Molecular cloning vectors of Saccharomyces: generalized cloning vectors.
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Cloning, Molecular↗
Biomedical subjects
R W West
Publications and source records attributed to R W West.
Cocarcinogenicity of saccharin and N-alkylnitrosoureas in cultured human diploid fibroblasts.
Previous attempts to transform human foreskin fibroblasts in vitro with N-methylnitrosourea (MNU) or N-ethylnitrosourea (ENU) have been unsuccessful, and concurrent treatment with cocarcinogens or tumor promotors and either MNU or ENU have also failed to produce a neoplastic response. The present study was undertaken to test the effect of sodium saccharin on MNU- or ENU-induced cell transformation. Saccharin alone was not effective in inducing the growth of colonies in soft agar (anchorage-independent growth). However, concurrent treatment with saccharin (50 micrograms/ml, nontoxic dose) and MNU or ENU (29 micrograms/ml or 44 micrograms/ml, respectively) was effective in inducing transformation (greater than 300 colonies/10(5) cells), but only when the cells were treated with saccharin after being released from a G1 block (amino acid deprivation) and followed by MNU or ENU treatment in early S phase. In contrast to results obtained with other chemical carcinogens, transformation frequencies induced by saccharin and MNU or ENU were only slightly decreased in the absence of insulin, which is normally required for growth in this system. Saccharin-MNU- or saccharin-ENU-treated cells that exhibited growth in soft agar also exhibited cellular invasiveness in 9-d-old embryonic chick skin in vitro. In addition, these cells reacted with a monoclonal antibody prepared against a molecular weight 115,000 sarcoma-cell surface-associated glycoprotein and also developed tumors in nude mice. These data demonstrate the cell-cycle-dependent cocarcinogenic potential of saccharin and MNU or ENU in cultured human skin fibroblasts.
Antigens, Neoplasm↗
Standardization of the tangent screen examination: some neglected parameters.
Usually two factors are not rigidly standardized in the routine tangent screen examination: (1) the stimulus characteristics of the carrier that fixes the spot to the wand, and (2) patient instructions about what aspects of the stimulus they should attend to. Differences in these factors within the range of what is commonly done in the tangent screen test can lead to significant differences in the locations of the isopters. There is also an interesting interaction between these two factors in establishing isopter size.
Adult↗
Explanation of Broca's pupillometer.
Broca's pupillometer offers a simple and effective means of measuring the diameter of the pupil. However, it is often used without proper attention to the limitations of its optics. Errors of measurement may result from uncorrected refractive error, accommodation, spectacle correction, and spherical aberration (SA). A simple means of minimizing these errors is to hold the pinholes (PH's) of the pupillometer as close to the cornea as is feasible.
Accommodation, Ocular↗
GAL1-GAL10 divergent promoter region of Saccharomyces cerevisiae contains negative control elements in addition to functionally separate and possibly overlapping upstream activating sequences.
The upstream activating sequence (UASG) of the adjacent and divergently transcribed GAL1 and GAL10 promoters of Saccharomyces cerevisiae regulates the induction of the corresponding genes in response to the presence of galactose. We constructed chimeric yeast promoters in which a different UAS, UASC from the iso-1-cytochrome c (CYC1) gene of S. cerevisiae, was fused at different locations upstream of GAL1 (UASC-GAL1 promoters) or GAL10 (UASC-GAL10 promoters) and used to monitor the activity of UASG in cells grown in the presence or absence of galactose. Though the CYC1 promoter is fully induced in yeast grown in glycerol medium, UASC-GAL chimeric promoters containing UASG were repressed as much as 400-fold (UASC-GAL1) or 1350-fold (UASC-GAL10) in this growth medium. Several distinct portions of the GAL1-GAL10 divergent promoter region blocked the UASC-induced expression of the GAL1 and GAL10 promoters, whereas others did not, suggesting that several distinct negative control elements are present that may repress transcription of GAL1 and GAL10 in the absence of galactose. The approximate locations of these negative control elements were delimited to sites adjacent to or possibly overlapping the sites at which the positive control protein GAL4 binds in UASG. Deletion derivatives of GAL4 that fail to induce transcription from the wild-type GAL promoters but retain the DNA binding domain significantly derepressed the expression of the UASC-GAL chimeric promoters. These results, combined with those of earlier studies, suggest the possibility that GAL4 normally induces transcription of GAL1 and GAL10 by blocking the activity of these negative control elements, in addition to stimulating transcription by a mechanism of positive control.
Chromosome Mapping↗
The effects of saccharin on the development of neoplastic lesions initiated with N-methyl-N-nitrosourea in the rat urothelium.
Saccharin has been reported to induce urinary bladder tumors in multigeneration rat feeding studies and to promote bladder carcinogenesis in rats initiated with known bladder carcinogens. To examine the dose-dependent effects of saccharin on tumor promotion, sodium saccharin was administered at six levels in the diet (5.0, 2.5, 1.0, 0.5, 0.1, and 0%) to female Sprague-Dawley rats which had received, by trans-urethral instillation into the bladder, either a single dose of saline or an initiating dose of N-methyl-N-nitrosourea (MNU), a potent direct-acting carcinogen. Additional groups with and without MNU treatment received sodium saccharin (2%) in the drinking water, acid-saccharin (5%) in the diet, or MNU, four weekly doses, as a positive control. Histopathologic examination of the urinary bladders from dead and moribund animals and from animals sacrificed after 102 weeks on dose was performed, and benign papillomas were commonly observed in those animals given MNU. A statistical analysis of the lesions indicated an increase in tumor incidence and a decrease in time to tumor with increasing saccharin dose in dead and moribund animals. This response was observed in the dose series of 0 to 2.5% saccharin in the diet. Dead and moribund animals which had received 5% sodium saccharin exhibited few tumors. An increasing incidence of tumors in MNU-treated control animals was observed during the final weeks of the study. Although this increase in background tumors in senescent animals complicated the interpretation of the total tumor incidences, the results in dead and moribund animals (about 60% of the total) indicated that saccharin served as a tumor promoter in this two-stage carcinogenesis model system by decreasing the latency period of the lesions.
Animals↗
Benzyl derivative facilitation of transcription in Escherichia coli at the ara and lac operon promoters: metabolite gene regulation (MGR).
A number of benzyl derivatives have been tested for their ability to induce the expression of the araBAD operon in an Escherichia coli K-12 strain. Those derivatives shown to be stimulatory include: benzoic acid (BA), para-amino benzoic acid (PABA), para-hydroxy benzoic acid (PHBA), ortho-amino benzoic acid (OABA), 3-hydroxy-4-methoxy phenylethylamine (MTA), and 4-hydroxy-3-methoxyphenol acetic acid (HVA). The araC gene product was necessary to facilitate the induction. To further characterize if the inductive effect was mediated at the level of transcription, an araBAD-tetracycline resistant (Tcr) operon fusion plasmid (pAP-B) was employed. Benzyl derivatives which induce expression of the araBAD operon in situ also induced a Tcr phenotype with pAP-B. Both indole acetic acid (IAA) and imidazole (IM), which were previously shown to circumvent the necessity for cAMP in the induction of the araBAD operon, also induced a Tcr phenotype with pAP-B. Induction of lac or other cAMP responding operons with the inducing molecules at the chromosomal level was not detectable when assessed by carbon utilization. However, a lacZYA-Tcr operon fusion plasmid (pLPI) did respond to IAA and several of the inducing benzyl derivatives. Catabolite repression of chromosomal araBAD expression was reversed when the exogenous concentration of OABA was elevated. Similar effects on the Tcr phenotypes conferred by pAP-B and pLP1 were observed when OABA or several other inducing benzyl derivatives were present exogenously.
Aldose-Ketose Isomerases↗
Histidine operon control region of Klebsiella pneumoniae: analysis with an Escherichia coli promoter-probe plasmid vector.
The control region for the histidine operon of Klebsiella pneumoniae was cloned and analyzed with the Escherichia coli promoter-probe plasmid pPV33. A restriction fragment which contained the his control region was identified by its ability to activate the tetracycline resistance (Tcr) gene on this vector. Expression of Tcr by bacteria containing the his promoter-active plasmid was found to be under the attenuation control of the his promoter. DNA sequence analysis of the his control region revealed a base sequence homology of approximately 86% of the analogous DNA sequences of E. coli and Salmonella typhimurium. Most of the base alterations in the K. pneumoniae DNA sequence were found to reside in regions flanking the transcriptional and translational regulatory sites.
Alcohol Oxidoreductases↗
Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG.
The GAL1 and GAL10 genes, separated by 680 base pairs and divergently transcribed on chromosome 2 of Saccharomyces cerevisiae, were separately fused to the lacZ gene of Escherichia coli so that beta-galactosidase synthesis in S. cerevisiae reflected GAL1 and GAL10 promoter function. Analysis of two sets of deletions defined a 75-base-pair sequence, located ca. midway between the transcription initiation regions of GAL1 and GAL10, that mediates GAL4-dependent induction of both genes. Deletion of various parts of this sequence (called the GAL upstream activating sequence or UASG) reduced GAL1 and GAL10 induction about equally. Sequences in the GAL10-proximal half of UASG in some sequence contexts functioned independently of sequences in the GAL1-proximal half of UASG. A 33-base-pair deletion of the GAL10-proximal half of UASG drastically reduced induction. Deletions between UASG and the GAL1 TATA box caused beta-galactosidase to be synthesized at an unexpectedly high basal level, that is, in the absence of galactose and GAL4 product. Some of these mutations also reduced the repression caused by glucose.
Base Sequence↗
The role of urinary physiological changes in the genesis of urothelial lesions in mice given 4-ethylsulfonylnaphthalene-1-sulfonamide, acetazolamide, and oxamide.
BALB/c female mice were administered several compounds, including 4-ethylsulfonylnaphthalene-1-sulfonamide, acetazolamide, and oxamide, in the diet for six weeks. Fresh urine samples were analyzed three times per week for pH, osmolality, micro-crystals, and protein; and a histopathological evaluation was made of the urothelium at the end of the six weeks test. Incidences of hyperplasia, nodular hyperplasia, vacuolization, ulceration and acute inflammation of the bladder urothelium appeared to be related to the osmolality of the urine and the micro-crystalluria experienced by the mice. Correlation coefficients between lesions and urinary osmolality or crystals were -0.69 (p less than 0.0001) and 0.31 (p less than 0.03), respectively, at the 5% significance level.
Acetazolamide↗
Urothelial lesions in mice given 4-ethylsulfonylnaphthalene-1-sulfonamide, acetazolamide, and oxamide.
4-Ethylsulfonylnaphthalene-1-sulfonamide, acetazolamide and oxamide were administered in the diet to female BALB/c mice for varying periods of time from three to eighty weeks. All three compounds induced lesions in the urothelium. In the bladder, these included simple hyperplasia, nodular hyperplasia, inflammation and calculi. Similar lesions were observed in the ureter and urethra, along with a novel lesion, diverticulum, in the ureter. The diverticular lesions existed as down-growths of the transitional epithelium which often extended from the mucosa through the muscle layers to the adventitial surface. The etiology of the lesions appeared to be related to urinary physiological alterations (crystalluria, calculi, hypoosmolality) caused by administration of the compounds.
Acetazolamide↗
Synaptic contact curvature: effects of differential rearing on rat occipital cortex.
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Animals↗
Construction and characterization of E. coli promoter-probe plasmid vectors. III. pBR322 derivatives with deletions in the tetracycline resistance promoter region.
Deletions of the promoter region for the tetracycline-resistance (Tcr) gene(s) of pBR322 were constructed in order to generate new promoter-probe plasmid cloning vectors. The deletions were constructed in vitro by exonuclease digestion at the HindIII site and blunt-end ligation of the digestion products. Plasmids which lost the HindIII site but retained the EcoRI site carried deletions ranging from 5 to 60 bp. Some of the plasmids lacked the nucleotide sequences required for initiation of transcription from the Tcr promoter and "anti-Tcr" promoter. Three of the promoter-deletion plasmids (containing deletions of 5-29 bp) formed tight-binding complexes with RNA polymerase in vitro, despite their tetracycline sensitive phenotype. One deletion plasmid, pPV33, retained three out-of-phase stop codons located between the promoter-cloning site (EcoRI) and the translational start codon for the tetracycline resistance gene. These features give pPV33 several advantages over previously described promoter-cloning vehicles.
Cloning, Molecular↗
Isolation and characterization of the hisG and hisD genes of Klebsiella pneumoniae.
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ATP Phosphoribosyltransferase↗
Saccharin effects on the urinary physiology and urothelium of the rat when administered in diet or drinking water.
Three groups of 4 rats each were given 5% saccharin in the diet, 4% saccharin in the drinking water, or control diet, and were observed for changes in urinary physiology and pathology. Rats receiving saccharin in the water showed a significantly increased urinary osmolality over control values. Rats receiving saccharin in the diet showed significant increases in food and water consumption, urine elimination, and urinary sediment. Only the latter rats exhibited histopathological changes consisting of two instances of mild hyperplasia and one instance of mild cellular vacuolization.
Animals↗
The rapid purification of T4 DNA ligase from a lambda T4 lig lysogen.
A procedure has been developed for the rapid purification of the enzyme T4 DNA ligase. The procedure involves the induction at 42 degrees C of a lambda lysogen containing the gene for T4 DNA ligase (Murray, N.E., Bruce, S.A., and Murray, K. (1979) J. Mol. Biol. 132, 493-504), followed by purification of the ligase activity by phosphocellulose and hydroxylapatite chromatography. This results in the purification of large amounts of ligase with very high specific activity. The enzyme is free of contaminating exo- and endonuclease activities and active in the ligation of DNA fragments possessing cohesive or blunt-end termini.
DNA Ligases↗
Isolation of E.coli promoters from the late region of bacteriophage T7 DNA.
Promotor sequences recognized by Escherichia coli RNA polymerase have been isolated from bacteriophage T7 DNA using the plasmid pBRH4. T7 DNA was digested with the restriction endonuclease Hae III, Alu I, and Eco RI* and the products of these digestions were ligated into the EcoRI site of pBRH4. Cloning of Hae III and Alu I-digested T7 DNA was achieved by blunt-end ligation of these fragments to the polymerized ends of Eco-RI-cleaved pBRH4. This converts blunt-end Eco RI fragments of T7 DNA into cohesive-end EcoRI fragments. Promoter-containing T7 restriction fragments were selected by activation of the tetracycline-resistance gene located on the plasmid vector. The genomic location of each T7 insert was determined and Hpa I-cleaved T7 DNA. Two promoter-active restriction fragments are thought to contain the C and E promoters of T7. However, the majority, of the promoter-active fragments cloned map within the late gene region of T7. In vitro binding studies indicate that E. coli RNA polymerase can form heparin resistant complexes with the cloned T7 DNA promoter fragments. These results suggest that while E. coli RNA polymerase may not participate directly in the transcription of late T7 genes, promoters for this enzyme are present in this region of the DNA.
Cloning, Molecular↗
Construction and characterization of E. coli promoter-probe plasmid vectors. II. RNA polymerase binding studies on antibiotic-resistance promoters.
The binding of Escherichia coli RNA polymerase to antibiotic-resistance promoters was examined using the nitrocellulose filter assay. Four filter-retainable HaeIII fragments were observed with pBR322 and the promoter-probe plasmids, pBRH1, pBRH2 and pBRH4. Of the three fragments studied, two were shown to carry promoters for the ampicillin (Ap) and tetracycline (Tc) resistance genes, while the third present in pBRH1 appears to be the promoter for colicin E1 immunity (Colimm). Although the formation of filter-retainable complexes involving the Tcr promoter was sensitive to high salt, Apr promoter complexes were not. It was also shown that plasmids containing only the "firm-binding" portion of the Tcr promoter could still bind RNA polymerase in vitro despite the fact that these plasmids confer no in vivo Tcr. Additional filter-binding experiments performed with AluI-digested pBR322 DNA revealed the presence of a fifth RNA polymerase binding site on pBR322. This site is probably the promoter for the 100 bp transcript thought to be involved in the initiation of plasmid replication. An analysis of the recombinant plasmid (pKTR25) which carries the Kan-B portion of the EcoRI kanamycin (Kn) resistance fragment revealed that this fragment contains two RNA polymerase binding sites. We believe that these sites are responsible for the insertional activation of the Tcr gene and may be the promoters for the Knr and fusidic acid (Fa) resistance genes.
DNA, Bacterial↗