Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-24
2002-06-25
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007210
Reexamination Certificate
active
06410241
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of molecular biology and functional genomics. More particularly, it concerns methods to screen genes or gene components for their functions or biological effects and methods of generating immune responses or immune reagents.
2. Description of Related Art
Genomic sequencing efforts are producing a wealth of data. Sequence information is being compiled not only from humans but also plants, animals and microbes. This abundance of data has spawned the need for new technologies for analyzing and functionally assessing the millions of genes that will now be available. The expanding repertoire of areas that functional genomics are applied should lead to new insights into evolution, reveal how cellular pathways integrate, and yield new drugs and vaccines. A current challenge is to develop the technologies that will enable these advancements.
For example, there are currently at least thirty microbial genome sequences in the public domain and additional projects underway. Most of these are pathogens of humans or commercial animals. One often expressed hope is that knowledge of these sequences will lead to the development of vaccines against these pathogens. Though computational analyses may be useful, the more sure-footed approach would be to functionally screen each gene from all the pathogens in animals for its value as a protective agent. However the time and expense of cloning thousands of genes from each pathogen then preparing appropriate reagents from them is prohibitive, using current methods.
In order to quickly and effectively assess the activity of any particular gene product or a physiological response to it, an assay method is required that avoids plasmids and bacterial cloning procedures. This ideal method would also permit the plethora of new genes from sequencing projects to be rapid screened in organisms, cells or cell-free systems.
SUMMARY OF THE INVENTION
The inventors have determined methods and compositions which allow for the production of linear expression elements (“LEEs”) and/or circular expression elements (“CEEs”) encompassing a complete gene (promoter, coding sequence, and terminator). These LEEs and CEEs can be directly introduced into and expressed in cells or an intact organism to yield expression levels comparable to those from a standard supercoiled, replicative plasmid.
In some general embodiments, the invention relates to methods of assaying for the production or regulation of expression of at least one polypeptide from a linear or circular nucleic acid segment comprising a promoter or putative promoter and an ORF or putative ORF. These methods may comprise: a) obtaining at least one linear or circular nucleic acid segment comprising a promoter or putative promoter and an ORF encoding a peptide or putative ORF; b) placing the nucleic acid segment under conditions conducive to expression of the polypeptide from the ORF; and c) assaying for the production or regulation of expression of a polypeptide from the ORF or putative ORF. In many preferred embodiments, the nucleic acid segment will comprise a terminator. However, in some applications, including those where the linear nucleic acid segment is assayed in a cell-free expression system, no terminator is required. In some cases, the linear nucleic acid is obtained by a PCR® process. In other situations, the linear nucleic acid may be cut out of a plasmid, chromosome, or other larger piece of nucleic acid; in these cases, the linear nucleic acid cut from the larger piece of nucleic acid will typically comprise a promoter and ORF, and in many cases a terminator, that are already in operable relationship.
Of course, those of skill in the art will understand that the promoter or putative promoter and any terminator will typically be placed in a operable relationship to the ORF or putative ORF, employing methods disclosed herein or know to those in the art.
In many cases, the linear nucleic acid segment is obtained by synthesis. In some preferred methods, the synthesis comprises non-covalent linkage of the promoter to the ORF. For example, this non-covalent linkage may be performed by a) obtaining a PCR® product comprising the nucleic acid segment, which PCR® product is obtained by amplification from at least one primer that has complementary stretches comprising deoxyuridines with uracil-DNA glycosylase to create overhangs to which the promoter can link; b) providing the promoter; and c) non-covalently linking the promoter to the nucleic acid segment to create the linear or circular expression element. In many cases a terminator will be non-covalently linked to the ORF, using a similar technique, although it is possible that the ORF or putative ORF may have a terminator incorporated into it, such that the addition of a terminator may not be required. In some presently employed embodiments, the primer that has complementary stretches comprising deoxyuridines. These stretches allow the use of uracil-DNA glycosylase, or another suitable enzyme to create overhangs to which the promoter can non-covalently link create the linear or circular expression element. The non-covalent linkage of a terminator to the ORF can be accomplished by much the same technique. Of course, it will be understood to those of skill in the art that other nucleotide/enzyme pairs may be used to perform this non-covalent linking, and that other techniques of non-covalent linking may be employed, so long as the purposes of the invention are accomplished. In some specific embodiments, the primer comprises the promoter and the terminator in divergent orientation, such that the step of non-covalently linking the promoter and the terminator to the ORF results in a circular expression element.
While the promoter may be of any origin that will work for the purposes of the invention, in some preferred embodiments, the promoter is a eukaryotic promoter. Likewise, the terminator may be of any source, but in may cases the terminator will be a eukaryotic terminator.
The nucleic acid segment containing the ORF, putative ORF, or any other nucleic acid segment which is comprised in a LEE or CEE may be obtained from any of a variety of sources. For example, it may be obtained by PCR®, from a linear nucleic acid that is cut out of a plasmid, or obtained by synthesis.
In some methods according to the invention, the nucleic acid segment forming LEE or CEE is placed into a cell so that it is under conditions conducive to expression of a polypeptide from the ORF or putative ORF. In some preferred embodiments, the linear nucleic acid is placed into a cell but not integrated into the cell's genome. The inventors have determined that integration into the genome is not required for expression of linear nucleic acids. Further, the inventors have determined that supercoiled plasmids are not required for expression of genes. In some embodiments, the cell is in cell culture, while in others, the cell is comprised in a tissue or an entire organism. All organisms are contemplated in this regard, including, but not limited to plant, animal, mammal, fish, bird, reptilian, human, rabbit, rat, hamster, mouse and other cells. The LEEs and CEEs of the invention may be placed into cells using any of the technologies described elsewhere in the specification. In some preferred embodiments, a LEE or CEE in injected into the cell. In some particularly preferred embodiments injection comprises microprojectile bombardment. In other embodiments, the LEE or CEE many be placed in a cell-free expression reaction.
Various preferred embodiments relate to methods of analyzing a nucleic acid sequence comprising: a) obtaining a nucleic acid segment; b) linking the nucleic acid segment to a promoter and a terminator to create a linear or circular expression element; c) providing the linear or circular expression element to a cell-free expression system or to a cell under conditions conducive to expression of any product encoded for by the nucleic acid segment; and d) analyzing any e
Johnston Stephen Albert
Sykes Kathryn F.
Board of Regents , The University of Texas System
Fulbright & Jaworski L.L.P.
Loeb Bronwen M.
Yucel Remy
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