Independent regulation of tumor necrosis factor and lymphotoxin production by human peripheral blood lymphocytes.

We present evidence that human peripheral blood lymphocytes, free of contaminating monocytes, rapidly produce high levels of tumor necrosis factor (TNF) when stimulated with phorbol diester and calcium ionophore, and lower but significant levels of TNF when stimulated with mitogens. These two types of inducers act preferentially on T cells, both CD4+ and CD8+. NK cells produce TNF only when stimulated with phorbol diester and calcium ionophore, and they do so at a much lower level than T cells. The procedures used in the purification of lymphocytes and the differential ability to respond to various inducers allow us to exclude that monocytes or basophils contaminating the lymphocyte preparation participate in the production of TNF. In particular, LPS, a potent inducer of TNF production from monocytes, is unable to induce significant levels of TNF in the lymphocyte preparations. The TNF produced by lymphocytes has antigenic, physicochemical, and biochemical characteristics identical to those of the TNF produced by myeloid cell lines or monocytes upon stimulation with LPS. LT is also produced by lymphocyte preparations. Production of TNF and LT proteins in response to the different inducers is paralleled by accumulation of cytoplasmic TNF and LT mRNA. Both at mRNA and at protein levels, stimulation of T lymphocytes with phorbol diester and calcium ionophore preferentially induces TNF, whereas mitogen stimulation preferentially induces LT. Our data suggest that the TNF and LT genes, two closely linked genes encoding two partially homologous proteins with almost identical biological functions, are independently regulated in lymphocytes.

Two domains of p53 interact with the TATA-binding protein, and the adenovirus 13S E1A protein disrupts the association, relieving p53-mediated transcriptional repression.

The tumor suppressor gene product p53 can activate and repress transcription. Both transcriptional activation and repression are thought to involve the direct interaction of p53 with the basal transcriptional machinery. Previous work has demonstrated an in vitro interaction between p53 and the TATA-binding protein that requires amino acids 20 to 57 of p53 and amino acids 220 to 271 of the TATA-binding protein. The present results show that a 75-amino-acid segment from the carboxy terminus of p53 also can bind to the TATA-binding protein in vitro, and this interaction requires amino acids 217 to 268 of the TATA-binding protein, essentially the same domain that is required for interaction with the amino-terminal domain of p53. A carboxy-terminal segment of p53 can mediate repression when bound to DNA as a GAL4-p53 fusion protein. The amino- and carboxy-terminal p53 interactions occur within the domain on the TATA-binding protein to which the adenovirus 13S E1A oncoprotein has previously been shown to bind. The 13S E1A oncoprotein can dissociate the complex formed between the carboxy-terminal domain of p53 and the TATA-binding protein and relieve p53-mediated transcriptional repression. These results demonstrate that two independent domains of p53 can potentially interact with the TATA-binding protein, and they define a mechanism--relief of repression--by which the 13S E1A oncoprotein can activate transcription through the TATA motif.

Transcription elongation factor SII interacts with a domain of the large subunit of human RNA polymerase II.

Genomic sequences for the large subunit of human RNA polymerase II corresponding to a part of the fifth exon were inserted into an expression vector at the carboxy-terminal end of the beta-galactosidase gene. The in-frame construct produced a 125-kilodalton fusion protein, containing approximately 10 kilodaltons of the large subunit of RNA polymerase II and 116 kilodaltons of beta-galactosidase. The purified bacterially produced fusion protein inhibited specific transcription from the adenovirus type 2 major late promoter, while beta-galactosidase had no effect. This effect of the fusion protein was during RNA elongation, not at the level of initiation, resembling the faithfully initiated but incomplete transcripts produced with purified factors in the absence of SII. Similarly, monoclonal antibody 2-7B, which reacts with the RNA polymerase II region represented in the fusion protein, inhibited specific transcription at the level of elongation in a whole-cell extract. Both monoclonal antibody 2-7B and the fusion protein, although unable to inhibit purified RNA polymerase II in a nonspecific transcription assay, selectively blocked the stimulation elicited by transcription elongation factor SII on the activity of the purified enzyme in vitro. This suggests that the fusion protein traps the SII in nonstimulatory interactions and that antibody 2-7B inhibits SII binding to RNA polymerase II. Thus, this suggests that an SII-binding contact required for specific RNA elongation resides within the fifth exon region of the largest RNA polymerase II subunit.

A DNA element that regulates expression of an endogenous retrovirus during F9 cell differentiation is E1A dependent.

The retinoic acid-induced differentiation of F9 cells into parietal endoderm-like cells activates transcription of the endogenous mouse retrovirus, the intracisternal A-particle (IAP). To investigate the elements that control IAP gene differentiation-specific expression, we used methylation interference, Southwestern (DNA-protein), and transient-transfection assays and identified the IAP-proximal enhancer (IPE) element that directs differentiation-specific expression. We find that the IPE is inactive in undifferentiated F9 cells and active in differentiated parietal endoderm-like PYS-2 cells. Three proteins of 40, 60, and 68 kDa bind to the sequence GAGTAGAC located between nucleotides -53 and -47 within the IPE. The 40- and 68-kDa proteins from both the undifferentiated and differentiated cells exhibit similar DNA-binding activities. However, the 60-kDa protein from differentiated cells has greater binding activity than that from undifferentiated cells, suggesting a role for this protein in F9 differentiation-specific expression of the IAP gene. The IAP gene is negatively regulated by the adenovirus E1A proteins, and the E1A sequence responsible for repression is located at the N terminus, between amino acids 2 and 67. The DNA sequence that is the target of E1A repression also maps to the IPE element. Colocalization of the differentiation-specific and E1A-sensitive elements to the same protein-binding site within the IPE suggests that the E1A-like activity functions in F9 cells to repress IAP gene expression. Activation of the IAP gene may result when the E1A-like activity is lost or inactivated during F9 cell differentiation, followed by binding of the 60-kDa positive regulatory protein to the enhancer element.

c-fos mediated stimulation of an AP-1 DNA binding activity in undifferentiated teratocarcinoma cell lines.

Undifferentiated F9 and PCC4 embryonal carcinoma (EC) cells contain low levels of AP-1 DNA binding activity. Upon differentiation induced by retinoic acid and cyclic AMP or in differentiated cell lines, AP-1 DNA binding activity can be readily detected. Minute amounts of 3T6 cells extracts, that by themselves were unable to show any binding to an AP-1 site, stimulate AP-1 DNA binding activity when added to the EC cell extracts, suggesting that components of the 3T6 extracts stimulate this DNA binding activity in F9 and PCC4 cell extracts. This enhancement of DNA binding activity requires the presence in the donor fraction (3T6 cells) of a thermostable protein(s) that possesses neither protein kinase nor phosphatase activities. The proteins responsible for stimulation in 3T6 extracts can be separated from the ones responsible for AP-1 binding by chromatography. 3T6 c-fos immunodepleted fractions are unable to activate AP-1 DNA binding activity in EC cell extracts, while c-jun depleted fractions activate normally. Moreover, in vitro translated c-fos, but not c-jun proteins, are able to stimulate binding in EC extracts. These data suggest an important role for c-fos protein in activation of a specific DNA binding transcriptional factor during cellular differentiation and provide a convenient in vitro assay for c-fos function.

Interactions between adenovirus E1A and members of the AP-1 family of cellular transcription factors.

We have studied interactions between bacterially produced E1A linked to Sepharose and the various DNA-binding proteins present in HeLa cell nuclear extracts (NE). DNA-binding activities and cross-reactive polypeptides recognizing the cAMP-responsive element (CRE) and the activator protein 1 (AP1) sites were bound to the E1A column, whereas nuclear factor 1 (NF1) and the activator protein 2 (AP2) DNA-binding activities were not retained by E1A. The binding activities that were retained belonged to the CRE and JUN protein family, as judged by Western blot analysis. Authentic CRE-BP1, c-Jun and c-Fos proteins produced by in-vitro translation also bound to the E1A column. However, efficient binding of in-vitro-translated CRE-BP1 and c-Fos proteins to E1A required preincubation with NE. We show here that immobilized E1A sequesters several cellular upstream transcription activators, and suggest a role for members of the AP1 family of transcription factors in E1A-mediated gene regulation.

Farnesyltransferase inhibitors are inhibitors of Ras but not R-Ras2/TC21, transformation.

Recent results from several laboratories including ours strongly suggest that farnesyltransferase (FT) inhibitors belonging to distinct chemical classes block growth of oncogenic Ras transformed cells at concentrations that do not affect the growth and viability of normal cells. This is despite blocking the farnesylation and thus the membrane association of Ras in both cell types. This is a paradox given the requirement for Ras function in normal cell growth. Recent evidence that R-Ras2/TC21 utilizes components of Ras signal transduction pathways to trigger cellular transformation (Graham et al., MCB 14, 4108-4115, 1994) prompted us to consider the possibility that R-Ras2/TC21 is involved in some aspects of the growth regulation of normal cells. If so, R-Ras2/TC21 may be compensating for Ras function in untransformed cells treated with FT inhibitors. In this study, we demonstrated that a cell active bisubstrate analog FT inhibitor, BMS-186511, completely blocked the function of oncogenic Ras, but did not affect the function of oncogenic R-Ras2/TC21, as determined by several criteria including inhibition of anchorage dependent and independent growth, reversal of transformed morphology and restoration of actin cytoskeleton. While it is known that TC21 protein becomes prenylated, it is not known whether it is farnesylated or geranylgeranylated. Our in vitro prenylation experiments indicate that R-Ras2/TC21 protein serves as a good substrate for FT as well as geranylgeranyltransferase I (GGTI) and thus provide the apparent molecular basis for these differences. Overall, these results, coupled with the ubiquitous expression of R-Ras2/TC21 in many cells including untransformed NIH3T3 cells, are consistent with the possibility that R-Ras2/TC21 may be one of the factors that render normal cells insensitive to the growth inhibitory action of FT inhibitors.

Use of thiotriphosphates for the study of in vitro initiation in adenovirus-infected cell nuclei.

The use of thiotriphosphates as a means of analyzing in vitro initiation products of adenovirus-infected cells is demonstrated for the RNA polymerase III transcript VA-RNA. Results suggest its usefulness in discriminating between adenosine- or guanosine-initiated transcripts and in selecting the 5'-oligonucleotide.

Shut-off of actin biosynthesis in adenovirus serotype-2-infected cells.

Adenovirus produces a dramatic shut-off of host protein synthesis after infection of HeLa cells. The level of actin messenger RNAs remained relatively unchanged after viral infection, when assayed by in vitro translation and two-dimensional gel electrophoresis analysis of the proteins or hybridization of the total cytoplasmic RNAs to the human actin gene. The distribution of actin mRNA in the polyribosomes is altered after adenovirus infection, with small polyribosomes and monoribosomes of the infected cells occupied by actin messages untranslatable in a rabbit reticulocyte lysate. The large polyribosomes still retain enough functional mRNAs to provide significant levels of actin protein in a rabbit reticulocyte in vitro translation system. In contrast, in homologous infected cell lysates, the translation of exogenous actin mRNA is greatly reduced when compared to uninfected HeLa cell lysates. In nuclease-treated uninfected or infected HeLa cell-free extracts, translation of viral mRNA is equally efficient and higher than that of actin mRNA. Thus, translational regulatory mechanisms which include inactivation of a part of the actin mRNA population accompanied by displacement to small polysomes and/or virus-induced modification of the cellular translational machinery to discriminate against cellular actin mRNA seem to account for the sharp reduction in actin protein synthesis of adenovirus-infected cells.

Actin mRNAs in HeLa cells. Stabilization after adenovirus infection.

We have carried out a comparative analysis of the expression of the actin genes in HeLa and adenovirus-infected HeLa cells. The rate of actin gene transcription was examined in these cells by pulse-labeling of the newly synthesized RNA and/or by in vitro transcription in nuclei isolated from uninfected or infected HeLa cells. In addition, accumulation of actin-specific heterogeneous nuclear RNA, and rate of appearance of the actin mRNAs in the cytoplasm were examined by dot and Northern blot analysis. The rate of actin gene transcription remained constant after infection of HeLa cells with adenovirus serotype 2, while the level of the actin precursor in the nuclei was slightly reduced. In the infected cells, newly synthesized actin mRNA enters the cytoplasm at a very reduced rate. The deficiency of transport does not affect the steady-state level of the messages in the cytoplasm. The half-life of cytoplasmic actin mRNAs was analyzed by traditional pulse-chase experiments and by a novel procedure using 5-6-diCl-1-beta-d-ribofuranosylbenzimidazole, which does not rely on labeled RNA. Both procedures gave identical results. Uninfected HeLa cells have actin mRNAs with relatively short half-lives, from less than six to 12 hours. In contrast, the half-lives of the actin-specific mRNAs, in the cytoplasm of adenovirus-infected cells, is greater than 14 to 24 hours. These observations suggest that, although the rate of transport of actin mRNAs to the cytoplasm is reduced upon infection with adenovirus, increased half-lives result in accumulation of actin mRNAs to normal levels in the cytoplasm.

Mechanism of action of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. II. A resistant human cell mutant with an altered transcriptional machinery.

To determine the role of DRB in transcription, we isolated a resistant (DRBR) HeLa cell mutant. After mutagenesis with N-methyl-N'-nitro-nitrosoguanidine, cell colonies able to grow at 20 micrograms DRB/ml (63 microM) were selected. One of these colonies, DRBR-1, was stable and able to grow at concentrations of DRB three to five times higher than tolerated by normal HeLa cells. The DNA of DRBR-1 was able to confer resistance to DRB to other HeLa cells by transfection. Uridine uptake was reduced by DRB to a similar extent in both wild-type and mutant cells. In contrast, transcription in the mutant cells, as measured by [3H]uridine incorporation into RNA in short pulses, was resistant to DRB. Cell-free extracts prepared from DRBR-1 cells are able to transcribe the epsilon-globin or the adenovirus 2 major late promoter genes at DRB concentrations that eliminate the transcriptional activity of HeLa cell extracts. Thus the transcriptional machinery of the mutant is altered. The presence of both DRB-resistant and DRB-sensitive transcriptional activities in extracts from DRBR-1 cells, grown in the presence of the drug, suggests constitutive expression of this cellular component. Efficient somatic cell hybridization with an alpha-amanitin-resistant RNA polymerase II mouse mutant indicates cross-complementation in vivo. This DRBR mutant provides a useful tool for the biochemical analysis of the mechanism of action of DRB on transcription. It also serves as a genetic handle for selection of the gene responsible for DRB resistance.

Mechanism of action of DRB. III. Effect on specific in vitro initiation of transcription.

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole, an adenosine analogue, has been used previously as an inhibitor of heterogeneous nuclear and messenger RNA synthesis. In an in vitro transcriptional system, we have detected inhibition of synthesis of full-length runoff RNAs at concentrations at which in vivo mRNA synthesis is inhibited. By hybridization of RNA synthesized in vitro to single-stranded DNA and gel analysis, we were able to reduce the background of the transcription reaction, detect DRB-induced inhibition of full-length runoff RNAs and DRB-insensitive transcription of short RNAs. To establish further the effect of DRB on initiation of transcription, preincubation experiments with template, whole cell extract and two initial nucleotides of the transcript were performed. Elongation was then measured as discrete-sized RNAs transcribed from the truncated template after addition of the other triphosphates (one of them labeled), in the presence or absence of DRB. An effect on initiation but not on elongation or termination was detected. Fingerprint analysis of these runoff RNAs indicates that the labeling of U in the presence of DRB is uniform throughout the molecule. A model to explain a novel interpretation of the action of DRB is presented.

Regulation of ribosomal RNA and 5s RNA synthesis in Drosophila melanogaster. I. Bobbed mutants.

Analysis of the rates and amounts of rRNA and 5s RNA synthesized in Drosophila melanogaster bobbed mutants was done by using acrylamide-gel electrophoresis. The results show that the amounts of rRNA synthesized are constant, although the rates of rRNA synthesis in bb's are reduced to 30% of the wild-type level. The rates of synthesis of 5s RNA were constant. The rate of synthesis of the two kinds of molecules that enter in equimolar amounts into the mature ribosome is non-coordinated.-The rates of rRNA synthesis were shown to be proportional to the length of the scutellar bristles, supporting the notion that in trichogen cells there is no developmental delay, but the size of the bristle depends directly on the rate of rRNA synthesis.

Isolation and characterization of human actin genes cloned in phage lambda vectors.

Using Drosophila and chicken actin probes, we have selected 14 human actin lambda recombinants from a genomic library. We present a restriction maps indicating the positions of the sequences homologous to actin and to an Alu probe. Restriction mapping has revealed that nine out of ten of these clones are distinct, indicating that actin is a multigene family. Hybrid elution of HeLa cell mRNA from filters containing the recombinant DNA, followed by in vitro translation and immunoprecipitation, as well as one- or two-dimensional protein analysis, shows that these recombinants code for actin. Hybridization back to human DNA digested with restriction enzymes shows that the EcoRI fragments of at least one of the lambda recombinants (lambda HA-5) result in similar-sized human DNA fragments in the intact genome. In nuclei, a 4.5-kb mRNA precursor to the cytoplasmic 1.9-kb mRNA can be detected by hybridization with genomic or cDNA probes, indicating the presence of additional sequences and RNA processing.

A rapid purification method for calf thymus casein kinase II.

We report here the rapid purification to homogeneity of a cyclic nucleotide-independent protein kinase sensitive to 5'6-dichloro-1-beta-D-ribofuranozylbenzimidazole (DRB), identical to the previously described casein kinase II, from lyophilized calf thymus by chromatography on phosphocellulose and Mono-Q FPLC columns.

The phosphorylation of nucleoplasmin by casein kinase-2 is resistant to heparin inhibition.

Highly purified preparations of casein kinase-2 from the nuclei of Xenopus laevis oocytes and from calf thymus can phosphorylate in vitro purified nucleoplasmin from X. laevis oocytes and eggs. The phosphorylation of nucleoplasmin by both kinase preparations is quite insensitive to heparin in contrast with casein phosphorylation which is completely abolished by heparin concentrations above 10 micrograms/ml. However, the phosphorylation of nucleoplasmin and casein are inhibited in a very similar fashion by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a well characterized specific inhibitor of casein kinase-2. Similarly, nucleoplasmin phosphorylation by the oocyte enzyme can be stimulated several-fold by spermine, another characteristic of this enzyme. These findings indicate that the phosphorylation of nucleoplasmin by purified casein kinase-2, while showing typical response to DRB and spermine, exhibits anomalous behavior in its resistance to heparin inhibition. It is possible that the large clusters of acidic amino acids in nucleoplasmin permit this substrate to interact with the enzyme more efficiently than other protein substrates. Heparin is generally considered a potent and specific inhibitor of casein kinase-2. This study, however, questions the validity of utilizing heparin inhibition as a criterion for casein kinase-2 involvement.

Erythropoietin-beta-D-galactosidase. The generation, purification and use of a fusion protein.

A human erythropoietin (Epo) cDNA fragment encoding the complete erythropoietin peptide sequence was fused to the 3'-end of the lacZ gene in the polylinker region of the high expression vector, pUR 278. Escherichia coli bacteria were transformed with the recombinant plasmid harboring the hybrid Epo-beta-D-galactosidase gene. After induction with isopropyl-thiogalactoside large amounts of the fusion protein, Epo-beta-D-galactosidase were synthesized in the transformed bacteria. The fusion protein was partially purified and shown to exhibit intact galactosidase enzymatic activity. Although no biological activity of the Epo counterpart of the fusion protein was detected both in an in vivo and in an in vitro bioassay, the fusion protein served as an effective antigen for the production of anti-erythropoietin antibodies. Antifusion protein antibodies raised in rabbits were shown to react with the intact human Epo molecule from erythropoietin producing culture supernatants. The affinity of these anti-fusion protein antibodies was sufficiently high to permit the development of a sensitive radioimmunoassay for human Epo. This fusion protein approach is a relatively straightforward and rapid method of generating antibodies with specificity for any protein encoded by a cloned eukaryotic gene.

Transforming region of 243R E1A contains two overlapping but distinct transactivation domains.

Conserved regions 1 and 2 as well as the amino terminus of E1A are required for the transforming activity of the E1A oncoprotein. We show here that the amino terminus of 243R E1A has transactivation activity when brought to a promoter in yeast. Recruitment to a specific promoter is essential. Mutagenesis studies correlated the transactivation function with the extreme amino terminus and the conserved region 1 of E1A. Cotransfection assays in rodent cells confirmed that two overlapping but distinguishable domains, amino acids 1-65 and 37-80, can transactivate independently when targeted to a promoter. We also observed that when recruited to the proliferating cell nuclear antigen (PCNA) promoter, the amino-terminal region was sufficient to transactivate the PCNA promoter. On the other hand, deletion of the amino terminus of E1A resulted in failure to induce PCNA expression. Fusion of VP16 with the amino-terminal-deleted E1A mutant was able to restore the ability to induce the PCNA promoter. We further show that the amino-terminal region also is required for 243R E1A to repress the transactivation mediated by a universal transactivator DBD.VP16 and DBD.E1A. This repression could be specifically relieved by overexpression of TBP but not TFIIB. In addition, we show that the amino terminus of E1A is involved in in vitro interaction with the TATA binding protein (TBP). Thus the amino-terminal transforming region of E1A may regulate cellular gene expression in species that are distant in evolution via a common mechanism, functionally targeting TBP.

The basic RNA polymerase II transcriptional machinery.

All genes encoding proteins in eukaryotes are transcribed by RNA polymerase II. The first step in analyzing transcriptional regulation requires understanding the general mechanisms of RNA polymerase II-specific gene transcription. The basal promoter, a template containing a TATA box devoid of upstream regulatory sequences, has been used to identify and characterize the factors which, together with RNA polymerase II, govern transcription in mammalian systems: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIG, TFIIH, and TFIIJ. Interactions between regulatory transcription factors and basal elements of the transcriptional machinery affect the transcriptional rate in a positive or negative fashion. As these multiple proteins are purified, and their coding sequences are isolated, we come closer to reproducing these processes in vitro with pure components, and thus to elucidating the complex interactions among them.

Preferential distribution of active RNA polymerase II molecules in the nuclear periphery.

We have combined immunogold labeling with the Miller spreading technique in order to localize proteins at the electron microscope (EM) level in whole mount nuclei from mouse and human fibroblasts. Anti-histone H1 antibody labels nuclei uniformly, indicating that the nuclear interior is accessible to both antibodies and gold conjugates. Anti-topoisomerase I antibody labels nucleoli intensely, in agreement with previous immunofluorescent and biochemical data. Two different antibodies against the large subunit of RNA polymerase II (pol II) show preferential labeling of the nuclear periphery, as do antibodies against lamin, a known peripheral nuclear protein. Treatment of cells with alpha-amanitin results in loss of virtually all RNA polymerase II staining, supporting the specificity of labeling. Finally, when nuclei are incubated in the presence of biotin-UTP (bio-UTP) under run-off transcription conditions, incorporation is preferentially located at the nuclear periphery. These results support the conclusions that transcriptionally active pol II molecules are non-uniformly distributed in fibroblast nuclei, and that their differential distribution mirrors that of total pol II.