Bacillus subtilis sigma factor sigma 29 is the product of the sporulation-essential gene spoIIG.

Evidence is presented that the sporulation-essential locus spoIIG codes for both sigma 29 and a structurally related protein, P31. This demonstrates that at least one specific Bacillus subtilis RNA polymerase binding protein provides a critical function in endospore formation. spoIIG-specific RNA is present in B. subtilis cultures that are synthesizing P31 and sigma 29 and is absent in those that are not. A monoclonal antibody specific for an antigenic determinant on P31/sigma 29 detected crossreacting proteins (P25/P21) but not P31 or sigma 29 in a Spo- B. subtilis strain with a mutation at the spoIIG locus (spoIIG41). The appearance of P25 and P21 occurs in this mutant at a time when P31 and sigma 29 would normally appear and suggests that they are homologous proteins. Transformation of the spoIIG41 strain with plasmid DNA carrying the structural gene for spoIIG complements the Spo- phenotype and results in the synthesis of P31, sigma 29, P25, and P21 at the appropriate times during sporulation. In Escherichia coli, the cloned spoIIG sequence encoded a protein that reacted with the anti-P31/sigma 29 monoclonal antibody and had the electrophoretic mobility of authentic P31.     

Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli.

The rpoS gene of Escherichia coli encodes a putative RNA polymerase sigma factor that is considered to be the central regulator of gene expression in stationary phase. The gene product (sigma 38) was overproduced using the cloned rpoS gene and purified to homogeneity. Reconstituted RNA polymerase holoenzyme (E sigma 38) was found to recognize in vitro a number of typical sigma 70-type promoters, including the lacUV5 and trp promoters. Some, however, were recognized exclusively or preferentially by E sigma 70, whereas at least one, fic, was favored by E sigma 38. Thus E. coli promoters can be classified into three groups: the first group is recognized by E sigma 70 and E sigma 38, but the second and third groups are recognized substantially by either E sigma 70 or E sigma 38 alone. In contrast to other minor sigma factors, sigma 38 shares a set of amino acid sequences common among the principal sigma factors of eubacteria and is therefore a member of the RpoD-related protein family. The intracellular level of sigma 38 was demonstrated to increase in vivo upon entry into stationary phase. These results together indicate that sigma 38 is a second principal sigma factor in stationary-phase E. coli.     

Correlation between the 32-kDa sigma factor levels and in vitro expression of Escherichia coli heat shock genes.

S-30 extracts from Escherichia coli cells were used to express heat shock (HS) and non-HS genes in vitro in a DNA-directed protein synthesis system. The S-30 extracts prepared from cells that have been shifted to 45 degrees C express HS genes in vitro approximately 8 times better than extracts from cells at 33 degrees C. In contrast, the expression of non-HS genes in extracts from heat-induced cells is only 40% of that seen in extracts from cells at 33 degrees C. These results correlate well with the levels of HS sigma factor and normal sigma factor bound to RNA polymerase. Thus, there was an 8-fold increase in the HS sigma factor and a 60% decrease in the normal sigma factor associated with RNA polymerase at the higher temperature. Part of the increase in the level of the HS sigma factor could be accounted for by a 3-fold increase in the level of HS sigma factor mRNA during heat induction.     

Direct evidence that hepatocyte growth factor is a hepatotrophic factor for liver regeneration and has a potent antihepatitis effect in vivo.

Hepatocyte growth factor, a potent mitogen for mature hepatocytes in vitro, seems to function as a hepatotrophic factor for liver regeneration. We examined the mitogenic effect of hepatocyte growth factor on mouse liver in vivo. The labeling index of hepatocytes was markedly increased when recombinant human hepatocyte growth factor was injected intravenously into mice subjected to 30% hepatectomy (control, 1.7% +/- 0.1%; 1 microgram hepatocyte growth factor, 6.4% +/- 1.3%; 5 micrograms hepatocyte growth factor, 18.3% +/- 0.2%) and into mice administered carbon tetrachloride (control, 12.7% +/- 1.0%; 1 microgram hepatocyte growth factor, 26.3% +/- 2.8%) or alpha-naphthylisothiocyanate (control, 0.4% +/- 0.1%; 1 microgram hepatocyte growth factor, 3.8% +/- 1.1%; 5 micrograms hepatocyte growth factor, 14.2% +/- 2.0%). In addition, weights of the remnant livers in mice given hepatocyte growth factor 60 hr after 30% hepatectomy were significantly greater than those of untreated control mice (control, 0.93 +/- 0.04 gm; 5 micrograms hepatocyte growth factor, 1.06 +/- 0.04 gm). Hepatocyte growth factor prevented any marked increase in the serum levels of liver enzymes and bilirubin when it was administered to mice also treated with alpha-naphthylisothiocyanate (control: ALT, 394 +/- 278 IU/L; lactate dehydrogenase, 2,644 +/- 1,109 IU/L; bilirubin, 9.6 +/- 2.6 mg/dl; and 5 micrograms hepatocyte growth factor: ALT, 135 +/- 7.9 IU/L; lactate dehydrogenase, 1,672 +/- 626 IU/L; bilirubin, 1.0 +/- 0.8 mg/dl). Our findings show that intravenously injected hepatocyte growth factor stimulates the growth of hepatocytes in mouse liver and protects the integrity of hepatocytes in vivo against hepatitis caused by hepatotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological evidence that the adrenal gland is a source of an endogenous digitalis-like factor

A study has been performed to investigate the tissue of origin of an endogenous plasma factor which has been previously shown to bind to an antidigoxin antibody raised in this laboratory. Adult male rats were used to provide tissue, which was homogenized ultrasonically and extracted on C18 minicolumns. The extracts were then assayed in a RIA for digoxin, and the ability of tissue extracts to displace radioiodinated digoxin from its antibody was determined. Consistent displacement was produced by extracts of whole adrenal gland, but not by whole brain and brain regions, anterior or posterior pituitary gland, skeletal muscle, aorta, testis, kidney, spleen, atrium, or ventricle. In some samples, extracts of liver also produced displacement of digoxin tracer. Further experiments were performed to determine whether bilateral adrenalectomy was able to influence plasma levels of the endogenous digitalis-like factor. Forty-eight hours after adrenalectomy, plasma levels of the factor were significantly reduced in whole plasma and ether extracts of plasma and were insignificantly lower in C18 extracts of plasma. These findings suggest that the endogenous factor we have studied previously in plasma may be derived from the adrenal gland.