CDNA formation

Expression of a tomato cDNA coding for phytoene synthase in Escherichia coli, ph)doene formation in vivo and in vitro, and functional analysis of the varions trimcated gene prodncts , J Biochem (Tokyo), 116, 980-85. 

In this chapter we report recent analytical applications of CL imaging for the detection of biospecific reactions in macrosamples such as microtiter plates of different format (96 or 384 wells), filter membranes and irregular surfaces represented by specimens related to the cultural heritage, and results obtained when the CCD detector is coupled with optical microscopy for enzyme localization, immunohistochemical reactions, and complementary DNA (cDNA) detection (Table 1). 

Since 1988, the methods that we use to isolate cDNAs of alkaloid biosynthesis have become ever more facile and sensitive, allowing for more efficient cDNA identification. We do not, however, yet understand enough about the cellular localization of alkaloid formation or about the nature of the catalysts to move completely away from enzymology and biochemistry and to use only molecular genetic techniques to dissect these biosynthetic pathways. Even our most recently successful cDNA isolations and identifications involved classical protein purification. We are beginning now to use proteomics and EST sequencing to identify natural product biosynthetic cDNAs, but these approaches are more feasible when a specialized cell/tissue type in which secondary metabolite biosynthetic pathways are active, can be isolated and used as a protein or RNA source. 

Angiotensin-II AT, Human cDNA Artherosderosis, cardiac hypertrophy, congestive heart failure, hypertension, myocardial infarction, renal disease, cancer, diabetes, obesity, glaucoma, cystic fibrosis, Alzheimer s disease, Parkinson s disease Smooth muscle contraction, cell proliferation and migration, aldosterone and ADH release, central and peripheral sympathetic stimulation, extracellular matrix formation, tubular sodium retention, neuroprotection... 

Moore KR, Blakely RD. 1994. Restriction site-independent formation of chimeras from homologous neurotransmitter-transporter cDNAs. BioTechniques 17 130. 

Ex situ (also known as spotted or printed) arrays have become very popular formats, especially for the building of custom noncommercial arrays used primarily by academic laboratories [see Association of Biomolecular Resource Facilities (ABRF) surveys on microarrays atwww.abrf.org]. The printed cDNA microarray was largely developed from gene expression work originating in the laboratories of RO. Brown and R.W. Davis at Stanford University (Schena et al., 1995). Plans for the construction of the microarrayer and split pin designs were available at the Brown lab website at http //cmgm.stanford.edu/ pbrown/mguide/index.html. This enabled researchers to prepare their own microarrays appropriate for their particular experiments. 

The advent of the polymerase chain reaction (PCR) made it possible to directly spot down cDNA amplicons onto membranes, giving rise to the Southern dot blot format. In fact, the dot blot should be regarded as one of the earliest, if not the first, array format (albeit a macroarray). Why did many abandon the membrane in favor of the glass substrate for DNA microarrays ... 

The induction of PAL activity at the onset of vascular differentiation can be shown by the use of plant tissue cultures (37-39). Xylem cells with secondary and lignified walls are differentiated over a time course of 3-14 days by the application of the plant growth factors naphthylene acetic acid (NAA) and kinetin in the ratio 5 1 (1.0 mg/liter NAA, 0.2 mg/liter kinetin) to tissue cultures of bean cells (Phaseolus vulgaris) (37,40). The time for differentiation varies with the type of culture, solid or suspension, and with the frequency and duration of subculture, but for any one culture it is relatively constant (37,41,42). At the time of differentiation when the xylem vessels form, the activity of PAL rises to a maximum. The rising phase of the enzyme activity was inhibited by actinomycin D and by D-2,4-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide (MDMP) applied under carefully controlled conditions (42). This indicated that both transcription and translation were necessary for the response to the hormones. Experiments using an antibody for PAL and a cDNA probe for the PAL-mRNA have also shown that there is an increase in the amount of transcript for PAL during the formation of lignin when Zinnia mesophyll cells are induced to form xylem elements in culture (Lin and Northcote, unpublished work). 

In order to prove that specific isozymes are, in fact, involved in lignin formation, each must first be purified to homogeneity and subjected to appropriate immunochemical and kinetic investigations. Some cautious optimism for the resolution of this problem can be hoped for, since an antibody of the isozyme from Nicotiana tabacum has been obtained and its cDNA cloned (75). It is to be hoped that the following can now be determined ... 

There are many branches to the flavonoid biosynthetic pathways, with the best characterized being those leading to the colored anthocyanins and proanthocyanidins (PAs) and the generally colorless flavones, flavonols, and isoflavonoids. Genes or cDNAs have now been identified for all the core steps leading to anthocyanin, flavone, and flavonol formation, as well as many steps of the isoflavonoid branch, allowing extensive analysis of the encoded enzymes (Table 3.1). In addition, several DNA sequences are available for the modification enzymes that produce the variety of structures known within each class of compound. 

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