Biomedical subjects
I Kruczek
Publications and source records attributed to I Kruczek.
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. For rapid cloning of the integrated promoter and adjacent cellular DNA sequences, these cell clones were fused to COS-1 cells, thereby providing SV40 large T antigen and the monkey cell permissive factor necessary for SV40 replication. Resulting from this fusion, the integrated plasmid and adjacent sequences were amplified to about 1000 extrachromosomal copies giving rise to an abundant pool of promoter elements which thus can be cloned into a plasmid very easily for further investigations. Promoter analyses of three clones in the chloramphenicol acetyltransferase transient expression assay demonstrated that the recombination with cellular DNA enables the initially defective SV40 promoter to express at wild type levels.
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. In many viral and nonviral eukaryotic genes, DNA methylation at highly specific sites in the promoter region can attain a regulatory function in gene expression. One of the important sites is the 5' C-C-G-G 3' sequence. The CAT-promoting activity of the early simian virus 40 promoter in plasmid pSV2 CAT is refractory to methylation by the Hpa II or Hha I DNA methyltransferase at 5' C-C-G-G 3' or 5' G-C-G-C 3' sequences, respectively, because this promoter lacks such sites. The CAT coding sequence of this plasmid carries four Hpa II and no Hha I sites. Methylation of the Hpa II sites in the coding region does not affect expression. The E1a promoter of Ad12 DNA comprising the leftmost 525 base pairs of the viral genome carries two 5' C-C-G-G 3' and three 5' G-C-G-C 3' sites upstream from the leftmost "TATA" signal. Methylation of the Hpa II or Hha I sites incapacitates this promoter. The promoter of protein IX gene of Ad12 DNA contains one 5' C-C-G-G 3' and one 5' G-C-G-C 3' site downstream and two 5' G-C-G-C 3' sites greater than 300 base pairs upstream from the TATA motif and probably outside the promoter. The protein IX promoter is not inactivated by methylation of these sites. These data demonstrate that critical 5' methylations in the promoter region decrease or eliminate transcription; methylations of sites too far upstream or probably any sites downstream from the TATA site do not affect expression.
DNA methylation and gene activity: the adenovirus system as a model.
Explore the source record for details and available documents.
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., 1982a ). The MspI sites in the early and in some of the late Ad12 genes in cell lines HA12 /7, T637 , and A2497 -3 have now been precisely mapped. The data presented here reveal that the promoter/leader and 5'-regions of the early genes are unmethylated both at MspI sites and at 5'-GCGC-3' (HhaI) sites. In some instances, e.g., in the E2a regions in all three lines, the main parts of the early genes are partly methylated, even though the genes are expressed. In cell line HA12 /7, the early region E3 is not expressed, and the promoter/leader and 5'-regions of this segment are fully methylated. All late regions are completely methylated. The results suggest that the state of methylation in the promoter/leader and 5'-regions of integrated adenovirus genes is important in the control of gene expression.
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.
The cloned EcoRI fragments of Autographa californica nuclear polyhedrosis virus DNA.
Explore the source record for details and available documents.
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.