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PubMed1995

Analysis of beta-globin mutations shows stable mixed chimerism in patients with thalassemia after bone marrow transplantation.

Beta-thalassemia major (TM) is caused by any of approximately 150 mutations within the beta-globin gene. To establish the degree of chimerism after bone marrow transplantation (BMT), we have performed molecular analysis of beta-globin mutations in 14 patients with TM over a period of 10 years. All patients underwent T cell-depleted allogeneic BMT from HLA-identical related donors, using either in vitro T-cell depletion with CAMPATH 1M and complement or in vivo depletion using CAMPATH 1G in the bone marrow collection bag. To date, at different time periods after BMT, seven patients have some degree of chimerism; six of these patients, all blood transfusion-independent, have donor cells in the range of 70% to 95%, with stable mixed chimerism (MC). The seventh patient has less than 10% donor cells with, surprisingly, only minimal transfusion requirements. The detection of beta-globin gene point mutation, as used here, is a highly specific and sensitive marker for engraftment and MC in patients with thalassemia. In light of its specificity, the method is applicable in all cases of TM, as it is independent of sex and other non-globin-related DNA markers. The high incidence of MC found in our patients may be a consequence of the pre-BMT T-cell depletion. Because MC was associated with transfusion independence, complete eradication of residual host cells for effective treatment of TM and possibly other genetic diseases may prove not to be essential.

Alleles↗
PubMed1994

Xenobiotics, chimerism and the induction of tolerance following organ transplantation.

The successful results seen after organ transplantation are largely attributable to the potency and specificity of modern immunosuppressive agents. Although drug-free unresponsiveness to graft alloantigens has not been routinely achieved in clinical practice, recent appreciation of the importance of cell chimerism, which develops after the migration from donor to host of leukocytes contained in solid organ grafts, has introduced a concept which may explain the mechanism of graft tolerance. Recent evidence has indicated that immunosuppressive drugs may have a common potential to induce graft tolerance, even though they act through diverse mechanisms, and that this potential may be mediated by a permissive effect on the migration and survival of donor-derived leukocytes. This review briefly examines the mechanisms by which immunosuppressive drugs function and analyses the different methods which these agents might use to induce chimerism associated with graft tolerance. Furthermore, we describe ongoing clinical studies in which the chimerism produced after solid organ transplantation is augmented with donor bone marrow in an attempt to facilitate the induction of tolerance.

Animals↗
PubMed1995

In vivo antitumor activity of T cells redirected with chimeric antibody/T-cell receptor genes.

In an effort to broaden the applicability of adoptive cellular immunotherapy toward nonmelanoma cancers, we have designed chimeric antibody/T-cell receptor genes composed of the variable domains from mAbs joined to T-cell receptor-signaling chains. We have demonstrated that T cells retrovirally transduced with these genes can recognize antibody-defined antigens and that this recognition leads to T-cell activation, specific lysis, and cytokine release. In this study, we have examined the in vivo activity of murine T cells transduced with a chimeric receptor gene (MOv-gamma) derived from the mAb MOv18, which binds to a folate-binding protein overexpressed on most human ovarian adenocarcinomas. Nude mice that were given i.p. implants of human ovarian cancer (IGROV) cells were treated 3 days later with i.p. murine tumor-infiltrating lymphocytes (TIL) derived from an unrelated tumor. Mice treated with MOv-gamma-transduced TIL (MOv-TIL) had significantly increased survival compared to mice treated with saline only, nontransduced TIL, or TIL transduced with a control anti-trinitrophenyl chimeric receptor gene (TNP-TIL). In another model, C57BL/6 mice were given i.v. injections of a syngeneic methylcholanthrene-induced sarcoma transduced with the folate-binding protein (FBP) gene. Three days later, mice were treated i.v. with various transduced murine TIL (derived from an unrelated tumor), followed by low-dose systemic interleukin 2. Eleven days after tumor injection, mice were sacrificed, and lung metastases were counted. In multiple experiments, mice receiving MOv-TIL had significantly fewer lung metastases than did mice treated with interleukin 2 alone, nontransduced TIL, or TNP-TIL. These studies indicate that T cells can be gene modified to react in vivo against tumor antigens, defined by mAbs. This approach is potentially applicable to a number of neoplastic and infectious diseases and may allow adoptive immunotherapy against types of cancer not previously amenable to cellular immunotherapy.

Animals↗
PubMed1995

Mixed xenogeneic chimerism and tolerance III. A nonlethal approach to induce donor-specific transplantation tolerance to simultaneous islet xenografts.

Previously, we reported that transplantation of rat bone marrow into lethally irradiated (950 rads) mouse recipients results in stable xenogeneic (rat-->mouse) chimerism and confers donor-specific transplantation tolerance for subsequent or simultaneous islet xenografts. For potential clinical application of chimerism to induce tolerance, it would be important to avoid the morbidity and mortality associated with lethal conditioning. Recently, we established a model to achieve durable multilineage xenogeneic chimerism using a nonlethal cytoreductive approach. We report here for the first time that donor-specific rat islet xenografts placed coincident with the bone marrow transplant are permanently accepted by nonlethally conditioned recipients. All recipients conditioned with 700 cGy of total body irradiation and transplanted with 40 x 10(6) F344 rat bone marrow cells repopulated as mixed donor/host chimeras. The chimeras exhibited permanent acceptance of donor islet xenografts, since donor-specific F344 (Rt1A1) rat islet xenografts were significantly prolonged (median survival time > 110 days), while MHC-disparate third-party WF (Rt1Au) rat islets were prolonged but rejected (median survival time = 33.2 days). The islets were functional to maintain normoglycemia and regulated in function to respond to an intraperitoneal glucose challenge. These data suggest that tolerance to donor-specific islet xenografts placed coincident with bone marrow transplantation can be achieved after nonlethal conditioning.

Animals↗
PubMed1995

Amino terminus and the first four membrane-spanning segments of the Arabidopsis K+ channel KAT1 confer inward-rectification property of plant-animal chimeric channels.

The Arabidopsis hyperpolarization-activated (inward-rectifying) K+ channel KAT1 is structurally more similar to animal depolarization-activated (outward-rectifying) K+ channels than to animal hyperpolarization-activated K+ channels. To gain insight into the structural basis for the opposite voltage dependences of plant inward-rectifying and animal outward-rectifying K+ channels, we constructed recombinant chimeric channels between the hyperpolarization-activated K+ channel KAT1 and a Xenopus depolarization-activated K+ channel. We report here that two of the chimeric constructs, which contain the first third of the KAT1 sequence, including the first four membrane-spanning segments (S1-S4) and the linker sequence between the fourth and fifth membrane-spanning segments, express functional channels that retain activation by hyperpolarization, but not depolarization. These two chimeric channels are no longer selective for K+. The chimeras are selective for cations over anions and are permeable to Ca2+. Therefore, unlike animal hyperpolarization-activated K+ channels, in which the carboxyl terminus is important for inward rectification induced by Mg2+ and polyamine block, the plant KAT1 channel has its major determinants for inward rectification in the amino-terminal region, which ends at the end of the S4-S5 linker.

Amino Acid Sequence↗
PubMed1995

Increase of bcr-abl chimeric mRNA expression in tumor cells of patients with chronic myeloid leukemia precedes disease progression.

The translocation t(9;22) in chronic myeloid leukemia (CML) generates a bcr-abl fusion gene that codes for an aberrant chimeric mRNA. Cell lines established from CML patients in blast crisis show higher expression of this aberrant bcr-abl transcript than cells from patients in chronic phase of the disease. This observation provided the stimulus to investigate whether increased expression of the aberrant bcr-abl fusion transcript is critical to the progression of CML from chronic phase to blast crisis. We have monitored the bcr-abl mRNA expression in 25 patients by serial quantitative polymerase chain reaction analyses during a follow-up period of 12 to 156 months after diagnosis, with a median observation time of 28 months. In all patients who have shown disease progression to accelerated phase (n = 4) or blast crisis (n = 7), an increase in bcr-abl mRNA expression was detected up to 16 months before laboratory or clinical parameters showed phenotypic transformation of the malignant clone. To investigate whether the elevated levels of bcr-abl mRNA reflected an increase in the proportion of leukemic cells in the samples analyzed or primarily enhanced transcriptional activity of the bcr-abl fusion gene, we performed quantitative analyses of the bcr-abl gene at the DNA level and of the Ph chromosome at the cytogenetic level and compared these data with steady-state bcr-abl mRNA levels. We show that increased levels of the bcr-abl transcript did not reflect increased proportions of leukemic cells but elevated steady-state levels of the chimeric mRNA in the malignant cells before disease progression. Therefore, our data strongly suggest that an increase of the chimeric mRNA expression in the leukemic cells precedes the phenotypic transformation of the malignant clone.

Adult↗
PubMed1995

Catalytic properties of NAD(P)H:quinone acceptor oxidoreductase: study involving mouse, rat, human, and mouse-rat chimeric enzymes.

NAD(P):quinone acceptor oxidoreductase (quinone reductase) (DT-diaphorase, EC 1.6.99.2) is involved in the process of reductive activation of cytotoxic antitumor quinones and nitrobenzenes. In this study, we initially examined the relative abilities of mouse, rat, and human quinone reductases to reduce two prodrugs, CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] and EO9 [5-(1-aziridinyl)-3-(hydroxymethyl)-2-(3-hydroxy-1-propenyl)-1- methyl-1H-indole-4,7-dione]. By using Escherichia coli-expressed quinone reductases and evaluating them under identical conditions, we confirmed previous finding showing that the human enzyme is not as effective as the rat enzyme in reducing CB 1954 and EO9, although the two enzymes have similar NAD(P)H-menadione reductase activities. Interestingly, although the amino acid sequence of mouse quinone reductase is more homologous to that of the rat enzyme, we found that the mouse enzyme behaves similarly to the human enzyme in its ability to reduce these compounds and to generate drug-induced DNA damage. To determine the region of quinone reductase that is responsible for the catalytic differences, two mouse-rat chimeric enzymes were generated. MR-P, a chimeric enzyme that has mouse amino-terminal and rat carboxy-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of rat quinone reductase, and RM-P, a chimeric enzyme that has rat amino-terminal and mouse carboxyl-terminal segments of quinone reductase, was shown to have catalytic properties resembling those of mouse quinone reductase. In addition, MR-P and RM-P were found to be inhibited by flavones with Ki values similar to those for rat and mouse quinone reductases, respectively. Based on these results, we propose that the carboxyl-terminal portion of the enzyme plays an important role in the reduction of cytotoxic drugs and the binding of flavones.

Amino Acid Sequence↗
PubMed1995

Characterization of donor chimerism, alloreactive host T cells and memory cell development in thymi from mice resistant to neonatal transplantation tolerance.

Certain B10 background mouse strains are resistant to tolerance induction after neonatal inoculation of class I/II MHC-disparate F1 hybrid cells. Despite initial thymic deletion of alloreactive cells, the majority (95%) of these mice reacquire the capacity to reject donor allografts. These current studies examined thymi of adult B10.S (H-2S/H-2E-) mice that neonatally received (B10.S x B10.A)F1 cells, before and after rejection or acceptance of B10.A (H-2k/d/H-2E+) skin grafts. Alloreactive thymic V beta 11+ and V beta 5+ T cells were often reduced in the injected recipients preceding allograft challenge. In some mice a single B10.A skin graft generated an increase in these T cell populations concurrent with allograft rejection. Injected mice that accepted B10.A skin grafts (i.e., tolerant) or that rejected two sequential B10.A grafts often had a reduction of these T cells. Thymic memory cells (CD44high+) were present before transplant in injected mice and reduced in the tolerant mice. Donor chimeric cells were identified in the majority of injected mice before transplant and diminished after B10.A graft application. A greater proportion of chimeric cells coexpressed CD11b or B220 in the tolerant mice. Thus, neonatally injected B10.S mice resistant to tolerance induction reacquire immunocompetent thymic T cells bearing characteristics of memory T cells that could mediate graft rejection. Finally, the initial presentation of chimeric cells (e.g., macrophages) that may efficiently present class I Ags through H-2E likely increases the possibility of adult tolerance.

Animals↗
PubMed1993

Targeting of T lymphocytes to Neu/HER2-expressing cells using chimeric single chain Fv receptors.

Cell surface molecules essential for the transformed phenotype or growth of malignant cells are attractive targets for anticancer immunotherapy. Antibodies specific to Neu/HER2, a human adenocarcinoma-associated growth factor receptor, were demonstrated to have tumor-inhibitory capacity. Yet, the inefficient accessibility of antibodies to solid tumors limits their clinical use. To redirect effector lymphocytes to adenocarcinomas, we constructed and functionally expressed in T cells chimeric single chain receptor genes incorporating both the Ag-binding domain of anti-Neu/HER2 antibodies and the zeta-signal-transducing subunit of the TCR/CD3 complex or the gamma-signal-transducing subunit of the Ig Fc receptor complex. Surface expression of the anti-Neu/HER2 chimeric genes in cytotoxic T cell hybridomas endowed them with specific Neu/HER2 recognition enabling their activation for IL-2 production and lysis of transformed cells overexpressing Neu/HER2. These chimeric genes hold promise for the immunotherapy of cancer.

Animals↗
PubMed1994

Small chimeric toxins containing only transforming growth factor alpha and domain III of Pseudomonas exotoxin with good antitumor activity in mice.

Chimeric toxins composed of transforming growth factor alpha (TGF alpha) fused to mutant forms of Pseudomonas exotoxin (PE) bind to the epidermal growth factor receptor and kill cells bearing epidermal growth factor receptors. Initially, the binding domain (Ia; amino acids 1-252) of PE was deleted and replaced with TGF alpha to make TGF alpha-PE40 in which TGF alpha is fused to domains II, Ib, and III of PE (amino acids 253-613). That drug is currently undergoing clinical study for the intravesical therapy of bladder cancer. To generate smaller molecules that would have increased tumor penetration, several deletion mutants were constructed. In one of these, TGF alpha was inserted near the carboxyl terminus of PE, and residues in domains II and Ib of PE (amino acids 253-279 and 365-380) were deleted so that the chimeric toxin did not need to be cleaved by an intracellular protease to be activated (Theuer et al., J. Biol. Chem., 267: 16872-16877, 1992). We have now constructed chimeric toxins which contain only domain III, yet still exhibit high cytotoxic activity on epidermal growth factor receptor-containing cells and produce substantial tumor regressions in mice bearing a human xenograft. The high cytotoxic activity of these severely truncated toxins provides new insights on the proposed functions of domains II and III of PE.

Adenosine Diphosphate Ribose↗
PubMed1994

Chimeric (mouse/human) anti-colon cancer antibody c30.6 inhibits the growth of human colorectal cancer xenografts in scid/scid mice.

The mouse monoclonal antibody, m30.6 (IgG2b), detects an antigenic determinant expressed predominantly on the surface of colorectal adenocarcinoma cells and has been shown previously to be a potentially useful therapeutic and diagnostic reagent for human colon cancer. We report the production and characterization of a mouse/human chimeric antibody, c30.6, with potent in vitro and in vivo antitumor activity. The genes encoding the variable domains for heavy and light chains were amplified by thermal cycling using degenerate oligonucleotide primers complementary to conserved immunoglobulin framework sequences. The gene segments were sequenced, subcloned into eukaryotic expression vectors containing human constant region genes (IgG1 and kappa), and cotransfected into nonsecreting Sp2/0 mouse myeloma cells. There were significant differences in the biological activities of the murine and chimeric antibodies. The i.p. administration of c30.6 but not of m30.6 produced a marked growth inhibition of s.c. 30.6+ COLO 205 tumors in scid/scid mice (approximately 40% reduction in tumor size, measured 21 days after tumor inoculation). Reduced tumor growth was not due to altered binding characteristics of c30.6 because: (a) the chimeric antibody was shown by flow cytometry to bind exclusively to cell lines that expressed the 30.6 determinant; (b) c30.6 was able to completely inhibit the binding of m30.6 on 30.6+ cells; and (c) the affinity of binding of the two antibodies was the same (Ka, approximately 1.50 x 10(8)). Up to 15% of the total injected antibody dose/g tissue was localized in 30.6+ tumors at 24 h, approximately 13% was present in the tumors at 48 h, and approximately 10% was present at 72 h. Furthermore, c30.6 demonstrated a shorter circulating half-life (53 h; m30.6, 72 h) when given i.p. to C57BL6 x BALB/cF1 mice. Unlike m30.6, c30.6 was also strongly active in antibody-dependent cell-mediated cytotoxicity against a range of 30.6+ tumor target cells in vitro. Up to 80% specific 51Cr release was achieved using either freshly isolated human peripheral blood mononuclear cells or 2-day-old interleukin 2-stimulated human peripheral blood mononuclear cells as effectors. The enhanced antitumor activity of c30.6 suggests that it might be a useful immunotherapeutic reagent for colorectal carcinoma.

Adenocarcinoma↗
PubMed1994

Detection of chimerism in subpopulations of cells by fluorescence in situ hybridization and immunofluorescent staining of cell surface antigens.

Fluorescence in situ hybridization (FISH) enables the visualization of the X and Y chromosomes in nuclei of interphase cells using probes which recognize repetitive sequences on the X and Y chromosomes. The process of cell preparation, hybridization and visualization of the probes within the nuclei lasts between 16 and 24 h. Because of these features the method is ideal for monitoring engraftment after sex-mismatched bone marrow transplant (BMT). So far, the FISH technique has been applied to assess the degree of chimerism in either unseparated peripheral blood cells or unseparated bone marrow cells. However, for early detection of rejection or relapse, the degree of chimerism in subpopulations of cells is probably more informative. A new FISH protocol was developed with the aim of simultaneously visualizing the surface antigens by fluorescent antibodies and X or Y chromosomes with fluorescent probes. After development of the new FISH protocol, it was applied to patient samples after sex-mismatched BMT. The degree of chimerism was assessed comparing the results of the new FISH protocol with a 'standard' FISH protocol which is employed both in our and other laboratories to detect chromosomes in interphase nuclei. Both methods gave similar results. The new FISH protocol was applicable for the detection of Y and X chromosomes. The clinical relevance of this new FISH protocol is illustrated by the results obtained on peripheral blood samples of five BMT patients who were mixed chimeras after sex-mismatched BMT. A clear distinction between engraftment of T and B cells was noted. In some patients the T cells were of donor origin while the B cells were of host origin.

Adult↗
PubMed1994

Chimeric muscarinic cholinergic:beta-adrenergic receptors that are functionally promiscuous among G proteins.

We evaluated the G protein selectivity of chimeric M1 and M2 muscarinic cholinergic receptors in which either the third intracellular (I3) loop or the N-terminal portion of this loop (the I3N peptide) was replaced by the corresponding sequence from the beta 1-adrenergic receptor. The chimeras retained agonist-dependent G protein regulatory activity, but were completely promiscuous among potential G protein targets. When expressed in transfected cells, the chimeric receptors activated adenylyl cyclase, the major target of the beta-adrenergic receptor, and activated phospholipase C via a pertussis toxin-insensitive G protein, presumably a Gq. Gs is not a target of either muscarinic receptor, and Gq is not a cellular target of either the M2 muscarinic or beta-adrenergic receptor. When co-reconstituted into phospholipid vesicles with purified G proteins, the chimeric receptors were completely nonselective among all G proteins tested. They activated Gi, G(o), Gz, and Gs with similar efficiencies. This promiscuity was largely suppressed, both in transfected cells and in reconstituted vesicles, by the additional replacement of the second intracellular (I2) loop of the beta-adrenergic receptor. Such double substitutions created receptors specific for Gs, the target of the beta-adrenergic receptor. These findings suggest that G protein specificity depends on the proper combination of multiple regions on a receptor's cytoplasmic surface. In addition, the promiscuous receptors described here may be useful for regulating novel G proteins whose natural regulators are not yet known.

Adenylate Cyclase Toxin↗
PubMed1993

5'-flanking region responsible for endosperm-specific expression of rice prolamine chimeric gene in transgenic tobacco.

The 5' upstream region (-680-(+)40) containing the complete signal peptide coding sequence of the rice seed storage prolamine gene was amplified in vitro with polymerase chain reaction from the genome of Chinese super rice cultivar Zhonghua 8. Physical map and DNA sequence analysis showed strong homology with the 5'-flanking region of rice prolamine gene reported by Kim in 1988. No changes in the signal peptide coding sequence and a long leader sequence with several small ORFs were found. Chimeric gene containing 5'-flanking region of the prolamine gene has been transcriptionally fused with the beta-glucuronidase reporter gene. The fusion junction was confirmed by both physical map and DNA sequence analysis. The resultant chimeric gene was used to transform the tobacco explants by Ti binary system of Agrobacterium tumefaciens LBA4404. With dot and Southern blotting hybridization, three transgenic tobacco plants with the copies of chimeric GUS genes as many as 20 were obtained. Histochemical analysis revealed that the GUS activity in the endosperm tissues of tobacco seeds at the developmental stage was about 20 DAP. No GUS activity was found in leaves, stems, roots and flowers of the transgenic tobacco plants. Therefore, we concluded that the 5'-upstream cis-elements from -680 to -18 were enough to confer the endosperm-specific and temporal expression of rice prolamine gene.

Base Sequence↗
PubMed1994

A chimeric protein comprised of IL-4 and Pseudomonas exotoxin is cytotoxic for activated human lymphocytes.

IL4-Pseudomonas exotoxin (IL4-PE4E) is a chimeric molecule in which human IL-4 is genetically fused to the mutated binding domain of Pseudomonas exotoxin. This molecule binds specifically to human IL-4 receptor-bearing cells. IL4-PE4E was extremely cytotoxic to highly purified anti-CD3-activated CD8+ T lymphocytes. The cytotoxic activity of this molecule was dependent on the activation state of CD8+ T cells: 3- and 4-day activated T cells were very susceptible to the cytotoxic activity of IL4-PE4E compared with 0- to 2-day activated cells. PHA-activated lymphocytes and PBL activated in mixed lymphocyte reaction were also highly sensitive to IL4-PE4E. CD16+ and/or CD56+ highly purified NK cells or highly purified, IL-2-activated NK cells were also very sensitive to the cytotoxic effect of IL4-PE4E. IL-2-activated LAK cells had little susceptibility after 1 day but were very sensitive to IL4-PE4E after 3 days. The cytotoxic effects of IL4-PE4E were mediated through a ligand receptor interaction because excess rIL-4 abrogated these effects as did a neutralizing Ab to human IL-4. A chimeric mutant protein that can bind to IL-4 receptors but lacks the ability to inhibit protein synthesis was not cytotoxic to activated lymphocytes. The IL4-PE4E-mediated cytotoxicity of activated T cells correlated with the level of expression of IL-4 receptors on these cells. CD8+ T cells activated for 3 days expressed the highest density of IL-4 receptors compared with 1- or 2-day activated cells. Among two chimeric toxins tested only IL4-PE4E was cytotoxic to 2-day anti-CD3-activated CD8+ T lymphocytes, whereas IL6-PE4E was not active at all. These studies suggest that human IL4 toxin could be a potent agent for the elimination of activated lymphocytes in allograft rejection, some autoimmune diseases, or treatment of lymphomas and leukemias.

ADP Ribose Transferases↗
PubMed1994

A reverse transcriptase-polymerase chain reaction detects heterogeneous chimeric mRNAs in leukemias with 11q23 abnormalities.

The MLL gene involved in 11q23 translocations found in the majority of infantile leukemias and some secondary leukemias makes fusion transcripts with genes such as LTG4 (chromosome 4), LTG9 (chromosome 9), and LTG19 (chromosome 19) as a result of reciprocal translocation. We have examined 25 cases of leukemias with 11q23 abnormalities by Southern blot analysis and the reverse transcriptase-polymerase chain reaction (RT-PCR). Using various primer pairs, chimeric mRNAs could be amplified in 6 of 7 leukemias with t(4;11), 6 of 8 leukemias with t(9;11) including secondary leukemia, 8 of 9 leukemias with t(11;19), and 1 with a deletion at 11q23. The chimeric mRNAs were heterogeneous and differential usage of the MLL exons was found, irrespective of the partner chromosomes. Sensitivity studies showed that a single clone with chimeric mRNA in 10(4) to 10(5) cells could be detected. These findings show that the present RT-PCR settings provide a rapid, accurate, and sensitive tool for diagnosing leukemias with 11q23 translocations and for monitoring response to therapy in these patients.

Adult↗
PubMed1994

Production of mouse V/human C chimeric kappa genes by homologous recombination in hybridoma cells. Analysis of vector design and recombinant gene expression.

Homologous recombination between transferred and chromosomal Ig genes in mouse hybridoma cells offers a general method of altering the chromosomal Ig genes in predetermined ways. Recombination is infrequent in hybridoma cells, and we have been interested in improving the methods for identifying and recovering the rare recombinants. We have used vectors that are designed to replace the mouse chromosomal C kappa segment with the human equivalent, so that recombinants produce mouse V/human C chimeric kappa-chains. We describe an enhancerless, replacement type vector that can be used with the herpes thymidine kinase counterselection to provide such enrichment that homologous recombinants constitute 15% of the selected G418-resistant, FIAU-resistant cells. We have also measured the level of chimeric kappa gene expression and found surprisingly that (1) it is very variable among transformants with the same recombinant gene structure, (2) there is no systematic difference in the level of production by recombinants that retain or have lost the J-C kappa intron enhancer, and (3) the amount of chimeric kappa mRNA in even the highest producing transformants is much less than the amount of the corresponding mouse kappa mRNA.

Animals↗
PubMed1993

Mapping of ligand binding sites of neuronal nicotinic acetylcholine receptors using chimeric alpha subunits.

We constructed a series of chimeric neuronal nicotinic acetylcholine (ACh) receptor (nAChR) alpha subunits to map the location of amino acid residues that determine the pharmacological properties of these receptors. The alpha 2 and alpha 3 subunits form pharmacologically distinct nAChRs upon expression, in combination with the beta 2 subunit, in Xenopus oocytes. The alpha 2 beta 2 subunit combination is insensitive to the nicotinic antagonist neuronal bungarotoxin (NBT) and is much more sensitive to nicotine than to ACh. In contrast, the alpha 3 beta 2 subunit combination is potently inhibited by NBT and is much less sensitive to nicotine than to ACh. Chimeric subunits were constructed by replacing portions of alpha 2 or alpha 3 with the analogous portion of the other alpha subunit. Pharmacological analysis of receptors formed by these chimeric subunits, in combination with beta 2, revealed that amino acid residues involved in determining NBT sensitivity were located within sequence segments 84-121, 121-181, and 195-215. Amino acid residues that determine agonist sensitivity were located within sequence segments 1-84 and 195-215. Within region 195-215, we used site-directed mutagenesis to demonstrate the importance of Gln-198 of alpha 3 (proline in alpha 2) in determining both the antagonist sensitivity and the agonist sensitivity of neuronal nAChRs.

Acetylcholine↗