Overall the low-throughput analysis confirmed the genome wide distribution profile gathered through the IC-Seq analysis. We found no unidentifiable sequence inserted at AAV-2 chromosomal junctions. Assessing both left and right ends of the AAV genome, viral breakpoints predominantly occurred in one hairpin of the inverted terminal repeat and AAV genomes were preferentially integrated as single copies.
We reasoned that additional insight regarding the integration of AAV-2 into human genomic DNA could be gleaned by low-throughput sequencing of complete viral-chromosomal junctions. In this study, junctions were assayed from wild-type AAV-2 infected HeLa cells processed through the Integrant-Capture Sequencing (IC-Seq) protocol (Figure 1A and B). AAV-2 generated from helper-free plasmid transfection (Applied Viromics) and applied at 1E4 viral genomes per cell. These conditions provide maximal integration efficiency with minimal residual episomal virus, as previously described [15, 16]. Since this protocol generates random chromosomal breaks using sonication and does not rely on locus-specific primers, it should be less biased than previous junction studies [15, 17, 18]. Primer sets to both the left and right portions of the AAV-2 genome were used to assess each biological replicate. The L1/L2 primer set was previously described [15, 19] and the R1/R2 oligonucleotides were five-prime modified from a previous study [20] to include [Bio-TEG]C and CGTTT respectively. Methods for IC-Seq are presented in references [15, 17, 18], and we hope to publish a step-by-step methods guide for future reference. Subsequent to the main phase of IC-Seq, sample pools were cloned into bacterial plasmids, and individual clones were sequenced.
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Interestingly, the data provided in this study offer insight into the question of whether wild-type AAV genomes integrate as single copies or concatamers. Previous work using Southern blotting to characterize integrations from several cell lines suggested that AAV integrates as head-to-tail concatamers [31]. The data analyzed in this study are one hundred unique sequences from a diverse cell population. Of the one hundred sequences that met our inclusion criteria, forty-six were intact viral sequence, thirty-six were direct viral-chromosomal events, fifteen were viral-viral recombinations and three sequences possessed both viral-viral and viral-chromosomal recombination. Therefore, 66.7% of all recombination events captured were between single viral genomes and human chromosomal DNA (Figure 3C). Additionally, we noted that 82% of all sequences were free of viral-viral recombinations (Figure 3D). Thus, analyzing both ends of integrated AAV-2 sequences, the data indicate viral genomes predominantly integrate into host DNA as single copies.
This study of complete viral-chromosomal junctions derived from cloning and sequencing IC-Seq DNA pools provides valuable insight into AAV integration. The structurally complex, repetitive, and GC-rich nature of these sequences may hinder capture of the entire junction-population. We have taken many steps to mitigate these effects. These steps included using: short sequences from random breaks, two primer sets, stringent sequence validation, robust polymerases, and high melting temperatures. Therefore, we believe that the junctions captured and analyzed in this study are not unduly influenced by sequence constraints, and present a valuable representation of the AAV-2 junction population. The insertion profile of AAV-2 maintained the same top three hotspots found using high-throughput technology and the distribution around AAVS1, the largest hotspot, was also quite similar. In the absence of Rep, the unique AAV-2 ITR structure is a target for cellular DNA repair and recombination pathways which can vary in a cell dependent manner [21, 30, 32, 33]. In the case of wild-type AAV-2, Rep binding to the RBE as well as the hairpin stem influences helicase activity [25]. Therefore, Rep, in concert with cellular DNA repair complexes, may contribute to formation of the internal stem-loop ITR recombination hotspot identified in this study. We anticipate that cell-specific differences in DNA repair proteins and Rep interacting proteins may also influence the integration profile to some extent. However, direct Rep-DNA interactions appear to play the dominant role in defining the genome-wide targets for AAV-2 integration [15, 19]. Finally, based on the population of junctions captured, AAV-2 genomes were found to predominately integrate as single genome copies, and viral-viral recombination was modest. This study may impact Rep-mediated gene therapy approaches and highlights how long read length, even on a modest scale, may serve to significantly augment the understanding of high-throughput data sets.
Besides of PML-RARA, additional gene mutations have been identified in APL and they possibly cooperated with PML-RARA to participate in the leukemogenesis and therapeutic resistance. At diagnosis, several studies demonstrated that FLT3-ITD/TKD mutation was the most common additional gene mutation. WT1, NRAS/KRAS, and ARID1A/ARID1B mutations were also frequent [16,17,18,19,20]. Though epigenetic modifiers, such as DNMT3A, TET2, ASXL1, and IDH1/2, mutations were relatively rare in APL, they conferred APL one poor prognosis characterized by short overall survival duration and disease-free survival duration [17]. At relapse, the gene mutational landscape has skewed, and the most remarkable change was the emerged large amount of PML or RARA mutation [16, 18, 19, 21, 22]. Besides, the frequency of RUNX1 mutation also elevated compared to it at primary diagnosis [16, 18]. However, FLT3-ITD/TKD mutation was still the most common, and WT1, NRAS/KRAS, and ARID1A/ARID1B mutations followed [19]. Notably, the increased total frequency of additional genetic mutations was strongly associated with the high Sanz score at diagnosis [17]. Consistently, APL with these mutations preferred to fall in high-risk group and develop to relapsed disease during the clinical course. These results indicated that there were two different pathways for gene mutations to mediate therapeutic resistance and promote APL relapse at least. The first was that PML or RARA mutation impaired the binding of PML-RARA to ATO or ATRA, respectively; the second was that additional gene mutations, especially FLT3-ITD/TKD mutation, cooperated with PML-RARA to drive APL progression. Therefore, besides of PML-RARA, additional gene mutations were also important in the pathogenesis of APL.
In vertebrates, whole genome duplication (WGD) events led to the expansion of the repertoire of rar and rxr genes [14]. Thus, while the cephalochordate amphioxus possesses only one rar and one rxr, the mouse genome encodes three rar genes and three rxr genes, called rarα, rarβ, and rarγ, and rxrα, rxrβ, and rxrγ, respectively. The rar and rxr repertoires have been expanded further in some bony fish (the teleosts), whose genomes have undergone an additional round of WGD [15-17]. Thus, the zebrafish genome encodes four rar genes (rarαa, rarαb, rarγa, and rarγb) and six rxr genes (rxrαa, rxrαb, rxrβa, rxrβb, rxrγa, and rxrγb). During chordate development, expression of rar and rxr genes is generally dynamic and detectable in most embryonic tissues [16-21]. Interestingly, paralogous rar and rxr genes in jawed vertebrates (that is, gnathostomes) show highly diverse expression patterns as well as divergent functions during development, indicating that vertebrate-specific genome duplications have mediated lineage-specific diversification of the developmental processes controlled by specific rar and rxr genes [17,21].
Surprisingly, while developmental roles of RA signaling have been extensively studied in gnathostomes and invertebrate chordates, much less is known about RA functions in cyclostomes [22-24], a group of jawless vertebrates comprising lampreys and hagfish and representing the phylogenetic sister group of the gnathostomes [25]. Cyclostomes are particularly appealing models for comparative studies, because they possess many vertebrate-specific features, such as neural crest derivatives, but lack key characters that are present in other vertebrates, such as the jaws [26-28]. In addition to the overall morphology, cyclostome genomes, when compared to those of gnathostomes, also exhibit both similarities and differences [29]. For instance, while lamprey genomes have very likely experienced the two rounds of WGDs characteristic of vertebrates [30-32], their genomes undergo dramatic remodeling during development, resulting in the elimination of hundreds of millions of base pairs (bp), including hundreds of genes, from somatic cell lineages [33,34].
In lampreys, some preliminary studies have been carried out to investigate the roles of RA signaling during embryonic development and have provided insights into the evolution of RA functions in the vertebrate lineage [22-24]. For example, it has been shown that RA treatments during gastrulation induce rostral truncations of both the brain and the pharynx, leading, in the severest cases, to embryos that consist only of trunk segments [22]. Previous work has also suggested that the genomes of the sea lamprey, Petromyzon marinus, the Japanese lamprey, Lethenteron japonicum, the Australian lamprey, Mordacia mordax, as well as of the inshore hagfish, Eptatretus burgeri, encode at least three rar genes [24,31], although phylogenetic analyses have failed to unambiguously assign orthologies between the cyclostome and gnathostome rars [31]. Furthermore, the expression patterns of lamprey rars have so far only been described for a single developmental stage, and the functions of these genes in the lamprey embryo still remain elusive [24]. 2ff7e9595c
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