Deletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA.

Lambda transducing phages containing portions of the phoA gene have been isolated and used to construct a deletion map of the phoA gene. The isolation of a plaque-forming lambda transducing phage carrying the entire phoA gene is also described. Two new methods for screening or selection of mutants that have altered levels of alkaline phosphatase activity are reported.     

Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli.

We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon insertions in essential genes.     

Diffusible Fraction of Serum Cholesterol and Atherogenesis

CHOLESTEROL is found in the blood as a structural component of lipoproteins concerned with the transport of other lipids¹. The high resolution nuclear magnetic resonance spectra of high density serum lipoproteins are similar to that observed when lipids are dissolved in organic solvents, or dispersed in water by bile salts or detergents, or in sonicated form. The lipid component in lipoproteins is therefore probably in an extremely fluid condition². If human serum is mixed with paraffin oil, some of the cholesterol diffuses into the oil without affecting the ultraviolet absorption spectrum of serum proteins. This procedure avoids any protein denaturing action used for cholesterol extraction^3–5. It therefore seems that serum cholesterol has two fractions, one strongly bound by lipoprotein structures and the other loosely bound and diffusible in an oil phase. In this article I designate the loosely bound fraction “diffusible”.     

One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coli secretion machinery.

The E. coli secE (prlG) gene codes for an integral cytoplasmic membrane protein which is part of the cell's secretory machinery. A deletion of nearly the entire gene renders the cell dependent on the presence of a complementing secE+ plasmid, indicating that the SecE protein is essential for growth. Deletions which remove carboxy-terminal sequences or substantial amounts near the amino-terminus of SecE can still complement the lethal deletion. This deletion analysis suggests that the essential domain of the SecE protein includes only a single one of its three hydrophobic membrane-spanning segments. Two of three dominant prlG signal sequence suppressors map to this segment. Consistent with the insensitivity of SecE to major structural changes, several cold-sensitive mutations cause lethality not because of any change in the protein, but because of a reduction in its level of expression. Our results suggest that higher levels of the protein are needed at the lower temperature. These findings are discussed in terms of the interactions between various components of the secretory machinery.     

Proper insertion of a complex membrane protein in the absence of its amino-terminal export signal

The MalF protein spans the Escherichia coli cytoplasmic membrane eight times. Deletion of the first transmembrane stretch of MalF, which acts as an export signal, results in a truncated protein that still exhibits high levels of maltose transport activity. These and additional results indicate that the orientation of a membrane protein is not determined by the amino-terminal export signal, topological information is distributed throughout the MalF protein, and insertion of a protein into the cytoplasmic membrane can occur nonsequentially.     

Membrane insertion of the Escherichia coli MalF protein in cells with impaired secretion machinery

The MalF protein is an integral membrane protein of Escherichia coli containing eight membrane-spanning stretches and a large periplasmic domain of approximately 180 amino acids. We have asked whether this protein is dependent for its membrane insertion on the bacterial secretion machinery specified by the sec genes. Using azide to inhibit the SecA protein and sec mutants to reduce the functioning of the machinery, we have studied the membrane assembly of MalF and beta-galactosidase and alkaline phosphatase fusions to MalF. In no case did we see an effect of reducing sec gene function on the insertion of MalF or fusion proteins. Selection for mutants that would cause internalization of a MalF-beta-galactosidase hybrid protein yielded no mutations in sec genes. Our results suggest that MalF can assemble in the membrane independently of the bacterial secretion machinery.     

The product of gene secC is involved in the synthesis of exported proteins in E. coli

To obtain additional mutants in the secretory apparatus of E. coli we have isolated suppressors of a mutant (secAts) that is temperature-sensitive for secretion. One of these, secC, can suppress the secretion defect of secA and has a phenotype of its own. At 23 degrees C, the secC mutant is cold-sensitive for growth and blocks the synthesis of transported proteins. The synthesis of at least one secreted protein, maltose-binding protein (MBP), can be restored by mutations that alter the hydrophobic region of the signal sequence of MBP. The phenotype of the secC mutant suggests that the SecC protein may be a component of the secretory apparatus of E. coli; it also supports the notion that in procaryotes secretion and gene expression are coupled. The secC gene maps at 68.5 minutes on the E. coli chromosome.