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71.
72.
Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells. 总被引:2,自引:0,他引:2 下载免费PDF全文
The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide. 相似文献
73.
The constitution and properties of phosphorylated and unphosphorylated C-terminal fragments of progastrin from dog and ferret antrum 总被引:1,自引:0,他引:1
Antibodies to the extreme C-terminal tryptic (nona-) peptide fragment of porcine progastrin have been used in radioimmunoassay to identify progastrin fragments in dog, ferret and pig antral mucosa extracts and to monitor their purification. In addition to previously characterised phosphorylated and unphosphorylated C-terminal tryptic peptides of porcine progastrin a minor form corresponding to the C-terminal octapeptide (i.e. des-Ser C-terminal nonapeptide) was isolated and characterised. The latter form together with phosphorylated and unphosphorylated forms of the nonapeptides were also isolated and chemically characterised from dog antrum, and the unphosphorylated nonapeptide was characterised from ferret antrum. The primary amino acid sequences of the dog, ferret and pig nonapeptides were identical. In ferret the unphosphorylated nonapeptide predominated, and in dog the phosphorylated form predominated; in pig both forms of the nonapeptide were well represented. Intact progastrin was identified in gel filtration eluates of extracts of all 3 species, but occurred only in relatively low concentrations. The nonapeptides did not stimulate acid secretion in the conscious gastric fistula rat and they did not modify the acid response to G17. Phosphorylation of progastrin-derived peptides is evidently well conserved across a range of species even though there appear to be differences in the relative proportions of phosphorylated and unphosphorylated forms. 相似文献
74.
Dual translational initiation sites control function of the lambda S gene. 总被引:17,自引:3,他引:14 下载免费PDF全文
Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1-Lys2-Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem-loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer-extension inhibition or 'toeprinting', we show that the two S start codons are served by two adjacent Shine-Dalgarno sequences. Moreover, the timing of lysis supported by the wild-type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in-frame initiation events have opposing function. 相似文献
75.
A cdc2-3w weel-50 double mutant of fission yeast displays a temperature-sensitive lethal phenotype that is associated with gross abnormalities of chromosome segregation and has been termed mitotic catastrophe. In order to identify new genetic elements that might interact with the cdc2 protein kinase in the regulation of mitosis, we have isolated revertants of the lethal double mutant. The suppressor mutations define six mcs genes (mcs: mitotic catastrophe suppressor) that are not allelic to any of the following mitotic control genes: cdc2, wee 1, cdc13, cdc25, suc1 or nim1. Each mcs mutation is recessive with respect to wild-type in its ability to suppress mitotic catastrophe. None confer a lethal phenotype as a single mutant but few of the mutants are expected to be nulls. A diverse range of genetic interactions between the mcs mutants and other mitotic regulators were uncovered, including the following examples. First, mcs2 cdc2w or mcs6 cdc2w double mutants display a cell cycle defect dependent on the specific wee allele of cdc2. Second, both mcs1 cdc25-22 or mcs4 cdc25-22 double mutants are nonconditionally lethal, even at a temperature normally permissive for cdc25-22. Finally, the characteristic suppression of the cdc25 phenotype by a loss-of-function wee1 mutation is reversed in a mcs3 mutant background. The mcs genes define new mitotic elements that might be activators or substrates of the cdc2 protein kinase. 相似文献
76.
A lethally malformed neonate with mosaic tetrasomy 12p is presented. This is the third reported case of mosaic tetrasomy 12p to have died in the neonatal period. These three babies have shown a consistent phenotype characterized by dysmorphic facies and large diaphragmatic hernia. Mosaic tetrasomy 12p is usually not detectable from lymphocyte investigation, indicating that chromosome studies from cultured fibroblasts should be undertaken in neonates with multiple malformations which include a diaphragmatic defect. 相似文献
77.
A J Ping A E Reeve D J Law M R Young M Boehnke A P Feinberg 《American journal of human genetics》1989,44(5):720-723
Beckwith-Wiedemann syndrome (BWS), characterized by multiorgan developmental abnormalities and predisposition to cancer, usually occurs sporadically, but small apparently dominant pedigrees have been described. Since rare patients show varying karyotypic abnormalities on the short arm of chromosome 11, it has been suggested that BWS may be related to the Wilms tumor gene on 11p13 or, alternatively, to growth factor genes on 11p15. We performed genetic linkage analysis on two BWS kindreds, using RFLPs for loci on 11p. BWS was linked to the insulin gene (11p15.5), with an overall maximum lod score of 3.60 (recombination fraction = .00). Linkage to D11S16 (11p13) could be excluded for recombination fractions less than or equal to .03. These results suggest that BWS defines a tumor-predisposition gene on 11p15. 相似文献
78.
Studies on the structure of NADH: ubiquinone oxidoreductase complex: topography of the subunits of the iron-sulfur protein component 总被引:2,自引:0,他引:2
Resolution of the mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) by chaotropic agents result in the separation of three building blocks of the enzyme, designated FP (flavoprotein), IP (iron-sulfur protein), and HP (hydrophobic protein). FP contains three subunits of Mr 51, 24, and 9 kDa; one FMN; and two iron-sulfur clusters. Immunochemical studies with monospecific antibodies to the FP subunits have indicated that all three subunits of FP protrude from the inner mitochondrial membrane on the matrix side, whereas no reactive epitopes from these subunits were found exposed on the cytosolic side [A.-L. Han, T. Yagi, and Y. Hatefi (1988) Arch. Biochem. Biophys. 267, 490-496]. IP contains six subunits of Mr 75, 49, 30, 18, 15, and 13 kDa and four iron-sulfur clusters. In the present study, immunochemical experiments (enzyme-linked immunosorbent assays and 125I-protein A labeling) were carried out with monospecific antibodies to the above IP subunits and with bovine Complex I, submitochondrial particles, mitoplasts, and intact mitochondria as sources of antigens. Results have indicated that all six IP subunits protrude from the inner mitochondrial membrane into the matrix, and that the 75-kDa subunit, and possibly the 15-kDa subunit, protrude in mitoplasts from the cytosolic side as well. No epitopes reactive toward the monospecific antibodies to the 49-, 30-, 18-, and 13-kDa subunits were detected in mitoplasts. 相似文献
79.
Differential susceptibility of type III erythrocytes of paroxysmal nocturnal hemoglobinuria to lysis mediated by complement and perforin 总被引:1,自引:0,他引:1
S Jiang P M Persechini W F Rosse B Perussia J D Young 《Biochemical and biophysical research communications》1989,162(1):316-325
Previous reports have suggested that a 65 kDa membrane protein, termed homologous restriction factor (HRF), in addition to protecting erythrocytes (E) against lysis by homologous complement (C), may also be involved in protecting cytolytic lymphocytes against lysis mediated by a pore-forming protein (PFP/perforin), one of their own lytic mediators. Here, we used HRF-deficient type III E of patients with paroxysmal nocturnal hemoglobinuria (PNH) to study their susceptibility to lysis mediated by homologous C and perforin, and compared it with lysis of HRF-bearing control or PNH type I E. We show that type III E of PNH patients are indeed more susceptible to lysis mediated by homologous C than control or type I E, but they are as susceptible to perforin-mediated lysis as type I E. In addition, all human E (type I or III) tested here are equally susceptible to lysis mediated by either human (homologous) or murine (heterologous) perforin. By immunoblot analysis, we confirm that type III E, in contrast to type I E, were deficient in the 65 kDa HRF. These results support the notion that homologous species restriction is seen in the C- but not in the lymphocyte perforin-system and argue against an active participation of HRF in protecting cells from perforin-mediated lysis. 相似文献
80.
Resonance Raman (RR) spectra from the hemoprotein subunit of Escherichia coli sulfite reductase (SiR-HP) are examined in the low-frequency (200-500 cm-1) region where Fe-S stretching modes are expected. In spectra obtained with excitation in the siroheme Soret or Q bands, this region is dominated by siroheme modes. Modes assignable to the Fe4S4 cluster are selectively enhanced, however, with excitation at 488.0 or 457.9 nm. The assignments are confirmed by observation of the expected frequency shifts in SiR-HP extracted from E. coli grown on 34S-labeled sulfate. The mode frequencies and isotopic shifts resemble those seen in RR spectra of other Fe4S4 proteins and analogues, but the breathing mode of the cluster at 342 cm-1 is higher than that observed in the other species. Spectra of various ligand complexes of SiR-HP reveal only slight sensitivity of the cluster terminal ligand modes to the presence of exogenous heme ligands, at variance with a model of ligand binding in a bridged mode between heme and cluster. Close examination of RR spectra obtained with siroheme Soret-band excitation reveals additional 34S-sensitive features at 352 and 393 cm-1. These may be attributed to a bridging thiolate ligand. 相似文献