Roger Chalkley - Academia.edu (original) (raw)
Papers by Roger Chalkley
Multiple domains for initiator binding proteins TFII-I and
Journal of Biological Chemistry, 1970
Proceedings of the National Academy of Sciences, 1976
Nucleic Acids Research, 1995
In the present study, we have explored further the organization of the TATA-less rat xanthlne deh... more In the present study, we have explored further the organization of the TATA-less rat xanthlne dehydrogenase/oxldase gene (XDH/XO). A DNase I hypersensitive site has been Identified which it colocalizes with the basal promoter reported previously [Chow et al. (1994) Nucleic Acids Res., 22,1846-1854]. Gel mobility shift assays Indicate the presence of multiple binding factors located In the promoter. At least six footprints were detected of which two have been shown to be C/EBP binding sites. Members of the C/EBP-a and C/EBP-p, but not C/EBP-5, family are able to bind to these two sites. Deletlonal and mutatlonal studies revealed that C/EBP binding Is not essential for the basal level of transcription Initiation of this promoter. Much of the transcrlptlonal activity resides in the-102 to-7 DNA fragment, which contains all Initator activity which acts unidlrectionally. Within this fragment, four putative initiator elements could be identified; Interestingly, the linear integrity of these Initiators is important for efficient transcription of the XDH/XO gene. Separation of the Initiators leads to a complete loss of transcription activity; however, this loss could be partially restored by the Introduction of an Sp1 binding site upstream of the separated initiators. Despite a difference In usage/frequency of initiation at the various initiators, primer extension analyses reveal similar positions for transcription initiations in both XDH/XO reporter constructs and in the endogenous XDH/XO gene. The differential usage of initiators may imply a possible post-transcriptional regulation for the XDH/XO gene.
Biochemistry, 1985
We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucl... more We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucleosomal structures. DNA synthesis was inhibited by incubating hepatoma tissue culture cells in medium containing 5.0 mM hydroxyurea for 40 min. During the final 20 min, the cells were pulsed with [3H]lysine to radiolabel the histones (all five histones are substantially labeled under these conditions). By two electrophoretic techniques, we demonstrate that histones H1, H2A, and H2B synthesized in the presence of hydroxyurea do not merely associate with the surface of the chromatin but instead exchange with preexisting histones so that for the latter two histones there is incorporation into nucleosome structures. On the other hand, H3 and H4 synthesized during this same time period appear to be only weakly bound, if at all, to chromatin. These two histones have been isolated from postnuclear washes and purified. Some possible implications of in vivo exchange are discussed.
We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. Th... more We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplasmin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S.
The EMBO Journal
We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. Th... more We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplanin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S.
Journal of Biological Chemistry
We have analyzed site occupancy in vivo with constructs containing one or more copies of the bind... more We have analyzed site occupancy in vivo with constructs containing one or more copies of the binding site for the yeast trans-activator, yIBF. The data indicate only a modest difference in site occupancy (at most 2-fold) even though multiple copies activate transcription several hundredfold more than one copy. Using studies in which we have mutated the IES2 sequence and slightly decreased its affinity for yIBF, we have also shown that synergistic activation of transcription is dependent on overall site occupancy. Finally, synergy declines as yIBF-binding sites are separated, and loss of synergy appears to be correlated with loss of a linking surface. These results are consistent with the simultaneous contact model of synergistic activation.
The EMBO Journal
Novobiocin, an inhibitor of prokaryotic DNA gyrase and eukaryotic type H topoisomerase enzymes, i... more Novobiocin, an inhibitor of prokaryotic DNA gyrase and eukaryotic type H topoisomerase enzymes, interferes with in vitro chromatin assembly using purified histones, DNA and nucleoplasmin. The target of inhibition is not topoisomerase II; this energy-independent assembly system lacks any ATP and Mg2+-dependent type II topoisomerase or gyrase activities. Rather, novobiocin interacts with histones, disrupting histone-histone associations required for octamer formation, and causing histones to precipitate from both nucleoplasmin-histone and histone-DNA complexes. Thus, novobiocin is able to generate 'dynamic' chromatin in vitro in the absence of ATP and Mg2+ by removing histones from previously assembled static chromatin, so that the DNA supercoils, previously constrained by conventional nucleosomes, become susceptible to removal by topoisomerase I.
Journal of Biological Chemistry
Exposure of HTC cells to 6 mM sodium butyrate for 20 h results in significantly reduced amounts o... more Exposure of HTC cells to 6 mM sodium butyrate for 20 h results in significantly reduced amounts of nuclear histone acetyltransferase. While the direct butyrate inhibition of histone deacetylases is rapidly reversed upon removal of cells from butyrate, histone acetyltransferase activity increases only gradually following release of cells from butyrate, requiring several hours to regain control cell levels. As a result, HTC cells released from a 20-h exposure to butyrate display normal kinetics of histone acetate turnover but markedly reduced rates of histone acetylation, the imbalance in these two processes produces histone hypoacetylation.
Journal of Biological Chemistry
We have studied the competitive binding of histones and the Rous sarcoma virus internal enhancer ... more We have studied the competitive binding of histones and the Rous sarcoma virus internal enhancer binding factor (IBF) factor (which recent studies indicate is almost certainly cEBP beta). We find that histones and IBF are incapable of forming a ternary complex with a 159-base pair (bp) fragment of DNA containing a single IBF binding site and that histones and factor are mutually exclusive in binding. We have analyzed the various physical parameters of binding, in an attempt to understand how the factor might establish an exclusive binding in the cell. The stability of the nucleosome and the factor-DNA complex have been determined, and in addition a minimum value for the affinity of the histone octamer has been computed. We find that in simple competition the IBF can successfully compete, only if the substrate DNA is shorter than 140 bp. The relevance of these results is discussed in terms of a kinetic model for successful factor competition during the replication of the factor bindi...
Journal of Biological Chemistry
Enhancer factor I (EFI) is a trans-acting factor which binds to the Rous sarcoma virus long termi... more Enhancer factor I (EFI) is a trans-acting factor which binds to the Rous sarcoma virus long terminal repeat enhancer and promoter at two inverted CCAAT-box motifs. We demonstrate that two forms of EFI DNA binding activity exist in nuclear extracts of avian cells. One form requires two heterologous components (EFIA)(EFIB) for high affinity, specific DNA binding activity, whereas a second form is not dependent on EFIB for binding and may be composed solely of EFIA, perhaps as a multimer. Both forms give rise to the same mobility shift in gel retardation assays, but the two forms can be separated chromatographically under buffer conditions which stabilize the two DNA binding activities. A cDNA for EFIA has been isolated from a rat liver cDNA expression library. The 1489-base pair EFIA cDNA encodes a 322-amino acid protein which is nearly identical to two previously described human DNA binding proteins. These are dbpB, a DNA binding protein of unknown specificity which binds to the epid...
Journal of Biological Chemistry
A cis-acting transcriptional activation sequence (IES2) from the Rous sarcoma virus internal enha... more A cis-acting transcriptional activation sequence (IES2) from the Rous sarcoma virus internal enhancer was found to stimulate transcription of a heterologous gene in Saccharomyces cerevisiae. A hamster protein (termed IBF) which binds to IES2 and stimulates transcription in vitro has previously been purified and was found to have a subunit molecular mass of 40,000 (Karnitz, L., Poon, D., Weil, P.A., and Chalkley, R. (1989) Mol. Cell. Biol. 9, 1929-1939). The identification and purification of the yeast homolog of IBF (yIBF) is reported here. Purified yIBF has a subunit molecular mass of 92,000. This protein functions as a trans-activator of transcription in a heterologous HeLa transcription extract in a cis-element sequence-dependent manner in vitro.
Journal of Biological Chemistry
The interaction of estradiol-17β with mature bovine endometrial tissue, and with isolated nuclei ... more The interaction of estradiol-17β with mature bovine endometrial tissue, and with isolated nuclei has been studied. The hormone binds to an insoluble nuclear fraction. This fraction contains membrane and evidence is presented to show that estradiol is bound to the nuclear membrane. Incubation of isolated nuclei and microsome fractions with estradiol-17β shows that the hormone binds essentially instantaneously to microsomes and nuclei. Such binding is non-saturable up to estradiol-17β concentrations as great as 2.5 x 10-6 moles per mg of membrane protein and it is likely that this interaction is not biologically significant. A second form of binding is observed in nuclei which is of higher affinity and saturable, with 507 ± 47 sites per nucleus. This class of binding sites is found to be blocked in cattle that are maintained in artificially induced estrus by feeding with diethylstilbesterol. The high affinity binding site is present only in the mature endometrium and is absent in nucl...
Journal of the Chemical Society C: Organic, 1970
ABSTRACT
Journal of the Chemical Society, Perkin Transactions 1, 1978
Aryloxysulphoxonium salts, easily accessible from diazonium salts and sulphonyl compounds, are ef... more Aryloxysulphoxonium salts, easily accessible from diazonium salts and sulphonyl compounds, are effective electrophiles. With aliphatic primary and secondary amines they react by attack on sulphur, to give a range of sulphurnitrogen compounds, including ...
Methods in Enzymology, 1968
ABSTRACT
Molecular and cellular biology, 1987
We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at... more We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at least two protein factors (EFI and EFII) that bound specifically to the Rous sarcoma virus (RSV) enhancer in vitro. These factors were differentially extracted from quail cell nuclei, recognized different nucleotide sequences in the U3 region of the RSV long terminal repeat, and appeared to bind preferentially to opposite DNA strands as monitored by the DNase I protection assay. The EFI- and EFII-protected regions within U3 corresponded closely to sequences previously demonstrated by deletion mutagenesis to be required for enhancer activity, strongly suggesting a functional significance for these proteins. Only weak homologies between other enhancer consensus sequence motifs and the EFI and EFII recognition sites were observed, and other viral enhancers from simian virus 40 and Moloney murine sarcoma virus did not compete effectively with the RSV enhancer for binding either factor.
The Journal of biological chemistry, Jan 10, 1975
The capacity to effectively label tumor cell hostones using very short pulses of [3-H]acetate and... more The capacity to effectively label tumor cell hostones using very short pulses of [3-H]acetate and [32-P]phosphate (1 to 10 min) has been developed. Four histone fractions F3, F2a1, F2a2, and F2b are extensively acetylated in short time periods. About 70% of the acetate accumulated on the histone during a short pulse is removed with a half-life of similar to 3 min. The rest of the metabolically active acetate is removed with a half-life of 30 to 40 min. Histones F2a1, F2a2, and F1 are acetylated at the NH2 terminus and this modification is metabolically stable. In short pulses, histones are labeled with 32-P in the order F2a2 greater than F1 greater than F3 greater than F2a1 greater than F2b. All fractions have a fairly rapid turnover time (t1/2 similar 20 to 40 min) except F1 phosphate which turns over some 5 times more slowly.
Multiple domains for initiator binding proteins TFII-I and
Journal of Biological Chemistry, 1970
Proceedings of the National Academy of Sciences, 1976
Nucleic Acids Research, 1995
In the present study, we have explored further the organization of the TATA-less rat xanthlne deh... more In the present study, we have explored further the organization of the TATA-less rat xanthlne dehydrogenase/oxldase gene (XDH/XO). A DNase I hypersensitive site has been Identified which it colocalizes with the basal promoter reported previously [Chow et al. (1994) Nucleic Acids Res., 22,1846-1854]. Gel mobility shift assays Indicate the presence of multiple binding factors located In the promoter. At least six footprints were detected of which two have been shown to be C/EBP binding sites. Members of the C/EBP-a and C/EBP-p, but not C/EBP-5, family are able to bind to these two sites. Deletlonal and mutatlonal studies revealed that C/EBP binding Is not essential for the basal level of transcription Initiation of this promoter. Much of the transcrlptlonal activity resides in the-102 to-7 DNA fragment, which contains all Initator activity which acts unidlrectionally. Within this fragment, four putative initiator elements could be identified; Interestingly, the linear integrity of these Initiators is important for efficient transcription of the XDH/XO gene. Separation of the Initiators leads to a complete loss of transcription activity; however, this loss could be partially restored by the Introduction of an Sp1 binding site upstream of the separated initiators. Despite a difference In usage/frequency of initiation at the various initiators, primer extension analyses reveal similar positions for transcription initiations in both XDH/XO reporter constructs and in the endogenous XDH/XO gene. The differential usage of initiators may imply a possible post-transcriptional regulation for the XDH/XO gene.
Biochemistry, 1985
We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucl... more We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucleosomal structures. DNA synthesis was inhibited by incubating hepatoma tissue culture cells in medium containing 5.0 mM hydroxyurea for 40 min. During the final 20 min, the cells were pulsed with [3H]lysine to radiolabel the histones (all five histones are substantially labeled under these conditions). By two electrophoretic techniques, we demonstrate that histones H1, H2A, and H2B synthesized in the presence of hydroxyurea do not merely associate with the surface of the chromatin but instead exchange with preexisting histones so that for the latter two histones there is incorporation into nucleosome structures. On the other hand, H3 and H4 synthesized during this same time period appear to be only weakly bound, if at all, to chromatin. These two histones have been isolated from postnuclear washes and purified. Some possible implications of in vivo exchange are discussed.
We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. Th... more We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplasmin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S.
The EMBO Journal
We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. Th... more We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplanin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S.
Journal of Biological Chemistry
We have analyzed site occupancy in vivo with constructs containing one or more copies of the bind... more We have analyzed site occupancy in vivo with constructs containing one or more copies of the binding site for the yeast trans-activator, yIBF. The data indicate only a modest difference in site occupancy (at most 2-fold) even though multiple copies activate transcription several hundredfold more than one copy. Using studies in which we have mutated the IES2 sequence and slightly decreased its affinity for yIBF, we have also shown that synergistic activation of transcription is dependent on overall site occupancy. Finally, synergy declines as yIBF-binding sites are separated, and loss of synergy appears to be correlated with loss of a linking surface. These results are consistent with the simultaneous contact model of synergistic activation.
The EMBO Journal
Novobiocin, an inhibitor of prokaryotic DNA gyrase and eukaryotic type H topoisomerase enzymes, i... more Novobiocin, an inhibitor of prokaryotic DNA gyrase and eukaryotic type H topoisomerase enzymes, interferes with in vitro chromatin assembly using purified histones, DNA and nucleoplasmin. The target of inhibition is not topoisomerase II; this energy-independent assembly system lacks any ATP and Mg2+-dependent type II topoisomerase or gyrase activities. Rather, novobiocin interacts with histones, disrupting histone-histone associations required for octamer formation, and causing histones to precipitate from both nucleoplasmin-histone and histone-DNA complexes. Thus, novobiocin is able to generate 'dynamic' chromatin in vitro in the absence of ATP and Mg2+ by removing histones from previously assembled static chromatin, so that the DNA supercoils, previously constrained by conventional nucleosomes, become susceptible to removal by topoisomerase I.
Journal of Biological Chemistry
Exposure of HTC cells to 6 mM sodium butyrate for 20 h results in significantly reduced amounts o... more Exposure of HTC cells to 6 mM sodium butyrate for 20 h results in significantly reduced amounts of nuclear histone acetyltransferase. While the direct butyrate inhibition of histone deacetylases is rapidly reversed upon removal of cells from butyrate, histone acetyltransferase activity increases only gradually following release of cells from butyrate, requiring several hours to regain control cell levels. As a result, HTC cells released from a 20-h exposure to butyrate display normal kinetics of histone acetate turnover but markedly reduced rates of histone acetylation, the imbalance in these two processes produces histone hypoacetylation.
Journal of Biological Chemistry
We have studied the competitive binding of histones and the Rous sarcoma virus internal enhancer ... more We have studied the competitive binding of histones and the Rous sarcoma virus internal enhancer binding factor (IBF) factor (which recent studies indicate is almost certainly cEBP beta). We find that histones and IBF are incapable of forming a ternary complex with a 159-base pair (bp) fragment of DNA containing a single IBF binding site and that histones and factor are mutually exclusive in binding. We have analyzed the various physical parameters of binding, in an attempt to understand how the factor might establish an exclusive binding in the cell. The stability of the nucleosome and the factor-DNA complex have been determined, and in addition a minimum value for the affinity of the histone octamer has been computed. We find that in simple competition the IBF can successfully compete, only if the substrate DNA is shorter than 140 bp. The relevance of these results is discussed in terms of a kinetic model for successful factor competition during the replication of the factor bindi...
Journal of Biological Chemistry
Enhancer factor I (EFI) is a trans-acting factor which binds to the Rous sarcoma virus long termi... more Enhancer factor I (EFI) is a trans-acting factor which binds to the Rous sarcoma virus long terminal repeat enhancer and promoter at two inverted CCAAT-box motifs. We demonstrate that two forms of EFI DNA binding activity exist in nuclear extracts of avian cells. One form requires two heterologous components (EFIA)(EFIB) for high affinity, specific DNA binding activity, whereas a second form is not dependent on EFIB for binding and may be composed solely of EFIA, perhaps as a multimer. Both forms give rise to the same mobility shift in gel retardation assays, but the two forms can be separated chromatographically under buffer conditions which stabilize the two DNA binding activities. A cDNA for EFIA has been isolated from a rat liver cDNA expression library. The 1489-base pair EFIA cDNA encodes a 322-amino acid protein which is nearly identical to two previously described human DNA binding proteins. These are dbpB, a DNA binding protein of unknown specificity which binds to the epid...
Journal of Biological Chemistry
A cis-acting transcriptional activation sequence (IES2) from the Rous sarcoma virus internal enha... more A cis-acting transcriptional activation sequence (IES2) from the Rous sarcoma virus internal enhancer was found to stimulate transcription of a heterologous gene in Saccharomyces cerevisiae. A hamster protein (termed IBF) which binds to IES2 and stimulates transcription in vitro has previously been purified and was found to have a subunit molecular mass of 40,000 (Karnitz, L., Poon, D., Weil, P.A., and Chalkley, R. (1989) Mol. Cell. Biol. 9, 1929-1939). The identification and purification of the yeast homolog of IBF (yIBF) is reported here. Purified yIBF has a subunit molecular mass of 92,000. This protein functions as a trans-activator of transcription in a heterologous HeLa transcription extract in a cis-element sequence-dependent manner in vitro.
Journal of Biological Chemistry
The interaction of estradiol-17β with mature bovine endometrial tissue, and with isolated nuclei ... more The interaction of estradiol-17β with mature bovine endometrial tissue, and with isolated nuclei has been studied. The hormone binds to an insoluble nuclear fraction. This fraction contains membrane and evidence is presented to show that estradiol is bound to the nuclear membrane. Incubation of isolated nuclei and microsome fractions with estradiol-17β shows that the hormone binds essentially instantaneously to microsomes and nuclei. Such binding is non-saturable up to estradiol-17β concentrations as great as 2.5 x 10-6 moles per mg of membrane protein and it is likely that this interaction is not biologically significant. A second form of binding is observed in nuclei which is of higher affinity and saturable, with 507 ± 47 sites per nucleus. This class of binding sites is found to be blocked in cattle that are maintained in artificially induced estrus by feeding with diethylstilbesterol. The high affinity binding site is present only in the mature endometrium and is absent in nucl...
Journal of the Chemical Society C: Organic, 1970
ABSTRACT
Journal of the Chemical Society, Perkin Transactions 1, 1978
Aryloxysulphoxonium salts, easily accessible from diazonium salts and sulphonyl compounds, are ef... more Aryloxysulphoxonium salts, easily accessible from diazonium salts and sulphonyl compounds, are effective electrophiles. With aliphatic primary and secondary amines they react by attack on sulphur, to give a range of sulphurnitrogen compounds, including ...
Methods in Enzymology, 1968
ABSTRACT
Molecular and cellular biology, 1987
We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at... more We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at least two protein factors (EFI and EFII) that bound specifically to the Rous sarcoma virus (RSV) enhancer in vitro. These factors were differentially extracted from quail cell nuclei, recognized different nucleotide sequences in the U3 region of the RSV long terminal repeat, and appeared to bind preferentially to opposite DNA strands as monitored by the DNase I protection assay. The EFI- and EFII-protected regions within U3 corresponded closely to sequences previously demonstrated by deletion mutagenesis to be required for enhancer activity, strongly suggesting a functional significance for these proteins. Only weak homologies between other enhancer consensus sequence motifs and the EFI and EFII recognition sites were observed, and other viral enhancers from simian virus 40 and Moloney murine sarcoma virus did not compete effectively with the RSV enhancer for binding either factor.
The Journal of biological chemistry, Jan 10, 1975
The capacity to effectively label tumor cell hostones using very short pulses of [3-H]acetate and... more The capacity to effectively label tumor cell hostones using very short pulses of [3-H]acetate and [32-P]phosphate (1 to 10 min) has been developed. Four histone fractions F3, F2a1, F2a2, and F2b are extensively acetylated in short time periods. About 70% of the acetate accumulated on the histone during a short pulse is removed with a half-life of similar to 3 min. The rest of the metabolically active acetate is removed with a half-life of 30 to 40 min. Histones F2a1, F2a2, and F1 are acetylated at the NH2 terminus and this modification is metabolically stable. In short pulses, histones are labeled with 32-P in the order F2a2 greater than F1 greater than F3 greater than F2a1 greater than F2b. All fractions have a fairly rapid turnover time (t1/2 similar 20 to 40 min) except F1 phosphate which turns over some 5 times more slowly.