ABSTRACT
A new gene (POLL) encoding a novel DNA polymerase (Pol lambda) has been identified at mouse chromosome 19. Murine Pol lambda, consisting of 573 amino acid residues, has a 32% identity to Pol beta, involved in nuclear DNA repair in eukaryotic cells. It is interesting that Pol lambda contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol beta and other DNA polymerases belonging to family X. Murine Pol lambda, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis. Pol lambda also conserves the critical residues of Pol beta required for its intrinsic deoxyribose phosphate lyase (dRPase) activity. The first 230 amino acid residues of Pol lambda, that have no counterpart in Pol beta, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair. Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol lambda specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes. These first evidences, although indirect, suggest a potential role of Pol lambda in DNA repair synthesis associated with meiosis.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Eukaryotic Cells/enzymology , Meiosis , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence , DNA Polymerase beta/chemistry , DNA Polymerase beta/metabolism , DNA Repair , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Eukaryotic Cells/cytology , Exons/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Humans , Introns/genetics , Male , Meiosis/genetics , Mice , Mice, Inbred C57BL , Models, Biological , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , RNA, Messenger/analysis , RNA, Messenger/genetics , Saccharomyces cerevisiae/enzymology , Sequence Alignment , Spermatids/enzymology , Spermatocytes/enzymology , Testis/cytology , Testis/enzymology , Testis/metabolismABSTRACT
We describe a new selectable marker for retroviral transduction and selection of human and murine cells. The molecule expressed on the cell surface of the transduced population is a truncated version of human growth hormone receptor (deltahGHR), capable of ligand (hGH) binding, but devoid of the domains involved in signal triggering. We demonstrate that the engineered molecule is stably expressed in the target cells as an inert protein unable to trigger proliferation or to rescue the cells from apoptosis after ligand binding. This new marker will probably have a wide application spectrum, since hGHR in the human adult is highly expressed only in liver cells, and lower levels have been reported in certain lymphocyte cell populations. The deltahGHR label has high biosafety potential, as it belongs to a well-characterized hormonal system that is nonessential in adults, and there is extensive clinical experience with hGH administration in humans. This record allows us to hypothesize the lack of relevant clinical consequences resulting from massive transgene expression caused by successful replacement of a large tissue with genetically transduced cells. We take advantage of the differential binding properties of several monoclonal antibodies (MAbs) in describing a cell rescue method in which the antibody used to select deltahGHR-transduced cells is eluted by competition with hGH or, alternatively biotinylated hGH is used to capture tagged cells. In the latter system, the final purified population would be recovered free of attached antibodies in hGH (a substance approved for human use)-containing medium, providing additional biosafety relative to currently existing methods that rely on the use of murine MAb to rescue genetically labeled cells.
Subject(s)
Biomarkers , Gene Transfer Techniques , Membrane Proteins/genetics , Receptors, Somatotropin/genetics , Retroviridae/genetics , Transduction, Genetic , Animals , Antibodies, Monoclonal/genetics , Blotting, Western , Cell Cycle/genetics , Cell Division/genetics , Cell Line , Cloning, Molecular , Dose-Response Relationship, Drug , Fetal Blood/metabolism , Flow Cytometry , Genome, Viral , Hematopoietic Stem Cells/immunology , Human Growth Hormone/immunology , Human Growth Hormone/metabolism , Humans , Mice , Mice, Inbred BALB C , Models, Immunological , Precipitin Tests , Proviruses/genetics , Time FactorsABSTRACT
The CCR5 chemokine receptor plays a crucial role in the initiation of in vivo HIV infection, acting as a critical coreceptor molecule for primary strains. Individuals with mutations in the CCR5 gene that reduce its level of expression are resistant to HIV-1 infection. Since these mutations are not associated with any known clinical condition, CCR5 may be an ideal target for anti-HIV therapy. We have designed an artificial hammerhead ribozyme, denoted RzR5-76, targeted to exon 2 of the human CCR5 mRNA. When RzR5-76 activity is induced in HEK 293 cells transfected with a CCR5 expression plasmid, the surface levels of this chemokine receptor are reduced up to 60%. The results indicate that this inhibitory effect is mainly due to the catalytic activity of the ribozyme and not to its antisense properties. These preliminary data suggest that intracellular ribozymes could be used in vivo to block HIV-1 entry into human cells.
