S-Adenosylmethionine (AdoMet or SAM) plays a pivotal role as a methyl donor in a myriad of biological and biochemical events. Although it has been claimed that AdoMet itself has therapeutic benefits, it remains to be established whether it can be taken up intact by cells. S-Adenosylhomocysteine (AdoHcy), formed after donation of the methyl group of AdoMet to a methyl acceptor, is then hydrolyzed to adenosine and homocysteine by AdoHcy hydrolase. This enzyme has long been a target for inhibition as its blockade can affect methylation of phospholipids, proteins, DNA, RNA, and other small molecules. Protein carboxymethylation may be involved in repair functions of aging proteins, and heat shock proteins are methylated in response to stress. Bacterial chemotaxis involves carboxymethylation and demethylation in receptor-transducer proteins, although a similar role in mammalian cells is unclear. The precise role of phospholipid methylation remains open. DNA methylation is related to mammalian gene activities, somatic inheritance, and cellular differentiation. Activation of some genes has been ascribed to the demethylation of critical mCpG loci, and silencing of some genes may be related to the methylation of specific CpG loci. Viral DNA genomes exist in cells as extrachromosomal units and are generally not methylated, although once integrated into host chromosomes, different patterns of methylation are correlated with altered paradigms of transcriptional activity. Some viral latency may be related to DNA methylation. Cellular factors have been found to interact with methylated DNA sequences. Methylation of mammalian ribosomal RNAs occurs soon after the synthesis of its 47S precursor RNA in the nucleolus before cleavage to smaller fragments. Inhibition of the methylation of rRNA affects its processing to mature 18S and 28S rRNAs. The methylation of 5'-terminal cap plays an important role in mRNA export from the nucleus, efficient translation, and protection of the integrity of mRNAs. Another important function of AdoMet is that it serves as the sole donor of an aminopropyl group that is conjugated with putrescine to form, first, the polyamine spermidine, and then spermine.
We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p4o. This The human parvovirus, adeno-associated virus (AAV), replicates in mammalian cells in the presence of helper functions provided by adenovirus or herpesvirus (10). When molecular clones containing the entire AAV type 2 (AAV2) genome in bacterial plasmids are transfected into mammalian cells in the presence of adenovirus, the AAV genome is excised from the plasmid and replicated to produce infectious AAV particles (38,50,52). This facilitated both the genetic analysis of AAV (27,(51)(52)(53)59) and the development of AAV as a eucaryotic expression vector (26,60,61).The AAV genome contains three transcription promoters, P5, Pi9, and P40, which yield overlapping transcripts (11). Genetic analysis showed that a major AAV reading frame (orf-2) which is accessible from P40 transcripts codes for a major portion of the AAV capsid proteins (27,59). Mutations in this region (cap-) allow normal synthesis of duplex replicating form DNA but prevent synthesis of progeny single-stranded DNA and infectious particles (27,53,59). A second major open reading frame (orf-1) in the left half of the genome is apparently accessible from either p5 or P19 transcripts and codes for one or more proteins (rep) required for AAV DNA replication (27,53,59). Mutations in this region (rep-) are DNA negative but can be complemented. Deletion of both terminal palindromes results in a cis-dominant (ori-) defect which reflects the presence of the AAV replication origins in the terminal repeat sequences (3,4,25,51,53).
Cultures of established rat fibroblasts transformed by the avian erythroblastosis virus were more susceptible to the cytopathic effect of the autonomous parvovirus minute virus of mice, prototype strain (MVMp), than were their untransformed homologs. This effect could be ascribed to the presence of a greater fraction of cells that were sensitive to the killing action of MVMp in transformed cultures than in their normal parents. Yet, transformed and normal lines were similarly efficient in virus uptake, DNA amplification, and capsid protein synthesis. In contrast, transformants accumulated 2.5to 3-fold greater amounts of all three major MVM mRNA species and nonstructural protein than did their normal progenitors. Thus, in this system transformation-associated sensitization of cells to MVMp appears to correlate primarily with an increase in their capacity for the expression of the viral transcription unit which encodes nonstructural proteins and is controlled by the P4 promoter. Consistently, a reporter gene was expressed at a higher level by transformed versus normal cultures, when placed under the control of the MVM P4 promoter. As infectious MVMp was produced in larger amounts by transformed cultures, a late step of the parvoviral cycle, such as synthesis, encapsidation of progeny DNA, or both, was also stimulated in the transformed cells.
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