Cell Fusion Experiments Research Articles

Biological Cell Processing Using Laser Beams.

This paper describes precise processing techniques of a single biological cell by using laser beams. A laser beam is focused to the order of its wavelength on the cell membrane or the microorganisms. This non-contact and precise biological cell micro-processing technique is mainly used for the injection of foreign substances such as DNA into cells and for cell fusion. In particular the laser induced cell fusion method has several advantages over other conventional fusion techniques. Our recent results of cell fusion experiments including all optical schemes in-corporating with optical trapping are reported and discussed.

Characterization of Chinese hamster ovary cells that are resistant to 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibition of low density lipoprotein-derived cholesterol metabolism.

The pharmacological agent U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibits the intracellular transport of low density lipoprotein (LDL)-derived cholesterol in Chinese hamster ovary (CHO) cells. LDL-derived cholesterol accumulates in the lysosomes of U18666A-treated cells causing delayed LDL-mediated regulation of cellular cholesterol metabolism and impaired movement of LDL-derived cholesterol to other cell membranes. As a result of impaired LDL-derived cholesterol transport, LDL-dependent growth of CHO cells is also inhibited by U18666A. By selecting for cell growth in the presence of U18666A, we have identified a CHO cell line, designated U18R, that is resistant to U18666A-inhibition of LDL-derived cholesterol trafficking. When compared to parental CHO cells, U18R cells are relatively resistant to U18666A inhibition of LDL-derived cholesterol transport as well as LDL-mediated regulation of cellular cholesterol metabolism. In cell fusion experiments, the U18666A resistance observed in U18R cells displays a dominant phenotype. Identification of the U18666A-resistant factor may provide important insights toward the understanding of intracellular LDL-derived cholesterol regulation and trafficking.

Comparative behavior of lysosomes and the pre-lysosome compartment (PLC) in in vivo cell fusion experiments

Interspecies cell fusion was used to compare protein intermixing within the mannose 6-phosphate receptor (MPR)-enriched pre-lysosome compartment (PLC) and within the MPR-negative lysosomal compartment. Both compartments were positive for lysosomal glycoprotein (lgp) membrane markers but were morphologically distinct. In most experiments, rat-mouse cell syncytia were formed by u.v.-inactivated Sindbis virus-mediated fusion. By immunogold electron microscopy of syncytia, extensive intermixing of species-specific lysosomal membrane proteins was observed in both lysosomes and PLC. At 3 h post cell fusion, multiple-label immunogold studies showed that 82% of the lysosome-like structures positive for the rat lysosomal membrane protein LIMP-I were also positive for the mouse lysosomal membrane protein mLAMP-1. By immunofluorescence, LIMP-I and mLAMP-1 co-localized with a t1/2 of 30 min after cell fusion; although the lgp-positive organelle populations had evidently interchanged their proteins, the lysosomal structures remained small, punctate bodies distributed throughout the syncytoplasm as observed in single cells. In contrast, the initially separate units of the PLC congregated with a t1/2 of 1 h to form large, pre-lysosome complexes associated with individual nuclear clusters. At the electron-microscope level, gold markers endocytized by the rat and mouse parent cells in a 1 h uptake followed by a 16-20 h chase co-localized in these extended PLC complexes, as did the membrane markers mLAMP-1 and LIMP-I. The density of labeling for rat MPR in the extended PLCs was markedly decreased, consistent with membrane fusions and dilution of the antigen upon congregation of the PLC compartments from the donor cells. The extended PLC complex behaved as a late endocytic compartment, as shown by co-localization of the MPR and rhodamine-dextran following a 10 min dextran uptake and a 50 min chase. These differences in behavior between lysosomes and the PLC in rat-mouse cell syncytia suggest that the pathway(s) of protein intermixing with respect to the two organelles may be different.

Behavior of a transitional tubulovesicular compartment at the cis side of the Golgi apparatus in in vivo fusion studies of mammalian cells

We have investigated the behavior in in vivo cell fusion experiments of a transitional compartment lying between the endoplasmic reticulum and Golgi apparatus to determine if the compartment, as recognized by the antibody G1/93, might congregate in a similar manner to Golgi apparatus [W. C. Ho et al. (1990) Eur. J. Cell Biol. 52, 315–327]. The distributions of the transitional tubulovesicular compartment, endoplasmic reticulum, and Golgi apparatus in HeLa cells were assessed by immunofluorescent staining using mouse monoclonal antibody G1/93, mouse monoclonal antibody HP 24, and rabbit anti-galactosyltransferase, respectively. In agreement with previous results [W. C. Ho et al. (1990) Eur. J. Cell Biol. 52, 315–327], the Golgi apparatus was observed to congregate gradually over a 3- to 6-h period, forming a large, extended, central Golgi complex in uv-inactivated Sindbis virus-fused HeLa cells. Concomitant with this was a marked congregation of the transitional tubulovesicular compartment. Congregation of the tubulovesicular compartment was not affected by cycloheximide. The endoplasmic reticulum retained its web-like distribution throughout the syncytoplasm and rimmed the nuclear periphery. Treatment of HeLa cells with nocodazole prior to fusion followed by incubation of the syncytia in drug-containing media blocked congregation of the G1/93-positive compartment. With this long-term nocodazole treatment, Golgi apparatus was dispersed into scattered Golgi elements and the G1/93 distribution was endoplasmic reticulum-like. These results suggest that the transitional tubulovesicular compartment recognized by G1/93 is normally structured on microtubules and microtubule organizing centers and may be considered to be a subcompartment of a greater, perinuclear, Golgi complex.

Genetic and molecular analysis of the Ah receptor and of Cyp1a1 gene expression

The Ah receptor is a soluble protein complex that mediates carcinogenesis by a wide range of environmental pollutants, including polycyclic aromatic hydrocarbons, heterocyclic amines, and polychlorinated aromatic compounds. The best understood activity of the receptor concerns its role in the induction of cytochrome P450IA1. We undertook a somatic cell genetic analysis of P450IA1 induction using the mouse hepatoma cell line, Hepa-1. Clones of Hepa-1 were isolated that are defective in induction of P450IA1. Evidence was obtained that the clones are mutational in origin. Cell fusion experiments demonstrated that a few of the mutants are dominant, while the majority are recessive. The dominant mutants were shown to synthesize a repressor of P450IA1 transcription. The recessive mutants were assigned to 4 complementation groups (probably corresponding to 4 different genes). Complementation group A corresponds to the P450IA1 structural gene. Mutations in the B, C and D genes all affect functioning of the Ah receptor. A ‘reverse selection procedure’, whereby cells that express P450IA1 inducibility can be selected from a majority population of cells lacking inducibility, was developed. The reverse selection procedure was used to isolate transfectants of representative recessive mutants in which the mutational defects are complemented by exogenously applied genomic DNA. A human DNA-derived transfectant of a C − mutant was used to clone the human C gene. The C gene is not the ligand-binding subunit of the Ah receptor but is a protein that is required for translocation of Ah receptor-ligand complexes from cytoplasm to nucleus. In analogous experiments the dominant gene from one of the dominant mutants was transfected into wild-type Hepa-1 cells. Success in transfecting the dominant gene should provide the means for cloning it.

Identification of two HSP70-related Xenopus oocyte proteins that are capable of recycling across the nuclear envelope.

Two 70-kD polypeptides, B3 and B4, are present in equivalent concentrations in the nucleus and cytoplasm of Xenopus oocytes. The objectives of this study were to determine if they (a) are members of the 70-kD family of heat shock proteins, and (b) recycle between the nuclear and cytoplasmic compartments. Evidence based on high-affinity binding to ATP, cross-reactivity of B3/B4-specific antibodies with rat hsc70, and a comparison of cyanogen bromide cleavage peptide maps with hsc70, verified that B3 and B4 are members of the 70-kD family of heat-shock proteins. Nuclear uptake studies were performed by microinjecting 125I-labeled B3/B4, rat hsc70, and BSA into the cytoplasm of oocytes, and examining their subsequent intracellular distributions. By 6 h postinjection, the nuclear concentration of B3/B4 and hsc70 were approximately 24-fold greater than BSA controls. It was also found that B3/B4-coated gold particles as large as 120A in diameter were able to enter the nucleus by passing through the pores. Nuclear efflux was analyzed by microinjecting the iodinated proteins directly into the oocyte nuclei. 2 h after nuclear injection, at least 46% of the B3/B4 and 60% of the hsc70 were found in the cytoplasmic fractions, compared with less than 10% for the BSA controls. Cell fusion experiments, in which labeled, anucleate oocyte vegetal hemispheres were fused, under oil, with nucleate unlabeled animal hemispheres, demonstrated that cytoplasmic B3 and B4 could enter the nucleus after equilibration was reached, arguing against the existence of separate nuclear and cytoplasmic populations. Collectively, these results show that B3, B4, and rat hsc70 are transported across the nuclear envelope and recycle between the nucleus and cytoplasm.

Temporary complementation of temperature‐sensitive mutants of the cell cycle by transfection with a wild‐type or a mutant cDNA of ADP/ATP translocase

A number of cell-cycle-specific temperature-sensitive (ts) mutants have been isolated from animal cells, especially Syrian hamster cells. These ts mutants, like cell cycle ts mutants of yeast, can be complemented by specific genes, some of which have been molecularly cloned. We have isolated a cDNA clone that complements TK-ts13 cells, but only temporarily. This clone, called B1, differs from a previously isolated clone (Sekiguchi et al.: EMBO Journal 7:1683-1687, 1988) that specifically complements ts13 cells. In addition, B1 also complemented temporarily three other ts mutants of the cell cycle, tsAF8, ts694, and ts550C cells. These mutants have different mutations since, in cell fusion experiments, they complement each other. Sequencing of the B1 cDNA clone revealed that it was a mutant of human ADP/ATP translocase in which some human sequences at the 5' end have been replaced by SV40 sequences. The wild-type translocase was less effective but could still increase the survival time of cell cycle ts mutants at the restrictive temperature. Using the polymerase chain reaction, it was possible to demonstrate that the B1 plasmid is expressed in TK-ts13 cells undergoing temporary complementation.

Use of SV40 transfected human kidney cells in cell fusion experiments

Use of SV40 transfected human kidney cells in cell fusion experiments

Generation of Monoclonal Antibodies to Alpha‐Fetoprotein and Application in Solid‐Phase Enzyme Immunoassay

By using an improved hybridoma technique, monoclonal antibodies specific to alpha-fetoprotein (AFP) were generated. After three subcutaneous immunizations and three intravenous boosters, cell fusion experiments were performed. The hybrid cells were initially cultured in a semisolid medium containing methylcellulose and later transferred to a liquid medium for subculture. Out of 800 colonies recovered after two cell fusion experiments, 16 were shown to exhibit affinity to AFP by radioimmunoassay. Six hybrid cell lines which showed high affinity and specificity were selected for further evaluation. From the results of a cross-matching procedure, two pairs of antibodies (AFP 3 and AFP 05; AFP 3 and AFP 013) reacting with discrete antigenic determinants were identified for preparing solid-phase sandwich enzyme immunoassay (EIA) kits. The association constants between AFP and these three antibodies (AFP 3, AFP 05, and AFP 013) were 2.0, 3.7, and 3.8 X 10(9) M-1, respectively. The immunoglobulin subclass of them was determined to be IgG1. The EIA procedure designed could be performed within 40 min in a one-stage incubation and 70 min in a two-stage incubation. The incubation time was shown to be equal to or shorter than that of any other known commercial kits and the sensitivity was less than 1 IU/ml. In order to avoid the high-dose hook effect which occurred in the one-stage incubation procedure, a two-stage incubation protocol was advised.

Generation of monoclonal antibodies to prostatic acid phosphatase isoenzyme 2 and application in solid-phase enzyme immunoassay.

Monoclonal antibodies specific to prostatic acid phosphatase (PAP) isoenzyme 2 were generated by using an improved hybridoma technique. After three subcutaneous immunizations and three intravenous boosters, cell fusion experiments were performed. The hybrid cells were first cultured in a semisolid medium containing methylcellulose and later transferred to a liquid medium for further subculture. Out of a total of 600 colonies recovered after two cell fusion experiments, 13 were shown to exhibit affinity to PAP isoenzyme 2 by radioimmunoassay. Nine hybrid cell lines which showed high affinity and specificity were established for further evaluation. Their immunoglobulin subclass was determined to be immunoglobulin G. The association constants between PAP isoenzyme 2 and each monoclonal antibody were determined by titration curve in radioimmunoassay (RIA). Three of them (PAP 1, PAP 03, and PAP 019) were shown to be over 1 X 10(9) M-1. From the results of a matrix cross-matching procedure, a pair of antibodies (PAP 03 and PAP 1) reacting with discrete antigenic determinants were identified for preparing a solid phase sandwich enzyme immunoassay (EIA) kit. The designed EIA procedure could be performed within 40 min in a one-stage incubation protocol. The assay time was shorter than that of other commercial RIA or EIA kits, and the sensitivity was 0.4 ng/ml which was comparable to that of RIA kits. The EIA kit was shown not to cross-react with human thyroid stimulating hormone, alpha-fetoprotein, carcinoembryonic antigen, and acid phosphatases derived from tissues other than prostate. Therefore, this design was a simple and rapid method with high sensitivity and specificity for determining PAP isoenzyme 2 in human serum.

Two distinct nuclear factors bind the conserved regulatory sequences of a rabbit major histocompatibility complex class II gene.

The constitutive coexpression of the major histocompatibility complex (MHC) class II genes in B lymphocytes requires positive, trans-acting transcriptional factors. The need for these trans-acting factors has been suggested by the reversion of the MHC class II-negative phenotype of rare B-lymphocyte mutants through somatic cell fusion with B cells or T-cell lines. The mechanism by which the trans-acting factors exert their effect on gene transcription is unknown. The possibility that two highly conserved DNA sequences, located 90 to 100 base pairs (bp) (the A sequence) and 60 to 70 bp (the B sequence) upstream of the transcription start site of the class II genes, are recognized by the trans-acting factors was investigated in this study. By using the gel electrophoresis retardation assay, a minimum of two proteins which specifically bound the conserved A or B sequence of a rabbit DP beta gene were identified in murine nuclear extracts of a B-lymphoma cell line, A20-2J. Fractionation of nuclear extract through a heparin-agarose column allowed the identification of one protein, designated NF-MHCIIB, which bound an oligonucleotide containing the B sequence and protected the entire B sequence in the DNase I protection analysis. Another protein, designated NF-MHCIIA, which bound an oligonucleotide containing the A sequence and partially protected the 3' half of this sequence, was also identified. NF-MHCIIB did not protect a CCAAT sequence located 17 bp downstream of the B sequence. The possible relationship between these DNA-binding factors and the trans-acting factors identified in the cell fusion experiments is discussed.

Two distinct nuclear factors bind the conserved regulatory sequences of a rabbit major histocompatibility complex class II gene.

The constitutive coexpression of the major histocompatibility complex (MHC) class II genes in B lymphocytes requires positive, trans-acting transcriptional factors. The need for these trans-acting factors has been suggested by the reversion of the MHC class II-negative phenotype of rare B-lymphocyte mutants through somatic cell fusion with B cells or T-cell lines. The mechanism by which the trans-acting factors exert their effect on gene transcription is unknown. The possibility that two highly conserved DNA sequences, located 90 to 100 base pairs (bp) (the A sequence) and 60 to 70 bp (the B sequence) upstream of the transcription start site of the class II genes, are recognized by the trans-acting factors was investigated in this study. By using the gel electrophoresis retardation assay, a minimum of two proteins which specifically bound the conserved A or B sequence of a rabbit DP beta gene were identified in murine nuclear extracts of a B-lymphoma cell line, A20-2J. Fractionation of nuclear extract through a heparin-agarose column allowed the identification of one protein, designated NF-MHCIIB, which bound an oligonucleotide containing the B sequence and protected the entire B sequence in the DNase I protection analysis. Another protein, designated NF-MHCIIA, which bound an oligonucleotide containing the A sequence and partially protected the 3' half of this sequence, was also identified. NF-MHCIIB did not protect a CCAAT sequence located 17 bp downstream of the B sequence. The possible relationship between these DNA-binding factors and the trans-acting factors identified in the cell fusion experiments is discussed.

Conditions for the appearance of maturation promoting factor following germinal vesicle disruption of prophase‐arrested starfish oocytes

AbstractIn this paper, we report that so‐called “incompetent” prophase‐arrested oocytes of Marthasterias glacialis and Asterias rubens fail to produce any aster or spindle when their nucleoplasm and cytoplasm are mixed via pressure or microinjection. We also show that the same incompetent oocytes always complete their maturation up to second polar body extrusion and female pronucleus formation following a 30 sec shaking treatment, which results at once in germinal vesicle (GV) disruption and produces the same mixing of their nuclear and cytoplasmic compartments.32P phosphate incorporation into proteins and cell fusion experiments indicates that this mechanical treatment induces an increased protein phosphorylation and triggers the formation of active maturation promoting factor (MPF) molecules which are not produced following a simple nucleoplasm transfer. While bovine serum albumin (BSA) had no effect on this process, the intracellular Ca2+antagonists (2‐[(2‐bist[Carboxymethyl]‐amino‐5‐methylphenoxy)‐methyl]‐6‐methoxy‐8‐bis[carboxy‐methyl]‐aminoquinolinetetrakis‐[acetoxymethyl]ester) (Quin 2‐AM) and 8(N‐N‐diethylamino) octyl‐3,4,5‐trimethoxybenzoate (TMB 8) were found to inhibit both hormone‐induced and shaking‐induced maturations. These results exclude the possibility that shaking might act by releasing arachidonic acid in the external medium. Instead, they suggest that such a mechanical treatment may trigger the same significant changes in Ca2+ concentration or compartmentalization that have been shown to occur following hormone stimulation and that seem to be required for activating resting female centrioles as well as latent MPF precursors and protein kinases.

Urea-Induced Meiosis Reinitiation in Oocytes of the Starfish, Marthasterias glacialis: (intracellular calcium/maturation promoting factor/polyethylene glycol-induced cell fusion/protein phosphorylation/starfish oocyte maturation).

In the absence of hormone stimulation, prophase-blocked oocytes of Marthasterias glacialis have been induced to undergo meiosis reinitiation up to female pronucleus formation by pulse incubation in isoosmotic urea-sea water solutions. Even when this procedure was not effective all along the breeding season, it could trigger full maturation when applied to so-called "incompetent oocytes" that did not complete maturation following microinjection-induced mixing of their nucleoplasm and cytoplasm. 32 P phosphate incorporation into proteins and cell fusion experiments demonstrate that this treatment produces an increased protein phosphorylation which appears tightly associated with the production of M-phase promoting factor (MPF). Instead, when oocytes are maintained in the inducing medium, dephosphorylation soon occurs and MPF is no longer present to support meiosis. Under these conditions, the GV-disrupted oocytes present a permanent nucleolus and do not form a meiotic spindle. The same cytological aspect was also obtained when the oocytes were treated in the presence of 90 μM emetine or 150 μM of the intracellular chelator Quin 2-AM. These data suggest that urea-induced maturation may involve an intracellular Ca2+ shift which would be required to activate both MPF precursor molecules and the resting female centers which stand in the animal cortex outside the nucleus and give rise to the poles of the first maturation spindle. They also show that nuclear disruption alone, without protein phosphorylation, cannot trigger meiosis reinitiation of incompetent oocytes.

Homing Receptors and Metastasis

As discussed in the preceding sections, there are several indications that the lymphocyte homing receptors involved in the normal process of lymphocyte recirculation are also relevant to the behavior of metastatic cells. Cell fusion experiments indicate that previously nonmetastatic cells can acquire metastatic capacity from fusion with normal lymphocytes. Murine T lymphomas that bear high levels of functional homing receptors can metastasize to peripheral lymphoid organs, whereas those lymphomas lacking homing receptors cannot. Virtually all lymph node metastases of lymphomas contain a high proportion of MEL-14hi cells, even if the primary tumor has been selected to be relatively deficient in these cells. Further investigations of the biology of lymphocyte homing receptors will reveal whether or not there are additional lymphocyte homing receptors and will clarify the role of lymphocyte homing receptors in metastasis. Antibodies against three lymphocyte homing receptors could therefore be useful for diagnosis and treatment of metastatic disease.

New clues about T-cell antigen receptor complex function

The emerging picture of the T-cell antigen receptor complex is that of an extraordinarily elaborate structure. The best-understood subunit is the Ti heterodimer, con- sisting of integral membrane glycoproteins bearing substantial structural and functional similarity to immunoglobulins 1. Somatic cell fusion and gene transfer experiments have shown that, in cells expressing the more common '~13' receptor, antigen specificity is deter- mined by the polymorphic Ti oLl~ subunit 2-4. While the function of the '~/~' form of the receptor is unknown, presumably its ligand reactivity is also specified by the Ti ~/~ subunit. Both types of Ti subunits are expressed on their respective cells in noncovalent association with an array of invariant proteins collectively referred to as the CD3 (formerly T3) complex. It is the function of the CD3 portion of the antigen receptor complex that remains a subject of speculation. The CD3 complex comprises at least four, and possibly five, different integral membrane proteins 1.s. The human CD3 complex contains the ~/, 6, e and disulfide-linked chains. The murine complex contains homologous com- ponents that are proposed to assemble as a ~/~e~2 complex6; an alternate form, comprising the ~/, a, e and chains with another component, p21 (in a disulfide- linked p21-~ dimer), has been observed in low abund- ance on a murine hybridoma 7. From their studies of the biosynthesis and assembly of the rnurine CD3-Ti complex, R. Klausner and colleagues have proposed that the ~/, ~ and e chains are produced in large excess, and that the ~ chain is the limiting compo- nent in the completion of an expressable CD3 complex 6. Their recent molecular cloning of the ~ chain has sug- gested a transmembrane topology that places most of the molecule on the cytoplasmic side of the plasma membrane, with a hydrophobic transmembrane domain and a short, highly charged extracellular segment 8. The other recognized CD3 chains appear to have prominent cytoplasmic and extracellular domains. The large intra- cellular portions of CD3 chains compared to the Ti chains (which have only 5-12 intracellular amino acids) have been suspected to allow CD3 to perform transmembrane signalling activities for the antigen-binding Ti subunit. In the absence of attractive alternatives, this hypothesis is widely held to be the correct interpretation of CD3 function, one that merely awaits experimental confirma- tion. What this model fails to address is the function of the large extracellular domains of the CD3 ~/, a and e chains, which could be envisaged interacting with Ti or binding a nonpolymorphic ligand provided by the antigen-presenting cell. The best-characterized signal transduction response to binding of ligands to the CD3-Ti complex is the acti- vation of the phosphatidylinositol (PI) second messenger system 9, in which hydrolysis of phosphatidylinositol 4,5- bisphosphate yields the second messengers diacyl- Howard Hughes Medical Institute, Division of Rheumatology/Im- munology, University of California San Francisco, 3rd and Parnassus Avenues, San Frar~cisco, CA 94143-0724, USA.

Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity.

A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795). The higher cholesterol ester content of 25-RA is due to an increase in the rate of cholesterol biosynthesis and low density lipoprotein receptor activity compared to wild-type Chinese hamster ovary cells, and not due to an abnormal acyl-CoA:cholesterol acyltransferase enzyme. The procedure to isolate cholesterol ester-deficient mutants utilizes amphotericin B, a polyene antibiotic known to bind to cholesterol and to form pore complexes in membranes. After incubation in cholesterol-free medium plus an inhibitor of endogenous cholesterol biosynthesis, 25-RA cells were found to be 50-500 times more sensitive to amphotericin B killing than were mutant cells containing reduced amounts of cholesterol ester. Twelve amphotericin B-resistant mutants were isolated which retained the 25-hydroxycholesterol-resistant phenotype. These mutants did not exhibit the perinuclear lipid droplets characteristic of 25-RA cells, and lipid analysis revealed a large (up to 40-fold) reduction in cellular cholesterol ester. The acyl-CoA:cholesterol acyltransferase activities of these cholesterol ester-deficient mutants were markedly lower than 25-RA when assayed in intact cells or in an in vitro reconstitution assay. The tightest mutant characterized, AC29, was found to have less than 1% of the parental acyl-CoA:cholesterol acyltransferase activity. These mutants all have reduced rates of sterol synthesis and lower low density lipoprotein receptor activity compared to 25-RA, probably as a consequence of their reduced enzyme activities. Cell fusion experiments revealed that the phenotypes of all the mutants examined are not dominant and that the mutants all belong to the same complementation group. We conclude that these mutants contain a lesion in the gene encoding acyl-CoA:cholesterol acyltransferase or in a gene encoding a factor needed for enzyme production.

Identification by cell fusion of gene sequences that interact with positive trans-acting factors.

Cell fusion experiments have implicated positive or negative regulatory factors in the cell type--specific expression of specialized endogenous genes. The inability to readily manipulate such genes has prevented characterization of the cis-acting DNA sequences that interact with these factors. A transfection-fusion technique, which combined stable gene transfer and formation of transient heterokaryons, was used to study this class of factors and their DNA binding sites. Messenger RNA directed by a quiescent, rat prolactin promoter region stably transferred into mouse fibroblasts was detected only after fusion to rat pituitary cells, implying that pituitary cells contain a positive cell type--specific factor or factors. Nuclear run-on assays showed that fusion activation is transcriptional. Fusion did not activate either a stably transferred rat growth hormone gene promoter or expression of the endogenous silent fibroblast prolactin or growth hormone genes. Analysis by 5'-deletion mutation identified a 30-base pair DNA sequence required for cell fusion activation of the rat prolactin promoter region. Comparison with previous results from direct cellular transfer of this region implies that transfection-fusion identifies novel regulatory DNA sequences.

The photodecomposition of aminopterin

Aminopterin is used in cell fusion experiments to select for hybrid cells by killing unfused cells which are deficient in enzymes for nucleotide salvage pathways. Aqueous aminopterin solutions exposed to fluorescent room light (wavelength greater than 300 nm) were found to lose cytoxicity. Inactivation was accompanied by changes in the ultraviolet absorption spectra of the solutions. The ultraviolet spectrum of an irradiated aminopterin solution is used to provide a quantitative measure of its cytotoxicity. Aminopterin appears to be photolytically cleaved at the C9-N10 methylene-anilino bond in a reaction analogous to that known for the photolysis of folic acid irradiated at 365 nm (Lowry et al., 1949).

Cell fusion by nasopharyngeal carcinoma-derived Epstein-Barr virus.

Cell fusion experiments using Epstein-Barr virus (EBV) obtained from nasopharyngeal carcinoma (NPC) hybrid cells (NPC-KT) were carried out to examine whether human adenoid epithelial cells (Ad-AH) could be fused with lymphoblastoid cells (BJAB, Raji, and A2L) superinfected or infected by EBV, and if Ad-AH nuclei of the fused cells could express EBV-associated nuclear antigen. The presence of NPC EBV could induce EBV early antigen in all three lymphoblastoid cell lines. By autoradiography, we found that NPC EBV could induce cell fusion between Ad-AH cells and tritiated thymidine-labeled lymphoblastoid cells. In addition, we found that Ad-AH nuclei of the fused cells expressed EBV-associated nuclear antigen four days after NPC EBV-mediated cell fusion of Ad-AH cells with lymphoblastoid cells.