7/13/2023 0 Comments Macvector chromatogram![]() When transformed into an mrfA null strain, the unmutated multimer construct was also inactive. Combinatorial mutations in the three MrfA binding sites within the 39-mer inactivated their in vitro binding, but mutation of any single site was sufficient to eliminate reporter expression. Multimerisation of an essential 39 nt distal subfragment of this region, and insertion next to basal promoter elements, generated a reporter construct that directs pstA-specific gene expression ( Senoo et al., 2012). These cells, the pstA cells, comprise the anterior half of the prestalk region and can be recognised by their ability to utilise a cap-site proximal region of the ecmA promoter ( Early et al., 1993). It was identified as the transcription factor that activates expression of a reporter construct which defines a subtype of stalk cell precursors. Dictyostelium MrfA is an orthologue of MRF ( Senoo et al., 2012). Overexpression of myelin gene regulatory factor (MRF), stimulates transcription of CNS myelin genes and mice lacking MRF in the oligodendrocyte lineage are defective in myelination. Recently, however, a transcription factor has been described that is required for the expression of a large number of mouse CNS myelin genes ( Emery et al., 2009). We also present evidence that the auto-proteolysis of MrfA occurs rapidly and constitutively in the ER and that its specific role in prestalk cell differentiation is conferred by the regulated nuclear translocation of the liberated fragment.ĭespite its essential role in vertebrate brain function the process of myelination within the central nervous system (CNS) is poorly understood at the molecular level. Instead, an auto-proteolytic cleavage mechanism, previously only described for the intramolecular chaperone domains of bacteriophage tail-spike proteins, processes MrfA and, by implication, the metazoan MRF proteins. We confirm this for MrfA but report a radically different mode of processing from that of paradigmatic tethered transcriptional regulators, which are cleaved within the transmembrane domain by a dedicated protease. We show that the MRFs contain a predicted transmembrane domain, suggesting that they are synthesised as membrane-tethered proteins that are then proteolytically released. USA 80, 726–730.MrfA, a transcription factor that regulates Dictyostelium prestalk cell differentiation, is an orthologue of the metazoan myelin gene regulatory factor (MRF) proteins. (1983) Rapid similarity searches of nucleic acid and protein databanks. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. (1985) Rapid and sensitive protein similarity searches. (1982) A high speed, high capacity homology matrix zooming through SV40 and polyoma. (1994) Issues in searching molecular sequence databases. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. (1978) Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. (1974) Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. (1984) The codon preference plot graphic analysis of protein coding sequences and prediction of gene expression. Gribskov, M., Devereux, J., and Burgess, R. (1982) Recognition of protein coding regions in DNA sequences. Oxford Molecular, Oxford, England.įickett, J. Oxford Molecular Group (1998) MacVector 6.5 User Guide. This process is experimental and the keywords may be updated as the learning algorithm improves. These keywords were added by machine and not by the authors. MacVector also comes with a module for contig assembly, called AssemblyLIGN. At the time of writing, the version of MacVector available was 6.5. It provides all of the most commonly used nucleic acid and protein analysis tools and also provides access via the Internet to the public Entrez databases at the National Center for Biotechnology Information (NCBI). It is an integrated comprehensive sequence analysis program that runs on the Macintosh. MacVector™, from Oxford Molecular Group, Campbell CA, is one such computer package. It is evident by now that efficiently performing the above operations on thousands or millions of base pairs by hand is so difficult as to be impossible, and computer programs that do sequence analysis are becoming more and more ubiquitous in laboratories practicing molecular biology. Whether a researcher is working on a genome project, or is cloning and characterizing a gene of interest, the ability to manipulate, analyze, and annotate sequence data is becoming increasingly important.
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