Biomedical subjects

S M Liao

Publications and source records attributed to S M Liao.

13 recordsLinked to original sources

PubMed2001

[Cluster analysis of Dendrobium by RAPD and design of specific primer for Dendrobium candidum].

OBJECTIVE: To analyze the genetic relationship of Dendrobium and design a specific primer to differentiate Dendrobium candidum effectively. METHOD: Random amplified polymorphic DNA (RAPD) technique was used to analyze the genetic relationships of 26 species of Dendrobium and one species of Ephemerantha. Dendrogram was constructed by UPGMA. According to the sequence of DNA fragment selected, Sangon 18 primer had been extended from 3' extreme to 20 bp in order to form a specific primer. RESULT AND CONCLUSION: This primer can be used to distinguish Dendrobium candidum from other Dendrobium effectively. Such a technique provides a new way for the identification of Chinese traditional medicines.

Cluster Analysis↗

PubMed2000

IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex.

Here we report the identification of a novel PMA-inducible IkappaB kinase complex, distinct from the well-characterized high-molecular weight IkappaB kinase complex containing IKKalpha, IKKbeta, and IKKgamma. We have characterized one kinase from this complex, which we designate IKKepsilon. Although recombinant IKKepsilon directly phosphorylates only serine 36 of IKBalpha, the PMA-activated endogenous IKKepsilon complex phosphorylates both critical serine residues. Remarkably, this activity is due to the presence of a distinct kinase in this complex. A dominant-negative mutant of IKKepsilon blocks induction of NF-kappaB by both PMA and activation of the T cell receptor but has no effect on the activation of NF-KB by TNFalpha or IL-1. These observations indicate that the activation of NF-kappaB requires multiple distinct IkappaB kinase complexes, which respond to both overlapping and discrete signaling pathways.

Amino Acid Sequence↗

PubMed1998

Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases.

Two cyclin-dependent kinases have been identified in yeast and mammalian RNA polymerase II transcription initiation complexes. We find that the two yeast kinases are indistinguishable in their ability to phosphorylate the RNA polymerase II CTD, and yet in living cells one kinase is a positive regulator and the other a negative regulator. This paradox is resolved by the observation that the negative regulator, Srb10, is uniquely capable of phosphorylating the CTD prior to formation of the initiation complex on promoter DNA, with consequent inhibition of transcription. In contrast, the TFIIH kinase phosphorylates the CTD only after the transcription apparatus is associated with promoter DNA. These results reveal that the timing of CTD phosphorylation can account for the positive and negative functions of the two kinases and provide a model for Srb10-dependent repression of genes involved in cell type specificity, meiosis, and sugar utilization.

Cell Cycle↗

PubMed1995

Association of an activator with an RNA polymerase II holoenzyme.

RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and four SRB proteins. The SRB proteins, which were identified through a selection for genes involved in transcription initiation by RNA polymerase II in vivo, are a hallmark of the holoenzyme. We report here the isolation and characterization of additional SRB genes. We show that the products of all nine SRB genes identified thus far are components of the RNA polymerase II holoenzyme and are associated with a holoenzyme subcomplex termed the mediator of activation. The holoenzyme is capable of responding to a transcriptional activator, suggesting a model in which activators function, in part, through direct interactions with the holoenzyme. Immunoprecipitation experiments with anti-SRB5 antibodies demonstrate that the acidic activating domain of VP16 specifically binds to the holoenzyme. Furthermore, the holoenzyme and the mediator subcomplex bind to a VP16 affinity column. These results provide a more complete description of the RNA polymerase II holoenzyme and suggest that this form of the transcription apparatus can be recruited to promoters via direct interactions with activators.

Amino Acid Sequence↗

PubMed1995

A kinase-cyclin pair in the RNA polymerase II holoenzyme.

The RNA polymerase II holoenzyme consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins. The genes encoding SRB proteins were isolated as suppressors of mutations in the RNA polymerase II carboxy-terminal domain (CTD). The CTD and SRB proteins have been implicated in the response to transcriptional regulators. We report here the isolation of two new SRB genes, SRB10 and SRB11, which encode kinase- and cyclin-like proteins, respectively. Genetic and biochemical evidence indicates that the SRB10 and SRB11 proteins form a kinase-cyclin pair in the holoenzyme. The SRB10/11 kinase is essential for a normal transcriptional response to galactose induction in vivo. Holoenzymes lacking SRB10/11 kinase function are strikingly deficient in CTD phosphorylation. Although defects in the kinase substantially affect transcription in vivo, purified holoenzymes lacking SRB10/11 kinase function do not show defects in defined in vitro transcription systems, suggesting that the factors necessary to elicit the regulatory role of the SRB10/11 kinase are missing in these systems. These results indicate that the SRB10/11 kinase is involved in CTD phosphorylation and suggest that this modification has a role in the response to transcriptional regulators in vivo.

Amino Acid Sequence↗

PubMed1991

RNA polymerase II carboxy-terminal domain contributes to the response to multiple acidic activators in vitro.

The largest subunit of RNA polymerase II contains a unique carboxy-terminal domain (CTD) that consists of repeats of the heptapeptide YSPTSPS. RNA polymerase II CTD truncation mutations affect the ability to induce transcription of a subset of yeast genes in vivo, and the lack of response to induction maps to the upstream activating sequences of these genes. Here, we report that progressive truncation of the yeast RNA polymerase II CTD causes progressive loss of trans-activator-dependent transcription in nuclear extracts but has little effect on elongation or termination. Specific transcription, which is reduced by up to 50-fold in these assays, can be restored in the defective nuclear extracts by adding purified wild-type RNA polymerase II. The defects in factor-dependent transcription are observed with templates that are assembled into nucleosomes as well as with templates that are not so assembled. Defects in factor-independent transcription are also observed, but these are not as profound as those observed in the presence of trans-activators. These results indicate that the RNA polymerase II CTD functions during transcription initiation and is required for normal levels of activated transcription in vitro.

Amino Acid Sequence↗

PubMed1990

Subunits shared by eukaryotic nuclear RNA polymerases.

RNA polymerases I, II, and III share three subunits that are immunologically and biochemically indistinguishable. The Saccharomyces cerevisiae genes that encode these subunits (RPB5, RPB6, and RPB8) were isolated and sequenced, and their transcriptional start sites were deduced. RPB5 encodes a 25-kD protein, RPB6, an 18-kD protein, and RPB8, a 16-kD protein. These genes are single copy, reside on different chromosomes, and are essential for viability. The fact that the genes are single copy, corroborates previous evidence suggesting that each of the common subunits is identical in RNA polymerases I, II, and III. Furthermore, immunoprecipitation of RPB6 coprecipitates proteins whose sizes are consistent with RNA polymerase I, II, and III subunits. Sequence similarity between the yeast RPB5 protein and a previously characterized human RNA polymerase subunit demonstrates that the common subunits of the nuclear RNA polymerases are well conserved among eukaryotes. The presence of these conserved and essential subunits in all three nuclear RNA polymerases and the absence of recognizable sequence motifs for DNA and nucleoside triphosphate-binding indicate that the common subunits do not have a catalytic role but are important for a function shared by the RNA polymerases such as transcriptional efficiency, nuclear localization, enzyme stability, or coordinate regulation of rRNA, mRNA, and tRNA synthesis.

Amino Acid Sequence↗

PubMed1990

RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.

Cell Cycle↗

PubMed1990

[The function of 100% O2 suction key in Bennett 7200 microprocessor ventilator].

Tracheal suctioning of a patient on a ventilator may induce hypoxemia. Bennett 7200 microprocessor ventilator has an "100% O2 suction" key which is claimed to provide 100% oxygen for two minutes and switch back automatically when this function is set. Four 7200 ventilators were tested to verify this function. We found that the mean lag time varied from 20.1 seconds to 103.3 seconds and the mean recovery time varied from 33.0 seconds to 134.6 seconds according to the difference of initial tidal volume, ventilator rate and fractional inspired oxygen setting. When the ventilator was on "100% O2 suction", the delivered tidal volume was less than the preset tidal volume. In order to hyper-oxygenate and hyper-inflate the patient to prevent suction-induced hypoxemia, those finding should be taken into consideration when the clinicians apply this function in clinical situation.

Evaluation Studies as Topic↗

PubMed1987

Bacteriophage P22 Mnt repressor. DNA binding and effects on transcription in vitro.

We have examined the binding of Mnt repressor to operator DNA in vitro and have determined how this binding affects the level of transcription from two nearby promoters, Pant and Pmnt. Mnt binds to a region of DNA that overlaps the startpoint of transcription of Pant and the -35 region of Pmnt. Mnt represses transcription in vitro from Pant and enhances transcription from Pmnt. Protection and interference experiments show that Mnt binds to a single, 17 base-pair operator site. The operator sequence and the protein-DNA contacts are symmetric. Mnt makes major groove contacts on both faces of the operator DNA. At pH 7.5, 200 mM-KCl, 22 degrees C, the Mnt tetramer binds operator with high affinity (Kd = 2.2 X 10(-11M) and the protein-DNA complex is quite stable (t1/2 = 48 min). Operator binding shows large dependencies on pH, salt concentration, and temperature.

Base Sequence↗

PubMed1987

Interaction of the bacteriophage P22 Arc repressor with operator DNA.

Are repressor binds to a single, partially symmetric, 21 base-pair operator site that is centered between the -10 and -35 regions of the Pant promoter. Protection and interference experiments show that Arc makes contacts with the operator on one side of the DNA helix. Although Arc is a small protein (53 residues/subunit), it makes contacts that are farther from the center of the operator than those made by many larger repressors. These extended contacts include the phosphate groups at the ends of the 21 base-pair site. Under standard conditions (pH 7.5, 100 mM-KCl, 3 mM-MgCl2, 22 degrees C) half-maximal operator binding is observed at an Arc concentration of 2.5 X 10(-9) M and the protein-DNA complex is very stable (t1/2 approximately equal to 80 min).

DNA, Viral↗

PubMed1987

Control of gene expression in bacteriophage P22 by a small antisense RNA. I. Characterization in vitro of the Psar promoter and the sar RNA transcript.

The characterization in vitro of a newly discovered promoter (Psar) in the bacteriophage P22 immI region is described. Psar is located within the ant gene and is directed toward the major immI promoter, Pant. The entire intercistronic region between the P22 arc and ant genes (69 bp) is transcribed. The initiation and termination of sar (small antisense regulatory) RNA transcription are unusual. Frequent abortive initiation occurs in the presence of all four NTPs; RNA products 3-13 nucleotides in length are produced in about 15- to 25-fold larger numbers than full-length transcripts. Termination of sar RNA synthesis occurs after transcription of the first and second Ts of a TTTA sequence following a region of hyphenated dyad symmetry. The effects of convergent transcription between Pant and Psar were investigated on linear and supercoiled templates. Active transcription from Pant interferes with full-length transcription from Psar; several factors that interfere with Pant initiation (e.g., Pant down-mutation, Mnt repressor protein, Arc repressor protein) result in indirect activation of sar RNA synthesis. The sar RNA pairs rapidly with ant mRNA to form a stable stoichiometric complex. The location and properties of Psar suggest an important regulatory function for sar RNA as a negative effector of ant expression. The results of Wu et al. (this issue) support this suggestion.

Base Sequence↗

PubMed1987

Control of gene expression in bacteriophage P22 by a small antisense RNA. II. Characterization of mutants defective in repression.

Phage P22 produces antirepressor protein early after infection from a transcript initiated at the Pant promoter. After the first few minutes of infection, transcription from Pant is repressed by a protein encoded by the arc gene. Antirepressor is not produced late in infection, even though the antirepressor gene, ant, is transcribed from the late operon promoter Plate. We describe the isolation of P22 mutants that synthesize antirepressor from the Plate transcript. The mutations inactivate a promoter Psar, which lies within the ant coding sequence and directs the synthesis of sar RNA, a small antisense regulatory RNA complementary to the ant ribosome binding site. Characterization of the Psar down-mutants shows that transcription from Psar interferes with synthesis of antirepressor from both the Plate and Pant transcripts. Since sar RNA represses synthesis of antirepressor in trans, we propose that sar RNA base-pairs with ant mRNA to inhibit antirepressor synthesis at a post-transcriptional level. The role and importance of sar RNA in P22 biology are discussed.

Gene Expression Regulation↗