Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-14
2001-11-27
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C536S023100, C536S024300, C530S350000, C530S324000, C530S325000
Reexamination Certificate
active
06322977
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a new protein and to the use of these sequences in the characterization, diagnosis, prevention, and treatment of conditions such as cancer and immune and reproductive disorders.
BACKGROUND OF THE INVENTION
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of biochemical and physiological mechanisms and metabolic pathways. Despite different evolutionary pressures, proteins that regulate the cell cycle in yeast, nematode, fly, rat, and man have common chemical or structural features and modulate the same general cellular activity. Comparisons of human gene sequences with those from other organisms where the structure and/or function may be known allow researchers to draw analogies and to develop model systems for testing hypotheses. These model systems are of great importance in developing and testing diagnostic and therapeutic agents for human conditions, diseases and disorders.
Tapasin is a 48-kDa transmembrane glycoprotein. It is found in the endoplasmic reticulum (ER) and displays a cytoplasmic retention signal. Tapasin is a member of the immunoglobulin (Ig) superfamily and is encoded by an major histocompatibility (MHC)-linked gene. The protein plays a critical functional role in MHC class I-restricted antigen processing. Tapasin mediates the interaction between the transporter associated with antigen processing (TAP) and newly synthesized MHC class I molecules by forming complexes with other chaperones such as calnexin and calreticulin. Up to four MHC class I-tapasin complexes bind and present molecules to each TAP molecule. (See Pamer and Cresswell (1998) Annu. Rev. Immunol. 16:323-58; Ortmann et al. (1997) Science 277:1306-9.) Tapasin is essential for human lymphocyte (HLA) A1, B8, and B4402 antigen presentation. Although tapasin is required for HLA-A2 molecules to bind TAP, its absence affects the overall efficiency of the process of loading HLA-A2 with optimal, stabilizing peptides. With its Ig_MHC binding signature (Y)xCx(V)xB, tapasin is a necessary cofactor in a multicomponent ‘peptide loading complex’ where lack of binding results in proteasome-mediated degradation (Lewis et al. (1998) Eur. J. Immunol. 28:3214-20). After analysis of mutant molecules which fail to bind tapasin or TAP, Suh et al. (1999; J. Immunol. 162:1530-40) also suggest a peptide-editing function for tapasin/TAP in addition to their role in enhancing peptide loading.
Correct antigen presentation to T lymphocytes is important in the infectious disease process. In a study of the mutant MHC class I molecule T134K (in which Thr134 was changed to Lys), Lewis and Elliott (1998; Curr. Biol. 8:717-20) reported that the point mutation disrupted, directly or indirectly, the interaction between MHC class I molecules and calreticulin. T134K molecules were tranported out of the ER as ‘empty’ MHC class I complexes rather than being retained and degraded and neither bound TAP nor presented viral antigens to T cells.
The discovery of a nucleic acid sequence encoding a tapasin-like protein provides new compositions which are useful in the characterization, diagnosis, prevention, and treatment of conditions such as cancer and immune and reproductive disorders.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a nucleic acid sequence encoding a mammalian protein, tapasin-like protein (TLP), which satisfies a need in the art by providing new compositions useful in the characterization, diagnosis, prevention, and treatment of conditions such as cancer and immune and reproductive disorders.
The invention provides an isolated and purified mammalian nucleic acid sequence comprising SEQ ID NO:1 or a fragment thereof (SEQ ID NOs:3-9). The invention further provides fragments homologous to the mammalian nucleic acid sequences from mouse and rat identified as SEQ ID NOs: 10-13 in the Sequence Listing. The invention also provides a substantially purified mammalian nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2 or a portion thereof.
The invention further provides an isolated and purified nucleic acid sequence or a fragment thereof which hybridizes under high stringency conditions to the nucleic acid sequence of SEQ ID NO:1. The invention also provides an isolated and purified nucleic acid sequence or a fragment thereof which is complementary to the nucleic acid sequence of SEQ ID NO:1. In one aspect, a single stranded complementary RNA or DNA sequence is used as a probe which hybridizes under high stringency conditions to the mammalian nucleic acid sequence or a fragment thereof.
The invention further provides a method for detecting a nucleic acid sequence in a sample, the method comprising the steps of hybridizing the complement of the nucleic acid sequence to at least one nucleic acid sequence of the sample, thereby forming a hybridization complex; and detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a nucleic acid sequence in the sample. In one aspect, the method further comprises amplifying the nucleic acid sequence prior to hybridization. The nucleic acid sequence or fragment thereof may comprise either an element or a target on a microarray. The invention also provides a method for using a nucleic acid sequence or a fragment thereof to screen a library of molecules to identify at least one molecule which specifically binds the nucleic acid sequence, the method comprising providing a library of molecules, combining the nucleic acid sequence with the library of molecules under conditions suitable to allow specific binding, and detecting specific binding, thereby identifying a molecule which specifically binds the nucleic acid sequence. Such libraries include DNA and RNA molecules, peptides, PNAs, proteins, and the like which are potential regulators of replication, transcription, and translation.
The invention also provides an expression vector containing at least a fragment of the nucleic acid sequence of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell. The invention further provides a method for producing a protein, the method comprising the steps of culturing the host cell under conditions suitable for the expression of the protein and recovering the protein from the host cell culture. The invention also provides an isolated and purified protein comprising the amino acid sequence of SEQ ID NO:2 or a portion thereof. Additionally, the invention provides a pharmaceutical composition comprising a substantially purified protein having the sequence of SEQ ID NO:2 or a portion thereof in conjunction with a suitable pharmaceutical carrier.
The invention further provides a method for using a portion of the protein to produce antibodies. The invention also provides a method for using a protein or a portion thereof to screen a library of molecules to identify at least one molecule which specifically binds the protein, the method comprising providing a library of molecules, combining the protein with the library of molecules under conditions suitable to allow specific binding, and detecting specific binding, thereby identifying a molecule which specifically binds the protein. In one aspect, a molecule identified using the method modulates the activity of the protein.
The invention further provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the natural mammalian nucleic acid sequence. The invention also provides a method for using SEQ ID NO:1 to produce a mammalian model system, the method comprises constructing a vector containing SEQ ID NO:1; introducing the vector into a suitable totipotent mammalian embryonic stem cell; selecting an embryonic stem cell with the vector integrated into genomic DNA; microinjecting the selected cell into a suitable mammalian blastocyst, thereby forming a chimeric blastocy
Baughn Mariah R.
Kaser Matthew R.
Lal Preeti
Celsa Bennett
Incyte Genomics Inc.
Incyte Genomics, Inc.
Murry Lynn E.
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