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Global health projects-a source of inspiration and collaboration for applied human biology-benefit scholars, governments, NGOs, and aid organizations. While such research is intended to improve population health, direct benefits to individuals and communities are often excluded from published works and/or not considered in study designs and framing. This exclusion is increasingly recognized as a colonial legacy that hinders global health equity, particularly for Indigenous and other marginalized populations. Collaboration and community engagement are avenues for addressing these injustices, but they require planning, intention, and resources. Drawing on our collective experience and ongoing dialogues about community engagement in human biology, we propose six recommendations to increase equity in global health research. These include: (1) Incorporating trusted local specialists and stakeholders at all project levels; (2) disseminating health information to participants in strengths-based and culturally meaningful ways and contributing to solutions wherever possible; (3) investing in local healthcare, research, and infrastructure; (4) making study results/data available to stakeholders; (5) working within data frameworks that respect community sovereignty; and, (6) applying culturally informed bioethics frameworks. Our discussion highlights persistent needs to address community rights and benefits and to dismantle colonial legacies within global health and human biology while recognizing structural barriers to implementing these needed changes, particularly within the context of global health projects wherein human biologists are not the main power brokers or resource holders. When interfacing with global health, human biologists must continue to pursue health equity and decolonization through implementing critical, culturally informed bioethics frameworks centering community engagement., (© 2025 Wiley Periodicals LLC.)

Much of the literature on eating disorders deals with Western subjects. Although the majority of those seen in clinical settings are Caucasians, reports from Asia suggest that anorexia nervosa and bulimia nervosa do occur in the Chinese, sparking debate as to whether or not it is the result of Westernization. This project begins with a review of the current literature on eating disorders in Chinese populations and the role of culture as a mediating factor. A psychodynamic conceptualization and the potential role of traumatic experiences are explored in the emergence of pathological eating habits. Research suggests that applying Western models for Chinese subjects with eating disorders may not always be appropriate.

An asymmetric polypurine-polypyrimidine cis-element located in the 5' region of the mouse vascular smooth muscle alpha-actin gene serves as a binding site for multiple proteins with specific affinity for either single- or double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to trans-activate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur alpha and Pur beta with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur alpha, Pur beta, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur alpha, Pur beta, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur alpha, Pur beta, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.

Tissue factor, the cellular initiator of blood coagulation, has been implicated as a determinant of metastatic potential in human melanoma cells. Here, we report that differential expression of tissue factor in murine melanoma cell lines of known metastatic behavior is mediated by AP-1-dependent and 12S E1A oncoprotein-repressible gene transcription. When compared to weakly metastatic C10 cells, highly metastatic M4 cells possessed elevated levels of tissue factor cofactor activity, transfected promoter activity, and heterodimeric AP-1 DNA-binding complexes containing Fra-1. Transient co-expression of the adenovirus E1A 12S oncoprotein strongly repressed transcription of an AP-1-driven tissue factor reporter gene indicating the additional requirement of N-terminal E1A-interacting coactivators. Stable expression of E1A mutants defective in CBP/p300-binding failed to suppress tissue factor expression and experimental metastasis by M4 cells while clones expressing wild type E1A exhibited greatly reduced tissue factor cofactor activity and metastatic potential in vivo. Overexpression of functional tissue factor in cells containing wild type E1A failed to restore the highly metastatic M4 phenotype suggesting that additional E1A-responsive and CBP/p300-dependent genes are required to facilitate metastasis of murine melanoma cells demonstrating high tissue factor expression and cofactor activity.

Serum-stimulation of quiescent mouse fibroblasts results in transcriptional activation of tissue factor (TF), the cellular initiator of blood coagulation. This requires the rapid entry of c-Fos into specific AP-1 DNA-binding complexes and can be strongly inhibited by the adenovirus EIA 12S gene product. In this study, we utilized a panel of E1A mutants deficient in cellular protein binding to analyse the molecular basis for EIA inhibition of a minimal, c-Fos-dependent TF promoter/ reporter construct in mouse AKR-2B fibroblasts. Mutations which impaired binding of the retinoblastoma tumor suppressor protein family members pRB, p107, and p130 relieved E1A-mediated inhibition of transcription in response to serum-stimulation or c-Fos overexpression. Inhibition was restricted to the G0 to G1 transition, consistent with the specificity of E1A for hypophosphorylated forms of RB proteins. Although E1A mutants deficient in CBP/p300 binding retained the ability to inhibit TF transcription, deletion of the amino-terminal portion of the CBP/p300 interaction domain was required to permit rescue of TF promoter activity by coexpression of pRB. Moreover, ectopic p107 could effectively substitute for pRB in relieving E1A-mediated repression. In primary mouse embryo fibroblasts, activity of the minimal AP-1-dependent TF promoter was suppressed in Rb(-/-) cells compared to parallel Rb(+/-) and Rb(+/+) transfectants. Ectopic expression of either pRB or p107 markedly enhanced TF promoter activity in Rb(-/-) fibroblasts. Collectively, these data imply that pRB and p107 can cooperate with c-Fos to activate TF gene transcription in fibroblasts and suggest a requirement for another, as yet unidentified, E1A-binding protein.

Stimulation of quiescent AKR-2B mouse fibroblasts with transforming growth factor beta1 results in uniform conversion to a myofibroblast-like phenotype as judged by a rapid accumulation of smooth muscle alpha-actin mRNA and protein. Because transcriptional regulation of the smooth muscle alpha-actin gene in these cells might be mediated by single-stranded DNA-binding proteins, we have examined the sensitivity of genomic DNA to chemical reagents with specificity for unpaired bases in a region of the promoter previously implicated in Puralpha, Purbeta, and MSY1 binding in vitro (Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238-14245). Our data reveal specific differences between purified DNA treated in vitro and nucleoprotein complexes treated in living cells. Although some differences were observed in quiescent cells, treatment with transforming growth factor beta1 resulted in the development of additional sensitivity within 1 h. This enhancement was most pronounced in bases immediately upstream of an MCAT enhancer element-containing polypurine-polypyrimidine tract. A TATA-proximal element of similar base distribution showed no such hyperreactivities. These results suggest that activation of the endogenous smooth muscle alpha-actin gene during myofibroblast conversion is accompanied by specific structural changes in the promoter that are consistent with a decline in single-stranded DNA repressor protein binding.

Amino acids 44-53 of mouse vascular smooth muscle alpha-actin are encoded by a region of exon 3 that bears structural similarity to an essential MCAT enhancer element in the 5' promoter of the gene. The single-stranded DNA-binding proteins, Puralpha, Purbeta, and MSY1, interact with each other and with opposite strands of the enhancer to repress transcription in fibroblasts (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436; Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238-14245). In this study, we employed both recombinant and fibroblast-derived proteins to demonstrate that all three proteins specifically interact with the mRNA counterpart of the exon 3 sequence in cell-free binding assays. When placed in the 5'-untranslated region of a reporter mRNA, the exon 3-derived sequence suppressed mRNA translation in transfected fibroblasts. Translational efficiency was restored by mutations that impaired mRNA binding of Puralpha, Purbeta, and MSY1, implying that these proteins can also participate in messenger ribonucleoprotein formation in living cells. Additionally, primary structure determinants required for interaction of Purbeta with single-stranded DNA, mRNA, and protein ligands were mapped by deletion mutagenesis. These experiments reveal highly specific protein-mRNA interactions that are potentially important in regulating expression of the vascular smooth muscle alpha-actin gene in fibroblasts.

Transcriptional activity of the mouse vascular smooth muscle alpha-actin gene in fibroblasts is regulated, in part, by a 30-base pair asymmetric polypurine-polypyrimidine tract containing an essential MCAT enhancer motif. The double-stranded form of this sequence serves as a binding site for a transcription enhancer factor 1-related protein while the separated single strands interact with two distinct DNA binding activities termed VACssBF1 and 2 (Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995) J. Biol. Chem. 270, 11310-11321; Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2936). VACssBF2 has been recently cloned and shown to consist of two closely related proteins, Puralpha and Purbeta (Kelm, R. J., Elder, P. K., Strauch, A. R., and Getz, M. J. (1997) J. Biol. Chem. 272, 26727-26733). In this study, we demonstrate that Puralpha and Purbeta interact with each other via highly specific protein-protein interactions and bind to the purine-rich strand of the MCAT enhancer in the form of both homo- and heteromeric complexes. Moreover, both Pur proteins interact with MSY1, a VACssBF1-like protein cloned by virtue of its affinity for the pyrimidine-rich strand of the enhancer. Interactions between Puralpha, Purbeta, and MSY1 do not require the participation of DNA. Combinatorial interactions between these three single-stranded DNA-binding proteins may be important in regulating activity of the smooth muscle alpha-actin MCAT enhancer in fibroblasts.

Transcriptional repression of the mouse vascular smooth muscle alpha-actin gene in fibroblasts and myoblasts is mediated, in part, by the interaction of two single-stranded DNA binding activities with opposite strands of an essential transcription enhancer factor-1 recognition element (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436). One of these activities, previously designated vascular actin single-stranded DNA-binding factor 2 includes two distinct polypeptides (p44 and p46) which specifically interact with the purine-rich strand of both the enhancer and a related element in a protein coding exon of the gene (Kelm, R. J., Jr., Sun, S., Strauch, A. R., and Getz, M. J. (1996) J. Biol. Chem. 271, 24278-24285). Expression screening of a mouse lung cDNA library with a vascular actin single-stranded DNA-binding factor 2 recognition element has now resulted in the isolation of two distinct cDNA clones that encode p46 and p44. One of these proteins is identical to Puralpha, a retinoblastoma-binding protein previously implicated in both transcriptional activation and DNA replication. The other is a related family member, presumably Purbeta. Comparative band shift and Southwestern blot analyses conducted with cellular p46, p44, and cloned Pur proteins synthesized in vitro and in vivo, establish identity of p46 with Puralpha and p44 with Purbeta. This study implicates Puralpha and/or Purbeta in the control of vascular smooth muscle alpha-actin gene transcription.

Stimulation of quiescent mouse fibroblasts with TGF-beta1 and certain other growth factors result in cooperative activation of tissue factor (TF) gene transcription, an event accompanied by the rapid entry of c-Fos into specific AP-1 DNA-binding complexes (Felts et al. (1995) Biochemistry 34, 12355-12362). Here, we demonstrate that the ability of TGF-beta1 to synergistically activate TF transcription in serum-stimulated fibroblasts is dependent upon both c-Fos and a promoter-specific factor with functional properties characteristic of transcriptional coactivators. Inhibition of TF promoter activity by an adenovirus E1A mutant deleted in an essential CREB binding protein (CBP) interaction domain suggests that this factor is distinct from the CBP/p300 family of transcriptional coactivators. Importantly, the ability of this factor to mediate molecular interactions with c-Fos required for transcriptional synergism is directly linked to TGF-beta1 signaling. These data suggest a model in which a component of the TF basal transcription complex functions to integrate multiple signaling pathways required for full transcriptional activation of TF in fibroblasts.

Tissue factor (TF), the cellular initiator of the protease blood coagulation cascade, has been shown to be expressed in a variety of solid tumors, particularly those of epithelial origin. However, the mechanisms that mediate TF expression in tumors, as well as the clinical implications of this expression, remain largely unknown. In this study, we examined the cytological distribution of TF in normal human breast tissue and breast carcinomas. Epithelial cells exhibited TF immunoreactivity with little obvious correlation with malignant progression from in situ lesions to invasive cancer. However, there was a strong correlation between progression to invasive cancer and the expression of TF antigen in cellular components of the stroma. TF-positive cells were particularly abundant in close proximity to infiltrating tumor cells and included both macrophages and myofibroblasts, as determined by double-immunofluorescent staining for TF and cell type-specific marker proteins. Double-immunofluorescent staining for TF and transforming growth factor beta (TGF-beta) revealed TGF-beta immunoreactivity both in tumor cells and in the extracellular matrix surrounding TF-positive stromal cells. To test the role of carcinoma cell-derived growth factors in the regulation of stromal cell TF activity, we examined the ability of conditioned media (CM) from breast carcinoma cell lines to stimulate TF activity in myofibroblast-like cells in vitro. Extracts from myofibroblasts exposed to CM displayed strong TF procoagulant activity. However, extracts from cells exposed to unconditioned media or CM pretreated with anti-TGF-beta antibodies did not. The induction of TF activity was also observed upon treatment of indicator cells with recombinant TGF-beta isoforms. Collectively, these data indicate that the recruitment and/or activation of TF-expressing stromal cells is an early event in progression to invasive breast cancer and likely occurs, in part, as a paracrine response to tumor cell-derived members of the TGF-beta family of growth factors.

Transcriptional repression of the murine vascular smooth muscle alpha-actin gene in fibroblasts results from the interaction of two sequence-specific single-stranded DNA binding activities (VACssBF1 and VACssBF2) with opposite strands of an essential transcriptional enhancer factor-1 (TEF-1) element (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436). Here, we identify a sequence element located within a protein-coding exon of the gene that bears structural similarity with the TEF-1 enhancer. This includes a 30-base pair region of purine-pyrimidine asymmetry encompassing a perfect 6-base pair GGAATG TEF-1 recognition motif. Unlike the enhancer, however, the exon sequence exhibits no TEF-1 binding activity nor does the pyrimidine-rich strand bind VACssBF1. However, VACssBF2 interacts equally well with the purine-rich strand of both the enhancer and the exon sequence. To test the ability of VACssBF2 to independently repress transcription, the exon sequence was placed upstream of a deletionally activated promoter containing an intact TEF-1 binding site. The exon sequence repressed promoter activity, whereas a mutant deficient in VACssBF2 binding did not. Moreover, VACssBF2 similarly repressed activator protein-1-dependent transcription of a heterologous tissue factor promoter. These results suggest that VACssBF2 possesses an intrinsic ability to disrupt enhancer function independently of the enhancer-binding proteins involved.

Serum stimulation of quiescent mouse fibroblasts results in transcriptional activation of tissue factor (TF), the cellular initiator of the protease cascade leading to blood coagulation. In this study, we demonstrate that two AP-1 DNA-binding elements located 200-220 bp upstream of the transcription start site are both necessary and sufficient to confer serum inducibility to the TF gene promoter in fibroblasts. Analysis of AP-1 DNA-binding complexes indicates that the predominant form of AP-1 activity in quiescent cells consists of an unidentified Fos-related protein and JunD. While c-Fos is notably absent from these preexisting complexes, serum stimulation results in the rapid entry of c-Fos into the TF AP-1 DNA-binding complexes. A similar induction of c-Fos DNA-binding activity occurs in cells treated with recombinant growth factors such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). Importantly, overexpression of JunD and c-Fos abrogates the requirement for serum in the stimulation of TF promoter activity in fibroblasts. Together, these data indicate that the entry of c-Fos into heterodimeric AP-1 DNA-binding complexes with JunD is a key event underlying serum-stimulated transcription of the TF gene in fibroblasts.

Transcriptional activity of the mouse vascular smooth muscle (VSM) alpha-actin promoter was governed by both cell type and developmental stage-specific mechanisms. A purine-rich motif (PrM) located as -181 to -176 in the promoter was absolutely required for activation in mouse AKR-2B embryonic fibroblasts and partially contributed to activation in undifferentiated mouse BC3H1 myoblasts. Transcriptional enhancer factor 1 recognized the PrM and cooperated with other promoter-binding proteins to regulate serum growth factor-dependent transcription in both myoblasts and fibroblasts. Two distinct protein factors (VAC-ssBF1 and VAC-ssBF2) also were identified that bound sequence-specifically to single-stranded oligonucleotide probes that spanned both the PrM and a closely positioned negative regulatory element. VAC-ssBF1 and BF2 binding activity was detected in undifferentiated myoblasts, embryonic fibroblasts, and several smooth muscle tissues in the mouse and human. A myoblast-specific protein (VAC-RF1) also was detected that bound double-stranded probes containing a CArG-like sequence that previously was shown to impart strong, cell type specific repression. The binding activity of transcription enhancer factor 1, VAC-RF1, and VAC-ssBF1 was significantly diminished when confluent BC3H1 myoblasts differentiated into myocytes and expressed VSM alpha-actin mRNA after exposure to serum-free medium. The results indicated that cell type-specific control of the VSM alpha-actin gene promoter required the participation of multiple DNA-binding proteins, including two that were enriched in smooth muscle and had preferential affinity for single-stranded DNA.

Transcriptional activation and repression of the vascular smooth muscle (VSM) alpha-actin gene in myoblasts and fibroblasts is mediated, in part, by positive and negative elements contained within an approximately 30-bp polypurine-polypyrimidine tract. This region contains binding sites for an essential transcription-activating protein, identified as transcriptional enhancer factor I (TEF-1), and two tissue-restrictive, sequence-specific, single-stranded-DNA-binding activities termed VACssBF1 and VACssBF2. TEF-1 has no detectable single-stranded-DNA-binding activity, while VACssBF1 and VACssBF2 have little, if any, affinity for double-stranded DNA. Site-specific mutagenesis experiments demonstrate that the determinants of VACssBF1 and VACssBF2 binding lie on opposite strands of the DNA helix and include the TEF-1 recognition sequence. Functional analysis of this region reveals that the CCAAT box-binding protein nuclear factor Y (NF-Y) can substitute for TEF-1 in activating VSM alpha-actin transcription but that the TEF-1-binding site is essential for the maintenance of full transcriptional repression. Importantly, replacement of the TEF-1-binding site with that for NF-Y diminishes the ability of VACssBF1 and VACssBF2 to bind to separated single strands. Additional activating mutations have been identified which lie outside of the TEF-1-binding site but which also impair single-stranded-DNA-binding activity. These data support a model in which VACssBF1 and VACssBF2 function as repressors of VSM alpha-actin transcription by stabilizing a local single-stranded-DNA conformation, thus precluding double-stranded-DNA binding by the essential transcriptional activator TEF-1.

The intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR) has been shown to be responsible for many of the pleiotropic intracellular effects resulting from ligand stimulation [W.S. Chen, C.S. Lazar, M. Poenie, R.Y. Tsien, G.N. Gill, and M.G. Rosenfeld, Nature (London) 328:820-823, 1987; A.M. Honegger, D. Szapary, A. Schmidt, R. Lyall, E. Van Obberghen, T.J. Dull, A. Ulrich, and J. Schlessinger, Mol. Cell. Biol. 7:4568-4571, 1987]. Recently, however, it has been shown that addition of ligand to cells expressing kinase-defective EGFR mutants can result in the phosphorylation of mitogen-activated protein kinase (R. Campos-González and J.R. Glenney, Jr., J. Biol. Chem. 267:14535-14538, 1992; E. Selva, D.L. Raden, and R.J. Davis, J. Biol. Chem. 268:2250-2254, 1993), as well as stimulation of DNA synthesis (K.J. Coker, J.V. Staros, and C.A. Guyer, Proc. Natl. Acad. Sci. USA 91:6967-6971, 1994). Moreover, mitogen-activated protein kinase has been shown to phosphorylate the transcription factor p62TCF in vitro, leading to enhanced ternary complex formation between p62TCF, p67SRF, and the c-fos serum response element (SRE) [H. Gille, A.D. Sharrocks, and P.E. Shaw, Nature (London) 358:414-417, 1992]. On the basis of these observations, we have investigated the possibility that the intrinsic tyrosine kinase activity of the EGFR may not be necessary for transcriptional activation mediated via p62TCF. Here, we demonstrate that a kinase-defective EGFR mutant can signal ligand-induced expression of c-fos protein and that a significant component of this induction appears to be mediated at the transcriptional level. Investigation of transcriptional activation mediated by the c-fos SRE shows that this response is impaired by mutations in the SRE which eliminate binding of p62(TCF). These data indicate that information inherent in the structure of the EGFR can be accessed by ligand stimulation independent of the receptor's catalytic kinase function.

Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways.

Segments of the 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene were assayed for promoter activity in transfected mouse BC3H1 myogenic cells and AKR-2B embryonic fibroblasts. The region between -150 and -191 that functions as a positive transcriptional element in myogenic and fibroblastic cells contains a mammalian-specific inverted CC(A/T)6GG-type consensus sequence. Expression was restricted to fully differentiated myogenic cells when an additional sequence spanning -191 to -224 was included in reporter gene constructs. This 33-base pair (bp) negative regulatory element is 70% conserved between the mouse and human genes and contains a 10-bp motif at its 3' end that only partially resembles a CC(A/T)6GG element. Retention of a GGGA motif at the 3' boundary of the 33-bp region is sufficient to maintain full transcriptional repression in fibroblasts and is partly responsible for repression in undifferentiated myoblasts. Complete muscle tissue-restrictive expression requires an additional 8 bp from the CC(A/T)6GG-like element immediately 5' to the GGGA motif, since replacement of this region with an unrelated 10-bp sequence completely eliminated restrictive transcriptional behavior in undifferentiated myoblasts. The distal portion of the 5'-flanking region between -224 and -1074 contains six E-box motifs (CANNTG) and mediates high level transcription only in postconfluent BC3H1 myoblasts. Analysis of reporter gene constructs including either the proximal E-box at -240 or all six E-boxes indicate that the five distal E-boxes are not required for high level transcription. A 724-bp segment of the 5'-flanking region consisting of the proximal E-box flanked upstream by a mammalian-specific 352-bp region was sufficient for maximal transcriptional activation in postconfluent BC3H1 myoblasts. Deletion of the 352-bp region restricts the early transcriptional response to high cell density in temporal studies of promoter activity during BC3H1 myogenic cell differentiation.

We have cloned a serum- and cycloheximide-inducible mRNA from AKR-2B murine fibroblasts which encodes a protein with significant sequence similarity to human tissue factor, a cellular initiator of the blood coagulation cascade. Information derived from this clone was used to establish the presence of a virtually identical sequence in mouse brain. Most importantly, cDNA-directed expression in a quail fibroblast cell line produced high levels of tissue factor procoagulant activity, confirming the identity of this protein as murine tissue factor. Additional studies demonstrate that transforming growth factor type beta 1 stimulates tissue factor gene transcription and is a potent inducer of tissue factor procoagulant activity in fibroblasts. Other tested mitogens such as platelet-derived growth factor, epidermal growth factor, and insulin were weak inducers. These results may reflect a role for transforming growth factor beta 1 in the maintenance of hemostasis or, alternatively, a role for tissue factor in cellular functions unrelated to blood coagulation.

Transforming growth factor type beta 1 (TGF-beta 1) is a pleiotropic regulator of cell growth and differentiation which can potentiate or otherwise modify cellular responses to different growth factors such as epidermal growth factor (EGF). Since cellular responses to peptide growth factors are mediated, in part, through the regulation of specific gene expression, we have studied the effect of TGF-beta 1 on the EGF-dependent transcriptional of a diverse panel of genes which included the fibronectin gene, the cytoskeletal beta- and gamma-actin genes, and the c-fos protooncogene. The addition of a combination of TGF-beta 1 (10 ng/ml) and EGF (10 ng/ml) to quiescent AKR-2B cells maintained in serum-free medium resulted in a strong synergistic stimulation of transcription which was not evident using either growth factor individually at concentrations of 20-100 ng/ml. This effect was evident within 10 min and did not require the continual presence of TGF-beta 1 in the culture medium. Increased responsiveness of some gene promoters to EGF stimulation was evident for up to 6 h following a brief 10-min exposure to TGF-beta 1. Similar exposure to either platelet-derived growth factor or fibroblast growth factor had little or no effect on EGF-stimulated transcription. These results indicate that the transcriptional response of specific genes to EGF can be stably altered as a consequence of exposure to TGF-beta 1.

Stimulation of quiescent AKR-2B mouse embryo cells with epidermal growth factor (EGF) results in a rapid and specific induction of actin mRNA sequences. These mRNAs include those coding for both beta- and gamma-cytoskeletal, but not alpha-skeletal muscle, actin isotypes. Elongation of nascent RNA chains in isolated nuclei (run-off transcription) demonstrates that the mRNA accumulation is preceded by an increase in actin gene transcription. This increase is transient, however, and is followed by a rapid attenuation of transcriptional activity. An inhibitor of protein synthesis, cycloheximide, was also found to induce beta- and gamma-actin mRNA accumulation. Furthermore, the simultaneous addition of EGF and cycloheximide produced a synergistic effect on actin sequences in both steady-state nuclear and polysomal RNA. Run-off transcription experiments demonstrate that this synergistic effect results from an increase in the magnitude and duration of actin gene transcription. It is also specific in that alpha-tubulin gene transcription is not similarly affected. These data suggest the existence of a specific labile repressor of actin gene transcription.

A cloned mouse DNA fragment containing an endogenous "virus-like" DNA (VL30 DNA) sequence was identified by virtue of its ability to hybridize to the virus-like RNA component of mixed-pseudotype AKR-murine leukemia virus virions, its lack of detectable sequence homology with cloned AKR-murine leukemia virus DNA, and its hybridization to a 5.6 kilobase pair (30S) cellular polyadenylic acid [poly(A)]-containing RNA species. Restriction enzyme mapping of the cloned mouse fragment revealed the presence of a 5- to 6-kilobase pair VL30 DNA segment flanked by non-VL30 segments of approximately 7 and 0.3 kilobase pairs. Southern blot analysis of VL30 DNA sequence organization in the DNA of two nontransformed mouse cell lines (AKR-2B, C3H/10T 1/2) and two chemically transformed derivatives (AKR-MCA, C3H/MCA-58) revealed 15 to 20 bands organized in an apparent strain-specific pattern. Within a given strain, however, no differences were detectable between the nontransformed cells and their chemically transformed counterparts. The expression of VL30 genes in the above cell lines was assayed by hybridization of 32P-labeled poly(A)-containing polysomal RNA to several internal restriction fragments derived from the cloned VL30 DNA sequence. The level of VL30 RNA was enhanced approximately 10-fold in both chemically transformed cell lines as compared to the nontransformed cell lines (under normal growth conditions). In addition, nontransformed AKR-2B cells maintained in the presence of purified epidermal growth factor exhibited similarly enhanced levels of VL30 RNA sequences in polysomal RNA. Since these cells displayed several growth characteristics of transformed cells but, in an epidermal growth factor-dependent and completely reversible fashion, these data suggest that the expression of VL30 genes is not simply incidental to chemically transformed cells but may be related to alterations in growth control.

Hybridization to synthetic oligonucleotides representing conserved regions in the promoter and first intron of several vertebrate beta-actin genes was used to discriminate between what appears to be a single functional beta-actin gene and numerous pseudogenes in the mouse genome. Sequences derived from the 5' end of this gene were shown to confer serum-inducible expression upon a heterologous reporter gene when transfected into mouse fibroblasts. Moreover, these sequences rendered reporter gene expression superinducible by a combination of serum and cycloheximide. These experiments indicate that the 5' end of the mouse beta-actin gene contains sequence elements which mediate the stimulatory effects of serum growth factors and which are responsive to both positive and negative regulators of gene expression.

The VL30 sequences of mouse DNA are a family of sequences with retrovirus-like structure which code for a 30S RNA transcript that can be packaged into the virions of murine leukemia viruses and thereby transmitted from cell to cell. A Southern blot analysis of these sequences revealed that multiple copies are present in the DNA of all mice examined, regardless of species or geographic origin. Considerable locus polymorphism was also apparent, and at least one of these polymorphisms appeared to reflect the differing chromosomal location of a complete VL30 sequence. These data indicated that VL30 elements are not recent additions to the mouse genome and suggested that the evolution of the VL30 multigene family has been accompanied by duplication and dispersion of VL30 sequences to diverse genomic sites. In addition, we reexamined the issue of genetic relatedness between mouse VL30 sequences and a physically similar family of virus-like elements in the rat genome. We found that many, if not all, rat and mouse VL30 loci contain regions of sequence homology. These data suggested that rodent VL30 sequences have evolved from a common ancestral sequence.

AKR-2B mouse embryo cells undergoing the serum-stimulated transition from a quiescent to a proliferating state exhibit an increase in the rate of hnRNA synthesis which appears to be mediated through an increase in the actual number of RNA polymerase II molecules. alpha-Amanitin, administered early in the prereplication interval following stimulation, effectively inhibits hnRNA synthesis, polysomal mRNA accumulation, polyribosome formation, and subsequent DNA synthesis, and cell division. Unexpectedly, alpha-amanitin treatment also produces almost complete inhibition of the synthesis of 45S rRNA precursor and the increase in accumulation of cytoplasmic rRNA following serum stimulation. In order to determine whether the inhibition of new ribosomal synthesis might in itself be sufficient to prevent serum-stimulated DNA synthesis, the effects of 5-fluorouridine (5-FU), a specific inhibitor of 45S rRNA processing, were investigated. If added within eight hours following serum stimulation, 5-FU was found to completely inhibit subsequent DNA synthesis. These results suggest that quiescent AKR-2B cells do not contain a sufficient excess of ribosomes to support the synthesis of proteins which are required for DNA synthesis in response to serum growth factors. Furthermore, an early polymerase II mediated synthesis of mRNA(s) coding for some factor(s) necessary for ribosomal gene transcription may be an essential step in the serum-stimulated synthesis of new ribosomes.

Five to six percent (by mass) of AKR-2B mouse embryo cell polysomal RNA consists of messenger RNA sequences which may exist in polyadenylated form. In the steady state, however, only 30--40% of these molecules are retained by extensive passage over oligo(dT)-cellulose, the remainder being present in the form of poly(A)-deficient analogues. Within experimental limits, these poly(A)-deficient analogues contain representatives of all poly(A)-containing mRNA sequences in these cells. An analysis of the kinetics of hybridization of cDNA probes enriched for either abundant or rare poly(A)-containing mRNA sequences suggests that the frequency distributions of poly(A)-containing and poly(A)-deficient analogues are dissimilar, and that a relationship exists between the intracellular frequency of a given mRNA sequence and the number of poly(A)-deficient analogues of that sequence. High frequency sequences appear to be enriched in the poly(A)-containing fraction, while low frequency sequences are predominately associated with the poly(A)-deficient fraction, thus, poly(A) may play a role in the regulation of mRNA frequency in the cytoplasm.

Stimulation of quiescent cultures of AKR-2B cells with transforming growth factor type beta (TGF beta) resulted in a transitory increase in actin cytoplasmic poly(A) + RNA. Levels of actin mRNA peaked approximately 4-8 hours subsequent to TGF beta addition and approached basal levels by 24 hours. The accumulation of actin transcripts was dose dependent and insensitive to inhibitors of protein synthesis; 1-3 ng/ml TGF beta induced maximal actin gene expression. Actin isotype-specific probes demonstrated that both beta- and gamma-cytoplasmic actins are induced by TGF beta.

The nucleotide sequence complexity of murine leukemia virus *MuLV)-related RNA has been measured by RNA-complementary DNA hybridization analysis in nuclear and cytoplasmic RNA isolated from liver and brain of low-leukemia-strain C57BL/6J mice of different ages. In these two tissues, an approximate 1.5- to 2-fold increase in the complexity of steadystate nuclear MuLV-related RNA sequences was observed as a function of age. Maximum complexity was observed with nuclear RNA extracts from old mice and corresponded to roughly 70 to 75% of the total MuLV genome. In contrast to the age-related increase in complexity of nuclear MuLV genome was detected in liver and brain steady-state cytoplasmic RNA, irrespective of animal age. These data suggest that control mechanisms regulating the transcription and/or stabilization of nuclear RNA transcripts of endogenous mouse MuLV-related genomes become less stringent with animal age even in low-tumor mouse strains. The data also support the existence of independent posttranscriptional mechanisms which prevent accumulation of these MuLV-related transcripts in steady-state cytoplasmic RNA and which do not seem to be as subject to the relaxation of stringency as a function of age.

Southern blot analyses using reduced stringency hybridization conditions have been employed to search for sequence homologies between rodent VL30 genes and murine leukemia virus (MuLV) proviruses. These constitute two classes of transposon-like elements previously believed to be genetically unrelated. Our results demonstrate that cloned representatives of both ecotropic and xenotropic-like proviruses share discrete regions of sequence homology with VL30 genes of both rat and mouse origin. These regions of homology exist in both 3' and 5' halves of the MuLV genome but do not include extensive portions of the long terminal repeat (LTR) or a 0.4 Kbp segment of the env gene specific for recently acquired ecotropic-type MuLV proviruses. DNA sequencing, however, revealed that the short inverted terminal repeat sequence of MuLV proviral LTRs is almost perfectly conserved at the terminus of an integrated mouse VL30 gene. These results suggest that recombination events with rodent VL30-type sequences occurred during early MuLV evolution. The strong conservation of the inverted terminal repeat sequence may reflect a common integration mechanism for VL30 elements and MuLV proviruses.

Derivative plots have been constructed for hybridization reactions between polysomal poly(A)-containing RNA and oligo(dT)-primed cDNA. In one method the derivative was calculated directly from the data, and in the other, from a non-linear least squares fit using 9-10 ideal components. In some cases these methods yield very similar results and strongly suggest that the hybridization data support discrete components. Reactions with two and four major components indicate that the often-reported three abundance class model is only one of several possibilities for eukaryotic cells. In other situations neither method strongly suggests the presence of discrete components (in one case even after enrichment of the cDNA population by kinetic fractionation), implying that the components are closely spaced or that the entire mRNA population of those cells may not exist as discrete abundance classes. The universal occurrence of discrete abundance classes should be critically reexamined.

The relationship between cell proliferation and the amount and diversity of polyribosome-associated poly(A)-containing messenger RNA [poly(A)+mRNA]has been investigated using a cloned AKR-mouse embryo cell culture system. The following results were obtained. First, an early response to the stimulation of proliferation of AKR-2B cells in culture is a rapid increase in the rate of accumulation of polyribosomal poly(A)+ mRNA. This results in a large increase in the total poly(A)+ mRNA content of rapidly proliferating cells compared to that found in resting cells. Second, the total amount of unique DNA sequence contributing to the poly(A)+ mRNA populations of both growing and resting cells is not detectably different. This corresponds to 9000-11,000 diverse gene equivalents of DNA and represents the transcription of 0.8-0.9% of the haploid mouse genome. Third, most of the increased poly(A)+ mRNA content of growing cells (greater than 90%) reflects an increased rate of production of polysomal mRNA species which are also found in resting cells. Fourth, growing cells appear to contain some species of poly(A)+ mRNA which are either absent or present in very low concentrations in non-growing cells. Within the limits of detection, however, all species of poly(A)+ mRNA present in non-growing cells are also present in growing cells.

The involvement of post-transcriptional mechanisms in the determination of the frequency distribution of messenger RNA sequences has been studied in cloned mouse embryo cells in culture. Hybridization kinetic experiments between poly(A)-containing RNA and complementary DNA have been used to study the alterations in frequencies which occur in those nuclear poly(A)-containing RNA sequences which are conserved in cytoplasmic poly-ribosomes. Sequences adjacent to nuclear poly(A) tracts are present in a much narrower frequency distribution in the nucleus than in polysomes, with a large proportion of the nuclear sequences present in an average frequency of about one molecule per cell. Few nuclear sequences appear to be present in more than ten copies per cell. A minimum of 70% of these sequences are also found in poly-ribosomal RNA but in greatly altered frequencies. Abundant sequences which comprise a major fraction of the poly(A)-containing polyribosomal RNA are derived from a small fraction of the nuclear poly(A)-adjacent RNA sequences. Very few nuclear poly(A)-adjacent sequences are present in a frequency characteristic of high abundance polysomal sequences. Conversely, poly(A)-containing polyribosomal RNA appears to contain few sequences which are present in as low a frequency as the majority of nuclear poly(A)-adjacent sequences. These observations suggest that post-transcriptional mechanisms play a major role in determining the steady-state frequency of polyribosome-associated messenger RNAs.

The possibility that chemical carcinogens may induce enhanced expression of endogenous C-type RNA tumor virus genes in the absence of intact virus particle production has been partially tested in a model system. Thie was accomplished by measuring the abundance and diversity of murine leukemia virus-related RNA sequences associated with the polyribosome fraction of nontransformed C3H/10T1/2 clone 8 cells and a 3-methylcholanthrene-transformed derivative clone. Although both clones are virus nonproducers, they were found to contain significant amounts of polyadenylate-containing murine leukemia virus-related RNA sequences; however, both the types and quantities of such sequences appear indistinguishable in both clones. These results suggest that expression of the corresponding gene sequences into RNA is not related to the maintenance of the transformed state in these chemically transformed cells.

A cDNA library, prepared from poly(A)+ RNA isolated from quiescent AKR-2B cells 4 hr after stimulation with epidermal growth factor in the presence of cycloheximide, was screened to identify RNA transcripts whose abundance is specifically increased as a primary response to growth stimulation. Approximately 40% of the inducible clones detected by this procedure corresponded to either cytoskeletal beta- or gamma-actin genes. One nonactin clone, designated c99, was found to be derived from an 8.5-kilobase RNA whose abundance began to increase as early as 30 min after stimulation. DNA sequencing established the identity of this RNA as fibronectin. Several additional mitogens were then tested and found to efficiently induce fibronectin mRNA. These included fetal calf serum, platelet-derived growth factor, and transforming growth factor type beta. For at least one inducer, fetal calf serum, the increase in mRNA was preceded by an increase in fibronectin gene transcription. This increase was rapid, reaching maximal levels within 10 min, and was accompanied by near-coordinate increases in both c-fos and beta-actin transcription. These results indicate that fibronectin is a member of a class of "early-response" genes, typified by c-fos and including beta-actin, whose rapid expression may be important in mediating cellular responses to peptide growth factors.

Complementary DNA (cDNA) has been transcribed by viral reverse transcriptase from poly(A)-containing nuclear RNA prepared from growing mouse Friend cells (clone M2). Annealing experiments with this cDNA have demonstrated that a large proportion of the poly(A) tracts in M2 cell nuclear RNA are adjacent to RNA sequences which are transcribed from nonrepetitive DNA in the mouse genome. The kinetics of hybridization of cDNA to template RNA indicate that nuclear poly(A)-containing RNA consists of at least two abundance classes, the more complex of which is transcribed from approximately 3 percent of the genome. Thus there are at least 5 times more unique DNA sequences represented in nuclear polyadenylated RNA than in polysomal polyadenylated RNA. Moreover, there is evidence for posttranscriptional mechanisms which alter the relative concentrations of some (at least) gene transcripts between nucleus and cytoplasm.

Complementary DNA probes prepared from total polysomal poly(A)+RNA populations were used to identify clones of mouse DNA containing sequences whose expression is specifically enhanced after epidermal growth factor (EGF) stimulation of quiescent mouse embryo cells in culture. Three such clones were isolated and used to study changes in the levels of clone-specific poly(A)+RNA in the polysomes of cells after mitogenic stimulation by EGF. RNA complementary to sequences present in these clones increased approximately equal to 10-fold as a fraction of the total poly(A)+RNA by 6 hr after stimulation. All three clones were found by hybridization criteria to contain sequences related to the class of mouse retrovirus or transposon-like elements termed VL30. These VL30-related sequences were further found to be complementary to EGF-inducible poly(A)+RNAs and enhanced expression was detectable as early as 1 hr after EGF stimulation. In contrast, nine additional clones, including an AKR-type murine leukemia provirus DNA clone, contained no detectable VL30 sequence elements and were complementary to poly(A)+RNA species whose relative concentration was essentially constant in quiescent and EGF-stimulated cells. Therefore, VL30 sequence elements appear distinct in that they encompass members whose expression is specifically regulated in response to a defined peptide growth factor.

The possibility of age-associated changes in DNA sequence in terms of amplification and rearrangement was examined in mouse spleen, liver and brain using the method of Southern transfer and filter hybridization. The DNA regions studied were at and around nine cloned sequences, most of which are known to move or amplify in certain situations. No detectable age-associated change, however, was observed in all DNA regions studied. These results suggest that widespread DNA sequence rearrangements or amplifications do not occur during the ageing process in mice.

DNA sequencing and blot hybridization analyses have been used to study the structure of a mouse VL30 gene and the molecular nature of VL30-related RNA which is induced upon the stimulation of cultured AKR mouse embryo cells with defined peptide growth factors. An integrated mouse VL30 gene was found to contain identical 601-base-pair long terminal repeats (LTRs) which were themselves terminated in short inverted repeats. The entire VL30 gene was flanked by a 4-base-pair direct repeat of cellular DNA. Thus, VL30 genes are structurally analogous to integrated forms of retrovirus proviruses and certain other classes of mobile genetic elements. The LTR sequence was found to contain putative promoter and polyadenylation signals and generally exhibited little sequence homology to murine leukemia virus proviral LTRs. Certain short regions of sequence conservation, however, were evident, including the inverted terminal repeat, LTR-adjacent regions corresponding to origins of murine leukemia virus proviral DNA synthesis, and a 36-base-pair direct repeat bearing homology to the 72-base-pair direct repeat (enhancer sequence) of the murine leukemia virus-related Moloney sarcoma virus. Upon mitogenic stimulation of quiescent cells with epidermal growth factor and insulin, a major 5.5-kilobase VL30-specific RNA complementary to both LTR and non-LTR sequences was rapidly induced. We conclude that a complete VL30 gene(s) is highly regulated by peptide growth factor binding to specific membrane receptors in these cells.

Transcription of the c-fos proto-oncogene and the cytoskeletal actin genes is induced within minutes of the addition of serum growth factors in a variety of cell types. Inhibitors of protein synthesis such as cycloheximide have been shown to dramatically potentiate the transcriptional response, an effect termed 'superinduction'. Although the stimulatory effect of serum has been shown to be transmitted through a cis-acting enhancer sequence termed a serum response element (SRE), the sequence element(s) responsible for mediating the effect of cycloheximide has not been identified. We now report that a synthetic copy of the c-fos SRE is sufficient to confer cycloheximide-dependent inducibility upon a heterologous promoter. This does not require the presence of serum, but several mutations in the SRE that impair serum-inducibility also impair cycloheximide-inducubility. These results imply that serum-responsive enhancer elements are negatively regulated by one or more labile proteins and that both positive and negative regulators of enhancer activity require a functional 'CArG box', a sequence domain previously implicated in muscle-specific transcription.