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1.
Ribonucleoprotein complexes composed of small molecular weight nuclear RNA (4--9 S) and proteins were isolated from hepatic nuclei of Rana catesbeiana (bullfrog) and the protein moiety of this nuclear ribonucleoprotein complex compared during different stages of development. SDS-polyacrylamide gel analysis of premetamorphic tadpoles and adult frog nuclear ribonucleoprotein complexes revealed that while the protein profiles of these two particles were very similar polypeptides of 47,000, 70,000, and 11,000 molecular weight were present in significantly higher concentrations in the frog ribonucleoprotein complexes. Comparison of the chromatin proteins isolated from these two developmental stages demonstrated that these three polypeptides of frog ribonucleoprotein were not contaminants from chromatin. Since these three polypeptides could not be preferentially extracted from the frog ribonucleoprotein complex by 0.5 M KCl or 1 M urea, it was unlikely that these polypeptides were bound nonspecifically to the ribonucleoprotein particle. Polypeptide analysis of the nuclear ribonucleoprotein complexes isolated from tadpoles immersed in the thyroid hormone L-thyroxine revealed an increase in two polypeptides of 37,000 and 45,000 molecular weight during metamorphosis. The absence of reduced amount of these two polypeptides in either the premetamorphic tadpole or adult frog demonstrated that their presence in Rana catesbeiana nuclear ribonucleoprotein was transient during development and specifically associated with tadpole metamorphosis. We conclude from these experiments that the nuclear ribonucleoprotein complex is a dynamic structure during Rana catesbeiana development and that specific changes in its protein composition are associated with discrete stages of amphibian development. 相似文献
2.
Abraham L. Kierszenbaum Munir Abdullah Hiroshi Ueda Laura L. Tres 《Molecular and cellular biochemistry》1989,86(2):171-179
We have previously reported that (a) polyclonal antisera raised against rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein recognize outer dense fiber polypeptides from rat sperm tail, and (b) protein S70 is antigenically related to polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein. We now report that polyclonal antisera generated against three different outer dense fiber polypeptides recognize (a) the putative antigen of the sperm tail and (b) Sertoli cell secretory protein S70 and its antigenically-related polypeptides. Immunogold electron microscopy shows that outer dense fibers of epididymal sperm crossreact with anti-S70 serum as well as with an antiserum raised against the polypeptide D complex of extracted outer dense fibers. Electron microscopy demonstrates that outer dense fibers consist of filamentous, coil-coiled units aligned side-by-side with each other. Results of this study strengthen the antigenic homology between Sertoli cell secretory proteins and outer dense fiber polypeptides of the sperm tail. 相似文献
3.
《Theriogenology》2013,79(9):2007-2019.e2
Although the existence of a complex population of mRNA in sperm is well documented, its role has not been completely elucidated. The objective of this study was to determine the relationship of mRNA abundance of sperm specific proteins and sire conception rates (SCR; a fertility index) in Holstein bulls. Samples of sperm from a single collection from commercial Holstein bulls (N = 34) were used to evaluate relative mRNA expression of adenylate kinase (AK) 1, integrin beta (IB) 5, Doppel, nerve growth factor, tissue inhibitors of metalloproteinases (TIMP) 2, lactate dehydrogenase C 1, small nuclear ribonucleoprotein polypeptide N, outer dense fiber 2, and phospholipase C zeta (PLCz) 1 in sperm. With the exception of lactate dehydrogenase C 1 and outer dense fiber 2, the mRNA abundances of these proteins were greater (P < 0.05) for high fertility (> +2 to ≤ 4 SCR) bulls compared with average (≥ 2 to ≤ +2) and low fertility (> −2 to ≤ −4) bulls. Of all the multivariate regression models tested, a combination of AK1, IB5, TIMP2, small nuclear ribonucleoprotein polypeptide N, and PLCz1 accounted for 97.4% of the variance in SCR scores. In the absence of PLCz1, the combination of AK1, IB5, Doppel, nerve growth factor, TIMP, and small nuclear ribonucleoprotein polypeptide N accounted for 96.6% of the variance in SCR scores. In addition, immunocytochemistry confirmed that the sperm-specific protein markers evaluated in this study were present in sperm. In conclusion, frozen-thawed semen from bulls with higher AK1, IB5, TIMP, small nuclear ribonucleoprotein polypeptide N 2 and PLCz1 mRNA abundances in the sperm had greater correlations with sire fertility index and may possess greater probabilities of siring calves. 相似文献
4.
In rats injected intraperitoneally with actinomycin D (2 mg/kg body weight) 12 h earlier, the yield of the 30S ribonucleoprotein particles isolated from liver nuclei by extraction with 0.1 M NaCl at pH 8.0 decreased by 60 per cent. The protein-to-RNA ratio of these particles increased to 32:1 from the ratio 4.4:1 found in the same particles isolated from the nuclei of liver of control rats. The particles isolated from the liver nuclei of rats injected with actinomycin D were depleted of all charge isomers of the two most prominent polypeptides (33,000 and 39,000 daltons) present in the particles of liver of control animals. The most abundant protein in these particles was a 43,000 dalton polypeptide. This polypeptide is the least prominent of the 3 major polypeptides present in the control particles. The same charge isomers of the 43,000 dalton polypeptide were present in the nuclear ribonucleoprotein particles isolated from the liver of control animals and from the liver of animals treated with actinomycin D 12 h earlier. In control animals the nuclear ribonucleoprotein monoparticles isolated from kidney contained 3 major polypeptides of the same molecular weight with the same distribution of their charge isomers as were present in the particles isolated from liver nuclei. The injection of actinomycin D 12 h earlier was without any effect on the protein composition of the 30S nuclear ribonucleoprotein particles of rat kidney. 相似文献
5.
Characterization of pre-messenger-RNA-containing nuclear ribonucleoprotein particles from avian erythroblasts. 总被引:2,自引:0,他引:2
Ribonucleoprotein particles have been isolated from duck erythroblast nuclei using a procedure designed to produce maximal cytoplasmic dispersion with minimal release of endogenous hydrolytic enzymes. The RNA extracted from the purified nuclear ribonucleoprotein fraction is shown to contain globin messenger RNA sequences at a concentration comparable to that present in total nuclear RNA. The polypeptide composition of this fraction revealed by electrophoresis in two dimensions is complex, consisting of at least 65 acidic species and 21 basic species. Several lines of evidence suggest that these are authentic components of nuclear ribonucleoprotein. The so-called 'core' proteins of nuclear ribonucleoprotein which were previously shown to migrate as a single band on low-pH urea gels, and as six bands on sodium dodecyl sulphate gels are here shown to be considerably more complex being resolved by two-dimensional electrophoresis into a group of 15 basic and 6 more and less neutral polypeptides. Isoelectric focusing of nuclear ribonucleoprotein under non-denaturing conditions suggests that these latter species are not uniformly distributed along the pre-messenger RNA molecule. 相似文献
6.
Janey Symington Theodore Gurney George L. Eliceiri 《Biochemical and biophysical research communications》1984,120(1):81-87
Whole nuclear and cytoplasmic fractions from HeLa cells were analyzed in protein gel blots probed with either monoclonal anti-Sm or polyclonal anti-(U1)RNP antibodies. The cells were fractionated by a nonaqueous procedure, to minimize proteolysis and artifactual leakage of nuclear components to the cytoplasmic fraction. Unexpectedly, more reactive proteins were detected in the nucleus than shown earlier in partially purified small nuclear ribonucleoprotein particles (snRNPs). In addition, reactive polypeptides were now found in the cytoplasm. These results are discussed in reference to the possibility that the nucleus and cytoplasm of adult somatic human cells may have a more complex than anticipated set of populations of polypeptides bearing Sm or RNP antigenic determinants, including some proteins that might not be in snRNP form. 相似文献
7.
G D Grothaus B J Packer M M Castilla M T Muller J P Kreier 《The Journal of parasitology》1984,70(6):849-858
In vitro, Plasmodium berghei infected erythrocytes incorporated 35S-methionine into 31 polypeptides with molecular weights from 21 kd to 300 kd. Hemoglobin and additional smaller molecular weight polypeptides were labelled with 35S-methionine by a population of uninfected, reticulocyte-rich rat erythrocytes. 3H-glucosamine was incorporated into at least 3 components by Plasmodium berghei infected erythrocytes. Uninfected, reticulocyte-rich rat erythrocytes did not incorporate 3H-glucosamine. Rabbit antisera against small, free plasmodia formed complexes which contained between 12 and 22 of the 31 labelled polypeptides in the 35S-methionine labelled antigen preparation. Rabbit antisera against soluble antigens washed from small, free plasmodia formed complexes containing many of the same labelled plasmodial polypeptides, however the reactions were particularly strong with those components which yielded polypeptides with molecular weights of 25 kd and 31 kd. Rabbit origin antisera against the 2 preparations did not form detectable complexes with the 3H-glucosamine labelled plasmodial components. Sera from rats undergoing progressive P. berghei infection formed complexes containing an increasing number of 35S-methionine labelled plasmodial polypeptides. Hyperimmune rat serum, the only serum protective upon passive transfer into mice, formed complexes containing 7 polypeptides with molecular weights of 35 kd, 75 kd, 80 kd, 92 kd, 100 kd, 150 kd and 190 kd. Antigens containing 1 or more of these polypeptides may be important in the induction of a protective antibody response against the parasite. 相似文献
8.
Formation of the perinuclear theca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides 总被引:1,自引:0,他引:1
The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus. 相似文献
9.
The nuclear matrix isolated from rat liver nuclei whose protein sulfhydryl groups were oxidised with the o-phenantroline-copper (OP-Cu) complex was enriched with a set of 32-44 kd polypeptides identified as core proteins of ribonucleoprotein particles (RNP). The most conspicuous protein in the nuclear matrix was a 36 kd protein present as a disulfide-linked homodimer. The propensity of protein 36 to be oxidised and form intermolecular associations suggests that it may contribute to the interaction of RNP particles with the nuclear matrix and thus to their spatial distribution in the nucleus. 相似文献
10.
It has been shown that small nuclear RNA (snRNA) species U1, U2, U4, U5, and U6 are found in the nucleus in the form of small nuclear ribonucleoprotein particles (snRNPs), and that anti-Sm antibodies react with snRNP polypeptides, which are associated with all five snRNAs. We report here a novel intranuclear complex, denoted “Sm cluster,” detected by immunostaining with monoclonal anti-Sm antibodies in HeLa cells. 相似文献
11.
The feeding of carcinogenic 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) in the early stages results in a change in the protein composition of the nuclear ribonucleoprotein particles of the rat liver. These particles are associated with newly synthesized RNA and it is assumed that they are involved in the processing and in the transport of this RNA. After 6 weeks of feeding of this azocarcinogen, the amount of one of the main polypeptides (apparent molecular weight 42 000) is decreased and after 10 weeks of feeding the particles are devoid of this polypeptide completely. Feeding of the non-carcinogenic p-aminoazobenzene (AB) is without any effect. The loss of this polypeptide is not characteristic for the malignant transformation. In the nuclear ribonucleoprotein particles isolated from hepatoma which has been induced by 3'-MeDAB this polypeptide is present in even higher proportion to other polypeptides than it is in particles isolated from liver cells of control animals. The 3'-MeDAB binds to the proteins of the liver nuclear ribonucleoprotein particles and interferes with the RNA processing. It is proposed that the changes in the composition of the protein moiety of the particles reflect changes in the population of liver cells leading finally to the selection of hepatoma cells which are resistant to the toxic effect of 3'-MeDAB on RNA processing. 相似文献
12.
Polypeptide components of Drosophila small nuclear ribonucleoprotein particles. 总被引:5,自引:1,他引:4
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In eukaryotes splicing of pre-mRNAs is mediated by the spliceosome, a dynamic complex of small nuclear ribonucleoprotein particles (snRNPs) that associate transiently during spliceosome assembly and the splicing reaction. We have purified snRNPs from nuclear extracts of Drosophila cells by affinity chromatography with an antibody specific for the trimethylguanosine (m3G) cap structure of snRNAs U1-U5. The polypeptide components of Drosophila snRNPs have been characterized and shown to consist of a number of proteins shared by all the snRNPs, and some proteins which appear to be specific to individual snRNP particles. On the basis of their apparent molecular weight and antigenicity many of these common and particle specific Drosophila snRNP proteins are remarkably conserved between Drosophila and human spliceosomes. By probing western blots of the Drosophila snRNP polypeptides with a number of antisera raised against human snRNP proteins, Drosophila polypeptides equivalent to many of the HeLa snRNP-common proteins have been identified, as well as candidates for a number of U1, U2 and U5-specific proteins. 相似文献
13.
Characterization of 20-S and 40-S non-polysomal cytoplasmic messenger ribonucleoprotein particles from rat liver 总被引:1,自引:0,他引:1
Two populations of free messenger ribonucleoprotein (mRNP) particles, sedimenting at 20 S and 40 S respectively, were isolated from a rat liver postpolysomal supernatant. After treatment with 0.5 M KCl and recentrifugation through a sucrose layer, the mRNP particles were characterized with respect to their low-molecular-weight RNA and protein components. 40-S and 20-S particles show very different RNA patterns. Four distinct low-molecular-weight RNA species of approximately 105, 139, 187 and 256 nucleotides were found as components of the 40-S mRNPs. The 20-S mRNP particles contain one major low-Mr RNA species of approximately 243 nucleotides and a characteristic pattern of low-Mr RNAs similar to the one found in nuclear ribonucleoprotein particles. In contrast to the low-Mr RNAs found in nuclear RNP particles most of the low-Mr RNA species present in 20-S and 40-S mRNP particles are rapidly labeled after [3H]orotate administration. Whereas the low-Mr RNA composition of 20-S and 40-S mRNP particles is very different, the protein patterns of both mRNP complexes are very similar. Six major polypeptides with the following molecular weights of 117000, 79800, 76700, 53800, 43900, 36300 and several minor ones were found in both 20-S and 40-S mRNPs. In a cell-free system from wheat germs neither 20-S nor 40-S mRNP particles stimulated the incorporation of [3H]leucine into proteins. However, phenol-extracted RNA from 20-S and 40-S mRNPs stimulated total protein synthesis 16-fold and 3-fold, respectively. Furthermore, the RNA from both mRNP pools directed the synthesis of albumin in vitro. 相似文献
14.
Karin A. Mattern Bruno M. Humbel Anton O. Muijsers Luitzen de Jong Roel van Driel 《Journal of cellular biochemistry》1996,62(2):275-289
The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa S3 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA. © 1996 Wiley-Liss, Inc. 相似文献
15.
16.
The isolation of total nuclear ribonucleoprotein particles fromDrosophila melanogaster embryos, using a pH 8.0, 01 M NaCl extraction of purified nuclei, is described. When the extract is fractionated on isokinetic sucrose gradients, at least six major classes of nuclear ribonucleoprotein complexes, differing in RNA and protein content as well as sedimentation behavior, are observed. The two largest complexes are preribosomal complexes. The remaining four major classes of RNPs sediment at roughly 6S, 8S, 12S and 30S. A minor class at 17S is also observed. The 30S fraction is 200–250 Å in width and appears to be analogous to the mammalian monoparticle. It is composed primarily of polypeptides at about 36 000 and 37 000 daltons, along with 1–2 kilobase RNA fragments. The 6S, 8S and 12S complexes contain a few discrete small nuclear RNAs from 80–600 bases in length, along with a small number of polypeptides, about 50 000, 52 000, 56 000 and 75 000 daltons. These novel complexes are of the order of a 100 Å in width (60–120 Å range). 相似文献
17.
A Martí M Matsuda R K Sihag G L Eliceiri 《Biochemical and biophysical research communications》1984,123(1):345-351
We have examined the polypeptide pattern of small nuclear ribonucleoprotein particles that react with monoclonal anti-Sm antibodies or polyclonal anti-(U1)RNP antibodies. The fresh nuclear extracts analyzed were prepared from human cells that had been pulse-chased with a mixture of 15 3H-labeled amino acids. In contrast to previous reports in the literature, the apparent molecular weights of the major polypeptides that remained in the 1 M NaCl-washed ribonucleoprotein-antibody complexes were approximately 80000, 55000, 28000, 25000, 14000 and 9000, when probed with monoclonal anti-Sm antibodies, and about 69000, 58000 and 35000, when polyclonal anti-(U1)RNP antibodies were used. 相似文献
18.
The composition of rat testis chromatin proteins in fractions produced by limited DNase II digestion followed by differential precipitation with MgCl2 has been studied. Over 50% of the acid-soluble proteins in the soluble chromatin fraction appeared to be quite similar to proteins which are associated with ribonucleoprotein (RNP) particles in HeLa cells. Although the ratios of the testis RNP protein components differed from those of HeLa RNP particles, the three major polypeptides were most similar to the HeLa components designated A2, B2, and C1. The soluble chromatin fraction was also enriched in the high mobility group proteins HMG1 and HMG2. 相似文献
19.
Munir Abdullah Laura L. Tres Hiroshi Ueda Ping-Chuan Hu Abraham L. Kierszenbaum 《Molecular and cellular biochemistry》1988,81(2):165-176
A high performance liquid chromatographic procedure has been used for the purification of rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein to determine their role during spermatogenesis. These two proteins display binding affinity for each other and appear antigenically related. We have observed that: 1. S70 and S45-S35 heterodimeric protein coelute during purification, 2. polyclonal antiserum raised against protein S70 recognizes common antigenic determinants in polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein, and 3. a monoclonal antibody that recognizes polypeptide S35 but does not crossreact with either protein S70 or polypeptide S45, immunoprecipitates the S70/S45-S35 heterodimeric protein complex. In immunofluorescent experiments, antisera raised against protein S70 and polypeptide components of S45-S35 heterodimeric protein immunoreact with two major sperm intracellular structures: the acrosome and periaxonemal outer dense fibers of sperm tail. Immunoreactivity was not detected on the sperm plasma membrane surface of unfixed, living sperm. Outer dense fibers extracted from sperm tails by a combined treatment with cetylthrimethylammonium bromide and 2-mercaptoethanol, yielded a characteristic polypeptide pattern. In immunoblotting experiments sperm tail polypeptides were recognized by polyclonal antisera raised against Sertoli cell secretory proteins. We conclude that Sertoli cell secretory proteins S70 and S45-S35 heterodimeric protein are antigenically related to each other and to keratin-like polypeptides from sperm tail. 相似文献
20.
Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: Further evidence for an in situ nuclear matrix 总被引:8,自引:0,他引:8
Harold C. Smith Robert L. Ochs Enrique A. Fernandez David L. Spector 《Molecular and cellular biochemistry》1986,70(2):151-168
Summary Polyclonal antibodies have been produced which react with a nuclear protein having a molecular weight of 107kD and a pl of 8.7–8.8 (designated p107). This protein is shown to be a component of the residual ribonucleoprotein (RNP) network of the nuclear matrix. P107 localized exclusively to the nuclear interior but not within nucleolar or chromatin domains. We have taken advantage of this unique probe to examine whether the RNP network of the isolated nuclear matrix has a physical counterpart in situ. We show that RNA, p107, divalent cations and the 28 kD Sm antigen of U-snRNPs are components of in situ macromolecular assemblies. While the morphology and intranuclear distribution of these assemblies are insensitive to the removal of chromatin, they are markedly altered by degradation of RNA. Digestion in situ of RNA in the presence of EDTA followed by extraction with high ionic strength buffers solubilized the components of these assemblies. Electron microscopic and immunobiochemical data are presented which support the concept that the residual RNP network of the nuclear matrix is an isolate of a pre-existing structure, and that perturbations in this internal network can be created by RNA degradation, depletion of essential metal ions and proteolysis.Abbreviations CRLM
polyclonal chicken antibody raised against rat liver nuclear matrix
- Sm
monoclonal antibody specific for the 28 kd protein antigen of U1, U2, U4, U5 and U6 snRNPs
- hnRNP
ribonucleoprotein particles containing hnRNA
- snRNP
ribonucleoprotein particles containing snRNA
- PBS
phosphate buffered saline
- PMSF
phenylmethylsulfonyl fluoride
- PAGE
polyacrylamide gel electrophoresis
- EDTA
ethylenediaminetetraacetic acid
- VRC
vanadium ribonucleoside complex
- BSA
bovine serum albumin
- DMSO
dimethylsulfoxide
- HS
high salt buffer
- LS
low salt buffer 相似文献