Jungnam Lee

Human Genomic Deletions Mediated by Recombination between Alu Elements

Recombination between Alu elements results in genomic deletions associated with many human genetic disorders. Here, we compare the reference human and chimpanzee genomes to determine the magnitude of this recombination process in the human lineage since the human-chimpanzee divergence approximately 6 million years ago. Combining computational data mining and wet-bench experimental verification, we identified 492 human-specific deletions (for a total of approximately 400 kb) attributable to this process, a significant component of the insertion/deletion spectrum of the human genome. The majority of the deletions (295 of 492) coincide with known or predicted genes (including 3 that deleted functional exons, as compared with orthologous chimpanzee genes), which implicates this process in creating a substantial portion of the genomic differences between humans and chimpanzees. Overall, we found that Alu recombination-mediated genomic deletion has had a much higher impact than was inferred from previously identified isolated events and that it continues to contribute to the dynamic nature of the human genome.

L1 recombination-associated deletions generate human genomic variation

Mobile elements have created structural variation in the human genome through their de novo insertions and post-insertional genomic rearrangements. L1 elements are a type of long interspersed element (LINE) that is dispersed at high copy numbers within most mammalian genomes. To determine the magnitude of L1 recombination-associated deletions (L1RADs), we computationally extracted L1RAD candidates by comparing the human and chimpanzee genomes and verified each of the L1RAD events by using wet-bench analyses. Through these analyses, we identified 73 human-specific L1RAD events that occurred subsequent to the divergence of the human and chimpanzee lineages. Despite their low frequency, the L1RAD events deleted ≈450 kb of the human genome. One L1RAD event generated a large deletion of ≈64 kb. Multiple alignments of prerecombination and postrecombination L1 elements suggested that two different deletion mechanisms generated the L1RADs: nonallelic homologous recombination (55 events) and nonhomologous end joining between two L1s (18 events). In addition, the position of L1RADs throughout the genome does not correlate with local chromosomal recombination rates. This process may be implicated in the partial regulation of L1 copy numbers by the finding that ≈60% of the DNA sequences deleted by the L1RADs consist of L1 sequences that were either directly involved in the recombination events or located in the intervening sequence between recombining L1s. Overall, there is increasing evidence that L1RADs have played an important role in creating structural variation.

Genomic rearrangements by LINE-1 insertion-mediated deletion in the human and chimpanzee lineages.

Long INterspersed Elements (LINE-1s or L1s) are abundant non-LTR retrotransposons in mammalian genomes that are capable of insertional mutagenesis. They have been associated with target site deletions upon insertion in cell culture studies of retrotransposition. Here, we report 50 deletion events in the human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of ∼18 kb of sequence from the human genome and ∼15 kb from the chimpanzee genome. Our data suggest that during the primate radiation, L1 insertions may have deleted up to 7.5 Mb of target genomic sequences. While the results of our in vivo analysis differ from those of previous cell culture assays of L1 insertion-mediated deletions in terms of the size and rate of sequence deletion, evolutionary factors can reconcile the differences. We report a pattern of genomic deletion sizes similar to those created during the retrotransposition of Alu elements. Our study provides support for the existence of different mechanisms for small and large L1-mediated deletions, and we present a model for the correlation of L1 element size and the corresponding deletion size. In addition, we show that internal rearrangements can modify L1 structure during retrotransposition events associated with large deletions.

Alu Recombination-Mediated Structural Deletions in the Chimpanzee Genome

With more than 1.2 million copies, Alu elements are one of the most important sources of structural variation in primate genomes. Here, we compare the chimpanzee and human genomes to determine the extent of Alu recombination-mediated deletion (ARMD) in the chimpanzee genome since the divergence of the chimpanzee and human lineages (∼6 million y ago). Combining computational data analysis and experimental verification, we have identified 663 chimpanzee lineage-specific deletions (involving a total of ∼771 kb of genomic sequence) attributable to this process. The ARMD events essentially counteract the genomic expansion caused by chimpanzee-specific Alu inserts. The RefSeq databases indicate that 13 exons in six genes, annotated as either demonstrably or putatively functional in the human genome, and 299 intronic regions have been deleted through ARMDs in the chimpanzee lineage. Therefore, our data suggest that this process may contribute to the genomic and phenotypic diversity between chimpanzees and humans. In addition, we found four independent ARMD events at orthologous loci in the gorilla or orangutan genomes. This suggests that human orthologs of loci at which ARMD events have already occurred in other nonhuman primate genomes may be “at-risk” motifs for future deletions, which may subsequently contribute to human lineage-specific genetic rearrangements and disorders.

Chromosomal Inversions between Human and Chimpanzee Lineages Caused by Retrotransposons

The long interspersed element-1 (LINE-1 or L1) and Alu elements are the most abundant mobile elements comprising 21% and 11% of the human genome, respectively. Since the divergence of human and chimpanzee lineages, these elements have vigorously created chromosomal rearrangements causing genomic difference between humans and chimpanzees by either increasing or decreasing the size of genome. Here, we report an exotic mechanism, retrotransposon recombination-mediated inversion (RRMI), that usually does not alter the amount of genomic material present. Through the comparison of the human and chimpanzee draft genome sequences, we identified 252 inversions whose respective inversion junctions can clearly be characterized. Our results suggest that L1 and Alu elements cause chromosomal inversions by either forming a secondary structure or providing a fragile site for double-strand breaks. The detailed analysis of the inversion breakpoints showed that L1 and Alu elements are responsible for at least 44% of the 252 inversion loci between human and chimpanzee lineages, including 49 RRMI loci. Among them, three RRMI loci inverted exonic regions in known genes, which implicates this mechanism in generating the genomic and phenotypic differences between human and chimpanzee lineages. This study is the first comprehensive analysis of mobile element bases inversion breakpoints between human and chimpanzee lineages, and highlights their role in primate genome evolution.

Effect of dietary fermented garlic by Weissella koreensis powder on growth performance, blood characteristics, and immune response of growing pigs challenged with Escherichia coli lipopolysaccharide

Two experiments were conducted to investigate the effect of fermented garlic by Weissella koreensis powder (WKG) on pig growth performance and immune responses after an Escherichia coli lipopolysaccharide (LPS) challenge. In Exp. 1, 120 growing barrows (23.5 ± 0.5 kg of BW and 56 d of age) were used in a 35-d experiment to determine the optimal amounts of WKG. Pigs were randomly allotted to 1 of 5 treatments with 6 replicate pens and 4 pigs per pen. Dietary treatments included 1) NC (negative control; basal diet without antibiotics), 2) PC (positive control; basal diet + 1 g of tylosin/kg), 3) WKG1 (basal diet + 1 g of WKG/kg), 4) WKG2 (basal diet + 2 g of WKG/kg), and 5) basal diet + 4 g of WKG/kg. At the end of the feeding period, 12 pigs each were selected from the NC and WKG2 treatment groups, and 6 pigs were injected with LPS (50 μg/kg of BW) and the other 6 pigs with an equivalent amount of sterile saline, resulting in a 2 × 2 factorial arrangement of treatments. Blood samples and rectal temperature data were collected at 0, 2, 4, 6, 8, and 12 h after challenge. The ADG of pigs fed WKG- and antibiotic-supplemented diets was greater (P<0.05) than NC from d 14 to 35 and the overall phase, but no dosage-dependent effects were observed. At the end of the experiment, the fecal E. coli count was linearly reduced by the increasing amounts of WKG at d 35 (P=0.01). Challenge with LPS increased white blood cell counts at 6 and 8 h (P<0.01) and depressed lymphocyte concentration at 4, 8, and 12 h (P<0.01). During challenge, LPS injection increased rectal temperature at 2, 4, 6, and 8 h postchallenge (P<0.05), and WKG2 alleviated (P<0.05) the increase in the temperature at 2 h postchallenge. The LPS injection increased plasma tumor necrosis factor-α and IGF-1 concentrations at 2, 4, 6, 8, and 12 h (P<0.01), whereas an alleviating effect of WKG was observed at 4, 6, and 8 h after LPS challenge (P<0.05). At 2, 4, and 6 h postchallenge, concentration of cluster of differentiation-antigen-4-positive cells and cluster of differentiation-antigen-8-positive cells (CD4(+) and CD8(+), respectively) increased in the LPS treatments (P<0.05), and the WKG2 boosted this effect (P<0.05). In conclusion, dietary supplementation of WKG2 in growing pigs can improve ADG and have a beneficial effect on the immune response during an inflammatory challenge.

Effects of phenyllactic acid on growth performance, intestinal microbiota, relative organ weight, blood characteristics, and meat quality of broiler chicks

This study was conducted to determine the effects of dietary supplementation with phenyllactic acid (PLA) on growth performance, intestinal microbiota, relative organ weight, blood characteristics, and meat quality in broilers. A total of 500 male broilers (BW = 46.3 g) were randomly allotted into 1 of the following 5 dietary treatments: 1) basal diet (CON), 2) basal diet + 44 mg/kg of avilamycin (ANT), 3) basal diet + 0.2% PLA (PLA0.2), 4) basal diet + 0.4% PLA (PLA0.4), 5) basal diet + 0.2% PLA + 44 mg/kg of avilamycin (PA). Chicks fed PLA had lower feed intake (FI) from d 0 to 7 (P < 0.05) than those fed CON and ANT. From d 21 to 35, BW gain was greater in ANT, PLA0.4, and PA diets than CON and PLA0.2 diets (P < 0.05), whereas the FI was lowest in the PLA0.4 diet. Feed efficiency was depressed (P < 0.05) by the antibiotics and PLA supplementation during d 0 to 7, whereas it was improved (P < 0.05) in the PLA and ANT diets during d 21 to 35, with the best value in PLA0.4.The population of Escherichia coli in the large intestine was lower in the ANT, PLA0.4, and PA groups than the CON and PLA0.2 groups (P < 0.05). The relative weights of gizzard, liver, spleen, bursa of Fabricius, breast, and abdominal fat were unaffected by any of the dietary supplementations. Treatment of PLA led to an increase (P < 0.05) in the concentrations of white blood cells and lymphocyte percentage. The yellowness of breast muscle decreased by ANT, PLA0.4, and PA treatment. In conclusion, PLA can improve growth performance when it is supplemented in finisher diet (d 21 to 35), whereas it can depress BW gain and FI in earlier days (d 0 to 7). In addition, PLA can also decrease the number of E. coli in the large intestine and improve the number of immune-related blood cells.

Human-Specific HERV-K Insertion Causes Genomic Variations in the Human Genome

Human endogenous retroviruses (HERV) sequences account for about 8% of the human genome. Through comparative genomics and literature mining, we identified a total of 29 human-specific HERV-K insertions. We characterized them focusing on their structure and flanking sequence. The results showed that four of the human-specific HERV-K insertions deleted human genomic sequences via non-classical insertion mechanisms. Interestingly, two of the human-specific HERV-K insertion loci contained two HERV-K internals and three LTR elements, a pattern which could be explained by LTR-LTR ectopic recombination or template switching. In addition, we conducted a polymorphic test and observed that twelve out of the 29 elements are polymorphic in the human population. In conclusion, human-specific HERV-K elements have inserted into human genome since the divergence of human and chimpanzee, causing human genomic changes. Thus, we believe that human-specific HERV-K activity has contributed to the genomic divergence between humans and chimpanzees, as well as within the human population.

Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase.

Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the pro-inflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs.

Human Genomic Deletions Generated by SVA-Associated Events

Mobile elements are responsible for half of the human genome. Among the elements, L1 and Alu are most ubiquitous. They use L1 enzymatic machinery to move in their host genomes. A significant amount of research has been conducted about these two elements. The results showed that these two elements have played important roles in generating genomic variations between human and chimpanzee lineages and even within a species, through various mechanisms. SVA elements are a third type of mobile element which uses the L1 enzymatic machinery to propagate in the human genome but has not been studied much relative to the other elements. Here, we attempt the first identification of the human genomic deletions caused by SVA elements, through the comparison of human and chimpanzee genome sequences. We identified 13 SVA recombination-associated deletions (SRADs) and 13 SVA insertion-mediated deletions (SIMDs) in the human genome and characterized them, focusing on deletion size and the mechanisms causing the events. The results showed that the SRADs and SIMDs have deleted 15,752 and 30,785 bp, respectively, in the human genome since the divergence of human and chimpanzee and that SRADs were caused by two different mechanisms, nonhomologous end joining and nonallelic homologous recombination.