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1.
Cancer Gene Ther ; 7(8): 1127-34, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10975673

ABSTRACT

We have previously described several novel chimeric immune receptors (CIRs) that redirect human T cells to kill malignant or HIV-infected cells. These CIRs comprise a cancer- or virus-specific ligand or single-chain antibody fused to the signaling domain of the T-cell receptor CD3-zeta subunit. Binding of the ligand- or antibody-based CIR to the target antigen (Ag) triggers T-cell-mediated cytolysis of the tumor- or virus-infected cell independent of target cell major histocompatibility complex class I expression. A new type of CIR was developed to mediate the lysis of cells that expressed one or more distinct viral or tumor Ags; three bispecific CIRs (BCIRs) were generated that recognized the carcinoembryonic Ag (CEA) and TAG-72 tumor Ags or, alternatively, distinct epitopes in the HIV envelope (HIVenv). T cells expressing the antitumoral Ag BCIR lysed both CEA- and TAG-72-expressing targets and did not kill Ag-negative targets or target cells expressing other members of the CEA family. Similarly, T cells expressing the anti-HIVenv BCIR lysed target cells expressing both the wild-type HIVenv and a mutant HIVenv that lacked the epitopes recognized by the monospecific CIRs. This approach permits the generation of T cells with a broader spectrum of activity capable of killing virus-infected cells and malignant cells and reduces the potential of progression of disease due to Ag loss variants.


Subject(s)
HIV/immunology , Receptors, Immunologic/immunology , Recombinant Fusion Proteins/immunology , T-Lymphocytes/immunology , Amino Acid Sequence , Antigens/immunology , Humans , Molecular Sequence Data
2.
Cancer Gene Ther ; 6(5): 437-46, 1999.
Article in English | MEDLINE | ID: mdl-10505854

ABSTRACT

Replication-deficient adenovirus vectors are efficient vehicles for delivering therapeutic genes into mammalian cells. However, the high doses required to produce effective gene transfer in vivo can also cause unwanted cellular toxicity. To improve replication-deficient adenovirus transgene expression while minimizing adverse reactions, we have tested polycationic compounds for their ability to enhance adenovirus adsorption. We demonstrate increased transgene expression after mixing adenovirus preparations with polycations, cationic lipids, and CaCl2 prior to transduction in vitro. An E1-deleted adenovirus vector was admixed with various polycations, and beta-galactosidase (beta-gal) activity was evaluated. The optimal polycation concentrations for augmenting adenovirus-mediated gene transfer were 5-10 microg/mL polybrene, 400 microg/mL protamine sulfate, 10 microg/mL N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP), 2.5 microg/mL Lipofectamine, and 62.5 mM CaCl2. Polycations enhanced beta-gal expression in three of six established cell lines. Similar results were obtained using primary tumor cell cultures, where beta-gal expression was increased 1.5- to 10.7-fold (mean = 3.6) by polybrene, 1.8- to 7.5-fold (mean = 3.4) by DOTAP, and 2.3- to 10.4-fold (mean = 4.8) by protamine sulfate. Adenovirus transduction efficiency in two primary leukemia isolates was improved by 3- and 4.5-fold. We were unable to demonstrate any benefit when adenovirus was admixed with protamine sulfate prior to intratumoral injection in a xenogeneic severe combined immunodeficient mouse melanoma tumor model. Further studies will determine whether polycations can improve intratumoral gene transfer.


Subject(s)
Adenoviridae/genetics , Lipids/pharmacology , Transduction, Genetic , Transgenes , Animals , Cations , Defective Viruses/genetics , Genetic Therapy , Humans , Mice , Neoplasms/therapy , Protamines/pharmacology , Tumor Cells, Cultured
3.
Gene Ther ; 6(3): 412-9, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10435091

ABSTRACT

Chimeric immune receptors (CIR) encompass tumor- or virus-specific ligands or antibodies fused to the signaling domains of either the T cell receptor or Fc receptor. T cells expressing these receptors recapitulate the cytopathic effects mediated by the T cell receptor and allow the targeting of tumor or virus infected cells in an MHC-independent manner. With this technology, large numbers of T cells with redirected target specificity can be generated. To define the structural features of recombinant CIRs required for optimal function, a panel of five closely related CIRs with identical target specificity were generated. These receptors recognized HIVenv through the single chain Fv (scFv) of an anti-gp 120 antibody. These scFv-zeta receptors were constructed to include alternative extracellular spacer and transmembrane protein domains derived from members of the immunoglobulin supergene family. The effect of these alternative extracellular protein domains on receptor stability, antigen affinity and T cell activity was assessed. We demonstrate that modifying the extracellular protein domains of the anti-HIVenv CIRs significantly impacted receptor stability and substrate binding affinity and that these effects, and not simply the level of cell surface expression, correlated most strongly with changes in CIR-mediated killing. These studies will aid in the rationale design of recombinant CIRs for the immunotherapy of viral infections, cancer and other diseases.


Subject(s)
HIV Envelope Protein gp120/immunology , Immunotherapy, Adoptive , Receptors, Antigen, T-Cell, gamma-delta/genetics , T-Lymphocytes, Cytotoxic/immunology , Blotting, Southern , Blotting, Western , Cloning, Molecular , Cytokines/analysis , Humans , Transfection
4.
Hum Gene Ther ; 10(2): 165-73, 1999 Jan 20.
Article in English | MEDLINE | ID: mdl-10022542

ABSTRACT

A chimeric immune receptor consisting of an extracellular antigen-binding domain derived from the CC49 humanized single-chain antibody, linked to the CD3zeta signaling domain of the T cell receptor, was generated (CC49-zeta). This receptor binds to TAG-72, a mucin antigen expressed by most human adenocarcinomas. CC49-zeta was expressed in CD4+ and CD8+ T cells and induced cytokine production on stimulation. Human T cells expressing CC49-zeta recognized and killed tumor cell lines and primary tumor cells expressing TAG-72. CC49-zeta T cells did not mediate bystander killing of TAG-72-negative cells. In addition, CC49-zeta T cells not only killed FasL-positive tumor cells in vitro and in vivo, but also survived in their presence, and were immunoprotective in intraperitoneal and subcutaneous murine tumor xenograft models with TAG-72-positive human tumor cells. Finally, receptor-positive T cells were still effective in killing TAG-72-positive targets in the presence of physiological levels of soluble TAG-72, and did not induce killing of TAG-72-negative cells under the same conditions. This approach is being currently being utilized in a phase I clinical trial for the treatment of colon cancer.


Subject(s)
Antibodies, Neoplasm/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Receptors, Immunologic/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Neoplasm/genetics , Antigens, Neoplasm/immunology , Cell Division/immunology , Fas Ligand Protein , Glycoproteins/immunology , Humans , Lymphocytes, Tumor-Infiltrating/cytology , Membrane Glycoproteins/immunology , Mice , Mice, Inbred NOD , Mice, SCID , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , T-Lymphocytes/cytology
5.
J Immunol ; 161(1): 375-84, 1998 Jul 01.
Article in English | MEDLINE | ID: mdl-9647246

ABSTRACT

TCR- and IgG-binding Fc receptors (Fc gamma R) mediate a variety of critical biologic activities including cytolysis via the structurally related zeta- and gamma-chains. In previous studies, we have described chimeric immune receptors (CIR) in which the ligand-binding domain of a heterologous receptor or Ab is fused directly to the cytoplasmic domain of the TCR zeta-chain. Such zeta-CIRs efficiently trigger cytotoxic function of both T and NK cells in a target-specific manner. In this report, we compared the ability of both zeta- and gamma-CIRs to activate the cytolytic function of two distinct classes of Fc gamma R-bearing effectors, NK cells and neutrophils. Mature neutrophils expressing zeta- and gamma-CIR were generated in vivo from murine hemopoietic stem cells following transplantation of syngeneic mice with retrovirally transduced bone marrow or in vitro from transduced human CD34+ progenitors following differentiation. Both zeta- and gamma-based CIRs were capable of activating target-specific cytolysis by both NK cells and neutrophils, although the zeta-CIR was consistently more efficient. The experimental approach described is a powerful one with which to study the role of nonlymphoid effector cells in the host immune system and permits the rational design of immunotherapeutic strategies that rely on harnessing multiple immune cell functions via CIR-modified hemopoietic stem cells or progenitors.


Subject(s)
Cytotoxicity, Immunologic/immunology , Killer Cells, Natural/immunology , Membrane Proteins/biosynthesis , Neutrophils/immunology , Receptors, Antigen, T-Cell, gamma-delta/biosynthesis , Receptors, Antigen, T-Cell/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/immunology , Signal Transduction/immunology , Adult , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Transplantation/immunology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cytotoxicity Tests, Immunologic , Epitopes, T-Lymphocyte/immunology , Female , Genetic Vectors/chemical synthesis , Humans , Killer Cells, Natural/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Mice, SCID , Moloney murine leukemia virus/genetics , Moloney murine leukemia virus/immunology , Neutrophils/cytology , Neutrophils/metabolism , Protein Structure, Tertiary , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell, gamma-delta/genetics , Receptors, Antigen, T-Cell, gamma-delta/immunology , Recombinant Fusion Proteins/genetics , Signal Transduction/genetics , Transduction, Genetic/immunology
6.
Gene Ther ; 4(5): 393-400, 1997 May.
Article in English | MEDLINE | ID: mdl-9274715

ABSTRACT

Extensive in vivo gene transfer studies in animal models and human gene therapy clinical trials with E1-deleted adenovirus vectors have demonstrated transience of transgene expression due to direct cytopathic effects of the vectors and host immune response to virally expressed proteins. In order to overcome these difficulties, we have recently developed packaging cell lines which support the growth of adenovirus vectors containing lethal deletions in both E1 and E4 gene regions. Here we demonstrate that use of E1/E4-deleted adenovirus vectors leads to prolonged in vivo transgene expression due to elimination of cytopathic effects and significant reduction of virus-specific immune response.


Subject(s)
Adenoviridae , Adenovirus E1 Proteins/genetics , Gene Deletion , Gene Transfer Techniques , Genetic Vectors , Adenovirus E4 Proteins/genetics , Animals , CD8-Positive T-Lymphocytes/immunology , Gene Expression , Immunity, Cellular , Liver/cytology , Liver/immunology , Mice , Mice, Inbred CBA , Transgenes
7.
Nat Genet ; 15(2): 146-56, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9020839

ABSTRACT

We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.


Subject(s)
Antibody Formation , Genes, Immunoglobulin , Transgenes , Amino Acid Sequence , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibody Affinity , Antibody Diversity , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Chromosomes, Artificial, Yeast/genetics , ErbB Receptors/immunology , Gene Rearrangement, B-Lymphocyte , Humans , Hybridomas/immunology , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Interleukin-8/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Species Specificity , Tumor Necrosis Factor-alpha/immunology
8.
J Exp Med ; 184(6): 2261-9, 1996 Dec 01.
Article in English | MEDLINE | ID: mdl-8976181

ABSTRACT

Gene modification of hematopoietic stem cells (HSC) with antigen-specific, chimeric, or "universal" immune receptors (URs) is a novel but untested form of targeted immunotherapy. A human immunodeficiency virus (HIV) envelope-specific UR consisting of the extracellular domain of human CD4 linked to the zeta chain of the T cell receptor (CD4 zeta) was introduced ex vivo into murine HSC by retroviral transduction. After transplantation into immunodeficient SCID mice, sustained high level expression of CD4 zeta was observed in circulating myeloid and natural killer cells. CD4 zeta-transplanted mice were protected from challenge with a lethal dose of a disseminated human leukemia expressing HIV envelope. These results demonstrate the ability of chimeric receptors bearing zeta-signaling domains to activate non-T cell effector populations in vivo and thereby mediate systemic immunity.


Subject(s)
Bone Marrow Transplantation/immunology , CD4 Antigens/immunology , Gene Products, env/immunology , HIV/immunology , Membrane Proteins/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , Animals , Cell Line , Cytotoxicity, Immunologic , DNA Primers , Female , Gene Products, env/biosynthesis , Graft Survival/immunology , Humans , Male , Mice , Mice, SCID , Neutrophils/physiology , Polymerase Chain Reaction , Transplantation, Heterologous/immunology
10.
Gene Ther ; 2(10): 775-83, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8750018

ABSTRACT

A cell line that provides the E1 as well as the E4 gene functions of human adenovirus 5 has been established by introduction of the full-length Ad5 E4 region into 293 cells. To avoid the E1A transactivation of the E4 gene expression, the E4 promoter was replaced by the mouse alpha inhibin promoter containing a cAMP response element. This cell line was used to generate E1/E4-deleted adenovirus vectors containing a lacZ gene in the E1 region under the control of mouse pgk promoter. The titer and the lacZ gene expression of E1/E4-deleted adenovirus vector were comparable to those of E1-deleted vectors. Evidence of cytopathic effect was absent following infection of nonpermissive cell lines with E1/E4-deleted adenovirus in vitro. Establishment of the 293-E4 cell line and the generation of E1/E4-deleted adenovirus vectors may prolong gene expression in vivo and significantly improve the safety of adenovirus vectors for human gene therapy.


Subject(s)
Adenoviruses, Human/genetics , Gene Deletion , Genetic Therapy/methods , Genetic Vectors , Adenovirus E1A Proteins/biosynthesis , Adenovirus E1A Proteins/genetics , Adenovirus E4 Proteins/biosynthesis , Adenovirus E4 Proteins/genetics , Base Sequence , Cell Line , Culture Techniques/methods , DNA Primers , Genes, Lethal , Genes, Viral , Humans , Kidney , Molecular Sequence Data , Oligonucleotide Probes , Polymerase Chain Reaction , Recombination, Genetic , Transcriptional Activation , Transfection
11.
Blood ; 84(9): 2878-89, 1994 Nov 01.
Article in English | MEDLINE | ID: mdl-7949163

ABSTRACT

We have developed an immunotherapeutic approach with potential application in the treatment of viral and malignant disease. We show that primary CD8+ T cells isolated from peripheral blood can be genetically modified by retroviral transduction to express high levels of universal (major histocompatibility complex-unrestricted) chimeric T-cell receptors specific for human immunodeficiency virus (HIV) antigens. Two classes of HIV-specific URs in which the antigen-binding domain is comprised of either CD4 or a single-chain antibody are capable of activating a number of T-cell effector functions in response to target cells, including cytolysis, in a highly sensitive and specific manner. Importantly, we have addressed a number of issues which, although particularly relevant to the clinical application of this approach in the treatment of HIV infection, may also impact on the potential of UR immunotherapy for other disease targets. The UR immunotherapeutic system is particularly suited for evaluation in the clinical setting.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , HIV Infections/therapy , HIV/immunology , Receptors, Antigen, T-Cell/immunology , Animals , Antigens, Viral/immunology , Cytotoxicity, Immunologic , HIV Antibodies/immunology , HIV Envelope Protein gp120/immunology , Immunity, Cellular , Immunotherapy , In Vitro Techniques , Lymphocyte Activation , Recombinant Fusion Proteins/immunology
12.
Blood ; 83(1): 43-50, 1994 Jan 01.
Article in English | MEDLINE | ID: mdl-8274751

ABSTRACT

We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was produced without the need to generate stable producer clones. kat expression vectors, which produce high levels of retroviral vector transcripts and retroviral packaging functions, were transfected into 293 cells followed by virus harvest 48 hours posttransfection. Viral titers as high as 3.8 proviral copies/cell/mL of frozen supernatant in 3T3 cells were obtained, 10- to 50-fold greater than transient viral titers reported using 3T3-based retroviral packaging lines. Cocultivation of primary human CD8+ T lymphocytes after transient transfection of 293 cells with kat plasmids resulted in transduction efficiencies of 10% to 40%, 5- to 10-fold greater compared to cocultivation with a high titer PA317 producer clone and significantly greater than previously reported results for transduction of primary human T lymphocytes with retroviral vectors. Virus produced using the kat system was shown to be free of detectable replication competent retrovirus by an extended provirus mobilization assay, demonstrating that this system is as safe as currently available stable packaging lines. The kat virus production system should be of general use for the rapid production of high titer viral supernatants, as well as for high-efficiency transduction hematopoietic cell types refractory to retroviral transduction.


Subject(s)
Retroviridae/genetics , T-Lymphocytes/physiology , Transduction, Genetic , 3T3 Cells , Animals , CD8 Antigens/analysis , Cell Line , Genetic Therapy , Humans , Mice , Plasmids , Retroviridae/growth & development , Transfection/methods
13.
Methods Mol Biol ; 7: 283-96, 1991.
Article in English | MEDLINE | ID: mdl-21416363

ABSTRACT

A sensitive method for quantitation of mRNA in gene transfer studies is mRNA protection using end-labeled DNA probes. In addition, this technique also provides structural information about the transcript under study (1). In vitro labeled antisense RNAcan be used as an alternative to end-labeled DNA probes (2,3. RNA probes have attained wide popularity because of the ease of synthesis and high yield of probe in a labeling reaction. This has been made possible by the recent characterization of single subunit bacteriophage RNA polymerases and their promoters (4,5). Currently, three different bacteriophage RNA polymerase/promoter combinations are in use. They are derived from bacteriophage SP6 of Salmonella typhimurium (3), bacteriophage T3 of E. coli (6), and bacteriophage T7 of E. coli (6). These RNA polymerases are ideal for in vitro synthesis of labeled transcripts because of their ease of purification, stability, high rate of polymerization (10 times faster than E. coli), and their high specificity resulting from the recognition of large promoter sequences.

14.
J Biol Chem ; 264(9): 5112-20, 1989 Mar 25.
Article in English | MEDLINE | ID: mdl-2925683

ABSTRACT

A quantitative determination of collagen expression was carried out in cultured chondrocytes obtained from a tissue that undergoes endochondral bone replacement (ventral vertebra) and one that does not (caudal sterna). The "short chain" collagen, type X is only expressed in the former while the other "short chain" collagen type IX, was primarily expressed in the latter. These two tissues also differ in that vertebral chondrocytes express moderate levels of both type I procollagen mRNAs which were translated into full length procollagen chains both in vivo and in vitro, while caudal sternal chondrocytes did not. The percent of collagen synthesis was about 50% in both cell types, but sternal cells expressed twice as much collagen as vertebral cells even though type II procollagen was more efficiently processed to alpha-chains in vertebral chondrocytes than in sternal chondrocytes. The number of type II procollagen mRNA molecules/cell was found to be about 2300 in vertebral chondrocytes and about 8000 in sternal cells, in good agreement with the results reported by Kravis and Upholt (Kravis, D., and Upholt, W. B. (1985) Dev. Biol. 108, 164-172). There were about 630 copies of type I procollagen mRNAs with an alpha 1/alpha 2 ratio of 1.6 in vertebral chondrocytes compared with 5100 copies and an alpha 1/alpha 2 ratio of 2.2 in osteoblasts, and less than 40 copies in sternal cells. Since the rate of type I collagen chain synthesis was 50 times greater in osteoblasts than in vertebral cells, type I procollagen mRNAs were about six times less efficiently translated in vertebral cells than in osteoblasts. The type I mRNAs in vertebral chondrocytes were polyadenylated and had 5' ends that were identical in osteoblasts, fibroblasts, and myoblasts. Moreover, type I mRNAs isolated from vertebral chondrocytes were translated into full length preprocollagen chains in vitro in rabbit reticulocyte lysates. Thus, chondrocytes isolated from cartilage tissues with different developmental fates differed quantitatively and qualitatively in total collagen synthesis, procollagen processing, and distribution of collagen types.


Subject(s)
Cartilage/analysis , Collagen/analysis , Embryonic and Fetal Development , Animals , Cartilage/embryology , Chick Embryo , Collagen/biosynthesis , Nucleic Acid Hybridization , Procollagen/metabolism , Protein Processing, Post-Translational , RNA, Messenger/metabolism , Spine , Sternum
15.
J Virol ; 62(10): 3795-801, 1988 Oct.
Article in English | MEDLINE | ID: mdl-3418785

ABSTRACT

A series of retrovirus vectors were constructed in which cellular promoter elements derived from the chicken beta-actin and human histone H4 genes were introduced within the proviral transcriptional unit of Moloney murine leukemia virus in order to promote expression of inserted sequences. Each of these vectors gave rise to high titer of virus capable of transferring the expected proviral structure to cells. Inclusion of normal 5' splice sequences or a portion of viral gag sequences in these constructions resulted in significant increases in virus titer. Each construction was transcriptionally active in NIH 3T3 cells and in undifferentiated F9 cells. One of the vectors, HSG-neo, which contained the human histone H4 promoter, was shown to be transcriptionally active in hematopoietic cells derived from long-term reconstituted bone marrow transplant recipients engrafted with transduced stem cells. These vectors should be of general use for obtaining efficient gene expression in embryonal and hematopoietic cells.


Subject(s)
Gene Expression Regulation , Genetic Vectors , Hematopoietic Stem Cells/microbiology , Moloney murine leukemia virus/genetics , Animals , Cell Line , DNA, Viral/genetics , Female , Genes, Viral , Mice , Nucleic Acid Hybridization , Promoter Regions, Genetic , RNA, Viral/genetics , Transcription, Genetic , Transfection
16.
J Biol Chem ; 262(27): 13323-32, 1987 Sep 25.
Article in English | MEDLINE | ID: mdl-2820966

ABSTRACT

Genomic clones corresponding to the amino-terminal propeptide and 5'-flanking sequences of the chicken pro-alpha 1(I) collagen gene were isolated as a first step in the identification of DNA sequences important for transcriptional regulation of the pro-alpha 1(I) collagen gene. Due to the failure to identify positive clones in either primary or amplified genomic libraries, a 5.1-kilobase pair StuI genomic fragment identified by Southern blotting was enriched by sucrose gradient fractionation of genomic DNA and cloned into lambda gt11. Comparison of the DNA sequence of the 5.1-kilobase pair StuI fragment to the DNA sequence of a cDNA clone encoding the amino-terminal propeptide, signal peptide, and the 5'-untranslated region identified the first four exons and most of the fifth. Exon size and intron position have been largely conserved between human and chicken alpha 1(I) genes. DNA sequence analysis of the region 5' to the transcription initiation site identified the canonical TATA and CAAT boxes. However, the 40-nucleotide pyrimidine stretch centered between -150 and -180 nucleotides, found in all previously isolated type I procollagen genes from chicken, mouse, and human, was absent in the chicken pro-alpha 1(I) collagen gene. This sequence corresponds to the in vivo DNase I hypersensitive site in the chicken pro-alpha 2(I) and mouse pro-alpha 1(I) collagen genes, as well as the in vitro S1 nuclease hypersensitive site in both chicken and mouse pro-alpha 2(I) collagen genes. Two unusual DNA sequences were identified within the chicken pro-alpha 1(I) collagen gene. Fifteen tandem repeats of the sequence GGGGAGA were identified within the first intron, 300 nucleotides 3' to the first exon. This sequence was identified due to its hypersensitivity to S1 nuclease in vitro in supercoiled plasmids. The second sequence located 5' to -180 contained at least 25 copies of a polymorphic, 23-base pair tandemly repeated sequence not identified in other type I procollagen genes. Both of these tandem repeat sequences were identified at other locations in the chicken genome by Southern blot hybridization.


Subject(s)
Genes , Procollagen/genetics , Promoter Regions, Genetic , Amino Acid Sequence , Animals , Base Sequence , Chickens , DNA Restriction Enzymes , Introns , Molecular Sequence Data , Repetitive Sequences, Nucleic Acid
17.
Gene ; 56(1): 71-8, 1987.
Article in English | MEDLINE | ID: mdl-3678834

ABSTRACT

As a first step in isolating the 5' end of the chicken pro alpha 1(I) collagen gene, we constructed cDNA clones complementary to the 5' end of the pro alpha 1(I) mRNA using synthetic oligodeoxynucleotides complementary to a conserved region within the N-terminal telopeptide as primers. cDNA clones corresponding to the 5'-untranslated region, signal peptide, N-propeptide and telopeptide were identified based on homology with the human pro alpha 1(I) collagen protein sequence, and on hybridization to pro alpha 1(I) mRNA on Northern blots. A comparison of the nucleotide sequence of these clones with the sequence of the 5' end of the pro alpha 2(I) collagen mRNA confirms that there is 84% homology in a 49-bp region surrounding the translation start point, and shows that there is 70% homology in the nucleotide sequences encoding the N-propeptide triple helical region of the two type-I collagen chains.


Subject(s)
DNA/genetics , Procollagen/genetics , RNA, Messenger/genetics , Animals , Base Sequence , Chickens , Cloning, Molecular , Humans , Molecular Sequence Data , Species Specificity
18.
Mol Cell Biol ; 5(6): 1415-24, 1985 Jun.
Article in English | MEDLINE | ID: mdl-4033659

ABSTRACT

Growth of embryonic chicken sternal chondrocytes in the presence of phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, resulted in a dramatic morphological change from spherical floating cells to adherent fibroblastic cells. This morphological change was accompanied by a quantitative switch from synthesis of cartilage-specific type II procollagen to type I procollagen. Type II procollagen mRNA levels decreased 10-fold in PMA-treated cells. Activation of type I collagen genes led to the accumulation of type I procollagen mRNA levels comparable to those of type II mRNA in these cells. However, only type I procollagen mRNA was translated. In addition to gene activation, unprocessed pro alpha 1(I) transcripts present at low levels in control chondrocytes were processed to mature mRNA species. Redifferentiation of PMA-treated chondrocytes was possible if cells were removed from PMA after the morphological change and cessation of type II procollagen synthesis but before detectable amounts of type I procollagen were synthesized. Production of type I collagen thus marks a late phase of chondrocyte "dedifferentiation" from which reversion is no longer possible. Redifferentiated cell populations contained 24-fold more pro alpha 1(II) collagen mRNA than pro alpha 1(I) collagen mRNA, but the rates of procollagen synthesis were comparable. This suggests that the PMA-mediated dedifferentiation of chondrocytes as well as their redifferentiation is under both transcriptional and posttranscriptional regulation.


Subject(s)
Cartilage/drug effects , Collagen/metabolism , Phorbols/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Animals , Cartilage/cytology , Cell Differentiation/drug effects , Cell Division/drug effects , Chick Embryo , Gene Expression Regulation/drug effects , Procollagen/genetics , Procollagen/metabolism , RNA, Messenger/analysis , Transcriptional Activation
19.
Mol Cell Biol ; 5(6): 1425-33, 1985 Jun.
Article in English | MEDLINE | ID: mdl-4033660

ABSTRACT

Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.


Subject(s)
Actins/metabolism , Cartilage/drug effects , Fibroblasts/drug effects , Phorbols/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Actins/genetics , Animals , Cartilage/cytology , Cartilage/metabolism , Cell Adhesion/drug effects , Cells, Cultured , Chick Embryo , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Regulation , Muscles/cytology , RNA, Messenger/analysis
20.
Proc Natl Acad Sci U S A ; 81(6): 1659-63, 1984 Mar.
Article in English | MEDLINE | ID: mdl-6324210

ABSTRACT

A site that is preferentially cleaved by the single-strand-specific endonuclease from Aspergillus oryzae was located in vitro 180 base pairs upstream from the 5' end of the chicken pro-alpha 2(I) collagen gene. It is found in supercoiled plasmids with a negative superhelical density of -0.024 or more but not in linear DNA molecules. The nuclease S1 sensitivity is retained in plasmids containing genomic fragments extending from position +8 to -285 (where +1 is the first transcribed base) and from -147 to -351 and also in a 5.7-kilobase EcoRI fragment that extends 1.6 kilobases 5' and 4.1 kilobases 3' to the 5' end of the gene. Analysis at the nucleotide level on a DNA sequence gel places the site at -181 to -182 on the sense strand and at -182 to -184 and -192 to -195 on the nonsense strand. These sites lie within a stretch of 42 pyrimidines interrupted by a single guanine and within the sequence T-C-C-C-T-C-C-C-T-T-C-C-T-C-C-C-T-C-C-C-T.


Subject(s)
Gene Expression Regulation , Procollagen/genetics , Animals , Chickens , DNA, Superhelical/genetics , Endonucleases , Genes , Genetic Linkage , Nucleic Acid Conformation , Operon , Plasmids , Single-Strand Specific DNA and RNA Endonucleases
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