RESUMEN
Whole genome comparisons of distantly related species effectively predict biologically important sequences--core genes and cis-acting regulatory elements (REs)--but require experimentation to verify biological activity. To examine the efficacy of comparative genomics in identification of active REs from anonymous, non-coding (NC) sequences, we generated a novel alignment of the human and draft zebrafish genomes, and contrasted this set to existing human and fugu datasets. We tested the transcriptional regulatory potential of candidate sequences using two in vivo assays. Strict selection of non-genic elements which are deeply conserved in vertebrate evolution identifies 1744 core vertebrate REs in human and two fish genomes. We tested 16 elements in vivo for cis-acting gene regulatory properties using zebrafish transient transgenesis and found that 10 (63%) strongly modulate tissue-specific expression of a green fluorescent protein reporter vector. We also report a novel quantitative enhancer assay with potential for increased throughput based on normalized luciferase activity in vivo. This complementary system identified 11 (69%; including 9 of 10 GFP-confirmed elements) with cis-acting function. Together, these data support the utility of comparative genomics of distantly related vertebrate species to identify REs and provide a scaleable, in vivo quantitative assay to define functional activity of candidate REs.
Asunto(s)
Regulación de la Expresión Génica , Genoma Humano , Elementos de Respuesta , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Secuencia Conservada , Embrión no Mamífero/metabolismo , Genómica , Humanos , Luciferasas de Luciérnaga/análisis , Luciferasas de Luciérnaga/genética , Luciferasas de Renilla/análisis , Luciferasas de Renilla/genética , Sustancias Luminiscentes , Transcripción Genética , Pez Cebra/embriologíaRESUMEN
Oligoclonal T cell expansions (TCE) are common in old humans and mice, but it is not known whether the T cell response to a specific antigen is more restricted in old vs. young animals. Herein, we describe an enhanced and prolonged response of tumor antigen-specific CD8 cells in old mice identified by K(d)/peptide tetramers and Vbeta10 staining. At the onset of the response CD8 T cell numbers and Vbeta10+CD8+ cells at the site of tumor injection were lower in old mice, hinting that control of initial tumor growth may not be optimal. As further evidence of a dysregulated response in old mice, antibody titers to the tumor were deficient and the CD8 tumor antigen-specific response was greater and more prolonged in the blood and spleen. Old mice selected a more oligoclonal TCR repertoire based on TCRbeta chain CDR3 length analysis and sequences. Persistent expansions of Vbeta10+CD8+ cells in old mice had memory/activation phenotypes. This induced tumor antigen-specific response may represent a model for the spontaneous TCE observed with aging and demonstrates that the CD8 response to a defined peptide/MHC antigen is indeed more oligoclonal in old mice.