Inactivation of SARS-CoV-2 in Serum Using Physical Methods.

Since 2019, many studies on coronavirus disease 2019, which has caused extensive damage as a pandemic, have been ongoing on a global scale. These include serological and biochemical studies using sera from patients and animal models. Testing with these sera must be performed after inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Heat treatment, UV irradiation, and/or gamma-ray irradiation have been used to inactivate viruses in the serum. Determining the inactivation conditions that ensure the inactivation of viruses and minimize the effect on test results after inactivation is important to ensure worker safety and the accuracy of test results. In this study, serum samples containing SARS-CoV-2 were subjected to heat, UV irradiation, and gamma irradiation to determine optimal inactivation conditions. The viral titers were below the detection limit after heating at 56°C for 1 h or 60°C for 15 min, UV-B irradiation with a transilluminator for 30 min, or gamma-ray irradiation with 60 Co at 10 kGy. These results provide useful information for safe serological and biochemical experiments.

Acetylation of the nuclear localization signal in Ku70 diminishes the interaction with importin-α.

Proteins are functionally regulated by various types of posttranslational modifications (PTMs). Ku, a heterodimer complex of Ku70 and Ku80 subunits, participates in DNA repair processes. Ku is distributed not only in the nucleus but also in the cytoplasm, suggesting that the function of Ku is regulated by its subcellular localization. Although Ku70 undergoes PTMs including phosphorylation or acetylation, it remains unknown whether the PTMs of Ku70 affect the subcellular localization of Ku. Using a cell-free pull-down assay technique, we show that Nε-acetylation of lysine residues in the synthetic peptide matched to Ku70's nuclear localization signal (NLS) reduces the peptide's interaction with the nuclear transport factor importin-α. The reduced interaction by acetylation was supported by molecular simulation analysis. In addition, when expressed in the endogenous Ku80-defective Chinese hamster ovary xrs-6 cells, some full-size human Ku70 mutants with substitutions of glutamine, a possible structural mimetic of Nε-acetyl-lysine, for lysine at the specific NLS positions exhibited no nuclear distribution. These findings imply that acetylation of particular lysine residues in the Ku70 NLS regulates nuclear localization of Ku.

Bitterness compounds in coffee brew measured by analytical instruments and taste sensing system.

We investigated the bitter compounds in coffee brews using multivariate analysis of the data obtained from analytical instrument and electronic taste sensor experiments. Coffee brews were prepared from coffee beans roasted to four different degrees. Each brew was fractionated into four fractions by liquid-liquid extraction. The relative amounts of 30 compounds in each fraction were analyzed by analytical instruments, and the bitterness response value of each fraction was analyzed by a taste sensor. Candidate bitter compounds in the coffee brews were identified with reference to their variable importance in projection and by coefficient of projection to latent structure regression (PLS-R) analysis. PLS-R analysis suggested that nicotinic acid, l-lactic acid, and nicotinamide contributed to the bitterness of the coffee brews. In fact, the coffee brews with added nicotinic acid, l-lactic acid, and nicotinamide had an increased bitterness response value compared to those without.

Artificial immunoglobulin light chain with potential to associate with a wide variety of immunoglobulin heavy chains.

Immunoglobulins play important roles in antigen recognition during the immune response, and the complementarity-determining region (CDR) 3 of the heavy chain is considered as the critical antigen-binding site. We previously developed a statistical protocol for the extensive analysis of heavy chain variable region repertoires and the dynamics of their immune response using next-generation sequencing (NGS). The properties of important antibody heavy chains predicted in silico by the protocol were examined by gene synthesis and antibody protein expression; however, the corresponding light chain that matches with the heavy chain could not be predicted by our protocol. To understand the dynamics of the heavy chain and the effect of light chain pairing on it, we firstly tried to obtain an artificial light chain that pairs with a broad range of heavy chains and then analyzed its effect on the antigen binding of heavy chains upon pairing. During the pre-B cell stage, the surrogate light chain (SLC) could pair with the nascent immunoglobulin µ heavy chains (Ig-µH) and promote them to function in the periphery. On the basis of this property, we designed several versions of genetically engineered "common light chain" prototypes by modifying the SLC structure. Among them, the mouse-derived VpreB1λ5Cκ light chain showed acceptable matching property with several different heavy chains without losing specificity of the original heavy chains, though the antigen affinities were variable. The extent of matching depended on the heavy chain; surprisingly, a specific heavy chain (IGHV9-3) could match with two different conventional Vκs (IGKV3-2*01 and IGKV10-96*01) without losing the antigen affinities, whereas another heavy chain (IGHV1-72) completely lost its antigen affinities by the same matching. Thus, the results suggested that the antigen recognition of the heavy chain is variably affected by the paired light chain, and that the artificial light chain, Mm_VpreB1λ5Cκ, has the potential to be a "common light chain", providing a novel system to analyze the effects of light chains in antigen recognition of heavy chains.

Enzymatic activity necessary to restore the lethality due to Escherichia coli RNase E deficiency is distributed among bacteria lacking RNase E homologues.

Escherichia coli RNase E (Eco-RNase E), encoded by rne (Eco-rne), is considered the global RNA decay initiator. Although Eco-RNase E is an essential gene product in E. coli, some bacterial species, such as Bacillus subtilis, do not possess Eco-RNase E sequence homologues. B. subtilis instead possesses RNase J1/J2 (Bsu-RNase J1/J2) and RNase Y (Bsu-RNase Y) to execute RNA decay. Here we found that E. coli lacking the Eco-rne gene (Δrne E. coli) was viable conditional on M9 minimal media by introducing Bsu-RNase J1/J2 or Bsu-RNase Y. We also cloned an extremely short Eco-RNase E homologue (Wpi-RNase E) and a canonical sized Bsu-RNase J1/J2 homologue (Wpi-RNase J) from Wolbachia pipientis, an α-proteobacterial endosymbiont of arthropods. We found that Wpi-RNase J restored the colony-forming ability (CFA) of Δrne E. coli, whereas Wpi-RNase E did not. Unexpectedly, Wpi-RNase E restored defective CFA due to lack of Eco-RNase G, a paralogue of Eco-RNase E. Our results indicate that bacterial species that lack Eco-RNase E homologues or bacterial species that possess Eco-RNase E homologues which lack Eco-RNase E-like activities have a modest Eco-RNase E-like function using RNase J and/or RNase Y. These results suggest that Eco-RNase E-like activities might distribute among a wide array of bacteria and that functions of RNases may have changed dynamically during evolutionary divergence of bacterial lineages.

PpsA-mediated alternative pathway to complement RNase E essentiality in Escherichia coli.

Escherichia coli cells require RNase E, encoded by the essential gene rne, to propagate. The growth properties on different carbon sources of E. coli cells undergoing suppression of RNase E production suggested that reduction in RNase E is associated with decreased expression of phosphoenolpyruvate synthetase (PpsA), which converts pyruvate to phosphoenolpyruvate during gluconeogenesis. Western blotting and genetic complementation confirmed the role of RNase E in PpsA expression. Adventitious ppsA overexpression from a multicopy plasmid was sufficient to restore colony formation of ∆rne E. coli on minimal media containing glycerol or succinate as the sole carbon source. Complementation of ∆rne by ppsA overproduction was observed during growth on solid media but was only partial, and bacteria showed slowed cell division and grew as filamentous chains. We found that restoration of colony-forming ability by ppsA complementation occurred independent of the presence of endogenous RNase G or second-site suppressors of RNase E essentiality. Our investigations demonstrate the role of phosphoryl transfer catalyzable by PpsA as a determinant of RNase E essentiality in E. coli.

Inactivation of Bombyx mori macula-like virus under physical conditions.

The Bombyx mori macula-like virus (BmMLV) is a member of the genus Maculavirus, family Tymoviridae, and contains a positive-sense single-stranded RNA genome. Previously, we reported that almost all B. mori-derived cell lines have already been contaminated with BmMLV via an unknown infection route. Since B. mori-derived cell lines are used for the baculovirus expression vector system, the invasion of BmMLV will cause a serious safety risk in the production of recombinant proteins. In this study, to determine the inactivation effectiveness of BmMLV, viruses were treated with various temperatures as well as gamma and ultraviolet (UV) light radiation. After these treatments, the virus solutions were inoculated into BmMLV-free BmVF cells. At 7 days postinoculation, the amount of virus in cells was evaluated by real-time reverse transcription PCR. Regarding heat treatment, conditions under 56°C for 3 h were tolerated, whereas infectivity disappeared after treatment at 75°C for 1 h. Regarding gamma radiation treatment, viruses were relatively stable at 1 kGy; however, their infectivity was entirely eliminated at a dose of 10 kGy. With 254 nm UV-C treatment, viruses were still active at less than 120 mJ/cm(2); however, their infectivity was completely lost at greater than 140 mJ/cm(2) UV-C radiation. These results provide quantitative evidence of the potential for BmMLV inactivation under a variety of physical conditions.

In vivo evidence that phenylalanine 171 acts as a molecular brake for translesion DNA synthesis across benzo[a]pyrene DNA adducts by human DNA polymerase κ.

Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG. In the present study, we established human cell lines that express wild-type Pol κ (POLK+/-), F171A (POLK F171A/-) or lack expression of Pol κ (POLK-/-) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (-)- or (+)-trans-anti-BPDE-N(2)-dG in the supF gene. The frequencies of mutations were in the order of POLK-/->POLK+/->POLK F171A/- both in (-)- and (+)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N(2)-dG by Pol κ and raise the possibility that the cognate substrates for Pol κ are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens.

Lipid transfer particle from the silkworm, Bombyx mori, is a novel member of the apoB/large lipid transfer protein family.

Lipid transfer particle (LTP) is a high-molecular-weight, very high-density lipoprotein known to catalyze the transfer of lipids between a variety of lipoproteins, including both insects and vertebrates. Studying the biosynthesis and regulation pathways of LTP in detail has not been possible due to a lack of information regarding the apoproteins. Here, we sequenced the cDNA and deduced amino acid sequences for three apoproteins of LTP from the silkworm (Bombyx mori). The three subunit proteins of the LTP are coded by two genes, apoLTP-II/I and apoLTP-III. ApoLTP-I and apoLTP-II are predicted to be generated by posttranslational cleavage of the precursor protein, apoLTP-II/I. Clusters of amphipathic secondary structure within apoLTP-II/I are similar to Homo sapiens apolipoprotein B (apoB) and insect lipophorins. The apoLTP-II/I gene is a novel member of the apoB/large lipid transfer protein gene family. ApoLTP-III has a putative conserved juvenile hormone-binding protein superfamily domain. Expression of apoLTP-II/I and apoLTP-III genes was synchronized and both genes were primarily expressed in the fat body at the stage corresponding to increased lipid transport needs. We are now in a position to study in detail the physiological role of LTP and its biosynthesis and assembly.

A possible overestimation of the effect of acetylation on lysine residues in KQ mutant analysis.

Acetylation of lysine residues, one of the most common protein post-transcriptional modifications, is thought to regulate protein affinity with other proteins or nucleotides. Experimentally, the effects of acetylation have been studied using recombinant mutants in which lysine residues (K) are substituted with glutamine (Q) as a mimic of acetyl lysine (KQ mutant), or with arginine (R) as a mimic of nonacetylated lysine (KR mutant). These substitutions, however, have not been properly validated. The effects lysine acetylation on Ku, a multifunctional protein that has been primarily implicated in DNA repair and cell survival, are characterized herein using a series of computer simulations. The binding free energy was reduced in the KQ mutant, while the KR mutant had no effect, which is consistent with previous experimental results. Unexpectedly, the binding energy between Ku and DNA was maintained at almost the same level as in the wild type protein despite full acetylation of the lysine residues. These results suggest that the effects of acetylation may be overestimated when the KQ mutant is used as a mimic of the acetylated protein.

Diversity in copy number and structure of a silkworm morphogenetic gene as a result of domestication.

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time.

Phenylalanine 171 is a molecular brake for translesion synthesis across benzo[a]pyrene-guanine adducts by human DNA polymerase kappa.

Human cells possess multiple specialized DNA polymerases (Pols) that bypass a variety of DNA lesions which otherwise would block chromosome replication. Human polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. To better understand the relationship between the structural features in the active site and lesion bypass by Pol κ, we mutated codons corresponding to amino acids appearing close to the adducts in the active site, and compared bypass efficiencies. Remarkably, the substitution of alanine for phenylalanine 171 (F171), an amino acid conserved between Pol κ and its bacterial counterpart Escherichia coli DinB, enhanced the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG 18-fold. This substitution affected neither the fidelity of TLS nor the efficiency of dCMP incorporation opposite normal guanine. This amino acid change also enhanced the binding affinity of Pol κ to template/primer DNA containing (-)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 functions as a molecular brake for TLS across BPDE-N(2)-dG by Pol κ and that the F171A derivative of Pol κ bypasses these DNA lesions more actively than does the wild-type enzyme.

Apolipophorin-III expression and low density lipophorin formation during embryonic development of the silkworm, Bombyx mori.

We examined the expression of apolipophorin-III (apoLp-III) during embryonic development of the silkworm Bombyx mori. ApoLp-III mRNA was first expressed 24h after oviposition, which corresponds to the time of germ band formation. The amount of apoLp-III in the eggs increased from day 2, peaked on day 4, and then gradually decreased until hatching (on day 9.5). ApoLp-III was apparently synthesized during early embryogenesis, as radioactive amino acids were incorporated into newly synthesized apoLp-III in three-day-old eggs. Moreover, radioactive apoLp-III was found only in the embryo and not in the extraembryonic tissue. KBr density gradient ultracentrifugation of egg homogenates showed that apoLp-III was associated with low-density lipophorin (LDLp). These results suggest that LDLp is required for the delivery of lipids for organogenesis during embryogenesis.

A CD36-related transmembrane protein is coordinated with an intracellular lipid-binding protein in selective carotenoid transport for cocoon coloration.

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.

Critical amino acids in human DNA polymerases eta and kappa involved in erroneous incorporation of oxidized nucleotides.

Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Poleta and Polkappa proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Poleta was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Poleta) to 7 : 1 (R61K). Similarly, Tyr112 in Polkappa was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polkappa) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Poleta, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Poleta and Polkappa might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.

Purification and expression analysis of imaginal disc growth factor in the silkworm, Bombyx mori.

In the present study, we purified and sequenced a homolog of the Drosophila imaginal disc growth factor (IDGF) from the hemolymph of Bombyx mori (BmIDGF). Antibodies against BmIDGF were produced and subsequently used in immunoblotting analyses. The immunoblotting analyses demonstrated an extremely high level of BmIDGF in the hemolymph throughout the period of rapid growth of the organs of B. mori. The results of RT-PCR showed that BmIDGF was predominantly expressed in fat bodies. Real-time RT-PCR revealed that BmIDGF transcripts in fat bodies were highly expressed during the feeding stage but significantly suppressed during the molting, wandering, and pupal stages. Starvation brought about a significant decline of BmIDGF mRNAs in the fat bodies and BmIDGF proteins in the hemolymph. After re-feeding, the BmIDGF transcripts in fat bodies and BmIDGF proteins in the hemolymph increased again. In addition, an immunocytochemical study revealed BmIDGF proteins on the surface of wing discs. The present findings suggest that the level of BmIDGF in the hemolymph was modulated by the fat body in response to nutritional conditions and that BmIDGF was transported to target organs through the hemolymph.

The steric gate amino acid tyrosine 112 is required for efficient mismatched-primer extension by human DNA polymerase kappa.

Human DNA is continuously damaged by exogenous and endogenous genotoxic insults. To counteract DNA damage and ensure the completion of DNA replication, cells possess specialized DNA polymerases (Pols) that bypass a variety of DNA lesions. Human DNA polymerase kappa (hPolkappa) is a member of the Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolkappa is characterized by its ability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) and thymine glycol] and efficiently extend primers with mismatches at the termini. hPolkappa is structurally distinct from E. coli DinB in that it possesses an approximately 100-amino acid extension at the N-terminus. Here, we report that tyrosine 112 (Y112), the steric gate amino acid of hPolkappa, which distinguishes dNTPs from rNTPs by sensing the 2'-hydroxy group of incoming nucleotides, plays a crucial role in extension reactions with mismatched primer termini. When Y112 was replaced with alanine, the amino acid change severely reduced the catalytic constant, i.e., k(cat), of the extending mismatched primers and lowered the efficiency, i.e., k(cat)/K(m), of this process by approximately 400-fold compared with that of the wild-type enzyme. In contrast, the amino acid replacement did not reduce the insertion efficiency of dCMP opposite BPDE-N(2)-dG in template DNA, nor did it affect the ability of hPolkappa to bind strongly to template-primer DNA with BPDE-N(2)-dG/dCMP. We conclude that the steric gate of hPolkappa is a major fidelity factor that regulates extension reactions from mismatched primer termini.

Carotenoid silk coloration is controlled by a carotenoid-binding protein, a product of the Yellow blood gene.

Mechanisms for the uptake and transport of carotenoids, essential nutrients for humans, are not well understood in any animal system. The Y (Yellow blood) gene, a critical cocoon color determinant in the silkworm Bombyx mori, controls the uptake of carotenoids into the intestinal mucosa and the silk gland. Here we provide evidence that the Y gene corresponds to the intracellular carotenoid-binding protein (CBP) gene. In the Y recessive strain, the absence of an exon, likely due to an incorrect mRNA splicing caused by a transposon-associated genomic deletion, generates a nonfunctional CBP mRNA, resulting in colorless hemolymph and white cocoons. Enhancement of carotenoid uptake and coloration of the white cocoon was achieved by germ-line transformation with the CBP gene. This study demonstrates the existence of a genetically facilitated intracellular process beyond passive diffusion for carotenoid uptake in the animal phyla, and paves the way for modulating silk color and lipid content through genetic engineering.

Molecular dynamics simulation of clustered DNA damage sites containing 8-oxoguanine and abasic site.

Clustered DNA damage sites induced by ionizing radiation have been suggested to have serious consequences to organisms, such as cancer, due to their reduced probability to be repaired by the enzymatic repair machinery of the cell. Although experimental results have revealed that clustered DNA damage sites effectively retard the efficient function of repair enzymes, it remains unclear as to what particular factors influence this retardation. In this study, approaches based on molecular dynamics (MD) simulation have been applied to examine conformational changes and energetic properties of DNA molecules containing clustered damage sites consisting of two lesioned sites, namely 7,8-dihydro-8-oxoguanine (8-oxoG) and apurinic/apyrimidinic (AP) site, located within a few base pairs of each other. After 1 ns of MD simulation, one of the six DNA molecules containing a clustered damage site develops specific characteristic features: sharp bending at the lesioned site and weakening or complete loss of electrostatic interaction energy between 8-oxoG and bases located on the complementary strand. From these results it is suggested that these changes would make it difficult for the repair enzyme to bind to the lesions within the clustered damage site and thereby result in a reduction of its repair capacity.

Integration of the 5' end of the retrotransposon, R2Bm, can be complemented by homologous recombination.

R2Bm is a non-long-terminal-repeat (non-LTR) retrotransposon that was identified at a specific target site in the 28S rRNA genes of the silkworm, Bombyx mori. Although in vitro analysis has revealed that the 3' end of R2Bm is integrated into the target site by means of target-primed reverse transcription (TPRT), the mechanism of the 5' end integration is not well understood. We established a novel in vivo system to assay the insertion mechanism of R2Bm using a cultured cell line, C65, and a baculovirus, AcNPV, as host and vector, respectively. The 3' end of R2Bm integrated at the target site in the rRNA genes of C65 cells when an AcNPV containing both the full-length 3' UTR and the entire open reading frame (ORF) of R2Bm was introduced while the 5' end integration was incorrect. The 5' end of R2Bm was integrated, however, when the 28S gene sequence upstream of the R2Bm target site was added to the R2Bm sequence. Thus, in our assay, homologous sequences were likely essential for the successful integration of the entire R2Bm into the host cell genome. We also demonstrated that the failure to integrate caused by a frame-shifted ORF was rescued by co-infection with a helper virus that contained only the R2Bm ORF. This indicates that R2 retrotransposition can be complemented in trans. These findings suggest that the host's mechanism for DNA repair may be necessary for the integration of the 5' end of R2Bm and that R2Bm protein may only have the ability to integrate the 3' end of the element by TPRT.