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S A Adam

Publications and source records attributed to S A Adam.

36 records · Page 2Linked to original sources
PubMed1991

Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import.

We have purified two major polypeptides of 54 and 56 kd from bovine erythrocytes that specifically bind the nuclear location sequence (NLS) of the SV40 large T antigen. When added to a permeabilized cell system for nuclear import, the purified proteins increase by 2- to 3-fold the nuclear accumulation of a fluorescent protein containing the large T antigen NLS. The import stimulation is saturable and dependent upon the presence of cytosol. Nuclear protein accumulation in vitro is sensitive to inactivation by N-ethylmaleimide (NEM). NEM inactivation can be overcome by addition of the purified NLS-binding proteins to the import system. NEM treatment of the purified proteins abolishes their ability to stimulate import but does not affect NLS binding. Our results indicate that the NLS-binding proteins are NEM-sensitive receptors for nuclear import. At least one other NEM-sensitive cytosolic activity and an NEM-insensitive cytosolic activity are also necessary for protein import in vitro.

Amino Acid Sequence↗
PubMed1990

Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors.

We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completely inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport.

Amino Acid Sequence↗
PubMed1989

Identification of specific binding proteins for a nuclear location sequence.

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex and are directed by specific nuclear location sequences (NLS) in the proteins. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells and the nuclear accumulation of chimaeric proteins or of non-nuclear proteins conjugated to synthetic peptides. The best-characterized NLS is the simian virus 40 large T-antigen sequence. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus.

Amino Acid Sequence↗
PubMed1989

The ribonucleoprotein structures along the pathway of mRNA formation.

Heterogeneous nuclear RNAs (hnRNAs), some of which are mRNA precursors, and the mature mRNAs are associated in eukaryotic cells with specific proteins to form ribonucleoprotein complexes (RNP). The RNP proteins are likely to play a major role in the formation, packaging, processing, and function of mRNA. The major proteins that interact with hnRNA and with mRNA were identified by photochemical RNA-protein cross-linking in intact cells and monoclonal antibodies to several of these proteins were produced. Using these antibodies the hnRNP proteins were characterized and the hnRNP complex was isolated from vertebrate cell nuclei. The hnRNP complex is a unitary structure of consistent, defined and conserved components. The proteins of the hnRNP complex were identified and the general organization of hnRNA and proteins in the hRNP complex were studied.

Animals↗
PubMed1987

Adenovirus proteins associated with mRNA and hnRNA in infected HeLa cells.

The proteins that interact with cytoplasmic and nuclear polyadenylated RNA in adenovirus type 5 (Ad5) infection of HeLa cells were examined by UV-induced RNA-protein cross-linking in intact cells. The Ad5 100-kilodalton late nonvirion protein (100K protein) was cross-linked to both host and viral polyadenylated cytoplasmic RNA (mRNA). The cross-linking of the 100K protein to mRNA appears to correlate with productive infection, because the protein is not cross-linked to mRNA in abortive infection of wild-type Ad5 in monkey cells (CV-1) even though normal amounts of it are produced. However, when CV-1 cells are infected with Ad5 hr404, and Ad5 mutant which overcomes the host restriction to wild-type Ad5 infection in these cells, the 100K protein is cross-linked to mRNA. To identify and obtain antibodies to RNA-contacting proteins, a mouse was immunized with oligo(dT)-selected cross-linked RNA-protein complexes from Ad5-infected cells and the serum was used for immunoblotting experiments. It was found that in addition to the 100K protein, the Ad5 72K DNA-binding protein is also associated with RNA in the infected cells. The 72K DNA-binding protein is cross-linked to polyadenylated nuclear RNA sequences. These findings indicate that adenovirus proteins interact with RNAs in the infected cell and suggest possible mechanisms for the effects of the virus on mRNA metabolism.

Adenoviruses, Human↗
PubMed1986

Interaction of mRNA with proteins in vesicular stomatitis virus-infected cells.

The interaction of mRNA with proteins in vesicular stomatitis virus (VSV)-infected cells was studied by photochemical cross-linking in intact cells. The major [35S]methionine-labeled proteins which became cross-linked by UV light to mRNA in uninfected and in VSV-infected HeLa cells were similar and had apparent mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to 135, 93, 72, 68, 53, 50, 43, and 36 kilodaltons. The proteins which were cross-linked in vivo specifically to the five mRNAs of VSV were labeled through radioactive nucleotides incorporated only into VSV mRNAs under conditions (5 micrograms of actinomycin D per ml) in which only VSV mRNAs are labeled. The same major mRNP proteins that became cross-linked to host mRNAs also became cross-linked to VSV mRNAs, although several quantitative differences were detected. Photochemical cross-linking and immunoblotting of cross-linked mRNPs with VSV antiserum demonstrated that in addition to host proteins VSV mRNAs also became cross-linked to the VSV-encoded N protein. The poly(A) segment of both host and VSV mRNAs was associated in vivo selectively with the 72-kilodalton polypeptide. The major proteins of mRNA-ribonucleoprotein complexes are therefore ubiquitous and common to different mRNAs. Furthermore, since the major messenger ribonucleoproteins interact also with VSV mRNAs even though these mRNAs are transcribed in the cytoplasm, it appears that nuclear transcription and nucleocytoplasmic transport are not necessary for mRNA to interact with these proteins.

Cross-Linking Reagents↗
PubMed1986

mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence.

We identified and produced antibodies to the major proteins that interact with poly(A)+ RNAs in the yeast Saccharomyces cerevisiae. The major proteins which were cross-linked by UV light to poly(A)+ RNA in intact yeast cells had apparent molecular weights of 72,000, 60,000, and 50,000. The poly(A) segment of the RNA was selectively cross-linked to the 72,000-molecular-weight protein (72K protein). Mice immunized with purified UV-cross-linked RNA-protein (RNP) complexes produced antibodies to the three major RNP proteins. A yeast genomic DNA library constructed in the lambda gt11 expression vector was screened with the anti-RNP serum, and recombinant bacteriophage clones were isolated. One recombinant phage, lambda YPA72.1, bearing a 2.5-kilobase insert, produced a large beta-galactosidase-RNP fusion protein. Affinity-selected antibodies from the anti-RNP serum on this fusion protein recognized a single 72K protein which was cross-linked to the poly(A) segment of RNA in the intact cell. Furthermore, the fusion protein of lambda YPA72.1 had specific poly(A)-binding activity. Therefore, lambda YPA72.1 encodes the 72K poly(A)-binding protein. Immunofluorescence microscopy showed that this protein was localized in the cytoplasm. Hybrid-selected mRNA translated in vitro produced the 72K poly(A)-binding protein, and mRNA blot analysis detected a single 2.1-kilobase mRNA. DNA blot analysis suggested a single gene for the poly(A)-binding protein. DNA sequence analysis of genomic clones spanning the entire gene revealed a long open reading frame encoding a 64,272-molecular-weight protein with several distinct domains and repeating structural elements. A sequence of 11 to 13 amino acids is repeated three times in this protein. Strikingly, this repeated sequence (RNP consensus sequence) is highly homologous to a sequence that is repeated twice in a major mammalian heterogeneous nuclear RNP protein, A1. The conservation of the repetitive RNP consensus sequence suggests an important function and a common evolutionary origin for messenger RNP and heterogeneous nuclear RNP proteins.

Amino Acid Sequence↗
PubMed1985

Immunogenicity and the vascular risk of oral contraceptives.

Data concerning circulating immune complexes were obtained for women who had had a pulmonary embolism, myocardial infarction, or cerebral thrombosis, and for 224 healthy controls. In women with pulmonary embolism who had used oral contraceptives concentrations of circulating immune complexes were significantly higher than in healthy controls (regardless of oral contraceptive use), or in those with pulmonary embolism who had never used these preparations. Concentrations of circulating immune complexes were not raised in myocardial infarction, but these women had major risk factors for ischaemic heart disease. The group of patients with cerebral thrombosis without risk factors tended to have high concentrations of circulating immune complexes. The data provide some confirmation that immunological mechanisms may play a role in thrombotic episodes associated with oral contraceptives, especially when they occur in the absence of risk factors for vascular disease.

Adolescent↗
PubMed1984

Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription.

Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 (38K) become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared. Immunofluorescence microscopy shows mostly cytoplasmic and some nuclear staining. These observations demonstrate that commonly used inhibitors of transcription affect the physical state of messenger ribonucleoproteins in vivo.

Camptothecin↗
PubMed1984

Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.

Animals↗
PubMed1983

Trends in induced abortion in England and Wales.

Analysis of routinely published abortion and fertility data for England and Wales between 1968 and 1980 shows that the age-specific abortion rates increased from 1968 until about 1973 when the rates peaked for all ages; rates then declined until 1977 but have subsequently returned to higher levels. Two factors are implicated: (1) the recent changes are related to parallel changes in the fertility rate; but (2) there is also a tendency for recent cohorts of women to resort to abortion more readily. These relationships are derived from analyses of fertility rates and abortion ratios, the proportion of conceptions that result in abortions. The results are discussed in terms of attitudes and practices related to birth control.

Abortion, Induced↗
PubMed1981

A case-control study of the possible association between oral contraceptives and malignant melanoma.

In a case-control study, we investigated 169 women aged 15-49 years with malignant melanoma notified to the Oxford and South Western cancer registries during the years 1971-1976, together with 507 matched controls. Data about medical, reproductive, drug and smoking histories were obtained both by reviewing general practitioner (GP) records and from the women themselves by postal questionnaires. There was no significant evidence of any overall increase in the risk of melanoma in oral contraceptive (OC) users (data from GP records-ever use vs never use, relative risk (RR) 1.34, 95% confidence limits 0.92-1.96; corresponding data from postal questionnaires-RR 1.13, limits 0.73-1.75). However, although not significant, the risk estimated from data in the postal questionnaires was higher in women who had used OCs for 5 years or more (use greater than or equal to 5 years vs never use, RR 1.57, limits 0.83-3.03). Previously demonstrated risk factors for melanoma, such as fair skin, blond or red hair and Celtic origin were found to be commoner in the cases than in the controls. Data from the Oxford/Family Planning Association contraceptive study were also examined. Unexpectedly there was a strong suggestion of a negative association between OC use and melanoma risk, but the analysis was based on only 12 women with the disease.

Adolescent↗
PubMed1981

Oral contraception and myocardial infarction revisited: the effects of new preparations and prescribing patterns.

All deaths from myocardial infarction occurring during 1978 in women aged 15-44 years in England and Wales have been investigated. Using a case-control approach, oral contraceptives were found to be associated with an approximately two-fold increase in risk of this condition, but the effect was apparent only in women with no known risk factors for ischaemic heart disease. Preparations containing 30 micrograms or less oestrogen were associated with the same risk as those containing 50 micrograms oestrogen, but this could be due to the fact that preparations containing less oestrogen tend to contain higher doses of progestogens. Oral contraceptives appear no longer to be prescribed for women with recognised coronary risk factors and this improved prescribing practice has probably resulted in a substantial decrease in mortality attributable to oral contraceptive use.

Adolescent↗