Selenoproteins, coding sequences and methods

Details Selenoproteins, coding sequences and methods Selenoproteins, coding sequences and methods

1996-07-12

2001-10-16

Houtteman, Scott W. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C530S400000, C530S350000, C536S023100, C536S023740

Reexamination Certificate

active

06303295

ABSTRACT:

FIELD OF THE INVENTION
The field of this invention is in the area of molecular biology, in particular, the area of coding sequences, nucleic acid secondary structure and gene expression and its regulation, and more specifically, with reference to proteins containing selenocysteine, sequences encoding proteins containing selenocysteine, and methods for identifying coding sequences for selenocysteine-containing proteins.
BACKGROUND OF THE INVENTION
Selenium (Se) is an essential micronutrient that is now known to be incorporated as selenocysteine in a number of selenoproteins, glutathione peroxidase (GPx) being the prototypical example. Selenocysteine is specifically encoded by the UGA codon, and inserted in peptide chains by a cotranslational mechanism that is able to override the normal function of UGA as a termination codon. In eukaryotes, efficient selenocysteine incorporation at UGA codons requires a cellular protein factor and a cis-acting structural signal usually located in the mRNA 3′-untranslated region (3′-UTR), consisting of a selenocysteine insertion sequence (SECIS) in a characteristic stem-loop structure [23,24]. The required protein factor is presumed to be present in certain cells types that express selenoproteins, such as liver cells, lymphocytes, macrophages, thrombocytes, and other blood cells. In such cell types, the presence of a SECIS element in an mRNA is necessary and sufficient for in-frame UGA codons to be translated as selenocysteine.
Dietary Se is critical for proper immune function, and a number of immunomodulatory effects of Se have been documented [Turner and Finch (1991)
Proc. Nutr. Soc.
50, 275-285]. Se supplementation increases immunoglobin G synthesis, increased chemotactic responses in neutrophils, and enhancement of both T cell cytotoxicity and proliferation in response to mitogens and antigens [Dhur et al.
Comp. Biochem. Physiol.
C. 96, 271-280 (1990), 28, 5, 29, 6 and 7]. Impairment of these immune functions can include reduced T cell counts, including reduced CD4+ T cell counts [4, 7, 16], and impaired lymphocyte proliferation and responsiveness [Dhur (1990) supra, Roy et al.
Proc. Soc. Exp. Biol. Med.
193, 143-148 (1990); 3-7]. These immunological effects are in addition to various specific disorders that have been associated with Se deficiency [8]. There is a progressive decline in plasma Se and Glutathione peroxidase in ARC and AIDS patients [9-17]. This decline approximately parallels T cell loss or stage of HIV infection, but seems to be particularly noticeable in the terminal stages of AIDS, where Se deficiency is one of the symptoms of the disease.
Over half of plasma Se is in the form of selenoprotein P; its mRNA has 10 UGA selenocysteine (SeC) codons, mostly concentrated in the C-terminal 125 amino acids. It has been suggested to serve as an antioxidant, a Se transport/storage protein, and it attaches to various cell types via a specific receptor. Another mammalian selenoprotein is the type I 5′-iodothyronine deiodinase involved in conversion of T4 thyroid hormone to T3. Se is critical in mammals, including humans, for the maintenance of glutathione-dependent antioxidant status and thyroid T3 hormone levels.
Viruses, and to a lesser extent bacteria, are under a powerful constraint to maximize the information content of their genomes. Some microorganisms have evolved mechanisms to maximize the number of genes and amount of protein coding information in a given length of nucleic acid. For example, by placing overlapping genes in different reading frames of the same nucleotide sequence, coding density can be increased. Another maximization mechanism exploits RNA splicing, a characteristic feature of eukaryotic genes, and possibly a trick that viruses incorporated from their hosts. Alternative RNA splicing allows the modular construction of different proteins containing a common module, avoiding duplication of precious genetic material.
Ribosomal frameshifting can result in the same sort of modular construction, for example in a −1 frameshift, by placing alternate 3′ “exons” (modules) in two different reading frames of the same oligonucleotide; when expressed, each is attached to a common module encoded in the 5′-region of the zero reading frame. Multiple frameshift mechanisms, although likely of very low probability, can result in other kinds of modular constructs.
Both overlapping genes and RNA splicing also present new opportunities for various forms of genetic regulation, beyond that attainable by transcriptional regulation alone. The regulation of RNA splicing, along with cotranslational mechanisms like frameshifting and termination suppression enable a more sophisticated yet economical control over the expression of a greater variety of gene products than that attainable with a simple linear arrangement of non-overlapping genes. Retroviruses, for example, utilize a number of elaborate transcriptional and translational control mechanisms in order to influence not only the timing of gene expression, but also to precisely balance the relative quantities of their various gene products. The latter is necessary because structural proteins (products of the retroviral gag and env genes) are needed in much greater quantity than the products of the pol gene, which encodes the viral enzymes protease, reverse transcriptase (RT), and integrase.
Understanding the mechanisms of gene regulation and the extent to which higher organisms, viruses and microorganisms use them, is important in understanding the mechanisms of infectious disease, especially those mechanisms associated with viral and retroviral agents, and those mechanisms associated with the regulation of gene expression in other organisms, including mammals, especially with respect to cancer. The present methods have broad utility in understanding gene regulation, thereby allowing control over gene expression by exploiting the control mechanisms via genetic engineering and/or gene therapy.
SUMMARY OF THE INVENTION
A general method for finding and isolating potential new coding sequences is provided. The method involves an analysis of RNA structure in relation to potential novel open reading frames (ORFs), including those containing UGA codons, the most leaky termination codon. Viral genomic or mRNA, as well as mammalian coding sequences, can be analyzed. UGA termination can be suppressed either by opal suppressor tRNAs (readthrough suppression) with insertion of a conventional amino acid, or by insertion of selenocysteine, under the cotranslational control of specialized RNA structural elements. This analysis reveals locations of potential frameshift sites, and thus novel coding sequences, genes or gene variants, encoded in ORFs that could not otherwise be expressed, because they lack initiation codons. Alternatively, translational readthrough can result in the synthesis of proteins of unexpected sequences. In general, the information thus obtained can be employed to generate predicted amino acid sequences for proteins encoded by these potential ORFs and to prepare nucleic acid constructs to express these proteins in in vitro translation/expression systems or in vivo in appropriately chosen genetically engineered organisms or cell lines. Application of this method has located a number of previously undetected ORFs that encode novel proteins, including selenoproteins as well as previously undetected ORFs which do not contain selenocysteine. The proteins encoded by the newly identified ORFs can then be examined by procedures well-known in the art for functional similarities to known proteins and tested for that functionality. This method is particularly useful in finding and isolating proteins and the ORFs encoding them which have regulatory function in their natural environment, for example, in the transcriptional or translational regulation of known genes and most particularly in the transcriptional or translational regulation of the known gene genes in which the ORF i

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