Linear transcription

Linear transcription in microbial systems is postulated by some molecular biologists, and there is some evidence for linear transcription in yeast. The work of Gorman et al. (1964) has shown that in hybrids between two species of yeast, the two different forms of /3-glucosidase, characteristic of each parent, but apparently subject to identical induction and repression, are synthesized at two definite but different points of the division cycle. Comparisons with the behavior of other genes provides a nice argument for linear transcription. 

Owing to the apparently frequent differences in physical properties of enzymes of remote mammalian species, similar experiments should be possible with interspecific somatic hybrids and should thus provide evidence on the vahdity of these speculations. In principle (and we emphasize in principle ), the approximate alignment due to linear transcription should result in more or less clearly spaced bursts of homologous enzymes in the cell cycle of synchronized populations. Coordination by an induction-repression mechanism should, on the contrary, result in the simultaneous synthesis of homologous enzymes. 

A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 13.15 depicts such an expression vector. Linearized recombinant vector DNA is transcribed in vitro using SPG RNA polymerase. Large amounts of RNA product can be obtained in this manner if radioactive ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made. 

These vectors typically have a polylinker adjacent to the SPG promoter. Successive rounds of transcription initiated by SPG RNA polymerase at its promoter lead to the production of multiple RNA copies of any DNA inserted at the polylinker. Before transcription is initiated, the circular expression vector is linearized by a single cleavage at or near the end of the insert so that transcription terminates at a fixed point. 

This linear scheme of signal transduction (Fig. 12) from hypothetical membrane receptors to [Ca " ] and IP3 increases, calcium-calmodulin interaction, kinases activation and gene transcription is clearly an oversimplification of the reality several receptors must exist that are connected to different transduction cascades that activate a series of defense genes. Cross-talking between the pathways further complicates the picture. However, this represents a starting model on which to elaborate more refined hypotheses. 

Using human hepatoma-derived cell lines Kong et al. [268] showed that berberine increased mRNA and protein as well as the function of hepatic linear low density lipoprotein receptor (LDLR). It does not stimulate the transcription of LDLR, as the LDLR promoter activity was not increased by this compound. Post-transcriptional regulation appears to be the main working mechanism underlying the effect of this alkaloid on LDLR expression. It was proposed that berberine can be used as a monotherapy to treat hypercholes-terolemic patients [268]. Very recently it was observed [269] that berberine reduces cholesterol and Upid accumulations in plasma as well as Uver. 

Because the templates compete for amplification and, in the case of reverse transcription PCR (RT-PCR), also for reverse transcription, any variable affecting amplification has the same effect on both. Thus, the ratio of PCR products reflects the ratio of the initial amounts of the two templates as demonstrated by the function C/W=C (l+ )"/Wi(l+ )n, where Cand Ware the amounts of competitor and wild-type product, respectively, and C and W are the initial amounts of competitor and wild-type template, respectively, (Clementi etal., 1993). From this linear relationship, it could be concluded that a single concentration of competitor could be sufficient for quantitating unknown amounts of wild-type templates. However, in practice, the precise analysis of two template species in very different amounts has proved difficult and cPCRs using three to four competitor concentrations within the expected range of wild-type template concentrations are usually performed. In a recent study of different standardization concepts in quantitative RT-PCR assays, coamplification on a single concentration of a competitor with wild-type template was comparable to using multiple competitor concentrations and was much easier to perform (Haberhausen et al, 1998). 

The non-linear connections between DNA regions brought together by coiling and which are subject to control by transcription factors. These are then concentration-dependent cooperative features internal to the code. 

We routinely use a mix of five mRNAs that are derived from the lys (ATCC no. 87482), trp (ATCC no. 87485), dap (ATCC no. 87486), thr (ATCC no. 87484), andphe (ATCC no. 87483) clones from the bacterium Bacillus subtilis cloned into a vector that contains a stretch of As. These RNAs are generated by in vitro transcription using a T3 in vitro transcription kit (e.g., MEGAscript from Ambion) of the linearized DNA template with the appropriate restriction enzyme. 

Data from Tee el al. (1993a). Saturation kinetics were not observed the plot of kobsd vs. [CD] was linear. Thus, k2 was obtained from the slope and KTS from kjk2 (9). "The entry for this compound in Tee (1989) was incorrect due to a transcription error. ... 

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