Transcript localization hybridization

Molecular biology involves the study of the major macromolecules, DNA, RNA, and protein. The central dogma ofmolecular biology is illustrated in Fig. 2. The central dogma shows the relationship among the macromolecules in the processes of transcription and translation. Figure 2 also gives the relationship between immunoelectron microscopy and in situ hybridization. In situ hybridization allows one to localize a specific nucleic acid sequence. Immunoelectron microscopy is an essential component to the technique of in situ hybridization when applied at the EM level. 

Perhaps one of the most exciting areas in neuroscience to which in situ hybridization has made significant contributions in recent years is the discovery that specific mRNA transcripts can be localized to certain compartments of neuronal cells (Steward and Schuman, 2003), such as axons (Tohda, 2003), dendrites (Ma and Morris, 2002 Bockers et al., 2004), cell bodies (Lu et al., 1998), and growth... 

Figure 4.4 Nucleolar localized anti-HIV-1 ribozyme. The anti-HIV-1 ribozyme was inserted within the apical loop of the small nucleolar RNA U16. The chimeric U16-ribozyme was placed behind the Pol III U6 snRNA promoter. Transcripts of the chimeric RNA localize to the nucleolus, as shown by the in situ hybridization and co-localization with the small nucleolar RNA U3. The in situ hybrids were performed in transfected 293 cells. The ribozyme probe was fluorescein labeled (green), whereas the U3 probe was labeled with Cy3 (red). Reproduced with permission from Michienzi et al. (2000). (see Color Plate 3)...

The 5-HT6 receptors are positively coupled to adenylate cyclase. In the absence of selective agonists and antagonists as well as radioligands, they have been initially localized in the rat brain by Northern blot analyses (218,219), in situ hybridization histochemistry (219-221), and quantitative reverse transcription followed by polymerase chain reaction (222) (see also ref. 210). These studies have established that the receptor mRNA is abundant in extrapyramidal areas such as the striatum, as well as in limbic areas such as the nucleus accumbens, olfactory tubercle, hippocampus, and hypothalamus. 

Staining of consecutive sections with histochemical reagents for protein and antibodies against myrosinase showed that myrosin cells actually contain myrosinase (Fig. 4.2D,E).18,43,44 By electron microscopy immunogold labelling studies, the enzyme was localized to the interior of the vacuoles of the myrosin cells (Fig. 4.2F).18,40,41 It has also been shown by several in situ hybridization experiments that myrosinase transcripts are located in these cells (Fig. 4.2G),6 demonstrating that the cellular localization of myrosinase is due to transcriptional regulation and not to a transport process. No evidence for transport of myrosinase currently exists. These methods for detection and identification of myrosin cells and myrosinase expression are illustrated (Fig. 4.2). For a historical overview of myrosinase localization studies, see Bones and Rossiter.45... 

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