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Complementary DNA transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded has been inserted into E. coli plasmid pSC 101 by the poly-dT/poly-dA “tailing” and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. Some measure of the amount and identity of inserted globin sequences has been determined by fingerprinting fragments of 32p-labeled globin RNA hybridized to the purified plasmid chimeras.

Publisher Summary This chapter discusses the mutation rates for mammalian genomes that have often been computed from rates of amino-acid substitution. The fact that rates differing by manyfold are obtained for different proteins emphasizes that in some proteins, such as the histones, most amino-acid substitutions are sufficiently deleterious to be eliminated during the course of evolution. It reviews that by measuring acceptance rates of silent mutations through direct nucleotide sequence comparisons between species, it can remove one of the most important limitations of previous estimates of the mutation rate, which have been based on amino-acid substitution data. The chapter also discusses that Haldane computed the number of genetic deaths required for a gene substitution to occur according to strict Darwinian principles. With reasonable assumptions about permissive levels of mortality associated with Darwinian evolution, he estimated that there could be about one gene substitution every 300 generations. To account for the observed maintenance of mammalian genomes in the face of Haldane's dilemma, it proposes two solutions—that is, by Truncation selection and Neutral mutations. Truncation selection provides a useful way of explaining accurate maintenance of the repetitive gene clusters and satellite DNAs, but it is difficult to imagine that truncation selection could effectively maintain the accuracy of the single-copy DNA coding for most proteins. It is suggested that only a small fraction of the genome, perhaps less than 2%, is kept accurate as single-copy DNA. Since most mammalian genomes contain large amounts of single-copy DNA, the implication is that most such sequences must be genetically drifting.

Publisher Summary This chapter reviews that cDNA serves as the template for synthesis of a variety of products used in various phases. The synthesis products include 32 P-labeled RNA for sequence analysis of fragments resulting from a cleavage at G residues, 32 P-labeled dC- or dT-substituted RNAs for specific cleavage at U or at C residues, and duplex gene copies for insertion into bacterial plasmids. It reviews that molecular cloning approach is applied to a wide variety of eukaryotic mRNAs. One of the advantages of the technique that is developed for the insertion of eukaryotic mRNA copies into bacterial plasmids is that a rigorous purification is not necessary; the act of cloning such material definitively eliminates all contaminating eukaryotic nucleic acid sequences. This should be especially useful to the researcher who, for instance, has a mixture of 10 different mRNA species that are difficult to separate. To obtain the corresponding sequences in pure form, it suffices to make cDNA copies of the mixture of mRNAs, convert this mixture of cDNAs to duplex gene copies, insert the DNA into bacterial plasmids and introduce the mixture of plasmids thus obtained into bacteria. Since each bacterial transformant can receive only one of the eukaryotic gene inserts, analysis of a few dozen bacterial clones should suffice to provide one with a clone for each of the interesting sequences. With such clones, it is possible to produce milligram quantities of these sequences in duplex form, sufficient for the most rapid sequencing techniques.

Publisher Summary Recent developments indicate that in vitro synthesis of ribosubstituted DNA may allow the relatively rapid determination of DNA sequences of small to moderate complexity. In vitro synthesis provides an advantage as one can synthesize the same ribosubstituted DNA with the 32 P labeling a different one of the four bases each time, so that nearest-neighbor labeling information will greatly simplify the subsequent analysis. The ready availability of small molecules such as the tRNAs and 5 S ribosomal RNA was extremely important in permitting the rapid development of RNA sequencing methods. In order to obtain very simple sequences for working out DNA sequencing techniques we have turned to the highly iterated satellite DNA fractions of higher organisms. This chapter describes the methods for synthesis and cleavage of ribosubstituted DNA and discusses the analysis of the sequences of individual fragments and the use of micrococcal nuclease to determine the sequence of larger fragments.