Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-06-04
2002-09-10
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Vector, per se
C435S183000, C435S069100, C435S440000, C536S023100, C536S023200, C536S023500
Reexamination Certificate
active
06448072
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates in general to the field of recombinant nucleic acids, polypeptides and other derived materials and, more particularly, to the identification, isolation and characterization of human transcription factors that are involved in the expression of human genes.
BACKGROUND OF THE INVENTION
Without limiting the scope of the invention, its background is described in connection with the isolation, characterization and use of human transcription factors that are expressed throughout the organism, as an example.
Unlike the nucleic acid polymerases of prokaryotes, purified RNA polymerase II from eukaryotes initiates transcription very poorly and essentially at random. One key difference between prokaryotic and eukaryotic polymerases is the need for accessory factors that provide for the accurate initiation of transcription. These factors are referred to as the “general” or “basal” transcription factors, in that they are required, in addition to RNA polymerase II, for the transcription of all eukaryotic protein coding genes. As such, the general transcription factors are expected to be active, or at least present, in all or most tissues. One such general factor is called transcription factor IID (TFIID) and is responsible in large part for promoter recognition. Other general transcription factors include TFIIA, TFIIB, TFIIE, TFIIF and TFIIH.
Appropriate levels of gene- and tissue-specific transcription is achieved by another set of factors called activator proteins. These factors are often composed of two domains, a sequence-specific DNA recognition domain and an activation domain. When bound to DNA, the activation domain facilitates the formation and function of a preinitiation complex that consists of the general transcription factors and RNA polymerase II. In this way it is possible to direct the selective transcription of genes in an appropriately regulated fashion.
The structure of a typical promoter for a eukaryotic gene consists of two general regions. The core promoter is located at or near the actual site of transcription initiation and often includes a TATA sequence element located at about 30 base pairs upstream of the initiation site. The other regions are defined as sequence elements which are recognized by activator proteins. These are often located at various distances further upstream, but may be also be located downstream relative to the core promoter of the gene being regulated. Interactions between bound regulatory factors and the preinitiation complex are responsible for the precisely regulated transcription of each individual gene.
TFIIA is an essential general transcription factor and the purified factor from higher eukaryotes consists of three subunits, designated alpha (35 kD), beta (19 kD) and gamma (12 kD). In humans, the alpha and beta subunits are encoded by DNA sequences present in the TFIIA&agr;/&bgr; cDNA, sometimes referred to as the ‘large’ subunit cDNA. These two subunits are post-translationally processed from a large 55 kD product of TFIIA&agr;/&bgr;. The gamma subunit is encoded by DNA sequences present in the TFIIA&ggr; cDNA, sometimes referred to as the ‘small’ subunit cDNA. This sequence is the subject of U.S. Pat. No. 5,562,117 issued to Moore and Rosen. TFIIA has multiple roles in transcription initiation by RNA polymerase II, including an ability to stabilize TBP-TATA element interactions, displace TBP-associated repressors and serve as a cofactor during the processes of transcription activation.
Most of the known human general transcription factors appear to be generally required in all tissues for gene expression by RNA polymerase II. Thus, these factors will be important as markers to evaluate disease states which may arise from inappropriately regulated gene expression and as pharmacological reagents and/or targets with which to modulate patterns of gene expression. Similarly, overexpression via gene therapy or other means should have broad effects on the expression of many or all cellular genes. In contrast, mutations in the genes for activator proteins, which are normally observed to control expression of a select set of genes, often in a tissue or developmentally restricted pattern, typically result in specific defects. Likewise, overexpression of activator proteins only affects expression of cellular genes which contain cognate recognition sequences.
Testis has important endocrine (hormonal) functions and is the site for the production of haploid spermatozoa from undifferentiated stem cells, a process called spermatogenesis. Mutations in some specialized transcriptional activator proteins, such as A-myb and CREM, cause male infertility and show defects in spermatogenesis. The identification of tissue-specific human general transcription factor would bridge an important gap between the generality for general transcription factor function and the specificity of gene-specific transcriptional activator protein function. If such factors were testis-specific, they would be expected to regulate patterns of gene expression that are important in the endocrine, spermatogenic and other functions of this organ. The present invention satisfies a need in the art for new compositions for polynucleotide sequences and encoded polypeptide products, immunological reagents and other derived materials in terms of providing unique reagents for the detection of defects in testis function such as idiopathic male infertility or other syndromes, for detection of dysfunctional patterns of gene expression and as reagents that can modulate gene expression.
SUMMARY OF THE INVENTION
The present invention includes DNA sequences that encode two structurally distinct isoforms of the human general transcription factor TFIIA &agr;/&bgr;. One of these sequences is denoted as ALF, for TFIIA &agr;/&bgr;-like factor, which is expressed predominantly in human testis. The second sequence contains ALF connected to a unique upstream sequence and is denoted as SALF, for Stoned B/TFIIA &agr;/&bgr;-like factor. The present invention is also direct to recombinant polypeptide products and other derived materials. The uses of the invention include, but are not necessarily limited to, the propagation and preparation of the ALF and SALF DNA, RNA and recombinant proteins, and use of these materials as reagents and markers to detect and/or modify the function of eukaryotic cells in normal and disease states.
The present invention may be used in the detection of the endogenous ALF and SALF RNAs in eukaryotic cells using hybridization, polymerase chain reactions, immunological analysis and other methods. The invention may also be used along with the endogenous ALF and SALF DNAs, RNAs and proteins as specific in vivo pharmacological targets to artificially modulate the expression of eukaryotic genes. Furthermore, the ALF, SALF and the variable carboxyl terminal end may be introduced in a normal or modified versions of the ALF and SALF genes for expression in eukaryotic cells in order to replace or augment endogenous transcription factor activities (gene therapy). The present invention may also be used as testis-specific antigens for contraceptive vaccine development.
The present invention, in a general and overall sense, concerns the isolation and characterization of a novel transcriptional factor gene, ALF and carboxy terminal variable region. One embodiment of the present invention is a purified nucleic acid segment that encodes a protein having an amino acid sequence as shown in
FIG. 2
, in accordance with SEQ ID NO.:2. Another embodiment of the present invention is a purified nucleic acid 25 segment, further defined as including a nucleotide sequence in accordance with SEQ ID NO.:1.
The present invention also concerns the isolation and characterization of a novel transcriptional factor gene, SALF and a carboxy terminal variable region. One embodiment of the present invention is a purified nucleic acid segment that encodes a protein having an amino acid sequence as shown in
FIG. 3
, in accordance with SEQ ID NO.:4. Another embod
Board of Regents , The University of Texas System
Flores Edwin S.
Gardere Wynne & Sewell LLP
Hutson Richard
Prouty Rebecca E.
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