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Y D Liao

Publications and source records attributed to Y D Liao.

At least 19 recordsLinked to original sources
PubMed2001

Crystallization and preliminary X-ray diffraction analysis of cytotoxic ribonucleases from bullfrog Rana catesbeiana.

RC-RNases are ribonucleases from Rana catesbeiana oocytes with pyrimidine-guanine sequence specificity. They also possess cell cytotoxicity and lectin activity. Protein crystals of three RC-RNase isozymes, RC-RNase 3, RC-RNase 4 and RC-RNase 6, were grown in various crystal systems under different conditions. Crystals of RC-RNase3 belong to the orthorhombic C222(1) space group, with unit-cell parameters a = 66.66, b = 97.38, c = 85.74 A. Crystals of RC-RNase 4 belong to the trigonal space group P3(1) or P3(2), with unit-cell parameters a = b = 32.22, c = 92.12 A. Crystals of RC-RNase 6 complexed with cytidylyl 2'-5' guanosine belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 61.80, c = 65.96 A.

Amphibian Proteins↗
PubMed2001

Rapid diversification of RNase A superfamily ribonucleases from the bullfrog, Rana catesbeiana.

We present sequences of five novel RNase A superfamily ribonuclease genes of the bullfrog, Rana catesbeiana. All five genes encode ribonucleases that are similar to Onconase, a cytotoxic ribonuclease isolated from oocytes of R. pipiens. With amino acid sequence data from 14 ribonucleases from three Rana species (R. catesbeiana, R. japonica, and R. pipiens), we have constructed bootstrap-supported phylogenetic trees that reorganize these ribonucleases into five distinct lineages--the pancreatic ribonucleases (RNases 1), the eosinophil-associated ribonucleases (RNases 2, 3, and 6), the ribonucleases 4, the angiogenins (RNases 5) and the Rana ribonucleases--with the Rana ribonucleases no more closely related to the angiogenins than they are to any of the other ribonuclease lineages shown. Further phylogenetic analysis suggests the division of the Rana ribonucleases into two subclusters (A and B), with positive (Darwinian) selection (dN/dS > 1.0) and an elevated rate of radical nonsynonymous substitution (dR) contributing to the rapid diversification of ribonucleases within each cluster. This pattern of evolution-rapid diversification via positive selection among sequences of a multigene cluster-bears striking resemblance to what we have described for the eosinophil-associated ribonuclease genes of the rodent Mus musculus, a finding that may have implications with respect the physiologic function of this unique family of proteins.

Amino Acid Sequence↗
PubMed2000

Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog).

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.

Amino Acid Sequence↗
PubMed1998

The Rana catesbeiana rcr gene encoding a cytotoxic ribonuclease. Tissue distribution, cloning, purification, cytotoxicity, and active residues for RNase activity.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes. It possesses both ribonuclease activity and cytotoxicity against tumor cells. We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored. An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers. Full-length cDNA was obtained by 5'- and 3'-RACE technique. The cDNA clone, named rcr gene, contained a 5'-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3'-untranslated region, and a polyadenylation site. The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression in Escherichia coli strain BL21(DE3). The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity. The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes. Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity. Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes. This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily.

Amino Acid Sequence↗
PubMed1996

Large-scale preparation of a ribonuclease from Rana catesbeiana (bullfrog) oocytes and characterization of its specific cytotoxic activity against tumor cells.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found only in R. catesbeiana (bullfrog) oocytes, but not in other organs. The protein is localized in the yolk granules of oocytes but not in other organelles, as detected by immunohistochemistry. More than 99% of RC-RNase was found in the yolk granule pellet when a mild separation method was employed under physiological conditions. The ribonuclease was purified by precipitation of yolk granules, extraction of RC-RNase with 0.09 M NaCl, selective removal of impurities by Hepes buffer, and chromatographies on phosphocellulose and carboxymethyl cellulose columns. Three milligrams of RC-RNase was purified from a 1-g pellet of yolk granules prepared from 2 g of ovary tissue. Therefore, 150 milligrams of RC-RNase could be obtained from a mature female bullfrog (600 g in weight) which had 100 g of ovary tissue. The properties of RC-RNase isolated from yolk granules tested so far are identical to those of RC-RNase isolated from the cytosolic fraction and similar to those of a sialic acid-binding lectin from bullfrog oocytes. To investigate the possible role of RC-RNase in the regulation of cell growth and differentiation during embryogenesis, its cytotoxic activity against various cell lines was examined. The degradation of ribosomal RNA was found in RC-RNase-treated HeLa cells. However, both events were not found in RNase A-treated HeLa cells. Therefore, RC-RNase is proposed to have both ribonucleolytic and cytotoxic activity and a specific receptor on the tumor cell surface is suspected to be involved in the recognition and binding, and possibly entry of RC-RNase.

Amino Acid Sequence↗
PubMed1996

The secondary structure of a pyrimidine-guanine sequence-specific ribonuclease possessing cytotoxic activity from the oocytes of Rana catesbeiana.

RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a sialic-acid-binding lectin purified from Rana catesbeiana (bullfrog) oocytes. This 111-amino acid protein exhibits cytotoxicity toward several tumor cell lines. In this paper we report the assignments of proton NMR resonances and the identification of the secondary structure deduced from NOE constraints, chemical shift index, 3JNH alpha and amide proton exchange rates. The protein was directly isolated from bullfrog oocytes; we were able to assign all but five of the amino acid backbone protons of the unlabeled protein by analyzing a large set of two-dimensional proton NMR spectra obtained at several temperatures and pH conditions. Our results indicate that the structure of RC-RNase is dominated by the presence of two triple-stranded antiparallel beta-sheets and three alpha-helices, similar to those of the pyrimidine family ribonucleases. Two sets of resonances were observed for 11 amide protons and 8 alpha-protons located in the loop-1 region, an alpha 2 helix, and three beta-strands, (beta 1, beta 3 and beta 4), suggesting the presence of nonlocalized multiple conformations for RC-RNase.

Amino Acid Sequence↗
PubMed1995

Immunocytochemical localization of ribonuclease in yolk granules of adult Rana catesbeiana oocytes.

To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.

Amphibian Proteins↗
PubMed1994

Yolk granules are the major compartment for bullfrog (Rana catesbeiana) oocyte-specific ribonuclease.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine guanine sequence-specific ribonuclease found only in R. catesbeiana (bullfrog) oocytes, not in other organs. An immunohistochemical assay showed that RC-RNase was present in the regular yolk granules, but not in forming yolk granules, yolk platelets, pigment granules, mitochondria clouds or the nucleus. The RC-RNase was restricted to the lateral amorphous area of the yolk granules, and was absent from the central area that has a vitellogenin crystal lattice. The RC-RNase was extracted from yolk granules by 0.5 M NaCl and purified by dialysis and affinity chromatography. Most of the RC-RNase (94%) was found in the yolk granules, the rest RC-RNase (6%) was found in the cytosol in the form of free RNase and latent RNase. The RC-RNase extracted from yolk granules was further analyzed by immunoprecipitation and RNase activity assay on an SDS/polyacrylamide gel. Our results suggest that the RC-RNase activity is regulated by both compartmentation and inhibitor binding.

Animals↗
PubMed1992

A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes.

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.

Amino Acid Sequence↗
PubMed1989

Zinc release from Xenopus transcription factor IIIA induced by chemical modifications.

Xenopus transcription factor IIIA (TFIIIA) contains two tightly bound intrinsic Zn2+ ions that are released through treatment with either p-(hydroxymercuri)benzenesulfonate (PMPS) or diethyl pyrocarbonate (DEP) as monitored by the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR). The inactivation of TFIIIA by DEP as detected by an in vitro 5S RNA gene transcription assay was correlated with the extent of modification of histidine residues and Zn2+ release. Following reaction with PMPS, the 7S particle was dissociated into free TFIIIA and 5S RNA. This dissociation could be correlated with the extent of modification of cysteine residues as well as the Zn2+ release. The dissociation of the 7S particle was reversed by the addition of excess thiol reagent. However, the reversibility could be inhibited by EDTA, suggesting that Zn2+ was required for the binding of TFIIIA to 5S RNA. In the presence of PMPS- or DEP-modified TFIIIA or Zn2+-depleted TFIIIA, the fluorescence emission maximum of the hydrophobic probe, 8-anilinonaphthalenesulfonate, was blue-shifted by 30 nm, while only less than a 10-nm blue shift was observed in the presence of either the 7S particle or TFIIIA. These results indicate that the two Zn2+ ions in TFIIIA are coordinated with the cysteine and histidine residues and are required for maintenance of the proper conformation of TFIIIA.

Animals↗
PubMed1988

An N-terminally fused Xenopus transcription factor IIIA synthesized in Escherichia coli is biologically active.

A 1.5-kilobase DNA fragment containing the Xenopus transcription factor IIIA (TFIIIA) gene was inserted into the prokaryotic expression vector pIN-III(A) containing the lpp/lac promoter. The recombinant DNA was introduced into Escherichia coli K-12 strain SB221. The expression TFIIIA gene was induced by isopropyl-beta-D-thiogalactopyranoside, which resulted in the synthesis of a recombinant TFIIIA with an extra 17 amino acids fused to its N terminus as predicted from the nucleotide sequence. The engineered gene product, purified to at least 90% homogeneity, retained its binding affinity to the intragenic control region of the 5 S RNA gene, as well as its activity to stimulate 5 S RNA gene transcription in vitro.

Amino Acid Sequence↗
PubMed1988

Purification of Xenopus transcription factor IIIA and 5 S RNA from 7 S ribonucleoprotein particle by ammonium sulfate precipitation.

A simple and efficient method for purifying Xenopus transcription factor IIIA from the 7 S particle has been developed by taking advantage of the differential solubilities of the protein factor and 5 S RNA in ammonium sulfate solution. Conditions under which ammonium sulfate dissociates the 7 S particle and selectively precipitates factor IIIA while the 5 S RNA moiety remains in the supernatant were found. The method simultaneously purifies, in a nondestructive manner, both factor IIIA and 5 S RNA in high yield. Purification proceeds through several ammonium sulfate precipitations of the 7 S particle. Factor IIIA obtained by this method contains no detectable RNA and is highly active as judged by DNase I footprinting and in vitro transcription of the 5 S RNA gene, as well as reconstitution with 5 S RNA to form the 7 S particle. The molar extinction coefficients of factor IIIA at 205 and 280 nm were determined from the ultraviolet absorption spectra measured with the purified protein.

Ammonium Sulfate↗
PubMed1987

Regulation of transcription of the Xp10 genome in bacteriophage-infected Xanthomonas campestris pv. oryzae.

Results of in vivo studies showed that the transcription of the Xp10 genome in Xp10-infected cells shifted from rifampin sensitivity to rifampin resistance. Results of in vitro studies showed that a rapid reduction of rifampin-sensitive RNA polymerase activity coincided with a rapid increase of rifampin-resistant RNA polymerase activity in cell extracts with time after infection. Host and Xp10-encoded RNA polymerases were purified, and the transcripts from these two enzymes were hybridized to the restriction fragments of Xp10 DNA. The RNA probe generated by host RNA polymerase hybridized strongly to the leftmost 25% of Xp10 DNA and weakly to the rightmost 75% of Xp10 DNA. The RNA probe generated by Xp10 RNA polymerase hybridized strongly to the rightmost 75% of Xp10 DNA and weakly to the leftmost 25% of Xp10 DNA. Studies with 32P-labeled RNA isolated at various intervals after infection did not reveal any evidence for early versus late differences in transcription.

Bacteriophages↗
PubMed1986

Loss of sigma-factor of RNA polymerase of Xanthomonas campestris pv. oryzae during phage Xp10 infection.

Ten min after infection of Xanthomonas campestris pv. oryzae by phage Xp10, a sharp decrease in the activity of the host RNA polymerase was observed. Host RNA polymerase from phage-infected and uninfected cells was purified, and their properties were compared. The enzyme from uninfected cells contained four polypeptides with Mr = 155,000, 155,000, 93,000, and 37,000, respectively, and assembled with a stoichiometry of alpha 2 beta beta' sigma. The enzyme from infected cells lacked the sigma-subunit. The enzyme from uninfected cells utilized Xp10 DNA and poly[d(A-T)] as templates, the enzyme from phage-infected cells failed to transcribe Xp10 DNA, but retained the ability to transcribe poly(A-T). The regions of the Xp10 genome transcribed by the two enzymes were also investigated. The enzyme from uninfected cells transcribed the leftmost 25-30% of the Xp10 genome. The enzyme from phage-infected cells also transcribed the same region, but the enzyme activity was very low. Other properties such as (a) the response to RNA polymerase inhibitors, (b) the effect of N-ethylmaleimide, (c) the requirement of Mg2+ and Mn2+, and (d) the optimum temperature and pH of the two enzymes were very similar.

Bacteriophages↗
PubMed1986

Characterization of phage-Xp10-coded RNA polymerase.

A bacteriophage-coded RNA polymerase was isolated from bacteriophage-Xp10-infected Xanthomonas campestris pv. oryzae. The enzyme was purified to homogeneity through precipitation by poly(ethylene glycol) and chromatography on DEAE-cellulose, heparin--Sepharose 4B and blue-dextran--Sepharose 4B. It is composed of a single polypeptide of Mr96,000. The enzyme preferred denatured Xp10 DNA, calf thymus DNA, host bacterium DNA and poly[d(A-T)] as templates. The optimal concentration of MgCl2 is 16 mM. The optimal temperature and pH are 37 degrees C and 8.0, respectively. The Km of ATP is 26 microM. DNA, MgCl2 and four ribonucleotides were required for enzyme activity. If ATP alone was present, half of the Xp10 RNA polymerase activity was retained. The enzyme activity was inhibited by KCl, spermidine, actinomycin D, heparin, blue dextran and ethidium bromide; it was resistant to rifampicin and streptovaricin. N-Ethylmaleimide did not affect the enzyme activity. The transcription site and product of Xp10 RNA polymerase upon Xp10 DNA were analyzed by DNA/RNA hybridization and polyacrylamide-agarose composite gel electrophoresis. The enzyme could specifically transcribe the late region of Xp10 genome and produce two RNA bands.

Adenosine Triphosphate↗