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The field of optical sensors has been a growing research area over the last three decades. A wide range of books and review articles has been published by experts in the field who have highlighted the advantages of optical sensing over other transduction methods. Fluorescence is by far the method most often applied and comes in a variety of schemes. Nowadays, one of the most common approaches in the field of optical biosensors is to combine the high sensitivity of fluorescence detection in combination with the high selectivity provided by ligand-binding proteins. In this chapter we deal with reviewing our recent results on the implementation of fluorescence-based sensors for monitoring environmentally hazardous gas molecules (e.g. nitric oxide, hydrogen sulfide). Reflectivity-based sensors, fluorescence correlation spectroscopy-based (FCS) systems, and sensors relying on the enhanced fluorescence emission on silver island films (SIFs) coupled to the total internal reflection fluorescence mode (TIRF) for the detection of gliadin and other prolamines considered toxic for celiac patients are also discussed herein.

Transcription of mouse genes coding for rRNA in CHO cells was promoter dependent at levels 3 to 10% of that of endogenous rRNA synthesis. Northern (RNA) and Si nuclease mapping analyses demonstrated that transcription proceeds through the entire gene segment coding for rRNA in transfected constructs and continues, at least in some cases, into the adjoining plasmid sequences. Si nuclease mapping also detected some processing cleavages in the transcripts, including those at the 3' terminus of 18S rRNA, those at the rapidly cleaved site at +650 in the external transcribed spacer, and those at a previously uncharacterized, rapidly cleaved site in the internal transcribed spacer. Deletion of sequences upstream or downstream from the promoter generally had no measurable effect on the level of transcription, but deletion of a 300-base-pair XhoI-XhoI fragment starting 1,287 base pairs from the transcription start site sharply increased the steady-state level of rRNA. Effects on processing were harder to test, because many intermediates are too unstable to detect even by Si nuclease mapping; however, the data suggest that RNAs with deletions in the external transcribed spacer are processed poorly at distal sites. Processing at some sites may thus depend on interactions involving distant segments of rRNA.The processing of mRNA precursors has been extensively analyzed in extracts and in cells (16,18). In contrast, comparable information on the processing of rRNA remains sparse. While it is known that rRNA processing in mammalian cells proceeds through a set of simple cleavages, no in vitro system which produces fully processed rRNAs has yet been devised. Even in vivo, the expression of transfected constructs of genes coding for rRNA (rDNA) is difficult to study because of the very high background of endogenous rRNA formation. One way to overcome this problem is to analyze the expression of transfected heterologous rDNA with probes unique to spacer sequences in the transfected rDNA (9). Even though rDNA transcription has been shown to be restricted by species-specific barriers (10, 14), it can be studied in cells or extracts of some closely related species (human and monkey [12], mouse and rat [20], and mouse and hamster [7]). In those cases, the spacer sequences are different enough to permit adequate discrimination of transcripts from endogenous and transfected rDNA. Recombinant rDNA plasmids. Figure 1 shows a schematic map of the subclones and deletion constructs used in the transient expression experiments. All plasmids used for transfection were cloned in the vector pUC18. Mouse clone numbers are as described elsewhere (2, 3). pCL3 was derived by subcloning the 3.2-kilobase (kb) SalI-SalI fragment of pMr974 (20) insert into the EcoRI site of clone 6 in the appropriate orientation. The resulting construct, containing the mouse rDNA promoter, 8 kb of upstream DNA, and an intact transcription unit through the first one-third of internal transcribed spacer 2 (ITS2), was the parent molecule for a number of constructs. pRD18...

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We have developed a way to fit yeast artificial chromosomes (YACs) with markers that permit the selection of stably transformed mamnalian cells, and have determined the fate and expression of such YACs containing the genes for human ribosomal RNA (rDNA) or glucose-6phosphate dehydrogenase (G6PD). The YACs in the yeast cell are "retrofitted" with selectable markers by homologous recombination with the URA3 gene of one vector arm. The DNA fragment introduced contains a LYS2 marker selective in yeast and a thymidine kinase (TK) marker selective in TK-deficient cells, bracketed by portions of the URA3 sequence that disrupt the endogenous gene during the recombination event. Analyses of transformed L-M TK-mouse cells showed that YACs containing rDNA or G6PD were incorporated in essentially intact form into the mammalian cell DNA. For G6PD, a single copy of the transfected YAC was found in each of two transformants analyzed and was fully expressed, producing the expected human isozyme as well as the heterodimer composed ofthe human gene product and the endogenous mouse gene product.To study gene expression, cloned fragments of DNA are often reintroduced into eukaryotic cells by transfection (1) or electroporation (2). Conventional vectors, however, have placed an upper limit on the size of DNA that can be studied, since inserts of no more than 40 kilobases (kb) can be accommodated, and it is now clear that many genes are larger than that. One way to circumvent this limitation has been provided by the development of yeast artificial chromosomes (YACs) as a cloning system, since DNA inserts in YACs can be as large as a megabase or more (3-7). We have designed a selective system to test the potential of transfected YACs for short-and long-term studies of gene expression.The test genes we have used are rDNA, encoding ribosomal RNA (rRNA), and the housekeeping gene G6PD, encoding glucose-6-phosphate dehydrogenase (G6PD). The number of natural variants detected for G6PD, now in excess of 300, is the largest known for any enzyme (8), providing unusually favorable material for studies of natural mutational variation and possible regulatory changes.Though the transcription units in the cases of rDNA and G6PD are well defined and are each contained in <20 kb, repeated searches have failed to recover the entire units in single A or cosmid clones. In contrast, YACs of 40-50 kb contain the entire genes (9, 10), and when a YAC containing G6PD was transfected into mammalian cells, the normal human isozyme was transiently expressed at easily detectable levels within 48 hr (11).Only a small number of cells take up DNA in standard assays with calcium phosphate-mediated transfections (1), and much of the DNA is either in the process of being degraded or is adventitiously stuck to cellular material. (14), which contains the URA3 gene, into pUC18 (15)]. The Stu I site is in the middle of the yeast URA3 gene of the plasmid, so that the net result of ligation is an 11-kb bacterial plasmid, TKLU2, containing the L YS2 and TK genes i...