If it looks like a clone and acts like a clone, is it not a clone?
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Bioethics↗
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Cloning name games: therapeutic cloning is still cloning.
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Beginning of Human Life↗
I, clone. The Three Laws of Cloning will protect clones and advance science.
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Cloning, Organism↗
Cloning Californians? Report of the California Advisory Committee on Human Cloning and recent cloning-related legislation.
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Advisory Committees↗
Cloning's slippery slope: how embryonic cloning leads to human cloning.
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Animals↗
Determinants of transmission success of individual clones from mixed-clone infections of the rodent malaria parasite, Plasmodium chabaudi.
Interactions between malaria parasite clones within mixed infections can have a profound effect on transmission and therefore the epidemiology of the disease. However, factors which determine the relative transmission success of individual clones from mixed infections are unknown. We have used two clones of the rodent malaria Plasmodium chabaudi to investigate changes in the clonal composition of asexual parasites over the course of mixed-clone infections in mice and how these relate to the clonal composition of transmission (oocyst) populations in mosquitoes. Clonal composition was determined using monoclonal antibody analyses for the asexual blood stage populations and PCR analysis of single oocysts for the transmission populations in mosquitoes. The relative frequency of the two clones changed dramatically during the course of the infection in mice, depending on their ratio in the inoculum. The clonal composition of parasites within mosquitoes most closely resembled that in the asexual infection at the time of transmission rather than that at any point earlier in the infection. These results provide no evidence that clones increase rates of gametocytogenesis in response to competitive suppression. Most likely, transmission success follows from asexual success in the later parts of the infection. The clone which dominated the earlier part of the infection, when most parasites are produced, did not necessarily dominate the transmission from the infection. The two clones differed in competitive ability and the data suggest that interactions with the host immune system may be a major factor in determining transmission success from mixed-clone infections.
Animals↗
Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by TH1 clones.
We have previously shown that at least two types of Lyt-1+, Lyt-2-, L3T4+ helper T cell clones can be distinguished in vitro by different patterns of lymphokine secretion and by different forms of B cell help. Evidence is presented here to show that one type of helper T cell clone (TH1) causes delayed-type hypersensitivity (DTH) when injected with the appropriate antigen into the footpads of naive mice. The antigen-specific, major histocompatability complex (MHC)-restricted footpad swelling reaction peaked at approximately 24 hr. Footpad swelling was induced by all TH1 clones tested so far, including clones specific for soluble, particulate, or allogeneic antigens. In contrast, local transfer of TH2 cells and antigen did not produce a DTH reaction, even when supplemented with syngeneic spleen accessory cells. Similarly, local transfer of an alloreactive cytotoxic T lymphocyte clone into appropriate recipients did not produce DTH. The requirements for the DTH reaction induced by TH1 cells were investigated further by using TH1 clones with dual specificity for both foreign antigens and M1s antigens. Although these clones responded in vitro to either antigen + syngeneic presenting cells, or M1s disparate spleen cells, they responded in vivo only to antigen + MHC and did not cause footpad swelling in an M1s-disparate mouse in the absence of antigen. Moreover, in vitro preactivation of TH1 or TH2 cells with the lectin concanavalin A was insufficient to induce DTH reactions upon subsequent injection into footpads. From these results, we conclude that the lack of DTH given by TH2 clones in vivo could be due to the inability of the TH2 cells to produce the correct mediators of DTH, or to a lack of stimulation of TH2 clones in the footpad environment.
Animals↗
Cloning of an unidentified T-cell receptor alpha chain gene from a vesicular stomatitis virus-specific helper T-cell clone.
A vesicular stomatitis virus glycoprotein-specific, class II restricted, CD4+ T-cell clone was obtained and the unidentified T-cell receptor alpha chain cloned in order to establish a T-cell receptor (TCR) alpha chain transgenic mouse line. Polymerase chain reaction (PCR) strategies have been developed to clone TCR chain genes starting from T-cell cDNA. There remain difficulties, however, in cloning the functional TCR alpha chain due to the complexity of the protocols applied if the variable (V) alpha region rearranged is not known. The strategy described here allows the identification and cloning of alpha chains that are not recognized by any of the anti-V alpha monoclonal antibodies available: three 5' consensus V alpha primers designed from all known V alpha gene sequences and a primer specific for the constant (C) alpha region were used and the PCR product sequenced. The T-cell clone displayed two alpha gene rearrangements, only one of which giving rise to a functional protein. The alpha chain used by the T-cell clone contained a V alpha 3.1 and a J alpha region which has been described only at the genomic level, but never in a functional TCR. The complete alpha chain gene was cloned by enriching the alpha chain-encoding cDNA by ligation-anchored PCR and using an alpha specific primer pair. The use of the present method, even if the sequence of the 5' untranslated (UT) region of the alpha chain is not known, is also discussed.
Amino Acid Sequence↗
Cloning of a fresh isolate of Plasmodium falciparum and drug sensitivity of the clones.
A freshly isolated strain of Plasmodium falciparum was cloned by limited dilution using a co-culture of infected erythrocytes on monolayers of functionally active rodent hepatocytes. 15 clones were isolated, and the anti-malarial activity of chloroquine, quinine, mefloquine and halofantrine against the clones, the original isolate, and a culture-adapted isolate was determined using a 48 h radioisotope microdilution method. The multiplication rates of all clones and the culture-adapted isolate were estimated by counting the number of parasitized cells on Giemsa-stained thin smears. Variations found in drug sensitivity and multiplication rate of different clones provided strong evidence of heterogeneity of a single strain parasite population. No morphological variation was detected by light microscopy. The use of a hepatocyte feeder layer improved the adaptation of cloned parasites to continuous culture conditions and thus enabled us to clone directly a fresh isolate, without losing clones during the culture-adaptation process.
Animals↗
Thyroid-specific T cells in the normal Wistar rat. II. T cell clones interact with cloned wistar rat thyroid cells and provide direct evidence for autoantigen presentation by thyroid epithelial cells.
Strains of rat differ in their susceptibility to experimental autoimmune thyroiditis (EAT). We recently observed that the normal Wistar rat has lymph node (LN) T cells which recognize the newly available cloned Wistar thyroid cell line (WRT) and/or rat thyroglobulin (rTg). We have now cloned thyroid-specific T cells and characterized their interaction with the WRT target cell. Twenty-three T cell clones were tested for their reactivity to syngeneic thymocytes, WRT cells alone, or WRT cells with thymocytes. All the clones were of the CD4+CD8- phenotype. Seven of 23 T cell clones proliferated in the presence of WRT cells alone or with the combination of WRT cells and thymocytes, exhibiting stimulation indices of 1.5 to 5. In all but one of the T cell clones responding to WRT cells alone was there no evidence that the additional presence of thymocytes supplied a stronger "second" proliferative signal than the WRT cells. These WRT-reactive clones which were able to be more extensively characterized were MHC class II restricted, secreted rat interferon (IFN)-gamma in response to WRT cell exposure, and one clone showed cross-reactivity with rTg antigen. Induction of WRT cell MHC class II antigen by prior treatment with IFN-gamma failed to further enhance the WRT cell-induced T cell proliferation. These data provide the first evidence for direct antigen presentation by thyroid epithelial cells (TECs) in the absence of other antigen-presenting cells. Furthermore, they provide evidence that TECs are able to provide the appropriate "second" signals required for T cell activation and successful autoantigen presentation.
Animals↗
Functional cloning vectors for use in directional cDNA cloning using cohesive ends produced with T4 DNA polymerase.
This paper describes the construction of 'Prime' cloning vectors, which include phage lambda and plasmid vectors useful for functional cloning in oocytes, yeast, and mammalian cells, and their use in a 'Prime' cloning system. The system takes advantage of the very active and precise 3' exonuclease activity of T4 DNA polymerase to produce single-stranded (ss) ends (cut-back) of vector and insert DNA. This results in the highly efficient directional cloning of cDNA and PCR-amplified DNA. The system obviates the need to digest insert DNA with a restriction endonuclease to unveil cloning sites, and thus eliminates the chance of internal digestion of the insert DNA. The cloning of PCR-amplified DNA, which is sometimes difficult, is made routine with this system. The 'Prime' sequence is included in vector cloning sites and cDNA and PCR primers. The 'Prime' sequence was chosen so that the ss sticky ends are nonpalindromic and will hybridize only to the appropriate partners. This makes cloning with the 'Prime' system very efficient, because neither the vector nor insert DNA is lost to unproductive self-hybridization.
Bacteriophage lambda↗
Cloning of a functional Salmonella SPI-1 type III secretion system and development of a method to create mutations and epitope fusions in the cloned genes.
Bacterial type III secretion systems have significant potential to be harnessed for beneficial purposes including vaccine development, anti-cancer therapies, strategies to counteract harmful bacteria-host interactions, and evolutionary studies. The ability to clone and manipulate type III secretion systems would allow researchers to perform novel experiments that would progress the biotechnological development of the potentially positive uses of these systems. Here, we report the cloning of the entire Salmonella pathogenicity island 1 (SPI-1) type III secretion system on a single DNA fragment that is contained on a self-transmissible plasmid vector for convenient transfer to alternate hosts. We demonstrate that the cloned SPI-1 type III system is functional for secretion and translocation via complementation of an S. typhimurium Delta SPI-1 strain. We also present a convenient method to construct mutations and epitope fusions in the cloned type III genes and demonstrate that the engineered substrate protein fusions are recognized by the cloned type III system. We transferred the cloned SPI-1 type III system into bacterial strains of different genera and found that there is a SPI-1 gene expression defect in these strains. The results describe a novel strategy for cloning and manipulation of bacterial secretion system gene clusters and provide a foundation for future studies to develop the beneficial uses of cloned type III secretion systems.
Bacterial Proteins↗
Limitations of serial cloning in mammals: unresolved donor-cell genomic integrity challenges broad claims of cloning limits.
Wakayama et al. describe an extraordinary 20-year serial cloning study in mice, concluding that serial cloning in mammals is ultimately limited by the accumulation of genetic anomalies. However, their whole-genome sequencing (WGS) analysis characterized selected cloned animals but did not include matched genomic profiling of the corresponding cumulus cell (CC)-donor mice, the source CC populations, or developmental stages. Because each reconstructed embryo originated from a single CC nucleus and re-cloned animals were used to advance the lineage, pre-existing somatic variation could have entered the lineage and subsequently been propagated. Consequently, variants detected in later generations cannot be assigned definitively to pre-existing donor-cell mosaicism, donor-cell handling, somatic cell nuclear transfer manipulation, or early embryogenesis. Thus, the observed decline cannot be attributed exclusively to genetic lesions arising during repeated cloning, but the unresolved genomic status of the lineage-founding donor cells remains a plausible but unproven contributor. The study therefore demonstrates the transmission and propagation of genetic lesions through serial cloning more directly than it establishes that all initiating lesions arose because of repeated cloning. Paired genomic profiling of donor-cell populations, embryos, and offspring would help resolve the origins of accumulated genetic lesions and determine whether donor-cell screening could extend serial cloning.
Animals↗
Functional heterogeneity in allospecific cytotoxic T lymphocyte clones. I. CTL clones express strong anti-self suppressive activity.
Five out of five allo-specific cytotoxic T lymphocyte (CTL) clones tested strongly suppressed the development of CTLs directed against the H-2 haplotype of the CTL clone and independent of the H-2 specificity recognized by the CTL clone. This was shown by including 100-1,000 cells from the five clones in one way mixed lymphocyte reaction (MLR) cultures in which the stimulator cells were of the same H-2 type as the CTL cells. When these cultures were assayed for cytotoxicity against the stimulator cell haplotype, the cytotoxic activity was decreased in a CTL cell dose-dependent manner by 50 to more than 90%. Suppression was usually not observed in MLR cultures where the CTL-H-2 type was identical with the responder cells or was different from both the responder or stimulator cells. Suppression was demonstrated not to be due to "cold" target inhibition at the time of cytotoxicity assay. Even if the added CTL were completely removed after 48-72 h of culture, significant suppression was obtained. Suppressive ability did not appear to be correlated with the level of allo-specific cytotoxic activity present in the CTL clones, but might involve direct killing of MLR precursor cells by cells in the added CTL clones. The suppression observed here, which is anti-self from the point of view of the added CTL clone, appears to be triggered by precursor cells in the MLR responder population recognizing MHC determinants on cells from the added CTL clone. This peculiar type of suppression, in which the regulator regulates on being recognized, has been christened the veto phenomenon and may play a role in maintenance of self tolerance.
Animals↗
Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies.
Lymphokine synthesis patterns of a panel of 19 T cell clones have been evaluated, using mRNA hybridization methods to examine 11 different mRNAs induced by Con A. The two types of CD4+ Th cell clone described previously were clearly distinguished by this procedure, and the differences between the two types have now been extended to six induced products. With minor exceptions, only Th1 clones synthesized mRNA for IL-2, IFN-gamma, and lymphotoxin, and only Th2 clones synthesized mRNA for IL-4, IL-5, and another induced gene, P600. Four more induced products were expressed preferentially but not uniquely by one or another type of clone: mRNAs for GM-CSF, TNF, and another induced, secreted product (TY5) were produced in larger amounts by Th1 clones, whereas preproenkephalin was preferentially expressed by Th2 clones. IL-3 was produced in similar amounts by both types of clone. mAbs were used to establish three bioassays that were functionally monospecific for IL-2, IL-3, and IL-4, and a new anti-IFN gamma mAb, XMG1.2, was used to establish an ELISA for IFN-gamma. These four assays were used to show that secreted protein and mRNA levels correlated well for all cell lines. The implications of these findings for normal T cells are discussed.
Animals↗
Helper activity for immunoglobulin synthesis of T helper type 1 (Th1) and Th2 human T cell clones: the help of Th1 clones is limited by their cytolytic capacity.
A large number of CD4+ human T helper type 1 (Th1) clones specific for purified protein derivative and of Th2 clones specific for the excretory/secretory antigen of Toxocara canis, derived from the same individuals, were analyzed for both cytotoxic capacity and helper function for immunoglobulin (Ig) synthesis. The great majority of Th1, but only a minority of Th2 clones exhibited cytolytic activity. All Th2 (noncytolytic) clones induced IgM, IgG, IgA, and IgE synthesis by autologous B cells in the presence of the specific antigen, and the degree of response was proportional to the number of Th2 cells added to B cells. Under the same experimental conditions, Th1 (cytolytic) clones provided helper function for IgM, IgG, and IgA, but not IgE, synthesis with a peak response at 1:1 T/B cell ratio. At higher T/B cell ratios, a strong decrease of Ig synthesis was observed. All Th1 clones lysed Epstein-Barr virus transformed autologous B cells pulsed with the specific antigen. The decrease of Ig production at high T/B cell ratios correlated with the lytic activity of Th1 clones against autologous antigen-presenting B cell targets. These data suggest that Th1 differ from Th2 human T cell clones not only for their profile of cytokine secretion, but also for cytolytic potential and mode of help for B cell Ig synthesis.
Antibody Formation↗
T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2-restricted and melanocyte-lineage-specific CTL clone.
HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA-A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth.
Amino Acid Sequence↗
Molecular cloning of alpha-amylase genes from Drosophila melanogaster. I. Clone isolation by use of a mouse probe.
A cloned alpha-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone lambda Dm32, representing class A, hybridizes within chromosome section 53CD. Clone lambda Dm65 of class B hybridizes within section 54A1-B1. Clone lambda Dm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for alpha-amylase. No RNA homologous to lambda Dm32 was detected. We suggest that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.
Animals↗