Pure Clones Research Articles

Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus

Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety.

Evidence of intra-varietal genetic variability in the vegetatively propagated crop oca (Oxalis tuberosa Mol.) in the Andean traditional farming system

Oxalis tuberosa is a vegetatively propagated tuber crop in the Andes. The peasants cultivate a great number of varieties for which genetic homogeneity has never been demonstrated. Morphological descriptors and ISSR markers were used to determine the intra-varietal diversity and the influence of the mode of conservation ex-situ vs in-situ. Molecular markers revealed an intra-varietal genetic diversity attesting that oca varieties are not pure clones. The morphological analysis was congruent with the peasant classification, contrary to the molecular markers. The comparison between both conservation strategies revealed a larger intra-varietal diversity in in-situ conditions and a genetic divergence between plants. The traditional practices are likely to be responsible of the intra-varietal polymorphism since the oca is propagated almost exclusively vegetatively. At the conservation level, differences could be explained by the sampling methods. A more integrated approach between genebanks and in-situ conservation is recommended to maintain the genetic resources of the species.

RNAi-dependent and -independent antiviral phenotypes of chromosomally integrated shRNA clones: Role of VASP in respiratory syncytial virus growth

Stable RNA interference (RNAi) is commonly achieved by recombinant expression of short hairpin RNA (shRNA). To generate virus-resistant cell lines, we cloned a shRNA cassette against the phosphoprotein gene of respiratory syncytial virus (RSV) into a polIII-driven plasmid vector. Analysis of individual stable transfectants showed a spectrum of RSV resistance correlating with the levels of shRNA expressed from different chromosomal locations. Interestingly, resistance in a minority of clones was due to mono-allelic disruption of the cellular gene for vasodilator-stimulated phosphoprotein (VASP). Thus, pure clones of chromosomally integrated DNA-directed RNAi can exhibit gene disruption phenotypes resembling but unrelated to RNAi.

Catalytic Site Modifications of TAP1 and TAP2 and Their Functional Consequences

The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.

High content selection of cells

Conventional methods for clonal analysis are mostly based on limiting dilution or FACS sorting. Purity of clones can only be verified retrospectively. This process is tedious and time-consuming. The Elektra™ improves and expedites the cell cloning process using IACS™ (Image Activated Cell Selection) for isolation of clonal cells providing high-content single cell information.A micro fluidic Sorter Chip within the Elektra™ uses CellProcessor technology, i. e. negative dielectrophoretic (nDEP) force to guide, cage and sort cells (about 500 to 1000 cells per run). The system is equipped with a highly sensitive CCD camera and a 40× lens. Cells are analyzed on-line and selected according to phase contrast, fluorescence and size while passing the micro device. Target cells are trapped and an image series can be acquired. Post process evaluation of the images enables quantitative analysis of e. g. subcellular fluorescence distribution and co-localization. Elektra™ produces micro titer plates containing single viable and 100% pure clones without the need for multiple iterations. This is demonstrated for different cell lines (e. g. CHO, U2-OS, hybridoma cells).The technology is shown to be compatible to label free detection techniques like impedance, electrorotation and dielectrophoretic cell deformation.

Genetic conflicts and intercellular heterogeneity

Genetic conflicts and intercellular heterogeneity

Molecular identification of distinct neurogenic and melanogenic neural crest sublineages.

Clonal and lineage analyses have demonstrated that although some neural crest cells have the ability to generate multiple cell types and display self-renewal ability, other crest cells generate a single or limited repertoire of cell types. However, it is not yet clear when, and in what order, crest cells become specified to adopt a particular fate. We report that the receptor tyrosine kinases TrkC and C-Kit are expressed by distinct neural crest subpopulations in vitro. We then analyzed the lineages of individual receptor-expressing crest cells and found that TrkC-expressing cells that have just emerged from the neural tube give rise to clones containing neurons or glial cells, or both, but never produce melanocytes. A short time later, TrkC-expressing cells only generate pure neuronal clones. By contrast, from their earliest appearance in neural tube outgrowths, C-Kit-expressing cells invariably give rise to clones containing only melanocytes. Our results directly demonstrate that distinct neurogenic and melanogenic sublineages diverge before or soon after crest cells emerge from the neural tube, that fate-restricted precursors are present in nascent neural crest populations and that these sublineages can be distinguished by their cell type-specific expression of receptor tyrosine kinases.

Functional characterization of three clones of the human intestinal Caco-2 cell line for dietary lipid processing

We aimed to improve the use of the human intestinal Caco-2 cell line for studying dietary lipid and cholesterol processing by using isolated pure clones (Chantret et al. 1994). Three clones (TC7, PD7 and PF11) were grown as monolayers on semi-permeable filters and compared for cell viability, fatty acid and cholesterol apical uptake or basolateral secretion, apolipoprotein B-48 basolateral secretion and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity. The TC7 clone showed the best viability upon apical incubation with mixed micelles and should be preferred for routine work. Short-term (3.0 h) rates of apical uptake of cholesterol were not different with the three clones, whereas the rate of apical uptake of oleic acid (18:1) was lower (P<0.05) with PF11 (250.6 nmol/mg) and the basolateral secretion of cholesterol and oleic acid was lower with the TC7 clone (0.40 and 29.1 nmol/mg respectively). The secretion of apolipoprotein B-48 basolaterally was about 2-fold lower than from PD7 clone. The basal levels of HMG-CoA reductase activity were significantly different (P<0.05; TC7>PF11 >PD7). The down-regulation of the enzyme activity was moderate (range 13.8-21.0%) and comparable in the presence of apical micellar cholesterol, but was much marked upon basolateral incubation with LDL (range 34.0-53.6%), especially for the PD7 clone. In conclusion, the Caco-2 clones characterized here proved to be particularly suitable for studying lipid nutrients processing. Because these three clones exhibit some different metabolic capabilities, they provide a new tool to study intestinal response to lipid nutrients.

Functional characterization of three clones of the human intestinal Caco-2 cell line for dietary lipid processing

We aimed to improve the use of the human intestinal Caco-2 cell line for studying dietary lipid and cholesterol processing by using isolated pure clones (). Three clones (TC7, PD7 and PF11) were grown as monolayers on semi-permeable filters and compared for cell viability, fatty acid and cholesterol apical uptake or basolateral secretion, apolipoprotein B-48 basolateral secretion and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity. The TC7 clone showed the best viability upon apical incubation with mixed micelles and should be preferred for routine work. Short-term (3·0 h) rates of apical uptake of cholesterol were not different with the three clones, whereas the rate of apical uptake of oleic acid (18 : 1) was lower (P&lt;0·05) with PF11 (250·6 nmol/mg) and the basolateral secretion of cholesterol and oleic acid was lower with the TC7 clone (0·40 and 29·1 nmol/mg respectively). The secretion of apolipoprotein B-48 basolaterally was about 2-fold lower than from PD7 clone. The basal levels of HMG-CoA reductase activity were significantly different (P&lt;0·05; TC7&gt;PF11&gt;PD7). The down-regulation of the enzyme activity was moderate (range 13·8–21·0 %) and comparable in the presence of apical micellar cholesterol, but was much marked upon basolateral incubation with LDL (range 34·0–53·6 %), especially for the PD7 clone. In conclusion, the Caco-2 clones characterized here proved to be particularly suitable for studying lipid nutrients processing. Because these three clones exhibit some different metabolic capabilities, they provide a new tool to study intestinal response to lipid nutrients.

Genetic conflict and conditional altruism in social aphid colonies.

Although kin selection is central to the modern study of social evolution, recent studies of social species have revealed that no simple relationship exists between levels of kinship and sociality. The soldier-producing aphids are unique among highly social animals because, barring movement by aphids between colonies, they occur in clonal groups of genetically identical individuals. Potentially, clonality simplifies efforts to understand social evolution in aphids by obviating issues of intragroup conflict. However, we report here high levels of clonal mixing and conflict in an aphid society. The gall-dwelling colonies of a social aphid species (Pemphigus obesinymphae) are not pure clones, but are invaded by large numbers of aphids from other clones. Intruders behave and develop selfishly once they have invaded a colony of nonkin. They refrain from risky defensive behaviors and accelerate their own development into reproductive rather than defensive stages. This conditionality in the social life of P. obesinymphae reveals complex dynamics and a degree of behavioral plasticity not previously known in aphid societies.

Plasmodium falciparum: Gene Mutations and Amplification of Dihydrofolate Reductase Genes in Parasites Grown in Vitro in Presence of Pyrimethamine

Thaithong, S., Ranford-Cartwright, L. C., Siripoon, N., Harnyuttanakorn, P., Kanchanakhan, N. S., Seugorn, A., Rungsihirunrat, K., Cravo, P. V. L., and Beale, G. H. 2001. Plasmodium falciparum: Gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of pyrimethamine. Experimental Parasitology98, 59–70. Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum were grown for periods of 22–46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold increases in resistance to the drug. With clone T9/94RC17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pure clones, all having increased resistance to pyrimethamine, and these sequences were compared with the sequence of the original pyrimethamine-sensitive clone. No changes in amino acid sequence were found to have occurred. Some resistant clones obtained by this method were then examined by pulsed-field gel electrophoresis, and the results indicated that there had been an increase in the size of chromosome 4. This was confirmed by hybridization of Southern blots with a chromosome 4-specific probe, the vacuolar ATPase subunit B gene, and a probe to DHFR. Dot-blotting with an oligonucleotide probe to DHFR confirmed that there had been increases up to 44-fold in copy number of the DHFR gene in the resistant strains. Resistant clones obtained by this procedure were then grown in medium lacking pyrimethamine for a period of nearly 2 years, and reversion nearly to the level of pyrimethamine sensitivity of the original clone T9/94RC17 was found to occur after about 16 months. Correspondingly, the chromosome 4 of the reverted population reverted to a size like that of the original sensitive clone T9/94RC17. The procedure of growing parasites in stepwise increases of pyrimethamine concentration was repeated with two other pyrimethamine-sensitive clones: TM4CB8-2.2.3 and G112CB1.1. (The DHFR gene of these clones encodes serine at position 108, in place of threonine as in clone T9/94RC17, and it was thought that this difference might conceivably affect the rate of mutation to asparagine at this position). Clones TM4CB8-2.2.3 and G112CB1.1 also responded by developing gradually increased resistance to pyrimethamine. However, in clone TM4CB8-2.2.3 a single mutation from Ile to Met at position 164 in the DHFR gene sequence was identified, and in clone G112CB1.1 there was a single mutation from Ala to Ser at position 16, but no mutations at position 108 were obtained in any of the clones studied here. In addition, chromosome 4 of clone TM4CB8-2.2.3 increased in size, presumably due to amplification of the DHFR gene. No increase in size was seen in clone G112CB1.1. We conclude that whereas some mutations producing changes in the amino acid sequence of the DHFR molecule may occur occasionally in clones or populations of P. falciparum grown in vitro in the presence of pyrimethamine, amplification of the DHFR gene following adaptation to growth in medium containing pyrimethamine occurs as a regular feature. The bearing of these findings on the development of pyrimethamine-resistant forms of malaria parasites in endemic areas is discussed.

Identification and Characterization of a Novel Family of Cyclic Nucleotide Phosphodiesterases

We report the cloning, expression, and characterization of a new family of cyclic nucleotide phosphodiesterase (PDE) that has unique kinetic and inhibitor specificities. A clone corresponding to the C terminus of this PDE was initially identified by a bioinformatic approach and used to isolate a cDNA that is likely full-length. This novel PDE, designated as MMPDE9A1, shows highest mRNA expression in kidney with lower levels in liver, lung, and brain. The mRNA size by Northern blot analysis is approximately 2.0 kilobases, and the cDNA encoding PDE9A1 is 1929 base pairs in length. The largest open reading frame predicts a protein of 534 amino acids with a molecular mass of 62,000 Da. When expressed in COS-7 cells, PDE9A1 activity was not inhibited well by either the nonselective inhibitor 3-isobutyl-1-methyl-xanthine or the new selective PDE5 inhibitor, sildenafil, but it is inhibited by the PDE1/5 inhibitor (+)-cis-5,6a, 7,8,9 hyl] phenylmethyl]-5-methyl-cylopent[4,5]imidao[2, 1-b]purin-49(3H)one (SCH51866) with an IC50 of 1.55 microM. This new phosphodiesterase is highly specific for cGMP. Its Km of approximately 0.07 microM for cGMP is the lowest yet reported for a PDE, being at least 40-170 times lower than that of PDE5 and PDE6, respectively.

Variability in schizonticidal drug susceptibility amongst clones and isolates of Plasmodium falciparum.

Plasmodium falciparum isolates from Malaysia, Africa and Thailand were cultured in vitro following the method of Trager and Jensen and subsequently cloned using the limiting dilution method of Rosario. These clones were presently characterized against three schizonticidal drugs, chloroquine, mefloquine and quinine, using the modified in vitro microtechnique. Results showed that all the clones derived from Gombak A isolate were chloroquine-resistant with average IC50 values ranging at 0.1377-1.0420 microM (0.007-0.058 mefloquine activity), sensitive to mefloquine at 0.0032-0.0103 microM and quinine at 0.0025-0.0428 microM (0.075-3.080 mefloquine activity). Similarly, the TGR clone displayed resistance to chloroquine at 0.1715-0.5875 microM (0.002-0.029 mefloquine activity) but were also sensitive to mefloquine at 0.0008-0.0058 microM and quinine at 0.0055-0.0700 microM (0.055-0.202 mefloquine activity). In contrast, four out of six Gambian clones were sensitive to chloroquine at 0.0047-0.0172 microM (0.122-0.617 mefloquine activity) but all were sensitive to mefloquine at 0.0008-0.0029 and 0.0016-0.0102 microM (0.096-1.813 mefloquine activity). In general, most of the clones displayed susceptibility patterns similar to that of their parent isolates against the three schizonticidal drugs except Gm/B2 and Gm/H5 Gambian clones were chloroquine-resistant at 0.3427 microM (0.006 mefloquine activity) and 0.2260 microM (0.004 mefloquine activity), respectively. Further results indicated that they were pure clones compared to their parent isolates as their schizonticidal drug susceptibilities were statistically different (p < 0.05) except Gm/C6 and TGR/B7 clones against mefloquine (p < 0.05).

Genetics of Cronartium ribicola. I. Axenic culture of haploid clones

Genetically pure lines of the mononucleate (haploid) stage of Cronartium ribicola can be grown by isolating mycelium from single spots on infected white pine needles or by culturing single basidiospores on defined media preconditioned by cell-free diffusates of massed germinating basidiospores. Although growth is initially slow, cultures can be increased rapidly by alternately subculturing from solid to liquid media and back again indefinitely. The ability to culture pure haploid clones has important implications for studying the genetic structure and sexual behavior of rust populations and the nature of virulence. Keywords: white pine blister rust, sugar pine, virulence.

Electrophoretic variations of enzyme, GDH (NADP-dependent glutamate dehydrogenase)(EC. 1.4.1.4) in characterizing clones and isolates of Malaysian Plasmodium falciparum.

Malaysian, African and Thai Plasmodium falciparum isolates were cultured in vitro by the Trager and Jensen method (1976; 1977) and were later cloned by the limiting dilution method (Rosario, 1981). Forty-eight clones were obtained and were characterized by electrophoretic variations of GDH (NADP-dependent glutamate dehydrogenase)(EC. 1.4.1.4). It was found that they were pure clones because they possessed either GDH-1 or GDH-2 unlike their parent isolates which exhibited both GDH-1 and GDH-2.

In vitro susceptibility studies of Plasmodium falciparum isolates and clones against type II antifolate drugs.

Six clones were derived from each Plasmodium falciparum isolate obtained from Malaysia, Africa and Thailand and were characterized against type II antifolate drugs, cycloguanil and pyrimethamine using the modified in vitro microtechnique. Results showed that these isolates were of a heterogeneous population, with 50% inhibitory concentrations of Gombak A clones at 0.0151-0.1450 and 0.0068-0.1158 microM, Gambian clones at 0.0056-0.1792 and 0.0004-0.0068 microM and TGR clones at 0.0103-0.0703 and 0.0776-0.3205 microM against cycloguanil and pyrimethamine, respectively. All clones displayed similar susceptibilities as their parent isolates except A/D3, A/D5, A/G4 and A/H7 clones which were sensitive to cycloguanil at 0.0735, 0.0151, 0.0540 and 0.0254 microM but Gm/B2 clone was resistant at 0.1792 microM, respectively. However, A/D3, TGR/B4, TGR/B7, TGR/C4, TGR/C7 and TGR/H2 clones were resistant to pyrimethamine at 0.1158, 0.1070, 0.1632, 0.1580, 0.2409 and 0.3205 microM, respectively. Further results indicated that they were pure clones compared to their parent isolates as their drug susceptibility studies were statistically different (p < 0.05).

Characterization and response to interleukin 1 and tumor necrosis factor of immortalized murine biliary epithelial cells

Background & Aims : Biliary epithelial cells are the target of numerous immune-mediated liver diseases, yet their role in pathogenesis remains unclear because of difficulties in obtaining pure preparations. The aim of this study was to establish pure clones of immortalized murine intrahepatic biliary epithelial cells. Methods : The transgenic mouse harboring the SV40 thermosensitive immortalizing mutant gene TsA58 under the control of the major histocompatibility complex class I promoter was used to establish conditionally immortalized intrahepatic bile duct cells by countercurrent centrifugal elutriation and clonal dilution. Results : Immortalized clones of cells expressing cytokeratin 19, which organized themselves into ductlike structures, were obtained. On electron-microscopic sections, cells were well differentiated and polarized. Cells proliferate in response to epidermal growth factor, interleukin 1α, and tumor necrosis factor α. Using the reverse-transcriptase polymerase chain reaction technique, these cells were found to contain messenger RNA, which encodes for the interleukin 1 and tumor necrosis factor receptors. Conclusions : The availability of unlimited numbers of pure bile duct cells that behave in an identical fashion to biliary epithelial cells from “normal” mice will allow for more rigorous studies of the behavior and function of this epithelium.

Myxoma virus and shope fibroma virus encode dual-specificity tyrosine/serine phosphatases which are essential for virus viability

Sequence analysis of the genomes of the Leporipoxviruses myxoma virus and Shope fibroma virus (SFV) led to thediscovery of open reading frames homologous to the vaccinia H1L gene encoding a soluble protein phosphatase with dual tyrosine/serine specificity. These viral phosphatase genes were subsequently localized to the myxoma BamHI-I fragment and the SFV BamHI-M fragment, and the resulting encoded proteins were designated l1L and M1L, respectively. The localization and orientation of the myxoma l1L and SFV M1L open reading frames within the well conserved central core of the viral genomes closely mirror that of the Orthopoxviruses vaccinia virus and variola virus. The myxoma l1L and SFV M1L phosphatases each contain the conserved tyrosine phosphatase signature sequence motif, (I/V)HCXAGXXR(S/T)G, including the active site cysteine, found previously to be essential for phosphotyrosine dephosphorylation. The vaccinia H1L phosphatase was originally shown to have the ability to dephosphorylate phosphotyrosyl and phosphoseryl residues in vitro. To assess whether this is a common feature of poxvirus phosphatases, myxoma 11 L was expressed as a GST-fusion protein, purified, and shown to dephosphorylate substrates containing tyrosine and serine phosphorylated residues, in a similar fashion to vaccinia H1L. A myxoma l1L variant, in which the active site cysteine 110 was mutated to serine, was expressed in a parallel fashion to the wild-type l1L protein and found to be completely deficient in its ability to dephosphorylate both phosphotyrosine and phosphoserine amino acids. In an attempt to ascertain the biological requirement for the myxoma l1L phosphatase, we constructed a recombinant myxoma virus containing a disrupted l1L open reading frame. This l1L mutant virus was able to successfully propagate in tissue culture only in the presence of a wild-type complementing gene, and pure virus clones containing only the disrupted allele were not viable. Thus, we conclude that the myxoma l1L dual specificity phosphatase is an essential factor for virus viability.

Cell lineage of larval and imaginal thoracic anlagen cells of Drosophila melanogaster, as revealed by single-cell transplantations

ABSTRACT We have analyzed the cell lineage of larval and imaginal cells in the thoracic ectoderm of the early embryo of Drosophila melanogaster, by homotopic transplantation of single cells in the region of 50 –60% egg length. Single cells were isolated prior to transplantation in an in vitro solution. The donors were ‘enhancer-trap’ lines in which the nuclei of all larval and imaginal cells exhibit a uniformly intense expression of the lacZ gene of E. coli. The transplantations were carried out from the blastoderm to the early gastrula stage, as a rule immediately after the onset of gastrulation (stage 6). It was found that at this time the cells of the thoracic ectoderm are not yet committed to form larval or imaginal structures, as indicated by the presence of clones overlapping all structures formed by the thoracic ectoderm, i.e. the nervous system, the larval epidermis, the tracheae and the imaginal discs. The average size of pure epidermal clones was five cells. In clones overlapping either other larval tissues or imaginal discs, the average number of epidermal cells was between three and four. The mean relative clone size was 1/5 of the size of the total structure for leg imaginal discs and 1/7 for the wing imaginal disc. We therefore infer that the precursors for the leg discs and wing disc on one side together number 22 cells in the blastoderm or early gastrula stage. These cells eventually give rise not only to precursors of the imaginal discs but usually also to larval epidermal and nervous-system cells, because most of the imaginal disc clones (80%) overlap larval tissue. The transplantations were not precisely homotopic; the fact that up to 10 cells were removed from the donor essentially rules out exact homotopy between donor and host sites, because a segment anlage is only about three cells wide. Nevertheless, the clones developed completely normal tissue together with the recipient cells. Although the clones have the capacity to extend over different ectodermal tissues and can include both imaginal discs in a given segment, no clones were found that clearly crossed larval or imaginal segment boundaries. We propose a model in which the segregation of the cells that are to differentiate into the imaginal tissues does not occur until the second postblastodermal mitosis

Modulation of spontaneous outgrowth of Epstein-Barr virus immortalized B-cell clones by granulocyte-macrophage colony-stimulating factor and interleukin-3

Spontaneous outgrowth of immortalized Epstein-Barr virus (EBV) infected B-cell clones will occur from cultures of peripheral blood mononuclear cells (PBMNCs) of some persons with a history of EBV infection. We determined that outgrowth of such clones may be reproducibly modulated by supplementation of cultures with the hematopoietic growth factors GM- CSF and interleukin-3 (IL-3). Continuous supplementation of cultures with GM-CSF facilitates emergence of immortalized B-cell clones, whereas supplementation with IL-3 completely prevents their emergence. The effect of GM-CSF may be direct, at least in part, as the proliferation of pure clones of EBV-transformed B lymphocytes was augmented in response to GM-CSF. An indirect mechanism appears to be responsible for the inhibition of transformed B-cell outgrowth in response to IL-3, as IL-3 had no inhibitory effect on proliferation of pure transformed B-cell clones and IL-3-mediated inhibition could be eliminated by antibody neutralization of gamma-interferon (gamma-IFN) or tumor necrosis factor-alpha (TNF-alpha) early in culture. The mechanisms of these effects deserve further study and may have clinical relevance to use of hematopoietic growth factors for support of bone marrow (BM) function in immunocompromised patients.