Levels Of DNA Binding Activity Research Articles

NF-κB p65 Subunit DNA-Binding Activity: Association with Depleted Antioxidant Levels in Breast Carcinoma Patients

NF-κB is recognized as a redox-sensitive transcription factor and has been implicated in cellular response to oxidative stress. The study was designed to correlate the changes in antioxidant status with the levels of nuclear factor-κB (NF-κB) p65 subunit DNA-binding activity in relation to lymph node involvement, tumor size, and staging in breast carcinoma patients. Case control study comprised of 40 breast carcinoma patients along with 40 age- and sex-matched healthy subjects as controls. Levels of enzymatic/nonenzymatic antioxidants along with the trace elements were measured to study the antioxidant status in the study subjects. Levels of NF-κB p65 subunit DNA-binding activity was estimated by ELISA assay. The levels of enzymatic, nonenzymatic antioxidants, and trace elements were found to be significantly depleted in breast carcinoma patients in comparison to healthy controls suggesting significantly decreased levels of antioxidant activity in the breast carcinoma patients. Also, these results indicate that antioxidant levels decrease progressively with the advancement of stage and subsequent progression of disease. DNA-binding activity of NF-κB p65 subunit was higher in breast cancer patients in comparison to normal healthy controls, and the activity was found to increase with the advancement of disease. Significant correlation was observed between the DNA-binding activity of NF-κB p65 subunit and antioxidant status in the patients. The logistic regression analysis revealed decreased levels of antioxidants and increased level of DNA-binding activity of NF-κB p65 subunit were significantly associated with incidence of breast carcinoma. Depleted antioxidant status and increased level of DNA-binding activity of NF-κB p65 subunit thus point clearly of an association in relation to disease progression, clinical stage, and cytological grade in the pathophysiology of breast carcinoma.

Constitutively lower expressions of CD54 on primary myeloma cells and their different localizations in bone marrow

To evaluate nuclear factor-kappaB (NF-kappaB) activity in primary myeloma cells from myeloma patients, we confirmed that the expression levels of CD54 showed a good correlation with the levels of DNA binding activity for NF-kappaB in human myeloma cell lines, and thus analyzed the expression levels of CD54 on CD38(++) plasma cell fractions as one of NF-kappaB activity. Primary myeloma cells unexpectedly showed constitutively lower expressions of CD54 than normal bone marrow (BM) plasma cells. Furthermore, the expression levels of CD54 on these plasma cells showed a significant correlation with the plasma levels of CXCL12 stromal cell-derived factor-1alpha (SDF-1alpha) in their BM aspirates, and the expressions of CXCR4, the receptor for CXCL12, decreased on primary myeloma cells compared with normal BM plasma cells. It was also confirmed that the addition of CXCL12 to the in vitro culture significantly induced the up-regulation of CD54 expression in primary myeloma cells. In addition, myeloma cells with lower expressions of CD54 were more unstable in the in vitro culture, resulting in a marked reduction of the viable cell number. In the immunohistochemical analysis of BM aspirates, myeloma cells with lower CD54 expression resided in the perivascular regions. Therefore, these data suggest that primary myeloma cells exhibit constitutively lower CD54 that might be partially regulated by CXCL12, and their localizations in the BM may be associated with the expression levels of CD54.

Biological Significance of Constitutive NF-κB Activity Levels in Human Myeloma Cell Lines

To elucidate the mechanism of constitutive NF-κB activity in human myeloma cell lines, constitutive expression of all the NF-κB family, RelA (p65), c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52), genes in different human myeloma cell lines, such as IL-6-independent (AMO1, NOP2), IL-6-dependent (ILKM2, ILKM8) and IL-6-responsive cell line (U266), were first checked by RT-PCR and western blot analysis. In the electrophoretic mobility shift assay (EMSA) for κB main consensus, total DNA binding activity levels for NF-κB were higher in IL-6-dependent, IL-6-responsive and IL-6-independent human myeloma cell lines respectively. Supershift analysis with anti-NF-κB family antibodies and immunoprecipitation followed by western blotting, confirmed that p50/p50, p50/RelB and p50/p52 as the principal player responsible for constitutive activation of NF-κB in IL-6-independent cell lines. However, beside these major dimers, p50/p65 dimer was also involved in the constitutive NF-κB activation in IL-6-dependent and IL-6-responsive cell lines. Moreover, on the basis of microarray data in human myeloma cell lines, we confirmed that the expression levels of NF-κB target genes, such as NFKB2, RELB, NFKBIA, MALT1 and CD54, showed correlation with the level of DNA binding activity for NF-κB. Therefore, these data suggest that the difference of total DNA-binding activities for NF-κB partly depended on the different p50/p65 activity among the IL-6-independent, IL-6-dependent and IL-6-responsive human myeloma cell lines, and further studies in the different NF-κB-dimers activity levels might contribute to the clarification of the regulation of NF-κB target genes expression in human myeloma cell lines.

Decreased Expression Levels of CD54 Associated with Decreased Activity of CXCL12 in Primary Myeloma Cells

In order to evaluate NF-kB activity in the primary myeloma cells, we examined the expressionlevels of CD54 by a flow cytometer. First, we confirmed that the expression levels of CD54 showed good correlation with the levels of DNA binding activity for NF-kB in human myeloma cell lines. Primary myeloma cells unexpectedly revealed lower expression levels of CD54 compared to normal BM plasma cells, while plasma cells from MGUS had high expression levels of CD54 as same as normal BM plasma cells. Furthermore, the expression levels of CD54 on these plasma cells showed the significantly positive correlation with the plasma levels of CXCL12 (SDF-1α) in their BM aspirates, and the expression levels of CXCR4, the receptor for CXCL12, decreased on primary myeloma cells compared to normal BM plasma cells. It was also confirmed that addition of CXCL12 in the in vitro culture significantly induced up-regulation of CD54 expressions in primary myeloma cells. Furthermore, myeloma cells with lower expressions of CD54 were unstable in the in vitro culture, resulting in the marked reduction of viable cell number; this was also the case where cell lines with lower or no expression of CD54 were sensitive to H2O2 treatment. Therefore, these data suggest that primary myeloma cells have lower expression levels of CD54, possibly lower NF-kB activities and different stability of in vitro survival from normal BM plasma cells, depending on expression levels of CD54.

Proteasome inhibitors can alter the signaling pathways and attenuate the P-glycoprotein-mediated multidrug resistance

Numerous signaling pathways were reported to be involved in the resistance for conventional cytotoxic drugs, although one of the main reasons is the overexpression of P-glycoprotein (P-gp) in multidrug resistant cancer cells. The overexpression of P-gp has been associated with the resistance to a wide range of anticancer drugs. Doxorubicin and paclitaxel are substrates of this transporter system and have an important role for the various human malignancies. In the present study, drug-sensitive MCF7 and multidrug resistant MCF7/ADR (characterized by overexpression of P-gp) human breast cancer cell lines were used as an experimental model. We have found that PS341 and MG132, proteasome inhibitors, reduced the degree of the multidrug resistance (MDR) in MCF7/ADR cells. This phenomenon was accompanied by a decrease in the IC50 value of doxorubicin and paclitaxel from 55.9 +/- 3.46 to 0.60 +/- 0.08 microM, and from 17.61 +/- 1.77 to 0.59 +/- 0.12 microM, respectively. The IC50 values of sensitive cells for doxorubicin and paclitaxel were about 0.42 and 0.83 microM, respectively. The effect of PS341 and MG132 on MCF7/ADR cells was associated with a significant decrease in both protein and gene levels of P-gp expression. Moreover, with regard to the expression of possible signal transduction pathways of mitogen-activated protein kinase (MAPK) related to the activation of mdr1, proteasome inhibitors did significantly influence the activation of these proteins. Western blot analysis revealed that 24 hr exposure of multidrug resistant MCF7/ADR cells with proteasome inhibitors did change the levels of DNA binding activity of nuclear factor-kappaB (NF-kappaB), pERK1/2, c-Jun, and p-c-Jun. In conclusion, we could remark that proteasome inhibitors (especially PS341) attenuate the resistance of MCF7/ADR cells for P-gp substrate drugs of doxorubicin and paclitaxel. Several proteins are supposed to be associated with the resensitization of the cells to conventional cytotoxic drugs, although decreased activity of P-gp is at least involved in the proteasome inhibitor-related resensitization. And influence with MAPK pathways, which have been reported to be associated with the regulation of P-gp, might be contributed to the resensitization brought by proteasome inhibitors.

The Small Nuclear RNA-activating Protein 190 Myb DNA Binding Domain Stimulates TATA Box-binding Protein-TATA Box Recognition

Human U6 small nuclear RNA (snRNA) gene transcription by RNA polymerase III requires cooperative promoter binding involving the snRNA-activating protein complex (SNAP(c)) and the TATA-box binding protein (TBP). To investigate the role of SNAP(c) for TBP function at U6 promoters, TBP recruitment assays were performed using full-length TBP and a mini-SNAP(c) containing SNAP43, SNAP50, and a truncated SNAP190. Mini-SNAP(c) efficiently recruits TBP to the U6 TATA box, and two SNAP(c) subunits, SNAP43 and SNAP190, directly interact with the TBP DNA binding domain. Truncated SNAP190 containing only the Myb DNA binding domain is sufficient for TBP recruitment to the TATA box. Therefore, the SNAP190 Myb domain functions both to specifically recognize the proximal sequence element present in the core promoters of human snRNA genes and to stimulate TBP recognition of the neighboring TATA box present in human U6 snRNA promoters. The SNAP190 Myb domain also stimulates complex assembly with TBP and Brf2, a subunit of a snRNA-specific TFIIIB complex. Thus, interactions between the DNA binding domains of SNAP190 and TBP at juxtaposed promoter elements define the assembly of a RNA polymerase III-specific preinitiation complex.

Phosphotyrosyl Peptides Block Stat3-mediated DNA Binding Activity, Gene Regulation, and Cell Transformation

Signal transducers and activators of transcription (STATs) comprise a family of cytoplasmic signaling proteins that participates in normal cellular responses to cytokines and growth factors. Frequently, however, constitutive activation of certain STAT family members, particularly Stat3, has accompanied a wide variety of human malignancies. To identify small molecule inhibitors of Stat3, we investigated the ability of the Stat3 SH2 domain-binding peptide, PY*LKTK (where Y* represents phosphotyrosine), to disrupt Stat3 activity in vitro. The presence of PY*LKTK, but not PYLKTK or PFLKTK, in nuclear extracts results in significant reduction in the levels of DNA binding activities of Stat3, to a lesser extent of Stat1, and with no effect on that of Stat5. Analyses of alanine scanning mutagenesis and deletion derivatives of PY*LKTK reveal that the Leu residue at the Y+1 position and a substituent at the Y-1 position (but not necessarily Pro) are essential for the disruption of active Stat3, thereby mapping the minimum active sequence to the tripeptide, XY*L. Studies involving bead-coupled PY*LKTK peptide demonstrate that this phosphopeptide directly complexes with Stat3 monomers in vitro, suggesting that PY*LKTK disrupts Stat3:Stat3 dimers. As evidence for the functional importance of peptide-directed inhibition of Stat3, PY*LKTK-mts (mts, membrane translocating sequence) selectively inhibits constitutive and ligand-induced Stat3 activation in vivo. Furthermore, PY*LKTK-mts suppresses transformation by the Src oncoprotein, which has been shown previously to require constitutive Stat3 activation. Altogether, we have identified a minimal peptide that inhibits Stat3 signaling and provides the conceptual basis for use of this peptide as a lead for novel peptidomimetic drug design.

Cyclical expression of egr-1/NGFI-A in the rat anterior pituitary: a molecular signal for ovulation?

Molecular genetic studies of egr-1/NGFI-A have recently established a key role for this immediate early gene in anterior pituitary development, and particularly in transcriptional regulation of the LH beta-subunit gene. These studies, using null mutant mice, addressed the developmental aspects of gonadotrophin gene regulation by egr-1 and, because of the limitations of this approach, did not address the role of egr-1 in adult reproductive physiology. Using the rat, we have now investigated the expression of egr-1 during the female reproductive cycle, and have found evidence of cyclical changes in anterior pituitary egr-1 expression, at mRNA, protein, and DNA binding activity levels. Specifically, we have shown that egr-1 mRNA is significantly elevated during proestrous, and conversely suppressed on the subsequent day of oestrous. We have also demonstrated significantly raised levels of an approximately 70 kDa Egr-1 immunoreactive protein band during the night of proestrous, together with markedly raised levels of a Egr-1 consensus sequence DNA binding complex. These studies are indicative of both direct egr-1 gene regulation by oestrogen, and also regulation by GnRH. Finally, we have sequenced previously uncharacterized regions of the egr-1 gene and found evidence of a potential oestrogen response element. Our findings are consistent with the hypothesis that Egr-1 forms at least part of the molecular signal for ovulation in the pituitary.

Differentiation-Dependent Prolactin Responsiveness and Stat (Signal Transducers and Activators of Transcription) Signaling in Rat Ovarian Cells

PRL activates an important cytokine signaling cascade that is obligatory for maintaining luteal cell function in the rat ovary. To determine when specific components of this cascade are expressed and can be activated by PRL, we analyzed the expression of receptor subtypes (short, PRL-R(s), and long, PRL-R(L)), the presence and kinetics of Stat (signal transducer and activator of transcription) activation using the PRL-response element (PRL-RE) of the alpha2M (alpha2-macroglobulin) gene, and the content and hormonal regulation of three specific modulators of cytokine signaling; the tyrosine phosphatases (SHP-1 and SHP-2), and the protein inhibitor of activated Stat3 (PIAS-3). These components were analyzed in differentiating granulosa/ luteal cells of hypophysectomized (H) rats and in corpora lutea of pregnant rats. Levels of PRL-R mRNAs increased as granulosa cells differentiated and reached maximal levels in luteal cells of pregnant rats where levels of PRL-R(s) approached those of PRL-R(L). The relative concentrations shifted from a 27-fold excess of PRL-R(L) in preovulatory granulosa cells to a 3.7-fold difference in luteal cells during midgestation. Despite the increased PRL-R(L) expression in differentiated granulosa cells, PRL did not stimulate detectable activation of Stats. Rather PRL activation of Stat5, principally Stat5b, occurred in association with luteinization. In contrast, granulosa cells of untreated immature and H rats contained a high level of DNA binding activity, which was shown to be comprised entirely of activated, phosphorylated Stat3. Treatment with estrogen and FSH reduced the amount of phosphorylated Stat3 and abolished its ability to bind DNA, an effect temporally related to increased PIAS-3. Expression of SHP-1 (but not SHP-2) was also hormonally regulated; SHP-1 mRNA and protein were high in granulosa cells of H rats, decreased by estrogen and FSH, and subsequently increased dramatically with luteinization. Of particular note, SHP-1 was localized in cytoplasm of granulosa cells in atretic follicles but was distinctly nuclear in luteal cells, indicative of different functional roles. Collectively, these results indicate that Stat3 and Stat5 are activated by distinct cytokine-signaling pathways modulated through differentiation-dependent transcriptional regulation of signaling pathway components and mediate distinct functional processes in the rat ovary: early follicle growth and atresia vs. luteinization.

The C-terminal hydrophobic repeat of Schizosaccharomyces pombe heat shock factor is not required for heat-induced DNA-binding.

The C-terminal hydrophobic repeat (CTR) of heat shock transcription factor (HSF) has been proposed to regulate DNA binding by intramolecular interactions with the leucine zipper motifs present in the HSF trimerization domain. Schizosaccharomyces pombe provides a useful model organism for the study of the regulation of HSF DNA binding because, unlike Saccharomyces cerevisiae, S. pombe hsf is highly heat shock inducible for DNA binding and contains a clear homology to the CTR. We examined the role that the CTR plays in the regulation of S. pombe hsf by constructing isogenic strains bearing deletion and point mutations in the chromosomal copy of hsf. Surprisingly, we found that point mutation of key hydrophobic amino acids within the CTR, as well as full deletion of it, yielded factors that show normal binding at normal growth temperatures and full levels of heat-induced binding. Deletion of the CTR did, however, slightly lower the temperature required for maximal activation. In contrast, a large deletion of the C-terminus, which removes close to a third of the coding sequence, was deregulated and bound DNA at control temperature. Several of the deletion mutants were significantly reduced in their level of expression, yet they showed wild-type levels of DNA binding activity following heat shock. These experiments demonstrate that appropriate regulation of the DNA binding activity of S. pombe hsf is not solely dependent upon the CTR, and imply that a feedback mechanism exists that establishes proper levels of DNA binding following heat shock despite mutations that significantly alter levels of total hsf.

Pattern of DNA binding of nuclear proteins to the proximal Agrobacterium rhizogenesrolC promoter is altered during somatic embryogenesis of carrot

Carrot cells cultured in vitro in a medium supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) proliferate as unorganized cell clusters. Upon removal of 2,4-D from the culture medium, these cells undergo somatic embryo formation through globular, heart, and torpedo stages. Since the proximal −255 bp upstream region of the rolC gene of the Ri plasmid confers somatic embryogenesis-related activation on the uidA gene in transgenic carrot cell culture, we investigated the interaction of nuclear proteins with the proximal −255 bp upstream sequences to characterize the mechanism of somatic embryogenesis-related activation. Gel retardation experiments revealed that there were several different profiles of the relative levels of DNA binding activities in nuclear protein extracts from calli, PEMs (proembryogenic masses), globular embryos, and heart/torpedo embryos. The binding activity associated with a fragment (−203 bp to −92 bp) of one protein (BI) was most abundant in globular embryos. Another DNA binding protein (AII) showed the highest DNA binding activity in calli, but had low binding activities in PEMs and globular embryos. Such an altered pattern of DNA binding activities of nuclear proteins may contribute to somatic embryogenesis-related activation of the rolC promoter. Competition experiments with oligonucleotides revealed that the BI protein interacts with AT-rich sequences.

The nuclear factor kappa-B signaling pathway participates in dysregulation of vascular smooth muscle cells in vitro and in human atherosclerosis.

In the lesions of atherosclerosis, vascular smooth muscle cells (SMC) display many functions characteristic of cytokine activation that likely contribute importantly to ongoing inflammation during human atherogenesis. The transcription factor nuclear factor kappa-B (NFkappaB) often mediates the effects of cytokines on target cells, but the identity of Rel family members important in human SMC activation remains uncertain. In vitro, human SMC express multiple Rel family members. Of these, dimers of p65 and p50, but not a putative SMC-Rel, comprise basal and inducible NFkappaB binding activities. SMC express two inhibitor proteins IkappaBbeta and IkappaBalpha. Interleukin-1beta stimulation caused transient loss of IkappaBalpha and a sustained decrease of IkappaBbeta that correlated with increased and persistent levels of p65/p50 protein and binding activity in the nucleus. SMC cultured under serum-free conditions displayed little NFkappaB activity, but addition of serum or platelet-derived growth factor did activate NFkappaB. In situ analyses showed no evidence for basal NFkappaB activity in SMC in vivo as nonatherosclerotic arteries did not contain nuclear p65 or p50 protein. However, the nuclei of intimal SMC within human atheroma did contain both Rel proteins. We conclude that (i) dimers of p65 and p50, but not SMC-Rel, comprise NFkappaB complexes in human SMC; (ii) stimulatory components in serum activate NFkappaB and likely account for previously reported "constitutive" NFkappaB activity in cultured SMC; and (iii) exposure to inflammatory cytokines may produce prolonged NFkappaB activation in SMC because of sustained decreases in the inhibitory subunit IkappaB-beta.

Differentiation of human fibroblasts to tissue macrophages by the Snyder-Theilen feline sarcoma virus is accompanied by nuclear accumulation of the tumor suppressor p53

Snyder-Theilen feline sarcoma virus (ST:FeSV)-transduced human fibroblasts differentiate into tissue macrophages with many of the properties of normal macrophages. These cells express high levels of the gag-fes tyrosine kinase fusion protein, p85(v-fes), and exhibit an elevated level of tyrosine phosphorylation. Expression of the macrophage phenotype is accompanied by increased levels of DNA-binding activity and nuclear accumulation of wild-type p53. The DNA-binding activity of the transcription factors Egr-1, CREB and Sp1, which are known to be involved in cell differentiation, is also increased in ST:FeSV-induced macrophages. These observations suggest that v-fes can activate signal transduction pathways normally involved in macrophage differentiation, and that transcription factors such as p53, further facilitate v-fes-induced terminal differentiation.

Differential effects of transforming growth factor-beta 1 on the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in young and old human fibroblasts.

The balance between the activities of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is an important control point in tissue remodeling. Previous studies have demonstrated elevated expression of the MMPs collagenase and stromelysin-1 by aged human diploid fibroblasts compared to early-passage cultures. We show here that aging cells display an altered response to transforming growth factor-beta 1 (TGF beta 1) that selectively affects MMP mRNA expression. In both young and old cells, phorbol myristoyl-13 acetate (PMA) induced the expression of transcripts of collagenase, stromelysin-1, gelatinase-B, TIMP-1, and TIMP-3. In young cells, TGF beta 1 reciprocally modulated PMA-induced MMP and TIMP gene expression leading to reduced levels of transcripts for the MMPs and augmented accumulation of TIMP-1 and TIMP-3 mRNAs. However, repressing effects of TGF beta 1 on collagenase, stromelysin-1, and gelatinase-B RNA expression were not apparent in old cells, though induction of the TIMP genes was unimpaired. By electrophoretic mobility shift analysis the nuclear transcription factors AP1 and serum response factor (SRF) showed reduced levels of DNA binding activities in old fibroblasts compared to young cells. A probe for the TGF beta-inhibitory element (TIE) gave equivalent levels of complexes with nuclear extracts from both types of cells, though of different mobilities. We conclude that the effects of TGF beta 1 on MMP and TIMP gene expression involve different cellular intermediaries, and suggest that altered composition or modification of TIE binding factors in aging cells may underlie the failure of TGF beta 1-mediated transcription repression. This mechanism may contribute to elevated constitutive expression of MMPs in old cells and to the connective tissue deterioration that accompanies the aging process.

Sp1 Is Phosphorylated and Its DNA Binding Activity Down-regulated upon Terminal Differentiation of the Liver

Using nuclear extracts prepared from rat liver it was demonstrated that binding of a transcription factor to site II of the D-site binding protein promoter could be induced by dephosphorylation of these extracts. Competition band shifts and supershift assays reveal this protein to be the general transcription factor Sp1. Phosphorylation of Sp1 appears to occur as a result of terminal differentiation of the liver. Proteins from both 1-day-old rat liver and adult liver undergoing regeneration have less of the phosphorylated form of Sp1 present with consequent increased DNA binding activity. Sp1 is similarly phosphorylated in brain, kidney, and spleen with phosphatase treatment of the extracts significantly increasing the level of DNA binding activity. Dephosphorylation of Sp1 results in a 10-fold increase in the affinity of Sp1 for its cognate site. Two-dimensional gel electrophoresis reveals that approximately 20% of the detectable protein appears to be in the phosphorylated form in adult liver extracts. Another protein with similar characteristics also appears to be present in the liver. Decreasing Sp1 DNA binding activity by phosphorylation may be an important mechanism for regulating gene expression, and possibly bringing about growth arrest during terminal differentiation.

A Temperature-Sensitive IE1 Protein of Autographa californica Nuclear Polyhedrosis Virus Has Altered Transactivation and DNA Binding Activities

The temperature-sensitive mutant tsB821 of Autographa californica nuclear polyhedrosis virus has two nucleotide transitions in the transregulatory ie 1 gene (Riblero et al. (1994) J. Virol. 68, 1075-1084). These mutations result in two conservative amino acid substitutions in the encoded protein. To determine whether the mutations affect the ability of IE1 to interact with viral enhancer elements, electrophoretic mobility shift assays were performed using extracts prepared from infected cells and a fragment of the viral enhancer element hr5. When binding reactions. Were assembled on ice, the extracts prepared from tsB821-infected cells showed wild-type levels of DNA binding activity, whether the infected cells were grown at the permissive or nonpermissive temperature. However, when binding reactions were incubated at 33°, enhancer binding activity was significantly reduced in the ts extract. This suggests that the ts phenotype results from reduced interactions of tsIE1 with the viral enhancer elements. Site-directed mutagenesis was used to construct plasmids encoding IE1 with both of the amino acid substitutions (tsIE1) and with each of the single substitutions. tsIE1 was expressed in transient assays and exhibited thermolabile enhancer binding activity. However, proteins with single substitutions did not accumulate to detectable levels and did not exhibit enhancer binding activity. Temperature shift-up experiments with tsB821 indicated that late genes were expressed at wt levels when cells were shifted to the nonpermissive temperature during the early phase. This suggests that IE1 is not directly and continuously required for expression of late genes. However, polyhedrin expression was decreased in the mutant-infected cells when the temperature was shifted during the early or late phases of infection. Together, these results suggest that IE1 may differentially affect expression of these two classes of genes.

Evidence for DNA and Protein Binding by Styrene and Styrene Oxide

Styrene is metabolized to styrene oxide, a direct-acting mutagen and carcinogen. Styrene oxide reacts with DNA mainly at the N-7 position in guanine, but also at other sites and with other bases. Substitution occurs at both the alpha- and beta-positions of the styrene molecule. Experiments with radiolabeled styrene and styrene oxide demonstrate that both have a low level of DNA binding activity in experimental animals. 32P-Postlabeling studies have demonstrated the potential of the technique to detect styrene-DNA adducts. Styrene oxide alkylates several nucleophilic sites in proteins, particularly cysteine sulfydryl, histidine imidazole, lysine amino, aspartic, and glutamic carboxylic groups, and the N-terminal position. In experimental animals, styrene oxide treatment results in cysteine adducts in hemoglobin and albumin, valine adducts in hemoglobin, and carboxylic acid adducts in hemoglobin. The extent of alkylation is low compared with that produced by ethylene oxide. The available evidence indicates, therefore, that styrene and styrene oxide have low DNA and protein binding activities in vivo. There is preliminary evidence for the presence of DNA adducts and for adducts in hemoglobin and albumin in blood cells of styrene-exposed workers. Nevertheless, the applicability and sensitivity of DNA and protein adduct detection methods for monitoring human exposure to styrene remain to be determined.

Purification of a novel factor which binds to the mouse α2 (I) collagen promoter

We have identified and purified a DNA binding protein which specifically binds to a segment of the mouse α2 (I) collagen promoter between −420 and −399 bp upstream of the start of transcription. Purification included heparin-agarose and sequence-specific DNA-affinity chromatography, followed by SDS-PAGE and renaturation of the DNA binding activity after elution from SDS-polyacrylamide gel. The DNA binding activity resides in two species of 42 kDa and 40 kDa, respectively. The levels of DNA binding activity of this factor, which has been tentatively designated as ColFl, are considerably higher in nuclear extracts of NIH-3T3 fibroblasts than in nuclear extracts from epidermal cells, lymphoid cells and transformed NIH-3T3 fibroblasts.

Different biological activities of the hetero- and homodimers formed by the 47- and 43-kilodalton proteins of transcription factor ATF/E4TF3

The transcription factor ATF/E4TF3 stimulates transcription from the adenovirus early region 4 (E4) promoter by binding to specific promoter elements. Among the multiple forms of ATF/E4TF3, two forms with molecular masses of 47 and 43 kDa, which are most active in transcription in vitro from the E4 promoter, have been purified to homogeneity from HeLa cells by sequence-specific DNA affinity chromatography and biochemically characterized. Each purified protein formed a homodimer. These two homodimers were easily altered into a heterodimer when mixed together in the absence, but not in the presence, of the specific DNA sequence. All of these dimers were able to activate transcription in vitro from the E4 promoter by binding to the specific DNA sequence. Their activities to bind to DNA or stimulate transcription were different. The ability of the 47-kDa homodimer to stimulate transcription in vitro from the E4 promoter was approximately nine and three times higher than the abilities of the 43 kDa homodimer and the heterodimer, respectively, at the same level of DNA-binding activity. However, the affinity of the 47-kDa homodimer for DNA was lower than that of the 43-kDa homodimer, and the heterodimer had intermediate affinity. These results are the first to show differential binding and transcriptional activation activities of the different dimers of ATF/E4TF3, using purified cellular proteins rather than cloned gene products.