Nucleotide correlation with protein

A simple way of evaluating the affect of a single nucleotide polymorphism on enzyme function is to perform transient transfection assays in COS-1 cells. Western blot analysis, with anti-UGTl A antibody, is used to determine protein expression levels of the variants, which often correlate with protein stability. Enzyme kinetic analysis is used to investigate the functional impact of amino acid substitutions. 

The extent of methylation of a gene is correlated with its ability to transcribe. Given that DNA methylation usually reduces transcription, two important, closely related questions remain unanswered How is methylation regulated in vivo How does methylation interfere with transcription Since methylation is known not to interfere with the elongation phase of RNA synthesis, it seems likely that methylation blocks initiation. The binding of polymerase and other regulatory proteins at the initiation locus is sensitive to modification of these nucleotides. The precise inhibition mechanisms, however, await further elucidation. 

Determining amino acid sequences of proteins by classic procedures is a tedious process. In addition, proteins are often insoluble (e.g., membrane proteins) or cannot be easily purified. However, if the protein s gene, mRNA, or cDNA of the mRNA are available, the amino acid sequence of the protein can be determined from nucleotide sequences rapidly and unambiguously using the universal genetic code. In this code, each amino acid in correlated with one or more nucleotide triplets in mRNA (see Chapter 12). mRNA is "transcribed" from DNA and can be used to synthesize complementary (cDNA) via an enzyme called reverse transcriptase. In many cases it is easier to isolate the mRNA or cDNA of a protein than the protein itself. 

The changes in cAMP induced by glucagon in isolated hepatocytes are well correlated with the changes in the activation state of the protein kinase [58,59]. This is illustrated in Fig. 2. Careful examination of the correlations between the increases in cAMP and cAMP-dependent protein kinase activity induced by very low concentrations of glucagon illustrates some cooperativity in the effect of the nucleotide on the kinase [59] consistent with the synergistic interaction between the... 

For the open reading frames, the nucleotide compositions were also correlated with the optimal growth temperatures. No direct relationship was observed between G-t-C content and temperature. However, as illustrated in Fig. 2, the G+C content of the protein-coding... 

A general outline of the bottom-up mass spectrometry approach for proteome analysis is presented in Figure 3. In general, the mass spectrometry is performed at the peptide level after digesting the protein to obtain the molecular mass and amino acid sequence-specific ions, which are correlated with similar information in the protein or nucleotide database.7 16 Based upon these measurements, the following approaches have evolved. 

Efficient initation of SL RNA synthesis required two sequence elements, one of which was centered approximately 50 nucleotides upstream from the transcriptional start site. Remarkably, the second sequence element was the 22nt SL sequence itself mutations in the SL sequence abolished transcription in vitro. DNase I footprinting showed that the SL sequence bound a protein factor the boundaries of the footprint exactly coincided with the SL sequence. Competition experiments indicated that binding of the factor was directly correlated with transcription (13). Further analysis has indicated that the 22nt binding factor is 60 kDa protein. It will be of considerable interest to see if this factor shares any homology with known transcription factors involved in RNA polymerase II transcription. 

Chiusano M.L., D Onofrio G., Alvarez-Valin F., Jabbari K., Colonna G., Bernard G. (1999). Correlations of nucleotide substitution rates and base composition of mammalian coding sequences with protein structure. Gene 238 23-31. 

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