The gene causing familial mediterranean fever maps to the short arm of chromosome 16 in Druze and Moslem Arab families

The gene causing familial Mediterranean fever maps to the short arm of chromosome 16 in Druze and Moslem Arab families.     

Mutations in the sarcosine dehydrogenase gene in patients with sarcosinemia

Sarcosinemia is an autosomal recessive metabolic trait manifested by relatively high concentrations of sarcosine in blood and urine. Sarcosine is a key intermediate in 1-carbon metabolism and under normal circumstances is converted to glycine by the enzyme sarcosine dehydrogenase. We encountered six families from two different descents (French and Arab), each with at least one individual with elevated levels of sarcosine in blood and urine. Using the “candidate gene approach” we sequenced the gene encoding sarcosine dehydrogenase (SARDH), which plays an important role in the conversion of sarcosine to glycine, and found four different mutations (P287L, V71F, R723X, R514X) in three patients. In an additional patient, we found a uniparental disomy in the region of SARDH gene. In two other patients, we did not find any mutations in this gene. We have shown for the first time that mutations in the SARDH gene are associated with sarcosinemia. In addition, our results indicate that other genes are most probably involved in the pathogenesis of this condition.     

Engineering Anthrax Toxin Variants That Exclusively Form Octamers and Their Application to Targeting Tumors

Anthrax toxin protective antigen (PA) delivers its effector proteins into the host cell cytosol through formation of an oligomeric pore, which can assume heptameric or octameric states. By screening a highly directed library of PA mutants, we identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application.     

Role of N-terminal amino acids in the potency of anthrax lethal factor

Anthrax lethal factor (LF) is a Zn(+2)-dependent metalloprotease that cleaves several MAPK kinases and is responsible for the lethality of anthrax lethal toxin (LT). We observed that a recombinant LF (LF-HMA) which differs from wild type LF (LF-A) by the addition of two residues (His-Met) to the native Ala (A) terminus as a result of cloning manipulations has 3-fold lower potency toward cultured cells and experimental animals. We hypothesized that the "N-end rule", which relates the half-life of proteins in cells to the identity of their N-terminal residue, might be operative in the case of LF, so that the N-terminal residue of LF would determine the cytosolic stability and thereby the potency of LF. Mutational studies that replaced the native N-terminal residue of LF with known N-end rule stabilizing or destabilizing residues confirmed that the N-terminal residue plays a significant role in determining the potency of LT for cultured cells and experimental animals. The fact that a commercially-available LF preparation (LF-HMA) that is widely used in basic research studies and for evaluation of vaccines and therapeutics is 3-fold less potent than native LF (LF-A) should be considered when comparing published studies and in the design of future experiments.     

The evolution of birds: an overview of the avian tree of life

The author provides a general overview of the molecular data used to reconstruct the avian tree of life, summarizes some highlights of the ensuing controversies, and reveals those taxonomic relationships that remain largely unchanged by molecular data.     

Survival of plasmid-containing strains ofEscherichia coli in soil: Effect of plasmid size and nutrients on survival of hosts and maintenance of plasmids

Several strains ofEscherichia coli, containing plasmids that ranged in size from 3.9 to 96 kb, were inoculated into soil at 10⁴–10⁵ cells/g soil. The initial total bacterial population was approximately 5×10⁷ cells/g soil, and the gram-negative bacterial population ranged from 10⁵ to 10⁶ cells/g soil. Changes in various populations were followed on selective media, and as few as 1 to 20 introduced colony forming units (CFU)/g soil could be detected. The introduced strains either remained at approximately 10⁴ CFU/g soil or dropped to undetectable levels during a 28-day incubation, depending on the host strain but not on the type or size of the plasmid. The decline of plasmid-containing strains was less rapid than that of the plasmidless hosts in the two host (PRC487 and 1666) and host-plasmid systems (PRC487[pACYC175] and 1666[pESO19]) that were compared. The addition of nutrients, initially or during the incubation, resulted in an increase in all bacteria and decreased the rate of decline of introduced strains. Some plasmid-containing strains could not be recovered from soil by direct plating on selective media but could be recovered after transfer from nonselective isolation media to selective media. These laboratory-cultured strains apparently became so debilitated in soil that they could not grow directly on antibiotic-containing media without resuscitation on a less stressful medium. With the exception of the 1666(pESO19) system, there appeared to be no loss of the plasmids in soil. These results indicated that the survival of some genetically engineered (i.e., plasmid-containing) bacteria in soil is primarily a function of the bacterial strain and not of the contained plasmid and is influenced by the nutritional state of the soil. No transfer of plasmids to indigenous bacteria was apparent in these studies.