RESUMO
Macronuclear DNA from Tetrahymena was examined in order to determine whether the pattern of adenine methylation changed with the transcriptional activity of nearby genes. The DNA from growing, starved and conjugating cells was digested with six restriction enzymes which are sensitive to methylation of adenine within their recognition site. Southern blots of the restricted DNAs were probed with seven cDNA clones and one genomic clone which are homologous to polyA+ RNAs, whose transcriptional activity varies with the physiological state of the cell. One of the cDNA clones, BC11, had not been described previously. It hybridized to a 1.3 kb transcript which was present in populations of starved and conjugating, but not in growing cells. On Southern blots of genomic DNA it hybridized to a complex pattern of bands which was highly polymorphic between the DNAs of closely related strains. It was estimated that between 137 and 272 sites were assayed for changes in methylation, including at least 27 sites which were known to be methylated. No differences were seen in the size of restriction fragments from cells in different physiological states. The data suggested that the methylation pattern, which is determined during macronuclear development, does not vary with the physiological state of the cell.
Assuntos
Adenina/metabolismo , DNA de Protozoário/metabolismo , Tetrahymena/genética , Animais , Northern Blotting , Southern Blotting , Clonagem Molecular , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Biblioteca Gênica , Metilação , Tetrahymena/crescimento & desenvolvimento , Tetrahymena/metabolismo , Transcrição GênicaRESUMO
In the ciliated protozoan Tetrahymena 10-20% of the DNA sequences are micronucleus (germ line) specific. Six members of a family of repeated mic-specific DNA sequences are homologous to a 1.5-kb poly(A)+ RNA. The transcript is present in mature cells starved in 10 or 60 mM Tris, in starved immature cells, and in stationary cells. RNA from log-phase and heat-shocked cells does not have detectable levels of the transcript. These data indicate that at least one germ line limited DNA sequence is transcribed in the micronucleus of Tetrahymena.
Assuntos
DNA/genética , Tetrahymena/genética , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Clonagem Molecular , Enzimas de Restrição do DNA , Células Germinativas/ultraestrutura , Temperatura Alta , Hibridização de Ácido Nucleico , Poli A/genética , RNA/genética , RNA Mensageiro , Tetrahymena/crescimento & desenvolvimento , Transcrição GênicaRESUMO
DNA from the amicronucleate Tetrahymena cell line BI3840 was probed for DNA sequences which are limited to the micronucleus in wild-type cells. Four micronucleus-specific DNA sequences were not detectable in DNA from the amicronucleate cell line. Two of the six micronucleus-specific DNA sequences tested hybridized to DNA from amicronucleate cells. Both the number of fragments homologous to these sequences and the intensity of hybridization were reduced in the DNA from the amicronucleate cells relative to DNA from a wild-type cell line, indicating that less than one micronucleus equivalent of the micronucleus-specific DNA sequences was retained in the amicronucleate cell line. Thus many micronucleus-specific DNA sequences were eliminated from the developing macronucleus of BI3840 as they are in wild-type cells, but in at least two cases the elimination was incomplete. In situ hybridization suggested that the DNA sequences which are limited to the micronucleus in wild-type cells are present in the macronucleus of the amicronucleate cell line. Southern blots of DNA from the amicronucleate cell line were also probed with DNA sequences which are retained in the macronucleus. At least two types of genome rearrangements occurred in the BI3840 macronucleus as they do in wild-type cells. No spurious rearrangements were observed.
