Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-28
2001-06-19
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S029000, C435S325000, C435S375000, C530S350000
Reexamination Certificate
active
06248532
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel polynucleotides encoding polypeptides which bind to cAMP regulatory DNA sequences.
BACKGROUND OF THE INVENTION
Extracellular signal transduction leading to specific gene expression is often carried out by a series of enzymatic reactions which ultimately modulate activity of nuclear transcription factors. In one such example, extracellular signaling alters cytoplasmic levels of adenosine 3′, 5′-monophosphate (cAP) which in turn modulates levels of active cAMP-dependent protein kinase (PKA). Once activated, PKA migrates into the nucleus and phosphorylates transcription factors which recognize DNA sequences common to genes that are regulated by cAMP signaling pathways. The common DNA sequence which permits cAMP regulation of gene expression has been designated the cAMP regulatory element (CRE) and the transcription factors which recognize and bind to the CRE are known as CRE-binding (CREB) proteins. It has been proposed that CREB proteins are ordinarily found in association with CRE DNA sequences and that the phosphorylation state of CREB determines the degree to which the protein is capable of inducing transcription of the associated gene. Once phosphorylated, CREB is able to bind a CREB-binding protein (CBP) which permits interaction of the complex with transcription factor TFIIB.
It is therefore apparent that regulation of the phosphorylation state of CREB is central to specific gene expression by cAM. The phosphorylation state of CREB, however, is not regulated solely by PKA. On the contrary, the degree of CREB phosphorylation is balanced between the activities of phophatases as well as kinases other than PKA. Thus, while CREB is a major participant in coordination of cAMP gene expression, CREB activity is subject to concurrent control by enzymes in other, non-cAMP related pathways.
Members in the CREB family of proteins contain conserved regions which carry out specific functions related to transcriptional activation. At the carboxy terminus, all CREB proteins have a leucine zipper region which permits dimerization of CREB with other CREB proteins or other heterologous transcription factor subunits. Adjacent the leucine zipper region, CREB proteins are characterized by a region designated the kinase inducible domain (KID) which is subject to phosphorylation by multiple kinases other than PKA, including for example, protein kinase C (PKC), casein kinase I (CKI) and casein kinase II (CKII), and possibly calcium-calmodulin dependent kinases I and II. At the amino terminus, CREB proteins each contain a DNA binding domain rich in basic amino acids. Despite seemingly subtle differences between proteins within the family, reports of variation in gene expression suggest that the proteins have unique physiological roles.
BRIEF SUMMARY OF THE INVENTION
In one respect the present invention provides purified and isolated polynucleotides (e.g., DNA sequences, RNA transcripts thereof and anti-sense oligonucleotides thereof) encoding a novel mouse cAMP regulatory element binding, designated mCREIBa, polypeptide well as polypeptide variants (including fragments and deletion, substitution, and addition analogs) thereof which display one or more DNA or protein binding activities, one or more specific gene transcription modulation activities, and/or immunological properties specific to mCREBa. DNA binding properties include recognition of specific DNA sequences through which mCREBa is able to modulate specific gene expression, while protein binding properties include interaction with various regulators of mCREBa activity, including any of a number of protein kinases, as well as interactions with specific and non-specific transcription factors. Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences. A presently preferred olynucleotide is set out in SEQ ID NO: 1. Plasmid pBSmb3, comprising the preferred cDNA of the invention, in
E. coli
strain DH5&agr;F′ was deposited on September 18, 1996 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, and assigned Accession Number 98171. Biological replicas (i.e., copies of isolated DNA sequences made in vivo or in vitro) of DNA sequences of the invention are contemplated. Also provided are autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating mCREBa sequences and especially vectors wherein DNA encoding mCREBa or a mCREBa variant is operatively linked to an endogenous or exogenous expression control DNA sequence.
According to another aspect of the invention, host cells, especially unicellular host cells such as procaryotic and eucaryotic cells, are stably transformed with DNA sequences of the invention in a manner allowing the desired polypeptides to be expressed therein. Host cells of the invention are conspicuously useful in methods for the large scale production of mCREBa and mCREBa variants wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown.
Novel mCREBa polypeptides of the invention may be obtained as isolates from natural cell sources, but, along with mCREBa variant products, are preferably produced by recombinant procedures involving host cells of the invention. A presently preferred amino acid sequence for a mCREBa polypeptide is set out in SEQ ID NO: 2. The recombinant products may be obtained in fully or partially phosphorylated or dephosphorylated forms, depending on the cell selected for recombinant expression and/or post-isolation processing. The mCREBa variants of the invention may comprise mCREBa fragments which include all or part of one or more of the domain regions having a biological or immunological property of mCREBa including, e.g., the ability to bind to a polypeptide or polynucleotide binding partner of mCREBa and/or inhibit binding of mCREBa to a natural binding partner. The mCREBa variants of the invention may also comprise polypeptide analogs wherein one or more of the specified amino acids is deleted or replaced: (1) without loss, and preferably with enhancement, of one or more biological activities or immunological characteristics specific for mCREBa; or (2) with specific disablement of a particular polypeptide/polypeptide or polypeptide/polynucleotide binding function. Analog polypeptides including additional amino acid (e.g., lysine or cysteine) residues that facilitate multimer formation are contemplated. Variant mCREBa polypeptides further include fusion polypeptides wherein all or part of mCREBa is expressed in conjunction with extraneous polypeptide sequences, including, but not limited to, for example poly-histidine tags, biotinylation tags, &bgr;-galactosidase chimera, or chimeric polypeptides including one or more the DNA binding or transactivating domains from various transcription factors.
Also comprehended by the present invention are antibody substances (e.g., monoclonal and polyclonal antibodies, antibody fragments, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like) and other binding proteins (e.g., polypeptides and peptides) which are specific (i.e., non-reactive with previously identified CREBa isoforms to which mCREBa is structurally related) for mCREBa or mCREBa variants. Antibody substances can be developed using isolated natural or recombinant mCREBa or mCREBa variants. Binding proteins of the invention are additionally useful for characterization of DNA or polypeptide binding site structure(s) (e.g., epitopes and/or sensitivity of binding properties to modifications in mCREBa amino acid sequence).
Binding proteins are useful, in turn, in compositions for immunization as well as for purifying polypeptides of the invention and identifying cells which express the polypeptides on their surfaces. They are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) DNA and/or polypepti
ICOS Corporation
Larson Thomas G.
LeGuyader John L.
Marshall O'Toole Gerstein Murray & Borun
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