amanitins and Anemia

The nucleotide sequences of the termini of two of the genomic segments of the negative strand RNA virus infectious salmon anaemia virus (ISAV) were determined. The sequence of the terminal 9 nucleotides at both ends of the viral RNAs was identical, and showed distinctive sequence homology with the conserved terminal sequences found in the orthomyxoviruses. For both ISAV genomic segments a computer-based secondary structure modelling indicated that the terminal 21-24 nucleotides were able to form self-complementary panhandle structures. Comparison with ISAV-derived mRNA sequences showed that ISAV mRNAs have heterogeneous 5'-ends, and are polyadenylated from a signal sequence 13-14 nucleotides downstream of the 5'-end terminus of the vRNA. Furthermore, the in vitro replication of ISAV was hindered by the RNA polymerase II inhibitor alpha-amanitin. These findings indicate that the mechanisms for replication of ISAV are similar to those of the orthomyxoviruses, and add to the previously reported structural similarities between ISAV and the orthomyxoviruses.

Topics: 3' Untranslated Regions; 5' Untranslated Regions; Amanitins; Anemia; Animals; Base Sequence; Cell Line; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fish Diseases; Fishes; Molecular Sequence Data; Nucleic Acid Conformation; Orthomyxoviridae; Orthomyxoviridae Infections; RNA Polymerase II; RNA, Messenger; RNA, Viral; Salmon; Sequence Alignment; Virus Replication

Accurate initiation of transcription in vitro requires, in addition to RNA polymerase II, factors present in soluble extracts of cultured cells. We have developed transcription system in vitro, which permits us to visualize the transcription-initiation complexes on a mouse beta globin restricted fragment from a recombinant beta globin bacteriophage DNA. Using the lambda fragments as internal controls this system has allowed us to assess the specificity of transcription with RNA polymerase II. Comparing extracts from cells and tissues expressing the globin genes (Friend cells, spleen and blood from phenylhydrazine-induced anemic mice) with those which do not (thymus, liver, PCC3 cells), we observed that specific initiation of transcription on the beta globin gene occurs only with soluble extracts from erythroid tissues. This tissue-specific transcription is partially sensitive to alpha-amanitin and occurs at the 5' end of the globin gene.

Topics: Amanitins; Anemia; Animals; DNA; Erythrocytes; Friend murine leukemia virus; Globins; Leukemia, Experimental; Mice; Mice, Inbred BALB C; RNA Polymerase II; Spleen; Tissue Extracts; Transcription Factors; Transcription, Genetic