MRNA sequence

Section 28 11 Three RNAs are involved m gene expression In the transcription phase a strand of messenger RNA (mRNA) is synthesized from a DNA tern plate The four bases A G C and U taken three at a time generate 64 possible combinations called codons These 64 codons comprise the genetic code and code for the 20 ammo acids found m proteins plus start and stop signals The mRNA sequence is translated into a prescribed protein sequence at the ribosomes There small polynucleotides called... 

What is the base sequence in the original DNA strand on which the mRNA sequence 1 in Problem 28.9 was made ... 

Give an mRNA sequence that will code for synthesis of metenkephalin. 

A problem with employment of ASON in a larger clinical setting is their poor uptake and inappropriate intracellular compartmentalization, e.g., sequestration in endosomal or lysosomal complexes. In addition, there is a need for a very careful selection of the ASON-mRNA pair sequences that would most efficiently hybridize. To date, several computer programs are used to predict the secondary and tertiary structures of the target mRNA and, in turn, which of the mRNA sequences are most accessible to the ASON. However, even with this sophisticated techniques, the choice of base-pairing partners still usually includes a component of empiricism. Despite these principal limitations, it has become clear that ASON can penetrate into cells and mediate their specific inhibitory effect of the protein synthesis in various circumstances. 

Recently, the related phenomenon of RNA interference (RNAi) has attracted much attention [5]. RNAi occurs when a short (generally 21 nucleotides in length) double-stranded RNA (dsRNA) catalyticaUy represses the translation of a fully complementary mRNA sequence. The process appears to proceed via a complex formed between the antisense RNA strand and a protein with RNase activity [6]. Upon binding to the target mRNA sequence, the ribonucleoprotein complex initiates cleavage of the mRNA transcript thus preventing translation of intact protein. After dissociation from the truncated mRNAs, the ribonucleoprotein complex is free to act on other intact mRNAs. Such small interfering RNAs (siRNAs) have... 

Dirks RW, van Gijlswijk RP, Tullis RH et al (1990) Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITC- and biotin-labeled oligonucleotides. J Flistochem Cytochem 38 467 173... 

Antisense agents are substances (in this case oligonucleotides) which are intended to prevent illnesses, for example by blocking certain mRNA sequences. 

Genetic code Sequence of nucleotides along the DNA and coded in triplets (codons) along the mRNA that determines the sequence of amino acids in protein synthesis. The DNA sequence of a gene can be used to predict the mRNA sequence, and subsequently to predict the amino acid sequence. 

Chloroplast protein synthesis is controlled largely at the post-transcriptional level [20,21] and can be repressed by the inclusion of antibiotics such as streptomycin in the sprouting medium. Streptomycin binds to the 16S rRNA and causes the ribosome to misread the mRNA sequence, producing incorrect and non-functional proteins [22]. 

An extension of the PCR method is RT-PCR. Here mRNA is extracted from the cells or tissue, converted to cDNA using the enzyme reverse transcriptase and PCR is then carried out This method is used to detect the expression of specific mRNA sequences in cells or tissues. 

Kl. Kawasaki, E. S., Clark, S. S., Coyne, M. Y., Smith, S. D., and McCormick, F. P., Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro. Proc. Natl. Acad. Sci. U.S.A. 85, 5698-5702 (1988). 

We performed a benchmark analysis on a set of genes for which the full mRNA and/or the pertinent protein sequence was documented in the literature. Instead of clustering ESTs without any template, we aligned them by a BLAST search (Altshul et al., 1997) to this set of master mRNA sequences and looked in these alignments for variant letters. We applied a set of filters as follows ... 

ESTs were aligned against the set of about 9,000 human mRNA sequences. Candidate cSNPs were extracted and subjected to various algorithmic quality filters, as described in the text. 

Currently, there is only one antisense drug on the market— Vitravene (active ingredient fomivirsen) for the treatment of cytomegalovirus (CMV)-induced retinitis (inflammation of the retina) in AIDS patients. Fomivirsen has 21 nucleotides complementary to a CMV mRNA sequence, which is necessary for the production of infectious virus. Two examples of experimental antisense drugs are provided in Exhibit 3.15, while Table 3.1 lists other antisense drugs in clinical phase. 

Generally differential display is used to compare levels of gene expression among experimental groups. However, this technique has also been used successfully to reveal mRNA sequence variation between samples. Eor example, differential display demonstrated that a cDNA fragment could be amphfied from samples derived from hippocampi of seizure-resistant but not seizure-prone rats (O Figure 17-2). A smaller... 

Fig. 3. Comparison of the amino acid sequences predicted from the a- and j3-tubulin mRNA sequences. Open circles indicate homologous, conservative substitutions, and boxed regions indicate areas of high homology. For completeness, the sequence of the first 25 amino acids of chick brain a-tubulin, deduced by the work of Luduena and Woodward (1973), is also included. Note that the asterisks indicate identical amino acids common to both polypeptide chain sequences. [Reproduced from Valenzuela et al. (1981). Nature (London) 289, 650-655.]...

Most genetic code tables designate the codons for amino adds as mRNA sequences (Figure 1-4-1). Important features of the genetic code include ... 

Ribozymes are enzymes that, in some cases, can cleave mRNA. A ribozyme is engineered so that it cleaves the mutant mRNA sequence, preventing its translation into a polypeptide. 

A selective method of preventing the expression of adhesion molecules or cytokines is the use of antisense oligonucleotides. These oligonucleotides are short sequences of nucleic acids complementary to mRNA sequences of specific proteins of interest. If delivered to the cytoplasmic compartment of cells these oligonucleotides are able to form a complex with their target mRNA. In this way the translation of mRNA into protein by ribosomes is inhibited. The subsequent mRNA degradation by RNAse H results in reduced expression of the protein (see also Chapter 5 for a description of antisense ohgonucleotides as therapeutic modalities). 

The codon combinations are shown in Table 14.2. A codon can be the DNA sequence in the coding strand or, alternatively, the related sequence found in mRNA. The table shows the mRNA sequences, since we shall be using these during consideration of protein synthesis. The DNA sequences merely have thymine (T) in place of uracil (U), as appropriate. The sequence is always listed from the 5 -end to the 3 -end. 

Off-target mRNAs with homology to target-mRNA Sequence comparison Comparison of siRNA sequences to EST databases (Is the EST expressed in the cell-line of interest )... 

Cross species functionality siRNA target conserved sequence Compare mRNA sequences of, e.g., mouse and hiunan and target 100% conserved motifs... 

---