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Novel host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. We found that a new Pseudomonas strain, Pseudomonas flavida IF-4, isolated from soil, carried two small cryptic plasmids, named pNI10 and pNI20. They were multi-copy, but not self-transmissible, and the genome size was 3.7 kb for pNI10 and 2.9 kb for pNI20. Several types of cloning vectors containing a kanamycin or streptomycin resistance (Kmr or Smr) gene were constructed from pNI10 and pNI20. These plasmid vectors were efficiently transformed into several strains of Pseudomonas at a frequency up to 4 x 10(5) transformants per 1 microgram plasmid DNA by the usual competent cell method. The vectors derived from pNI10 replicated not only in Pseudomonas but also in some other Gram-negative enteric bacteria such as Escherichia coli, Enterobacter aerogenes, and Proteus mirabilis.

A practicable enzyme immunoassay for measurement of thyroxine and triiodothyronine in serum was developed, involving a separation method based on the thiol-disulfide interchange reaction. Serum samples at alkaline pH were incubated for 1 h with antibodies and antigens labeled with beta-D-galactosidase. Each reaction mixture was then passed through a 0.1-mL column containing (anti-IgG) antibody immobilized on Sepharose 4B, to which the anti-IgG antibodies were coupled by means of a disulfide bond (3 g of IgG fraction per liter). The column was washed and the bound form of the label then was eluted from the column with a buffer containing dithiothreitol (25 mmol/L) by splitting the disulfide bonds between the anti-IgG antibody molecules and Sepharose matrix. From the enzyme activity in the eluate, concentrations of thyroxine or triiodothyronine in serum could be determined. Values obtained by this method and those obtained by a radioimmunoassay correlated well (thyroxine, r = 0.97, slope = 1.1 y-intercept = -0.94 microgram/L, n = 70; triiodothyronine, r = 0.98, slope = 0.91, y-intercept = 0.067 microgram/L, n = 77.