In vivo stimulation of H-2Dd expression following RadLV infection of thymocytes: increased transcription and DNA-binding activity to sequences 5' of the Dd gene.
Early studies showed that resistance to RadLV-induced leukaemia is mediated by gene(s) in the H-2D region of the MHC. Furthermore, these experiments correlated disease resistance with changes in H-2 expression occurring very early after virus inoculation. In the present study, we have begun to study at the molecular level this stimulation of H-2Dd class I expression in thymocytes of resistant mouse strains following infection by RadLV. The resistant strain of B10.T(6R) mice is used in these studies. When these infected thymocytes are assayed by fluorescence-activated cell sorting analysis, we can detect increased levels of H-2Dd expression on the surface of the thymocytes as early as 12 days following intrathymic injection of RadLV. RNA was prepared and examined by Northern blot analysis; H-2 mRNA levels are shown to be elevated on the order of four-fold. Nuclei were prepared from normal and infected thymocytes and the run-off transcripts were analysed by slot-blot hybridization. The rate of H-2 mRNA transcription is shown to be two- to three-fold higher in RadLV-infected thymocytes at 14 days post-infection when compared to that of normal thymocytes. These data demonstrate that elevation of H-2 surface expression following RadLV infection is the result of transcriptional activation. Extracts have been prepared from both normal and infected B10.T(6R) thymocytes and have been used in gel mobility assays in order to detect the interaction of potential trans-acting regulatory factors with sequences 5' of the H-2Dd gene. Specific binding occurs in both extracts, but the assay shows that the extracts differ both quantitatively and qualitatively; the extracts from infected thymocytes bind to additional sequences and to a higher degree than that from normal thymocytes. DNase I protection analysis locates a number of protein-binding sites, some of which are protected by extracts of either origin and some of which are only protected by extracts from infected cells. Two of these sequences are similar to the previously recognized consensus recognition sequences for the binding of AP-1 and NF-chi B. Oligonucleotides have been synthesized for both the genomic sequences being protected from DNase I digestion as well the published consensus sequences. While the DNA-binding activity in infected thymocytes for both AP-1 and NF-chi B-binding sites is increased, the binding to the genomic "AP-1 like" binding site is activated to a considerably greater level.(ABSTRACT TRUNCATED AT 400 WORDS)
Brown
G D
GD
Department of Pathology, New York University Medical Center, NY 10016.
Nobunaga
T
T
Morris
D R
DR
Meruelo
D
D
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D015870:Gene Expression; D006183:H-2 Antigens / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D016721:Radiation Leukemia Virus / Q000254:growth & development*; D013601:T-Lymphocytes / Q000382:microbiology*; D014157:Transcription Factors / Q000032:analysis; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D006183:H-2 Antigens; D012333:RNA, Messenger; D014157:Transcription Factors