-x, a bcl-2-related gene that can function as a bcl-2-independent regulator of programmed cell death (apoptosis). Alternative splicing results in two distinct bcl-x mRNAs. The protein product of the larger mRNA, bcl-xL, is similar in size and predicted structure to Bcl-2. When stably transfected into an IL-3-dependent cell line, bcl-xL inhibits cell death upon growth factor withdrawal at least as well as bcl-2. Surprisingly, the second mRNA species, bcl-xS, encodes a protein that inhibits the ability of bcl-2 to enhance the survival of growth factor-deprived cells. In vivo, bcl-xS mRNA is expressed at high levels in cells that undergo a high rate of turnover, such as developing lymphocytes. In contrast, bcl-xL is found in tissues containing long-lived postmitotic cells, such as adult brain. Together these data suggest that bcl-x plays an important role in both positive and negative regulation of programmed cell death.

Alternative Splicing↗
PubMed1993

Antigenic and molecular properties of Murayama virus isolated from cynomolgus monkeys: the virus is closely related to avian paramyxovirus type 2.

A new virus that belonged to a member of paramyxovirus was isolated from cynomolgus monkeys showing respiratory disorders about 20 years ago and was named Murayama virus (MrV). Interestingly, it showed no serological relationship with the mammalian paramyxoviruses tested. On the other hand, it was related to Yucaipa (YuV) and Bangor virus (BaV) belonging to avian paramyxovirus type 2 (PMV2). In analysis using anti-MrV monoclonal antibodies, MrV showed the closest relationship with YuV, and furthermore some conserved epitopes were found among avian paramyxovirus, MrV, YuV, BaV, and Newcastle disease virus (NDV). Subsequently, the nucleotide sequences of the F and HN genes of MrV were determined. In comparison with the deduced amino acid sequence of MrV and other paramyxoviruses, MrV showed the highest homology with NDV and higher similarity to HPIV2 group rather than to HPIV1 group. The present study clearly indicates that MrV belongs to PMV2 adapting to monkeys.

Amino Acid Sequence↗
PubMed1993

A position-dependent transcription-activating domain in TFIIIA.

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

ATP-Binding Cassette Transporters↗