Number Of Fusion Events Research Articles

Abstract P3-13-08: Fusion analysis including NTRK fusion in breast cancers (BC): From RNASeq data analysis from 629 BC tissue samples

Abstract Background: Neutrotrophin receptor tyrosine kinase (NTRK) gene fusions (NTRK1, NTRK2, or NTRK3) are oncogenic drivers of various tumor types. The NTRK fusion was detected in less than 5% of breast, colorectal, lung or any other types of cancers. However, large scaled next generation sequencing data for NTRK fusion in breast cancer have not existed. In this study, we performed RNASeq and fusion analysis including NTRK genes. Methods: We prospectively collected BC tumor tissues from the translational research conducted in Samsung Medical Center. Fusion was predicted from RNAseq using the following seven softwares(SWs): ChimeraScan, DeFuse, MapSplice, TophatFusion, STAR.Arriba, STAR.fusion, and STAR.SEQR. To remove false-positive fusion calls, calls less than 3 left/right spanning reads and 10 total supporting reads were removed. After filtering out the blacklist fusion calls which were recurrently detected, calls commonly predicted in more than two SWs were analyzed. Results: In total 629 BC samples, 613 samples were finally analyzed after quality control (QC) of RNASeq. According to immunohistochemistry (IHC) profile, 356(58.7%) was hormone receptor (HR) positive human epidermal growth factor receptor 2 (HER2) negative BC, 42 (6.9%) of HR positive HER2 positive BC, 53 (8.7%) of HR negative, HER2 positive and 155(25.6%) of triple negative BC (TNBC). PAM50 prediction informed that 174(28.9%) of luminal A, 151(24.6%) of luminal B, 170 (27.7%) of basal-like, 85 (13.9%) of HER2-enriched and 33 (5.4%) of normal. In median number of fusion events, 12 was called by ChimeraScan (Interquartile range [IQR]: 5, 33), 57 by DeFuse (IQR: 33, 82), eight by Mapsplice (IQR: 5, 12), two by TophatFusion (IQR: 0, 4), five by STAR.Arriba (IQR: 2, 12), two by STAR.fusion (IQR:0, 5) and three by STAR.SEQR fusion caller (IQR: 1, 7) after call filtering. After initial fusion call, we excluded the results from ChimeraScan and DeFuse fusion callers because of discrepancy of number of called fusion events. In five fusion callers, median number of fusion events was eight (IQR :2,20) per BC sample. In terms of NTRK fusion, we detected NTRK2-BANCR fusion event in one TNBC patients (1/425, 0.2%). This fusion was detected in four of five SWs for fusion detection with significant number of supporting reads in RNASeq. NTRK2-BANCR fusion was the out-of-frame fusion, which C-terminal truncated protein kinase domain of NTRK2 and its partner long non-coding RNA BANCR was combined and RNA expression of this fusion was increased. Other fusion events of BCs were NCOR2-PARP4 (3.7%), BRD4-NWD1 (3.7%), ESR1-RGS17 (1.8%), FGFR1-TACC1 (0.2%) and MKRN1-BRAF (0.2%). In BC subtype according to IHC, fusion events were more frequently observed in TNBC compared with other subtypes regardless of the type of fusion filters. In terms of intrinsic subtype, fusion events were most frequently observed in basal like type and least in normal like intrinsic subtype (all ps<0.05, respectively). Conclusions: In this large scaled RNASeq data analysis, a few fusion events were observed in BC patients. Prevalence of NTRK was extremely rare. Additional investigation including functional validation would be followed. Citation Format: Ji-Yeon Kim, Kyunghee Park, Woong-Yang Park, Jeong Eon Lee, Seok Won Kim, Seok Jin Nam, Jonghan Yu, Young-Hyuck Im, Jin Seok Ahn, Yeon Hee Park. Fusion analysis including NTRK fusion in breast cancers (BC): From RNASeq data analysis from 629 BC tissue samples [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-13-08.

Epiphyseal fusion and dental development in Pan paniscus with comparisons with Pan troglodytes.

Compared with frequent studies of skeletal development in chimpanzees, relatively little is known about bonobo skeletal development. This study seeks to explore the relationship between skeletal and dental development in both species of Pan. New data are presented for fusion sites not previously observed in bonobos. In a sample of 34 Pan paniscus and 168 Pan troglodytes subadults, state of fusion was recorded for 30 epiphyseal fusion sites using a three-stage system of unfused, midfusion, and complete fusion based on Wintheiser, Clauser, and Tappen. Stage of dental development for permanent mandibular dentition was assessed using the Demrijian, Goldstein, and Tanner method. These data allowed for comparisons of both species of Pan and the two subspecies of P. troglodytes. The sequence of fusion events was generally consistent between the two species, but some exceptions may exist for the knee and ankle. The number of fusion events that occurred after complete dental mineralization was similar in both species. No statistically significant differences were found in the fusion timing for the subspecies of P. troglodytes. Bolter and Zihlman suggested that fusion at the acetabulum occurs earlier in Pan paniscus, while fusion of epiphyses at the knee are delayed, compared with P. troglodytes. Our data do not indicate earlier fusion of the acetabulum, but fusion events at the knee may complete later relative to dental mineralization in Pan pansicus. Compared with Homo sapiens, both P. troglodytes and Pan paniscus demonstrate later completion of epiphyseal fusion relative to dental mineralization.

Search for a minimal machinery for Ca2+-triggered millisecond neuroexocytosis

Neurons have the remarkable ability to release a batch of neurotransmitters into the synapse immediately after an action potential. This signature event is made possible by the simultaneous fusion of a number of synaptic vesicles to the plasma membrane upon Ca2+ entry into the active zone. The outcomes of both cellular and in vitro studies suggest that soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) and synaptotagmin 1 (Syt1) constitute the minimal fast exocytosis machinery in the neuron. Syt1 is the major Ca2+-sensor and orchestrates the synchronous start of individual vesicle fusion events while SNAREs are the membrane fusion machinery that dictates the kinetics of each single fusion event. The data also suggest that Ca2+-bound Syt1 is involved in the upstream docking step which leads to an increase in the number of fusion events or the size of the release, leaving the SNARE complex alone to carry out membrane fusion by themselves.

Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs

The large gap in time scales between membrane fusion occurring in biological systems during neurotransmitter release and fusion observed between model membranes has provoked speculations over a large number of possible factors that might explain this discrepancy. One possible reason is an elevated lateral membrane tension present in the presynaptic membrane. We investigated the tension-dependency of fusion using model membranes equipped with a minimal fusion machinery consisting of syntaxin 1, synaptobrevin and SNAP 25. Two different strategies were realized; one based on supported bilayers and the other one employing sessile giant liposomes. In the first approach, isolated patches of planar bilayers derived from giant unilamellar vesicles containing syntaxin 1 and preassembled SNAP 25 (ΔN-complex) were deposited on a dilatable PDMS sheet. In a second approach, lateral membrane tension was controlled through the adhesion of intact giant unilamellar vesicles on a functionalized surface. In both approaches fusion efficiency increases considerably with lateral tension and we identified a threshold tension of 3.4 mN m−1, at which the number of fusion events is increased substantially.

Live Imaging of Myogenesis in Indirect Flight Muscles in Drosophila.

The indirect flight muscles (IFMs) are the largest muscles in the fly, making up the bulk of the adult thorax. IFMs in Drosophila are generated during pupariation by fusion of hundreds of muscle precursor cells (myoblasts) with larval muscle templates (myotubes). Prominent features, including the large number of fusion events, the structural similarity to vertebrate muscles, and the amenability to the powerful genetic techniques of the Drosophila system make the IFMs an attractive system to study muscle cell fusion. Here we describe methods for live imaging of IFMs, both in intact pupae, and in isolated IFMs ex-vivo. The protocols elaborated upon here were used in the manuscript by ( Segal et al., 2016 ).

Abstract 1539: Fusion events in NSCLC cell lines identified by whole transcriptome analysis

Abstract Since the discovery of Philadelphia chromosome, human chromosomal rearrangements consequence the great deal within the distinct area of cancer biomarker discovery and targeted therapy. These events are commonly generated by breaking and re-joining the segments on either inter- or intra-chromosomal locations, creating an error within DNA repair mechanism responsible for oncogenic properties and, one of the reasons, causing cancer. Also, alteration in the programing of gene regulation changes the transcriptome structure resulting pathological conditions. Recent advances in the cancer epidemiology predicted the highest death rates within Europe and Korea due to lung cancer representing the leading cause of death within cancer patients. Therefore, we have sequenced the whole transcriptome following the highly sensitive path of RNA-seq with the combination of bioinformatics tools and experimental confirmation. The mRNA was subjected on Illumina HiSeq2000 for paired-end (PE) sequencing and bioinformatics analyses were conducted on Linux based software tools. The PE reads were aligned with Ensemble reference human genome version hg19 using TopHat2 and bowtie1. The fusion events were predicted using two different software packages, deFuse and Tophat-Fusion, and expression analysis was conducted using Cufflinks and R-package cumeRbund. The predicted fusion break-points were confirmed by target specific sanger-sequencing. Through the RNA-seq analysis, we identified a number of fusion events shared by oncogenes in non-small cell lung cancer (NSCLC) cell-lines including EML4:ALK fusion identified in recently established gefitinib resistant SNU2292 cell-line and some novel ones. Moreover, a significant increase in the expression of cell-line specific fusion genes was also observed. Our result will be helpful not only for discovering novel biomarkers but also for understanding the tumorigenesis of NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Seung-Hyun Jung, Yeun-Jun Chung. Fusion events in NSCLC cell lines identified by whole transcriptome analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1539. doi:10.1158/1538-7445.AM2014-1539

Glia and the Synapse: Plasticity and Disease

Glia and the Synapse: Plasticity and Disease

A GALA lipopeptide mediates pH- and membrane charge dependent fusion with stable giant unilamellar vesicles

Peptides capable of mediating fusion between lipid membranes are widely observed in nature, and have attracted considerable attention in the liposome drug delivery field. However, studies that are proving the benefit of small synthetic fusion peptides as components in drug delivery systems remain sporadic and there is a strong need to characterize and increase our understanding of the membrane fusion properties of these peptides. Many fusion studies have focused on the ability of free peptides in solution that mediate fusion between liposomes. For drug delivery purposes it is a necessity to attach the peptides to the surface of the liposome drug delivery carrier, without impairing the peptide functionality. Here we present the synthesis and characterization of a new diacylated derivative of the previously described fusion peptide GALA. Two myristoyl chains were attached to the GALA peptide through the 1,2-diamino propanoic acid (Dap) moiety, yielding the lipopeptide dimyristoyl-Dap-GALA (DMDGALA). We have investigated DMDGALA as a component in large unilamellar vesicles (LUVs) and demonstrate pH-triggered fusion of peptide containing LUVs with stable target giant unilamellar vesicles (GUVs), which were used as simple mimics of cell membranes. The number of fusion events was large at pH 5.0, which is a physiologically relevant pH-range for a drug delivery system.

Membrane fusion of pH-sensitive liposomes – a quantitative study using giant unilamellar vesicles

Drug delivery systems based on liposome carriers that are designed for induction of membrane fusion in response to the microenvironment have been investigated for more than two decades. However, most studies have focused on self-fusion among large unilamellar vesicles (LUVs) in solution, a system with limited biological relevance which suffers from averaging effects and non-trivial interpretation of results. Here we present a fusion assay capable of visualizing and quantifying heterogeneous fusion between fusogenic liposomes and stable giant unilamellar vesicles (GUVs), suitable for studying both lipid and content mixing. Fusion was visualized and quantified using fluorescence microscopy and a FRET-based lipid mixing assay and the number of fusion events with single GUVs was estimated. Fusogenic pH-sensitive oleic acid (OA):DOPE liposomes were used as a model system and fusion was quantified with giant vesicles containing 5 or 10 mol% positively charged lipids. It was shown that the number of fusion events with single GUVs after 30 min at room temperature is approximately 100 or 200 per 100 μm2 of GUV surface, for 5 or 10 mol%, respectively. Furthermore, the mixing of the aqueous content during fusion was visualized and quantified, which showed that the content of the fusogenic liposomes is transferred essentially without leakage. Fusion was confirmed to be pH-dependent and is enhanced by electrostatic attraction between the fusing vesicles, which was demonstrated by the absence of fusion in experiments performed with negatively charged GUVs.

Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

BackgroundA variant of the CDKAL1 gene was reported to be associated with type 2 diabetes and reduced insulin release in humans; however, the role of CDKAL1 in β cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from β cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice.Principal FindingsTotal internal reflection fluorescence imaging of CDKAL1 KO β cells showed that the number of fusion events during first-phase insulin release was reduced. However, there was no significant difference in the number of fusion events during second-phase release or high K+-induced release between WT and KO cells. CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca2+ concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K+ (KATP) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5 between WT and CDKAL1 KO islets.Conclusions/SignificanceWe provide the first report describing the function of CDKAL1 in β cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in β cells by facilitating ATP generation, KATP channel responsiveness and the subsequent activity of Ca2+ channels through pathways other than CDK5-mediated regulation.

A targeted siRNA screen to identify SNAREs required for constitutive secretion in mammalian cells.

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry-based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein-specific siRNA screen (38 SNAREs, 4 SNARE-like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post-Golgi SNAREs SNAP-29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP-29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP-29-depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19-interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP-23, SNAP-25, SNAP-29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post-Golgi-specific R-SNARE in this process.

Biophysical Properties of Mitochondria Undergoing Fusion

Emerging evidence shows the importance of genes controlling mitochondrial fusion in physiology and their deregulation in neurodegenerative and metabolic disorders. However, apart from ΔΨm, the biophysical properties of the fusion-competent mitochondria remain elusive. To evaluate the conditions of contact formation and fusion, we used organelle-targeted fluorescent proteins, including photoactivatable GFP, which allow tracking of individual mitochondria. In H9c2 cells, almost every mitochondrion is aligned with microtubules that provide the primary tracks for mitochondrial movement. Our results show that ∼90% of fusion events involve moving mitochondria. However, fusion occurs irrespective of mitochondrial speed. Furthermore, ∼80% of fusion events involve the tip portion of the mitochondrion, whereas only ∼50% of these events involve organelle side. Nocodazol, a microtubule disrupting agent that inhibits mitochondrial movements decreases the fusion frequency and changes mitochondrial fusion sites. Strikingly, 80-90% of the physical contacts between adjacent mitochondria did not result in fusion events. To evaluate whether the fusion efficacy depends on the spacing between the outer and inner mitochondrial membranes we used drugs that alter the matrix volume. Valinomycin, a K+ ionophore that induces matrix swelling evoked a decrease in mitochondrial motility leading to fewer contacts among mitochondria but the number of fusion events was maintained, indicating an increase in fusion efficacy. This change occurred at a low valinomycin concentration (0.25nM) that did not affect ΔΨm or Opa1 cleavage. Nigericin (0.5μM), a K+/H+ ionophore that induces shrinkage of the matrix elicited fusion inhibition and mitochondrial aggregation. Importantly, no motility inhibition or Opa1 cleavage occurred at the same time and the ΔΨm was increased. These results suggest that mitochondrial fusion is facilitated by mitochondrial motility, the key determinant of the inter-mitochondrial encounter numbers and preferentially involves the front-tip of the moving organelle. In addition, fusion efficacy depends on the mitochondrial matrix volume.

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 μm2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

Adiponectin induces insulin secretion in vitro and in vivo at a low glucose concentration

A decrease in plasma adiponectin levels has been shown to contribute to the development of diabetes. However, it remains uncertain whether adiponectin plays a role in the regulation of insulin secretion. In this study, we investigated whether adiponectin may be involved in the regulation of insulin secretion in vivo and in vitro. The effect of adiponectin on insulin secretion was measured in vitro and in vivo, along with the effects of adiponectin on ATP generation, membrane potentials, Ca2+ currents, cytosolic calcium concentration and state of 5'-AMP-activated protein kinase (AMPK). In addition, insulin granule transport was measured by membrane capacitance and total internal reflection fluorescence (TIRF) analysis. Adiponectin significantly stimulated insulin secretion from pancreatic islets to approximately 2.3-fold the baseline value in the presence of a glucose concentration of 5.6 mmol/l. Although adiponectin had no effect on ATP generation, membrane potentials, Ca2+ currents, cytosolic calcium concentrations or activation status of AMPK, it caused a significant increase of membrane capacitance to approximately 2.3-fold the baseline value. TIRF analysis revealed that adiponectin induced a significant increase in the number of fusion events in mouse pancreatic beta cells under 5.6 mmol/l glucose loading, without affecting the status of previously docked granules. Moreover, intravenous injection of adiponectin significantly increased insulin secretion to approximately 1.6-fold of baseline in C57BL/6 mice. The above results indicate that adiponectin induces insulin secretion in vitro and in vivo.

IGF-I increases bone marrow contribution to adult skeletal muscle and enhances the fusion of myelomonocytic precursors

Muscle damage has been shown to enhance the contribution of bone marrow–derived cells (BMDCs) to regenerating skeletal muscle. One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC). However, the molecular components responsible for this injury-related response remain largely unknown. In this paper, we show that delivery of insulin-like growth factor I (IGF-I) to adult skeletal muscle by three different methods—plasmid electroporation, injection of genetically engineered myoblasts, and recombinant protein injection—increases the integration of BMDCs up to fourfold. To investigate the underlying mechanism, we developed an in vitro fusion assay in which co-cultures of MMCs and myotubes were exposed to IGF-I. The number of fusion events was substantially augmented by IGF-I, independent of its effect on cell survival. These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

Embraces and Grooming in Captive Spider Monkeys

Relatively little information is available regarding the role of social grooming and embraces in spider monkeys that live in fission-fusion societies in which individuals are usually split into subgroups. We investigated whether embraces and grooming have similar roles in captive spider monkeys by examining their occurrence in two contexts. One context was fusion, i.e. when the monkeys moved into an area of the enclosure where other monkeys were present, in which individuals from different subgroups were expected to exchange greeting behavior. The other context was the access to young infants, in which females were expected increase their friendly behavior toward mothers. We collected data by observing all individuals within a subgroup and via focal animal sampling. We found that overall embraces occurred more frequently than grooming and that their distributions were not correlated. The frequency of embraces was positively associated with the number of fusion events per observation, whereas the frequency of grooming bouts was not. Furthermore, embraces were more frequent following initial approaches after fusion versus subsequent approaches, and the figure was higher than the corresponding one for grooming. Mothers received more embraces after than before the birth of their infants, whereas there was no such effect for grooming. Embraces, but not grooming, play a role in the regulation of social relationships in spider monkeys. Embraces may serve as signals of benign disposition in contexts that are likely to be associated with tension, such as fusion and access to infants.

Docking and fusion of insulin secretory granules in SUR1 knock out mouse β-cells observed by total internal reflection fluorescence microscopy

To explore how the sulfonylurea receptor (SUR1) is involved in docking and fusion of insulin granules, dynamic motion of single insulin secretory granules near the plasma membrane was examined in SUR1 knock-out (Sur1KO) β-cells by total internal reflection fluorescence microscopy. Sur1KO β-cells exhibited a marked reduction in the number of fusion events from previously docked granules. However, the number of docked granules declined during stimulation as a consequence of the release of docked granules into the cytoplasm vs. fusion with the plasma membrane. Thus, the impaired docking and fusion results in decreased insulin exocytosis from Sur1KO β-cells.

Reactor Prospects of Muon-Catalyzed Fusion of Deuterium and Tritium Concentrated in Transition Metals

It is conjectured that the number of fusion events catalyzed by a single muon is orders of magnitude greater for deuterium and tritium concentrated in a transition metal than in gaseous form and that the recent observation of 2.5-MeV neutrons from a D/sub 2/O electrolytic cell with palladium and titanium cathodes can thereby be interpreted in terms of cosmic muon-catalyzed deuterium-deuterium fusion. This suggests a new fusion reactor reactor consisting of deuterium and tritium concentrated in transition metal fuel elements in a fusion core that surrounds an accelerator-produced muon source. The feasibility of net energy production in such a reactor is established in terms of requirements on the number of fusion events catalyzed per muon. The technological implications for a power reactor based on this concept are examined. The potential of such a concept as a neutron source for materials testing and tritium and plutonium production is briefly discussed.

Estimate of fusion through bose degeneracy

An estimate is made of the fusion probability for an aggregate of D2 molecules that have suffered bose degeneracy. A WKB analysis for the tunneling of deuterons through the electron intermolecular barrier gives the penetration rate $$R = (2E/h)\exp \left[ { - \frac{8}{3}\sqrt {\frac{{2mV_0 x_0^2 }}{\hbar }} \left( {1 - \frac{E}{{V_0 }}} \right)^{3/2} } \right]$$ In this expression,V0 andx0 are the potential barrier height and width, respectively, andE is the zero point energy of a deuteron in its ground molecular state. With molecules assumed effectively to be adjacent in the bose condensate, the number of fusion events per mole is found to be vanishingly small.

Electron microscopic study of Bacillus subtilis protoplast fusion.

When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope. The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation. This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion. When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria. Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation. Fusion in this case is apparently completed after plating on the wall-regeneration medium. After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered. These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J. Bacteriol. 137:1340--1345, 1979).