Preintegration Sites Research Articles

Plant DNA Repair and Agrobacterium T-DNA Integration.

Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, at its junctions with plant DNA, deletions of T−DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T−DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T−DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T−DNA integration into the plant genome.

Frequency of retrotransposon human endogenous retrovirus-K113 and a preliminary analysis of some microbial clues in bipolar disorder.

Aim: The aim of our study was to investigate whether the retrotransposon human endogenous retrovirus (HERV)-K113 could be related with bipolar disorder or not. As a second and a preliminary aim, we also conducted bacterial screening in whole blood in a limited number of samples. Patients & methods: Three separate PCR reactions including the preintegration sites and sites within the viral sequences were performed for HERV-K113 detection. Bacterial screening was performed with SSCP/sequencing analysis. Results & conclusion: No difference was observed in terms of the frequency of retrotransposon HERV-K113 in Turkish bipolar disorder patients and healthy controls. SSCP/sequencing and alignment analysis for bacterial screening reflected the possible presence of different bacteria. We strongly recommend the broadened retrotransposon and microbial diversity analyses in bipolar disorder for future studies.

Integration of HIV testing services into family planning services: a systematic review

BackgroundDespite significant interest in integrating sexual and reproductive health (SRH) services into HIV services, less attention has been paid to linkages in the other direction. Where women and girls are at risk of HIV, offering HIV testing services (HTS) during their visits to family planning (FP) services offers important opportunities to address both HIV and unwanted pregnancy needs simultaneously.MethodsWe conducted a systematic review of studies comparing FP services with integrated HTS to those without integrated HTS or with a lower level of integration (e.g., referral versus on-site services), on the following outcomes: uptake/counseling/offer of HTS, new cases of HIV identified, linkage to HIV care and treatment, dual method use, client satisfaction and service quality, and provider knowledge and attitudes about integrating HTS. We searched three online databases and included studies published in a peer-reviewed journal prior to the search date of June 20, 2017.ResultsOf 530 citations identified, six studies ultimately met the inclusion criteria. Three studies were conducted in Kenya, and one each in Uganda, Swaziland, and the USA. Most were in FP clinics. Three were from the Integra Initiative. Overall rigor was moderate, with one cluster-randomized trial. HTS uptake was generally higher with integrated sites versus comparison or pre-integration sites, including in adjusted analyses, though outcomes varied slightly across studies. One study found that women at integrated sites were more likely to have high satisfaction with services, but experienced longer waiting times. One study found a small increase in HIV seropositivity among female patients testing after full integration, compared to a dedicated HIV tester. No studies comparatively measured linkage to HIV care and treatment, dual method use, or provider knowledge/attitudes.ConclusionsGlobal progress and success for reaching SRH and HIV targets depends on progress in sub-Saharan Africa, where women bear a high burden of both unintended pregnancy and sexually transmitted infections, including HIV. While the evidence base is limited, it suggests that integration of HTS into FP services is feasible and has potential for positive joint outcomes. The success and scale-up of this approach will depend on population needs and health system factors.

Insertionally polymorphic sites of human endogenous retrovirus-K (HML-2) with long target site duplications

BackgroundHuman endogenous retroviruses (HERVs) belong to the LTR-retrotransposon family, where the complete HERV sequence contains two long terminal repeats (LTRs) located at each end. Intact LTRs possess highly conserved transcriptional promoter and enhancer sequences, so analyses of HERV insertional polymorphisms are expected to provide greater insights into human genomic variation compared with the conventional analysis of single nucleotide variations. High-throughput sequencing technology is developing but genome-wide investigations of HERVs are methodically challenging, and thus a comprehensive understanding of HERV insertional polymorphisms and target site duplications (TSDs) remains elusive.ResultsWe identified five human-specific insertionally polymorphic sites in HERVK (HML-2), one of the HERV subgroups, by extracting HML-2-deleted sequences from the genomic structural variation database, which we successfully characterized and then updated the existing catalogue of HML-2 insertional polymorphisms. The insertionally polymorphic states were confirmed in a small Japanese population by genomic PCR analysis for four of the five sites identified. Sequencing of the preintegration sites clearly showed that the HML-2 site located at 7p21.2 had 250-base pair (bp) TSDs, which is one of the longest TSDs in HML-2. In addition to these five sites, another insertionally polymorphic site for a non-human-specific HML-2 site was also identified at 6p25.2, which was flanked by 111-bp TSDs and the corresponding ERV locus was also annotated in the genome of non-human primates.ConclusionsOur analysis demonstrated the existence of HERV insertions flanked by unconventionally long TSDs, including those with lengths as high as 250 bp. This suggests that the length range of retroviral TSDs is larger than considered previously, which might help to understand how retroviral integration occurs in the host genome.

Whole genome computational comparative genomics: A fruitful approach for ascertaining Alu insertion polymorphisms

Alu elements are the most active and predominant type of short interspersed elements (SINEs) in the human genome. Recently inserted polymorphic (for presence/absence) Alu elements contribute to genome diversity among different human populations, and they are useful genetic markers for population genetic studies. The objective of this study is to identify polymorphic Alu insertions through an in silico comparative genomics approach and to analyze their distribution pattern throughout the human genome. By computationally comparing the public and Celera sequence assemblies of the human genome, we identified a total of 800 polymorphic Alu elements. We used polymerase chain reaction-based assays to screen a randomly selected set of 16 of these 800 Alu insertion polymorphisms using a human diversity panel to demonstrate the efficiency of our approach. Based on sequence analysis of the 800 Alu polymorphisms, we report three new Alu subfamilies, Ya3, Ya4b, and Yb11, with Yb11 being the smallest known Alu subfamily. Analysis of retrotransposition activity revealed Yb11, Ya8, Ya5, Yb9, and Yb8 as the most active Alu subfamilies and the maintenance of a very low level of retrotransposition activity or recent gene conversion events involving S subfamilies. The 800 polymorphic Alu insertions are characterized by the presence of target site duplications (TSDs) and longer than average polyA-tail length. Their pre-integration sites largely follow an extended “NT-AARA” motif. Among chromosomes, the density of Alu insertion polymorphisms is positively correlated with the Alu-site availability and is inversely correlated with the densities of older Alu elements and genes.

Genomic deletions and precise removal of transposable elements mediated by short identical DNA segments in primates

Insertion of transposable elements is a major cause of genomic expansion in eukaryotes. Less is understood, however, about mechanisms underlying contraction of genomes. In this study, we show that retroelements can, in rare cases, be precisely deleted from primate genomes, most likely via recombination between 10- to 20-bp target site duplications (TSDs) flanking the retroelement. The deleted loci are indistinguishable from pre-integration sites, effectively reversing the insertion. Through human-chimpanzee-Rhesus monkey genomic comparisons, we estimate that 0.5%-1% of apparent retroelement "insertions" distinguishing humans and chimpanzees actually represent deletions. Furthermore, we demonstrate that 19% of genomic deletions of 200-500 bp that have occurred since the human-chimpanzee divergence are associated with flanking identical repeats of at least 10 bp. A large number of deletions internal to Alu elements were also found flanked by homologies. These results suggest that illegitimate recombination between short direct repeats has played a significant role in human genome evolution. Moreover, this study lends perspective to the view that insertions of retroelements represent unidirectional genetic events.

Analysis of the human Alu Ye lineage

BackgroundAlu elements are short (~300 bp) interspersed elements that amplify in primate genomes through a process termed retroposition. The expansion of these elements has had a significant impact on the structure and function of primate genomes. Approximately 10 % of the mass of the human genome is comprised of Alu elements, making them the most abundant short interspersed element (SINE) in our genome. The majority of Alu amplification occurred early in primate evolution, and the current rate of Alu retroposition is at least 100 fold slower than the peak of amplification that occurred 30–50 million years ago. Alu elements are therefore a rich source of inter- and intra-species primate genomic variation.ResultsA total of 153 Alu elements from the Ye subfamily were extracted from the draft sequence of the human genome. Analysis of these elements resulted in the discovery of two new Alu subfamilies, Ye4 and Ye6, complementing the previously described Ye5 subfamily. DNA sequence analysis of each of the Alu Ye subfamilies yielded average age estimates of ~14, ~13 and ~9.5 million years old for the Alu Ye4, Ye5 and Ye6 subfamilies, respectively. In addition, 120 Alu Ye4, Ye5 and Ye6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity.ConclusionThe Alu Ye lineage appears to have started amplifying relatively early in primate evolution and continued propagating at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Detailed sequence analysis of several Alu pre-integration sites indicated that multiple types of events had occurred, including gene conversions, near-parallel independent insertions of different Alu elements and Alu-mediated genomic deletions. A potential hotspot for Alu insertion in the Fer1L3 gene on chromosome 10 was also identified.

Straightening out the LINEs: LINE-1 orthologous loci

The L1Hs preTa subfamily of long interspersed elements (LINEs) originated after the divergence of human and chimpanzee and is therefore found only in the human genome. Thirty-three of the 254 L1Hs preTa elements are polymorphic for the absence/presence of the insertion, making them useful markers for studying human population genetics. The problem of homoplasy, however, can diminish the value of LINEs as phylogenetic and population genetic markers. We examined anomalous orthologous sites in a range of nonhuman primates. Only two cases of other mobile elements inserting near the preintegration sites of L1Hs preTa elements were observed: an AluY insertion in Chlorocebus and an L1PA8 insertion in Aotus. Sequence analysis showed that both elements were clearly distinguishable from their human counterparts. We conclude that L1 elements can continue to be regarded as essentially homoplasy-free genetic characters.

Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans

Background: Endogenous retroviruses contribute to the evolution of the host genome and can be associated with disease. Human endogenous retrovirus K (HERV-K) is related to the mouse mammary tumor virus and is present in the genomes of humans, apes and cercopithecoids (Old World monkeys). It is unknown how long ago in primate evolution the full-length HERV-K proviruses that are in the human genome today were formed.Results: Ten full-length HERV-K proviruses were cloned from the human genome. Using provirus-specific probes, eight of the ten were found to be present in a genetically diverse set of humans but not in other extant hominoids. Intact preintegration sites for each of these eight proviruses were present in the apes. A ninth provirus was detected in the human, chimpanzee, bonobo and gorilla genomes, but not in the orang-utan genome. The tenth was found only in humans, chimpanzees and bonobos. Complete sequencing of six of the human-specific proviruses showed that full-length open reading frames for the retroviral protein precursors Gag–Pro–Pol or Env were each present in multiple proviruses.Conclusions: At least eight full-length HERV-K genomes that are in the human germline today integrated after humans diverged from chimpanzees. All of the viral open reading frames and cis-acting sequences necessary for HERV-K replication must have been intact during the recent time when these proviruses formed. Multiple full-length open reading frames for all HERV-K proteins are present in the human genome today.

Mechanism of chromosomal integration of transgenes in microinjected mouse eggs: sequence analysis of genome-transgene and transgene-transgene junctions at two loci

Production of transgenic animals is a key technique in modern biology, but the process of chromosomal integration of transgenes in microinjected eggs is still not fully understood. To gain information on the mechanisms involved in this process, we have cloned two transgene loci and their corresponding pre-integration sites and compared the junction sequences with the parental nucleotide (nt) sequences. No extensive DNA rearrangements were detected at these loci: only simple deletions (caused by the integration of the transgene concatemers) were present in the host genome. Analysis of three transgene-transgene junctions within the concatemers showed that ‘nibbling’ of ends (up to 3 nt) had occurred at some ends prior to joining. At all four genome-transgene junctions, short homologies of 1 to 3 nt were found, and at least three of these junctions were associated with the consensus sequence for topoisomerase-I cleavage sites. Moreover, three of the four integration junctions occurred in the terminal regions of the injected sequence, at positions only a few nt away from the ends. These results suggest that linear, but not circular, concatemers were preferentially integrated at their ends utilizing short homologies to the host genome.

CDNA genes formed after infection with retroviral vector particles lack the hallmarks of natural processed pseudogenes.

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.

CDNA Genes Formed after Infection with Retroviral Vector Particles Lack the Hallmarks of Natural Processed Pseudogenes

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.

Varied interactions between proviruses and adjacent host chromatin.

Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15d RBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described.

Varied interactions between proviruses and adjacent host chromatin.

Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15d RBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described.