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S T Jacob

Publications and source records attributed to S T Jacob.

At least 55 records · Page 3Linked to original sources
PubMed1991

Polyadenylation of SV40 late pre-mRNA is dependent on phosphorylation of an essential component associated with the 3' end processing machinery.

We investigated whether phosphorylation of the essential components involved in the 3' end processing of mRNAs was required for mRNA polyadenylation. The proteins in HeLa nuclear extract were dephosphorylated with alkaline phosphatase, which is known to remove the phosphate moieties from serine and tyrosine. The dephosphorylated extract was used for analyzing cleavage-dependent polyadenylation of SV40 late pre-mRNA. The phosphatase treatment of the extract completely blocked the polyadenylation reaction, whereas dephosphorylation of the extract did not inhibit the cleavage reaction. Since the cleavage depends upon functional integrity of the specificity factor, it is unlikely that the phosphorylated state of the latter factor is required for the 3' end processing. Sodium vanadate, a potent inhibitor of alkaline phosphatase, markedly reduced the inhibitory effect of the phosphatase on the polyadenylation reaction. Dephosphorylation of the extract also prevented formation of the polyadenylation-specific complex with pre-mRNA, whereas the cleavage-specific complexes were formed under this condition. The Mn-dependent polyadenylation, which is largely poly(A) extension reaction, was relatively resistant to the phosphatase treatment. These data indicate that phosphorylation of a key factor is essential for the 3' end processing of pre-mRNA, and suggest that the factor may be poly(A) polymerase.

Alkaline Phosphatase↗
PubMed1990

Tissue and species distribution of liver type and tumor type nuclear poly(A) polymerases.

Previous studies in this laboratory have identified two distinct nuclear poly(A) polymerases, a 48 kDA tumor type enzyme and a 36-38 kDA liver type enzyme. To investigate the tissue and species specificity of these enzymes, nuclear extracts were prepared from various rat tissues, pig brain and two human cell lines. These as well as whole cell extract from yeast were probed for the two enzymes by immunoblot analysis using polyclonal anti-tumor poly(A) polymerase antibodies or autoimmune sera which contain antibodies specific for the liver type enzyme. Results indicate that both tumor and liver type enzymes are conserved across species ranging from rat to human. The yeast enzyme does not appear to be immunologically related to the liver or the tumor type poly(A) polymerase. The liver type enzyme appears to be specific for normal tissues whereas the tumor type enzyme is detected only in tissues in a "tumorigenic" state or cell lines originating from tumor tissues.

Animals↗
PubMed1990

Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements.

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

Animals↗
PubMed1989

Polyadenylate polymerases from normal and cancer cells and their potential role in messenger RNA processing: a review.

Two structurally and immunologically distinct species of nuclear polyadenylate [poly(A)] polymerases have been characterized. One of these enzymes is relatively absent in normal tissues but is predominant in primary and transplanted tumors and transformed cell lines. The presence of the tumor type enzyme in fetal liver, but not in regenerating liver, suggests that it is an oncofetal protein. Antibodies against the tumor-type poly(A) polymerases are present in the sera of rats bearing tumors and in some cancer patients. These antibodies are also found in the sera of rats fed hepatocarcinogen even before preneoplastic nodules were visible, which suggests that elicitation of these antibodies is an early event in neoplastic transformation. Autoantibodies against both liver-type and tumor-type poly(A) polymerase are also present in some rheumatic autoimmune sera. Polyclonal antibodies against purified enzyme from a rat hepatoma, which exhibit a single band upon immunoblot analysis, were used in cell-free extracts to study the role of poly(A) polymerase in the 3'-end processing of pre-mRNA. These studies showed that the antibodies blocked both endonucleolytic cleavage and poly(A) addition at the cleavage site and complex formation between factors in the extract and pre-mRNA. Independent studies in other laboratories have demonstrated that both the cleavage and poly(A) polymerase activities require the same component for their function. These observations suggest that both cleavage and polyadenylation reactions are tightly coupled in a functional complex.

Animals↗
PubMed1989

Interaction of a positive regulatory factor(s) with a 106-base pair upstream region controls transcription of metallothionein-I gene in the liver.

The differential transcription of the cloned mouse metallothionein-I (MT-I) gene in tissues was studied in unfractionated and fractionated nuclear extracts from rat liver and brain. MT-I gene transcription was 10-fold greater in liver nuclear extract than in brain nuclear extract, whereas the level of transcription of the histone H4 gene was almost identical in both tissue extracts. 5' Deletion analysis of upstream sequences revealed that a 106-base pair (bp) region located between the -148- and -42-bp positions with respect to the transcription start site was responsible for the higher level of expression of the MT-I gene in the liver. Preincubation of the liver extract with the 106-bp fragment resulted in a significant decrease in MT-I gene transcription in the liver extract. In contrast, MT-I gene transcription in the brain nuclear extract was not altered by preincubation with the 106-bp upstream sequence. Mixing liver and brain extract did not diminish MT-I gene transcription normally occurring in liver nuclear extract. Preincubation of brain nuclear extract with the MT-I gene had no inhibitory effect on transcription of MT-I gene in liver nuclear extract. These studies suggest that neither an inhibitor nor a negative trans-acting factor in the brain is responsible for the differential transcription of MT-I gene; rather a positive regulatory factor(s) in the liver which interacts with the 106-bp upstream region contributes to the higher level of MT-I gene expression in this tissue.

Animals↗
PubMed1989

A 37-base pair element in the far upstream spacer region can enhance transcription of rat rDNA in vitro and can bind to the core promoter-binding factor(s).

Previous studies in this laboratory have demonstrated that a 174-base pair (bp) rat rDNA spacer region located more than 2 kilobase pairs upstream of the initiation site, can enhance rat rDNA transcription in vitro independent of its orientation or distance or when inserted downstream of the initiation site. Further dissection of this region showed that transcription of a rDNA fragment containing just 37 bp of the spacer sequence, located between -2.183 and -2.219 kilobase pairs upstream of the initiation site, is 8-fold greater than that of the rDNA fragment devoid of the spacer element. Electrophoretic mobility shift assay demonstrated specific interaction of the 37-bp DNA fragment with a cellular protein(s). The spacer DNA competed for essential transcription factors as demonstrated by the absence of transcription following preincubation of the extract with the 37-bp fragment. Similar competition was also observed when a 58-bp PolI promoter was substituted for the enhancer fragment. The binding of the factor(s) to the enhancer element was not altered when coding and noncoding strands of the 37-bp oligodeoxynucleotide were used separately in the competition assay. Since the 37-bp enhancer region and the core promoter do not exhibit any significant sequence homology, the factor(s) appears to interact with these cis-acting elements in a sequence-independent manner.

Adenocarcinoma↗
PubMed1989

A cis-acting sequence within the rat ribosomal DNA enhancer region can modulate RNA polymerase II-directed transcription of the metallothionein I gene in vitro.

Plasmids were constructed by inserting a 557-bp or 174-bp spacer fragment of rat ribosomal (r)DNA containing an enhancer element(s) at -148 bp upstream from a cloned mouse metallothionein gene (pMT-I). Transcription of these plasmids in a fractionated nuclear extract from a rat hepatoma resulted in 5 to 20-fold stimulation of MT-I gene transcription. This enhancement occurred independent of orientation of the enhancer or its distance from the metallothionein gene promoter or in the presence of the MT-I gene enhancer, and was sensitive to low levels of alpha-amanitin. Stimulation of MT-I gene transcription under the direction of the rDNA spacer element also occurred in HeLa nuclear extract, albeit to a smaller extent. Prior incubation of the nuclear extract with the 557-bp or 174-bp fragment resulted in as much as 5- to 10-fold stimulation of MT-I gene transcription. No significant effect on MT-I gene transcription was observed following preincubation with other DNAs. Preincubation of the extract with three subfragments of the 174-bp spacer inhibited MT-I gene transcription, which suggests that the majority of the 174-bp domain is required for binding to the negative regulatory factor(s) for MT-I gene transcription and that the subfragments can only interact with the positive core promoter-binding factor. The 37-bp subfragment, which has been shown to interact with a positive rDNA trans-acting factor, could also interact with a positive polymerase II (pol II) trans-acting factor. These studies have demonstrated that the 174-bp rat rDNA spacer element containing the pol I enhancer can also modulate pol II-directed transcription.

Animals↗
PubMed1989

Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor.

To determine the role of poly(A) polymerase in 3'-end processing of mRNA, the effect of purified poly(A) polymerase antibodies on endonucleolytic cleavage and polyadenylation was studied in HeLa nuclear extracts, using adenovirus L3 pre-mRNA as the substrate. Both Mg2+- and Mn2+-dependent reactions catalyzing addition of 200 to 250 and 400 to 800 adenylic acid residues, respectively, were inhibited by the antibodies, which suggested that the two reactions were catalyzed by the same enzyme. Anti-poly(A) polymerase antibodies also inhibited the cleavage reaction when the reaction was coupled or chemically uncoupled with polyadenylation. These antibodies also prevented formation of specific complexes between the RNA substrate and components of nuclear extracts during cleavage or polyadenylation, with the concurrent appearance of another, antibody-specific complex. These studies demonstrate that (i) previously characterized poly(A) polymerase is the enzyme responsible for addition of the poly(A) tract at the correct cleavage site and probably for the elongation of poly(A) chains and (ii) the coupling of these two 3'-end processing reactions appears to result from the potential requirement of poly(A) polymerase for the cleavage reaction. The results suggest that the specific endonuclease is associated with poly(A) polymerase in a functional complex.

Adenoviridae↗
PubMed1989

Glucocorticoid-induced stimulation of ribosomal gene transcription in rat hepatoma cells is mediated by modification of RNA polymerase I or an associated factor.

An in vitro transcription system was developed from H411EC3 (H4) hepatoma cells, which mimics the in vivo up-regulation by glucocorticoid hormones on ribosomal RNA (rRNA) synthesis. Ribosomal DNA (rDNA) transcription in extracts derived from H4 cells grown in the presence of 100 nM triamcinolone acetonide was 4- to 5-fold greater than that in extracts derived from cells grown in the absence of glucocorticoid. This effect was not a general stimulation by the steroid, as RNA polymerase II transcription of the metallothionein-1 gene which lacked a glucocorticoid responsive element was unaffected. The increased transcription in hormone-treated extracts was also independent of differential ribonuclease activities or inhibitors as ascertained by the inclusion of ribonuclease inhibitor and mixing experiments, respectively. Chromatography of H4 cell extracts on heparin-sepharose followed by transcription complementation analysis, showed that the hormone-induced stimulatory activity eluted with the fraction (TFIA) which contains RNA polymerase I (Pol I). Immunoblot analysis with specific anti-Pol I antibody showed similar subunit profiles in the absence and presence of the hormone. The presence of a Pol I enhancer element in addition to the rDNA promoter did not further modify the glucocorticoid-induced transcription. These results indicate that the glucocorticoid-mediated effects could be observed in cell extracts which accurately initiate transcription of cloned rat rDNA. Moreover, the alterations of rDNA transcription by the hormone is effected by a factor which elutes with fraction TFIA.

Animals↗
PubMed1988

Association of newly synthesized poly(A) polymerase with four distinct polypeptides.

Nuclear poly(A) polymerase was isolated from [35S]methionine-labeled hepatoma McA-RH 7777 cells and subjected to DEAE-Sephadex chromatography. Flow-through and low salt wash fractions containing poly(A) polymerase activity were pooled and subjected to immunoblot analysis using anti-tumor type poly(A) polymerase antibodies and a biotinylated second antibody. The immune complex contained a single 48-kDa polypeptide band corresponding to the tumor-type enzyme. When immunoprecipitations were carried out using the same fraction and antibodies, at least five 35S-methionine-labeled proteins with approximate molecular masses of 74, 48, 35, 30, and 22 kDa were observed. Pulse-chase studies did not indicate a precursor-product relationship between the immunoprecipitated proteins. Preimmune sera did not react with poly(A) polymerase or other components in the protein complex. These data show that poly(A) polymerase exists as part of a complex with at least four other polypeptides and suggest that these polypeptides may be involved in the cleavage and/or polyadenylation reactions.

Animals↗
PubMed1988

Association of poly(A) polymerase with U1 RNA.

Previous studies (Stetler, D. A., and Jacob, S. T. (1984) J. Biol. Chem. 259, 7239-7244) have shown that poly(A) polymerase from adult rat liver (liver-type) is structurally and immunologically distinct from the corresponding rat hepatoma (tumor-type) enzyme. When hepatoma 7777 (McA-RH 7777) cells were labeled with [32P]inorganic phosphate, followed by immunoprecipitation with anti-hepatoma poly(A) polymerase antibodies and analysis of the RNAs in the immunoprecipitate, only one labeled small nuclear RNA corresponding to U1 RNA was found. Preimmune sera did not form a complex with U1 RNA. Hepatoma poly(A) polymerase antisera did not immunoprecipitate U1 RNA or any other small nuclear RNA from a cell line (H4-11-EC3) which does not contain the tumor-type poly(A) polymerase. Immunoblot analysis of hepatoma 7777 nuclear extract or purified poly(A) polymerase with anti-ribonucleoprotein antisera did not show any cross-reactivity of the latter sera with poly(A) polymerase. The major RNA immunoprecipitated from the hepatoma nuclear extracts using trimethyl cap (m3G) antisera corresponded to the RNA immunoprecipitated with poly(A) polymerase antisera. These data indicate that U1 RNA is closely associated with poly(A) polymerase and suggest the potential involvement of this RNA in the cleavage/polyadenylation of mRNA precursor.

Animals↗
PubMed1988

Inhibition of RNA polymerase I-directed transcription by novobiocin. Potential use of novobiocin as a general inhibitor of eukaryotic transcription initiation.

The effect of novobiocin, a coumarin class antibiotic, on rat ribosomal gene (rDNA) transcription in a fractionated extract derived from adenocarcinoma ascites cells (fraction DE-B) was studied. This drug inhibited transcription of rDNA by blocking initiation of transcription, whereas it had no effect on the elongation of the rRNA transcript. Order of addition experiments indicated that the novobiocin effect was at a step(s) in preinitiation complex formation. Preincubation of fraction DE-B with ATP before exposure to this antibiotic prevented inhibition of rDNA transcription. Since novobiocin has been shown to inhibit RNA polymerases II- and III-directed transcription of linear DNAs by interfering with a step(s) in the initiation reaction, these data suggest that initiation of transcription of all classes of RNA is inhibited by novobiocin by a mechanism independent of its effect on DNA topoisomerase II.

Adenocarcinoma↗
PubMed1987

Novobiocin inhibits initiation of RNA polymerase II-directed transcription of the mouse metallothionein-I gene independent of its effect on DNA topoisomerase II.

The requirement for ATP hydrolysis in the initiation of RNA polymerase II (Pol II)-directed transcription and the relationship between ATP and novobiocin action led us to investigate whether novobiocin could inhibit transcription of the mouse metallothionein-I (MT-I) gene. Novobiocin inhibited the MT-I gene transcription in a fractionated rat hepatoma nuclear extract in a dose-dependent manner by direct interaction with a nuclear factor(s). This interaction prevented formation of stable preinitiation complexes but did not affect elongation of MT-I mRNA. Preincubation of the nuclear extract with ATP prevented the action of novobiocin on MT-I gene transcription. Although novobiocin is known to inhibit DNA topoisomerase II, VM-26, a specific inhibitor of this enzyme had no effect on the transcription. These results indicate that novobiocin blocks the Pol II-directed transcription by inhibiting formation of preinitiation complexes at an ATP-dependent step.

Animals↗
PubMed1987

Production of monoclonal antibodies against RNA polymerase I from nonimmunized autoimmune MRL/lpr mice and their use in rDNA transcription analysis.

A hybridoma secreting monoclonal antibodies against RNA polymerase I was produced by the fusion of myeloma cells with spleen cells from a nonimmunized MRL/lpr mouse which is known to produce autoantibodies to RNA polymerase I. The antibodies (McAb-2D11) belong to the IgG2b subclass, reacted specifically with the second largest (120 kDa) subunit of RNA polymerase I, and inhibited accurate transcription of cloned rat rDNA in a fractionated cell extract following immunoprecipitation of RNA polymerase I. McAb-2D11 did not inhibit RNA polymerase II-mediated transcription of the mouse metallothionein-I gene. Immunocytochemical procedures with biotinylated second antibody demonstrated specific immunolocalization of RNA polymerase I in the nucleus. These studies have (a) presented direct evidence that autoantibodies to functional RNA polymerase I are produced in a murine model of systemic lupus erythematosus, (b) demonstrated specificity of the monoclonal antibody for RNA polymerase I, and (c) provided a useful tool for the purification of RNA polymerase I and/or transcription factor(s) associated with RNA polymerase I.

Animals↗
PubMed1987

An enhancer element in the far upstream spacer region of rat ribosomal RNA gene.

To determine whether far upstream 5'-flanking sequences control rat rDNA transcription, we constructed plasmids containing several 5' and 3' deletions within the nontranscribed spacer region. In vitro transcription of these plasmids identified three enhancer regions, designated A, B, and C, which can dramatically stimulate transcription from the core promoter. Further analysis of region B showed that the enhancer element lies between -2.183 and -2.357 kilobase pairs upstream of the initiation site. The plasmid containing the 174-base pair enhancer element could stimulate rRNA gene transcription as much as 10-20-fold relative to transcription of the plasmid containing only the core promoter. This enhancer was not another promoter domain and could function irrespective of its orientation or distance from the promoter or when inserted downstream of the initiation site. Computer analysis of known sequences of enhancer regions A and C did not reveal any significant homology between these DNA segments and the 174-base pair enhancer element. Competition assay demonstrated that the enhancer element B forms a stable complex with the transcription factor(s) and that interaction between the enhancer and the factor(s) was essential for the stimulation of rDNA transcription. This is the first report of a mammalian ribosomal rDNA enhancer element that exhibits the characteristics of an RNA polymerase II enhancer.

Animals↗
PubMed1987

Autoantibodies against RNA polymerase I in scleroderma and Sjögren's syndrome sera.

Anti-RNA polymerase I antibodies were detected by radioimmunoassay in the sera of 8 out of 9 Sjögren's syndrome patients, 11 out of 19 individuals with scleroderma, and 19 out of 19 systemic lupus erythematosus patients. The results of the radioimmunoassay were confirmed by demonstrating the ability of the patients' IgG to inhibit RNA polymerase I activity in vitro. Sera from four patients in each category were also tested for reaction with individual subunits of RNA polymerase I. All 4 SLE sera, 3 out of 4 scleroderma sera and 1 out of 4 Sjögren's syndrome sera contained antibodies against the 65 kDa (S3) subunit of RNA polymerase I in addition to antibodies against one other subunit. Sera from the remaining scleroderma and Sjögren's syndrome patients tested in this assay contained only anti-S3 antibodies. These results demonstrate that anti-RNA polymerase I antibodies are characteristic of a variety of rheumatic autoimmune sera.

Autoantibodies↗
PubMed1987

Accurate transcription of mouse metallothionein-I gene in a fractionated nuclear extract from a rat hepatoma.

Nuclear extract from Morris hepatoma 3924A was fractionated by DEAE-Sephadex chromatography. The fraction eluting with 300 mM (NH4)2SO4 (DE-C) was used for transcribing cloned mouse metallothionein-I (MT-I) gene in a run-off assay. This fraction contained the majority of RNA polymerase II as well as the transcription factor(s). Accuracy of MT-I DNA transcription was confirmed by S1 nuclease mapping. Low concentrations (1 microgram/ml) of alpha-amanitin inhibited the reaction, indicating that RNA polymerase II directed the transcription. Unfractionated nuclear extracts from the hepatoma or a rat mammary adenocarcinoma as well as whole cell extract obtained from the mammary tumor also transcribed MT-I gene. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-C greater than whole cell extract derived from rat mammary adenocarcinoma cells greater than nuclear extract derived from rat hepatoma or rat mammary adenocarcinoma cells. These studies have demonstrated that a fractionated nuclear extract obtained from a tissue supports efficient and accurate RNA polymerase II-mediated transcription of MT-I DNA.

Adenocarcinoma↗