Search PubMed⌕ Search

Biomedical subjects

J B Cohen

Publications and source records attributed to J B Cohen.

At least 55 records · Page 3Linked to original sources
PubMed1997

A splicing enhancer in the 3'-terminal c-H-ras exon influences mRNA abundance and transforming activity.

Analysis of cDNA clones previously identified an optional intron in the 3'-untranslated region of the human H-ras gene. A possible correlation was observed between failure to remove this intron and overexpression of the gene, suggesting that splicing of the intron may require a specific titrable factor. The splicing signals at the end of the intron deviate from the consensus and may be inefficient, but we noticed that the adjacent exon downstream has a purine-rich region reminiscent of purine-rich splicing enhancers in other genes that stimulate the removal of weak, flanking introns. We show here that the purine-rich region of H-ras has splicing-enhancer activity in the homologous as well as a heterologous context. Interestingly, although the affected intron is outside the coding region, inversion or deletion of the enhancer reduced the transforming activity of oncogenic H-ras alleles severalfold. Experiments with corresponding cDNA constructs suggested that this is not a consequence of the altered structures of the mRNAs produced when the enhancer is inverted or deleted. Instead, we propose that the region controls an additional pre-mRNA processing event besides splicing of the terminal intron. Our work indicates that the purine-rich region may play an important role in the control of H-ras activity.

Animals↗
PubMed1997

U1 small nuclear RNA-promoted exon selection requires a minimal distance between the position of U1 binding and the 3' splice site across the exon.

Both experimental work and surveys of the lengths of internal exons in nature have suggested that vertebrate internal exons require a minimum size of approximately 50 nucleotides for efficient inclusion in mature mRNA. This phenomenon has been ascribed to steric interference between complexes involved in recognition of the splicing signals at the two ends of short internal exons. To determine whether U1 small nuclear ribonucleoprotein, a multicomponent splicing factor that is involved in the first recognition of splice sites, contributes to the lower size limit of vertebrate internal exons, we have taken advantage of our previous observation that U1 small nuclear RNAs (snRNAs) which bind upstream or downstream of the 5' splice site (5'SS) stimulate splicing of the upstream intron. By varying the position of U1 binding relative to the 3'SS, we show that U1-dependent splicing of the upstream intron becomes inefficient when U1 is positioned 48 nucleotides or less downstream of the 3'SS, suggesting a minimal distance between U1 and the 3'SS of approximately 50 nucleotides. This distance corresponds well to the suggested minimum size of internal exons. The results of experiments in which the 3'SS region of the reporter was duplicated suggest an optimal distance of greater than 72 nucleotides. We have also found that inclusion of a 24-nucleotide miniexon is promoted by the binding of U1 to the downstream intron but not by binding to the 5'SS. Our results are discussed in the context of models to explain constitutive splicing of small exons in nature.

Base Sequence↗
PubMed1997

The alternative H-ras protein p19 displays properties of a negative regulator of p21Ras.

In previous work, we demonstrated that expression of mammalian H-ras genes is controlled by alternative splicing. Mutational analyses indicated that most H-ras premessenger RNA (pre-mRNA) is not processed into mRNA for the usual p21Ras protein, but is recognized instead for processing through an alternative pathway that would produce mRNA for a hypothetical p19Ras protein, which would be a truncated form of p21 with a different carboxyl-terminus. We have raised the possibility that p19 could be a negative regulator of p21, but only small amounts of mature p19 mRNA could be detected and we had no evidence that the protein could be made. We now show by transient and stable transfection experiments that p19 can be produced from complementary DNA (cDNA) expression vectors in human 293 cells, which express early adenoviral genes. However, p19 cDNA inhibited the formation of drug-selected colonies of Rat-1 cells and surviving colonies produced little p19. Colony formation and p19 expression were increased when the cDNA had mutations in the putative effector domain of p19 or when oncogenes believed to act downstream of p21Ras, such as adenoviral E1A, were included in the transfections. These results indicate that p19 can act as an inhibitor of p21. We also show that authentic p19 can be abundantly expressed in 293 cells from H-ras gene constructs with mutations favoring the alternative splicing pathway, which suggests that regulated splicing could support overproduction of p19 under natural conditions. Our work indicates that mutations abolishing alternative H-ras splicing may not only contribute to oncogenesis by increasing the production of p21, but also by interfering with the production of an antiproliferative H-ras activity.

Alternative Splicing↗
PubMed1996

U1 snRNA promotes the selection of nearby 5' splice sites by U6 snRNA in mammalian cells.

Communication between exon boundaries is a central feature of the exon definition model of pre-mRNA splice-site selection and an exon-bridging interaction involving U1 small nuclear RNA (snRNA) paired with the 5' splice site (5'ss) has been identified previously. It has become increasingly clear, however, that the 5'ss is not defined relative to the base-pairing interaction with U1, suggesting that a connection in the proposed line of communication between exon boundaries is missing. To explore this issue, we have first sought to characterize the role in mammalian 5'ss selection of a previously suggested base-pairing interaction with U6 snRNA. Using transfection experiments, we show that mutations at positions 5 and 6 of a 5'ss associated with an internal exon can be suppressed by compensatory changes in the first two positions of a conserved hexanucleotide of U6 RNA. The specificity of the effect was established by covariation experiments as well as by experiments with two splice sites arranged in tandem. Suppression of 5'ss mutations by U6 was more efficient when U1 could pair nearby than when pairing was restored further away and individual U1 RNAs stimulated U6-defined proximal sites more efficiently than distal sites. These results are interpreted to suggest that U1 acts to direct 5'ss choice by U6 to matching sequences nearby. Our work supports a central role for base-pairing with U6 snRNA in mammalian 5'ss selection and suggests how the interaction may be established properly despite the limited complementarity involved.

Animals↗
PubMed1996

Cloning and characterization of the human homologue of a dystrophin related phosphoprotein found at the Torpedo electric organ post-synaptic membrane.

Dystrophin is the protein product which is absent in Duchenne muscular dystrophy (DMD). In mammalian skeletal muscle, dystrophin is found in association with several integral and peripheral membrane proteins, forming a complex known as the dystrophin glycoprotein complex (DGC). In an expressed sequence tag (EST) database search to identify new dystrophin related genes, we isolated EST00891 which showed 57% homology to the cysteine-rich domain of dystrophin and localized to 18q12.1-12.2. This EST is also highly homologous (90%) to the Torpedo californica post-synaptic 87 kDa phosphoprotein. Screening human adult brain and skeletal muscle cDNA libraries with this EST resulted in cloning multiple cDNAs which encode several splice forms all homologous to the C-terminal domain of dystrophin. The largest open reading frame isolated shows 94% homology (86% identity) to the Torpedo 87 kDa protein and 50% homology to the cysteine-rich and carboxy-terminal domains of dystrophin. The other cDNAs isolated encode smaller splice forms of this gene which we have named dystrobrevin. The tissue distribution of dystrobrevin mRNA shows five distinct transcripts which are preferentially expressed between different tissues. In addition, antibodies against either the Torpedo 87 kDa protein or human dystrobrevin demonstrate that at least three of the splice forms are translated as proteins in human brain tissue extracts.

Alternative Splicing↗
PubMed1996

Base pairing at the 5' splice site with U1 small nuclear RNA promotes splicing of the upstream intron but may be dispensable for slicing of the downstream intron.

We previously reported that exon skipping in vivo due to point mutations in the 5' splice site (5'ss) signal of an internal mammalian exon can be prevented by coexpression of U1 small nuclear RNAs, termed shift-U1s, with complementarity to sequence upstream or downstream of the mutated site. We now show by S1 nuclease protection experiments that a typical shift-U1 restores splicing of the upstream intron, but not necessarily of the down stream intron. This indicates that the normal 5'ss sequence acts as an enhancer for splicing of the upstream intron, that it owes this activity to base pairing with U1, and that the enhancer activity is reproduced by base pairing of U1 with other sequences in the area. Shift-U1s are dispensable when the 3'ss sequence of the upstream intron is improved, which suggests that base pairing of U1 with sequences at or near the downstream end of the exon normally functions by compensating for a weakness in the upstream 3'ss. Accordingly, U1 appears to be involved in communication across the exon, but our data indicate at the same time that extensive base pairing between U1 and the 5'ss sequence is not necessary for accurate splicing of the downstream intron. These findings are discussed in relation to the coordinate selection exon termini proposed by the exon definition model.

Alternative Splicing↗
PubMed1996

Phenotypic heterogeneity associated with the splicing mutation in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

One mutation frequently identified in 21-hydroxylase deficiency is the intron 2 splicing mutation, in which the normal polymorphic C or A at nucleotide 655 has been converted to G. Using allele-specific oligonucleotide hybridization, single strand conformational polymorphism analysis, and heteroduplex analyses, we identified 38 individuals from 21 different families who had 2 deleterious mutations. All were homozygous or compound heterozygotes for the splicing mutation. Comparison of the phenotypic features with the molecular genotypes shows phenotypic heterogeneity extending from classical salt-losing 21-hydroxylase deficiency to asymptomatic. Single strand conformational polymorphism analysis followed by DNA sequence analysis revealed numerous sequence variations in intron 2, most commonly at nucleotides 601 and 683. Transient transfection experiments show that the 3'-portion of intron 2 is sufficient to transfer the effect of the 655c/a-->g mutation to a chimeric heterologous gene. Clinical correlations and initial transfection studies suggest that sequence variations at nucleotides 601 and 683 do not correlate with clinical severity or substantially affect splicing. In summary, a single nucleotide change, 655c/a-->g, alters the splice acceptor site at the intron 2/exon 3 boundary. The molecular basis of the phenotypic heterogeneity associated with the mutation remains to be elucidated.

Adrenal Hyperplasia, Congenital↗
PubMed1996

Smokers' knowledge and understanding of advertised tar numbers: health policy implications.

OBJECTIVES: This article examines health policy implications of providing smokers with numerical tar yield information in cigarette advertising. METHODS: Results of a national probability telephone survey regarding smokers' knowledge and understanding of numerical tar yields and deliveries are reported. RESULTS: Few smokers knew the tar level of their own cigarettes (the exception being smokers of 1- to 5-mg tar cigarettes), and a majority could not correctly judge the relative tar levels of cigarettes. Smokers were unsure whether switching to lower-tar cigarettes would reduce their personal health risks. Many smokers relied on absolute numbers in making trade-offs between number of cigarettes smoked and their tar levels, thus confusion machine-rated tar-yields with actual amounts ingested. CONCLUSIONS: The wisdom of the present method of providing tar and nicotine numbers in ads and recommendations for modifying the test protocol are now under discussion. This research indicates that these tar numbers and their implications are poorly understood. The paper recommends revisions in tar ratings to make them more useful and a required statement on cigarette packages to more explicitly relate tar levels to major health risks.

Adult↗
PubMed1994

Suppression of mammalian 5' splice-site defects by U1 small nuclear RNAs from a distance.

One of the earliest events in the process of intron removal from mRNA precursors is the establishment of a base-pairing interaction between U1 small nuclear (sn) RNA and the 5' splice site. Mutations at the 5' splice site that prevent splicing can often be suppressed by coexpression of U1 snRNAs with compensatory changes, but in yeast, accurate splicing is not restored when the universally conserved first intron base is changed. In our mammalian system as well, such a mutation could not be suppressed, but the complementary U1 caused aberrant splicing 12 bases downstream. This result is reminiscent of observations in yeast that aberrant 5' splice sites can be activated by U1 snRNA from a distance. Using a rapid, qualitative protein expression assay, we provide evidence that 5' splice-site mutations can be suppressed in mammalian cells by U1 snRNAs with complementarity to a range of sequences upstream or downstream of the site. Our approach uncouples in vivo the commitment-activation step of mammalian splicing from the process of 5' splice-site definition and as such will facilitate the genetic characterization of both.

Alternative Splicing↗
PubMed1994

Active-site peptides of acetylcholinesterase of Electrophorus electricus: labelling of His-440 by 1-bromo-[2-14C]pinacolone and Ser-200 by tritiated diisopropyl fluorophosphate.

To characterize the structure of the active site of acetylcholinesterase (AChE) from the electric organ of E. electricus, we identified sites of incorporation of two active-site affinity labels, [3H]diisopropyl fluorophosphate ([3H]DFP), and 1-bromo-2-[14C]pinacolone ([14C]BrPin). AChE was isolated, purified, inactivated and digested with trypsin, and peptides containing 3H or 14C were purified by reverse-phase HPLC and characterized by N-terminal sequence analysis. [3H]DFP, labelling Ser-200, was found in a single peptide, QVTIFGESAGAASVGMHLLSPDSR, 83% identical with the sequence from Thr-193 to Arg-216 deduced for AChE of T. californica, with Gln, Ala, Leu, and Asp in place of Thr-193, Gly-203, Ile-210 and Gly-214, respectively, and 87% identical with that from bovine and human brain AChEs. Inactivation by [14C]BrPin led to two radioactive peptides. One, ASNLVWPEWMGVIHGYEIEFVFGLPLEK, was 96% identical with that extending from Ala-427 to Lys-454 of T. californica. Release of 14C in cycle 14 established reaction of [14C]BrPin with active-site His-440, protected by 5-trimethylammonio-2-pentanone (TAP). The other peptide, LLXVTENIDDAER, 77% homologous with that of T. californica extending from Leu-531 to Arg-543, had label associated with the third cycle, not protected by TAP, corresponding to Asn-533. The slow inactivation of eel AChE by reaction of [14C]BrPin at His-440 contrasts with that of AChE from T. nobiliana, where it reacts rapidly with a free cysteine, Cys-231, not present in eel AChE. For both AChEs, inactivation by BrPin prevents subsequent reaction with [3H]DFP, and prior inactivation by DFP does not prevent reactions with [14C]BrPin.

Acetylcholinesterase↗
PubMed1994

Identifying the lipid-protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.

To identify amino acid residues of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used the photoactivatable, hydrophobic probe 3-trifluoromethyl-3-(m-[125I]-iodophenyl)diazirine([125I]TID). The pattern of [125I]TID incorporation into the M3 and M4 hydrophobic segments of each subunit was the same both in the presence and absence of the agonist carbamoylcholine and in the presence of an excess of nonradioactive TID, consistent with nonspecific photoincorporation from the lipid-protein interface. [125I]TID reacted with five residues in alpha-M4 [Blanton, M.P., & Cohen, J. B. (1992) Biochemistry 31, 3738-3750] but with only two or three residues in M4 segments of beta-, gamma-, and delta-subunits. In delta-M3, [125I]TID reacted with Met-293, Ser-297, Gly-301, Val-304, and Asn-305 as well as with Ile-288 preceding M3. Residues at corresponding positions were labeled in beta-M3 (Met-285, Ile-289, Phe-293) and in gamma-M3 (Phe-292, Leu-296, Met-299, and Asn-300) as well as gamma-Ile-283. Within alpha-M3, Phe-284 and Ser-287 were labeled. The periodicity of labeled residues provides the first direct evidence that M3 as well as M4 segments of each subunit are organized as transmembrane alpha-helices each with substantial contact with lipid. In addition, in alpha-M1 [125I]TID reacted nonspecifically with Cys-222, Leu-223, Phe-227, and Leu-228, a pattern of incorporation inconsistent with the labeling pattern expected either for a "face" of an alpha-helix or a beta-sheet.

Amino Acid Sequence↗
PubMed1994

Agonist-induced photoincorporation of a p-benzoylphenylalanine derivative of substance P into membrane-spanning region 2 of the Torpedo nicotinic acetylcholine receptor delta subunit.

The neuropeptide substance P acts, at micromolar concentrations, as a noncompetitive antagonist of nicotinic acetylcholine receptors (AChRs) of both neuronal and muscle subtypes. The mechanism of this inhibition has been shown to be most consistent with stabilization of a nonconducting desensitized state of the AChR, via binding to a site distinct from both the agonist site and the high affinity noncompetitive antagonist site. We have used a radioiodinated photoreactive analogue of substance P, containing the amino acid p-benzoyl-L-phenylalanine in place of the Phe8 residue of substance P, to identify the sites of interaction of substance P within the Torpedo california AChR. AChR-rich membrane suspensions were photolabeled in the absence or presence of the agonist carbamylcholine and/or nonradioactive substance P, and incorporation into AChR subunits was assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of agonist 125I incorporation was detected in each subunit and was insensitive to substance P, whereas in the presence of carbamylcholine there was a 2-fold increase in photoincorporation into the AChR delta subunit that was inhibited by the addition of an excess of substance P. The sites of specific photoincorporation in the delta subunit were initially mapped by use of Staphylococcus aureus V8 protease to a 14-kDa fragment extending from delta Ile-192 to Glu-280. Further fragmentation of this 14-kDa fragment with trypsin and S. aureus V8 protease established that the sites of specific incorporation were restricted to the region delta Ser-253 to Glu-280, which contains the membrane-spanning region 2 that is known to form the lining of the ion channel. These results establish that in the presence of agonist at least a part of the undecapeptide substance P binds within the ion channel in the desensitized state of the AChR, and it is likely that the binding of substance P to this site is responsible for the action of substance P as a noncompetitive AChR antagonist.

Affinity Labels↗
PubMed1994

HIV-related oral manifestations in two cohorts of women in San Francisco.

The goals of this study were to compare the prevalence of oral lesions in women infected with human immunodeficiency virus (HIV) and HIV-negative women, and to determine the association of oral lesions with route of HIV transmission and with level of immunosuppression in infected women. As part of a prospective 4-year study, oral examinations and blood tests were performed, at 6-month intervals, on 176 HIV-infected women and on 117 HIV-negative women at risk for HIV infection. We evaluated participants for the following oral conditions: hairy leukoplakia, candidiasis, ulcers, warts, non-Hodgkin's lymphoma, Kaposi's sarcoma, and parotid enlargement. As previously reported in men, the prevalence of oral lesions was significantly higher among HIV-infected (22%) than HIV-negative women (3%) [odds ratio (OR) = 8.2; 95% confidence interval (CI) 2.8, 23.5], particularly candidiasis (14%) and hairy leukoplakia (10%). Among HIV-infected women with CD4 cell count nadir > or = 200 cells/microliters, the prevalence of hairy leukoplakia was higher among those infected heterosexually than among injection drug users (OR = 5.5; 95% CI: 1.5; 19). The OR for the association between oral lesions and CD4 cell count nadir (< 200 vs. > 500 cells/microliters) was 8.9 (95% CI: 2.6, 30), indicating a strong positive association with level of immunosuppression.

Adult↗
PubMed1993

Labeling of cysteine 231 in acetylcholinesterase from Torpedo nobiliana by the active-site directed reagent, 1-bromo-2-[14C] pinacolone. Effects of 2,2'-dipyridyl disulfide and other sulfhydryl reagents.

Acetylcholinesterase (AcChE, EC 3.1.1.7) was isolated from the electric organ of T. nobiliana and treated with the active-site-directed alkylating agent 1-bromo-2-[14C]pinacolone ([14C]BrPin), or with BrPin, which acts initially as a competitive inhibitor, Ki = 0.18 mM, and then inactivates the enzyme, k2 = 1.8 x 10(-4) s-1. AcChE aliquots were digested with trypsin and fractionated by reversed phase high performance liquid chromatography. Inactivation caused a decrease in one absorption peak and an increase in another, identified as the peptide beginning at Ala-222 and extending to Arg-242. 5-Trimethylammonio-2-pentanone, a competitive inhibitor, isosteric with acetylcholine, retarded the inactivation and decreased the quantity of labeled peptide. On sequencing, the 14C label was found associated with Cys-231. This was confirmed by comparison with synthesized S-pinacolonylcysteine, by study of effects of blocking the sequencing by o-phthalaldehyde, and by inactivation by 2,2'-dipyridyl disulfide (2-PDS), a thiol-specific reagent that acts initially as a competitive inhibitor, Ki = 0.042 mM, and then inactivates the enzyme, k2 = 5.0 x 10(-4) s-1. This is retarded by 5-trimethylammonio-2-pentanone, and prior inactivation by 2-PDS prevents subsequent reaction of [14C]BrPin in the active site. BrPin inactivates AcChEs from Electrophorus electricus and from human erythrocyte, but 2-PDS does not. Neither reagent inactivates butyrylcholinesterases from human and horse serum.

2,2'-Dipyridyl↗
PubMed1993

The 87K postsynaptic membrane protein from Torpedo is a protein-tyrosine kinase substrate homologous to dystrophin.

Postsynaptic peripheral membrane proteins at the neuromuscular junction have been proposed to participate in the immobilization of the nicotinic acetylcholine receptor at the synapse. An 87 kd cytoplasmic peripheral membrane protein has been demonstrated to colocalize with the nicotinic acetylcholine receptor in the Torpedo electric organ and at the mammalian neuromuscular junction. We have cloned the cDNA encoding the 87K protein from Torpedo electric organ, and the predicted protein sequence is homologous to the C-terminal domains of dystrophin, the protein product of the Duchenne muscular dystrophy gene. The 87K protein displays a restricted pattern of expression detected only in electric organ, brain, and skeletal muscle. Analysis of the in vitro and in vivo phosphorylation of the 87K protein indicates that it is multiply phosphorylated on serine, threonine, and tyrosine residues. The 87K protein is in a complex with other proteins associated with the postsynaptic membrane, including dystrophin and a 58 kd protein. These results suggest that the 87K protein is involved in the formation and stability of synapses and is regulated by protein phosphorylation.

Amino Acid Sequence↗
PubMed1993

U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

Alternative Splicing↗