Avian Leukosis Virus Activation of an Antisense RNA Upstream of TERT in B-Cell Lymphomas.

UNLABELLED: Avian leukosis virus (ALV) induces tumors by integrating its proviral DNA into the chicken genome and altering the expression of nearby genes via strong promoter and enhancer elements. Viral integration sites that contribute to oncogenesis are selected in tumor cells. Deep-sequencing analysis of B-cell lymphoma DNA confirmed that the telomerase reverse transcriptase (TERT) gene promoter is a common ALV integration target. Twenty-six unique proviral integration sites were mapped between 46 and 3,552 nucleotides (nt) upstream of the TERT transcription start site, predominantly in the opposite transcriptional orientation to TERT Transcriptome-sequencing (RNA-seq) analysis of normal bursa revealed a transcribed region upstream of TERT in the opposite orientation, suggesting the TERT promoter is bidirectional. This transcript appears to be an uncharacterized antisense RNA. We have previously shown that TERT expression is upregulated in tumors with integrations in the TERT promoter region. We now report that the viral promoter drives the expression of a chimeric transcript containing viral sequences spliced to exons 4 through 7 of this antisense RNA. Clonal expansion of cells with ALV integrations driving overexpression of the TERT antisense RNA suggest it may have a role in tumorigenesis. IMPORTANCE: The data suggest that ALV integrations in the TERT promoter region drive the overexpression of a novel antisense RNA and contribute to the development of lymphomas.

A comparative physical map reveals the pattern of chromosomal evolution between the turkey (Meleagris gallopavo) and chicken (Gallus gallus) genomes.

BACKGROUND: A robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species. RESULTS: The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes. CONCLUSION: The BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.

Defining the turkey MHC: identification of expressed class I- and class IIB-like genes independent of the MHC-B.

The MHC of the turkey (Meleagris gallopavo) is divided into two genetically unlinked regions; the MHC-B and MHC-Y. Although previous studies found the turkey MHC-B to be highly similar to that of the chicken, little is known of the gene content and extent of the MHC-Y. This study describes two partially overlapping large-insert BAC clones that genetically and physically map to the turkey MHC chromosome (MGA18) but to a region that assorts independently of MHC-B. Within the sequence assembly, 14 genes were predicted including new class I- and class IIB-like loci. Additional unassembled sequences corresponded to multiple copies of the ribosomal RNA repeat unit (18S-5.8S-28S). Thus, this newly identified MHC region appears to represent a physical boundary of the turkey MHC-Y. High-resolution multi-color fluorescence in situ hybridization studies confirm rearrangement of MGA18 relative to the orthologous chicken chromosome (GGA16) in regard to chromosome architecture, but not gene order. The difference in centromere position between the species is indicative of multiple chromosome rearrangements or alternate events such as neocentromere formation/centromere inactivation in the evolution of the MHC chromosome. Comparative sequencing of commercial turkeys (six amplicons totaling 7.6 kb) identified 68 single nucleotide variants defining nine MHC-Y haplotypes. Sequences of the new class I- and class IIB-like genes are most similar to MHC-Y genes in the chicken. All three loci are expressed in the spleen. Differential transcription of the MHC-Y class IIB-like loci was evident as one class IIB-like locus was only expressed in some individuals.

Telomere biology of the chicken: a model for aging research.

Division-dependent telomere shortening correlating with age triggers senescence on a cellular level and telomere dysfunction can facilitate oncogenesis. Therefore, the study of telomere biology is critical to the understanding of aging and cancer. The domestic chicken, a classic model for the study of developmental biology, possesses a telomere genome with highly conserved aspects and distinctive features which make it uniquely suited for the study of telomere maintenance mechanisms, their function and dysfunction. The purpose of this review is to highlight the chicken as a model for aging research, specifically as a model for telomere and telomerase research, and to increase its utility as such by describing developments in the study of chicken telomeres and telomerase in the context of related research in human and mouse.

Genetic variation exists for telomeric array organization within and among the genomes of normal, immortalized, and transformed chicken systems.

This study investigated telomeric array organization of diverse chicken genotypes utilizing in vivo and in vitro cells having phenotypes with different proliferation potencies. Our experimental objective was to characterize the extent and nature of array variation present to explore the hypothesis that mega-telomeres are a universal and fixed feature of chicken genotypes. Four different genotypes were studied including normal (UCD 001, USDA-ADOL Line 0), immortalized (DF-1), and transformed (DT40) cells. Both cytogenetic and molecular approaches were utilized to develop an integrated view of telomeric array organization. It was determined that significant variation exists within and among chicken genotypes for chromosome-specific telomeric array organization and total genomic-telomeric sequence content. Although there was variation for mega-telomere number and distribution, two mega-telomere loci were in common among chicken genetic lines (GGA 9 and GGA W). The DF-1 cell line was discovered to maintain a complex derivative karyotype involving chromosome fusions in the homozygous and heterozygous condition. Also, the DF-1 cell line was found to contain the greatest amount of telomeric sequence per genome (17%) as compared to UCD 001 (5%) and DT40 (1.2%). The chicken is an excellent model for studying unique and universal features of vertebrate telomere biology, and characterization of the telomere length variation among genotypes will be useful in the exploration of mechanisms controlling telomere length maintenance in different cell types having unique phenotypes.

Telomerase gene expression in the chicken: Telomerase RNA (TR) and reverse transcriptase (TERT) transcript profiles are tissue-specific and correlate with telomerase activity.

Telomerase is the specialized enzyme which replicates the telomeres, thus maintaining the integrity of the chromosome ends; in absence of enzyme activity telomere lengths decrease, ultimately impacting genome stability. In this study, we examined the mRNA expression of both enzyme components, the RNA template (TR) and catalytic subunit (TERT) during growth and development of the chicken to better understand mechanisms which regulate telomerase activity in vertebrates. Quantitative real-time PCR was used to establish transcript profiles for six ages ranging from pre-blastula to two-year old adults. Organ-specific profiles were established for brain, heart, liver, intestine, spleen and gonad. The pre-blastula and gastrula stages exhibited very high transcript levels of both telomerase components; organs from the embryos and adult showed transcript levels either similar or down-regulated relative to the early differentiation embryo stages. Organs which are known to become negative for telomerase activity between the embryo and adult stages (brain, heart, liver) exhibited down-regulation of TR and either no change or an increase in TERT transcripts. Whereas, organs which maintain high telomerase activity even in adults (intestine, spleen, gonad), generally exhibited up-regulation of transcripts for both components. However, there were some tissue-specific differences between telomerase-positive tissues. These results show that TERT and TR transcript levels correlate with telomerase activity profiles and suggest that TR is the rate-limiting component in telomerase-negative tissues.