PA leadership: Implementation, structure, and sustainability.

Physician associate/assistant (PA) leaders are essential to healthcare and critical to a healthcare organization's success. In this article, the second of a two-part series on PA leadership, we summarize methods for implementing and sustaining PA leadership opportunities and organizational structures.

Cloning and analysis of a gene from Streptomyces lividans 66 encoding a novel secreted protease exhibiting homology to subtilisin BPN'.

Amino-terminal degradation has been observed for many of the secreted heterologous proteins produced by S. lividans 66. We, therefore, set out to characterize the relevant proteinases and their genes.

Isolation and characterization of a gene encoding a chymotrypsin-like serine protease from Streptomyces lividans 66.

A gene encoding a Streptomyces lividans homologue of the chymotrypsin-like serine protease (SAM-P20) of Streptomyces albogriseolus was isolated using the Streptomyces griseus prtB gene as a hybridization probe. Nucleotide sequence analysis of a representative clone uncovered the possible presence of a sequence of 900 nucleotides encoding 300 amino acids, including a putative "prepro" region of 115 amino acids. Alignment of the predicted amino acid sequence of this putative gene with other members of the family of Streptomyces extracellular chymotrypsin-like proteases indicated a high degree of homology in all cases, especially with the SAM-P20 protease.

Isolation and characterization of two genes encoding proteases associated with the mycelium of Streptomyces lividans 66.

A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-beta-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered.

Cloning and characterization of a gene encoding a secreted tripeptidyl aminopeptidase from Streptomyces lividans 66.

The gene encoding a tripeptidyl aminopeptidase (Tap) from Streptomyces lividans was cloned by using a simple agar plate activity assay. Overexpression of the cloned gene results in the production of a secreted protein which has an apparent subunit molecular weight of 55,000 and is responsible for the major amino-terminal degradative activity in culture broths of S. lividans strains.

The aminopeptidase N-encoding pepN gene of Streptomyces lividans 66.

The gene (pepN) encoding an aminopeptidase N (PepN) has been cloned from Streptomyces lividans. This was done using either leucine-beta-naphthylamide or arginine-beta-naphthylamide in a liquid overlayer on colonies growing on agar medium to screen for overproduction of the ability to hydrolyse the substrates. The nucleotide sequence of pepN was determined and shown to encode a 95-kDa protein, which displayed significant homology to PepN proteins from other organisms.

Intracellular aminopeptidases in Streptomyces lividans 66.

We have investigated the aminopeptidase activities present in Streptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues.

Cloning of genetic loci involved in endoprotease activity in Streptomyces lividans 66: a novel neutral protease gene with an adjacent divergent putative regulatory gene.

A skimmed-milk clearing assay was used to identify, in a multicopy Streptomyces lividans 66 genomic library, DNA fragments that lead to increased expression of protease activity in S. lividans 66. Three independent loci were identified.

In vitro expression of two proteins from overlapping reading frames in a eukaryotic DNA sequence.

The in vitro expression of two distinct proteins from overlapping reading frames in a sequence of rainbow trout genomic DNA has been demonstrated. In vitro transcription of DNA sequences, cloned in a plasmid under the control of Salmonella phage 6 polymerase promoter, led to the synthesis of two distinct and functional mRNAs corresponding to the protamine mRNA and also to another overlapping mRNA, termed Y. These mRNAs were translated in an mRNA-dependent rabbit reticulocyte lysate cell free system which synthesized the corresponding protein products.

Functional prokaryotic gene control signals within a eukaryotic rainbow trout protamine promoter.

Following the construction of a series of pSV2-cat derived plasmids containing the chloramphenicol acetyltransferase (CAT) gene under the control of a eukaryotic trout protamine promoter, it was noted that Escherichia coli, transformed with these plasmids, developed resistance to chloramphenicol (CM). This result suggested that the eukaryotic trout protamine promoter possessed significant prokaryotic promoter activity. Modification of the trout protamine promoter region by removing the region containing the eukaryotic Goldberg-Hogness box in the plasmid p525-cat increased the expression of the CAT gene almost to the wild-type level and conferred strong CM resistance.