WT Hosts Research Articles

A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by l-threonine

In Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum, homoserine dehydrogenase (HD), the enzyme after the branch point of the threonine/methionine and lysine biosynthetic pathways, is allosterically inhibited by l-threonine. To investigate the regulation of the C. glutamicum HD enzyme by l-threonine, the structural gene, hom, was mutated by UV irradiation of whole cells to obtain a deregulated allele, hom dr. l-Threonine inhibits the wild-type (wt) enzyme with a K i of 0.16 mM. The deregulated enzyme remains 80% active in the presence of 50 mM l-threonine. The hom dr gene mutant was isolated and cloned in E. coli. In a C. glutamicum wt host background, but not in E. coli, the cloned hom dr gene is genetically unstable. The cloned hom dr gene is overexpressed tenfold in C. glutamicum and is active in the presence of over 60 mM l-threonine. Sequence analysis revealed that the hom dr mutation is a single nucleotide (G 1964) deletion in codon 429 within the hom reading frame. The resulting frame-shift mutation radically alters the structure of the C terminus, resulting in ten amino acid (aa) changes and a deletion of the last 7 aa relative to the wt protein. These observations suggest that the C terminus may be associated with the l-threonine allosteric response. The hom dr mutation is unstable and probably deleterious to the cell. This may explain why only one mutation was obtained despite repeated mutagenesis.

Developmental control of the excitability of muscle: transplantation experiments on a myotonic mouse mutant.

Developmental aspects of an animal model of myotonia, the mouse mutant called "arrested development of righting response" (ADR phenotype), were studied. Adult ADR muscle is characterized by a low chloride conductance of the membrane, leading to hyperexcitability, and by a low parvalbumin content. The myotonic hyperexcitability (as measured by the extent of "aftercontractions") of ADR muscle increased steeply between postnatal days 9 and 18, by which time it had approached the adult level. To study the tissue autonomy of the myotonic phenotype, muscle grafts were performed in all four combinations between ADR and wildtype (WT phenotype) donors and hosts. In most experiments, the relative contributions of donor and host to the regenerated muscles were determined by an allelic marker (glucose phosphate isomerase). In WT and ADR hosts, ADR grafts showed myotonic responses that in WT nude mouse hosts were incomplete and similar to those of juvenile ADR muscle. In no case did grafts from WT donors show any myotonia. This shows that the myotonic ADR phenotype is based on an intrinsic muscle property most likely related to the plasma membrane. The parvalbumin contents of grafted muscles, when compared with those of untransplanted muscles, indicated graft-host interaction in the expression of this secondary phenotypic property.