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Glycine betaine, which functions as an osmoprotectant, is accumulated to high intracellular concentrations in Escherichia coli at high osmolarity. We demonstrate the presence of a high-affinity, binding protein dependent transport system for glycine betaine, which is encoded by the proU region. We show the osmotically regulated synthesis of a 32 kDa periplasmic protein that is a glycine betaine binding protein with a KD of 1.4 microM. ProU-mediated glycine betaine transport is osmotically stimulated at the level of gene expression. The osmolarity of the medium also regulates the activity of the transport system, while binding of glycine betaine to its binding protein is independent of the osmolarity. We also find a second glycine betaine transport system that is dependent on proP and exhibits a lower substrate affinity. Like ProU, this system is regulated at two levels: both gene expression and the activity of the transport system are osmotically stimulated. Using lambda plac Mu-generated lacZ operon and gene fusions, we find that expression of the proU region is osmotically regulated at the level of transcription. We cloned a part of the proU region together with the phi(proU-lacZ)hyb2 gene fusion into a multicopy plasmid and show that the DNA sequences required in cis for osmotic regulation are present on the plasmid.

Genetics plays a role, to a greater or lesser extent, in all diseases. Variations in our DNA and differences in how that DNA functions (alone or in combinations), alongside the environment (which encompasses lifestyle), contribute to disease processes. This review explores the genetic basis of human disease, including single gene disorders, chromosomal imbalances, epigenetics, cancer and complex disorders, and considers how our understanding and technological advances can be applied to provision of appropriate diagnosis, management and therapy for patients.

Cyclin D1 in cooperation with its major catalytic partners, cyclin-dependent kinases cdk4 and cdk6, facilitates progression through the G 1 phase of the eukaryotic cell cycle, in part through phosphorylation of the retinoblastoma protein. Cyclin D1's oncogenic properties have been suggested by its cooperation with ras or adenovirus E1a to transform cultured cells, as well its overexpression in transgenic mice that leads to breast cancer. Activated by a number of dierent mechanisms in human cancers, the cyclin D1 gene is frequently ampli®ed in squamous epithelial cancers derived from the head/neck and esophageal regions. In order to study the functional consequences of cyclin D1 overexpression in these squamous epithelial speci®c sites, we have linked the Epstein-Barr virus ED-L2 promoter to the human cyclin D1 cDNA and utilized this transgene to generate founder lines. This transgene is transcribed speci®cally in the tongue, esophagus and forestomach, all sharing a strati®ed squamous epithelium. The transgene protein product localizes to the basal and suprabasal compartments of these squamous epithelial tissues, and mice from dierent lines develop dysplasia, a prominent precursor to carcinoma, by 16 months of age in contrast to age-matched wild-type mice. This transgenic model is useful in demonstrating cyclin D1 may be a tumor initiating event in aero-upper digestive squamous epithelial tissues.

We have identified a novel c-Jun N-terminal kinase (JNK)-interacting protein, Sab, by yeast two-hybrid screening. Sab binds to and serves as a substrate for JNK in vitro, and was previously found to interact with the Src homology 3 (SH3) domain of Bruton's tyrosine kinase (Btk). Inspection of the sequence of Sab reveals the presence of two putative mitogen-activated protein kinase interaction motifs (KIMs) similar to that found in the JNK docking domain of the c-Jun transcription factor, and four potential serine-proline JNK phosphorylation sites in the C-terminal half of the molecule. Using deletion and site-directed mutagenesis, we demonstrate that the most N-terminal KIM in Sab is essential for JNK binding, and that, as with c-Jun, physical interaction with JNK is necessary for Sab phosphorylation. Interestingly, confocal immunocytochemistry and cell fractionation studies indicate that Sab is associated with mitochondria, where it co-localizes with a fraction of active JNK. These and previously reported properties of Sab suggest a possible role in targeting JNK to this subcellular compartment and/or mediating cross-talk between the Btk and JNK signal transduction pathways.

A class of trans-acting mutations, which alter the osmoregulated expression of the Escherichia coli proU operon, maps at 27 min on the chromosome in a locus we have called osmZ. Mutations in osmZ are allelic to bglY, pilG and virR, affect gene expression, increase the frequency of the site-specific DNA inversion mediating fimbrial phase variation, stimulate the formation of deletions, and influence in vivo supercoiling of reporter plasmids. We have cloned the osmZ+ gene, mapped it at 1307 kb of the E. coli restriction map, identified its gene product as a 16 kDa protein, and determined the nucleotide sequence of the osmZ+ gene. The deduced amino acid sequence for OsmZ predicts a protein of 137 amino acid residues with a calculated molecular weight of 15,530. The primary sequence of OsmZ is identical to that of H-NS (H1a), a DNA-binding protein that affects DNA topology and is known to be associated with the bacterial nucleoid. Thus, osmZ is the structural gene for the H-NS (H1a) protein. The nucleotide sequence of osmZ is almost identical to that of hns; however, hns was incorrectly located at 6.1 min on the E. coli linkage map. Increased osmZ gene dosage leads to cell filament formation, altered gene expression, and reduced frequency of fimbrial phase variation. Our results suggest that the nucleoid-associated DNA-binding protein H-NS (H1a) plays a critical role in gene expression and in determining the structure of the genetic material.

The OmpC and OmpF porins are major outer membrane proteins of Escherichia coli and Salmonella typhimurium. Their expression is affected by many environmental factors and by mutations in a variety of independent genes. The pair of regulatory proteins, OmpR and EnvZ, are required for normal porin expression. Despite intensive investigation, the mechanisms by which porin expression is regulated remain unclear. Mutations which alter supercoiling, as well as inhibitors of DNA gyrase, show that porin expression is extremely and specifically sensitive to the level of DNA supercoiling. Our data lead us to suggest that environmentally induced changes in DNA supercoiling may play a role in determining the level of porin expression. These findings have implications for current models of porin regulation.