Construction of a multiframe vector to express coding sequences in Escherichia coli.

Cloning of foreign DNA fragments for coding sequence analysis in Escherichia coli usually involves sets of three vectors. To simplify this, we constructed an expression vector named pMFV7 containing three ATG codons in different frames downstream of a Shine-Dalgarno sequence, assuming that the ribosome can use any of the three start codons in an alternative manner. Translation beginning at either of the start codons would drive the expression of any coding fragment cloned downstream. To test the feasibility of this proposal, we cloned DNA fragments of the lacZ gene in each of the possible reading frames downstream from pMFV7 start codons. Sequence analysis of the N-terminus regions around the fusion sites indicates that ribosomes indeed initiate translation at each of the three initiation codons. In one case, levels of beta-galactosidase activity depended largely on the N-terminus of the translation products. We conclude that pMFV7 may be useful for expressing coding sequences regardless of their reading frame.

Purification, characterization, and complete amino acid sequence of a trypsin inhibitor from amaranth (Amaranthus hypochondriacus) seeds.

A protein proteinase inhibitor was purified from a seed extract of amaranth (Amaranthus hypochondriacus) by precipitation with (NH4)2SO4, gel-filtration chromatography, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. It is a 69-amino acid protein with a high content of valine, arginine, and glutamic acid, but lacking in methionine. The inhibitor has a relative molecular weight of 7400 and an isoelectric point of 7.5. It is a serine proteinase inhibitor that recognizes chymotrypsin, trypsin, and trypsin-like proteinase activities extracted from larvae of the insect Prostephanus truncatus. This inhibitor belongs to the potato-I inhibitor family, showing the closest homology (59.5%) with the Lycopersicum peruvianum trypsin inhibitor, and (51%) with the proteinase inhibitor 5 extracted from the seeds of Cucurbita maxima. The position of the lysine-aspartic acid residues present in the active site of the amaranth inhibitor are found in almost the same relative position as in the inhibitor from C. maxima.

[Proteins associated with actin: profilin in the trophozoites of Entamoeba histolytica]. / Proteínas asociadas a la actina: la profilina en los trofozoítos de Entamoeba histolytica.

The presence and distribution of profilin, an actin binding protein that regulates the actin organization, in trophozoites of Entamoeba histolytica, strain HM1-IMSS was examined using polyclonal antibodies against calf spleen profilin. The results shows: (1) in Entamoeba extracts a peptide that comigrates with spleen profilin and representing 0.86% of the total cells proteins, (2), this peptide is immunoprecipitated by antibodies antiprofilin, and (3) stained cells with the antibodies shows the protein distributed in cortical region associated to plasma membrane and th cytoplasm. These results show that a peptide similar to spleen profilin is present in the trophozoites of E. histolytica, supporting the idea that the profilin has been conserved through evolution. The presence of this protein would be crucial in the reversible control of soluble and polymerized actin required in the amoeboid movement.