A protocol is described for making a soluble whole-cell extract from yeast (Saccharomyces cerevisiae) that supports active and specific transcription initiation by RNA polymerases I, H, and Im. Specific initiation by polymerase I decreases in high-density cultures, paralleling the decrease in abundance of the endogenous 35S rRNA precursor. This extract should be useful for studying the molecular mechanisms that regulate rRNA transcription in yeast.The yeast Saccharomyces cerevisiae has emerged as the preeminent eukaryote for many studies of transcriptional regulation, due to both the ease of its genetic manipulation as well as the ability to inexpensively grow large amounts of the organism for biochemical fractionation. In the case of RNA polymerase I in yeast, definition of cis-acting regulatory elements has been made possible by the development of genetic approaches that circumvent the problems raised by the repetitive nature of the ribosomal genes (1, 2). Study of the trans-acting factors controlling transcription by RNA polymerase I has been hampered, however, by the lack of in vitro transcription extracts capable of supporting specific transcription initiation at the site employed in vivo.This situation is now changing. Riggs and Nomura (3) have reported a whole-cell polymerase I extract from yeast and Lue and Kornberg (4) have described a polymerase I extract made from yeast nuclei. And in this paper we wish to report our own, independently developed procedure for accomplishing the same goal. The method that we describe is rapid, produces extracts from whole cells without prior isolation of nuclei or chromatographic fractionation, and can be adapted for large amounts of yeast. We also show that polymerase I initiation activity in this extract varies with the growth state of the cell. This type of extract should therefore be useful in studying the mechanisms coordinating ribosomal gene transcription with cell growth. MATERIALS AND METHODSStrains. Transcription extracts were normally prepared from the multiply protease-deficient S. cerevisiae strain BJ2168 (leu2, trpl, ura3-52, prbl-1122 provided by S. Hahn,

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The structure of the ribosomal gene promoter from Saccharomyces cerevisiae has been analyzed in a whole cell in vitro extract. The promoter contains at least two essential domains, an upstream domain located at the 5' boundary near position -150 and a core promoter domain around the site of transcription initiation at + 1. The upstream domain augments transcription in vitro but is not absolutely required. Maintenance of correct spacing between the two domains is critical. The in vitro analysis agrees well with prior in vivo analysis and it appears that the yeast promoter has a structure very similar to that of vertebrate ribosomal gene promoters.

Protein nanoarrays are addressable ensembles of nano-scale protein domain on solid surfaces. This method can serve as a useful platform for ultraminiaturized bioanalysis. In this study, we investigated single molecular nanopatterning and molecular interaction of proteins that were immobilized on Prolinker surface of gold-coated silicon wafer by using dip-pen nanolithography (DPN) method. Contact force and humidity were optimized at 0.01 nN and 80%, respectively. The domain features of protein nanoarrays were developed at the contact time of 5 s. The optimized conditions for the nanoarray process were applied to create protein nanoarray using integrin alpha(v)beta3 and angiogenin. Constructed protein nanoarrays using integrin alpha(v)beta3 have single molecular monolayer with regular domain shape (height 15 +/- 5 nm). The changed height value due to the single molecular interaction between integrin alpha(v)beta3 and vitronectin was approximately 30 +/- 5 nm on Prolinker surface as measured with atomic force microscopy tip. Taken together, these results suggest that protein nanoarray on Prolinker surface fabricated by well-controlled DPN process can be used to analyze single molecular interaction of protein.

Anti-inflammatory effects of an ethanol extract of Angelica gigas (EAG; 50, 160, or 500 mg/kg) were investigated in a carrageenan-induced air pouch inflammation model. Injection of 1 ml of carrageenan (1%) into mouse air pouches markedly increased the exudate volume and exudate albumin concentration, which were significantly attenuated by oral pretreatment with EAG. EAG also markedly reduced carrageenan-induced infiltrations of neutrophils, monocytes, and lymphocytes, but did not influence eosinophils or basophils. Carrageenan dramatically increased levels of tumor necrosis factor-a and interleukin-6, which might be derived from the infiltrated cells. It also elevated nitric oxide, and slightly increased prostaglandin E 2 . EAG pretreatment significantly lowered tumor necrosis factor-a and nitric oxide, but did not alter interleukin-6 or prostaglandin E 2 levels. These results indicate that EAG attenuates some inflammatory responses by blocking the tumor necrosis factor-a-nitric oxide pathway, and that EAG could be a promising anti-inflammatory drug candidate for inflammatory diseases.