Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-10
2004-12-21
Leffers, Gerry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S007100, C435S320100, C435S235100, C435S069100, C536S023100, C536S024100, C530S350000
Reexamination Certificate
active
06833239
ABSTRACT:
1. BACKGROUND OF THE INVENTION
“Congenital heart disease” refers to defects in the heart and major great vessels produced by abnormalities at various stages of fetal development and present at birth, but which may not be diagnosed until later. The incidence of such anomalies is 1/120 live births (The Merck Manual of Diagnosis and Therapy, 16
th
Ed. (1992) p. 2051). “Atrial septal defect” is form of congenital heart disease in which there is an opening in the septum that normally separates the atria. The typical murmur of atrial septal defect is usually present after age 1 yr., when pulmonary blood flow has increased significantly.
Many congenital heart diseases have a genetic basis. However, surgery offers the only therapeutic option for many of these disorders. In addition, current identification and diagnosis of congenital heart disease depends on the recognition of affected cardiac function, such as heart murmurs representing turbulent flow, altered systemic and pulmonary blood flow, shunting in either direction, and evidences of altered work load of the cardiac chambers. Routine history, physical examination, ECG, and chest x-ray are usually performed for specific anatomic diagnosis, with supportive and confirmatory data from echocardiography, cardiac catheterization, angiocardiography and other laboratory data.
Improved therapies and diagnostics for genetically based congenital heart diseases are needed.
2. SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of a novel human gene, which encodes a novel human protein. These newly identified genes and proteins are referred to therein as “FKHL7”. FKHL7 is a monomeric DNA binding protein that shares core binding site (RTAAYA) with four other FKHL7-like proteins. In addition, the forkhead domain of this protein shows strong homology to the human gene, FKHL14 and the mouse genes Fkh1 and Fkh14 by BLASTN analysis.
A 9.8 kb subclone of BAC471g19 was partially sequenced and determined to contain the entire coding region of FKHL7 as well as 5′ and 3′ untranslated sequences (SEQ ID NO. 1). The human FKHL7 coding sequence is 1.7 kb in size and contains no introns. The 1659 bp open reading frame (SEQ ID NO. 3) encodes a 553 amino acid polypeptide (SEQ ID NO. 2). The COOH-terminal domain contains several stretches of homopolymeric runs of alanine and glycine. The FKHL7 coding region contains 5 recognition sites for the restriction enzyme NotI. A BLASTN screen of the public dbEST database with the FKHL7 genomic sequence yields only partial human and mouse cDNA coverage of this gene. Based on the analysis of cDNA clones identified in the public databases, there is evidence for the utilization of at least two different polyadenylation signals within the 3′ untranslated region.
Human FKHL7 is most abundantly expressed during embryogenesis and of the adult tissue tested, significant expression was observed in adult eye, heart, kidney and lung, while relatively little to no expression was observed in adult skeletal muscle, spleen or liver.
In one aspect, the invention features isolated FKHL7 nucleic acid molecules. In one embodiment, the FKHL7 nucleic acid is from a vertebrate. In a preferred embodiment, the FKHL7 nucleic acid is from a mammal, e.g. a human. In an even more preferred embodiment, the nucleic acid has the nucleic acid sequence set forth in SEQ ID NO. 1 or 3 or a portion thereof. The disclosed molecules can be non-coding, (e.g. a probe, antisense, or ribozyme molecule) or can encode a functional FKHL7 polypeptide (e.g. a polypeptide which functions as either an agonist or antagonist of at least one bioactivity of the human FKHL7 polypeptide). In one embodiment, the nucleic acid of the present invention can hybridize to a vertebrate FKHL7 gene or to the complement of a vertebrate FKHL7 gene. In a further embodiment, the claimed nucleic acid can hybridize with a nucleic acid sequence shown in
FIG. 1
(SEQ ID NOS. 1 and 3) or a complement thereof. In a preferred embodiment, the hybridization is conducted under mildly stringent or stringent conditions.
In further embodiments, the nucleic acid molecule is an FKHL7 nucleic acid that is at least about 70%, preferably about 80%, more preferably about 85%, and even more preferably at least about 90% or 95% homologous to the nucleic acid shouts as SEQ ID NOS. 1 or 3 or to the complement of the nucleic acid shown as SEQ ID NOS 1 or 3.
The invention also provides probes and primers comprising substantially purified oligonucleotides, which correspond to a region of nucleotide sequence which hybridizes to at least about 6, at least about 10, at least about 15, at least about 20, or preferably at least about 25 consecutive nucleotides of the sequence set forth as SEQ ID NO. 1 or SEQ ID NO. 3 or complements of the sequence set forth as SEQ ID NOS. 1 or 3 or naturally occurring mutants or allelic variants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto, which is capable of being detected.
For expression, the subject nucleic acids can be operably linked to a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter (e.g., for constitutive expression or inducible expression) or transcriptional enhancer sequence. Such regulatory sequences in conjunction with an FKHL7 nucleic acid molecule can provide a useful vector for gene expression. This invention also describes host cells transfected with said expression vector whether prokaryotic or eukaryotic and in vitro (e.g. cell culture) and in vivo (e.g. transgenic) methods for producing FKHL7 proteins by employing said expression vectors.
In another aspect, the invention features isolated FKHL7 polypeptides, preferably substantially pure preparations, e.g. of plasma purified or recombinantly produced polypeptides. The FKHL7 polypeptide can comprise a full length protein or can comprise smaller fragments corresponding to one or more particular motifs/domains, or fragments comprising at least about 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 510, 520, 530 or 540 amino acids in length. In particularly preferred embodiments, the subject polypeptide is capable of binding to an upstream region of a gene and/or otherwise regulating expression of a gene.
In a preferred embodiment, the polypeptide is encoded by a nucleic acid, which hybridizes with the nucleic acid sequence represented in SEQ ID NOS. 1 or 3. In a further preferred embodiment, the FKHL7 polypeptide is comprised of the amino acid sequence set forth in SEQ ID NO. 2. The subject FKHL7 protein also includes within its scope modified proteins, e.g proteins which are resistant to post-translational modification, for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or asparagine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with intracellular proteins involved in signal transduction.
The FKHL7 polypeptides of the present invention can be glycosylated, or conversely, by choice of the expression system or by modification of the protein sequence to preclude glycosylation, reduced carbohydrate analogs can also be provided. Glycosylated forms can be obtained, for example, based on derivatization with glycosaminoglycan chains.
In yet another preferred embodiment, the invention features a purified or recombinant polypeptide, which has the ability to modulate, e.g., mimic or antagonize, an activity of a wild-type FKHL7 protein. Preferably, the polypeptide comprises an amino acid sequence identical or homologous to a sequence designated in SEQ ID NO. 2.
Another aspect of the invention features chimeric molecules (e.g., fusion proteins) comprising an FKHL7 protein. For instance, the FKHL7 protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the FKHL7 polypeptide. A preferred FKHL7 fus
Alward Wallace L. M.
Nishimura Darryl
Patil Shiva
Sheffield Val C.
Stone Edwin M.
Fulbright & Jaworski LLP
Leffers Gerry
The University of Iowa Research Foundation
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