Insertion Of Long Terminal Repeat Research Articles

Fine mapping and candidate gene analysis of CRA8.1.6, which confers clubroot resistance in turnip (Brassica rapa ssp. rapa)

Clubroot disease poses a significant threat to Brassica crops, necessitating ongoing updates on resistance gene sources. In F2 segregants of the clubroot-resistant inbred line BrT18-6-4-3 and susceptible DH line Y510, the genetic analysis identified a single dominant gene responsible for clubroot resistance. Through bulk segregant sequencing analysis and kompetitive allele-specific polymerase chain reaction assays, CRA8.1.6 was mapped within 110 kb (12,255–12,365 Mb) between markers L-CR11 and L-CR12 on chromosome A08. We identified B raA08g015220.3.5C as the candidate gene of CRA8.1.6. Upon comparison with the sequence of disease-resistant material BrT18-6-4-3, we found 249 single-nucleotide polymorphisms, seven insertions, six deletions, and a long terminal repeat (LTR) retrotransposon (5,310 bp) at 909 bp of the first intron. However, the LTR retrotransposon was absent in the coding sequence of the susceptible DH line Y510. Given the presence of a non-functional LTR insertion in other materials, it showed that the LTR insertion might not be associated with susceptibility. Sequence alignment analysis revealed that the fourth exon of the susceptible line harbored two deletions and an insertion, resulting in a frameshift mutation at 8,551 bp, leading to translation termination at the leucine-rich repeat domain’s C-terminal in susceptible material. Sequence alignment of the CDS revealed a 99.4% similarity to Crr1a, which indicate that CRA8.1.6 is likely an allele of the Crr1a gene. Two functional markers, CRA08-InDel and CRA08-KASP1, have been developed for marker-assisted selection in CR turnip cultivars. Our findings could facilitate the development of clubroot-resistance turnip cultivars through marker-assisted selection.

A PAX3 insertion in the Celestial breed and certain feline breeding lines with dominant blue eyes.

During the last 60 years many inherited traits in domestic outbred cats were selected and retained giving birth to new breeds characterised by singular coat or morphological phenotypes. Among them, minimal white spotting associated with blue eyes was selected by feline breeders to create the Altai, Topaz, and Celestial breeds. Various established breeds also introduced this trait in their lineages. The trait, that was confirmed as autosomal dominant by breeding data, was first described in domestic cats from Kazakhstan and Russia, in British shorthair and British longhair from Russia, and in Maine Coon cats from the Netherlands, suggesting different founding effects. Using a genome-wide association study we identified a single region on chromosome C1 that was associated with the minimal white spotting and blue eyes phenotype (also called DBE by breeders for dominant blue eyes) in the French Celestial breed. Within that region we identified Paired Box3 (PAX3) as the strongest candidate gene, since PAX3 is a key regulator of MITF (Melanocyte-Inducing Transcription Factor) and PAX3 variants have been previously identified in various species showing white spotting with or without blue eyes including the mouse and the horse. Whole genome sequencing of a Celestial cat revealed an endogenous retrovirus LTR (long terminal repeat) insertion within PAX3 intron 4 known to contain regulatory sequences (conserved non-coding element [CNE]) involved in PAX3 expression. The insertion is in the vicinity of CNE2 and CNE3. All 52 Celestial and Celestial-mixed cats with a DBE phenotype presented the insertion, that was absent in their 22 non-DBE littermates and in 87 non-DBE cats from various breeds. The outbred Celestial founder was also heterozygous for the insertion. Additionally, the variant was found in nine DBE Maine Coon cats related to the Celestial founder and four DBE Siberian cats with an uncertain origin. Segregation of the variant in the Celestial breed is consistent with dominant inheritance and does not appear to be associated with deafness. We propose that this NC_018730.3:g.206974029_206974030insN[395] variant represents the DBECEL (Celestial Dominant Blue Eyes) allele in the domestic cat.

Endogenous feline leukemia virus long terminal repeat integration site diversity is highly variable in related and unrelated domestic cats

Endogenous retroviruses (ERV) are indicators of vertebrate evolutionary history and play important roles as homeostatic regulators. ERV long terminal repeat (LTR) elements may act as cis-activating promoters or trans-activating enhancer elements modifying gene transcription distant from LTR insertion sites. We previously documented that endogenous feline leukemia virus (FeLV)-LTR copy number variation in individual cats tracks inversely with susceptibility to virulent FeLV disease. To evaluate FeLV-LTR insertion characteristics, we assessed enFeLV-LTR integration site diversity in 20 cats from three genetically distinct populations using a baited linker-mediated PCR approach. We documented 765 individual integration sites unequally represented among individuals. Only three LTR integration sites were shared among all individuals, while 412 sites were unique to a single individual. When primary fibroblast cultures were challenged with exogenous FeLV, we found significantly increased expression of both exogenous and endogenous FeLV orthologs, supporting previous findings of potential exFeLV-enFeLV interactions; however, viral challenge did not elicit transcriptional changes in genes associated with the vast majority of integration sites. This study assesses FeLV-LTR integration sites in individual animals, providing unique transposome genotypes. Further, we document substantial individual variation in LTR integration site locations, even in a highly inbred population, and provide a framework for understanding potential endogenous retroviral element position influence on host gene transcription.

Large-scale long terminal repeat insertions produced a significant set of novel transcripts in cotton.

Genomic analysis has revealed that the 1,637-Mb Gossypium arboreum genome contains approximately 81% transposable elements (TEs), while only 57% of the 735-Mb G. raimondii genome is occupied by TEs. In this study, we investigated whether there were unknown transcripts associated with TE or TE fragments and, if so, how these new transcripts were evolved and regulated. As sequence depths increased from 4 to 100 G, a total of 10,284 novel intergenic transcripts (intergenic genes) were discovered. On average, approximately 84% of these intergenic transcripts possibly overlapped with the long terminal repeat (LTR) insertions in the otherwise untranscribed intergenic regions and were expressed at relatively low levels. Most of these intergenic transcripts possessed no transcription activation markers, while the majority of the regular genic genes possessed at least one such marker. Genes without transcription activation markers formed their+1 and -1 nucleosomes more closely (only (117±1.4)bp apart), while twice as big spaces (approximately (403.5±46.0) bp apart) were detected for genes with the activation markers. The analysis of 183 previously assembled genomes across three different kingdoms demonstrated systematically that intergenic transcript numbers in a given genome correlated positively with its LTR content. Evolutionary analysis revealed that genic genes originated during one of the whole-genome duplication events around 137.7 million years ago (MYA) for all eudicot genomes or 13.7 MYA for the Gossypium family, respectively, while the intergenic transcripts evolved around 1.6 MYA, resultant of the last LTR insertion. The characterization of these low-transcribed intergenic transcripts can facilitate our understanding of the potential biological roles played by LTRs during speciation and diversifications.

Epigenetic and chromosomal features drive transposon insertion in Drosophila melanogaster.

Transposons are mobile genetic elements prevalent in the genomes of most species. The distribution of transposons within a genome reflects the actions of two opposing processes: initial insertion site selection, and selective pressure from the host. By analyzing whole-genome sequencing data from transposon-activated Drosophila melanogaster, we identified 43 316 de novo and 237 germline insertions from four long-terminal-repeat (LTR) transposons, one LINE transposon (I-element), and one DNA transposon (P-element). We found that all transposon types favored insertion into promoters de novo, but otherwise displayed distinct insertion patterns. De novo and germline P-element insertions preferred replication origins, often landing in a narrow region around transcription start sites and in regions of high chromatin accessibility. De novo LTR transposon insertions preferred regions with high H3K36me3, promoters and exons of active genes; within genes, LTR insertion frequency correlated with gene expression. De novo I-element insertion density increased with distance from the centromere. Germline I-element and LTR transposon insertions were depleted in promoters and exons, suggesting strong selective pressure to remove transposons from functional elements. Transposon movement is associated with genome evolution and disease; therefore, our results can improve our understanding of genome and disease biology.

Allele-biased expression of the bovine APOB gene associated with the cholesterol deficiency defect suggests cis-regulatory enhancer effects of the LTR retrotransposon insertion

The insertion of an endogenous retroviral long terminal repeat (LTR) sequence into the bovine apolipoprotein B (APOB) gene is causal to the inherited genetic defect cholesterol deficiency (CD) observed in neonatal and young calves. Affected calves suffer from developmental abnormalities, symptoms of incurable diarrhoea and often die within weeks to a few months after birth. Neither the detailed effects of the LTR insertion on APOB expression profile nor the specific mode of inheritance nor detailed phenotypic consequences of the mutation are undisputed. In our study, we analysed German Holstein dairy heifers at the peak of hepatic metabolic load and exposed to an additional pathogen challenge for clinical, metabolic and hepatic transcriptome differences between wild type (CDF) and heterozygote carriers of the mutation (CDC). Our data revealed that a divergent allele-biased expression pattern of the APOB gene in heterozygous CDC animals leads to a tenfold higher expression of exons upstream and a decreased expression of exons downstream of the LTR insertion compared to expression levels of CDF animals. This expression pattern could be a result of enhancer activity induced by the LTR insertion, in addition to a previously reported artificial polyadenylation signal. Thus, our data support a regulatory potential of mobile element insertions. With regard to the phenotype generated by the LTR insertion, heterozygote CDC carriers display significantly differential hepatic expression of genes involved in cholesterol biosynthesis and lipid metabolism. Phenotypically, CDC carriers show a significantly affected lipomobilization compared to wild type animals. These results reject a completely recessive mode of inheritance for the CD defect, which should be considered for selection decisions in the affected population. Exemplarily, our results illustrate the regulatory impact of mobile element insertions not only on specific host target gene expression but also on global transcriptome profiles with subsequent biological, functional and phenotypic consequences in a natural in-vivo model of a non-model mammalian organism.

CsMYB184 regulates caffeine biosynthesis in tea plants.

CsMYB184 regulates caffeine biosynthesis in tea plants.

Reactivation of transposable elements following hybridization in fission yeast.

Hybridization is thought to reactivate transposable elements (TEs) that were efficiently suppressed in the genomes of the parental hosts. Here, we provide evidence for this “genomic shock hypothesis” in the fission yeast Schizosaccharomyces pombe. In this species, two divergent lineages (Sp and Sk) have experienced recent, likely human-induced, hybridization. We used long-read sequencing data to assemble genomes of 37 samples derived from 31 S. pombe strains spanning a wide range of ancestral admixture proportions. A comprehensive TE inventory revealed exclusive presence of long terminal repeat (LTR) retrotransposons. Sequence analysis of active full-length elements, as well as solo LTRs, revealed a complex history of homologous recombination. Population genetic analyses of syntenic sequences placed insertion of many solo LTRs before the split of the Sp and Sk lineages. Most full-length elements were inserted more recently, after hybridization. With the exception of a single full-length element with signs of positive selection, both solo LTRs and, in particular, full-length elements carry signatures of purifying selection indicating effective removal by the host. Consistent with reactivation upon hybridization, the number of full-length LTR retrotransposons, varying extensively from zero to 87 among strains, significantly increases with the degree of genomic admixture. This study gives a detailed account of global TE diversity in S. pombe, documents complex recombination histories within TE elements, and provides evidence for the “genomic shock hypothesis.”

Safety and Efficacy Evaluation of Recombinant Marek's Disease Virus with REV-LTR.

Recently, chickens vaccinated with the CVI988/Rispens vaccine showed increased tumor incidence. Moreover, many strains of Marek’s disease virus (MDV) that were naturally integrated with the long terminal repeat (LTR) of the avian reticuloendotheliosis virus (REV) have been isolated, which means it is necessary to develop a new vaccine. In this study, two LTR sequences were inserted into Rispens to construct a recombinant MDV (rMDV). Then, the safety and efficacy of rMDV were evaluated separately in chickens. The growth rate curves showed that the insertion of REV-LTR into MDV enabled a faster replication in vitro than Rispens. Chickens immunized with high or repeated dose rMDV had no MD clinical signs. Further, no tumor, tissue lesions, or evident pathological changes were observed in the chicken organs. Polymerase chain reaction (PCR) and virus isolation revealed that rMDV had the ability to spread horizontally to non-immunized chickens and had no impact on the environment. After five passages in chickens, there were no obvious lesions, and the LTR insertion was stable. There were also no deletions or mutations, which indicates that rMDV is safe in chickens. In addition, rMDV has an advantage over Rispens against vvMDV Md5 at low doses. All results demonstrate that the transgenic strain of rMDV with REV-LTR can be used as a live attenuated vaccine candidate.

Gossypium Genomics: Trends, Scope, and Utilization for Cotton Improvement.

Cotton (Gossypium spp.) is the most important natural fiber crop worldwide. The diversity of Gossypium species also provides an ideal model for investigating evolution and domestication of polyploids. However, the huge and complex cotton genome hinders genomic research. Technical advances in high-throughput sequencing and bioinformatics analysis have now largely overcome these obstacles, bringing about a new era of cotton genomics. Here, we review recent progress in Gossypium genomics based on whole genome sequencing, resequencing, and comparative genomics, which have provided insights about the genomic basis of fiber biogenesis and the landscape of cotton functional genomics. We address current challenges and present multidisciplinary genomics-enabled breeding strategies covering the breadth of high fiber yield, quality, and environmental resilience for future cotton breeding programs.

The molecular basis for host responses to Marek's disease viruses integrated with different retro-viral long terminal repeat

Integration of retro-viral long terminal repeat (LTR) into the Marek's disease virus (MDV) genome can occur both in co-cultivation cell cultures and naturally in dual infected chickens. It is clear that the LTR insert is associated with the pathogenicity of MDV. The objective of this study was to compare the host responses to MDV with a different retro-viral LTR insert. Gene-chip containing chicken genome was employed to investigate the gene transcription profile of chicken embryo fibroblasts cells, and 795 genes were differentially expressed in chicken embryo fibroblasts infected with GX0101 with a reticuloendotheliosis virus LTR insert as compared to GX0101-ALV-LTR significantly. The differentially expressed genes were mostly associated with the regulation of transcription and the development of multiple organs. Based on the bio functions of the differential genes, infection of GX0101 was predicated with a greater development disorder of multiple systems, resulting in higher growth retardation, mortality, tumorigenicity, and immunosuppression in chickens than GX0101-ALV-LTR. Collectively, our results provided valuable insights into elucidation of the possible relationship between retro-viral LTR insert and the observed phenotypes caused by MDV recombinant viruses.

Insertion of a solo LTR retrotransposon associates with spur mutations in 'Red Delicious' apple (Malus×domestica).

Insertion of a solo LTR, which possesses strong bidirectional, stem-specific promoter activities, is associated with the evolution of a dwarfing apple spur mutation. Spur mutations in apple scions revolutionized global apple production. Since long terminal repeat (LTR) retrotransposons are tightly related to natural mutations, inter-retrotransposon-amplified polymorphism technique and genome walking were used to find sequences in the apple genome based on these LTRs. In 'Red Delicious' spur mutants, a novel, 2190-bp insertion was identified as a spur-specific, solo LTR (sLTR) located at the 1038th nucleotide of another sLTR, which was 1536 bp in length. This insertion-within-an-insertion was localized within a preexisting Gypsy-50 retrotransposon at position 3,762,767 on chromosome 4. The analysis of transcriptional activity of the two sLTRs (the 2190- and 1536-bp inserts) indicated that the 2190-bp sLTR is a promoter, capable of bidirectional transcription. GUS expression in the 2190-bp-sense and 2190-bp-antisense transgenic lines was prominent in stems. In contrast, no promoter activity from either the sense or the antisense strand of the 1536-bp sLTR was detected. From ~150kb of DNA on each side of the 2190 bp, sLTR insertion site, corresponding to 300kb of the 'Golden Delicious' genome, 23 genes were predicted. Ten genes had predicted functions that could affect shoot development. This first report, of a sLTR insertion associated with the evolution of apple spur mutation, will facilitate apple breeding, cloning of spur-related genes, and discovery of mechanisms behind dwarf habit.

Transcriptional and Bioinformatic Analysis Provide a Relationship between Host Response Changes to Marek's Disease Viruses Infection and an Integrated Long Terminal Repeat.

GX0101, Marek's disease virus (MDV) strain with a long terminal repeat (LTR) insert of reticuloendotheliosis virus (REV), was isolated from CVI988/Rispens vaccinated birds showing tumors. We have constructed a LTR deleted strain GX0101ΔLTR in our previous study. To compare the host responses to GX0101 and GX0101ΔLTR, chicken embryo fibroblasts (CEF) cells were infected with two MDV strains and a gene-chip containing chicken genome was employed to examine gene transcription changes in host cells in the present study. Of the 42,368 chicken transcripts on the chip, there were 2199 genes that differentially expressed in CEF infected with GX0101 compared to GX0101ΔLTR significantly. Differentially expressed genes were distributed to 25 possible gene networks according to their intermolecular connections and were annotated to 56 pathways. The insertion of REV LTR showed the greatest influence on cancer formation and metastasis, followed with immune changes, atherosclerosis, and nervous system disorders in MDV-infected CEF cells. Based on these bio functions, GX0101 infection was predicated with a greater growth and survival inhibition but lower oncogenicity in chickens than GX0101ΔLTR, at least in the acute phase of infection. In summary, the insertion of REV LTR altered the expression of host genes in response to MDV infection, possibly resulting in novel phenotypic properties in chickens. Our study has provided the evidence of retroviral insertional changes of host responses to herpesvirus infection for the first time, which will promote to elucidation of the possible relationship between the LTR insertion and the observed phenotypes.

Abstract 2789: Associations between insertional polymorphisms of human endogenous retrovirus K113 and K115 and breast cancer risk in African American and European American women

Abstract Background: Human endogenous retroviruses (HERVs) are remnants of ancient virus infection. The majority of them are disabled due to mutation and/or deletion. However, HERV K113 and K115 have been shown to have full-length insertion in human genome and retain the ability to encode functional virus proteins in some individuals. Considering the potential role of HERVs in carcinogenesis and a high genetic homology between HERV K and mouse mammary tumor virus, the study is designed to investigate the distribution of HERV K113 and K115 in African American (AA) and European American (EA) women, and their association with breast cancer risk. Methods: Built on a funded multi-center case-control study, the Women's Circle of Health Study, the study included 1242 cases (608 AA and 634 EA) and 1422 controls (783 AA and 639 EA). PCR followed by fragment analysis was used for insertional polymorphism assay. For each HERV, three PCRs were performed to determine whether the insertion is partial or full length. Indeed, a subset of individuals showed insertion of long terminal repeat (LTR) of HERV, instead of whole virus insertion. The distribution of insertional polymorphisms was compared between AA and EA as well as between cases and controls. Logistic regression was used to calculate odds ratio (OR) and 95% confidence interval (CI) with adjustment for age at diagnosis and proportion of European ancestry. Results: For both K113 and K115, with or without including LTR insertion, the distribution was significantly different between AA and EA women. A two-fold higher prevalence of HERVs was observed in AA women, showing 51.5% of individuals with at least one copy of either K113 or K115 compared to 23.3% in EA women among controls. The prevalence of HERVs was inversely associated with proportions of European ancestry, showing a decrease from averagely 66% European ancestry in individuals without insertion to 42.9%, 23.2%, 13.3% with one, two, or more insertions of HERV K113 or K115, respectively. Between cases and controls, the prevalence of K113 was slightly higher in controls, but not for K115. Furthermore, both K113 and K115 insertion were significantly associated with the reduced risk of breast cancer in both AA and EA women. Near 50% reduction of breast cancer risk was observed in AA and EA women with homozygous insertion of K113 (combined OR, 0.45; 95% CI, 0.25-0.80). A trend of a reduction in breast cancer risk with an increase of copy number of HERV K insertion was observed in both AA and EA women. However, there was no association with breast cancer subtypes. Conclusion: This is the first study to document the prevalence of HERV K113 and K115 in AA and EA population, and to report an inverse association with breast cancer risk. Validation of the findings in a relatively large study is warranted in the future. Funded by 5R03CA156645, K07CA148888, P01151135, and R01CA100598. Citation Format: Li Tang, Steven R. Gregory, David Tritchler, Gary R. Zirpoli, Song Yao, Warren Davis, Gregory L. Ciupak, Yasmin Thanavala, Elisa V. Bandera, Christine B. Ambrosone. Associations between insertional polymorphisms of human endogenous retrovirus K113 and K115 and breast cancer risk in African American and European American women. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2789. doi:10.1158/1538-7445.AM2015-2789

Influence of avian leukosis virus long terminal repeat on biological activities of Marek's disease virus

GX0101 was the first reported field strain of recombinant Marek's disease virus (MDV) that contained a long terminal repeat (LTR) from the reticuloendotheliosis virus (REV). It is a very virulent MDV strain, with relatively high horizontal transmission ability. The REV LTR in GX0101 genome was proved to decrease the pathogenicity but increase the potential for horizontal transmission of the virus. Here we constructed a recombinant MDV GX0101-ALV-LTR to study stability of avian leukosis virus (ALV) LTR at the REV LTR insertion site in GX0101 genome and its influence on biological activities of the recombinant virus. The results showed that GX0101-ALV-LTR was able to replicate stably both in vitro and in vivo. ALV LTR remained stable in chickens infected either by inoculation with the recombinant virus GX0101-ALV-LTR or by horizontal transmission, as well as in cell culture. The pathogenic properties of GX0101-ALV-LTR virus were evaluated in infected specific-pathogen-free chickens. The present study demonstrated that the GX0101-ALV-LTR virus had a weaker inhibitory effect on the growth rates of the infected chickens and induced weaker immunosuppressive effects. Horizontal transmission ability of the GX0101-ALV-LTR virus appeared to be similar with its parental virus GX0101. In short, ALV LTR was stable in GX0101 after replacing REV LTR, and the recombinant virus showed similar horizontal transmission ability but decreased pathogenicity.

Evidence for a Retroviral Insertion in TRPM1 as the Cause of Congenital Stationary Night Blindness and Leopard Complex Spotting in the Horse

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ2=1022.00, p<<0.0005), and CSNB, testing 43 horses (χ2=43, p<<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.

Insertion of Reticuloendotheliosis Virus Long Terminal Repeat into the Genome of CVI988 Strain of Marek's Disease Virus Results in Enhanced Growth and Protection

Marek's disease (MD) is a lymphoproliferative disease of chickens caused by serotype 1 MD virus (MDV). Vaccination of commercial poultry has drastically reduced losses from MD, and the poultry industry cannot be sustained without the use of vaccines. Retrovirus insertion into herpesvirus genomes is an efficient process that alters the biological properties of herpesviruses. RM1, a virus derived from the virulent JM strain of MDV, by insertion of the reticuloendotheliosis (REV) long terminal repeat (LTR), was attenuated for oncogenicity but retains properties of the parental virus, such as lymphoid organ atrophy. Here we show that insertion of the REV LTR into the genome of vaccine strain CVI988 resulted in a virus (CVRM) that replicated to higher levels than parental CVI988 in cell culture and that remained apathogenic for chickens. In addition, CVRM showed protection indices similar or superior to those afforded by CVI988 virus in laboratory and field protection trials, indicating that it could be developed as a safe and efficacious vaccine to protect against very virulent plus MDV.

Sequence analysis of the whole genome of a recombinant Marek’s disease virus strain, GX0101, with a reticuloendotheliosis virus LTR insert

Marek's disease virus Chinese strain GX0101, isolated in 2001, is the first reported recombinant gallid herpesvirus type 2 (GaHV-2) field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. We constructed an infectious bacterial artificial chromosome (BAC) clone of GX0101, which showed characteristics very similar to those of the parental virus in replication and pathogenicity. Using the GX0101 BAC clone, the complete genome of GX0101 was sequenced and analyzed. The length of the GX0101 genome is 178,101bp, and it contains only one REV-LTR insert at a site 267bp upstream of the sorf2 gene.

Origins of Albino and Hooded Rats: Implications from Molecular Genetic Analysis across Modern Laboratory Rat Strains

Albino and hooded (or piebald) rats are one of the most frequently used laboratory animals for the past 150 years. Despite this fact, the origin of the albino mutation as well as the genetic basis of the hooded phenotype remained unclear. Recently, the albino mutation has been identified as the Arg299His missense mutation in the Tyrosinase gene and the hooded (H) locus has been mapped to the ∼460-kb region in which only the Kit gene exists. Here, we surveyed 172 laboratory rat strains for the albino mutation and the hooded (h) mutation that we identified by positional cloning approach to investigate possible genetic roots and relationships of albino and hooded rats. All of 117 existing laboratory albino rats shared the same albino missense mutation, indicating they had only one single ancestor. Genetic fine mapping followed by de novo sequencing of BAC inserts covering the H locus revealed that an endogenous retrovirus (ERV) element was inserted into the first intron of the Kit gene where the hooded allele maps. A solitary long terminal repeat (LTR) was found at the same position to the ERV insertion in another allele of the H locus, which causes the so called Irish (hi) phenotype. The ERV and the solitary LTR insertions were completely associated with the hooded and Irish coat patterns, respectively, across all colored rat strains examined. Interestingly, all 117 albino rat strains shared the ERV insertion without any exception, which strongly suggests that the albino mutation had originally occurred in hooded rats.

Insertion of reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of a very virulent Marek's disease virus alters its pathogenicity

Co-cultivation of the JM/102W strain of Marek's disease virus (MDV) with reticuloendotheliosis virus (REV) resulted in the generation of a recombinant MDV containing the REV long terminal repeat (LTR) named the RM1 strain of MDV, a strain that was highly attenuated for oncogenicity but induced severe bursal and thymic atrophy. We hypothesize that the phenotypic changes were solely due to the LTR insertion. Furthermore, we hypothesize that insertion of REV LTR into an analogous location in a different MDV would result in a similar phenotypic change. To test these hypotheses, we inserted the REV LTR into a bacterial artificial chromosome (BAC) clone of a very virulent strain of MDV, Md5, and designated the virus rMd5-RM1-LTR. The rMd5-RM1-LTR virus and the rMd5 virus were passaged in duck embryo fibroblast cells for up to 40 passages before pathogenicity studies. Susceptible chickens were inoculated intra-abdominally at hatch with the viruses rMd5-RM1-LTR, rMd5 BAC parental virus, wild-type strain Md5, or strain RM1 of MDV. The rMd5-RM1-LTR virus was attenuated at cell culture passage 40, whereas the rMd5 BAC without RM1 LTR retained its pathogenicity at cell culture passage 40. Using polymerase chain analysis, the RM1 LTR insert was detected in MDV isolated from buffy coat cells collected from chickens inoculated with rMd5-RM1-LTR, but only at 1 week post inoculation. The data suggest that the presence of the RM1 LTR insert within MDV genome for 1 week post inoculation with virus at hatch is sufficient to cause a reduction in pathogenicity of strain Md5 of MDV.