Giant DNA Research Articles

Targeting of lactate dehydrogenase C dysregulates the cell cycle and sensitizes breast cancer cells to DNA damage response targeted therapy.

The cancer testis antigen (CTA) lactate dehydrogenase C (LDHC) is a promising anticancer target with tumor‐specific expression and immunogenicity. Interrogation of breast cancer patient cohorts from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) indicate that upregulation of LDHC expression correlates with unfavorable prognosis. Although the role of LDHC is well characterized in spermatocytes, its role in tumors remains largely unknown. We investigated whether LDHC is involved in regulating genomic stability and whether it could be targeted to affect tumor cellular fitness. Silencing LDHC in four breast cancer cell lines significantly increased the presence of giant cells, nuclear aberrations, DNA damage, and apoptosis. LDHC‐silenced cells demonstrated aberrant cell cycle progression with differential expression of cell cycle checkpoint and DNA damage response regulators. In addition, LDHC silencing‐induced microtubule destabilization, culminating in increased mitotic catastrophe and reduced long‐term survival. Notably, the clonogenicity of LDHC‐silenced cells was further reduced by treatment with the poly (ADP‐ribose) polymerase (PARP) inhibitor olaparib and with the DNA‐damaging drug cisplatin. This study supports the therapeutic potential of targeting LDHC to mitigate cancer cell survival and improve sensitivity to agents that cause DNA damage or inhibit its repair.

Quantitative conversion of biomass in giant DNA virus infection

Bioconversion of organic materials is the foundation of many applications in chemical engineering, microbiology and biochemistry. Herein, we introduce a new methodology to quantitatively determine conversion of biomass in viral infections while simultaneously imaging morphological changes of the host cell. As proof of concept, the viral replication of an unidentified giant DNA virus and the cellular response of an amoebal host are studied using soft X-ray microscopy, titration dilution measurements and thermal gravimetric analysis. We find that virions produced inside the cell are visible from 18 h post infection and their numbers increase gradually to a burst size of 280–660 virions. Due to the large size of the virion and its strong X-ray absorption contrast, we estimate that the burst size corresponds to a conversion of 6–12% of carbonaceous biomass from amoebal host to virus. The occurrence of virion production correlates with the appearance of a possible viral factory and morphological changes in the phagosomes and contractile vacuole complex of the amoeba, whereas the nucleus and nucleolus appear unaffected throughout most of the replication cycle.

How the Circular and Linear Conformational Fluctuations of Giant DNA Molecules Change with the Viscosity of the Solvent

How the Circular and Linear Conformational Fluctuations of Giant DNA Molecules Change with the Viscosity of the Solvent

The high-resolution structure of a UDP-L-rhamnose synthase from Acanthamoeba polyphaga Mimivirus.

For the field of virology, perhaps one of the most paradigm-shifting events so far in the 21st century was the identification of the giant double-stranded DNA virus that infects amoebae. Remarkably, this virus, known as Mimivirus, has a genome that encodes for nearly 1,000 proteins, some of which are involved in the biosynthesis of unusual sugars. Indeed, the virus is coated by a layer of glycosylated fibers that contain d-glucose, N-acetyl-d-glucosamine, l-rhamnose, and 4-amino-4,6-dideoxy-d-glucose. Here we describe a combined structural and enzymological investigation of the protein encoded by the open-reading frame L780, which corresponds to an l-rhamnose synthase. The structure of the L780/NADP+ /UDP-l-rhamnose ternary complex was determined to 1.45 Å resolution and refined to an overall R-factor of 19.9%. Each subunit of the dimeric protein adopts a bilobal-shaped appearance with the N-terminal domain harboring the dinucleotide-binding site and the C-terminal domain positioning the UDP-sugar into the active site. The overall molecular architecture of L780 places it into the short-chain dehydrogenase/reductase superfamily. Kinetic analyses indicate that the enzyme can function on either UDP- and dTDP-sugars but displays a higher catalytic efficiency with the UDP-linked substrate. Site-directed mutagenesis experiments suggest that both Cys 108 and Lys 175 play key roles in catalysis. This structure represents the first model of a viral UDP-l-rhamnose synthase and provides new details into these fascinating enzymes.

Advantages and Limits of Metagenomic Assembly and Binning of a Giant Virus.

Giant viruses have large genomes, often within the size range of cellular organisms. This distinguishes them from most other viruses and demands additional effort for the successful recovery of their genomes from environmental sequence data. Here, we tested the performance of genome-resolved metagenomics on a recently isolated giant virus, Fadolivirus, by spiking it into an environmental sample from which two other giant viruses were isolated. At high spike-in levels, metagenome assembly and binning led to the successful genomic recovery of Fadolivirus from the sample. A complementary survey of the major capsid protein indicated the presence of other giant viruses in the sample matrix but did not detect the two isolated from this sample. Our results indicate that genome-resolved metagenomics is a valid approach for the recovery of near-complete giant virus genomes given that sufficient clonal particles are present. However, our data also underline that a vast majority of giant viruses remain currently undetected, even in an era of terabase-scale metagenomics.IMPORTANCE The discovery of large and giant nucleocytoplasmic large DNA viruses (NCLDV) with genomes in the megabase range and equipped with a wide variety of features typically associated with cellular organisms was one of the most unexpected, intriguing, and spectacular breakthroughs in virology. Recent studies suggest that these viruses are highly abundant in the oceans, freshwater, and soil, impact the biology and ecology of their eukaryotic hosts, and ultimately affect global nutrient cycles. Genome-resolved metagenomics is becoming an increasingly popular tool to assess the diversity and coding potential of giant viruses, but this approach is currently lacking validation.

Metavirome and its functional diversity analysis through microbiome study of the Sikkim Himalayan hot spring solfataric mud sediments.

Viruses are the most prodigious repertory of the genetic material on the earth. They are elusive, breakneck, evolutionary life particles that constitute a riveting concealed world. Environmental viruses have been obscurely explored, and hence, such an intriguing world of viruses was studied in the Himalayan Geothermal Belt of Indian peninsula at Sikkim corridor through hot springs. The hot springs located at the North Sikkim district were selected for the current study. The solfataric mud sediment samples were pooled from both the hot springs. The virus community showed significant diversity among the two hot springs of Yume Samdung. Reads for viruses among the mud sediments at Old Yume Samdung hot springs (OYS) was observed to be 11% and in the case of New Yume Samdung hot springs (NYS) it was 6%. Both the hot springs were abundant in dsDNA viromes. The metavirome reads in both the OYS and NYS hot spring mud sediments showed the predominance of Caudovirales; Herpesvirales; Ortervirales among which viral reads from Siphoviridae, Myoviridae, Phycodnaviridae and Podoviridae were abundantly present. Other viral communities belonged to families like Baculoviridae, Mimiviridae, Parvoviridae, Marseilleviridae etc. Interestingly, in the case of NYS, the unassigned group reads belonged to some unclassified giant DNA viruses like genera Pandoravirus and Pithovirus. Other interesting findings were - reads for Badnavirus having ds (RT-DNA) was exclusively found in NYS whereas Rubulavirus having ss(-)RNA was exclusively found in OYS sample. This is the first ever report on viruses from any hot springs of Sikkim till date.

Biochemical analysis of a sugar 4,6‐dehydratase from Acanthamoeba polyphaga Mimivirus

The exciting discovery of the giant DNA Mimivirus in 2003 challenged the conventional description of viruses in a radical way, and since then, dozens of additional giant viruses have been identified. It has now been demonstrated that the Mimivirus genome encodes for the two enzymes required for the production of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, namely a 4,6-dehydratase and an aminotransferase. In light of our long-standing interest in the bacterial 4,6-dehydratases and in unusual sugars in general, we conducted a combined structural and functional analysis of the Mimivirus 4,6-dehydratase referred to as R141. For this investigation, the three-dimensional X-ray structure of R141 was determined to 2.05 Å resolution and refined to an R-factor of 18.3%. The overall fold of R141 places it into the short-chain dehydrogenase/reductase (SDR) superfamily of proteins. Whereas its molecular architecture is similar to that observed for the bacterial 4,6-dehydratases, there are two key regions where the polypeptide chain adopts different conformations. In particular, the conserved tyrosine that has been implicated as a catalytic acid or base in SDR superfamily members is splayed away from the active site by nearly 12 Å, thereby suggesting that a major conformational change must occur upon substrate binding. In addition to the structural analysis, the kinetic parameters for R141 using either dTDP-d-glucose or UDP-d-glucose as substrates were determined. Contrary to a previous report, R141 demonstrates nearly identical catalytic efficiency with either nucleotide-linked sugar. The data presented herein represent the first three-dimensional model for a viral 4,6-dehydratase and thus expands our understanding of these fascinating enzymes.

Virus goes viral: an educational kit for virology classes

BackgroundViruses are the most numerous entities on Earth and have also been central to many episodes in the history of humankind. As the study of viruses progresses further and further, there are several limitations in transferring this knowledge to undergraduate and high school students. This deficiency is due to the difficulty in designing hands-on lessons that allow students to better absorb content, given limited financial resources and facilities, as well as the difficulty of exploiting viral particles, due to their small dimensions. The development of tools for teaching virology is important to encourage educators to expand on the covered topics and connect them to recent findings. Discoveries, such as giant DNA viruses, have provided an opportunity to explore aspects of viral particles in ways never seen before. Coupling these novel findings with techniques already explored by classical virology, including visualization of cytopathic effects on permissive cells, may represent a new way for teaching virology. This work aimed to develop a slide microscope kit that explores giant virus particles and some aspects of animal virus interaction with cell lines, with the goal of providing an innovative approach to virology teaching.MethodsSlides were produced by staining, with crystal violet, purified giant viruses and BSC-40 and Vero cells infected with viruses of the genera Orthopoxvirus, Flavivirus, and Alphavirus. Slides with amoebae infected with different species of giant viruses and stained with hemacolor reagents were also produced.ResultsStaining of the giant viruses allowed better visualization of the viral particles, and this technique highlights the diversity in morphology and sizes among them. Hemacolor staining enabled visualization of viral factories in amoebae, and the staining of infected BSC-40 and Vero cell monolayers with crystal violet highlights plaque-forming units.ConclusionsThis kit was used in practical virology classes for the Biological Sciences course (UFMG, Brazil), and it will soon be made available at a low-cost for elementary school teachers in institutions that have microscopes. We hope this tool will foster an inspiring learning environment.

Four high-quality draft genome assemblies of the marine heterotrophic nanoflagellate Cafeteria roenbergensis

The heterotrophic stramenopile Cafeteria roenbergensis is a globally distributed marine bacterivorous protist. This unicellular flagellate is host to the giant DNA virus CroV and the virophage mavirus. We sequenced the genomes of four cultured C. roenbergensis strains and generated 23.53 Gb of Illumina MiSeq data (99–282 × coverage per strain) and 5.09 Gb of PacBio RSII data (13–45 × coverage). Using the Canu assembler and customized curation procedures, we obtained high-quality draft genome assemblies with a total length of 34–36 Mbp per strain and contig N50 lengths of 148 kbp to 464 kbp. The C. roenbergensis genome has a GC content of ~70%, a repeat content of ~28%, and is predicted to contain approximately 7857–8483 protein-coding genes based on a combination of de novo, homology-based and transcriptome-supported annotation. These first high-quality genome assemblies of a bicosoecid fill an important gap in sequenced stramenopile representatives and enable a more detailed evolutionary analysis of heterotrophic protists.

Design of DNA Origami Diamond Photonic Crystals.

Self-assembled photonic crystals have proven to be a fascinating class of photonic materials for nonabsorbing structural colorizations over large areas and in diverse relevant applications, including tools for on-chip spectrometers and biosensors, platforms for reflective displays, and templates for energy devices. The most prevalent building blocks for the self-assembly of photonic crystals are spherical colloids and block copolymers (BCPs) because of the generic appeal of these materials, which can be crafted into large-area 3D lattices. However, because of the intrinsic limitations of these structures, these two building blocks are difficult to assemble into a direct rod-connected diamond lattice, which is considered to be a champion photonic crystal. Here, we present a DNA origami-route for a direct rod-connected diamond photonic crystal exhibiting a complete photonic bandgap (PBG) in the visible regime. Using a combination of electromagnetic, phononic, and mechanical numerical analyses, we identify (i) the structural constraints of the 50 megadalton-scale giant DNA origami building blocks that could self-assemble into a direct rod-connected diamond lattice with high accuracy, and (ii) the elastic moduli that are essentials for maintaining lattice integrity in a buffer solution. A solution molding process could enable the transformation of the as-assembled DNA origami lattice into a porous silicon- or germanium-coated composite crystal with enhanced refractive index contrast, in that a champion relative bandwidth for the photonic bandgap (i.e., 0.29) could become possible even for a relatively low volume fraction (i.e., 16 vol %).

A Puzzling Anomaly in the 4-Mer Composition of the Giant Pandoravirus Genomes Reveals a Stringent New Evolutionary Selection Process.

Pandoraviridae is a rapidly growing family of giant viruses, all of which have been isolated using laboratory strains of Acanthamoeba The genomes of 10 distinct strains have been fully characterized, reaching up to 2.5 Mb in size. These double-stranded DNA genomes encode the largest of all known viral proteomes and are propagated in oblate virions that are among the largest ever described (1.2 μm long and 0.5 μm wide). The evolutionary origin of these atypical viruses is the object of numerous speculations. Applying the chaos game representation to the pandoravirus genome sequences, we discovered that the tetranucleotide (4-mer) "AGCT" is totally absent from the genomes of 2 strains (Pandoravirus dulcis and Pandoravirus quercus) and strongly underrepresented in others. Given the amazingly low probability of such an observation in the corresponding randomized sequences, we investigated its biological significance through a comprehensive study of the 4-mer compositions of all viral genomes. Our results indicate that AGCT was specifically eliminated during the evolution of the Pandoraviridae and that none of the previously proposed host-virus antagonistic relationships could explain this phenomenon. Unlike the three other families of giant viruses (Mimiviridae, Pithoviridae, and Molliviridae) infecting the same Acanthamoeba host, the pandoraviruses exhibit a puzzling genomic anomaly suggesting a highly specific DNA editing in response to a new kind of strong evolutionary pressure.IMPORTANCE Recent years have seen the discovery of several families of giant DNA viruses infecting the ubiquitous amoebozoa of the genus Acanthamoeba With double-stranded DNA (dsDNA) genomes reaching 2.5 Mb in length packaged in oblate particles the size of a bacterium, the pandoraviruses are currently the most complex and largest viruses known. In addition to their spectacular dimensions, the pandoraviruses encode the largest proportion of proteins without homologs in other organisms, which is thought to result from a de novo gene creation process. While using comparative genomics to investigate the evolutionary forces responsible for the emergence of such an unusual giant virus family, we discovered a unique bias in the tetranucleotide composition of the pandoravirus genomes that can result only from an undescribed evolutionary process not encountered in any other microorganism.

Diversification of giant and large eukaryotic dsDNA viruses predated the origin of modern eukaryotes

Giant and large eukaryotic double-stranded DNA viruses from the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) assemblage represent a remarkably diverse and potentially ancient component of the eukaryotic virome. However, their origin(s), evolution, and potential roles in the emergence of modern eukaryotes remain subjects of intense debate. Here we present robust phylogenetic trees of NCLDVs, based on the 8 most conserved proteins responsible for virion morphogenesis and informational processes. Our results uncover the evolutionary relationships between different NCLDV families and support the existence of 2 superclades of NCLDVs, each encompassing several families. We present evidence strongly suggesting that the NCLDV core genes, which are involved in both informational processes and virion formation, were acquired vertically from a common ancestor. Among them, the largest subunits of the DNA-dependent RNA polymerase were transferred between 2 clades of NCLDVs and proto-eukaryotes, giving rise to 2 of the 3 eukaryotic DNA-dependent RNA polymerases. Our results strongly suggest that these transfers and the diversification of NCLDVs predated the emergence of modern eukaryotes, emphasizing the major role of viruses in the evolution of cellular domains.

Stable mutants of restriction-deficient/modification-proficient Bacillus subtilis 168: hub strains for giant DNA engineering.

Bacillus subtilis 168 has been explored as a platform for the synthesis and transmission of large DNA. Two inherent DNA incorporation systems, natural transformation and pLS20-based conjugation transfer, enable rapid handling of target DNA. Both systems are affected by the Bsu restriction-modification system that recognizes and cleaves unmethylated XhoI sites, limiting the choice of target DNA. We constructed B. subtilis 168 with stable mutation for restriction-deficient and modification-proficient (r-m+). It was demonstrated that the r-m+ strains can incorporate and transfer synthesized DNA with multiple XhoI sites. These should be of value as hub strains to integrate and disseminate giant DNA between B. subtilis 168 derivatives.

The Roles of Electrostatic Interactions in Capsid Assembly Mechanisms of Giant Viruses.

In the last three decades, many giant DNA viruses have been discovered. Giant viruses present a unique and essential research frontier for studies of self-assembly and regulation of supramolecular assemblies. The question on how these giant DNA viruses assemble thousands of proteins so accurately to form their protein shells, the capsids, remains largely unanswered. Revealing the mechanisms of giant virus assembly will help to discover the mysteries of many self-assembly biology problems. Paramecium bursaria Chlorella virus-1 (PBCV-1) is one of the most intensively studied giant viruses. Here, we implemented a multi-scale approach to investigate the interactions among PBCV-1 capsid building units called capsomers. Three binding modes with different strengths are found between capsomers around the relatively flat area of the virion surface at the icosahedral 2-fold axis. Furthermore, a capsomer structure manipulation package is developed to simulate the capsid assembly process. Using these tools, binding forces among capsomers were investigated and binding funnels were observed that were consistent with the final assembled capsid. In addition, total binding free energies of each binding mode were calculated. The results helped to explain previous experimental observations. Results and tools generated in this work established an initial computational approach to answer current unresolved questions regarding giant virus assembly mechanisms. Results will pave the way for studying more complicated process in other biomolecular structures.

Viva lavidaviruses! Five features of virophages that parasitize giant DNA viruses.

Viruses are obligate intracellular parasites and thus cannot replicate outside a living cell. Whereas a susceptible and permissive host cell is sufficient for most viruses to produce infectious progeny, some viruses such as satellite viruses require two different biological entities for their replication. In contrast to most satellite viruses with small RNA-based genomes, the lavidaviruses (or virophages) that replicate in eukaryotic microbes (protists) are comparable to adenoviruses in terms of particle size and genetic complexity. Lavidaviruses only replicate during a co-infection with a giant DNA virus (GV; see, e.g., [1]), and the first described virophage, Sputnik, was serendipitously discovered during a search for new GVs [2].

Horizontal Transfer of a Retrotransposon from the Rice Planthopper to the Genome of an Insect DNA Virus.

Horizontal transfer of genetic materials between virus and host has been frequently identified. Three rice planthoppers, Laodelphax striatellus, Nilaparvata lugens, and Sogatella furcifera, are agriculturally important insects because they are destructive rice pests and also the vector of a number of phytopathogenic viruses. In this study, we discovered that a small region (∼300 nucleotides [nt]) of the genome of invertebrate iridescent virus 6 (IIV-6; genus Iridovirus, family Iridoviridae), a giant DNA virus that infects invertebrates but is not known to infect planthoppers, is highly homologous to the sequences present in high copy numbers in these three planthopper genomes. These sequences are related to the short interspersed nuclear elements (SINEs), a class of non-long terminal repeat (LTR) retrotransposons (retroposons), suggesting a horizontal transfer event of a transposable element from the rice planthopper genome to the IIV-6 genome. In addition, a number of planthopper transcripts mapped to these rice planthopper SINE-like sequences (RPSlSs) were identified and appear to be transcriptionally regulated along the different developmental stages of planthoppers. Small RNAs derived from these RPSlSs are predominantly 26 to 28 nt long, which is a typical characteristic of PIWI-interacting RNAs. Phylogenetic analysis suggests that IIV-6 acquires a SINE-like retrotransposon from S. furcifera after the evolutionary divergence of the three rice planthoppers. This study provides further examples of the horizontal transfer of an insect transposon to virus and suggests the association of rice planthoppers with iridoviruses in the past or present.IMPORTANCE This study provides an example of the horizontal transfer event from a rice planthopper genome to an IIV-6 genome. A small region of the IIV-6 genome (∼300 nt) is highly homologous to the sequences presented in high copy numbers of three rice planthopper genomes that are related to the SINEs, a class of retroposons. The expression of these planthopper SINE-like sequences was confirmed, and corresponding Piwi-interacting RNA-like small RNAs were identified and comprehensively characterized. Phylogenetic analysis suggests that the giant invertebrate iridovirus IIV-6 obtains this SINE-related sequence from Sogatella furcifera through a horizontal transfer event in the past. To the best of our knowledge, this is the first report of a horizontal transfer event between a planthopper and a giant DNA virus and also is the first evidence for the eukaryotic origin of genetic material in iridoviruses.

Repulsive/attractive interaction among compact DNA molecules as judged through laser trapping: difference between linear- and branched-chain polyamines

It is well known that polyamines induce a folding transition from an elongated coil to a compact globule state for giant DNA larger than several tens of kbp (kilo base pairs). Here, we studied the interaction between compact DNA molecules in the presence of linear and branched-chain isomers of polyamines. We compared the stability of the assembly among plural number of compact DNA molecules generated by laser trapping. As a result, the assembly of compact DNAs with a linear-chain polyamine is stable even after the laser is switched off. On the other hand, the assembly of DNAs with a branched-chain polyamine disperses into individual compact DNAs when the laser is switched off. Thus, compact DNAs with linear- and branched-chain polyamines attract and repel each other, respectively. This difference in the effects of linear and branched polyamines is discussed in terms of the steric interaction between negatively charged double-strand DNA and cationic polyamines.

Mimivirus-Encoded Nucleotide Translocator VMC1 Targets the Mitochondrial Inner Membrane

Mimivirus (Acanthamoeba polyphaga mimivirus) was the first giant DNA virus identified in an amoeba species. Its genome contains at least 979 genes. One of these, L276, encodes a nucleotide translocator with similarities to mitochondrial metabolite carriers, provisionally named viral mitochondrial carrier 1 (VMC1). In this study, we investigated the intracellular distribution of VMC1 upon expression in HeLa cells and in the yeast Saccharomyces cerevisiae. We found that VMC1 is specifically targeted to mitochondria and to the inner mitochondrial membrane. Newly synthesized VMC1 binds to the mitochondrial outer-membrane protein Tom70 and translocates through the import channel formed by the β-barrel protein Tom40. Derivatization of the four cysteine residues inside Tom40 by N-ethylmaleimide caused a delay in translocation but not a complete occlusion. Cell viability was not reduced by VMC1. Neither the mitochondrial membrane potential nor the intracellular production of reactive oxygen species was affected. Similar to endogenous metabolite carriers, mimivirus-encoded VMC1 appears to act as a specific translocator in the mitochondrial inner membrane. Due to its permeability for deoxyribonucleotides, VMC1 confers to the mitochondria an opportunity to contribute nucleotides for the replication of the large DNA genome of the virus.

Structural basis for the inhibition of poxvirus assembly by the antibiotic rifampicin

Poxviruses are large DNA viruses that cause disease in animals and humans. They differ from classical enveloped viruses, because their membrane is acquired from cytoplasmic membrane precursors assembled onto a viral protein scaffold formed by the D13 protein rather than budding through cellular compartments. It was found three decades ago that the antibiotic rifampicin blocks this process and prevents scaffold formation. To elucidate the mechanism of action of rifampicin, we have determined the crystal structures of six D13-rifamycin complexes. These structures reveal that rifamycin compounds bind to a phenylalanine-rich region, or F-ring, at the membrane-proximal opening of the central channel of the D13 trimer. We show by NMR, surface plasmon resonance (SPR), and site-directed mutagenesis that A17, a membrane-associated viral protein, mediates the recruitment of the D13 scaffold by also binding to the F-ring. This interaction is the target of rifampicin, which prevents A17 binding, explaining the inhibition of viral morphogenesis. The F-ring of D13 is both conserved in sequence in mammalian poxviruses and essential for interaction with A17, defining a target for the development of assembly inhibitors. The model of the A17-D13 interaction describes a two-component system for remodeling nascent membranes that may be conserved in other large and giant DNA viruses.

Far rapid synthesis of giant DNA in the Bacillus subtilis genome by a conjugation transfer system

Bacillus subtilis offers a platform for giant DNA synthesis, which is mediated by the connection of overlapping DNA segments called domino DNA, in the cloning locus of the host. The domino method was successfully used to produce DNA fragments as large as 3500 kbp. However, domino DNA is limited to <100 kbp because of size restrictions regarding the transformation (TF) of B. subtilis competent cells. A novel conjugal transfer (CT) method was designed to eliminate the TF size limit. The CT method enables rapid and efficient domino reactions in addition to the transfer of giant DNA molecules of up to 875 kbp to another B. subtilis genome within 4 hours. The combined use of the TF and CT should enable significantly rapid giant DNA production.