An IS4-encoded protein is synthesized in minicells

Abstract

A protein of Mr 47,000 is synthesized in Escherichia coli minicells, when these harbor a multicopy plasmid carrying IS4 in either orientation and between different flanking sequences. The protein corresponds to the sequence predicted from the known DNA sequence of IS4, as shown by partial N-terminal radiolabel protein sequence analysis. Its apparent molecular weight, however, as determined from its electrophoretic mobility in SDS polyacrylamide gels, is smaller than predicted. When compared with other plasmid-encoded proteins, the IS4-encoded protein is synthesized in minicells in small amounts. Its synthesis has not been detected in a DNA-dependent cell-free system.

Synthesis and overproduction of the 5A protein of insertion sequence IS5

“…Other workers have detected the synthesis of very low levels of a 37-kDa 5A protein in vitro and in minicells by using an independent copy of IS5 which had inserted into the A cI gene in a single orientation (13). These observations are consistent with the low level of production seen for other insertion sequenceencoded proteins (13)(14)(15)(16)19).…”

“…These observations are consistent with the hypothesis that the 5A protein may be degraded into one or more lowermolecular-weight products. While further experiments are needed to clarify the nature and origin of these species, it is interesting to note that both the Mu transposase and the putative IS4 transposase may be processed to lower-molecular-weight forms (2,19).…”

Structural and functional similarities between the replication region of the Yersinia virulence plasmid and the RepFIIA replicons

“…The minicell-producing strain E. coli DS410 was transformed with the appropriate plasmids. Isolation and labeling by [35S]methionine (NEN Du Pont, Berkeley, Calif.) were performed as described by Trinks et al (49). The products were analyzed by sodium dodecyl Sequencing of DNA.…”

Limited proteolysis of lactose permease from Escherichia coli

“…Radiolabeling of E. coli minicells Minicells were prepared, stored and used essentially as described [21]. Derivatives of strain DS410 were grown in the presence of streptomycin (100 pg/ml) and tetracycline (12.5 pg/ml).…”

“…After 30 rnin at room temperature, samples were incubated overnight at 4°C and subsequently with S. aureus cells (10min at room temperature, 30min at 4°C). Final sediments of washed S. aureus cells carrying immunocomplexes were resuspended in sample buffer, incubated at 37 "C for 60 min, and subjected to SDS-PAGE on 11 -20% gradient gels [21].…”