Major repeat components covering one‐third of the ginseng (Panax ginseng C.A. Meyer) genome and evidence for allotetraploidy

Ginseng (Panax ginseng) is a famous medicinal herb, but the composition and structure of its genome are largely unknown. Here we characterized the major repeat components and inspected their distribution in the ginseng genome. By analyzing three repeat‐rich bacterial artificial chromosome (BAC) sequences from ginseng, we identified complex insertion patterns of 34 long terminal repeat retrotransposons (LTR‐RTs) and 11 LTR‐RT derivatives accounting for more than 80% of the BAC sequences. The LTR‐RTs were classified into three Ty3/gypsy (PgDel, PgTat and PgAthila) and two Ty1/Copia (PgTork and PgOryco) families. Mapping of 30‐Gbp Illumina whole‐genome shotgun reads to the BAC sequences revealed that these five LTR‐RT families occupy at least 34% of the ginseng genome. The Ty3/Gypsy families were predominant, comprising 74 and 33% of the BAC sequences and the genome, respectively. In particular, the PgDel family accounted for 29% of the genome and presumably played major roles in enlargement of the size of the ginseng genome. Fluorescence in situ hybridization (FISH) revealed that the PgDel1 elements are distributed throughout the chromosomes along dispersed heterochromatic regions except for ribosomal DNA blocks. The intensity of the PgDel2 FISH signals was biased toward 24 out of 48 chromosomes. Unique gene probes showed two pairs of signals with different locations, one pair in subtelomeric regions on PgDel2‐rich chromosomes and the other in interstitial regions on PgDel2‐poor chromosomes, demonstrating allotetraploidy in ginseng. Our findings promote understanding of the evolution of the ginseng genome and of that of related species in the Araliaceae.     

Characterization of a Novel Class of Interspersed LTR Elements in Primate Genomes: Structure, Genomic Distribution, and Evolution

Retrovirus-like sequences and their solitary (solo) long terminal repeats (LTRs) are common repetitive elements in eukaryotic genomes. We reported previously that the tandemly arrayed genes encoding U2 snRNA (the RNU2 locus) in humans and apes contain a solo LTR (U2-LTR) which was presumably generated by homologous recombination between the two LTRs of an ancestral provirus that is retained in the orthologous baboon RNU2 locus. We have now sequenced the orthologous U2-LTRs in human, chimpanzee, gorilla, orangutan, and baboon and examined numerous homologs of the U2-LTR that are dispersed throughout the human genome. Although these U2-LTR homologs have been collectively referred to as LTR13 in the literature, they do not display sequence similarity to any known retroviral LTRs; however, the structure of LTR13 closely resembles that of other retroviral LTRs with a putative promoter, polyadenylation signal, and a tandemly repeated 53-bp enhancer-like element. Genomic blotting indicates that LTR13 is primate-specific; based on sequence analysis, we estimate there are about 2,500 LTR13 elements in the human genome. Comparison of the primate U2-LTR sequences suggests that the homologous recombination event that gave rise to the solo U2-LTR occurred soon after insertion of the ancestral provirus into the ancestral U2 tandem array. Phylogenetic analysis of the LTR13 family confirms that it is diverse, but the orthologous U2-LTRs form a coherent group in which chimpanzee is closest to the humans; orangutan is a clear outgroup of human, chimpanzee, and gorilla; and baboon is a distant relative of human, chimpanzee, gorilla, and orangutan. We compare the LTR13 family with other known LTRs and consider whether these LTRs might play a role in concerted evolution of the primate RNU2 locus. Received: 29 September 1997 / Accepted: 16 January 1998     

Retrotransposon-based insertion polymorphism markers in mango

Retrotransposons are major components of eukaryotic genomes and are present in high copy numbers. We developed retrotransposon-based insertion polymorphism (RBIP) markers based on long terminal repeat (LTR) sequences and flanking genome regions by using shotgun genome sequence data of mango (Mangifera indica L.). Three novel LTR sequences were identified based on two LTR retrotransposon structural features; a 5′ LTR located upstream of the primer binding site and a 3′ LTR showing high sequence similarity to the 5′ LTR. Starting with 377 unique sequences containing both 3′ LTR and downstream genome region sequences, we developed 82 RBIP markers that were applied to DNA fingerprinting of 16 mango accession. Five RBIP markers were enough to distinguish all 16 accessions. Our result showed that LTR identification from shotgun genome sequences was effective for development of retrotransposon-based DNA markers without whole-genome sequence information. We discuss application of the developed RBIP markers for identification of genetic diversity and construction of a genetic linkage map.     

Comparison of enzymatic activities of the HIV-1 and HFV integrases to their U5 LTR substrates.

The human immunodeficiency virus type 1 (HIV-1) and human foamy virus (HFV) integrase proteins were overexpressed in Escherichia coli, and purified to a near homogeneity by one- or two-step purification scheme. The endonucleolytic, integration, and disintegration activities for the HIV-1 and HFV integrases were characterized in vitro. The endonucleolytic activities for the HIV-1 and HFV integrases were found only on their own substrates, respectively, indicating that the cognate U5 LTR sequences in the substrates is critical for specific cleavage. However, the integration and disintegration activities showed less specificity on the substrate usage. Our results suggest that the disintegration activity have more preference for substrates based on Y-shaped structure rather than on viral donor DNA sequence.     

Discovery of a novel long terminal repeat (LTR2i_SS) in Sus Scrofa

Long terminal repeat (LTR) retrotransposons are transposable elements flanked by 5′/3′ LTRs. They have a structure similar to endogenous retroviruses, but they lack the envelope (env) gene making them non‐infectious. Long terminal repeats are motif‐rich sequences and can act as bidirectional promoters or enhancers to regulate or inactivate genes by insertion. In this study, we identified a new chimeric LTR subfamily, LTR2i_SS, in the pig genome. This chimeric LTR family appears to be the ancestral form of the previously described LTR2_SS family. LTR2_SS appears to have deleted ~300 bp of un‐annotated, ancestral sequence from LTR2i_SS. We identified no functional provirus sequences for either of these LTR types. LTR2i_SS sequences have been exapted into the untranslated regions of two protein‐coding gene mRNAs. Both of these genes lie within previously mapped pig quantitative trait loci.     

LTR Retrotransposon-Gene Associations in Drosophila melanogaster

Thirty-three percent (228/682) of all long terminal repeat (LTR) retrotransposon sequences (LRSs) present in the sequenced Drosophila melanogaster genome were found to be located in or within 1000 bp of a gene. Recently inserted LTR retrotransposons are significantly more likely to be located in or within genes than are older, fragmented LTR retrotransposon sequences, indicating that most LRS-gene associations are selected against over evolutionary time. LRSs associated with conserved genes (homologenes) are especially prone to negative selection. In contrast, fragmented LRSs that have persisted in the genome over long spans of evolutionary time are preferentially associated with genes involved in signal transduction and other newly evolved functions. Reviewing Editor: Dr. Juergen Brosius     

Retrovirus-related sequences in human DNA: detection and cloning of sequences which hybridize with the long terminal repeat of baboon endogenous virus

Human DNA sequences which hybridized with the long terminal repeats (LTR) of baboon type C virus M7 were detected by non-stringent blot hybridization. About 7 to 10 discrete bands of the LTR-related sequences were commonly observed in the DNAs from four independent human cell lines after digestion with either Eco RI, Hind III or Bam HI. The amounts of these sequences were more abundant in tumor cell lines than in a non-malignant cell line. The human sequences related to the M7 LTR seemed to be located at relatively specific sites on the cell DNA. The human DNA clones which hybridized with M7 LTR were detected in the human DNA library described by Lawn et al. (Cell 15, 1157-1174, 1978), at a frequency of about 300 per haploid genome. Five clones were isolated which shared different extent of homology with M7 LTR and whose restriction maps were totally different one another. The DNA structures of two of them resembled the genome of retroviruses. These results suggest the presence of various types of the LTR-related sequences in human DNA: some of them might represent endogenous virus genomes of human cells.     

Global heterochromatic colocalization of transposable elements with minisatellites in the compact genome of the pufferfish Tetraodon nigroviridis

Because of its unusual high degree of compaction and paucity of repetitive sequences, the genome of the smooth pufferfish Tetraodon nigroviridis is the subject of a well-advanced sequencing project. An astonishing diversity of transposable elements not found in the human and the mouse has been observed in the genome of T. nigroviridis. Due to the difficulty of assembling repeat-rich regions, the whole genome shotgun sequencing approach will probably fail to reveal the general organisation of this compact vertebrate genome. Therefore, in order to gain new insights into the global distribution pattern of repeated DNA in the genome of T. nigroviridis, we have reconstructed partial/complete repetitive sequences from data generated by the genome project and performed double-colour fluorescent in situ hybridization (FISH) analysis for representatives of three major categories of repeated sequences including two minisatellites (ms100 and ms104), two DNA transposons (Tol2 and Buffy1) and two non-long terminal repeat (LTR) retrotransposons (Rex3 and Babar). We show that DNA transposons and retroelements very frequently colocalize with minisatellites and mostly accumulate within heterochromatic regions. These results, which have not been reported so far for the fugu Takifugu rubripes, show that repeated elements are generally excluded from gene-rich regions in T. nigroviridis and underline the extreme degree of compartmentalization of this compact genome. The genome organization of the pufferfish is clearly different from that observed in humans, where repeated sequences make up an important fraction of euchromatic DNA, and is more similar to that observed in the fruit fly Drosophila melanogaster.     

High-throughput retrotransposon-based genetic diversity of maize germplasm assessment and analysis

Maize is one of the world’s most important crops and a model for grass genome research. Long terminal repeat (LTR) retrotransposons comprise most of the maize genome; their ability to produce new copies makes them efficient high-throughput genetic markers. Inter-retrotransposon-amplified polymorphisms (IRAPs) were used to study the genetic diversity of maize germplasm. Five LTR retrotransposons (Huck, Tekay, Opie, Ji, and Grande) were chosen, based on their large number of copies in the maize genome, whereas polymerase chain reaction primers were designed based on consensus LTR sequences. The LTR primers showed high quality and reproducible DNA fingerprints, with a total of 677 bands including 392 polymorphic bands showing 58% polymorphism between maize hybrid lines. These markers were used to identify genetic similarities among all lines of maize. Analysis of genetic similarity was carried out based on polymorphic amplicon profiles and genetic similarity phylogeny analysis. This diversity was expected to display ecogeographical patterns of variation and local adaptation. The clustering method showed that the varieties were grouped into three clusters differing in ecogeographical origin. Each of these clusters comprised divergent hybrids with convergent characters. The clusters reflected the differences among maize hybrids and were in accordance with their pedigree. The IRAP technique is an efficient high-throughput genetic marker-generating method.

     

A rare event of insertion polymorphism of a HERV-K LTR in the human genome

Human endogenous retroviruses (HERVs), which constitute a significant part of the human genome, might have a serious impact on primate evolution. Over a hundred insertions of HERV-K(HML-2) family members distinguish the human genome from other primate genomes. However, only three cases of insertion polymorphisms have been reported so far, all for endogenous HERV-K proviruses. This suggests that some retroviral integrations occurred rather recently in human genome evolution. In this report, we describe a very rare case of true insertion polymorphism of a solitary HERV-K LTR in the human genome. Distribution of the LTR-containing allele was tested in 5 Africans and 83 individuals from three Russian populations. The allele frequency appeared to be relatively high in populations of both European and Asian origin. The detected polymorphic LTR could be a useful molecular genetic marker of the corresponding genomic region.     

Sequences in pol Are Required for Transfer of Human Foamy Virus-Based Vectors

A series of vectors with heterologous genes was constructed from HSRV1, an infectious clone of human foamy virus (HFV), and transfected into baby hamster kidney cells to generate stably transfected vector cell lines. Two cis-acting sequences were required to achieve efficient rescue by helper virus. The first element was located at the 5′ end upstream of position 1274 of the proviral DNA. Interestingly, a mutation in the leader sequence which decreased the ability to dimerize in vitro inhibited transfer by helper HFV. A second element that was important for vector transfer was located in the pol gene between positions 5638 and 6317. Constructs lacking this element were only poorly transferred by helper HFV, even though their RNA was produced in the vector cell lines. This finding rules out the possibility that the observed lack of transfer was due to RNA instability. A minimal vector containing only these two elements could be successfully delivered by helper HFV, confirming that all essential cis-acting sequences were present. The presence of a sequence described as a second polypurine tract in HFV was not necessary for transfer. Our data identified the minimal sequence requirements for HFV vector transfer for the development of useful vector systems.     

Human endogenous retrovirus K106 (HERV-K106) was infectious after the emergence of anatomically modern humans

HERV-K113 and HERV-K115 have been considered to be among the youngest HERVs because they are the only known full-length proviruses that are insertionally polymorphic and maintain the open reading frames of their coding genes. However, recent data suggest that HERV-K113 is at least 800,000 years old, and HERV-K115 even older. A systematic study of HERV-K HML2 members to identify HERVs that may have infected the human genome in the more recent evolutionary past is lacking. Therefore, we sought to determine how recently HERVs were exogenous and infectious by examining sequence variation in the long terminal repeat (LTR) regions of all full-length HERV-K loci. We used the traditional method of inter-LTR comparison to analyze all full length HERV-Ks and determined that two insertions, HERV-K106 and HERV-K116 have no differences between their 5' and 3' LTR sequences, suggesting that these insertions were endogenized in the recent evolutionary past. Among these insertions with no sequence differences between their LTR regions, HERV-K106 had the most intact viral sequence structure. Coalescent analysis of HERV-K106 3' LTR sequences representing 51 ethnically diverse individuals suggests that HERV-K106 integrated into the human germ line approximately 150,000 years ago, after the emergence of anatomically modern humans.