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41.
42.
Mutations in Pol1 Increase the Mitotic Instability of Tandem Inverted Repeats in Saccharomyces Cerevisiae 总被引:5,自引:1,他引:4
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Tandem inverted repeats (TIRs or hairpins) of 30 and 80 base-pair unit lengths are unstable mitotically in yeast (Saccharomyces cerevisiae). TIR instability results from deletions that remove part or all of the presumed hairpin structure from the chromosome. At least one deletion endpoint is always at or near the base of the hairpin, and almost all of the repaired junctions occur within short direct sequence repeats of 4 to 9 base pairs. The frequency of this event, which we call ``hairpin excision,' is influenced by chromosomal position, length of the inverted repeats, and the distance separating the repeat units; increasing the distance between the inverted repeats as little as 25 base pairs increases their chromosomal stability. The frequency of excision is not affected by representative rad mutations, but is influenced by mutations in certain genes affecting DNA synthesis. In particular, mutations in POL1/CDC17, the gene that encodes the large subunit of DNA polymerase I, increase the frequency of hairpin deletions significantly, implicating this protein in the normal maintainance of genomic TIRs. 相似文献
43.
By screening total human DNA with probes derived from the small polydisperse circular (spc) DNA fraction of cultured human cells, we identified three clones that carry long stretches of -satellite DNA. Further experiments have shown that the three sequences belong to at least two different -satellite subfamilies, which are characterized by different higher order subunits. Members of one of these subfamilies are located in the cytological satellites of all acrocentric chromosomes, whereas members of another are located on the short arms of the acrocentrics on both sides of the stalk regions and also in the centromeric regions of chromosomes 1 and 9. This is the first time that -satellite sequences obtained from the spcDNA of human cells have been assigned to -satellite subfamilies that are organized as long arrays of tandemly arranged higher order monomers. This indicates that -satellite sequences can be excised from their chromosomal loci via intrastrand-recombination processes.This paper is dedicated to Prof. Dr. Ulrich Wolf on his 60th birthday, January 1993 相似文献
44.
X Xiao G Hintermann AL Demanin J Piret 《Journal of industrial microbiology & biotechnology》1996,16(4):261-262
Streptomyces glaucescens is shown to possess -lactamase activity which is inhibitable by clavulanate. This is important in regard to its use as a cloning host for enzymes of \-lactam biosynthesis. 相似文献
45.
Mutations in PMR1 suppress oxidative damage in yeast cells lacking superoxide dismutase. 总被引:11,自引:2,他引:9
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P J Lapinskas K W Cunningham X F Liu G R Fink V C Culotta 《Molecular and cellular biology》1995,15(3):1382-1388
Mutants of Saccharomyces cerevisiae lacking a functional SOD1 gene encoding Cu/Zn superoxide dismutase (SOD) are sensitive to atmospheric levels of oxygen and are auxotrophic for lysine and methionine when grown in air. We have previously shown that these defects of SOD-deficient yeast cells can be overcome through mutations in either the BSD1 or BSD2 (bypass SOD defects) gene. In this study, the wild-type allele of BSD1 was cloned by functional complementation and was physically mapped to the left arm of chromosome VII. BSD1 is identical to PMR1, encoding a member of the P-type ATPase family that localizes to the Golgi apparatus. PMR1 is thought to function in calcium metabolism, and we provide evidence that PMR1 also participates in the homeostasis of manganese ions. Cells lacking a functional PMR1 gene accumulate elevated levels of intracellular manganese and are also extremely sensitive to manganese ion toxicity. We demonstrate that mutations in PMR1 bypass SOD deficiency through a mechanism that depends on extracellular manganese. Collectively, these findings indicate that oxidative damage in a eukaryotic cell can be prevented through alterations in manganese homeostasis. 相似文献
46.
G R Fink J C Marshall P W Halligan C D Frith R S Frackowiak R J Dolan 《Proceedings. Biological sciences / The Royal Society》1997,264(1381):487-494
Neuropsychological evidence indicates that the global aspect of complex visual scenes is preferentially processed by the right hemisphere, and local aspects are preferentially processed by the left hemisphere. Using letter-based hierarchical stimuli (Navon figures), we recently demonstrated, in a directed-attention task, lateralized neural activity (assessed by positron emission tomography) in the left prestriate cortex during local processing, and in the right prestriate cortex during global processing. Furthermore, temporal-parietal cortex was critically activated bilaterally in a divided-attention task that involved varying the number of target switches between local and global levels of letter-based hierarchical stimuli. Little is known about whether such stimulus categories influence such hemispheric lateralization. We now present data on brain activity, derived from positron emission tomography, in normal subjects scanned during either local or global processing of object-based hierarchical stimuli. We again observe attentional modulation of neural activity in prestriate cortex. There is now greater right-sided activation for local processing and greater left-sided activation for global processing, which is the opposite of that seen with letter-based stimuli. The results suggest that the relative differential hemispheric activations in the prestriate areas during global and local processing are modified by stimulus category. 相似文献
47.
Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 104 pollen grains were GUS+. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS+ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS
ß-glucuronidase
- CaMV
Cauliflower Mosaic Virus
- MCS
multicellular structure
- NPTII
neomycin phosphotransferase
- PEG
polyethylene glycol
- X-gluc
5-bromo-4-chloro-3-indolyl glucuronide
- DAPI
4,6-diamidino-2-phenylindole
- Tris
Tris(hydroxymethyl)aminomethane hydrochloride
- EDTA
ethylenedinitrilo tetraacetic acid, disodium salt dihydrate 相似文献
48.
Experimental support for the use of fluid aqueous organic solvent systems and subzero temperatures in mechanistic studies of -galactosidase is presented. The enzyme was stable and retained catalytic activity and structural integrity in 50% aqueous dimethyl sulfoxide and 60% aqueous methanol at 0°C; at lower temperatures higher concentrations of cosolvent may be successfully used. The effects of dimethyl sulfoxide on the catalytic and structural properties of the enzyme were investigated in detail. For the -galactoside-catalyzed h ydrolysis ofo-nitrophenyl--D-galactoside the value ofk
cat decreased in a linear manner with increasing cosolvent concentration, whereasK
m increased exponentially. The decrease ink
cat paralleled the decrease in water concentration, consistent with rate-limiting hydrolysis of a galactosylenzyme intermediate. The increase inK
m is attributed to less favorable partitioning of the substrate to the active site in the cryosolvent compared to aqueous solution. ThepH*-rate profile for this reaction at 0°C in 50% dimethyl sulfoxide was similar to that in aqueous solution, withpK*1=5.8 andpK*2=8.0. Linear Arrhenius plots, with energies of activation of 13.9 and 16.0 kcal mol–1, respectively, were obtained for the -galactosidase-catalyzed hydrolysis ofo-nitrophenyl- andp-nitrophenyl--D-galactosides in 50% dimethyl sulfoxide at temperatures to –57°C. Examination of the intrinsic fluorescence and ultraviolet spectra of the enzyme as a function of increasing cosolvent concentration showed no evidence for structural perturbation up to and including 50% dimethyl sulfoxide at 0°C. We conclude that these cryosolvent systems are suitable for mechanistic investigations of -galactosidase, in particular for trapping intermediates at subzero temperatures. 相似文献
49.
A procedure is described for detecting enzymatically active yeast histidinol dehydrogenase-antibody complexes in immunodiffusion agar. The method employs a coupled dye system, consisting of a tetrazolium salt and a phenazine methosulfate intermediate, that produces an insoluble formazan and stains active precipitin lines red. The specificity of the reaction is indicated by its dependence on substrate and by its dependence on an intact HIS4C region, based on observations with mutant forms of the yeast HIS4 multifunctional protein. 相似文献
50.
Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus. 总被引:110,自引:0,他引:110
A Marshak-Rothstein P Fink T Gridley D H Raulet M J Bevan M L Gefter 《Journal of immunology (Baltimore, Md. : 1950)》1979,122(6):2491-2497
Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site. 相似文献