Epicentre Technologies Corporation

Lubrizol Genetics, Inc.

Mycogen Plant Science, Inc.

Leslie M Hoffman

Jerome J Jendrisak

Darin J Haskins

Robert E Smith

Michael J Fiandt

Gregory A Hawkins

The present invention provides compositions and methods for controlling the copy number for a broad range of plasmids and uses thereof. Disclosed is a host cell for conditional control of copy number of a plasmid, which host cell comprises a poly(A) polymerase gene that is operably joined to a conditionally inducible promoter, and a method for cloning and stably maintaining a DNA sequence encoding a heterologous polypeptide in the host cell.

Compositions and methods for controlling copy number for a broad range of plasmids and uses thereof

A method for retrofitting DNA in a single-copy or high-copy vector, such as a fosmid or BAC, whereby an artificial transposon is used to introduce a conditional multi-copy origin of replication ('ori') into the DNA in said vector. Following random in vitro or in vivo transposition of the ori-containing transposon into DNA in the single-copy or low-copy vector, the resulting insertion clones are introduced into a special host strain that contains a gene which encodes a polypeptide required for replication from the multi-copy ori. However, since the gene for this polypeptide is expressed from a tightly-regulated inducible promoter, the polypeptide is not expressed in the absence of inducer. On addition of inducer to the culture medium, the host cell synthesizes the polypeptide, which in turn activates replication from the multi-copy ori, thereby increasing the amount of clone DNA synthesized by the cell.

Methods and compositions for amplifying DNA clone copy number

It can be difficult to achieve efficient transformation of many strains of bacterial cells due in part to the presence of one or more restriction and modification (R-M) systems in the cells that restricts unmodified transforming DNA. Phage T7 OCR protein is a potent inhibitor of Type I R-M systems. Methods are disclosed for improving transformation efficiency of Eubacterial and Archaebacterial cells having an R-M system by introducing into the cells an inhibitor of the restriction activity. For example, addition of 1&#x2013;5 micrograms of T7 OCR protein to 50 microliters of electrocompetent cells having a Type I R-M system prior to electroporation significantly increased transformation efficiency by unmodified plasmids, fosmid clones, and artificial transposons comprising synaptic complexes. Other inhibitors or restriction activity, such as phage-encoded proteins and proteins encoded by conjugative plasmids, as well as other disclosed inhibitors, also can be used to improve transformation (and transposition) efficiency. Host cells with heritable improved transformation efficiency can be made by transforming a cell having an R-M system with an expressible gene which encodes an inhibitor of the restriction activity, which gene can be conditionally expressible. Transient expression of a gene on an unmodified transforming DNA can also be improved by in vivo inhibition of restriction in host cells having an R-M system. Kits and compositions for carrying out the methods of the invention and host cells made using the methods are also disclosed.

DNA transformation efficiency by inhibiting host cell restriction

A method of characterizing a nucleic acid molecule is disclosed. The method comprises synthesizing DNA in the presence of a reaction mixture comprising a nucleic acid template, a primer molecule, an enzyme that extends the primer so that a DNA molecule may be synthesized, four canonical deoxynucleoside triphosphates and at least one non-canonical deoxynucleoside triphosphate. The non-canonical deoxynucleoside triphosphate is incorporated into the synthesized DNA in place of a portion of only one canonical deoxynucleoside triphosphate. The synthesized DNA is treated with an N-glycosylase that excises a base portion of the non-canonical deoxynucleoside triphosphate from the synthesized DNA. The DNA is then treated in such a manner that the phosphodiester backbone of the DNA is broken at the abasic site, thus creating at least two DNA fragments. The fragments are separated according to size.

Method for characterizing nucleic acid molecules

A method of analyzing a DNA molecule is disclosed. In one embodiment the method comprises the steps of exposing a DNA molecule to an effective amount of a chemical modification reagent wherein the reagent converts guanine to 8-hydroxyguanine. The oxidized product is then exposed to a DNA glycosylase enzyme and the DNA molecule is cleaved at the site of the 8-hydroxyguanine. The fragments are then resolved by electrophoresis and the position of guanine residues within the DNA molecule is determined. In a preferred embodiment of the present invention, the modification reagent is a thiazine dye and the enzyme is FPG protein.

Method of identifying nucleic acid molecules

The present invention discloses dicot cells containing monocot seed storage protein. Construction of genes encoding monocot seed storage proteins which are expressible in dicot cells and transformation of such genes into plant cells is also taught. Furthermore, methods and DNA molecules useful for producing plant cells containing monocot seed storage proteins are also disclosed. The invention is exemplified by combination of a 15 kD zein structural gene from a Zea mays gene with a promoter and a polyadenylation site derived from a Phaseolus vulgaris phaseolin gene.

Monocot seed storage proteins in dicots

The present invention discloses plant cells which contain modified 7S legume seed storage protein. Modification of 7S seed storage proteins which are expressible in plant cells and transformation of such genes into plant cells is also taught. Furthermore, methods and DNA molecules useful for producing plant cells containing modified 7S seed storage proteins are also disclosed. The invention is exemplified by insertion of an oligonucleotide encoding 15 amino acid residues, including 6 methionines, into a Phaseolus vulgaris phaseolin gene, thereby tripling its content of sulfur-containing amino acids.

Modified 7S legume seed storage proteins

The present invention discloses dicot cells containing monocot seed storage protein. Construction of genes encoding monocot seed storage proteins which are expressible in dicot cells and transformation of such genes into plant cells is also taught. Furthermore, methods and DNA molecules useful for producing plant cells containing monocot seed storage proteins are also disclosed. The invention is exemplified by combination of a 15 kD zein structural gene from Zea mays gene with a promoter and a polyadenylation site derived from a Phaseolus vulgaris phaseolin gene.

Methods and compositions for the construction of a recombinant vector containing plant or animal lectin DNA sequences, such vector being capable of being replicated, transcribed and translated in single cell hosts. The gene coding for the plant or animal lectin may be inserted into an expression vector which enables efficient production of the lectin protein.

Growing community of inventors

Madison, WI, United States of America

Leslie M Hoffman