Chimeric Gene Research Articles

Reliable Scarless Fusion of Genes through Overlap Extension PCR to Develop Chimeric Genes

Reliable Scarless Fusion of Genes through Overlap Extension PCR to Develop Chimeric Genes

Detection of neural connections with ex vivo MRI using a ferritin-encoding trans-synaptic virus

The elucidation of neural networks is essential to understanding the mechanisms of brain functions and brain disorders. Neurotropic virus-based trans-synaptic tracing tools have become an effective method for dissecting the structure and analyzing the function of neural-circuitry. However, these tracing systems rely on fluorescent signals, making it hard to visualize the panorama of the labeled networks in mammalian brain in vivo. One MRI method, Diffusion Tensor Imaging (DTI), is capable of imaging the networks of the whole brain in live animals but without information of anatomical connections through synapses. In this report, a chimeric gene coding for ferritin and enhanced green fluorescent protein (EGFP) was integrated into Vesicular stomatitis virus (VSV), a neurotropic virus that is able to spread anterogradely in synaptically connected networks. After the animal was injected with the recombinant VSV (rVSV), rVSV-Ferritin-EGFP, into the somatosensory cortex (SC) for four days, the labeled neural-network was visualized in the postmortem whole brain with a T2-weighted MRI sequence. The modified virus transmitted from SC to synaptically connected downstream regions. The results demonstrate that rVSV-Ferritin-EGFP could be used as a bimodal imaging vector for detecting synaptically connected neural-network with both ex vivo MRI and fluorescent imaging. The strategy in the current study has the potential to longitudinally monitor the global structure of a given neural-network in living animals.

Expression of Hemagglutinin-Neuraminidase (HN) and Fusion (F) Epitopes of Newcastle Disease Virus (NDV) in Chlamydomonas reinhardtii

Owing to their unique characteristics which combines the properties of both prokaryotes and eukaryotes, microalgae have emerged as an ideal platform for heterologous production of recombinant proteins including subunit vaccines. In an attempt to develop recombinant vaccine against Newcastle Disease, an agrobacterium-mediated genetic transformation was carried out to express a chimeric gene construct including Hemagglutinin-Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) in Chlamydomonas reinhardtii. Four tandem repeat of HN epitope with 96bp length followed by three tandem repeat of F epitope of NDV with 153bp length were used. Microalgal cells (Chlamydomonas reinhardtii) were co-cultivated with Agrobacterium tumefaciens cells harboring foreign gene construct and then transferred to selection medium. Single colonies representing putative transformation events were screened in selection medium enriched with kanamycin. PCR assay confirmed integration of F-HN sequence in microalgal nuclei. RT-PCR assay showed that the F-HN sequence was expressed in transformed colonies. Finally, translation of the foreign gene was confirmed by protein dot blotting, western blot and Elisa assay. The results of this experiment may contain both research and practical implications.

CCDC6, a gene product in fusion with different protoncogenes, as a potential chemotherapeutic target.

Cancer, a deadly disease is characterized by abnormal cell growth with the potential to invade to other parts of the body. Most cancers start due to changes at gene level that happen over a person's lifetime when DNA repair system becomes faulty. CCDC6, one of the players in DNA repair system acts as a tumor suppressor gene. It was originally identified in chimeric genes caused by chromosomal translocation involving RET proto-oncogene in some thyroid tumors. Different fusion chimers with different proto-oncogenes like RET are known for CCDC6 which hampered its function. Further, CCDC6 is recognized as a pro-apoptotic phosphoprotein, which is an ATM substrate responsive to genotoxic stress. In this article, we reviewed the published literature to characterize CCDC6 fusions with proto-oncogenes and the role of natural phytochemicals which can potentially alter CCDC6 activity and thus can prove beneficial for cancer patients.

Elucidating genomic patterns and recombination events in plant cybrid mitochondria.

Cybrid plant mitochondria undergo homologous recombination, mainly BIR, keep a single allele for each gene, and maintain exclusive sequences of each parent and a single copy of the homologous regions. The maintenance of a dynamic equilibrium between the mitochondrial and nuclear genomes requires continuous communication and a high level of compatibility between them, so that alterations in one genetic compartment need adjustments in the other. The co-evolution of nuclear and mitochondrial genomes has been poorly studied, even though the consequences and effects of this interaction are highly relevant for human health, as well as for crop improvement programs and for genetic engineering. The mitochondria of plants represent an excellent system to understand the mechanisms of genomic rearrangements, chimeric gene formation, incompatibility between nucleus and cytoplasm, and horizontal gene transfer. We carried out detailed analyses of the mtDNA of a repeated cybrid between the solanaceae Nicotiana tabacum and Hyoscyamus niger. The mtDNA of the cybrid was intermediate between the size of theparental mtDNAs and the sum of them. Noticeably, most of the homologous sequences inherited from both parents were lost. In contrast, the majority of the sequences exclusive of a single parent were maintained. The mitochondrial gene content included a majority of N. tabacum derived genes, but also chimeric, two-parent derived, and H. niger-derived genes in a tobacco nuclear background. Any of these alterations in the gene content could be the cause of CMS in the cybrid. The parental mtDNAs interacted through 28 homologous recombination events and a single case of illegitimate recombination. Three main homologous recombination mechanisms were recognized in the cybrid mitochondria. Break induced replication (BIR) pathway was the most frequent. We propose that BIR could be one of the mechanisms responsible for the loss of the majority of the repeated regions derived from H. niger.

Dissecting structure/function relationship of Streptococcus mutans membrane protein chaperones/insertases, YidC1 and YidC2

BackgroundApproximately, one third of most bacterial open reading frames encode membrane proteins; therefore, understanding the machinery that translocates these proteins to their functional location is of great interest. Most membrane proteins are translocated co‐translationally by the signal recognition particle (SRP) pathway via the SecYEG translocon. Additionally the Oxa/Alb/YidC (mitochondria/chloroplast/bacteria) family of membrane‐localized protein chaperone/insertases assists in membrane protein translocation in a Sec‐dependent manner, or independently if the proteins are small with two or fewer transmembrane (TM) domains. Most gram‐negative bacteria contain a single yidC whose deletion is lethal. Most gram‐positive bacteria contain two or more YidCs. The cariogenic bacterium S. mutans encodes YidC1 and YidC2, each with similar 5 TM domain topologies. Deletion of yidC2 results in a severe phenotype similar to disruption of the SRP pathway including impaired growth, sensitivity to acid, osmotic, and oxidative stresses, and loss of competence and mutacin production. Deletion of DyidC1 leads to increased localization of surface proteins and hyperadherence. The goal of this study is to better understand the structure and function of YidC1 and YidC2 as related to their known phenotypes.MethodsThe significance of the two cytoplasmic loops, C1 and C2, of YidC1 and YidC2 were evaluated in domain swapping experiments in which DNA segments encoding one or both C1 and C2 domains of yidC1 were incorporated into yidC2 and vice versa. Chimeric genes were integrated into the non‐essential gtfA locus of the DyidC1 and DyidC2 strains. Next the residual endogenous yidC was deleted, thus generating strains with a sole chimeric YidC for phenotypic analyses. In addition, a naturally‐acquired W138R point mutation within yidC1 was identified in a phenotypically‐reverted DyidC2 mutant; therefore, the significance of this residue was evaluated as well.Results and ConclusionsAn S. mutans strain producing a chimeric YidC2 harboring the C1 loop of YidC1 was similar to the DyidC2 strain with respect to sensitivity to acid and osmotic stress suggesting a functional contribution of YidC2's C1 loop to stress tolerance. Transfer of YidC2 C1 into YidC1 enabled YidC1 to rescue acid and osmotic stress sensitivity. Also, a deliberately engineered W138R point mutation within YidC1 enabled it to rescue growth and stress phenotypes of the DyidC2 mutant. Sequence comparison revealed that the YidC1 tryptophan 138 residue corresponds to serine 152 in YidC2, the only polar residue within its TM3. Additionally, engineered W138K/S mutations within YidC1 also enabled rescue of DyidC2‐associated phenotypes. These data reveal that functional activities unique to YidC2 reside within both its C1 domain and TM3 that can be transferred to YidC1.Support or Funding InformationNIH grant: R01 DE08007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel in vivo system to monitor tRNA expression based on the recovery of GFP fluorescence and its application for the determination of plant tRNA expression

We developed a novel assay system to quantitatively detect amber codon suppression by tRNAs expressed in plant cells. The assay was based on recovery of the expression of the green fluorescent protein (GFP) as a reporter, in which a fourth Lys codon (AAG) was changed to a premature amber codon TAG, designated as GFP/amber. Plasmids carrying GFP/amber, suppressor tRNA, and red fluorescent protein (RFF) as an internal control, respectively, were introduced into onion epidermal cells to monitor cell numbers with GFP and RFP fluorescence. First, an amber suppressor tRNASer from tobacco (NtS2) to suppress a TAG codon in GFP mRNA was examined, leading to the recovery of GFP fluorescence. Second, we used two different tRNAs (i.e., AtY3II-am and AtY3II-amiG7), both of which are intron-containing amber suppressor tRNAsTyr, the former impaired precursor-tRNA splicing but the latter did not, as confirmed previously using two different approaches (Szeykowska-Kulinska and Beier, 1991; Akama and Beier, 2003). As expected, coexpression of GFP/amber with AtY3II-am gave no green fluorescence, but significant fluorescence was observed with AtY3II-amiG7. Then, we applied this system for the analysis of 5′-regulatory sequences of the tRNAGln gene family from Arabidopsis. A 5′-flanking sequence of each of the 17 tRNAGln genes was fused to a coding region of an amber suppressor tRNASer gene (NtS2/amber) and its 3′-flanking sequence. Chimeric tRNASer gene, GFP/amber, and RFP were coexpressed, and the GFP or RFP fluorescence intensity was determined in cells using laser-scanning microscopy. In parallel, 17 kinds of original Arabidopsis tRNAGln genes and their chimeric genes with NtS2/amber were all analyzed in cell-free nuclear extract (Yukawa et al., 1997). Comparison of in vitro and in vivo expression of these chimeric tRNA genes displayed generally similar results, accompanied by a wide range of variance in the expression of each gene. Nevertheless, the expression patterns of several genes were clearly the opposite of each other comparing between the two different system, demonstrating the importance of in vivo systems in the study on tRNA expression in plants.

Potential glycoengineered anti-Burkholderia vaccines by exploiting the bacterial O-glycosylation machinery

Previously, we discovered a protein O-glycosylation (ogc) cluster conserved in all Burkholderia species, which glycosylates proteins with a trisaccharide glycan. Sera from Burkholderia-infected patients produce anti-glycan antibodies, suggesting that the Burkholderia protein glycosylation pathway can be exploited for potential vaccine development. Here, we successfully produced two prototypes of anti-Burkholderia vaccines: a recombinant glycoprotein-based vaccine and an E. coli LPS-display vaccine. To generate the former, we constructed a plasmid carrying a chimeric gene encoding three glycosylation sequons fused to the cholera toxin B subunit. The presence of the glycan was observed in recombinant proteins expressed in B. cenocepacia parental strain, but not in proteins expressed by the glycosyltransferase-deficient ΔpglLstrain, as determined by SDS-PAGE and fluorescent lectin blots. For the development of an E. coli LPS-display vaccine, we constructed a plasmid expressing the ogc cluster, which was introduced into an E. coli strain unable to synthesize O-antigen but carrying the O-antigen ligase WaaL. Our results show that the LPS of this strain contained an additional moiety consistent with the B. cenocepacia trisaccharide glycan, as demonstrated by silver-stained LPS gels and lectin blot. This extra moiety was not detected in aΔwaaL mutant. These results suggest that the plasmid was able to provide the necessary functions for the synthesis and membrane translocation of the lipid-linked trisaccharide, which became a substrate for the WaaL ligase and incorporation into the E. coli LPS. Therefore, we demonstrate that the O-glycosylation pathway can be manipulated for the construction of potential anti-Burkholderia vaccines.

Gene expression profiles of human granulosa cells treated with bioequivalent doses of corifollitropin alfa (CFA) or recombinant human follicle-stimulating hormone (recFSH)

Using recombinant DNA technologies, a chimeric gene containing the coding sequences of follicle stimulating hormone (FSH) β-subunit and C-terminal peptide of the human chorionic gonadotrophin (hCG) β-subunit have been designed to generate a new gonadotrophin named corifollitropin alfa (CFA). CFA has longer elimination half-life and slower rate of absorption compared with FSH, which makes CFA a long-acting hormone employed as a substitute of the recombinant FSH (recFSH) in the controlled ovarian stimulation (COS). The purpose of this study is to compare the gene expression profiles elicited by bioequivalent doses of CFA or recFSH in primary cultures of human granulosa cells (hGCs). Gonadotrophins exert their functions by binding FSH receptors (FSHRs), activating signaling pathways that increase the cyclic adenosine monophosphate (cAMP) intracellular content. Bioequivalence has been defined as the dose/duration of gonadotrophin treatment able to promote the same amount of intracellular cAMP. hGCs were treated with different doses of either gonadotrophin and the cAMP was measured after different incubation times to establish the bioequivalence. Results obtained by comparing the bioequivalent treatments, showed that CFA is more effective than recFSH in inducing aromatase gene expression after 6 and 24 h from the initial stimulation in agreement with its long-acting characteristic.

Rotavirus Infection Alters Splicing of the Stress-Related Transcription Factor XBP1.

XBP1 is a stress-regulated transcription factor also involved in mammalian host defenses and innate immune response. Our investigation of XBP1 RNA splicing during rotavirus infection revealed that an additional XBP1 RNA (XBP1es) that corresponded to exon skipping in the XBP1 pre-RNA is induced depending on the rotavirus strain used. We show that the translation product of XBP1es (XBP1es) has trans-activation properties similar to those of XBP1 on ER stress response element (ERSE) containing promoters. Using monoreassortant between ES+ ("skipping") and ES- ("nonskipping") strains of rotavirus, we show that gene 7 encoding the viral translation enhancer NSP3 is involved in this phenomenon and that exon skipping parallels the nuclear relocalization of cytoplasmic PABP. We further show, using recombinant rotaviruses carrying chimeric gene 7, that the ES+ phenotype is linked to the eIF4G-binding domain of NSP3. Because the XBP1 transcription factor is involved in stress and immunological responses, our results suggest an alternative way to activate XBP1 upon viral infection or nuclear localization of PABP.IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. Here we show that infection with several rotavirus strains induces an alternative splicing of the RNA encoding the stressed-induced transcription factor XBP1. The genetic determinant of XBP1 splicing is the viral RNA translation enhancer NSP3. Since XBP1 is involved in cellular stress and immune responses and since the XBP1 protein made from the alternatively spliced RNA is an active transcription factor, our observations raise the question of whether alternative splicing is a cellular response to rotavirus infection.

Molecular and Functional Evolution at the Odorant Receptor Or22 Locus in Drosophila melanogaster.

Insect odorant receptor (Or) genes determine the responses of sensory neurons that mediate critical behaviors. The Drosophila melanogaster Or22 locus represents an interesting example of molecular evolution, with high levels of sequence divergence and copy number variation between D. melanogaster and other Drosophila species, and a corresponding high level of variability in the responses of the neuron it controls, ab3A. However, the link between Or22 molecular and functional diversity has not been established. Here, we show that several naturally occurring Or22 variants generate major shifts in neuronal response properties. We determine the molecular changes that underpin these response shifts, one of which represents a chimeric gene variant previously suggested to be under natural selection. In addition, we show that several alternative molecular genetic mechanisms have evolved for ensuring that where there is more than one gene copy at this locus, only one functional receptor is generated. Our data thus provide a causal link between the striking levels of phenotypic neuronal response variation found in natural populations of D. melanogaster and genetic variation at the Or22 locus. Since neuronal responses govern animal behavior, we predict that Or22 may be a key player in underlying one or more olfactory-driven behaviors of significant adaptive importance.

Development of Stable Rotavirus Reporter Expression Systems.

Engineered recombinant viruses expressing reporter genes have been developed for real-time monitoring of replication and for mass screening of antiviral inhibitors. Recently, we reported using a reverse genetics system to develop the first recombinant reporter rotaviruses (RVs) that expressed NanoLuc (NLuc) luciferase. Here, we describe a strategy for developing stable reporter RVs expressing luciferase and green or red fluorescent proteins. The reporter genes were inserted into the open reading frame of NSP1 and expressed as a fusion with an NSP1 peptide consisting of amino acids 1 to 27. The stability of foreign genes within the reporter RV strains harboring a shorter chimeric NSP1-reporter gene was greater than that of those in the original reporter RV strain, independent of the transgene inserted. The improved reporter RV was used to screen for neutralizing monoclonal antibodies (MAbs). Sequence analysis of escape mutants from one MAb clone (clone 29) identified an amino acid substitution (arginine to glycine) at position 441 in the VP4 protein, which resides within neutralizing epitope 5-1 in the VP5* fragment. Furthermore, to express a native reporter protein lacking NSP1 amino acids 1 to 27, the 5'- and 3'-terminal region sequences were modified to restore the predicted secondary RNA structure of the NSP1-reporter chimeric gene. These data demonstrate the utility of reporter RVs for live monitoring of RV infections and also suggest further applications (e.g., RV vaccine vectors, which can induce mucosal immunity against intestinal pathogens).IMPORTANCE Development of reporter RVs has been hampered by the lack of comprehensive reverse genetics systems. Recently, we developed a plasmid-based reverse genetics system that enables generation of reporter RVs expressing NLuc luciferase. The prototype reporter RV had some disadvantages (i.e., the transgene was unstable and was expressed as a fusion protein with a partial NSP1 peptide); however, the improved reporter RV overcomes these problems through modification of the untranslated region of the reporter-NSP1 chimeric gene. This strategy for generating stable reporter RVs could be expanded to diverse transgenes and be used to develop RV transduction vectors. Also, the data improve our understanding of the importance of 5'- and 3'-terminal sequences in terms of genome replication, assembly, and packaging.

Prognostic significance of chimeric fusion gene analysis in pediatric acute megakaryoblastic leukemia

Acute megakaryoblastic leukemia in children without Down syndrome (non-DS AMKL) is considered to be a poor prognostic subtype in acute myeloid leukemia. Recently, some chimeric fusion genes were found in pediatric non-DS AMKL; therefore, we attempted to detect chimeric fusion genes RBM15-MKL1, CBFA2T3-GLIS2, and NUP98-KDM5A from 10 pediatric non-DS AMKL diagnostic samples using polymerase chain reaction and Sanger sequencing methods. Two samples were positive for RBM15-MKL1, four had CBFA2T3-GLIS2, and only one case had NUP98-KDM5A. Both RBM15-MKL1-positive patients showed long-term remission after chemotherapy. The eight RBM15-MKL1-negative patients received hematopoietic stem cell transplantation (HSCT). In four CBFA2T3-GLIS2-positive patients, three had HSCT without complete remission and two of themdied. Additional treatment stratification depending on chimeric fusion genes and development of new therapeutic drugs are required for non-DS AMKL.

Characterization of the mitochondrial genome of the MAX1 type of cytoplasmic male-sterile sunflower

BackgroundMore than 70 cytoplasmic male sterility (CMS) types have been identified in Helianthus, but only for less than half of them, research of mitochondrial organization has been conducted. Moreover, complete mitochondrion sequences have only been published for two CMS sources – PET1 and PET2. It has been demonstrated that other sunflower CMS sources like MAX1, significantly differ from the PET1 and PET2 types. However, possible molecular causes for the CMS induction by MAX1 have not yet been proposed. In the present study, we have investigated structural changes in the mitochondrial genome of HA89 (MAX1) CMS sunflower line in comparison to the fertile mitochondrial genome.ResultsEight significant major reorganization events have been determined in HA89 (MAX1) mtDNA: one 110 kb inverted region, four deletions of 439 bp, 978 bp, 3183 bp and 14,296 bp, respectively, and three insertions of 1999 bp, 5272 bp and 6583 bp. The rearrangements have led to functional changes in the mitochondrial genome of HA89 (MAX1) resulting in the complete elimination of orf777 and the appearance of new ORFs - orf306, orf480, orf645 and orf1287. Aligning the mtDNA of the CMS sources PET1 and PET2 with MAX1 we found some common reorganization features in their mitochondrial genome sequences.ConclusionThe new open reading frame orf1287, representing a chimeric atp6 gene, may play a key role in MAX1 CMS phenotype formation in sunflower, while the contribution of other mitochondrial reorganizations seems to appear negligible for the CMS development.

Chronic Myeloid Leukemia: Existing Therapeutic Options and Strategies to Overcome Drug Resistance.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused due to translocation between chromosome 9 and 22 leading to a chimeric gene product known as Bcr-Abl. Bcr-Abl fusion protein has constitutively activated Abl tyrosine kinase activity which is responsible for the uncontrolled proliferation in CML The tyrosine kinase inhibitors (TKIs) such as Imatinib, Dasatinib, and Nilotinib are the current first-line treatments approved by the United States Food and Drug Administration (US FDA) for the treatment of the disease. Despite the spectacular progress made over the decade with the TKIs, patients develop resistance to these TKIs. In such cases, stem cell transplant therapy, which is limited by donor availability, is the only proven cure for the patients. This highlights the need for the development of new strategies for CML treatment. The Bcr-Abl point mutations, including the gatekeeper T315I mutations, are the principal cause for the development of resistance to TKIs. However, other mechanisms are also involved in the failure of TKI therapy. This review outlines the Bcr-Abl dependent and independent mechanism of TKIs resistance development and the strategies used to overcome drug resistance, such as the development of ATP site and allosteric site inhibitors. Binding mode and structural elements of Bcr-Abl inhibition are discussed with emphasis on pathways involved in this complex disease to determine alternative strategies and combination therapies.

A Novel Chimeric Mitochondrial Gene Confers Cytoplasmic Effects on Seed Oil Content in Polyploid Rapeseed (Brassica napus)

Cytoplasmic effects (CEs) have been discovered to influence a diverse array of agronomic traits in crops, and understanding the underlying mechanisms can help accelerate breeding programs. Seed oil content (SOC) is of great agricultural, nutritional, and economic importance. However, the genetic basis of CEs on SOC (CE-SOC) remains enigmatic. In this study, we use an optimized approach to sequence the cytoplasmic (plastid and mitochondrial) genomes of allotetraploid oilseed rape (Brassica napus) cultivars, 51218 and 56366, that bear contrasting CE-SOC. By combining comparative genomics and genome-wide transcriptome analysis, we identify mitochondria-encoded orf188 as a potential CE-SOC determinant gene. Functional analyses in the model system Arabidopsis thaliana and rapeseed demonstrated that orf188 governs CE-SOC and could significantly increase SOC, strikingly, through promoting the yield of ATP. Consistent with this finding, transcriptional profiling with microarray and RNA sequencing revealed that orf188 affects transcriptional reprogramming of mitochondrial energy metabolism to facilitate ATP production. Intriguingly, orf188 is a previously uncharacterized chimeric gene, and the presence of this genetic novelty endows rapeseed with positive CE-SOC. Our results shed light on the molecular basis of CEs on a key quantitative trait in polyploid crops and enrich the theory of maternal control of oil content, providing new scientific guidance for breeding high-oil rapeseed germplasms.

Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

Parvoviruses are highly attractive templates for the engineering of safe, efficient, and specific gene therapy vectors, as best exemplified by adeno-associated virus (AAV). Another candidate that currently garners increasing attention is human bocavirus 1 (HBoV1). Notably, HBoV1 capsids can cross-package recombinant (r)AAV2 genomes, yielding rAAV2/HBoV1 chimeras that specifically transduce polarized human airway epithelia (pHAEs). Here, we largely expanded the repertoire of rAAV/BoV chimeras, by assembling packaging plasmids encoding the capsid genes of four additional primate bocaviruses, HBoV2–4 and GBoV (Gorilla BoV). Capsid protein expression and efficient rAAV cross-packaging were validated by immunoblotting and qPCR, respectively. Interestingly, not only HBoV1 but also HBoV4 and GBoV transduced pHAEs as well as primary human lung organoids. Flow cytometry analysis of pHAEs revealed distinct cellular specificities between the BoV isolates, with HBoV1 targeting ciliated, club, and KRT5+ basal cells, whereas HBoV4 showed a preference for KRT5+ basal cells. Surprisingly, primary human hepatocytes, skeletal muscle cells, and T cells were also highly amenable to rAAV/BoV transduction. Finally, we adapted our pipeline for AAV capsid gene shuffling to all five BoV isolates. Collectively, our chimeric rAAV/BoV vectors and bocaviral capsid library represent valuable new resources to dissect BoV biology and to breed unique gene therapy vectors.

RNA Polymerase II-Dependent Transcription Initiated by Selectivity Factor 1: A Central Mechanism Used by MLL Fusion Proteins in Leukemic Transformation.

Cancer cells transcribe RNAs in a characteristic manner in order to maintain their oncogenic potentials. In eukaryotes, RNA is polymerized by three distinct RNA polymerases, RNA polymerase I, II, and III (RNAP1, RNAP2, and RNAP3, respectively). The transcriptional machinery that initiates each transcription reaction has been purified and characterized. Selectivity factor 1 (SL1) is the complex responsible for RNAP1 pre-initiation complex formation. However, whether it plays any role in RNAP2-dependent transcription remains unclear. Our group previously found that SL1 specifically associates with AF4 family proteins. AF4 family proteins form the AEP complex with ENL family proteins and the P-TEFb elongation factor. Similar complexes have been independently characterized by several different laboratories and are often referred to as super elongation complex. The involvement of AEP in RNAP2-dependent transcription indicates that SL1 must play an important role in RNAP2-dependent transcription. To date, this role of SL1 has not been appreciated. In leukemia, AF4 and ENL family genes are frequently rearranged to form chimeric fusion genes with MLL. The resultant MLL fusion genes produce chimeric MLL fusion proteins comprising MLL and AEP components. The MLL portion functions as a targeting module, which specifically binds chromatin containing di-/tri-methylated histone H3 lysine 36 and non-methylated CpGs. This type of chromatin is enriched at the promoters of transcriptionally active genes which allows MLL fusion proteins to selectively bind to transcriptionally-active/CpG-rich gene promoters. The fusion partner portion, which recruits other AEP components and SL1, is responsible for activation of RNAP2-dependent transcription. Consequently, MLL fusion proteins constitutively activate the transcription of previously-transcribed MLL target genes. Structure/function analysis has shown that the ability of MLL fusion proteins to transform hematopoietic progenitors depends on the recruitment of AEP and SL1. Thus, the AEP/SL1-mediated gene activation pathway appears to be the central mechanism of MLL fusion-mediated transcriptional activation. However, the molecular mechanism by which SL1 activates RNAP2-dependent transcription remains largely unclear. This review aims to cover recent discoveries of the mechanism of transcriptional activation by MLL fusion proteins and to introduce novel roles of SL1 in RNAP2-dependent transcription by discussing how the RNAP1 machinery may be involved in RNAP2-dependent gene regulation.

Acute myeloid leukemia with t(10;11)(p11-12;q23.3): Results of Russian Pediatric AML registration study.

Translocations involving the KMT2A gene (also known as MLL) are frequently diagnosed in pediatric acute leukemia cases with either lymphoblastic or myeloid origin. KMT2A is translocated to multiple partner genes, including MLLT10/AF10 localizing at chromosomal band 10p12. KMT2A-MLLT10 is one of the common chimeric genes diagnosed in acute leukemia with KMT2A rearrangement (8%), especially in acute myeloid leukemia (AML; 18%). MLLT10 is localized in very close proximity to two other KMT2A partner genes at 10p11-12-NEBL and ABI1, so they could not be distinguished by conventional cytogenetics. In this work, we present a cohort of 28 patients enrolled into Russian Pediatric AML registration study carrying rearrangements between chromosomal regions 11q23.3 and 10p11-12. G-banding, FISH, reverse transcription PCR, and long-distance inverse PCR were used to characterize the KMT2A gene rearrangements in these patients. We demonstrate that 25 patients harbor the KMT2A-MLLT10 rearrangement, while three patients show the rare KMT2A rearrangements (2× KMT2A-NEBL; 1× KMT2A-ABI1). Therefore, the combination of cytogenetic and molecular genetic methods is of high importance in diagnosing cases with t(10;11)(p11-12;q23.3).

Solubility enhancement of Cry2Aa crystal through carboxy-terminal extension and synergism between the chimeric protein and Cry1Ac.

It was reported that the highly conserved C-terminal region of Bacillus thuringiensis Cry1A protoxins was very important for parasporal crystal formation and solubility feature in alkaline environment. In order to improve the solubilization efficiency of Cry2Aa crystal, the coding sequences of Cry2Aa protein and the C-terminal half of Cry1Ac were fused seamlessly through Red/ET homologous recombination and expressed in an acrystalliferous B. thuringiensis strain under the control of the cry1Ac promoter and terminator. Microscopic observation revealed that the recombinant strain containing the chimeric gene cry2Aa-1Ac produced distinct parasporal inclusion with semispherical to approximately cuboidal shape during sporulation. SDS-PAGE analysis showed that this strain expressed stable 130-kDa Cry2Aa-1Ac chimeric protein, which was confirmed to be the correctly expressed product by LC-MS/MS. The chimeric protein inclusion could be effectively dissolved at pH 10.5 and activated by trypsin like the parental Cry1Ac crystal. While, the parental Cry2Aa crystal exhibited very low solubility under this condition. Bioassays against third-instar larvae of Helicoverpa armigera proved that the chimeric protein was more toxic than Cry2Aa. Additionally, synergistic effect was clearly detected between the chimeric protein and Cry1Ac against H. armigera, while there was only additive effect for the combination of wild Cry2Aa and Cry1Ac. These results indicated that the developed chimeric protein might serve as a potent insecticidal toxin used in the field against lepidopteran pests.