Approaches to Gene Identification

FASTAI BLAST I PSI-BLAST I SSEARCH I S W SEARCH b DD8J Vector Screening System hew 

The DNA secondary databases offer analytical results (e.g. gene motifs, splice sties, transcription regulators) derived form the primary databases of INSDC, some of which are listed in Table 15.8. 

All primary sequence databases provide tools for essential sequence analyses. Many servers are also available on the web to perform useful computation on DNA/RNA sequences and structures. These web servers (Table 15.9) provide an array of diverse computational genomic tools. 

Genomics conducts stractural and functional studies of genomes (McKusick, 1997 Shapiro and Harris, 2000 Starkey and Elaswarapu, 2001). The former deals with the determination of DNA sequences and gene mapping, while the latter is concerned with the attachment of functional information to existing structural knowledge about DNA sequences. 

5S rRNA http //biobase.ibch.poznan.pl/5Sdata/ 5S rRNA sequences 

The basic information flow for an overall gene identification protocol follows  

Promoters. The promoter is an information-rich signal that regulates transcription, especially for eukaryotes. Computer recognition of promoters (Fickett and Hatzigeorgiou, 1997) is important partly for the advance it may provide in gene identification. Available programs include those depending primarily on simple 

Translation Initiation Site. In eukaryotes, if the transcription start site is known, and there is no intron interrupting the 5 UTR, Kozak s rule (Kozak, 1996) probably will locate the correct initiation codon in most cases. Splicing is normally absent in prokaryotes, yet because of the existence of multicitronic operons, promoter location is not the key information. Rather, the key is reliable localization of the ribosome binding site. The TATA sequence about 30 bp from the transcription start site may be used as a possible resource. 

Termination Signals. The polyadenylation and translation termination signals also help to demarcate the extent of a gene (Fickett and Hatzigeorgiou, 1997). 

---

A sequence-based approach to the identification of differentially expressed genes through comparative analysis. Allows simultaneous analysis of sequences that derive from different cell populations or tissues. This is not a chip-based method. Identification of sequences relies on completeness of public sequence databases and, therefore, can only be used to analyse known genes. 

A number of investigators have taken a genetic approach to the identification of genes and proteins involved in auxin response, particularly in Arabidopsis [47]. The sensitivity of Arabidopsis seedlings to low concentrations of exogenous auxin has been the basis for extensive screens for auxin response mutants. At least nine genes have been defined in these screens and four have been characterized at the molecular level (Table 1). The product of the AUXl gene probably functions in cellular auxin influx and will not be discussed further here [56]. 

Genome mapping of sites implicated in DNA-protein interaction. Specific DNA-protein interactions play a key role in gene expression and DNA replication. Methylation provides an important approach to the identification and characterization of the DNA sequences involved in such interactions. The rationale is that endogenous or artificially introduced MTases methylate all genomic targets except... 

A scientific procedure that turns the classical pharmacology approach upside down. Instead of finding the elusive receptor for a known hormone or transmitter what classical pharmacology aims at, reverse pharmacology is initiated through the discovery of the receptor gene and aspires to the identification of the receptors unknown ligand. 

Cell-free translation system, used for the identification of cloned genes and gene expression, has been investigated extensively as a preparative production system of commercially interesting proteins after the development of continuous-flow cell-free translation system. Many efforts have been devoted to improve the productivity of cell-free system [1], but the relatively low productivity of cell-free translation system still limits its potential as an alternative to the protein production using recombinant cells. One approach to enhance the translational efficiency is to use a condensed cell-free translation extract. However, simple addition of a condensed extract to a continuous-flow cell-free system equipped with an ultrafiltration membrane can cause fouling. Therefore, it needs to be developed a selective condensation of cell-free extract for the improvement of translational efficiency without fouling problem. 

Expressed sequence tag (EST) analysis of cDNAs from specific plant tissues has proved to be a valuable tool for the identification of genes for secondary metabolite biosynthesis.36 We have used this approach to identify two distinct sequences predicted to encode OSCs from cDNA libraries from roots of diploid oat (Avena strigosa).35 One of these sequences is highly homologous to cycloartenol... 

SEQUENCE-BASED APPROACHES TO ALKALOID BIOSYNTHESIS GENE IDENTIFICATION... 

---