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1.
Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing. 总被引:13,自引:6,他引:7
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Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process. 相似文献
2.
Antisera elicited by immunization of rabbits with 4'-aminomethyl-trioxsalen (AMT)-modified poly(A,U) complexed with methylated bovine serum albumin was characterized in competition radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). AMT-poly(A,U) was over 10,000-fold more reactive than unmodified poly(A,U) or AMT alone. The antiserum cross-reacted to varying extents with AMT-modified-RNA's and -DNA's. The presence of AMT-uridine usually assured strong reactivity. The amino group of AMT contributed to antibody binding to a small degree. Binding was not significantly affected by high ionic strength, suggesting that binding does not involve ion pair formation. Murine encephalomyocarditis virus replicative intermediates, as well as cellular RNA and DNA were modified by psoralen in intact cells, suggesting that EMCV RNA and cellular RNA's in intact cells possess detectable stretches of base pairs. The antibodies described here will be useful in studying the secondary and tertiary structure of RNA's in vitro and in intact cells. 相似文献
3.
cDNA structures and regulation of two interferon-induced human Mx proteins. 总被引:33,自引:2,他引:31
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M Aebi J F?h N Hurt C E Samuel D Thomis L Bazzigher J Pavlovic O Haller P Staeheli 《Molecular and cellular biology》1989,9(11):5062-5072
Human cells treated with interferon synthesize two proteins that exhibit high homology to murine Mx1 protein, which has previously been identified as the mediator of interferon-induced cellular resistance of mouse cells against influenza viruses. Using murine Mx1 cDNA as a hybridization probe, we have isolated cDNA clones originating from two distinct human Mx genes, designated MxA and MxB. In human fibroblasts, expression of MxA and MxB is strongly induced by alpha interferon (IFN-alpha), IFN-beta, Newcastle disease virus, and, to a much lesser extent, IFN-gamma, MxA and MxB proteins have molecular masses of 76 and 73 kilodaltons, respectively, and their sequences are 63% identical. A comparison of human and mouse Mx proteins revealed that human MxA and mouse Mx2 are the most closely related proteins, showing 77% sequence identity. Near their amino termini, human and mouse Mx proteins contain a block of 53 identical amino acids and additional regions of very high sequence similarity. These conserved sequences are also present in a double-stranded RNA-inducible fish gene, which suggests that they may constitute a functionally important domain of Mx proteins. In contrast to mouse Mx1 protein, which accumulates in the nuclei of IFN-treated mouse cells, the two human Mx proteins both accumulate in the cytoplasm of IFN-treated cells. 相似文献
4.
Repair response of human fibroblasts to bleomycin damage 总被引:1,自引:0,他引:1
The ability of human fibroblasts to repair the specific types of DNA damage caused by bleomycin (BLM) was examined in whole-cell experiments. The method utilized for analysis was alkaline sucrose-gradient centrifugation of DNA. The results of these studies show that a repair pathway exists for the damage produced in DNA by bleomycin. DNA from BLM-treated cells shows a decrease in molecular weight, caused by chemical or enzymatic incision at sites of drug action. If the drug is removed, the DNA rapidly returns to high molecular weight, demonstrating reformation of damaged DNA. This repair response to BLM-damage was also confirmed in fibroblasts isolated from patients with putative DNA-repair defects. We observed that the response (to BLM) of cells from patients with Fanconi anemia was altered in that the fall in molecular weight of DNA from treated cells was not as great as that observed in other cell strains after drug treatment. 相似文献
5.
Binding of the gamma-subunit of retinal rod-outer-segment phosphodiesterase with both transducin and the catalytic subunits of phosphodiesterase.
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The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane. 相似文献
6.
Nuclear pore proteins are involved in the biogenesis of functional tRNA. 总被引:24,自引:3,他引:21
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G Simos H Tekotte H Grosjean A Segref K Sharma D Tollervey E C Hurt 《The EMBO journal》1996,15(9):2270-2284
Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA. 相似文献
7.
Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast 总被引:33,自引:5,他引:28
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R P Jansen E C Hurt H Kern H Lehtonen M Carmo-Fonseca B Lapeyre D Tollervey 《The Journal of cell biology》1991,113(4):715-729
NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo. 相似文献
8.
The pharmacology of N6-endonorbornan-2-yl-9-methyladenine (N0861), a new selective antagonist of adenosine at the A1 adenosine receptor subtype (A1-AdoR), was studied in vivo using a canine model. First, the pharmacokinetics of N0861 were determined in anesthetized dogs. The time-dependent decay of plasma levels of N0861 fitted a two-compartment polyexponential model with alpha-phase t1/2 = 3.80 min and beta-phase t1/2 = 80.55 min. Secondly, the effect of N0861 on the negative chronotropic and vasodilatory actions of adenosine in the canine heart were determined. N0861 attenuated the negative chronotropic action of adenosine (1-6 mumol/kg; rapid bolus into the right atrium) on sinus node pacemaker activity in a dose-dependent manner (pA2 = 4.23). For example, the maximal prolongation of sinus cycle length induced by 6 mumol/kg adenosine was 82 +/- 13% under baseline conditions and 57 +/- 10, 34 +/- 5 and 34 +/- 6% during infusion of N0861 at incremental rates leading to plasma levels of 7.75 +/- 1.02, 14.15 +/- 0.87, and 19.71 +/- 1.83 micrograms/mL, respectively. In contrast, N0861 did not inhibit but had a tendency to potentiate the vasodilatory action of adenosine (thought to be mediated by the A2 adenosine receptor subtype (A2-AdoR)) on the left anterior descending and circumflex coronary arteries. These data indicate that two different receptors, similar to the typical A1-AdoR and A2-AdoR, mediate the electrophysiologic and vasodilatory actions of adenosine in the canine heart, respectively, and that N0861 is a selective antagonist of adenosine at A1-AdoR in the canine heart in vivo. 相似文献
9.
The periplasmic histidine transport system (permease) of Escherichia coli and Salmonella typhimurium is composed of a soluble, histidine-binding receptor located in the periplasm and a complex of three membrane-bound proteins of which one, HisP, was shown previously to bind ATP. These permeases are energized by ATP. HisP is a member of a family of membrane transport proteins which is conserved in all periplasmic permeases and is presumed to be involved in coupling the energy of ATP to periplasmic transport. In this paper the nature of the ATP-binding site of HisP has been explored by identification of some of the residues that come into contact with ATP. HisP was derivatized with 8-azido-ATP (N3ATP). Both the underivatized and the derivatized forms of HisP were solubilized, purified, and digested with trypsin. The resulting tryptic peptides were resolved by high pressure liquid chromatography, and peptides modified by N3ATP were isolated and sequenced. Two peptides, X and Z, spanning amino acid residues 16-23 and 31-45, were found to contain sites of N3ATP attachment at His19 and Ser41, respectively. Both peptides are close to the amino-terminal end of HisP; peptide Z is located in one of the well conserved regions comprising the nucleotide-binding consensus motifs of the energy-coupling components of these permeases. These consensus motifs are found in many purine nucleotide-binding proteins. The relationship between the location of these residues and the overall structure of the ATP-binding site is discussed. 相似文献
10.
Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery. 总被引:57,自引:8,他引:57
M Carmo-Fonseca D Tollervey R Pepperkok S M Barabino A Merdes C Brunner P D Zamore M R Green E Hurt A I Lamond 《The EMBO journal》1991,10(1):195-206
The organization of the major snRNP particles in mammalian cell nuclei has been analysed by in situ labelling using snRNA-specific antisense probes made of 2'-OMe RNA. U3 snRNA is exclusively detected in the nucleolus while all the spliceosomal snRNAs are found in the nucleoplasm outside of nucleoli. Surprisingly, U2, U4, U5 and U6 snRNAs are predominantly observed in discrete nucleoplasmic foci. U1 snRNA is also present in foci but in addition is detected widely distributed throughout the nucleoplasm. An anti-peptide antibody specific for the non-snRNP splicing factor U2AF reveals it to have a similar distribution to U1 snRNA. Co-localization studies using confocal fluorescence microscopy prove that U2AF is present in the snRNA-containing foci. Antibody staining also shows the foci to contain snRNP-specific proteins and m3G-cap structures. The presence of major components of the nuclear splicing apparatus in foci suggests that these structures may play a role in pre-mRNA processing. 相似文献