Expressed sequence tags and encoded human proteins

Details Expressed sequence tags and encoded human proteins Expressed sequence tags and encoded human proteins

2000-02-24

2004-08-31

Martinell, James (Department: 1631)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C536S023100

Reexamination Certificate

active

06783961

ABSTRACT:

BACKGROUND OF THE INVENTION
The estimated 50,000-100,000 genes scattered along the human chromosomes offer tremendous promise for the understanding, diagnosis, and treatment of human diseases. In addition, probes capable of specifically hybridizing to loci distributed throughout the human genome find applications in the construction of high resolution chromosome maps and in the identification of individuals.
In the past, the characterization of even a single human gene was a painstaking process, requiring years of effort. Recent developments in the areas of cloning vectors, DNA sequencing, and computer technology have merged to greatly accelerate the rate at which human genes can be isolated, sequenced, mapped, and characterized. Cloning vectors such as yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) are able to accept DNA inserts ranging from 300 to 1000 kilobases (cb) or 100-400 kb in length respectively, thereby facilitating the manipulation and ordering of DNA sequences distributed over great distances on the human chromosomes. Automated DNA sequencing machines permit the rapid sequencing of human genes. Bioinformatics software enables the comparison of nucleic acid and protein sequences, thereby assisting in the characterization of human gene products.
Currently, two different approaches are being pursued for identifying and characterizing the genes distributed along the human genome. In one approach, large fragments of genomic DNA are isolated, cloned, and sequenced. Potential open reading frames in these genomic sequences are identified using bioinformatics software. However, this approach entails sequencing large stretches of human DNA which do not encode proteins in order to find the protein encoding sequences scattered throughout the genome. In addition to requiring extensive sequencing, the bioinformatics software may mischaracterize the genomic sequences obtained. Thus, the software may produce false positives in which non-coding DNA is mischaracterized as coding DNA or false negatives in which coding DNA is mislabeled as non-coding DNA.
An alternative approach takes a more direct route to identifying and characterizing human genes. In this approach, complementary DNAs (cDNAs) are synthesized from isolated messenger RNAs (mRNAs) which encode human proteins. Using this approach, sequencing is only performed on DNA which is derived from protein coding portions of the genome. Often, only short stretches of the cDNAs are sequenced to obtain sequences called expressed sequence tags (ESTs). The ESTs may then be used to isolate or purify extended cDNAs which include sequences adjacent to the EST sequences. The extended cDNAs may contain all of the sequence of the EST which was used to obtain them or only a portion of the sequence of the EST which was used to obtain them. In addition, the extended cDNAs may contain the full coding sequence of the gene from which the EST was derived or, alternatively, the extended cDNAs may include portions of the coding sequence of the gene from which the EST was derived. It will be appreciated that there may be several extended cDNAs which include the EST sequence as a result of alternate splicing or the activity of alternative promoters. Alternatively, ESTs having partially overlapping sequences may be identified and contigs comprising the consensus sequences of the overlapping ESTs may be identified.
In the past, these short EST sequences were often obtained from oligo-dT primed cDNA libraries. Accordingly, they mainly corresponded to the 3′ untranslated region of the mRNA. In part, the prevalence of EST sequences derived from the 3′ end of the mRNA is a result of the fact that typical techniques for obtaining cDNAs, are not well suited for isolating cDNA sequences derived from the 5′ ends of mRNAs. (Adams et al.,
Nature
377:3-174, 1996, Hillier et al.,
Genome Res
. 6:807-828, 1996).
In addition, in those reported instances where longer cDNA sequences have been obtained, the reported sequences typically correspond to coding sequences and do not include the full 5′ untranslated region (5′ UTR) of the mRNA from which the cDNA is derived. 5′ UTRs are often involved in the regulation of gene expression, by affecting either the stability or translation of mRNAs. Indeed, 5′ UTRs may contain several features known to affect the initiation of translation: (i) the distance between the cap structure and the initiation codon, (ii) the presence of cis-acting elements which may be either linear sequences such as polypyrimidine tracts (Kaspar et al,
J. Biol. Chem
. 267, 508-514, 1992; Severson et al.,
Eur J Biochem
229:426-32, 1995) or secondary structures such as IREs (Rouault and Klausner,
Curr Top Cell Regul
35:1-19, 1997), and (iii) upstream open reading fraes or uORFs (Geballe and Morris,
Trends Biotech Sci
19:159-64, 1994). Thus, regulation of gene expression may be achieved through the use of alternative 5′ UTRs. For instance, the translation of the tissue inhibitor of metalloprotease mRNA is enhanced in mitogenically activated cells through modification of the start codon of an uORF in its 5′ UTR using an alternative promoter (Waterhouse et al,
J Biol Chem
. 265:5585-9. 1990). Furthermore, modification of 5′ UTR through mutation, insertion or translocation events may even be implied in pathogenesis. For instance, the fragile X syndrome, the most common cause of inherited mental retardation, is partly due to an insertion of multiple CGG trinucleotide's in the 5′ UTR of the fragile X mRNA resulting in the inhibition of protein synthesis via ribosome stalling (Feng et al,
Science
268:731-4, 1995). An aberrant mutation in regions of the 5′ UTR known to inhibit translation of the proto-oncogene c-myc was shown to result in upregulation of C-myc protein levels in cells derived from patients with multiple myelomas (Willis et al,
Curr Top Microbiol Immunol
224:269-76, 1997). However, the use of oligo-dT primed cDNA libraries does not allow the isolation of complete 5′ UTRs since such obtained incomplete sequences may not include the first exon of the mRNA, particularly in situations where the first exon is short. Furthermore, they may not include some exons, often short ones, which are located upstream of splicing sites. Thus, there is a need to obtain sequences derived from the 5′ ends of mRNAs.
While many sequences derived from human chromosomes have practical applications, approaches based on the identification and characterization of those chromosomal sequences which encode a protein product are particularly relevant to diagnostic and therapeutic uses. In some instances, the sequences used in such therapeutic or diagnostic techniques may be sequences which encode proteins which are secreted from the cell in which they are synthesized, as well as the secreted proteins themselves, are particularly valuable as potential therapeutic agents. Such proteins are often involved in cell to cell communication and may be responsible for producing a clinically relevant response in their target cells. In fact, several secretory proteins, including tissue plasminogen activator, G-CSF, GM-CSF, erythropoietin, human growth hormone, insulin, interferon-&agr;, interferon-&bgr;, interferon-&ggr;, and interleukin-2, are currently in clinical use. These proteins are used to treat a wide range of conditions, including acute myocardial infarction, acute ischemic stroke, anemia, diabetes, growth hormone deficiency, hepatitis, kidney carcinoma, chemotherapy-induced neutropenia and multiple sclerosis. For these reasons, extended cDNAs encoding secreted proteins or portions thereof represent a valuable source of therapeutic agents. Thus, there is a need for the identification and characterization of secreted proteins and the nucleic acids encoding them.
In addition to being therapeutically useful themselves, secretory proteins include short peptides, called signal peptides, at their amino termini which direct their secretion. These signal

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