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The current focus of many functional genomic studies is on the elucidation of gene regulatory networks. The functional analyses of transcription factors and their DNA-binding sites, in conjunction with genome-wide expression profiling, are crucial in understanding of gene regulatory networks. This paper describes an efficient and easy method for characterizing the DNA-binding sequence specificity of novel plant transcription factors. This new method is based on the fusion of a DNA-binding protein (DBP) to 6×His-tagged cellulase D (CELD), which serves both as a means for affinity purification of DBP-DNA complex in the selection of binding sites from a pool of biotinylated random-sequence oligonucleotides and as a reporter for measurement of DNA-binding activity. Thus, it eliminates the use of radioactivity and gel electrophoresis techniques currently used for purification of DBP-DNA complexes and assays of DNA-binding activity. The effectiveness of this method was demonstrated by the success of simultaneous selection of the binding sites of nine plant DBPs from four superfamilies (AP2, bHLH, NAC and MYB). The high-throughput capacity of CELD-based DNA-binding assays allows the quantitative analysis of the binding sequence specificity from a large number of DBP-selected oligonucleotides. The binding sequence specificity of three novel transcription factors (rice OsbHLH66, wheat TaNAC69 and TaMYB80), determined with this method, is presented. This new method provides the capacity of high-throughput analysis on the DNA-binding sequence specificity of a large number of putative transcription factors, predicted on the basis of conserved DNA-binding domains. [ABSTRACT FROM AUTHOR]

Oral Diseases (2012) 18, 147-152 Objective: The Family with sequence similarity 5 member C (FAM5C) has been suggested to contribute in aggressive periodontitis. However, there is no data regarding its role in chronic periodontitis. The aim of this study was to evaluate the FAM5C expression in chronic periodontitis and to study association of FAM5C with key immunoinflammatory markers. Material and Methods: Gingival biopsies were harvested from periodontally healthy subjects (n = 10) and chronic periodontitis subjects (n = 15). The levels of mRNA of FAM5C, interleukin (IL)-17, IL-6, IL-23, IL-10, IL-4, interferon-γ, toll-like receptor (TLR)-2, TLR-4, osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), tumor necrosis factor (TNF)-α, transforming growth factor-β, transcription factor forkhead box p3, and transcription factor orphan nuclear receptor C2 were evaluated by real-time polymerase chain reaction. Results: FAM5C mRNA levels were not different between periodontally healthy and diseased tissues (P > 0.05). Gene expressions of IL-17, TNF-α, OPG, RANKL, TLR-2, and TLR-4 were higher in periodontitis, when compared to periodontally healthy sites (P < 0.05), while no differences between groups were observed for the other genes evaluated (P > 0.05). There were no correlations between the gene expression of FAM5C and the other immunoinflammatory markers ( P > 0.05). Conclusion: Within the limits of this study, it seems that FAM5C expression does not contribute to chronic periodontitis. [ABSTRACT FROM AUTHOR]

A seven-week "gene to protein" laboratory sequence is described for an undergraduate biochemistry laboratory course. Student pairs were given the task of introducing a point mutation of their choosing into the well studied protein, enhanced green fluorescent protein (EGFP). After conducting literature searches, each student group chose the mutation they wanted to introduce into EGFP. Students designed their sequence-specific mutagenic primers and constructed their desired mutation. The resulting EGFP mutant proteins were expressed in E. coil, purified and characterized. This laboratory sequence connected the major concepts of molecular biology and biochemistry, while incorporating the thrill of novel discovery in an undergraduate-level biochemistry laboratory course. [ABSTRACT FROM AUTHOR]

This article discusses the difficulty that students have in understanding molecular genetics. An analytical framework is presented to illustrate the mapping of this education across oniologically distinct levels and a physical level containing hierarchically organized biophysical entities such as proteins, cells and tissues. Studies in the article evaluate the perception that students have of this mapping process, which is far less constrained than the scientific understandings of the genetic information. Also evaluated is the perception that students have in regards to the different functions of proteins, their relationship to genes, and the role proteins have in mediating the effects of the genetic information.

The yeast non­Mendelian factor [ETA+] is lethal in the presence of certain mutations in the SUP35 and SUP45 genes, which code for the translational release factors eRF3 and eRF1, respectively. One such mutation, sup35-2, is now shown to contain a UAG stop codon prior to the essential region of the gene. The non­Mendelian inheritance of [ETA+] is reminiscent of the yeast [PSI+] element, which is due to a self-propagating conformation of Sup35p. Here we show that [ETA+] and [PSI+] share many characteristics. Indeed, like [PSI+], the maintenance of [ETA+] requires the N-terminal region of Sup35p and depends on an appropriate level of the chaperone protein Hsp104. Moreover, [ETA+] can be induced de novo by excess Sup35p, and [ETA+] cells have a weak nonsense suppressor phenotype characteristic of weak [PSI+]. We conclude that [ETA ] is actually a weak, unstable variant of [PSI ]. We find that although some Sup35p aggregates in [ETA+] cells, more Sup35p remains soluble in [ETA+] cells than in isogenic strong [PSI+] cells. Our data suggest that the amount of soluble Sup35p determines the strength of translational non-sense suppression associated with different [PSI+] variants. [ABSTRACT FROM AUTHOR]