The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported. This is the first characterised example of a mycobacterial insertion element. IS900 consists of 1451bp of which 66% is G + C. It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity. A single open reading frame (ORF 1197) coding for 399 amino acids is predicted. This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2). It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family. IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases.

We report on the incorporation of radiolabelled sulphate into proteoglycan in the 'in situ'-perfused rat liver. After 5 min virtually all of the [35S]sulphate was incorporated into heparan sulphate; no partially sulphated precursors were detected. Pulse-chase experiments, followed by centrifugation in gradients of sucrose and metrizamide, showed that, at 5 min, the heparan sulphate was associated predominantly with the Golgi membranes. Over the next 20 min, intact proteoglycan appeared at the plasma membrane. At intermediate times the heparan sulphate was detected simultaneously in two distinct populations of membrane vesicles. Whether the heparan sulphate in these two populations has two different destinies (e.g. plasma membrane or secretion) is not yet clear. Subfractionation of the Golgi membranes showed that the N-sulphotransferase co-purified with the heparan [35S]sulphate and was separable from the galactosyltransferase of glycoprotein synthesis, confirming that the Golgi membrane system is functionally segregated. Subfractionation also permitted an almost 100-fold purification of the N-sulphotransferase over the homogenate: this will provide an excellent starting material for isolation and further characterization of the enzyme.

The effect of various parameters on the electric shock-mediated permeabilization and transfection of CHO cells has been investigated. Up to 70% of the cells can be maintained transiently permeable to erythrosin B for periods of at least 1 h at 20 degrees C. Electrical conditions optimal for transient permeabilization were also optimal for efficient DNA transfection by pSV2neo. However, the DNA must be present during exposure to the electric field for efficient transformation. The same requirement existed for voltage-induced DNA toxicity. The results suggest that DNA moves into the cells by electrophoresis, not by simple diffusion. Based on these observations a simple, rapid procedure for optimizing the conditions for electric shock-mediated DNA transfer into cells has been developed.