A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.

A random-primed complementary DNA library was constructed from plasma containing the uncharacterized non-A, non-B hepatitis (NANBH) agent and screened with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents.

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Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome

A bacterial gene (neo) conferring resistance to neomycin-kanamycin antibiotics has been inserted into SV40 hybrid plasmid vectors and introduced into cultured mammalian cells by DNA transfusion. Whereas normal cells are killed by the antibiotic G418, those that acquire and express neo continue to grow in the presence of G418. In the course of the selection, neo DNA becomes associated with high molecular weight cellular DNA and is retained even when cells are grown in the absence of G418 for extended periods. Since neo provides a marker for dominant selections, cell transformation to G418 resistance is an efficient means for cotransformation of nonselected genes.

Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.

A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 10(5) plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete alpha- of beta-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full- or nearly full-length cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's.

High-efficiency cloning of full-length cDNA.

A striking feature of the human genome is the dearth of CpG dinucleotides (CpGs) interrupted occasionally by CpG islands (CGIs), regions with relatively high content of the dinucleotide. CGIs are generally associated with promoters; genes, whose promoters are especially rich in CpG sequences, tend to be expressed in most tissues. However, all working definitions of what constitutes a CGI rely on ad hoc thresholds. Here we adopt a direct and comprehensive survey to identify the locations of all CpGs in the human genome and find that promoters segregate naturally into two classes by CpG content. Seventy-two percent of promoters belong to the class with high CpG content (HCG), and 28% are in the class whose CpG content is characteristic of the overall genome (low CpG content). The enrichment of CpGs in the HCG class is symmetric and peaks around the core promoter. The broad-based expression of the HCG promoters is not a consequence of a correlation with CpG content because within the HCG class the breadth of expression is independent of the CpG content. The overall depletion of CpGs throughout the genome is thought to be a consequence of the methylation of some germ-line CpGs and their susceptibility to mutation. A comparison of the frequencies of inferred deamination mutations at CpG and GpC dinucleotides in the two classes of promoters using SNPs in human-chimpanzee sequence alignments shows that CpGs mutate at a lower frequency in the HCG promoters, suggesting that CpGs in the HCG class are hypomethylated in the germ line.

/pdf/a-genome-wide-analysis-of-cpg-dinucleotides-in-the-human-6vc4xs6k4u.pdf

A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters.

Cultured monkey (TC7) and mouse (3T6) cells synthesize an Excherichia coli enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT; 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase, EC 2.4.2.22), after transfection with DNA vectors carrying the corresponding bacterial gene, Ecogpt. In contrast to mammalian cells, which do not efficiently use xanthine for purine nucleotide synthesis, cells that produce E. coli XGPRT can synthesize GMP from xanthine via XMP. After transfection with vector-Ecogpt DNAs, surviving cells producing XGPRT can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purine nucleotide synthesis. Cells transformed for Ecogpt arise with a frequency of 10(-4) to 10(-5); they appear to be genetically stable in as much as there is no discernible decrease in XGPRT formation or loss on their ability to grow in selective medium after propagation in nonselective medium. Although several of the vector-gpt DNAs can replicate in monkey and mouse cells, none of the transformants contain autonomously replicating vector-gpt DNA. Rather, the gpt transformants contain one to five copies of the transfecting DNA associated with, and most probably integrated into, cellular DNA sequences. In several transformants, vector-coded gene products for which there was no selection are also synthesized. This suggests that recombinant DNAs containing Ecogpt as a selective marker can be useful for cotransformation of nonselectable genes.

https://www.pnas.org/content/pnas/78/4/2072.full.pdf

Paul Berg

Papers

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.

High-efficiency cloning of full-length cDNA.

A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters.

Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase