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S T Jacob

Publications and source records attributed to S T Jacob.

At least 37 records · Page 2Linked to original sources
PubMed1996

Rat ribosomal RNA gene can utilize primate RNA polymerase I transcription machinery: lack of absolute species specificity in rDNA transcription.

The transcriptional activity of rodent ribosomal RNA gene (rDNA) in the primate cell was examined in the light of reported species specificity of eukaryotic ribosomal RNA synthesis. The present study showed that rat rDNA can be transcribed in HeLa nuclear extract whereas mouse rDNA was not transcribed in the heterologous extract. Rat and mouse rDNA transcription factors were interchangeable with respect to efficiency and accuracy of transcription. The initiation of rat ribosomal gene transcription by RNA polymerase I occurred at the +1 site in the heterologous extract. Initiation of transcription at the correct site also occurred in vivo following transfection of cloned rat rDNA into the primate (COS-7) cells. These data indicate that rat ribosomal RNA gene can be expressed in the primate system in vitro and in vivo. The absolute lack of species specificity in rDNA transcription has been discussed based on the present data and other reports.

Animals↗
PubMed1996

Heat shock selectively inhibits ribosomal RNA gene transcription and down-regulates E1BF/Ku in mouse lymphosarcoma cells.

The effect of heat shock on RNA polymerase I (pol I)-directed transcription of the rRNA gene was studied in S-100 extract derived from mouse lymphosarcoma cells, and by in vivo labelling of rRNA. Exposure of cells to 42 degrees C for 2 h resulted in complete inhibition of rRNA synthesis in vivo. Pol I transcription was inhibited by 50% within 2 h of heat shock and was abolished after 3 h exposure at 42 degrees C. Under this condition, the core-promoter-binding activity of the factor (CPBF) that modulates pol I transcription was unaffected. In contrast, the promoter-binding activity of enhancer-1-binding factor, a protein related to the Ku autoantigen, which is involved in pol I transcription initiation, was reduced by 50 and 90% after 2 and 3 h of heat shock respectively. Western-blot analysis with antibodies specific for the two subunits of Ku protein showed the absence of p72 subunit after 3 h of heat shock. Under this condition, pol II transcription from the adenovirus major late promoter and pol III transcription of 5 S RNA gene remained unaffected. Mixing experiments ruled out the possibility that the inhibition of transcription was due to activation of nucleases or other inhibitors. This is the first report to show selective down-regulation of pol I transcription in vitro by heat shock and of the potential involvement of a pol I transcription factor in this process.

Adenoviridae↗
PubMed1995

The RNA polymerase I promoter-activating factor CPBF is functionally and immunologically related to the basic helix-loop-helix-zipper DNA-binding protein USF.

Analysis of the core promoter sequence of the mammalian ribosomal RNA genes revealed an E-box-like sequence (CACGCTG) to which the upstream stimulatory factor (USF) binds. Because the core promoter binding factor (CPBF) binds specifically to the core promoter sequence of the ribosomal RNA gene (Liu, Z., and Jacob, S. T. (1994) J. Biol. Chem. 269, 16618-16626) and resembles USF in some respects, we explored the potential relationship between USF and CPBF. Competition electrophoretic mobility shift assay using labeled core promoter probe and several unlabeled competitor oligonucleotides showed that USF can indeed bind to the core promoter and that only those oligonucleotides with an E-box sequence could compete in the promoter-protein complex formation characteristic of CPBF. This complex formation was thermostable, a unique property of USF. Furthermore, antibodies raised against USF cross-reacted with the 44-kDa component of rat CPBF. To prove further the relationship between CPBF and USF, we examined the effects of the unlabeled USF and ribosomal gene core promoter oligonucleotides on polymerase (pol) I and pol II transcription. Both oligonucleotides inhibited rDNA transcription as well as transcription from the adenovirus major late promoter. Only the oligonucleotides that contain the E-box sequence competed in the transcription. These data indicate that the promoter sequence of mammalian ribosomal RNA gene contains an USF-binding site, that the 44-kDa polypeptide of CPBF is related to the 44/43-kDa polypeptide of human USF, and that USF or USF-related protein can transactivate both pol I and pol II promoters.

Animals↗
PubMed1995

E1BF/Ku interacts physically and functionally with the core promoter binding factor CPBF and promotes the basal transcription of rat and human ribosomal RNA genes.

We have previously characterized an RNA polymerase (pol) I transcription factor, E1BF, from rat cells. This protein is immunologically related to Ku autoantigen and is required in pol-I directed transcription of rodent ribosomal RNA gene (rDNA). Glycerol density gradient fractionation and in situ UV cross-linking analysis of the purified factor showed directly that it consists of a heterodimer of 85 and 72 kDa polypeptides. E1BF also interacted with the human core promoter and augmented transcription of human rDNA as much as fivefold in HeLa nuclear extract, whereas transcription from adenovirus major late promoter, CMV or SV40 early promoters by pol II and of U6 and 5S RNA genes by pol III were either unaffected or minimally inhibited by the antibodies. Purified rat E1BF partially restored the suppression of human rDNA transcription by anti-Ku antibodies. Immunoprecipitation of rat cell extract with the anti-Ku antibodies followed by SDS-PAGE of the precipitated proteins and Southwestern analysis showed that E1BF interacts with CPBF, a core promoter binding factor. When the majority of CPBF and E1BF was removed from the reaction mixture by preincubation with a core promoter oligo nucleotide fragment, rDNA transcription was severely impaired. Addition of exogenous CPBF or E1BF to such a reaction resulted in significant restoration of the transcription, whereas inclusion of both factors caused further enhancement of rDNA transcription. These data demonstrate that E1BF is a basal pol I transcription factor that interacts with a core promoter binding factor both physically and functionally, and that is not a general pol II or pol III transcription factor.

Adenoviridae↗
PubMed1994

Enhancer 1 binding factor (E1BF), a Ku-related protein, is a growth-regulated RNA polymerase I transcription factor: association of a repressor activity with purified E1BF from serum-deprived cells.

Previous studies from this laboratory have demonstrated that the enhancer 1 binding factor (E1BF), a Ku-related protein, purified from the serum-enriched cells functions as a positive factor in an RNA polymerase (pol I) transcription system. We have now shown that E1BF purified from the serum-deprived cells (E1BFs) can inhibit rDNA transcription completely in a fractionated extract from the cells grown in serum-enriched medium. The suppression of transcription was overcome by the addition of control E1BF (E1BFc). Immunoprecipitation of purified E1BFs by the anti-Ku monoclonal antibody and addition of the supernatant to the transcription reaction mixture prevented the inhibition significantly, whereas immunoprecipitation with the control mouse IgG did not restore the transcription. The transcriptional repressor activity associated with the final DNA affinity column fractions copurified with E1BF. Neither the amount of E1BF nor its promoter binding activity was altered following serum depletion. E1BFs selectively inhibited the initiation of rDNA transcription. The inhibitory activity of E1BFs was not due to a nonspecific RNase activity. These data suggest that E1BF is post-translationally modified following serum starvation of cells, and that the repressor activity of E1BFs is largely responsible for the down-regulation of pol I transcription in serum-deprived cells.

Animals↗
PubMed1994

Characterization of a protein that interacts with the rat ribosomal gene promoter and modulates RNA polymerase I transcription.

An RNA polymerase I core promoter binding factor (CPBF) was purified to apparent homogeneity from rat adenocarcinoma ascites cells by chromatographic fractionation on a series of columns including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides with molecular masses of 44,000 and 39,000 daltons. The binding of the factor to the promoter was demonstrated by Southwestern blotting, UV cross-linking and electrophoretic mobility shift assay. The specificity of its binding to the core promoter was confirmed by competitive electrophoretic mobility shift assay using several unlabeled oligo probes in the assay. The addition of increasing amounts of purified CPBF to the in vitro transcription reaction that contains a limiting quantity of the factor resulted in dramatic stimulation of RNA polymerase I (pol I) transcription of rat ribosomal RNA gene. The transcription stimulatory activity associated with the purified CPBF fractions co-purified with the core promoter binding activity in an electrophoretic mobility shift assay. Finally, in a reconstitution transcription system which is devoid of the factor and is incapable of ribosomal gene transcription, purified CPBF could trans-activate the pol I promoter. These data indicate that CPBF is a novel pol I promoter binding factor required for ribosomal gene transcription.

Adenocarcinoma↗
PubMed1994

Effects of repetitive and non-repetitive rat rDNA enhancer elements on in vivo transcription by RNA polymerases I and II.

Previous study has demonstrated that a far upstream 174-bp spacer sequence of the rat rRNA-encoding (rDNA) gene can function as an enhancer in vitro in an orientation- and distance-independent manner [Dixit et al., J. Biol. Chem. 262 (1987) 11616-11622]. To demonstrate that this element can also function in vivo, two rat rDNA-cat plasmids, one with the 174-bp element and the other without this sequence, were constructed and transfected into CHO cells. Primer extension analysis of the transcripts produced after transfection showed that transcription initiation occurred at the +1 site of the rDNA. The 174-bp sequence stimulated the rat polI promoter activity in cis 4-5-fold over the control (with the promoter alone). This RNA polymerase (polI) enhancer also stimulated the mouse metallothionein-I (MT-I) and SV40 promoter activities in vivo, irrespective of its distance and orientation. Further dissection of the 174-bp element revealed that the stimulatory activity on the RNA polymerase II (polII) promoter resides within the 37-bp and 43-bp domains at the 3' end of the 174-bp element. Unlike this spacer enhancer, the 130-bp repeat element (RE) proximal to the rat promoter [Ghosh et al., Gene 125 (1993) 217-222] was unable to modulate the polII promoter activity in vivo. These data show that while the non-repetitive enhancer sequence of rat rDNA is interchangeable for the polI and polII promoters, the RE is polI-specific.

Animals↗
PubMed1994

Specific inhibition of pre-ribosomal RNA processing in extracts from the lymphosarcoma cells treated with 5-fluorouracil.

To elucidate the molecular mechanism by which the potent anticancer drug, 5-fluorouracil (5-FUra), inhibits cell proliferation, the effect of its metabolite, 5-fluorouridine triphosphate, on transcription of rat rRNA gene and processing of pre-rRNA was investigated in S-100 extract from the mouse lymphosarcoma cells. The in vitro processing of pre-rRNA substrate synthesized from the T3 promoter occurred at the correct primary processing site. Replacement of UMP with 5-fluorouridine monophosphate in the rRNA substrate did not affect the pre-rRNA processing. Similar result was obtained when coupled transcription-processing was studied. When the coupled reaction was examined using extracts from the cells treated with 5-FUra, rRNA processing was abolished whereas transcription of rRNA gene was unaffected. Treatment of cells with thymidine along with 5-FUra did not reverse the inhibitory effect of the drug on rRNA processing. In contrast to the effect on rRNA processing, treatment of cells with 5-FUra did not impede the 3' end processing of pre-mRNA. These data show that inhibition of pre-rRNA processing is a major mechanism of action of 5-FUra and suggest that the activity and/or synthesis of a trans-acting factor(s) involved in this reaction is altered by the anticancer drug.

Animals↗
PubMed1994

Enhancer 1 binding factor, a Ku-related protein, is a positive regulator of RNA polymerase I transcription initiation.

We have previously characterized a protein, enhancer 1 binding factor (E1BF), from rat cells that can modulate RNA polymerase I-directed transcription of the rat rRNA gene in vitro. E1BF, a heterodimeric DNA binding protein composed of 72-kDa and 85-kDa subunits, is related to the human Ku autoantigen with respect to immunological and certain structural properties. To establish the direct role of E1BF in transcription, we investigated the effect of anti-Ku antibodies on RNA polymerase I-directed transcription in rat and mouse cell extracts. These antibodies, one directed against the 70-kDa Ku subunit and the other against a peptide fragment of this subunit, dissociated the E1BF heterodimer into its two subunits. The DNA-protein complex formed in the presence of the antibodies contained only the 72-kDa subunit. Preincubation of the extracts with these antibodies resulted in an almost complete inhibition of transcription. The reduced transcription was observed when either linear or circular template was used. The inhibitory effect of the antibodies was greatest when added prior to preinitiation complex formation and was minimized significantly when added after establishment of the initiation complex. The repression of rRNA gene transcription was overcome by the addition of purified E1BF. This study demonstrates that E1BF, a Ku-related protein, is required for RNA polymerase I-directed transcription, the 72-kDa subunit is the major DNA binding polypeptide, the factor acts primarily in the formation of the preinitiation complex, and heterodimerization of its two subunits is crucial for maintaining the functional integrity of the protein.

Animals↗
PubMed1993

Characterization of the 130-bp repeat enhancer element of the rat ribosomal gene: functional interaction with transcription factor E1BF.

The 130-bp repetitive element (RE) of the rat rDNA (ribosomal RNA-encoding gene) intergenic spacer stimulated the synthesis of rRNA four- to sixfold, in comparison with that of the promoter alone, both in vivo and in vitro, when ligated to the rat rDNA promoter. The addition of increasing amounts of highly purified E1BF (enhancer-1 binding factor), which binds to the rat rDNA promoter and an upstream nonrepetitive enhancer element [Zhang and Jacob, Mol. Cell. Biol. 10 (1990) 5177-5186], to an in vitro transcription system resulted in enhancement of rDNA transcription from the recombinant plasmids containing the promoter or promoter-RE. However, E1BF-mediated stimulation of transcription under the influence of the RE continued at higher concentrations of E1BF than did the control transcription from the promoter alone. The binding affinity of E1BF for the RE was comparable to its affinity for the nonrepetitive far upstream enhancer element previously characterized in our laboratory. The sequences protected by E1BF in the RE differed from those protected by UBF (upstream control element-binding factor), a well characterized pol I transcription factor. These data suggest that E1BF belongs to a class of transcription factors which interact with the promoter and spacer cis-acting RE to modulate rDNA transcription.

Animals↗
PubMed1993

Characterization of the factor E1BF from a rat hepatoma that modulates ribosomal RNA gene transcription and its relationship to the human Ku autoantigen.

We have purified the DNA binding protein E1BF (Enhancer 1 Binding Factor) from the nuclei of Morris hepatoma 3924A. This protein, which consists of two polypeptides of 72 kDa and 85 kDa, binds to several DNA sequences involved in the regulation of rDNA transcription. UV-crosslinking studies show that interaction of the protein with DNA is primarily through the 72 kDa polypeptide. Purified E1BF can also modulate pol I-directed transcription in a cell-free systems derived from rat and mouse. E1BF is related to the human autoantigen Ku as demonstrated by immunological cross-reactivity with monoclonal antibodies directed against Ku p70 in Western blot and gel mobility shift assays.

Animals↗
PubMed1993

Control of gene expression by lipophilic hormones.

Regulation of gene expression in response to steroids, thyroid hormone, and retinoids is mediated by an impressive array of intracellular receptors. Sequence analysis showed that the hormone receptors comprise a large superfamily of ligand-responsive transcription factors. Upon binding to hormones, the receptors interact with specific hormone response elements located in the promoters of numerous genes. Promoter-bound receptors communicate with distinct receptors and/or additional members of the transcriptional machinery, frequently evoking protein-protein interactions. Ultimately, this results in the induction of complex gene systems that control hormone-induced processes such as differentiation, cell growth, and homeostasis. In addition to the genes transcribed by RNA polymerase II, the lipophilic hormones, particularly glucocorticoids, can also modulate RNA polymerase I-directed transcription of the ribosomal gene. For both transcription systems, activation and repression of genes in response to hormones have been reported. Finally, the involvement of hormone receptors in tumorigenesis has been discussed. It is likely that receptor studies will have major implications in the diagnosis and therapy of diseases such as leukemia.

Animals↗
PubMed1993

Identification of a sequence within the mouse metallothionein-I gene promoter mediating its basal transcription and of a protein interacting with this element.

Previous studies in our laboratory have shown that a trans-acting factor, which binds to a 106 bp sequence in the mouse metallothionein-I (MT-I) gene, is responsible for the relatively high level of MT-I gene transcription in the liver. Using electrophoretic mobility shift assay, we have now identified a 26 bp sequence within the 106 bp region, which interacts with a trans-activating factor in the liver nuclear extract. This sequence, designated MRE-c', is located between positions -135 and -110 with respect to the transcription start site and comprises the metal regulatory element MRE-c and part of its 5' and 3' flanking sequences. UV cross-linking and Southwestern analysis showed that a protein of an apparent molecular mass of 33,000 specifically interacts with MRE-c'. Deletion of the MRE-c' region resulted in a six- to sevenfold decrease in the MT-I promoter activity, as measured by reduction in chloramphenicol acetyltransferase activity. A comparison of other regulatory domains of the MT-I gene and the potential factors interacting with these sequences indicates that MRE-c' and probably the 33 kDa polypeptide are involved in the constitutive transcription of the MT-I gene.

Animals↗
PubMed1991

Ara-ATP impairs 3'-end processing of pre-mRNAs by inhibiting both cleavage and polyadenylation.

Previous studies have demonstrated that Ara-ATP can inhibit poly(A) polymerase activity by competing with ATP. To elucidate the mechanism of action of this compound, its effect on the cleavage and polyadenylation of two specific substrates, SV40L and adenovirus L3 pre-mRNAs, was studied in HeLa nuclear extracts. Unlike cordycepin 5' triphosphate, Ara-ATP inhibited both cleavage and poly(A) addition. Addition of poly(A) polymerase fraction devoid of any other factors required for the processing reactions overcame the inhibitory effect on cleavage as well as polyadenylation of pre-mRNAs. These data suggest that Ara-ATP inhibits both cleavage and polyadenylation reactions by interacting with the ATP-binding site on poly(A) polymerase, the activity of which is essential for the cleavage reaction. Ara-ATP also blocked formation of the post-cleavage and polyadenylation-specific complexes, which further confirmed the inhibitory effect of the ATP analog on the two tightly coupled 3'-end processing reactions.

Adenoviridae↗
PubMed1991

E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription.

We previously described the purification and characterization of E1BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E1BF was selectively phosphorylated. The labeled phosphate could be removed from the E1BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E1BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with [gamma-32P] ATP resulted in the selective labeling of the 79-kDa band. The E1BF-associated protein kinase did not phosphorylate casein or histone H1. Fraction DE-B, a preparation containing RNA polymerase I and all polymerase I transcription factors (including E1BF), lost polymerase I transcriptional activity when treated with phosphatase. The phosphatase-induced inactivation of polymerase I activity associated with fraction DE-B could be reversed by the addition of purified E1BF. Treatment of purified E1BF with heat, SDS, or an ATP affinity analog eliminated its capacity to reactivate dephosphorylated fraction DE-B. These data demonstrate that (i) polymerase I promoter-binding factor E1BF contains an intrinsic substrate-specific protein kinase and (ii) E1BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.

Animals↗
PubMed1991

Potential role of poly(A) polymerase in the assembly of polyadenylation-specific RNP complexes.

To elucidate the mechanism by which poly(A) polymerase functions in the 3'-end processing of pre-mRNAs, polyadenylation-specific RNP complexes were isolated by sedimentation in sucrose density gradients and the fractions were analyzed for the presence of the enzyme. At early stages of the reaction, the RNP complexes were resolved into distinct peaks which sedimented at approximately 18S and 25S. When reactions were carried out under conditions which support cleavage or polyadenylation, the pre-mRNA was specifically assembled into the larger 25S RNP complexes. Polyclonal antibodies raised against the enzyme purified from a rat hepatoma, which have been shown to inhibit cleavage and polyadenylation (Terns, M., and Jacob, S. T., Mol. Cell. Biol. 9:1435-1444, 1989) also prevented assembly of the 25S polyadenylation-specific RNP complexes. Furthermore, formation of these complexes required the presence of a chromatographic fraction containing poly(A) polymerase. UV cross-linking analysis indicated that the purified enzyme could be readily cross-linked to pre-mRNA but in an apparent sequence-independent manner. Reconstitution studies with the fractionated components showed that formation of the 25S RNP complex required the poly(A) polymerase fraction. Although the enzyme has not been directly localized to the specific complexes, the data presented in this report supports the role of poly(A) polymerase as an essential component of polyadenylation-specific complexes which functions both as a structural and enzymatic constituent.

Antibodies↗
PubMed1991

Anti-poly(A) polymerase antibodies in the sera of tumor-bearing rats can inhibit specific cleavage and polyadenylation reactions.

Sera from rats bearing Morris hepatoma 3924A for 7 weeks contained antibodies against the 48 kd tumor-type poly(A) polymerase, whereas sera from normal rats or rats bearing the tumor for shorter periods did not exhibit immunoreactivity against the enzyme. Purified IgG from the sera of the tumor-bearing rats inhibited both cleavage at the poly(A) site and polyadenylation of adenovirus L3 RNA in nuclear extracts derived from HeLa cells. By contrast, IgG from normal rats did not block the 3'-terminal processing reaction. Control or immune IgG had no effect on the transcription of ribosomal gene in the extracts derived from H-4 hepatoma cells. These data demonstrate the functional specificity of the anti-poly(A) polymerase antibodies found in the sera of the tumor-bearing rats.

Animals↗