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1.
In vitro assembly of U1 snRNPs.   总被引:47,自引:10,他引:37       下载免费PDF全文
J Hamm  M Kazmaier    I W Mattaj 《The EMBO journal》1987,6(11):3479-3485
An efficient system for the in vitro assembly of U1 snRNPs is described. RNA-protein interactions in a series of U1 snRNA mutants assembled both in vivo and in vitro were studied in order to verify the accuracy of the system. Two discrete protein binding sites are defined by immunoprecipitation with antibodies against different protein components of the U1 snRNP and a newly developed protein sequestering assay. The U1 snRNP-specific proteins 70K and A require only the 5'-most stem-loop structure of U1 snRNA for binding, the common U snRNP proteins require the conserved Sm binding site (AUnG). Interactions between these two groups of proteins are detected. These results are combined to derive a model of the U1 snRNP structure. The potential use of the in vitro system in the functional analysis of U1 snRNP proteins is discussed.  相似文献   
2.
A I Lamond  B Sproat  U Ryder  J Hamm 《Cell》1989,58(2):383-390
We have used oligonucleotides made of 2'-OMe RNA to analyze the role of separate domains of U2 snRNA in the splicing process. We show that antisense 2'-OMe RNA oligonucleotides bind efficiently and specifically to U2 snRNP and demonstrate that masking of two separate regions of U2 snRNA can inhibit splicing by affecting different steps in the spliceosome assembly pathway. Masking the 5' terminus of U2 snRNA does not prevent U2 snRNP binding to pre-mRNA but blocks subsequent assembly of a functional spliceosome. By contrast, masking of U2 sequences complementary to the pre-mRNA branch site completely inhibits binding of pre-mRNA. Hybrid formation at the branch site complementary region also triggers a specific change which affects the 5' terminus of U2 snRNA.  相似文献   
3.
Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5'-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.  相似文献   
4.
Monoclonal antibodies to proteins important in phototransduction in the frog rod outer segment have been obtained. These include 6 different antibodies to rhodopsin, 50 to a guanine nucleotide binding protein (G-protein; 40,000 daltons), and 2 to cytoplasmic proteins. The antigens used were Percoll-purified rod outer segments, a rod outer segment soluble protein fraction, or a soluble plus peripheral membrane protein fraction. Antibodies were assayed by solid phase assay using a fluorogenic detection system. Proteins to which antibodies bound were assayed on Western blots, and the sensitivities of three different detection systems were compared. Most antibodies bound to only one rod outer segment protein band on Western blots. Immunofluorescence microscopy demonstrated binding of both anti-rhodopsin and anti-G-protein to isolated frog rod outer segments. Antibodies were purified from either culture supernatants or ascites fluid on protein A affinity columns. Two purified anti-G-protein antibodies have binding affinities to 125I-labeled G-protein of less than 10(-6) M-1. Of 11 antibodies to frog or bovine G-protein tested in solid phase and Western blot assays, all bind to the alpha rather than the beta or gamma subunits. Procedures developed here are being used in preparing other antibodies that affect reactions in the phototransduction pathway.  相似文献   
5.
6.
Male and female Aedes aegypti (L.) mosquitoes of the laboratory strain ROCK were irradiated with 130 mw of argon 514.5 nm laser microbeams for 0.04, 0.25, 0.4, and 0.5 s, respectively. Egg production, percentage hatch, and productivity (average number of adults surviving after 3 wk) were used to assess mutagenic effects. Mortality was high for males in all laser radiation groups and increased with time of exposure. Except for the group treated for 0.25 s, significant reductions in total F1 progeny also were demonstrated for all other experimentals when male parents were exposed to laser radiation. Females showed a high mortality when subjected to 0.4- and 0.5-s laser radiation. No F1 progeny were produced when parental females were exposed for 0.25, 0.4, and 0.5 s. Numbers of F1 progeny from females exposed to 0.04 s of laser radiation were significantly reduced. A comparison of weekly mean number of progeny showed that the important differences in productivity occurred during the first and second week, respectively, when either male or female adult parents were subjected to laser radiation.  相似文献   
7.
The ability of series of U1 snRNAs and U6 snRNAs to migrate into the nucleus of Xenopus oocytes after injection into the cytoplasm was analyzed. The U snRNAs were made either by injecting U snRNA genes into the nucleus of oocytes or, synthetically, by T7 RNA polymerase, incorporating a variety of cap structures. The results indicate that nuclear targeting of U1 snRNA requires both a trimethylguanosine cap structure and binding of at least one common U snRNP protein. Using synthetic U6 snRNAs, it is further demonstrated that the trimethylguanosine cap structure can act in nuclear targeting in the absence of the common U snRNP proteins. These results imply that U snRNP nuclear targeting signals are of a modular nature.  相似文献   
8.
Monomethylated cap structures facilitate RNA export from the nucleus   总被引:71,自引:0,他引:71  
J Hamm  I W Mattaj 《Cell》1990,63(1):109-118
RNA export from the nucleus has been analyzed in Xenopus oocytes. U1 snRNAs made by RNA polymerase II were exported into the cytoplasm, while U1 snRNAs synthesized by RNA polymerase III, and therefore with a different cap structure, remained in the nucleus. Export of the polymerase II-transcribed RNAs was inhibited by the cap analog m7GpppG. Spliced mRNAs carrying monomethylguanosine cap structures were rapidly exported, while hypermethylated cap structures delayed mRNA export. The export of a mutant precursor mRNA unable to form detectable splicing complexes was also significantly delayed by incorporation of a hypermethylated cap structure. The results suggest that the m7GpppN cap structure is likely to be a signal for RNA export from the nucleus.  相似文献   
9.
    
The limited proteolytic pattern of transducin,G t , and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the t subunit were identified. The t subunit in the GTPS bound form was cleaved into a major 38 kD fragment, whereas t -GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The t subunit was not cleaved and only a small portion of t was digested into several fragments. In order to determine which proteolytic fragment of t still contained the carboxyl terminal region, chymotrypsinization was carried out usingG t previously32P-labeled at Cys347 by petrussis toxin-catalyzed ADP-ribosylation. The32P-label was mainly associated with the t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not32P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of t , at Leu15 and Leu19. Chymotrypsin removed 15–19 amino acid residues from the amino terminus of t , generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp207 in a conformation-dependent manner. Trp207 of t -GTPS was resistant to proteolysis but t -GDP and the 38 kD fragments of t -GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus. This proves that the environment of Trp207 changes when GTP or GTPS is bound, leading to its inaccessibility to chymotrypsin.  相似文献   
10.
A new homogeneous catalyst system has been developed for the oxidation of olefins to carbonyls — ethylene to acetaldehyde and higher olefins to ketones. The catalyst system was first developed for the oxidation of ethylene to acetaldehyde in Wacker-type acetaldehyde plants. The aqueous catalyst solution has three key components. A palladium(II) catalyst oxidizes the olefin to the carbonyl, which is analogous to the Wacker system but with only a fraction of its palladium. Keggin phosphomolybdovanadates of the general formula PMo(12–x) V x O 40 (3+x)– provide a dioxygen-reversible vanadium(V)/vanadium(IV) redox agent for palladium(O) reoxidation, which is analogous to the copper(II)/copper(I) chlorides in the Wacker system. Chloride at centimolar concentrations, lacking in earlier reported palladium and polyoxometalate catalyst systems, is essential to maintain stable palladium(II) catalyst activity. Kinetic characterization and reaction engineering provided ethylene and oxygen reaction rates comparable to those obtained with the Wacker catalyst. A new, efficient method of preparing aqueous phosphomolybdovanadate solutions was developed for laboratory and large-scale production. This paper describes the catalyst system and its reactions with emphasis on the polyoxometalate chemistry.  相似文献   
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