Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-05-06
2002-01-08
Clark, Deborah J. R. (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024310, C536S023100, C435S006120, C435S070100, C435S320100, C435S325000
Reexamination Certificate
active
06337392
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to transcriptional control elements that are tissue specific. More particularly, the invention relates to nucleic acids that contain tissue-specific transcriptional control elements. The invention also relates to methods of using these control elements including for the identification modulators of tissue-specific protein expression.
BACKGROUND OF THE INVENTION
The transcription factor Pax-6 is known to have a critical role in development of the eye in a number of species [Quiring et al.,
Science
265:785-789 (1994)]. In Drosophila, homozygous mutation of the Pax-6 orthologue eyeless results in missing eye structures. In humans, heterozygous Pax-6 mutations give the ocular defects aniridia (iris hypolasia [Jordan et al.,
Nat. Genet
. 1:328-332 (1992)]) and Peters' anomaly (a combination of iris hypolasia and lens-corneal epithelium attachment [Hanson et al.,
Nature Genetics
, 6:168-173 (1994)]). Analogous defects are seen in the Small eye mice, also heterozygous for a Pax-6 mutation [Hill et al.,
Nature
354:522-525 (1991)]. Homozygous mutation in mice is lethal and results in severe cranio-facial abnormalities including absence of eyes [Hill et al.,
Nature
354:522-525 (1991)].
Gain-of-function experiments also demonstrate a central role for Pax-6 in development of the eye. In Xenopus, expression of the transcription factor Pax-6 directs induction of the lens [Altmann et al.,
Dev. Biol
. 185:119-123 (1997); U.S. application Ser. No. 08/846,463 filed May 1, 1997 hereby incorporated by reference in its entirety]. Analogously, overexpression of Drosophila Pax-6 in imaginal discs results in the development of ectopic compound eyes [Gehring et al.,
Genes to Cells
1: 11-15 (1996)]. Remarkably, this activity can be mimicked by Pax-6 from a number of species including, squid [Tomarev et al.,
Proc. Natl. Acad. Sci. U.S.A
. 94:2421-2426 (1997)] and ascidian [Glardon et al.,
Development
124:817-825 (1997)] arguing that Pax-6 function has been conserved across more than 40 million years of evolution [Gehring et al.,
Genes to Cells
1:11-15 (1996)].
In animal cap experiments, Pax-6 can induce the expression of the lens-specific marker &bgr;B1-crystallin without inducing the general neural marker NCAM. Ectopic Pax-6 expression also results in the formation of ectopic lenses in whole embryos indicating that in vertebrates, as well as Drosophila [Halder, et al.,
Science
, 267:1788-1792 (1995)], Pax-6 can direct the development of major components of the eye. According to NCAM staining, ectopic lenses formed in the whole embryo are only sometimes associated with neural tissue. Furthermore, treatments giving rise to anterior neural tissue result in the expression of both &bgr;B 1-crystallin and Pax-6.
In the mouse, Pax-6 has a complex pattern of expression that includes multiple components of the developing eye, as well as neural tube, forebrain neuroepithelium, olfactory epithelium and bulbs, pancreas and pituitary [Grindley et al.,
Development
121:1433-1422 (1995); Walther et al.,
Development
113:1435-1449 (1991)]. In the eye, Pax-6 is expressed in the presumptive and mature lens, retina and corneal epithelium [Grindley et al.,
Development
121:1433-1442 (1995); Walther et al.,
Development
113:1435-1449 (1991)]. This expression pattern is conserved in different vertebrate species including in Xenopus [Li et al.,
Development
, 124:603-615 (1997)] and chick [Li et al.,
Dev. Biol
., 162:181-194 (1994)].
Although it would be valuable to be able to express selected proteins solely in the lens and/or corneal epithelium, heretofore no lens and/or corneal epithelium specific transcriptional control element has been identified. Therefore, there is a need to identify such a lens and/or corneal epithelium transcriptional control element. Furthermore, there is a need to identify agents that can modulate lens and/or corneal epithelium development including naturally occurring transcription factors.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.
SUMMARY OF THE INVENTION
The present invention provides a lens transcription control element that can be used to identify factors, both naturally occurring and synthetic, that will modulate lens and/or corneal epithelium development and function. This lens transcription control element can be used to direct transcription in the lens and/or corneal epithelium
The present invention therefore provides a recombinant nucleic acid comprising a lens and corneal epithelium specific transcriptional control element (LCE) that hybridizes with a nucleic acid having the nucleotide sequence of SEQ ID NO:1 or a functional derivative thereof. In another embodiment the recombinant nucleic acid contains a LCE that hybridizes with a nucleic acid having the nucleotide sequence of SEQ ID NO:3. In a preferred embodiment of this type the hybridization is performed under standard conditions. In a more preferred embodiment the hybridization is performed under stringent conditions.
The present invention also provides a recombinant nucleic acid comprising a lens and corneal epithelium specific transcriptional control element (LCE) that hybridizes with a nucleic acid having the nucleotide sequence of SEQ ID NO:2 or a functional derivative thereof. In another embodiment the recombinant nucleic acid contains a LCE that hybridizes with a nucleic acid having the nucleotide sequence of SEQ ID NO:4. In a preferred embodiment of this type the hybridization is performed under standard conditions. In a more preferred embodiment the hybridization is performed under stringent conditions.
In one embodiment, the lens specific transcriptional control element contains between 100 to 800 nucleotides. In a preferred embodiment the LCE contains 200 to 600 nucleotides. In a more preferred embodiment the LCE contains 250 to 450 nucleotides. In a particular embodiment, the LCE contains 341 nucleotides. In a preferred embodiment of this type, the LCE has a nucleotide sequence of SEQ ID NO:3. In another embodiment, the LCE has a nucleotide sequence of SEQ ID NO:4.
The LCE of the present invention is preferably a vertebrate control element. In one such embodiment the LCE is a xenopus control element. In an alternative embodiment the LCE is a mammalian control element. In one such embodiment, the mammalian control element is a murine control element. In a preferred embodiment of this type the LCE is a Pax-6 conserved element (PACE) having the nucleotide sequence of SEQ ID NO:1. In an alternative embodiment the PACE has the nucleotide sequence of SEQ ID NO:3. In another such embodiment the mammalian control element is a human control element. In a preferred embodiment of this type the LCE is a Pax-6 conserved element (PACE) having the nucleotide sequence of SEQ ID NO:2. In an alternative embodiment the PACE has the nucleotide sequence of SEQ ID NO:4.
The present invention also includes nucleotide probes that hybridize with a nucleic acid having the nucleotide sequence of SEQ ID NO:1. In a related embodiment the present invention provides a nucleotide probe that hybridizes with a nucleic acid having the nucleotide sequence of SEQ ID NO:2. In yet another embodiment the nucleotide probe hybridizes with the nucleotide sequence of SEQ ID NO:3. In still another embodiment the nucleotide probe hybridizes with the nucleotide sequence of SEQ ID NO:4. In a preferred embodiment the hybridization is done under standard hybridization conditions. In a more preferred embodiment the hybridization is performed under stringent conditions.
The present invention further provides a recombinant DNA molecule that comprises a recombinant nucleic acid that contains an LCE of the present invention and a coding sequence operatively linked to the recombinant nucleic acid. In a preferred embodiment of this type t
Altmann Curtis R.
Chow Robert L.
Hemmati-Brivanlou Ali
Lang Richard A.
Williams Sonya
Clark Deborah J. R.
Paras, Jr. Peter
The Rockefeller University
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