Cloning techniques

Berger, S. L., and Kimmel, A. R., eds., 1987. Guide to Molecular Cloning Techniques. Methods in Enzymology, Volume 152. New York Academic Press. 

It had long been expected that an iron transporter would be found at the basolateral membrane of the enterocyte responsible for the exportation of iron from the enterocytes into the portal vein circulation, and it turns out that three groups appear to have made this discovery almost simultaneously (McKie et ah, 2000 Donovan et ah, 2000 Abboud and Haile, 2000). The first of these, IREG1, was originally identified by subtractive cloning techniques (McKie et ah, 1998), and isolated and... 

Volume 152. Guide to Molecular Cloning Techniques Edited by Shelby L. Berger and Alan R. Kimmel... 

Since that time, over 35 heterotrimeric G protein subunits have been identified by a combination of biochemical and molecular cloning techniques [1-4]. In addition to G Gs and G , the other types of G protein in brain are designated G0, Golf, G, Gz, Gq and G 16. Moreover, for most of these G proteins, multiple subtypes show unique distributions in the brain and peripheral tissues. G proteins are now divided into four main categories (Table 19-1) the Gs family stimulates adenylyl cyclase the G family (which includes G0, G, Gz) can inhibit... 

A unique characteristic of plasmids is that thousands are produced within each bacterium, to the point that up to 40% of the total DNA of the bacterium may in fact be plasmid DNA. Hence, a single piece of human DNA that heretofore could only be obtained in low concentrations can be recombined with a plasmid, and the DNA sequence multiplied by a million- or a billion-fold (i.e., cloned). Use of cloning techniques may produce many grams of a particular human protein, instead of the few molecules that are produced in normal cells. 

Because the fMet-Leu-Phe receptor is present only at low levels in neutrophils (-12 x 10 15 g of receptor per cell), it has proved difficult to purify and characterise. Researchers have therefore turned to molecular cloning techniques to gain insight into the molecular structure of this receptor. This approach itself has not been easy because, in the absence of an antibody that specifically binds to the receptor, or else without some amino acid sequence data that can be used to synthesise oligonucleotide probes, cDNA libraries cannot be screened to isolate relevant clones. Therefore, experimental systems in which functional fMet-Leu-Phe receptors are expressed on the surfaces of transfected cells have been used. Two main systems have been utilised expression of mRNA injected into Xenopus laevis oocytes and cDNA cloning into the COS-cell expression vector. 

In addition to the large, heterotrimeric G-proteins that have been shown to play a role in signal transduction, a superfamily of low-molecular-weight (LMW) GTP-binding proteins, which are structurally related to ras, has been shown also to play important roles in the regulation of cell function. These proteins were initially identified by molecular cloning techniques and... 

Xhang, G. and Gurlu, V. (1997) Detection of early-stage apoptosis in three adherent mammalian cell lines using the ApoAlert Annexin V Apoptosis Kit. Clone-techniques Xn, 24,25. 

There are many uses of recombinant DNA. As noted above, one technique that produces recombinant DNA is called cloning. In one cloning technique used for the production of the sheep Dolly in 1996, the DNA nucleus from a female s egg was replaced with a nucleus from another sheep. The egg was placed in the uterus of a third animal, known as the surrogate mother. Dolly is nearly genetically identical to the animal from which the nucleus was obtained but not genetically related to the surrogate mother. 

Interaction of ACh with the postsynaptic ganglionic cell muscarinic receptor is responsible for slowly developing depolarization, the slow EPSP, which has a longer latency than the fast EPSP and a duration of 30 to 60 seconds. The slow EPSP is due to inhibition of a voltage-dependent K+ current called the M current, and inhibition of the M current involves activation of G proteins. At least five types of muscarinic receptors (Mj, M2, M3, M4 and M5) have been identified using functional studies and at least five subtypes (mj, m2, m3, rru, and m5) identified by molecular cloning techniques. The Mj receptor, which appears responsible for inhibiting the M current, can be blocked by atropine. 

Although the role of the terpenes in the plants that produce them is still a matter of debate, evidence of an anti-herbivore function for many of them is accumulating. Modern DNA technology, gene splicing, and cloning techniques will undoubtedly make it possible to incorporate suitable defensive allelochemlcals into selected crop plants in order to minimise crop devastation by insect herbivores. However, it is necessary to understand the fund-... 

The first group of cytokines discovered, the interferons (IFNs), were followed by the colony-stimulating factors (CSFs, discussed in Chapter 33). The latter regulate the proliferation and differentiation of bone marrow progenitor cells. Most of the more recently discovered cytokines have been classified as interleukins (ILs) and numbered in the order of their discovery. Cytokines are produced using gene cloning techniques. 

Reverse transcriptases have become important reagents in the study of DNA-RNA relationships and in DNA cloning techniques. They make possible the synthesis of DNA complementaiy to an mRNA template, and synthetic DNA prepared in this manner, called complementary DNA (cDNA), can be used to clone cellular genes (see Fig. 9-14). 

Immobilization has other advantages it can slow enzyme deactivation by inhibiting protease attack and minimizing shear, interfacial, temperature, or solvent denaturation. As for the scarcity of some potentially very useful enzymes, it may be only a temporary problem. The development of cloning techniques, and probably the very increase in demand will result in lower prices. One spectacular instance is sialyl aldolase (see Table I). Industrial production of this enzyme by the gene-cloned strain of Escherichia coli has been reported.1,2 Sialylaldolase is now available from Toyobo at a moderate price. 

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