Genetic mapping of the Gs-alpha subunit gene (GNAS1) to the distal long arm of chromosome 20 using a polymorphism detected by denaturing gradient gel electrophoresis.
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Base Sequence↗
Biomedical subjects
W T Hsieh
Publications and source records attributed to W T Hsieh.
Gene mapping of human cathepsins and cystatins.
Chromosomal locations of human cathepsin B, stefin A and kininogen have been determined. Using techniques of somatic cell hybrids and polymerase chain reaction, cathepsin B has been mapped to 8p22, stefin A to 3cen-q21, and kininogen to 3q26-qter. Comparing this data with currently available mapping loci, it can be concluded that many members of cathepsins are dispersed in different chromosomes, whereas members of the cystatin family are restricted to only two human chromosomes, 3 and 20.
Cathepsin B↗
Genetic and physical mapping of the human cannabinoid receptor gene to chromosome 6q14-q15.
A cDNA encoding a G protein-coupled receptor that appears to mediate the behavioral effects of cannabinoids, the psychoactive ingredients of marijuana, has recently been cloned from rat cerebral cortex and expressed. We have now determined the genomic location of the human cannabinoid receptor gene (CNR) by a combination of genetic linkage mapping and chromosomal in situ hybridization. The segregation pattern of a CNR DNA polymorphism was analyzed in 508 individuals from two or three generations of 40 families. Linkage of CNR to chromosome 6 centromeric loci and to DNA markers on the long and short arms was detected. CNR was tightly linked to D6S27, which is known to be located at 6q (log10 odds ratio [lod score, Zmax] of 10.54 at a recombination fraction [theta] of 0.02). Close linkage was suggested between CNR and CGA, the locus for the alpha subunit of human chorionic gonadotropin (Zmax = 2.71 at theta = 0). Moreover, CNR was linked to the two markers 308/BamHI (theta = 0.14) and 308/TaqI (theta = 0.20) defining locus D6Z1, an extended, highly repetitive, and highly conserved sequence localized exclusively to centromeres of all chromosomes and enriched on chromosome 6. In situ hybridization using a biotinylated cosmid probe localizes the gene to 6q14-q15, thereby confirming the linkage analysis and defining a precise alignment of the genetic and cytogenetic maps.
Cannabinoids↗
A TaqI DNA polymorphism in the human cathepsin B gene (CTSB).
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Cathepsin B↗
Manic depressive illness not linked to factor IX region in an independent series of pedigrees.
We studied seven informative kindreds segregating for manic depressive illness (MDI), consistent with X-chromosome transmission of the trait (families do not show affective disease in both a father and a son), using markers mapped to the region of Xq27-Xq28. The lod scores were consistently below -2 in the region extending from about 10 cM centromeric from the Factor IX locus (F9) to the colorblindness region. This study does not replicate previous reports of linkage of MDI to Factor IX (Xq27) and colorblindness region (Xq28) chromosomal markers in other kindreds.
Bipolar Disorder↗
In vivo existence of left-handed DNA.
A genetic-biochemical assay has been developed to investigate the in vivo existence and consequences of unusual DNA structures. Left-handed DNA was shown to exist in living Escherichia coli. The EcoRI methyltransferase gene (temperature-sensitive) was cloned to serve as a probe for perturbed GAATTC sites in vivo. This plasmid was cotransformed with different plasmids containing inserts that had varying capacities to form left-handed helices or cruciforms with a target EcoRI site in the center or at the ends of the inserts. Inhibition of methylation in vivo was found for the stable inserts with the longest left-handed helices. In vitro methylation with the purified M.EcoRI enzyme agreed with the in vivo results.
Bacterial Proteins↗
The effects of field inversion electrophoresis on small DNA fragment mobility and its relevance to DNA polymorphism research.
The electrophoretic mobility of several DNA size markers of molecular lengths from 1.23 x 10(2) to 2.36 x 10(4) base pairs has been investigated in gels of 1% and 1.3% agarose (w/v) by field inversion gel electrophoresis (FIGE), in vertical slabs. Pulsing times studied were in the range of 0.3/0.1 ms to 300/100 ms. (Pulsing times are given in milliseconds, as X/Y, where X is the forward pulsing time and Y is the reverse time.) FIGE differentially retards DNA migration: this effect is more marked for shorter pulsing times, and varies as a function of molecular length of the DNA fragment, down to fragments as small as about 1.7 kb, with the pulsing times we used. Several FIGE conditions were found which generate improved resolution of DNA fragments in different size ranges. DNA separation improves by more than a factor of two for fragments of 23.1-9.4 kb (with 300/100 ms pulsing) and for fragments of 4.4-2.0 kb (3/1 ms). FIGE does not seem to have a marked resolution enhancing effect on DNA sized from 9.4-4.4 kb. An example of improved detection of closely spaced bands on Southern blots is shown.
Blotting, Southern↗
Left-handed DNA in vivo.
Left-handed DNA is shown to exist and elicit a biological response in Escherichia coli. A plasmid encoding the gene for a temperature-sensitive Eco RI methylase (MEco RI) was cotransformed with different plasmids containing inserts that had varying capacities to form left-handed helices or cruciforms with a target Eco RI site in the center or at the ends of the inserts. Inhibition of methylation in vivo was found for the stable inserts with the longest left-handed (presumably Z) helices. In vitro methylation with the purified MEco RI agreed with the results in vivo. Supercoil-induced changes in the structure of the primary helix in vitro provided confirmation that left-handed helices were responsible for this behavior. The presence in vivo of left-handed inserts elicits specific deletions and plasmid incompatibilities in certain instances.
Cloning, Molecular↗
Influence of negative supercoiling and of the proximity of left-handed Z-DNA on the Escherichia coli lactose repressor-operator interaction.
The influence of negative supercoiling and of flanking (dC-dG) tracts in either right-handed B- or left-handed Z-structures on the interaction of the Escherichia coli lac repressor was investigated. The operator was embodied within the lac control sequence, which was 95, 59, or 29 base pairs in length. Thus, the (dC-dG) regions (in either B- or Z-conformations) were at different distances from the repressor-binding site. Surprisingly, the presence of the promoter sequence (-59 to -20 relative to the +1 transcription start site) of the lac operator region increases the binding affinity of lactose repressor to the operator at high negative supercoil densities. This influence of the promoter region on the binding was abolished when the flanking (dC-dG) tracts were in the left-handed Z-DNA conformation. In contrast, minimal differences in the binding affinities were observed between plasmids containing shorter operator fragments (59 or 29 base pairs), whether the flanking (dC-dG) tracts were in right-handed B- or left-handed Z-forms. The promoter region may be directly involved in the repressor-operator complex in a previously unrecognized manner or may exert structural influence on the operator region. In general, increasing the amount of negative supercoiling increases the binding affinity and decreases the dissociation rate constants for the three operator-containing fragments, both with and without flanking Z-DNA tracts. Thus, the lac operator region possesses a previously unrecognized structural pliability, as influenced by negative supercoiling and neighboring sequences and/or conformations, which modulates its biological properties.
DNA, Superhelical↗
Influence of sequence and distance between two operators on interaction with the lac repressor.
The influence of additional operator or pseudooperator sequences on the lactose repressor-operator interaction has been investigated. Results of kinetic and equilibrium binding measurements suggest an important in vivo role for the Z-gene pseudooperator in repressor-operator binding; the formation of a ternary, looped complex is indicated by the influence of secondary operator sites on binding parameters. Although the binding affinity of the Z-gene pseudooperator [Oz] is only approximately 1/30 that observed for the primary operator [O], the binding affinity to DNA containing both Oz and O is significantly higher than either sequence alone or the sum of the two. This synergistic effect is enhanced further by replacing the pseudooperator sequence [Oz] with the primary operator sequence and results in an even stronger ternary complex in plasmids with duplicate primary sites. The distance between the center of the two primary operators affects the formation of a ternary complex in the linear DNA molecules. Decreased dissociation rate constants were observed with spacing of operator-like sequences between 300 and 500 base pairs (bp). Minimal influence of the second operator on repressor binding is observed when the operators are separated by approximately 4000 or approximately 100 bp. The significant influence of distance on kinetic and equilibrium parameters was demonstrated by measurements on plasmid pRW1511 [Oi-O-PL-Oz] cleaved with restriction enzymes either in the polylinker region to place Oi-O and Oz on opposite ends of the linear plasmid or outside this region to maintain the sites within 500 bp. These results are consistent with the formation of operator-repressor-pseudooperator ternary complex to generate a looped DNA structure.
Base Sequence↗
Influence of supercoiling and sequence context on operator DNA binding with lac repressor.
The dissociation of the repressor-operator complex from a series of negatively supercoiled plasmid DNAs was examined as a function of the sequence context, orientation, and spacing. The plasmids were grouped into four classes, each with common sequence context. The highest dissociation rate constants were observed for the plasmids containing only a single operator (or pseudooperator) sequence, while approximately 10-fold lower rate constants were measured for plasmids with the I gene pseudooperator in conjunction with either the Z gene pseudooperator or the primary operator. Comparison of the behavior of these two classes of plasmids demonstrated the importance of two operator sequences and supported a model of DNA loop formation to stabilize the repressor-operator complex (Whitson, P. A., and Matthews, K. S. (1986) Biochemistry 25, 3845-3852; Whitson, P. A., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 3852-3858; Whitson, P. A., Hsieh, W. T., Wells, R. D., and Matthews, K. S. (1987) J. Biol. Chem. 262, 4943-4946; Krämer, H., Niemöller, M., Amouyal, M., Revet, B., von Wilcken-Bergmann, B., and Müller-Hill, B. (1987) EMBO J. 6, 1481-1491). The third class, with intermediate dissociation rate constants, was comprised of plasmids which contained the primary operator and the higher affinity pseudooperator normally located in the Z gene. Neither the additional presence of the I gene pseudooperator nor the orientation of the primary operator relative to the Z gene pseudooperator significantly affected the dissociation rate constants. The binding characteristics of this group of plasmids demonstrated the essential role of the Z gene pseudooperator in the formation of intramolecular ternary complex and suggested an in vivo function for this pseudooperator. Plasmids containing two primary operator sequences were the class with lowest dissociation rate constants from lac repressor, and minimal effects of salt or spacing on dissociation of this class were observed. These data are consistent with formation of an intramolecular complex with a looped DNA segment stabilized by the combination of increased local concentration of binding sites and torsional stresses on the DNA which favor binding in supercoiled DNA.
Base Sequence↗
Supercoiling facilitates lac operator-repressor-pseudooperator interactions.
The binding affinity of the Escherichia coli lactose repressor to operator-containing plasmids was increased by negative supercoiling of the DNA. The increased affinities observed were dependent on the sequence context of the DNA as well as the degree of supercoiling. Dissociation rate constants for plasmids containing a single operator site decreased as a function of the negative supercoil density. However, the presence of pseudooperators in the plasmid DNA in addition to the primary operator sequence resulted in a significant decrease in the operator-plasmid dissociation rate at higher negative supercoil densities. Approximately eight ionic interactions were determined for both the supercoiled plasmids and the linear DNAs examined. These results suggest that the stabilization provided by the topology of supercoiled DNA affects the nonionic component of the protein-DNA interaction. The ability to form a ternary complex of protein with two DNA segments is increased by the presence of multiple operator-like sites on the DNA. Furthermore, supercoiling DNA with multiple operator-like sequences profoundly diminishes the dissociation rate and results in a remarkably stable ternary, presumably looped complex (t1/2 approximately 28 h). These data suggest a critical role in vivo for DNA topology and pseudooperator(s) in transcriptional regulation of the lac operon.
DNA, Bacterial↗
Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.
The cysteine proteinase cathepsin B is one member of the lysosomal acid hydrolases. Based on the peptide sequence of rat liver cathepsin B, an oligonucleotide mixture containing 128 different 17-mers was synthesized and used as a probe to screen adult and fetal human liver cDNA libraries. A recombinant clone with a 1540-nucleotide insert was identified from the fetal library, and DNA sequence analysis confirmed that this clone encodes human cathepsin B. The clone, designated pCB-1, has sequences for 81% of the coding region (for amino acid residues 50-252) together with approximately equal to 880 nucleotides of the 3' untranslated region of the mRNA. The DNA sequence also shows that the predicted carboxyl terminus of the coding sequence is longer than the mature protein by 6 amino acid residues. Southern blot analysis of restriction enzyme digests of human placental DNA revealed a simple pattern of hybridizing fragments using the cathepsin B coding sequence as probe. The result suggests that there is a single copy of cathepsin B gene per haploid genome.
Amino Acid Sequence↗
Lactose repressor protein modified with dansyl chloride: activity effects and fluorescence properties.
Chemical modification using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) has been used to explore the importance of lysine residues involved in the binding activities of the lactose repressor and to introduce a fluorescent probe into the protein. Dansyl chloride modification of lac repressor resulted in loss of operator DNA binding at low molar ratios of reagent/monomer. Loss of nonspecific DNA binding was observed only at higher molar ratios, while isopropyl beta-D-thiogalactoside binding was not affected at any of the reagent levels studied. Lysine residues were the only modified amino acids detected. Protection of lysines-33 and -37 from modification by the presence of nonspecific DNA correlated with maintenance of operator DNA binding activity, and reaction of lysine-37 paralleled operator binding activity loss. Energy transfer between dansyl incorporated in the core region of the repressor protein and tryptophan-201 was observed, with an approximate distance of 23 A calculated between these two moieties.
Carbon Radioisotopes↗
Utilization of glutamate accumulating bacterial cells for production of ribonucleotides.
Corynebacterium glutamicum cells are industrially used for glutamate production. However, the waste that contains microbial cells, cellular debris, residual sugars, ammonia and metabolites seriously pollutes the environment. The cells are recovered and utilized for ribonucleotide production so that the pollution caused by the cells is eliminated. Nucleic acid is extracted from the cells and is hydrolyzed with nuclease P1 from Penicillium citrinum. The hydrolysate is fractionated with Dowex-50 and Dowex-1 into 5'-CMP, 5'-UMP, 5'-GMP and 5'-AMP. The products are characterized by electrophoresis, ultraviolet light absorbance, and 5'-nucleotidase.
5'-Nucleotidase↗
Releasing of intracellular glucoamylase from Aspergillus niger.
A method is presented for the release of glucoamylase (alpha-D-1,4-glucan glucohydrolase, E. C. 3,2,1, 3) from mycelia of Aspergillus niger. The process involves (a) suspension of mycelia at the stationary phase in water, and (b) homogenization and agitation of the suspension. The released intracellular enzyme is further purified by DEAE-cellulose.
Aspergillus niger↗
Tetranitromethane modification of the tyrosine residues of the lactose repressor.
Repressor protein modified with N-ethylmaleimide has been used to determine the exclusive effects of tyrosine nitration by tetranitromethane. Since modification of proteins with tetranitromethane generally results in both cysteine oxidation and tyrosine nitration, N-ethylmaleimide has been used to protect the cysteines in the repressor against oxidation in subsequent tetranitromethane reactions. Nitration of tyrosine residues in repressor previously reacted with N-ethylmaleimide results in loss of both specific and nonspecific DNA-binding activities. Na2S2O4 reduction of tetranitromethane-modified protein restores partial operator DNA-binding and complete nonspecific DNA-binding capability. Residues primarily affected are tyrosines 7 and 17, which are both in the NH2 terminus. Inter- and intramolecular cross-links which are observed in the modified protein can be minimized by altering reaction conditions; the cross-links present occur between sites located in the NH2 termini. Modification of the core protein also results in loss of the operator DNA-binding capacity, and subsequent reduction restores partial operator-binding activity. Both operator and nonspecific DNA-binding capabilities of the repressor protein are protected by the presence of nonspecific DNA during the tetranitromethane modification, and simultaneously the extent of nitration is decreased.
Binding Sites↗
Comparison of purified lactate dehydrogenases from normal rat liver and Morris hepatomas in rats and in culture.
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Amino Acids↗