Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-01-14
2004-10-05
Canella, Karen A. (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500, C536S024310, C435S252300, C435S254110, C435S320100, C435S325000
Reexamination Certificate
active
06800750
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to molecular biology and medicine and more specifically to Mcl-1 gene regulatory elements, to a variant Mcl-1 polypeptide, the expression of which induces apoptosis of a cell, and to methods of modulating apoptosis of a cell by modulating expression of the variant Mcl-1 polypeptide.
2. Background Information
Growth and development of an organism requires that cell proliferation and cell death be tightly regulated such that the organism develops into a normal, healthy individual. Such regulation ensures that cells are present in the position and at the time they are required, and are removed when their function has been performed or they are no longer necessary or are detrimental to the organism. A mechanism of programmed cell death, also referred to a apoptosis, has evolved and contributes, in part, to the regulation of development by inducing cells to die at the appropriate time. Similarly, various self-renewing tissues in an organism, for example, skin and intestinal epithelium in a mammalian organism, are subject to continual replacement. In order to maintain homeostasis in an organism, programmed cell death acts as a balance to cell proliferation such that the renewing tissue is maintained in a functional form.
It has become clear that dysregulation of apoptosis is involved in various pathological conditions. For example, many cancers are characterized by a defect in the apoptotic process, such that the number of dying cells in a tissue is decreased below its normal level, resulting in an imbalance of cell death and cell proliferation and consequent growth of a tumor. In addition, neurodegenerative diseases are believed to be caused, at least in part, due to the induction of apoptosis, resulting in death of neuronal cells.
In view of the importance of apoptosis to the health and well being of most organisms, including humans, a great deal of effort has been directed to identifying the cellular molecules and pathways involved in the apoptotic process. As a result, several gene products that modulate the apoptotic process have been identified, and can be separated generally into two categories, each of which contains members that can function to either inhibit or induce programmed cell death. One category of gene products that modulate apoptosis includes the members of the Bcl-2 family of proteins. Bcl-2, is the best characterized member of this family and inhibits apoptosis when overexpressed in cells. Other members of this gene family include, for example, Bax, Bak, Mcl-1, Bcl-x
L
, Bcl-x
S
, and Bad. Some of these proteins, including Bcl-2, acts to prevent apoptosis, whereas others such as Bax, Bcl-x
S
and Bak augment apoptosis.
The second category of gene products that modulate the apoptotic process includes the family of aspartate-specific cysteine proteases (ASCPs; caspases). These proteases are related genetically to the Ced-3 gene product that was initially shown to be required for programmed cell death in the roundworm,
C. elegans
. The ASCPs family of proteases includes, for example, human interleukin-1-&bgr; converting enzyme (ICE), ICH-1
L
, ICH-1
S
, CPP32, Mch2, Mch3, ICH-2 and ICE
rel
sup-III. One common feature of these gene products is that they are cysteine proteases with specificity for substrate cleavage at Asp-X bonds. Although these proteases generally induce cell death when expressed in cells, several alternative structural forms such as ICE&dgr;, ICE&egr;, ICH-1
S
and Mch2&bgr; can function to inhibit apoptosis.
In addition to the Bcl-2 and ASCP gene family members that have been identified, it is likely that other as yet unidentified gene products exist that are important in mammalian cell death. For example, in addition to Ced-3, another
C. elegans
gene, Ced-4, exists and also is required for programmed cell death in
C. elegans
. However, a mammalian homolog of this protein has not yet been identified. The physiological control mechanisms that regulate programmed cell death and the mechanisms by which the cell death pathways interact with other physiological processes within the organism also have not yet been fully defined. The identification of genes and gene products involved in the regulation of apoptosis can provide a means to modulate the apoptotic process in cells, thus allowing the development, for example, of methods for intervening in pathological conditions associated with abnormally increased or decreased levels of apoptosis, including during the growth, development and differentiation of cells in an organism. Thus, a need exists to identify genes and gene products involved in the apoptotic process in an organism. The present invention satisfies this need and provides additional advantages.
SUMMARY OF THE INVENTION
The present invention relates to substantially pure nucleotide sequences that act as regulatory elements for expression of an Mcl-1 gene. An Mcl-1 gene regulatory element includes at least about twenty contiguous nucleotides of the nucleotide sequence set forth as nucleotides 1495 to 1657 of SEQ ID NO: 1 (positions −162 to +1 of the Mcl-1 gene 5′-flanking sequence as shown in FIG.
3
B). Mcl-1 gene regulatory elements are exemplified herein by a nucleotide sequence that includes nucleotides 1513 to 1564 of SEQ ID NO: 1 (positions −135 to −92 of 3B), as well as by a nucleotide sequence that includes nucleotides 1495 to 1550 of SEQ ID NO: 1 (positions −162 to −107 of FIG.
3
B); nucleotides 1495 to 1564 of SEQ ID NO: 1 (positions −162 to −92); nucleotides 1495 to 1606 of SEQ ID NO: 1 (positions −62 to −51); nucleotides 1513 to 1550 of SEQ ID NO: 1 (positions −135 to −107); nucleotides 1513 to 1606 of SEQ ID NO: 1 (positions −135 to −51); nucleotides 1550 to 1657 of SEQ ID NO: 1 (positions −107 to +1); and nucleotides 1606 to 1657 of SEQ ID NO: 1 (positions −51 to +1). A particularly useful Mcl-1 gene regulatory element is exemplified by the sequence shown as nucleotides 1495 to 1657 in SEQ ID NO: 1, which correspond to position −162 to +1 in FIG.
3
B. The present invention also relates to a vector that contains an Mcl-1 gene regulatory element as exemplified above. The vector can be an expression vector, and can contain a heterologous nucleic acid molecule operatively linked to the Mcl-1 gene regulatory element. A host cell containing such a vector also is contemplated.
The present invention also relates to a substantially pure Mcl-1 gene, which is a nucleic acid molecule that encodes an Mcl-1 polypeptide, and includes nucleotides 1727 to 3884 of SEQ ID NO: 1 (see, also, FIG.
1
). Additional nucleic acid molecules of the invention are exemplified by a nucleic acid molecule containing nucleotides 1657 to 3884 of SEQ ID NO: 1; or containing nucleotides 1495 to 3884 of SEQ ID NO: 1; or containing 1 to 8253 of SEQ ID NO: 1 (see, also, FIG.
1
). Nucleotide sequences complementary to the disclosed nucleic acid molecules also are contemplated.
The present invention further provides a substantially pure polynucleotide encoding the Mcl-1s/&Dgr;TM amino acid sequence, which is set forth in SEQ ID NO: 3 (see, also,
FIG. 4A
, lower sequence); as well as a nucleotide sequence complementary to such an encoding polynucleotide. A polynucleotide of the invention is exemplified by a polynucleotide containing nucleotides 1727 to 2414 of SEQ ID NO: 1 operatively linked to nucleotides 3768 to 3884 of SEQ ID NO: 1, wherein the linked sequence encodes the polypeptide of SEQ ID NO: 3. A vector, which can be an expression vector, containing such a polynucleotide, which can be a polydeoxyribonucleotide or a polyribonucleotide, also is contemplated, as are host cells that contain such a vector.
In addition, the present invention relates to oligonucleotides, which contain at least ten contiguous nucleotides of SEQ ID NO: 1 and can hybridize specifically either to a splice junction of the disclosed Mcl-1 gene or to a polynucleotide en
Bingle Colin D.
Craig Ruth W.
Whyte Moira
Canella Karen A.
Dartmouth College
Gray Cary Ware & Freidenrich LLP
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