Rapid rescue of cellular transcriptional activator elements by amplification of a single copy selection gene.

Cellular transcriptional activator sequences from a Syrian hamster cell line (baby hamster kidney (BHK] were rescued by a double selection procedure. An enhancer-deficient SV40 promoter was linked to the neomycin resistance (NEO) gene and transfected into BHK cells. Genomic DNA fragments of G418-resistant cell clones containing multiple copies of integrated plasmid DNAs were used for a second transfection of BHK cells, resulting in the genomic integration of a single copy plasmid which expresses the NEO gene efficiently.

Expression of the chloramphenicol acetyltransferase gene in mammalian cells under the control of adenovirus type 12 promoters: effect of promoter methylation on gene expression.

The effect of DNA methylation at specific promoter sites on gene expression was tested by using a sensitive and quantitative assay system. The plasmid pSVO CAT contains the prokaryotic gene chloramphenicol acetyltransferase (CAT) and a HindIII site in front of it for experimental promoter insertion. Upon insertion into pSVO CAT, the E1a and protein IX gene promoters from adenovirus type 12 (Ad12) DNA were capable of mediating CAT expression upon transfection in mouse cells.

The unmethylated state of the promoter/leader and 5'-regions of integrated adenovirus genes correlates with gene expression.

An inverse correlation has been established between the levels of DNA methylation at 5'-CCGG-3' (MspI/HpaII) sites in specific genes of integrated viral DNA in adenovirus type 12 (Ad12)-transformed hamster cell lines and the extent to which these genes are expressed ( Sutter and Doerfler , 1979, 1980). In general, early genes are transcribed into mRNA, while late genes are permanently switched off in these cell lines. Adenovirus type 2 genes methylated in vitro at 5'-CCGG-3' sites are not transcribed upon microinjection into nuclei of Xenopus laevis oocytes - unmethylated genes are expressed ( Vardimon et al.

Cloned fragments of human adenovirus type-12 DNA.

The following restriction endonuclease fragments of human adenovirus type 12 (Ad12) DNA have been cloned in plasmid or bacteriophage lambda vectors using standard protocols: the EcoRI-A*, -B, -D, -E, and -F fragments, the BamHI-B, -C, -D, -F, -G, -H, and -I fragments, the HindIII-F and -I fragments, and the PstI-A, -D, -F, -G, and -H fragments. The EcoRI-A* fragment comprises the right terminal 5 kb of Ad12 DNA including the terminal 143 bp.

Mutants of adenovirus type 12 after adaptation to growth in tumor cell lines. II. Reproducible acquisition of additional sequences after adaptation to a human cervical cancer line.

Adaptation of adenovirus type 12 to growth in a human cervical cancer line (C4/1) results in the reproducible selection of viral mutants carrying additional DNA sequences at the right-hand end of the genome which range in size from 190 to 400 base pairs. The viral mutants have selective growth advantage in comparison to the non-affected wild type, even after subsequent passages in human KB cells, commonly used for adenovirus propagation. Detailed restriction endonuclease analysis of one of the mutant DNAs shows that the additional sequences are located within the terminal 195 base pairs of the right end of the genome.