In vivo analysis of the relationships between the splicing and homing activities of a group I intron-encoded I-ScaI/bi2-maturase of Saccharomyces capensis produced in the yeast cytoplasm.

The I-ScaI/bi2-maturase of Saccharomyces capensis acts as a specific homing endonuclease promoting intron homing, and as a maturase promoting intron splicing. Using the universal code equivalent of the mitochondrial gene encoding the I-ScaI/bi2-maturase, a number of truncated forms of the synthetic gene were constructed, shortened on either side, as were several mutated alleles of the protein. The shortest translation product that fully retains both activities in vivo corresponds to 228 codons of the C-terminal region of the bi2 intron-encoded protein, whereas proteins resulting from more extensive deletions either at the N-terminus or at the C-terminus (up to 73 and four residues, respectively) were able to complement wholly the lack of endogenous maturase, but all lost the endonuclease activity.

Purification and characterization of the DNA cleavage and recognition site of I-ScaI mitochondrial group I intron encoded endonuclease produced in Escherichia coli.

The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs. Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain. It acts as a specific homing endonuclease I- Sca I promoting intron mobility and as a maturase promoting intron splicing.

Mimicking lipid-binding-induced conformational changes in the human apolipoprotein E N-terminal receptor binding domain effects of low pH and propanol.

We studied the effects of n-propanol and pH on the structure of the apolipoprotein E3 N-terminal receptor binding domain, apo E3(1-191), to determine whether conditions similar to those occurring near lipid surfaces (decreased dielectric constant and pH) can mimic lipid-induced conformational changes in apo E3. The addition of 30% n-propanol, at pH 7, induces a conformational change in apo E3(1-191) as shown by changes in the intrinsic tryptophan fluorescence and by an increase in the Stokes radius of the majority of the protein from 3.0 to 4.

Cytoplasmic and periplasmic production of human apolipoprotein E in Escherichia coli using natural and bacterial signal peptides.

To understand the toxicity of high levels of heterologous human serum apolipoprotein E (ApoE) in Escherichia coli, as well as to prepare a system for producing the structural domains of this protein, plasmids were constructed in which the coding sequence of the N-terminal domain or all of ApoE followed E. coli or human apolipoprotein signal peptides (SP) or the N-terminal eleven amino acids (f10) of the gene 10-encoded protein of phage T7. High levels of production of the 22-kDa N-terminal domain (22K) of ApoE were obtained either as an f10::22K fusion protein, or using the natural SP, or SP derived from the periplasmic protein, alkaline phosphatase (PhoA), or from the outer membrane protein A (OmpA).

Purification and characterization of the in vitro activity of I-Sce I, a novel and highly specific endonuclease encoded by a group I intron.

Group I intron encoded proteins represent a novel class of site specific double strand endonucleases. The endonuclease activity of this class of proteins has been first demonstrated in vivo for I-Sce I which is encoded by a mitochondrial intron of Saccharomyces cerevisiae. Assays using crude cell extracts have shown that I-Sce I can be used in vitro as a restriction endonuclease potentially useful for recombinant DNA technology owing to its large recognition sequence (18 nucleotides).