Ikaros: A T cell pathway regulatory gene

Details Ikaros: A T cell pathway regulatory gene Ikaros: A T cell pathway regulatory gene

1996-09-05

2001-05-08

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S325000, C536S023100, C536S023500

Reexamination Certificate

active

06228611

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to the Ikaros gene and to the differentiation and generation of T cells.
The generation of the T cell repertoire from a progenitor stem cell proceeds through a differentiation pathway in which the later intrathymic steps are well documented while the early extrathymic events are only poorly characterized. One of the earliest definitive T cell differentiation markers is the CD3&dgr; gene of the CD3/TCR complex.
SUMMARY OF THE INVENTION
The Ikaros gene, a gene active in the early differentiation of lymphocytes, e.g. T cells and B cells, has been discovered. The gene encodes a family of unique zinc finger proteins, the Ikaros proteins. The proteins of the Ikaros family are isoforms which arise from differential splicing of Ikaros gene transcripts. The isoforms of the Ikaros family generally include a common 3′ exon (Ikaros exon E7, which includes amino acid residues 283-518 of the mouse Ikaros protein represented by SEQ ID No. 198, and amino acid residues 229-461 of the human Ikaros protein represented by SEQ ID No. 196) but differ in the 5′ region. The Ikaros family includes all naturally occurring splicing variants which arise from transcription and processing of the Ikaros gene. Eight such isoforms are described herein. The Ikaros family may also includes other isoforms, including those generated by mutagenesis and/or by in vitro exon shuffling. The naturally occurring Ikaros proteins can bind and activate (to differing extents) the enhancer of the CD3&dgr; gene, and are expressed primarily if not solely in T cells in the adult. The expression pattern of this transcription factor during embryonic development suggests that Ikaros proteins play a role as a genetic switch regulating entry into the T cell lineage. The Ikaros gene is also expressed in the proximal corpus striatum during early embryogenesis in mice.
In general, the invention features, nucleic acid, e.g., DNA, preferably a purified DNA, including (or consisting essentially of) a sequence which encodes a peptide including (or consisting essentially of) one or more Ikaros exons. In preferred embodiments: the Ikaros exon is any of E1/2, E3, E4, E5, E6, or E7; the purified DNA does not encode exon E7.
In other preferred embodiments: the encoded peptide further includes a second Ikaros exon; the second exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7 and the second exon is any of E1/2, E3, E4, E5, E6.
In other preferred embodiments: the encoded peptide further includes a third Ikaros exon; the third exon is any of E1/2, E3, E4, ES, E6, or E7; the first exon is E7, said second exon is E3, and the third exon is E1/2; the peptide is Ikaros isoform 5.
In other preferred embodiments: the encoded peptide further includes a fourth Ikaros exon; the fourth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E6, the third exon is E4, and the fourth exon is E1/2; the first exon is E7, the second exon is E4, the third exon is E3, and the fourth exon is El/2; the peptide is Ikaros isoform 3 or4.
In other preferred embodiments: the encoded peptide further includes a fifth Ikaros exon; the fifth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E6, the third exon is E5, the fourth exon is E4, and the fifth exon is E1/2; the peptide is Ikaros isoform 2.
In preferred embodiments: the encoded peptide further includes a sixth Ikaros exon; the sixth exon is any of E1/2, E3, E4, E5, E6, or E7; the first exon is E7, the second exon is E6, the third exon is E5, the fourth exon is E4, the fifth exon is E3, and the sixth exon is E1/2; the peptide is Ikaros isoform 1.
In preferred embodiments: the sequence of the encoded Ikaros exon is essentially the same as that of a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity; the DNA sequence which encodes the Ikaros exon is at least 85%, more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98 or 99% homologous with DNA encoding a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity, e.g., Ikaros exon encoded by DNA from any of SEQ ID NOS: 2-8 or SEQ ID NO:165; the sequence which encodes an Ikaros exon hybridizes under high or low stringency to a nucleic acid which encodes a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity, e.g., an Ikaros exon with the same, or essentially the same, amino acid sequence as an Ikaros exon of any of SEQ ID NOS: 195-201 or SEQ ID NO: 153 or SEQ ID NO: 202; the amino acid sequence of the encoded Ikaros exon is at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 200 amino acid residues in length; the encoded Ikaros amino acid sequence is at least 50% more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity; the encoded Ikaros exon is essentially equal in length to a naturally occurring Ikaros exon, or a fragment thereof having Ikaros activity; the amino acid sequence of the encoded Ikaros exon is at least 80%, more preferably at least 85%, yet more preferably at least 90%, yet more preferably at least 95%, and a most preferably at least 98 or 99% homologous with a naturally occurring Ikaros exon sequence, or a fragment thereof having Ikaros activity, e.g., an Ikaros exon sequence of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO:197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201 or SEQ ID NO:202; the encoded Ikaros exon amino acid sequence is the same, or essentially the same, as that of a naturally occurring Ikaros exon, or a fragment of the sequence thereof, e.g., an Ikaros exon described in any of SEQ ID NOS: 195-201 or SEQ ID NO: 202, and the peptide has Ikaros peptide activity.
In preferred embodiments the Ikaros encoding DNA includes at least two exons and: the DNA can be represented by the general formula A-B-C-D-E, wherein A represents Exon 3 or is absent, B represents Exon 4 or is absent, C represents Exon 5 or is absent, D represents Exon 6 or is absent, and E represents Exon 7 or is absent; the polypeptide includes at least two of said exons; the encoded polypeptide includes at least one exon containing a zinc finger domain; the encoded polypeptide includes at least one exon selected from E3, E4 or E5.
In other embodiments, the Ikaros encoding DNA includes a sequence represented by the general formula {Ex
1
-Ex
2
. . . Ex
n
} wherein each of Ex
1
through Ex
n
represents any of the Ikaros Exons 1/2, 3, 4, 5, 6 or 7, and n is an integer from zero to 10, more preferably an integer from zero to 5, In preferred embodiments: the polypeptide is a combination of 2 or more Ikaros exons, the combination of which may or may not naturally occur; the polypeptide includes at least two of said exons and is represented by the formula Ex
1
-Ex
2
; the polypeptide includes at least three of said exons and is represented by the formula Ex
1
-Ex
2
-Ex
3
; the polypeptide includes at least four of said exons and is represented by the formula Ex
1
-Ex
2
-Ex
3
-Ex
4
; the polypeptide includes at least five of said exons and is represented by the formula Ex
1
-Ex
2
-Ex
3
-Ex
4
-Ex
5
; the polypeptide includes at least six of said exons and is represented by the formula Ex
1
-Ex
2
-Ex
3
-Ex
4
-Ex
5
-Ex
6
; the polypeptide includes at least one exon containing a zinc finger domain; the polypeptide includes at least one exon selected from E3, E4 or E5.
In preferred embodiments: the exons in the encoded peptide are arranged in the same relative linear order as found in a naturally occurring isoform, e.g., Ikaros isoform 1, e.g., in a peptide having the exons E3 and E7, E3 is located N-terminal to E7; the linear order of the encoded exons is different from that found in a naturally occurring isoform, e.g., in Ikaros isoform 1, e.g., in a peptide having exons E3, E5, and E7, the direction N-terminal to C

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