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COOL Cloning Insertion of short DNA sequences into plasmids is a widely applied technique, but it can be complicated by lack of precision and the need to screen and separate clones in order to iden...

Summary In an effort to understand how an accurate level of stress-specific expression is obtained, we studied the promoter of the yeast HSP104 gene. Through 5¢ deletions, we defined a 334 bp fragment upstream of the first coding AUG as sufficient and essential for maximal basal activity and a 260 bp fragment as sufficient and essential for heat shock responsiveness. These sequences contain heat shock elements (HSEs) and stress response elements (STREs) that cooperate to achieve maximal inducible expression. However, in the absence of one set of factors (e.g. in msn2Dmsn4D cells) proper induction is obtained exclusively through HSEs. We also show that HSP104 is constitutively derepressed in ras2D cells. This derepression is achieved exclusively through activation of STREs, with no role for HSEs. Strikingly, in ras2Dmsn2Dmsn4D cells the HSP104 promoter is also derepressed, but in this strain derepression is mediated through HSEs, showing the flexibility and adaptation of the promoter. Thus, appropriate transcription of HSP104 is usually obtained through cooperation between the Msn2/4/STRE and the HSF/ HSE systems, but each factor could activate the promoter alone, backing up the other. Transcription control of HSP104 is adaptive and robust, ensuring proper expression under extreme conditions and in various mutants.

Critical cellular processes such as DNA replication, DNA damage repair, and transcription are mediated and regulated by DNA-binding proteins. Many efforts have been invested therefore in developing methods that monitor the dynamics of protein-DNA association. As older techniques such as DNA footprinting, and electrophoretic mobility shift assays (EMSA) could be applied mostly in vitro, the development of the chromatin immunoprecipitation (ChIP) method, which allows quantitative measurement of protein-bound DNA most accurately in vivo, revolutionized our capabilities of understanding the mechanisms underlying the aforementioned processes. Furthermore, this powerful tool could be applied at the genomic-scale providing a global picture of the protein-DNA complexes at the entire genome.The procedure is conceptually simple; involves rapid crosslinking of proteins to DNA by the addition of formaldehyde to the culture, shearing the DNA and immunoprecipitating the protein of interest while covalently bound to its DNA targets. Following decrosslinking, DNA that was coimmunoprecipitated could be amplified by PCR or could serve as a probe of a genomic microarray to identify all DNA fragments that were bound to the protein.Although simple in principle, the method is not trivial to implement and the results might be misleading if proper controls are not included in the experiment. In this chapter, we provide therefore a highly detailed protocol of ChIP assay as is applied successfully in our laboratory. We pay special attention to describe every small detail, in order that any investigator could readily and successfully apply this important and powerful technology.

In an effort to understand how an accurate level of stress-specific expression is obtained, we studied the promoter of the yeast HSP104 gene. Through 5' deletions, we defined a 334 bp fragment upstream of the first coding AUG as sufficient and essential for maximal basal activity and a 260 bp fragment as sufficient and essential for heat shock responsiveness. These sequences contain heat shock elements (HSEs) and stress response elements (STREs) that cooperate to achieve maximal inducible expression. However, in the absence of one set of factors (e.g. in msn2Deltamsn4Delta cells) proper induction is obtained exclusively through HSEs. We also show that HSP104 is constitutively derepressed in ras2Delta cells. This derepression is achieved exclusively through activation of STREs, with no role for HSEs. Strikingly, in ras2Deltamsn2Deltamsn4Delta cells the HSP104 promoter is also derepressed, but in this strain derepression is mediated through HSEs, showing the flexibility and adaptation of the promoter. Thus, appropriate transcription of HSP104 is usually obtained through cooperation between the Msn2/4/STRE and the HSF/ HSE systems, but each factor could activate the promoter alone, backing up the other. Transcription control of HSP104 is adaptive and robust, ensuring proper expression under extreme conditions and in various mutants.

The broad expression pattern of the G protein-coupled P2Y receptors has demonstrated that these receptors are fundamental determinants in many physiological responses, including neuromodulation, vasodilation, inflammation, and cell migration. P2Y receptors couple either G(q) or G(i) upon activation, thereby activating different signaling pathways. Ionotropic ATP (P2X) receptors bind extracellular nucleotides, a signal which is transduced within the P2X protein complex into a cation channel opening, which usually leads to intracellular calcium concentration elevation. As such, this family of proteins initiates or shapes several cellular processes including synaptic transmission, gene expression, proliferation, migration, and apoptosis. The ever-growing range of applications for antibodies in the last 30 years attests to their major role in medicine and biological research. Antibodies have been used as therapeutic tools in cancer and inflammatory diseases, as diagnostic reagents (flow cytometry, ELISA, and immunohistochemistry, to name a few applications), and in widespread use in biological research, including Western blot, immunoprecipitation, and ELISPOT. In this article, we will showcase several of the advances that scientists around the world have achieved using the line of antibodies developed at Alomone Labs for P2Y and P2X receptors.