University of Massachusetts

Other

Peter E Newburger

Jack L Leonard

Sherman M Weissman

Jon Goguen

Yatindra Prashar

Subrahmanyam V Yerramilli

The present invention comprises a method to identify granulocytic cell genes that are differentially expressed upon exposure to a pathogen or in a sterile inflammatory disease by preparing a gene expression profile of a granulocytic cell population exposed to a pathogen or isolated from a subject having a sterile inflammatory disease and comparing that profile to a profile prepared from quiescent granulocytic cells. The present invention is particularly useful for identifying cytokine genes, genes encoding cell surface receptors and genes encoding intermediary signaling molecules. The invention also includes methods to identify a therapeutic agent that modulates the expression of at least one gene in a granulocytic population. Genes which are differentially expressed during neutrophil contact with a pathogen, such as a virulent bacteria, or that are differentially expressed in a subject having a sterile inflammatory disease are of particular importance.

Process to study changes in gene expression in granulocytic cells

Methods of controlling the in vivo and in vitro expression of a heterologous protein by transfecting a cell with a first nucleic acid encoding the heterologous polypeptide, wherein at least one codon of mRNA transcribed from the first nucleic acid is replaced by the codon UGA, and a second nucleic acid operably linked to the first nucleic acid, the second nucleic acid directing the translation of the UGA codon as selenocysteine only when the cell can obtain selenium from the medium in which it is grown; and growing the cell under conditions in which the production of the polypeptide is controlled by the level of selenium available to the cell.

Post transcriptional gene regulation by selenium

The invention provides methods for determining the structure of a polypeptide by a) transfecting a cell with (i) a first nucleic acid encoding the polypeptide, wherein at least one, specific codon of mRNA transcribed from the first nucleic acid is replaced by the codon UGA, and (ii) a second nucleic acid, operably linked to the first nucleic acid, that directs the translation of the UGA codon as selenocysteine only when the cell can obtain selenium from the medium in which it is grown; b) growing the cell in selenium-containing growth medium under conditions in which the cell incorporates at least one selenocysteine residue into the polypeptide at a specific location; c) isolating the polypeptide from the cell or the growth medium; d) forming a crystal of the polypeptide; and e) performing X-ray crystallography on the crystal, wherein the selenocysteine residue is used to determine the structure of the polypeptide.

Growing community of inventors

Waban, MA, United States of America

Peter E Newburger