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1.
Soc Work ; 46(4): 341-9, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11682975

ABSTRACT

This article explores administrator and staff perceptions of mission-critical agency capacity in a predominantly Hispanic region that has a high degree of acculturation and elevated use of alcohol, tobacco, and other drugs. The domains explored are financial resources, proposal development, agency policies, organizational structure, communication, leadership, planning, and networking capabilities. Although significant differences were found among all eight domains, both staff and administrators concurred regarding the two areas of least capacity--financial resources and proposal development--and the two areas of greatest capacity--planning and networking capabilities. The authors suggest that agreement about the ranking of the domains is the most important finding rather than the differences between administrators and staff. A discussion of the practice and educational implications concludes the article.


Subject(s)
Community Health Services/organization & administration , Hispanic or Latino/psychology , Substance Abuse Treatment Centers/organization & administration , Substance-Related Disorders/prevention & control , Administrative Personnel , Community Health Services/statistics & numerical data , Cultural Diversity , Health Care Surveys , Hispanic or Latino/statistics & numerical data , Humans , Population Surveillance , Program Evaluation/methods , Small-Area Analysis , Social Work/standards , Southwestern United States/epidemiology , Substance Abuse Treatment Centers/statistics & numerical data , Substance-Related Disorders/epidemiology , Surveys and Questionnaires
2.
J Biol Chem ; 276(43): 39508-11, 2001 Oct 26.
Article in English | MEDLINE | ID: mdl-11551897

ABSTRACT

Methylation of mammalian DNA by the DNA methyltransferase enzyme (dnmt-1) at CpG dinucleotide sequences has been recognized as an important epigenetic control mechanism in regulating the expression of cellular genes (Yen, R. W., Vertino, P. M., Nelkin, B. D., Yu, J. J., el-Deiry, W., Cumaraswamy, A., Lennon, G. G., Trask, B. J., Celano, P., and Baylin, S. B. (1992) Nucleic Acids Res. 20, 2287-2291; Ramchandani, S., Bigey, P., and Szyf, M. (1998) Biol. Chem. 379, 535-5401). Here we show that interleukin (IL)-6 regulates the methyltransferase promoter and resulting enzyme activity, which requires transcriptional activation by the Fli-1 transcription factor (Spyropoulos, D. D., Pharr, P. N., Lavenburg, K. R., Jackers, P., Papas, T. S., Ogawa, M., and Watson, D. K. (1998) Mol. Cell. Biol. 15, 5643-5652). The data suggest that inflammatory cytokines such as IL-6 may exert many epigenetic changes in cells via the regulation of the methyltransferase gene. Furthermore, IL-6 regulation of transcription factors like Fli-1, which can help to direct cells along opposing differentiation pathways, may in fact be reflected in part by their ability to regulate the methylation of cellular genes.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/biosynthesis , Gene Expression Regulation, Neoplastic/drug effects , Interleukin-6/pharmacology , Leukemia, Erythroblastic, Acute/enzymology , Proto-Oncogene Proteins , Cell Differentiation , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , DNA-Binding Proteins/metabolism , Humans , K562 Cells , Megakaryocytes/cytology , Mutagenesis, Site-Directed , Promoter Regions, Genetic , Proto-Oncogene Protein c-fli-1 , Trans-Activators/metabolism , Transcriptional Activation/drug effects
3.
Soc Work ; 46(3): 203-14, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11495366

ABSTRACT

This article introduces a new qualitative spiritual assessment instrument. It reviews existing qualitative assessment tools and presents a new multidimensional spiritual assessment framework. The instrument consists of two components: a spiritual history in which consumers relate their spiritual life story in a manner analogous to a family history and an interpretive framework to assist practitioners in eliciting and synthesizing the full potentiality of strengths extant in clients' spiritual lives. Common spiritual strengths the framework is designed to evoke are discussed, and a number of interventions based on prevalent spiritual strengths are suggested.


Subject(s)
Interview, Psychological , Religion and Psychology , Social Work/methods , Humans
4.
Mol Biotechnol ; 16(1): 67-76, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11098469

ABSTRACT

Analysis of promoter and enhancer DNA sequences provides the researcher with valuable information regarding the expression patterns of genes. Insertion of small DNA fragments containing the regulatory sequence of interest into vectors carrying reporter genes allows for the accurate quantitative analysis of the gene's expression patterns and responses to various stimuli. The use of bioluminescent reporter genes provides a simple, rapid, and inexpensive system that generates virtually no toxic or radioactive waste products. In addition, bioluminescent reporter vectors are more sensitive than previous methods such as the chloramphenicol acetyl transferase (CAT) systems, that require the use of hazardous chemicals and isotopically labeled reagents.


Subject(s)
Genes, Reporter , Luciferases/genetics , Transcriptional Activation , Transfection/methods , Animals , COS Cells , Chlorocebus aethiops , Electroporation , Enhancer Elements, Genetic , Genetic Vectors , Humans , Jurkat Cells , Luciferases/analysis , Luminescent Measurements , Promoter Regions, Genetic , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , T-Lymphocytes
5.
J Marital Fam Ther ; 26(2): 217-28, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10776608

ABSTRACT

This paper develops a new diagrammatic spiritual assessment tool, the spiritual ecomap, for use with individuals, couples, and families. While a genogram portrays a family's history over time, a spiritual ecomap provides a valuable supplement by depicting a family's current relationships to critical ecological systems in space. The spiritual ecomap is based upon an anthropological framework conceptualized in the spiritual formation tradition and can be used with families from diverse spiritual traditions. I use a case study to familiarize the reader with the instrument, and offer suggestions for its application.


Subject(s)
Family Relations , Marital Therapy/methods , Religion and Psychology , Humans , Individuality
6.
J Biol Chem ; 275(7): 4541-4, 2000 Feb 18.
Article in English | MEDLINE | ID: mdl-10671476

ABSTRACT

T lymphocyte activation is highlighted by the induction of interleukin-2 (IL-2) gene expression, which governs much of the early lymphocyte proliferation responses. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPARgamma mRNA expression was found in human peripheral blood T lymphocytes, raising the possibility of PPARgamma involvement in the regulation of T cell function. Here we show that PPARgamma ligands, troglitazone and 15-deoxy-Delta(12,14) prostaglandin J(2), but not PPARalpha agonist Wy14643, inhibited IL-2 production and phytohemagglutinin-inducible proliferation in human peripheral blood T-cells in a dose-dependent manner. This inhibitory effect on IL-2 was restricted to the PPARgamma2-expressing, not the PPARgamma-lacking, subpopulation of transfected Jurkat cells. The activated PPARgamma physically associates with transcriptional factor NFAT regulating the IL-2 promoter, blocking NFAT DNA binding and transcriptional activity. This interaction with T-cell-specific transcription factors indicates an important immunomodulatory role for PPARgamma in T lymphocytes and could suggest a previously unrecognized clinical potential for PPARgamma ligands as immunotherapeutic drugs to treat T-cell-mediated diseases by targeting IL-2 gene expression.


Subject(s)
Lymphocyte Activation/drug effects , Nuclear Proteins , Peroxisome Proliferators/pharmacology , Receptors, Cytoplasmic and Nuclear/agonists , T-Lymphocytes/drug effects , Thiazolidinediones , Transcription Factors/agonists , Base Sequence , Cell Line , Chromans/pharmacology , DNA Probes , DNA-Binding Proteins/metabolism , Humans , Interleukin-2/biosynthesis , Interleukin-2/genetics , NFATC Transcription Factors , Pyrimidines/pharmacology , Receptors, Cytoplasmic and Nuclear/metabolism , T-Lymphocytes/metabolism , Thiazoles/pharmacology , Transcription Factors/metabolism , Troglitazone
7.
Biochem J ; 339 ( Pt 2): 435-42, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10191277

ABSTRACT

The polo-like kinase (Plk) family has been shown to have an important role in the regulation of the cell-division cycle, especially in organization of the spindle structure, in species from fungi to humans. Recent reports have demonstrated that in mammalian cells Plk is associated with components of the anaphase-promoting complex and a peptidyl-prolyl isomerase, Pin1. To characterize a putative Plk-containing complex, we fractionated mitotic cell lysates on a gel-filtration column. The Plk complex was eluted from the column at molecular sizes ranging from 669 to 2500 kDa in the presence of detergent and high concentrations of salt. Specific associations of Plk with alpha-, beta- and gamma-tubulins in both interphase and mitotic cells were shown by reciprocal immunoprecipitations and immunoblottings and were independent of the microtubule polymerization state, whereas binding assays in vitro indicated that Plk interacts with alpha- and beta-tubulins directly. In addition, mitotic Plk was able to phosphorylate associated tubulins in vitro. Finally, we show that the kinase domain of the Plk molecule is both required and sufficient for its binding to tubulins in vivo. The specific interaction between Plk and tubulins might provide a molecular basis for the physiological functions of Plk in regulating the cell cycle, particularly in establishing the normal bipolar spindle.


Subject(s)
Protein Serine-Threonine Kinases/metabolism , Tubulin/metabolism , Amino Acid Sequence , Animals , Base Sequence , Chromatography, Gel , Chromatography, Ion Exchange , DNA Primers , Interphase , Mice , Mitosis , Phosphorylation , Precipitin Tests , Protein Binding , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/isolation & purification , Serine/metabolism , Threonine/metabolism , Tubulin/isolation & purification , Tumor Cells, Cultured
8.
J Med Primatol ; 27(2-3): 73-80, 1998.
Article in English | MEDLINE | ID: mdl-9747946

ABSTRACT

We have compared nef gene sequences isolated by PCR from peripheral blood lymphocyte DNA of macaques which had been inoculated with either biologically or molecularly cloned SIV(Mne). Two samples from each animal obtained either early after infection (week 2-8) or after significant CD4+ depletion (week 21-137) were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two more in all but one. Two of the common exchanges are located about 40 residues apart in the Nef core sequence, but are in proximity on the tertiary structure as judged by computer modelling using the structure of the HIV Nef core protein as a guide. Most recurring in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIVsm/HIV-2 groups. Animals inoculated with virus already containing the "late version" nef gene developed a more aggressive disease. The macaque adapted (MA)nef conferred a threefold higher infectivity to the cloned virus, but had no effects on CD4 downregulation. Propagation of virus with MAnef in tissue culture resulted in the rapid emergence of variants with newly attenuated nef. These findings suggest that the selective pressure on nef in vivo and in vitro are different.


Subject(s)
Gene Products, nef/genetics , Genes, nef , Genetic Variation , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , Amino Acid Sequence , Animals , Cells, Cultured , DNA Primers , Gene Products, nef/chemistry , Lymphocytes/virology , Macaca nemestrina , Models, Molecular , Molecular Sequence Data , Polymerase Chain Reaction , Protein Conformation , Sequence Alignment
9.
J Biol Chem ; 273(25): 15727-33, 1998 Jun 19.
Article in English | MEDLINE | ID: mdl-9624170

ABSTRACT

Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-2) and simian immunodeficiency viruses and has important roles in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In order to identify Nef binding proteins and map their site(s) of interaction, we targeted a highly conserved acidic sequence at the carboxyl-terminal region of Nef sharing striking similarity with an acidic sequence at the c-Raf1-binding site within the Ras effector region. Here, we used deletion and site-specific mutagenesis to generate mutant Nef proteins fused to bacterial glutathione S-transferase in in vitro precipitation assays and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence motif containing the core sequence Asp-Asp-X-X-X-Glu (position 174-179). Significantly, we demonstrate that substitution of the nonpolar glycine residue for either or both of the conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background completely abrogated binding of c-Raf1 in vitro. In addition, lysates from a permanent CEM T-cell line constitutively expressing the native HIV-1 Nef protein was used to coimmunoprecipitate a stable Nef-c-Raf1 complex, suggesting that molecular interactions between Nef and c-Raf1, an important downstream transducer of cell signaling through the c-Raf1-MAP kinase pathway, occur in vivo. This interaction may account for the Nef-induced perturbations of T-cell signaling and activation pathways in vitro and in vivo.


Subject(s)
Gene Products, nef/metabolism , HIV-1 , Proto-Oncogene Proteins c-raf/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites/genetics , Conserved Sequence , Gene Products, nef/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Mapping , Tumor Cells, Cultured , nef Gene Products, Human Immunodeficiency Virus
10.
Oncogene ; 14(2): 213-21, 1997 Jan 16.
Article in English | MEDLINE | ID: mdl-9010223

ABSTRACT

The ETS gene products are a family of transcriptional regulatory proteins that contain a highly conserved and structurally unique DNA binding domain, termed the ETS domain. Several ETS proteins bind to DNA as monomers, however it has been shown that the DNA binding activity is enhanced or modulated in the presence of other factors. By differential display and whole genome PCR techniques, we have recently shown that the Erg1 gene is a target for ETS proteins. The Egr1 promoter contains multiple ETS binding sites, three of which exist as parts of two serum response elements (SREI and SREII). The SRE is a cis-element that regulates the expression of many growth factor responsive genes. ELK1 and SAP1a have been shown to form ternary complexes with SRF on the SRE located in the c-fos promoter. Similarly, we examined whether the ELK1, SAP1a, FLI1, EWS-FLI1, ETS1, ETS2, PEA3 and PU.1 proteins can form ternary complexes with SRF on the Egr1 SREI and II. Our results demonstrate that indeed ELK1, SAPla, FLI1 and EWS-FLI1 are able to form ternary complexes with SRF on Egr1 SREs. In addition, ELK1 and SAP1a can also form quarternary complexes on the Egr1 SREI. However, the proteins ETS1, ETS2, PEA3 and PU.1 were unable to form ternary complexes with SRF on either the Egr1 or c-fos SREs. Our data demonstrate that FLI1 and EWS-FLI1 constitute new members of a subgroup of ETS proteins that can function as ternary complex factors and further implicate a novel function for these ETS transcription factors in the regulation of the Egr1 gene. By amino acid sequence comparison we found that, in fact, 50% of the amino acids present in the B-box of SAP1a and ELK1, which are required for interaction with SRF, are identical to those present in both FLI1 (amino acids 231- 248) and EWS-FLI1 proteins. This B-box is not present in ETS1, ETS2, PEA3 or PU.1 and these proteins were unable to form ternary complexes with SRF and Egrl-SREs or c-fos SRE. Furthermore, deletion of 194 amino terminal amino acids of FLI1 did not interfere with its ability to interact with SRF, in fact, this truncation increased the stability of the ternary complex. The FLI1 protein has a unique R-domain located next to the DNA binding region. This R-domain may modulate the interaction with SRF, providing a mechanism that would be unique to FLI1 and EWS-FLI1, thus implicating a novel function for these ETS transcription factors in the regulation of the Egr1 gene.


Subject(s)
DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Ribonucleoproteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites/genetics , Heterogeneous-Nuclear Ribonucleoproteins , Molecular Sequence Data , Polymerase Chain Reaction/methods , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Protein c-fli-1 , Proto-Oncogene Proteins c-ets , RNA-Binding Protein EWS , Rabbits , Serum Response Factor , ets-Domain Protein Elk-1 , ets-Domain Protein Elk-4
11.
Oncogene ; 12(6): 1199-1204, 1996 Mar 21.
Article in English | MEDLINE | ID: mdl-8649821

ABSTRACT

p53 is an extensively studied tumor suppressor gene implicated in the genesis of a large number of varied tumors. However, the pathways of regulation for the wild-type p53 gene and its product are as yet unknown. In situ hybridization analyses of ETS1 and ETS2 expression during mouse embryogenesis, have shown a pattern similar to that of p53 gene expression. Significantly, we have identified several ETS-binding sites (EBS) in the promoter regions of the human and mouse p53 genes. In the human promoter two of these EBS are present in the form of a palindrome, with the two EBS cores being separated by four nucleotides. This report shows that the EBS palindrome of the human p53 promoter has a high affinity for ETS1 and ETS2 and that such binding interaction intracellularly is able to activate the transcription of a CAT reporter gene by 5-10-fold using COS cells. To investigate whether the spacing between the two EBS cores influences the DNA binding activity, we synthesized oligonucleotides with increasing distances (4,12,16, and 20 bases respectively) between the two EBS cores of the palindrome. We observed an inverse correlation between an increasing distance in the two EBS cores of the palindrome and the ETS1 and ETS2 DNA binding activity respectively. Interestingly, optimal DNA binding activity was observed when the distance between the two EBS cores was four bases, identical to that which occurs in the natural promoter. Furthermore we show that the p53 mRNA is expressed at higher levels in NIH3T3 cells overexpressing ETS2 gene product, suggesting that the ETS2 transcription factor is a likely candidate for regulating the expression of p53 in vivo.


Subject(s)
DNA/metabolism , Gene Expression Regulation/physiology , Genes, p53 , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , 3T3 Cells/metabolism , 3T3 Cells/physiology , Animals , Base Sequence , Binding Sites , Cell Line, Transformed , Chloramphenicol O-Acetyltransferase/genetics , DNA-Binding Proteins/metabolism , Humans , Methylation , Mice , Molecular Sequence Data , Promoter Regions, Genetic/physiology , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Protein c-ets-2 , Proto-Oncogene Protein c-fli-1 , Proto-Oncogene Proteins c-ets , RNA, Messenger/metabolism , Repressor Proteins/metabolism , Trans-Activators/metabolism , Transcriptional Activation/physiology
12.
Oncogene ; 12(1): 11-8, 1996 Jan 04.
Article in English | MEDLINE | ID: mdl-8552380

ABSTRACT

ETS is a family of transcription factors that contain a highly conserved ETS DNA binding domain. Various members of the ETS family are expressed in cells of hematopoietic lineage. ETS-1, ETS-2 and ERGB/FLI-1 are expressed at high levels in T-lymphocytes. HIV-1 infects T-cells and it has been shown that its LTR contains binding sites for various transcription factors. In this study we show that the HIV-1 core enhancer is directly regulated by ERGB/FLI-1 protein positively, as well as, negatively, depending upon the presence or absence of accessory factors in different cell types. In addition, we show that the ETS-1 transactivation activity is enhanced upon dephosphorylation of the Calmodulin-dependent Protein Kinase II phosphorylation site located in exon VII. Finally, we demonstrate that the spacing between the two EBS cores in palindromic or direct repeat sites play a crucial role in binding of ETS proteins to DNA.


Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Enhancer Elements, Genetic , HIV Long Terminal Repeat , Molecular Sequence Data , Phosphorylation , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Protein c-fli-1 , Proto-Oncogene Proteins c-ets , Spodoptera
14.
AIDS Res Hum Retroviruses ; 11(1): 65-79, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7734198

ABSTRACT

The human immunodeficiency virus type 2 (HIV-2) Nef protein expressed in Escherichia coli forms highly stable homooligomeric complexes in vitro. Similarly, the native protein synthesized in the persistently infected H9 T cell line also forms stable homooligomers in vivo. To determine whether homooligomer formation is mediated by the leucine zipper-type sequence located in the middle region of the protein, site-directed mutagenesis was used to introduce double and triple point mutations at heptad leucine positions L1, L2, and L4 within the HIV-2NIHZ Nef protein sequence. Here, we show that substitution of a serine residue for the L1 (residue 108) and L2 (residue 115) heptad leucines, and a glutamine residue for the L4 (residue 129) heptad leucine, did not prevent Nef homooligomer formation in vitro. However, a more drastic substitution of alpha-helix-breaking proline residue for the L2 and L4 heptad leucines significantly abrogated ability of the protein to form stable homooligomers. In addition, because significantly higher levels of the Nef oligomers were consistently observed under the nonreducing SDS-PAGE condition, site-specific mutagenesis was also used to examine the role of cysteine residues in generating disulfide-linked Nef dimers in vitro. Here, we also show that single cysteine-to-glycine substitutions at positions 28, 32, or 55 drastically reduced covalent Nef dimer formation and thermal stability of the Nef protein in vitro. Therefore, these results demonstrate that the leucine zipper-type motif in the HIV-2 Nef protein mediates stable homooligomer formation in vitro, and also establish a role for covalent disulfide bonds in the formation of linked Nef dimers and thermal stability of the monomer Nef in vitro.


Subject(s)
Gene Products, nef/genetics , HIV-2/metabolism , Leucine Zippers/genetics , Amino Acid Sequence , Base Sequence , Cysteine/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Products, nef/biosynthesis , Gene Products, nef/chemistry , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligonucleotides , Recombinant Fusion Proteins/biosynthesis , nef Gene Products, Human Immunodeficiency Virus
15.
Gene ; 143(2): 303-4, 1994 Jun 10.
Article in English | MEDLINE | ID: mdl-8206394

ABSTRACT

STX-related genes (encoding syntaxin) have been identified in rat and yeast; however, no human STX gene has been isolated thus far. Here, we describe the nucleotide sequence of the first human STX gene isolated from a placental library. It encodes a 297-amino-acid (aa) protein and is 89% identical to the aa sequence of rat STX 4a.


Subject(s)
Membrane Proteins , Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Humans , Molecular Sequence Data , Proteins/chemistry , Qa-SNARE Proteins , Rats , Sequence Homology, Amino Acid
16.
Oncogene ; 9(2): 469-77, 1994 Feb.
Article in English | MEDLINE | ID: mdl-7507230

ABSTRACT

EndoA is a type II keratin and with EndoB (type I keratin), constitutes intermediate filaments in various simple epithelial tissues. EndoA is developmentally regulated and has an enhancer that is located at the 3'- end of the gene. This enhancer contains two single and five dual Ets binding sites. Thus far, no other promoter or enhancer has been shown to contain as many potential clustered Ets binding sites. To study the transcriptional regulation of EndoA by the ETS family proteins, we amplified the EndoA enhancer fragment from mouse genomic DNA by PCR, and cloned it into the pBLCAT2 vector upstream from the CAT reporter gene. Several pBLCAT-ENDOA clones were sequenced to verify the presence of all the ETS binding sites. Clones that did not show any point mutations in the ETS binding sites were chosen to study the transcription regulation by ETS1, ETS2 and ERGB/FLI-1 gene products. EMSA results indicated that the ETS1, ETS2 and ERGB/FLI-1 proteins bind to the enhancer sequence, and DNase I protection data demonstrated that the ETS proteins protect all seven EBS core sequences. Cotransfection of the COS cells with the pBLCAT-ENDOA construct, along with increasing amounts of different ETS expression vectors, resulted in a significant induction of CAT reporter gene expression. Previously, we have shown that the overexpression of the ETS1 gene transforms NIH3T3, and these transformed cells (7AQS2.1) produce high levels of ETS1 protein (Seth & Papas, 1990). In this report, we show that the undifferentiated P19 EC cells do not express detectable levels of ETS1; however, an elevated level of ETS1 is expressed in differentiated derivatives of these cells. We therefore used these two cell lines to examine the activity of the EndoA enhancer with the ETS1 product. Transfection of the pBLCAT-ENDOA construct alone in undifferentiated P19 EC cells results in very low CAT gene expression; however, upon differentiation with retinoic acid the level of CAT gene activity increases dramatically. Similarly, an increase in CAT expression from the same construct (pBLCAT-ENDOA) was also observed in 7AQS2.1 cells. Our results therefore indicate that the EndoA enhancer is regulated by ETS proteins via interaction with multiple ETS-binding site sequences.


Subject(s)
Enhancer Elements, Genetic/genetics , Gene Expression Regulation/genetics , Keratins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Transcription Factors , Animals , Base Sequence , Binding Sites , Cell Line , DNA/analysis , DNA/genetics , DNA/metabolism , Electrophoresis, Polyacrylamide Gel , Fibroblasts/chemistry , Fibroblasts/cytology , Fibroblasts/metabolism , Genes, Reporter/genetics , Genetic Vectors , Insecta , Keratins/analysis , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Protein Binding , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins c-ets , Transcriptional Activation , Transfection , Tretinoin/pharmacology
17.
AIDS Res Hum Retroviruses ; 9(12): 1225-31, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8142139

ABSTRACT

The entire nef gene sequence of HIV-2, NIH-Z strain, has been cloned into the pJL6 expression vector and used for the synthesis of a 23-kDa protein in E. coli. The expressed protein is a fusion between the N-terminal 13 amino acids of the cII gene, 8 amino acids resulting from the ligation procedure, and the 180 amino acids that comprise the HIV-2 Nef sequence from the NIH-Z strain. The bacterially expressed Nef protein has been purified to apparent homogeneity on analytical scale (10-20 micrograms) by a combination of sequential detergent extraction, gel filtration, and reversed-phase high-performance chromatography. The expressed Nef protein is highly susceptible to proteolysis (chymotryptic-like activity) and this property accounts for the low yield obtained by gel filtration and RP-HPLC. Larger amounts (> 100 micrograms) of the purified Nef protein have been produced by a purification procedure that employs sequential detergent extraction, chromatography on Q-Sepharose in the presence of 7 M urea, and chromatography on hydroxylapatite, also in 7 M urea. The purified HIV-2 Nef protein has been used for the production of polyclonal and monoclonal antibodies. The milder method of purification should facilitate structure-function studies of the Nef protein and its role in the life cycle of HIV.


Subject(s)
Gene Products, nef/genetics , Gene Products, nef/isolation & purification , HIV-2/genetics , Amino Acid Sequence , Bacteriophage lambda/genetics , Chromatography , Cloning, Molecular , Escherichia coli/genetics , GTP-Binding Proteins/genetics , Gene Expression , Gene Products, nef/immunology , Genes, Viral , Genetic Vectors , HIV Antibodies/blood , HIV Infections/immunology , HIV Infections/microbiology , HIV-2/immunology , Humans , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , nef Gene Products, Human Immunodeficiency Virus
18.
AIDS Res Hum Retroviruses ; 9(10): 1017-23, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8280476

ABSTRACT

ets is a multigene family and its members share a common ETS DNA-binding domain. ETS proteins activate transcription via binding to a purine-rich GGAA core sequence located in promoters/enhancers of various genes, including several that are transcriptionally active in T cells. The ETS1, ETS2, and ERBG/Hu-FLI-1 gene expression pattern also suggests a role for these genes in cells of hematopoietic lineage. The HIV-1 LTR core enhancer contains two 10-base pair direct repeat sequences (left and right) that are required for regulation of HIV-1 mRNA expression by host transcription factors, including NF kappa B. Two ETS-binding sites are present in the core enhancer of all the HIV-1 isolates reported so far. In our studies, we utilized HIV-1 HXB2 and HIV-1 Z2Z6 core enhancers because the Z2Z6 strain has a single point mutation flanking the right ETS-binding site. We demonstrate that the ETS1, ETS2, and ERGB/Hu-FLI-1 proteins can trans-activate transcription from both the HXB2 and Z2Z6 core enhancer when linked to a reporter (cat) gene. In addition, we show that the DNA binding and trans-activation with the Z2Z6 core enhancer is at least 40-fold higher than that observed with the HXB2 core enhancer. Further, we provide evidence that the marked increase in binding and trans-activation with Z2Z6 core enhancer sequences is due to the substitution of a flanking T residue in HXB2 TGGAA) by a C residue in Z2Z6 (CGGAA) isolate, thus generating an optimal ETS-binding core (CGGAA) sequence.


Subject(s)
Gene Expression Regulation, Viral , HIV Long Terminal Repeat/genetics , HIV-1/genetics , Trans-Activators/metabolism , Transcription, Genetic , Animals , Base Sequence , Cells, Cultured , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic/genetics , Humans , Molecular Sequence Data , Moths/cytology , Multigene Family , Transcriptional Activation
19.
AIDS Res Hum Retroviruses ; 7(8): 697-706, 1991 Aug.
Article in English | MEDLINE | ID: mdl-1931237

ABSTRACT

Analysis of the predicted amino acid sequences of the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and of the related simian immunodeficiency virus (SIV) nef gene products (Nef) reveals the presence of a conserved leucine zipper-like repeat with the characteristic 4,3 arrangement of mainly hydrophobic amino acids in the middle (core) region of the proteins, but lacking the basic (DNA binding) domain characteristic of DNA-binding leucine zipper (bZIP) proteins. Also, at the C-terminus of the Nef proteins is a highly acidic sequence (net charge of -5 to -8) stretched over about 40 amino acids, and contains two predicted alpha-helices separated by a beta-turn linker sequence with sequence homology to known activation domains of acidic transcriptional activation factors. Moreover, within this acidic region of transcriptional activators and the homologous sequence within the second Nef alpha-helix, is a potential transcriptional activation consensus sequence (TACS) bounded by a pair of acidic amino acids (aspartic or glutamic acids) at the N-terminus and a highly invariant phenylalanine (hydrophobic), often followed by an acidic (aspartic) residue, at the C-terminus of the sequence. These findings strongly implicate Nef proteins as belonging to a class of non-DNA-binding leucine zipper acidic transcription factors, and provide a structural basis for new approaches to studying Nef function.


Subject(s)
Gene Products, nef/chemistry , HIV-1 , HIV-2 , Leucine Zippers , Simian Immunodeficiency Virus , Trans-Activators/chemistry , Amino Acid Sequence , Molecular Sequence Data , Sequence Alignment , nef Gene Products, Human Immunodeficiency Virus
20.
Virology ; 179(1): 504-7, 1990 Nov.
Article in English | MEDLINE | ID: mdl-2219736

ABSTRACT

Human T-lymphocytic cell line H9 infected with the HTLV-IIIB isolate of human immunodeficiency virus type 1 (HIV-1) synthesizes two forms of the Nef protein (p25 and p27) that differ both in molecular weight and charge. Different subpopulations of viruses were isolated from the HTLV-IIIB stock which induce expression of only p25 or p27. Cells infected with HIV-1 derived from the HXB3 clone of the HTLV-IIIB isolate made only the p25 species, whereas the 8E5/LAV cell line which harbors a single defective LAV provirus produces only the p27 species. These findings are consistent with the notion that the HTLV-IIIB isolate consists of at least two distinct variants with different nef genes, one specifying p25 and the other encoding p27. After a considerable number of passages in culture, H9 cells chronically infected with the HTLV-IIIB isolate produced high levels of p25 and lower levels of p27. Passages in culture appear to select for a subpopulation of virus variants that specify high levels of p25 Nef expression.


Subject(s)
Gene Products, nef/isolation & purification , HIV-1/genetics , Cell Line , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Gene Products, nef/biosynthesis , Humans , Molecular Weight , nef Gene Products, Human Immunodeficiency Virus
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