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Dna from human sperm cells can be isolated with size and purity sufficient for use in recombinant DNA research. The DNA averages 100 000 base pairs (bp) in size (about 70.10(6) daltons) and is free of somatic cell, bacterial, and viral DNA. It can therefore be cloned under P2 + EK2 conditions as stipulated in the 1978 NIH Guidelines for recombinant DNA research.
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The construction of several recombinant plasmid derivatives containing novel triple-block DNA sequence insertions is described. The protocol for these constructions involves synthesis of a heterogenous mixture of block oligomer duplexes, : formula: (see text), using pancreatic deoxyribonuclease and terminal transferase. The synthetic duplexes were mixed with linearized and dG-tailed vectors and the DNA mixture used to transform E. coli. Triple-block sequences of the type dGidAjdCk.dGkdTjdCi, characterized by DNA sequencing, were inserted into the Bam HI site of pBR322 and next to the lac wild-type and UV5 promoter regions in pRW26 and pRW28. Similarly, sequences were inserted into the Sma I site of pACYC189 and could be excised by cleavage with Sma I since the procudure regenerates the recognition site. The approach provides a technique for the synthesis of a large family of defined sequence triple-block polymers in essentially unlimited amounts. Although these inserts contain sequences which have the potential for forming stable hairpin structures, the recombinant plasmids are stable and appear to replicate normally.
2500 recombinant plasmids containing insertions of yeast nuclear DNA have been cloned in Escherichia coli. It can be calculated that about 85% of the yeast genome is represented in this collection. The clones have been characterized by hybridization to purified RNA species. Of the 2000 clones examined, 75 contain insertions of yeast ribosomal DNA, 201 contain insertions of yeast tRNA genes, and 26 contain DNA sequences that are complementary to abundant mRNA species.
In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.
The hereditary characters of organisms are bound up in the DNA of their genomes. Genetic information is expressed by a complicated mechanism. The process by which this occurs and the control of the degree of expression in bacteria and a number of the other lower organisms are known only in broad outline. There usually is no exchange of DNA between organisms of unrelated species in nature. "Foreign" DNA can be linked to bacterial plasmid DNA and multiplied in the laboratory as a recombinant DNA molecule in bacteria using the recently discovered so-called restriction enzymes. Expression of bacterial DNA can thus be accomplished in a different, unrelated bacterium. In principle, this means that the natural barriers between unrealated species of organisms may be passed by. Human and animal DNA can also be multiplied in bacteria by this method. Previous recombinant DNA studies showed that the mode of expression of genetic information in the higher organisms differs markedly from that in bacteria. The initially sanguine expectations regarding the practical use of recombinant DNA research, for instance in the production of biologically important substances by bacteria, will therefore possibly not be realized at short notice. On the other hand this technique has added considerably to the knowledge of expression of genes within a short space of time. This is particularly true of the mechanisms of pathogenicity of E. coli, the bacterium which plays such an important role in veterinary medicine. The recombinant DNA technique is briefly reviewed in the present paper. The uses are broadly outlined, and one of these, which could be of importance in veterinary medicine, is described in greater detail. International discussions of potential hazards, and therefore also of the admissibility of this type of experiment, are griefly summarized.
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