The genes for alkaline protease (apr(BamP]) and neutral protease (npr[BamP]) from Bacillus amyloliquefaciens have been isolated and expressed in Bacillus subtilis. The DNA sequences of aprfBamP] and npr[BamP] revealed, in each case, the presence of a large open reading frame. The inferred amino acid sequence of either gene contained a signal sequence and an additional polypeptide sequence ('pro' sequence) preceding the mature protein. Based on DNA sequence, the start point of translation has been identified as amino acid residue-107 for apr{BamP] and-221 for npr[BamP]. To demonstrate that the start point of translation of apr{BamP] in vivo is probably at codon-107, codon-103 (AAA) was changed to an ochre (TAA) by site-directed mutagenesis. Alkaline protease was produced from this ochre mutant derivative of apr[BamP] only when the host strain was Su+. The presence of a pro sequence may be common to all of the secreted proteases from bacilli.

A RIA procedure for measuring progesterone (PROG), 5 alpha-pregnane-3,20-dione (5 alpha-DH PROG), and 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-TH PROG) has been developed and validated by GLC/mass spectrometry. Measurements were made in intact and adrenalectomized (ADX) male rats, in cyclic, pregnant, spayed, and spayed-ADX females, and in both males and spayed females injected with PROG. The predominant contribution of the ovary to the concentrations of 3 alpha,5 alpha-TH PROG in plasma and brain, was indicated by its larger levels in females, in particular during pregnancy, and by its presence in ovarian tissue and disappearance after ovariectomy. An additional adrenal origin in both males and females was shown. Neither PROG nor 5 alpha-DH PROG disappeared from brain, contrary to plasma, after combined adrenalectomy and gonadectomy, thus suggesting that PROG might be synthetized de novo in brain. However, the concentrations of 3 alpha,5 alpha-TH PROG in plasma and brain of female rats were positively correlated with the concentrations of PROG in plasma, indicating that plasma PROG was the major precursor of 3 alpha,5 alpha-TH PROG. The direct formation of 3 alpha,5 alpha-TH PROG from PROG in brain was strongly suggested by the increased 3 alpha,5 alpha-TH PROG/PROG ratios in brain vs. plasma, when measured in control females, and after injection of PROG to both males and OVX females. It was previously reported that 3 alpha,5 alpha-TH PROG is a sedative/anxiolytic steroid, as a result of its binding to gamma-aminobutyric acid (GABA)A receptors and allosteric potentiation of GABAcergic neurotransmission. Its concentrations in brain reach indeed the neuroactive range in cyclic and pregnant females, and are compatible with a physiological role of this neurosteroid.

No abstract

Excitatory amino acids (EAAs) are important neurotransmitters in the vertebrate central nervous system. Electrophysiological and ligand-binding studies indicate that at least three different receptor subtypes for EAAs exist--N-methyl-D-aspartate, kainate and quisqualate receptor subtypes--on the basis of the preferred agonist of the receptors. We recently purified a kainate-binding protein (KBP) from frog (Rana pipiens berlandieri) brain by domoic acid (a high-affinity kainate analogue) affinity chromatography, and showed that the kainate-binding activity was associated with a protein of relative molecular mass 48,000 (Mr 48 K). The pharmacological properties and the anatomical distribution of KBP were consistent with those of a kainate receptor-ionophore complex. We have now isolated a complementary DNA encoding KBP of Mr 48 K. The deduced amino-acid sequence of the KBP has similar hydrophobic profiles to those found in other ligand-gated ion channel subunits, and shows some amino-acid sequence similarities to the corresponding regions of brain nicotinic acetylcholine receptor subunits. Localization of the KBP messenger RNAs by in situ hybridization histochemistry is compatible with the results of immunohistochemistry and receptor autoradiography studies. COS-7 cells transfected with the cDNA encoding the KBP show high-affinity kainate-binding activity with pharmacological properties similar to those of the biochemically purified KBP. These results provide the first molecular characterization of an EAA-binding site and raise the possibility that the KBP cDNA encodes a ligand-binding subunit of a kainate receptor-ionophore complex.

A segment of Bacillus subtilis deoxyribonucleic acid (DNA) previously cloned in Escherichia coli contains a gene (the 0.4-kilobase [kb] gene) whose transcription is activated at an early stage of spore development. To-map the genetic location of the 0.4-kb gene, we constructed a hybrid plasmid that inserts a chloramphenicol resistance determinant into the B. subtilis chromosome by recombination at a site of homology between cloned B. subtilis DNA and the chromosome. This hybrid plasmid (p1949-2) was constructed from the E. coli plasmid pMB9, the B. subtilis chloramphenicol resistance plasmid pCM194 (whose replication function was inactivated), and B. subtilis DNA from the vicinity of the 0.4-kb gene. Transformation of B. subtilis cells to drug resistance by p1949-2 was dependent upon the B. subtilis RecE+ phenotype and resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the 0.4-kb gene region of the chromosome. Chloramphenicol resistance in cells transformed by p1949-2 was mapped to the purA-cysA region of the B. subtilis chromosome, a region that contains several sporulation genes and the origin of DNA replication. In addition, DNA adjacent to the 0.4-kb gene was shown to contain the wild-type allele of a genetic marker (tms-26) from that region.