Identification in Regulation and Gene Expression

Molecular biology and the equipment available for research have rapidly increased the sequencing of large portions of the genomes of different species. Currently several bacterial genomes, as well as those of some simple eukaryotes (e.g., Saccharomyces cerevisiae, or baker’s yeast) and more complex eukaryotes (C. elegans and Drosophila) have been sequenced in full. Various online sites and databases have aided bioinformaticians to do this [1]. The speed in which the Human Genome Project was carried out is truly amazing and can only depict on what is yet to come

Various famous sequence databases, such as GenBank and EMBL, NCBI have been growing at exponential rates. The information present is these data base is enormous. This deluge of information has necessitated the careful storage, organization and indexing of sequence information. Information science has been applied to biology to produce the field called bioinformatics. Internal ribosome entry sites (IRESes) of viral mRNAs comprise several hundred nucleotides.

These nucleotides are highly structured, there is of proof indicating that very short nucleotide sequences, both naturally occurring and synthetic, can similarly mediate internal initiation of translation. In this study, they performed deletion and mutational analyses of IRES contained within the 720-nucleotide (nt) 5′ leader of the Rbm3 mRNA and demonstrated that this IRES is highly modular, with at least 9 discrete cis-acting sequences. The different cis-acting sequences are included a 22-nt IRES module, a 10-nt enhancer, and 2 inhibitory sequences. This 22-nt sequence could function as an IRES when tested in isolation, and was demonstrated that it did not enhance translation by functioning as a transcriptional promoter, enhancer, or splice site [9].

In all there were 4 main cis-acting sequences which were further confirmed by their mutation in the context of the full IRES. But in this experiment, one of the inhibitory cis-acting sequences was contained within an upstream open reading frame (uORF), because of this containment the activity was probably masked by translation of this uORF. Further binding studies revealed that all 4 cis-acting sequences could bind specifically to distinct cytoplasmic proteins.

Not only this but also the 22-nt IRES module was shown to bind specifically to 40 S ribosomal subunits. My research is not on cis acting sequences but on the sequence of IRES in Menduca sexta mid-gut. The result seen in this experiment carried out show that different types of cis-acting sequences mediate or adjust translation of the Rbm3 mRNA. Such an outcome suggests that one of the IRES modules contained within the 5′ leader facilitates translation initiation. This is done by basically binding directly to 40 S ribosomal subunits. Hence it can be seen that here as well IRES plays an important role and can be used in such situations.

4.2 Further to this study, the optimal use of IRES in expression vectors was also done [10]. IT was seen that in the second cistron constructed bicistronic mRNAs is generally not translated unless special sequence named IRES is added. The addition of this IRES (Internal ribosomal site) is mandatory for expression. This is seen in higher eukaryotes where you do not find natural bicistronic mRNAs. Ribosome’s generally are cap dependent and cannot be recruited without 5′ UTR [11]. But in this case it was seen that ribosome was conscripted independently. This report deals with IRES found in HTLV-1 genome.

A brief description of this is mentioned in this report. Study revealed that this IRE along with IRES seen in polio virus and encephalomyocarditis virus works optimally when they are added about 100 nucleotides after the termination codon of the first cistron. One factor that can be critically evaluated in this experiment that was carried out is that these IRES when added after 300 nucleotides up to 500 nucleotides spaces became totally inefficient. These results do not match or coincide with the standard admitted results that are seen otherwise. They are potent translator simulators. Their effects are emphasized in cells in which the normal mechanism of translation initiation is inhibited.

My project deals with the expression of IRES. I have to yet find out how this expression is carried out or how this expression is spotted. 4.3 In this study, the molecular mechanism of IRES mediated initiation and how they are actually used under certain physiological conditions by specific mRNAs for translation is explained [12]. It is stated under mitosis, apoptosis, hypoxia and some other viral infections how translation occurs or is repressed. In most eukaryotic mRNAs initiation of translation commences with 5′ end-dependent recruitment of 40S ribosomal subunits to the mRNA. The mRNA is thought to be scanned in the 5′ to 3′ direction by certain initiation factors nd 40S subunit carrying initator methionine-tRNA. The scan through the 5′ to 3′ direction until an appropriate start codon is encountered at which stage a 60S subunit joins to form an 80S ribosome that can decode the RNA into protein (Kozak 1989; Hershey and Merrick 2000).

IRESs are a small subset of mRNAs, usually in the 5′ NTR, that enable end-independent initiation to occur. IRES-containing mRNAs are not subjected to many of the regulatory mechanisms that control recruitment of most mRNAs to the translation apparatus. Different mechanisms are explained for instance; The canonical scanning mechanism of translation initiation, Internal initiation, Experimental test for IRES activity.

Recently there are discoveries on IRES elements viral as well as cellular RNAs. Not all the discoveries are yet published but work on these element is being carried out. There is certain factor that must be applied to an RNA sequence to be termed IRES. The study states that the first, important factor is, the integrity of the translated dicistronic mRNA, which contains a suspected IRES element in the intergenic region, has to be examined to evaluate other possible reasons for translation of the downstream cistron. Care has to be taken if the enzymatic assays are used to monitor translation. The reason for doing this is that small amounts of broken dicistronic RNAs could yield functionally monocistronic RNA which in turn could translated into an active enzyme [13].

The second factor is IRES-mediated translation of the second cistron, that must be independent of translation of the first cistron. It’s important so that the mechanism of termination-reinitiation is ruled out. A particular convincing demonstration of IRES-mediated initiation involves the insertion of a suspected IRES into a circularized RNA, engineered to contain a single continuous open reading frame, so that multiple rounds of translation of the circle can be used as a measure of its integrity (Chen and Sarnow 1995).

4.4 One more literature on a cDNA clone encoding a subunit of the tobacco hornworm Manduca sexta (Ms) hemolymph (serum) ferritin (Fer) has been identified and sequenced [14]. This is in relation to my project as this study is done on Manduca sexta from which I have picked up the IRES element. In this experiment if we construe the amino acid sequence. It shows approximately 50% similarity to vertebrate Fer subunit sequences, and the nucleotide sequence contains a stem-loop structure in the 5′ untranslated region that could serve as an iron-responsive element (IRE). There is a system in which the IRE through the stem-loop which exhibits high identity to vertebrate IRE that play an essential role in the control of Fer synthesis.

My work is not on the Fer synthesis but it revolves round the element. This (Ms) Fer subunit lacks one of the three Tyr residues required for the rapid biomineralization of iron shown in vertebrate heavy-chain Fer. The ferroxidase comprised by the respective amino acid is not conserved, suggesting that the Ms Fer subunit more closely resembles the vertebrate light-chain subunit. A new technique called the Northern blot analyses indicate that the fer mRNA is expressed in the midgut, fat body and hemocytes, with the greatest expression in the midgut.

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