Assignment of the human GABA transporter gene (GABATHG) locus to chromosome 3p24-p25.
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Animals↗
Biomedical subjects
L H Guo
Publications and source records attributed to L H Guo.
[Cell-specific expression of AFP gene is dependent on some nuclear proteins].
AFP is an oncodevelopmental protein. Its level decreases abruptly after birth and reaches almost undetectable level during normal adult life. However, reexpression of the gene can be observed during hepatocarcinogenesis. To further understand mechanism of regulating AFP expression, we checked several restriction enzyme map of 5' terminal and flanking sequences of AFP gene. There are no differences among adult rat liver, fetal liver and hepatoma cells. Using +2(-)-255 bp sequence probe of AFP gene to do southwestern blotting assay, the result showed that the gene-active cells, such as hepatoma cells, contained binding-proteins which were apparently lacking in adult rat liver, lung, spleen, heart and kidney cells. While the fractions of nuclear proteins from adult rat liver cells were devoid of any stimulatory effect on transcription, those of binding-proteins from hepatoma stimulated the transcription of AFP gene in vitro. The hepatoma binding-proteins can rescue transcription activity of fraction of nuclear proteins from adult rat liver cells. These results indicate that cell-specific expression of AFP gene is regulated by protein-factors.
Animals↗
[Preparation of antibodies against zip product and the distribution of zip protein in Drosophila embryos].
Zip gene is required during the late neurogenesis of Drosophila. Molecular analysis of zip gene revealed that it encodes an integral membrane glycoprotein, functioning as a cellular recognition and/or adhesion molecule. It has been cloned into an expression plasmid pWR 590. By expression of this plasmid in E. coli, a fusion protein of zip with lacZ has been purified and used to prepare polyclonal antibodies from rabbits. After being identified by West blotting, antibodies were used to react with the whole mount embryos of Drosophila. The results of this antibody labelling experiment showed that zip proteins are fundamentally synthesized after germ band shortening, supporting our previous predication that zip gene is involved in the late neurogenesis and that antibodies against zip recognize lateral neural fascicles as well as neurons in CNS, proving that zip protein expressed in CNS may play a role in the establishment and maintenance of neural fasciculation.
Animals↗
Molecular cloning and structure of the human (GABATHG) GABA transporter gene.
A cDNA molecule encoding the human GABA transporter was synthesized by means of polymerase chain reaction (PCR) technique and used as probe for selecting a human genomic DNA fragment encoding GABA transporter. A positive clone harboring the whole gene was obtained from a human lymphocyte genomic library through utilizing the genomic 'walking' technique. The clone, designated as pHGAT, harbours a DNA fragment of about 39 kb in length inserted into the BamHI site in cosmid pWE15. The gene covers about 25 kb in length and is constituted by four EcoRI restricted fragments which are 13.7 kb, 3.1 kb, 4.2 kb and 7.2 kb long, respectively. The genomic clone contains 15 introns, including two introns prior to the initiator methionine (i.e., the translation start site is in exon 3). Eleven exons encode the twelve transmembrane regions in the transporter protein. Thus as in the case for a number of other membrane proteins, there appears to be a strong tendency for the putative transmembrane domains to be encoded by separate exons. It is noted that the structure of the human GABA transporter gene reported here differs from the mouse gene which is contains 12 introns.
Base Sequence↗
[Studies on genetic engineering of human insulin-purification and characterization of human proinsulin and insulin].
E. coli DH 5 alpha cells harboring a plasmid pWR 590-BCA 4 for fused human proinsulin production were cultured. The fused human proinsulin was isolated from the fermented cells and then subjected it to cleavage with BrCN. The cleaved product was then converted to crude proinsulin-S-sulfonate using oxidative sulfitolysis. The isolation of human proinsulin-S-sulfonate was accomplished by ion exchange chromatography on QAE-sephadex A-25, followed by gel filtration on sephadex G-50. The purified human proinsulin-S-sulfonate was folded using a disulfide interchange method. The folding mixture was then chromatographed on sephadex G-50 and purified proinsulin was obtained. The proinsulin was then converted to human insulin and C-peptide by a combination cleavage with trypsin and carboxypeptidase B. The total yield of human insulin was about 5 mg/L The Zinc insulin crystals were obtained with amorphous human insulin using citrate method. The amino acid composition N-terminal sequences as well as C-terminal amino acid residues are in agreement with expected results. The hypoglycemic activity of purified human insulin is 26-27 U/mg, as judged by mouse convulsion assay, and the RIA activity is about 99% of that of porcine insulin.
Cloning, Molecular↗
[Synthesis cloning and expressions in E coli of human insulin A and B chain genes].
Human insulin A and B chain genes were designed and synthesized by using a rapid and simple method. The synthesized A and B chain genes were cloned separately. The expression (plasmids) pWR 590-HIA and pWR 590-HIB were constructed, and the two plasmids can direct the synthesis of the approximately 590 amino acid-long truncated beta-galactosidases fused to human insulin A or B chains. The fused A or B chain proteins were isolated from the fermented cells and cleaved with BrCN. The resulting mixtures were sulfonated and the sulfonated A and B chains were purified. Human insulin was obtained by using an A and B chain combination method.
Amino Acid Sequence↗
[Studies on the function of Ser579 and Arg580 in beta-subunit of penicillin G acylase with the method of site-specific mutagenesis].
According to the comparison of amino acid sequence between PGA (Penicillin G Acylase) and PBPs (Penicillin Binding Protein), We suggest that No. 565-595 peptide fragment in beta-subunit of PGA may be a substrate-binding site of enzyme. Plasmid pTZGA was constructed by cloning the 2.6 kb PGA gene of pWGA into phagemid pTZ18U The technique of site-specific mutagenesis was used to study the role of residue No. 579 (Ser) and No. 580 (Arg) of PGA. Four kinds of mutants were obtained (Ser579-->Gly579, Arg580-->Gly580, Arg580-->Glu580, Arg580-->Lys580), both Glu580 and Gly580 mutants showed no activity of enzyme and Lys580 mutant remained 30% and Gly579 mutant kept 70% activity of wilde type. The same protein expression of four mutants according to the results of ELISA indicate that mutation does not affect the expression of PGA, but Arg580 residue may be essential for substrate-binding or catalysis of PGA.
Amino Acid Sequence↗
Direct electrochemistry of proteins. Investigations of yeast cytochrome c mutants and their complexes with cytochrome b5.
Direct electrochemistry of site-specific mutants of yeast iso-1-cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge-plane pyrolytic graphite (EPG) and bis(4-pyridyl)-disulphide-modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a variety of electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile-Cys102Thr greater than Cys102Thr greater than Asn52Ala-Cys102Thr. Mid-point potentials follow the order: Arg13Ile (+60 +/- 5 mV vs. standard calomel electrode) greater than Cys102Thr (+40 +/- 5 mV) greater than Lys27Gln (+30 +/- 5 mV) approximately Lys72Asp (+30 +/- 5 mV) greater than Asn52Ala-Cys102Thr (+15 +/- 5 mV) greater than Asn52Ile-Cys102Thr (-10 +/- 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical response are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single-site variation could modify or abolish direct electrochemistry in the ternary adduct.
Animals↗
[Effect of mutagenesis at Ser 177 residue in penicillin G acylase on activity of the enzyme].
The technique of cassette and site-specific mutagenesis were used to study the role of residue No. 177 in penicillin G acylase (PGA, EC 3.5.1.11). Ser is conserved at residue No. 177 in all penicillin binding proteins. We got a series of mutants in which the amino acid at residue No. 177 was replaced by other amino acids through the site-specific and cassette mutagenesis, and we characterized the mutants by colony hybridization, NIPAB paper test and DNA sequence analysis. These mutants all show no activity of enzyme, even if the Ser residue was replaced by Thr, Gly and Ala respectively. The results show that Ser residue may be essential for substrate-binding or catalysis of PGA.
Escherichia coli↗
[Effect of temperature on the expression of penicillin G acylase gene at the level of post-translational processing].
The expression of penicillin G acylase (PGA) gene is sensitive to temperature. Cells of E. coli DH 5 alpha harboring a plasmid pWGA carrying a gene for PGA produce active PGA at low level at 37 degrees C, but can synthesize significant amount of the enzyme below 30 degrees C. Active PGA is formed from a 94 KD precursor processed through remove of a signal peptide and a spacer peptide to yield an enzyme that contain a 23 KD (alpha) and 65 KD (beta) subunit. This paper reported the effect of temperature on transcription, translation, and post-translational processing of PGA gene expression. We detected the transcription of PGA gene by Northern blot, and found the amount of PGA mRNA at 37 degrees C much more than that 30 degrees C when the cells of DH 5 alpha (pWGA) were cultured. It is evident that the precursor processing step(s) is sensitive to temperature.
Enzyme Precursors↗
Direct electrochemistry of protein-protein complexes involving cytochrome c, cytochrome b5, and plastocyanin.
The direct electrochemistry of the cytochrome c/cytochrome b5 and cytochrome c/plastocyanin complexes has been investigated at edge-plane graphite and modified gold electrode surfaces, which are selective for one of the two components of the complex. Electrochemical response of one protein at an otherwise electrostatically unfavorable electrode surface was achieved in the presence of the other protein, and the calculated heterogeneous electron-transfer rate constant and diffusion coefficient were found to be in good agreement with the values determined previously from the electrochemistry of the individual proteins [Armstrong, F. A., Hill, H. A. O., & Walton, N. J. (1988) Acc. Chem. Res. 21, 407 and references therein]. A dynamic model of the protein-protein-electrode ternary complex is proposed to explain the promotion effect, and this model is supported by a study comparing the electrochemical responses of covalent and electrostatic cytochrome c/plastocyanin complexes. It is also suggested that the behavior of protein-protein complexes at electrode surfaces could be related to that of the complexes associated with biological membranes.
Cytochrome c Group↗
Direct voltammetry of the Chromatium vinosum enzyme, sulfide:cytochrome c oxidoreductase (flavocytochrome c552).
The electrochemistry of the enzyme, sulfide:cytochrome c oxidoreductase, also known as flavocytochrome c552 from the purple sulfur bacterium, Chromatium vinosum, has been studied using several modified electrodes. Direct electron transfer between the heme of the flavocytochrome and an electrode is observed in the presence of a redox-inactive cationic species which promotes the voltammetry of the enzyme. Quasi-reversible electron transfer was achieved using the aminoglycoside, neomycin, as a promoter at either a modified gold or polished edge-plane graphite electrode. Further evidence for direct electron transfer is provided by the catalytic response of the enzyme at the electrode in the presence of substrate. Also reported is the direct spectroelectrochemistry of flavocytochrome c552 at an optically transparent thin layer gold electrode modified with Cys-Glu-Cys in the presence of neomycin.
Chromatium↗
[Whole nucleotide sequence of penicillin G acylase gene and its flanking region from E. coli].
Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)
Amidohydrolases↗
An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid.
A strategy for kilo-base sequencing of a target DNA cloned in plasmid pWR34 is described. A long target DNA is progressively shortened from one end, by digestion with BAL31 nuclease or exonuclease III and nuclease S1, followed by cleaving off the shortened vector DNA. The family of the shortened target DNA molecule is next cloned in between the StuI site on one end, and a cohesive-ended restriction site on the other end, within the polylinker region of pWR34. DNA fragments cloned into this plasmid are sequenced directly by using a synthetic oligonucleotide primer, which binds to one side of the polylinker region using the dideoxynucleotide chain-termination method. The plasmid DNA, easily obtained by adoption of a rapid mini-preparation, is usually pure enough for direct DNA sequencing. Thus, both strands of any DNA several thousand base pairs in length can be completely sequenced (using two different primers) with ease within a short time, without the need for constructing a physical map.
Base Sequence↗
Exonuclease III: use for DNA sequence analysis and in specific deletions of nucleotides.
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Base Sequence↗
Rapid methods for sequencing DNA.
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Base Sequence↗
Synthesis of the 3'-half molecule of yeast alanine tRNA.
This paper deals with the synthesis of the 3'-half molecule of yeast alanine transfer RNA (tRNAAlay) by ligation with T4 RNA ligase of three component oligonucleotide fragments corresponding to nucleotides 36-45(I), 46-57(II) and 58-76(III) in succession extending from the 3'-end to the 5'-end. First, in a ratio of acceptor to donor at 1.5 to 1, we adopted a method of three successive reactions, namely, the 5'-phosphorylation of the nonadecamer (III), ligation with the dodecamer (II) and the 5'-phosphorylation of the ligation product formed; with one isolation step and obtained the 5'-phosphorylated 31mer(46-76) (IV) in an overall yield of 70%. Then the 31mer(IV) as a donor was ligated with 3 times of decamer (I) to form the 41mer(36-76) (V), the 3'-half molecule of tRNAAlay. The yield was 67%. After 5'-phosphorylation, (V) was ligated with the natural 5'-half molecule to form the semi-synthetic tRNAAlay, which was biologically active, i.e. accepting and transferring (3H)-alanine into proteins.
Base Sequence↗
Assay of biological activity of synthetic yeast alanine transfer RNA (tRNAAlay).
The biological activity of the synthetic tRNAAlay was studied with an extremely sensitive method. tRNAAlay accepted alanine in the presence of rat liver aminoacyl-tRNAAlay-synthetase (this was called the accepting activity). The aminoacylated tRNAAlay was conveniently precipitated by ethanol with good recovery. The efficiency of transferring alanine from the aminoacylated tRNAAlay into the protein was determined in in vitro rabbit reticulocyte lysate cell-free protein-synthesizing system (this was called the incorporation activity). Both accepting and incorporation activities could be determined in one assay with only 5-7 pmoles of tRNAAlay either in ligation mixture or in purified form. Our results show that the accepting activities of the synthetic products were 51.6-65.6% and 91.3-106.0% of that of natural and reconstituted natural tRNAAlay respectively. The efficiency of the incorporation of alanine in the aminoacylated tRNAAlay into the protein was 61.6-63.1%, corresponding to 90.6-91.7% and 97.2-115.8% of that of the natural and the reconstituted natural tRNAAlay respectively.
Animals↗