Paul Berg

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

Circular (e.g. simian virus 40) and linear (e.g. λ phage) DNAs have been labeled to high specific radioactivities (>108 cts/min per μg) in vitro using deoxynucleoside [α-32P]triphosphates (100 to 250 Ci/mmol) as substrates and the nick translation activity of Escherichia coli DNA polymerase I. The reaction product yields single-stranded fragments about 400 nucleotides long following denaturation. Because restriction fragments derived from different regions of the nick-translated DNA have nearly the same specific radioactivity (cts/min per 10[su3] bases), we infer that nicks are introduced, and nick translation is initiated, with equal probability within all internal regions of the DNA. Such labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.

High-efficiency cloning of full-length cDNA.

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 cDNAs over those with truncated cDNAs.

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

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.

Comparison of intron-dependent and intron-independent gene expression.

Recombinant simian virus 40 viruses carrying rabbit beta-globin cDNA failed to express the beta-globin sequence unless an intron was included in the transcription unit. The addition of either beta-globin IVS1 or IVS2 caused a 400-fold increase in RNA production. Stable beta-globin RNA production required sequences in IVS2 that were very close to the splice sites and that coincided with those needed for mRNA splicing. In addition to the recombinant viruses, intron-dependent expression was observed with both replicating and nonreplicating plasmid vectors in short-term transfections of cultured animal cells. Unlike transcriptional enhancer elements, IVS2 failed to increase stable RNA production when it was placed downstream of the polyadenylation site. Using a plasmid vector system to survey different inserted sequences for their dependence on introns for expression, we found that the presence of IVS2 stimulated the expression of these sequences 2- to 500-fold. Sequences from the transcribed region of the herpes simplex virus thymidine kinase gene, a gene that lacks an intervening sequence, permitted substantial intron-independent expression (greater than 100-fold increase) in the plasmid vector system.

Studies on the binding of RNA polymerase to polynucleotides

Abstract A simple, rapid method for detecting the binding of RNA polymerase to native DNA has been developed. Whereas native DNA and RNA polymerase, separately, pass through Millipore membrane filters, a complex of the two is quantitatively retained. With this technique the stoichiometry and some properties of the complex between native T7 DNA and RNA polymerase have been measured; we estimate that there are 35 to 70 binding sites for RNA polymerase per mole of T7 DNA. Other polynucleotides block the formation of the T7 DNA-RNA polymerase complex if added to RNA polymerase before but not after the T7 DNA. Measurements of the effect of polynucleotide concentration on the extent of inhibition of complex formation between T7 DNA and RNA polymerase suggest that the number of binding sites of RNA polymerase to nucleic acids depends upon the primary and secondary structure of the polynucleotides.

A prudent path forward for genomic engineering and germline gene modification

A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed Genome engineering technology offers unparalleled potential for modifying human and nonhuman genomes. In humans, it holds the promise of curing genetic disease, while in other organisms it provides methods to reshape the biosphere for the benefit of the environment and human societies. However, with such enormous opportunities come unknown risks to human health and well-being. In January, a group of interested stakeholders met in Napa, California (1), to discuss the scientific, medical, legal, and ethical implications of these new prospects for genome biology. The goal was to initiate an informed discussion of the uses of genome engineering technology, and to identify those areas where action is essential to prepare for future developments. The meeting identified immediate steps to take toward ensuring that the application of genome engineering technology is performed safely and ethically.

Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors.

A mouse complementary deoxyribonucleic acid segment coding for the enzyme dihydrofolate reductase has been cloned in two general classes of vectors containing simian virus 40 deoxyribonucleic acid: (i) those that can be propagated as virions in permissive cells and (ii) those that can be introduced into and maintained stably in various mammalian cells. Both types of vectors express the mouse dihydrofolate reductase by using signals supplied by simian virus 40 deoxyribonucleic acid sequences. Moreover, plasmid vectors carrying the complementary deoxyribonucleic acid segment can complement Chinese hamster ovary cells lacking dihydrofolate reductase.

Analysis of Gene Targeting and Intrachromosomal Homologous Recombination Stimulated by Genomic Double-Strand Breaks in Mouse Embryonic Stem Cells

ABSTRACT To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.

Recognition of tRNA by aminoacyl tRNA synthetases

A new method is described for the detection of tRNA in complex with an aminoacyl tRNA synthetase. Escherichia coli isoleucyl tRNA synthetase complexes only with tRNA Ile and tyrosyl tRNA synthetase complexes exclusively with tRNA Tyr . ATP and amino acid are not required to establish the complex, which also forms equally well whether the tRNA is acylated or not. Quantitative analysis of the binding curves suggests a single binding site per enzyme particle which binds either aminoacylated or unacylated tRNA, both tRNAs having an association constant of approximately 10 8 liters/mole. The binding technique also provides a short and direct procedure for obtaining tRNA highly enriched for a selected acceptor.

Recognition of tRNA by isoleucyl-tRNA synthetase: Effect of substrates on the dynamics of tRNA-enzyme interaction☆

An interaction between the isoleucine catalytic site and the tRNA recognition site of isoleucyl-tRNA synthetase (Escherichia coli) was detected using an assay for the binding of tRNA to enzyme. The effect of isoleucine is to increase the rate at which tRNA enters and leaves its binding site by sixfold without, therefore, a large effect on the equilibrium constant for tRNA binding. This effect requires neither transfer of isoleucine to tRNA nor activation of isoleucine as isoleucyl-AMP; the binding of isoleucine alone suffices. It appears from these data that release of aminoacyl-tRNA is the rate-limiting step in the acylation, or transfer, reaction. The apparent maximum rate constant for association of tRNA and enzyme is relatively large, 6 × 106 m−1 sec−1 (17 °C).