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SummaryA plasmid, designated pMK, containing the structural gene for thymidine kinase from herpes simplex virus (HSV) fused to the promoter/regulatory region of the mouse metallothionein-I gene, was injected into the pronucleus of fertilized one-cell mouse eggs; the eggs were subsequently reimplanted into the oviducts of pseudopregnant mice. The first experiment produced 19 offspring, one of which expressed high levels of HSV thymidine kinase activity in the liver and kidney. pMK DNA sequences were detected in equal amounts in several tissues of the expressing mouse as well as in three mice that did not express HSV thymidine kinase activity. In all cases, several copies of the pMK plasmid were tandemly duplicated and integrated into mouse DNA. It appears as though multiple copies of the intact plasmid were fused by homologous recombination either before or after integration at a single site in the mouse genome. The overall efficiency of obtaining somatic expression of thymidine kinase in experiments performed to date is about 10% (4/41), and twice this number have integrated pMK DNA. This procedure not only provides a means of introducing new genes into mice, but it will also be a valuable system for studying tissue-specific regulation of gene expression.

Microorganisms are efficient degraders of starch, chitin, and the polysaccharides in plant cell walls. Attempts to purify hydrolases led to the realization that a microorganism may produce a multiplicity of enzymes, referred to as a system, for the efficient utilization of a polysaccharide. In order to fully characterize a particular enzyme, it must be obtained free of the other components of a system. Quite often, this proves to be very difficult because of the complexity of a system. This realization led to the cloning of the genes encoding them as an approach to eliminating other components. More than 400 such genes have been cloned and sequenced, and the enzymes they encode have been grouped into more than 50 families of related amino acid sequences. The enzyme systems revealed in this manner are complex on two quite different levels. First, many of the individual enzymes are complex, as they are modular proteins comprising one or more catalytic domains linked to ancillary domains that often include one or more substrate-binding domains. Second, the systems are complex, comprising from a few to 20 or more enzymes, all of which hydrolyze a particular substrate. Systems for the hydrolysis of plant cell walls usually contain more components than systems for the hydrolysis of starch and chitin because the cell walls contain several polysaccharides. In general, the systems produced by different microorganisms for the hydrolysis of a particular polysaccharide comprise similar enzymes from the same families.

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A scheme is proposed for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants. These enzymes are predominantly L L-1,4-glycanases. The scheme is based on the classification of the catalytic domains of glycoside hydrolases into families of related amino acid sequences. The new designation for an enzyme indicates its family and, because all members of a family have these characteristics in common, its three-dimensional fold and stereospecificity of hydrolysis. The scheme is intended to simplify comparison of the systems of enzymes produced by different microorganisms for the hydrolysis of plant cell walls.z 1998 Federation of European Biochemical Societies.

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