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The carboxyl proteases are so called because they have two catalytically essential aspartate residues. They were formerly called acid proteases because most of them are active at low pH. The best-known member of the family is pepsin, which has the distinction of being the first enzyme to be named (in 1825, by T. Schwann). Other members are chymosin (rennin) cathepsin D Rhizopus-pepsin (from Rhizopus chinensis) penicillinopepsin (from Penicillium janthinel-lum) the enzyme from Endothia parasitica and renin, which is involved in the regulation of blood pressure. These constitute a homologous family, and all have an Mr of about 35 000. The aspartyl proteases have been thrown into prominence by the discovery of a retroviral subfamily, including one from HIV that is the target of therapy for AIDS. These are homodimers of subunits of about 100 residues.156,157 All the aspartyl proteases contain the two essential aspartyl residues. Their reaction mechanism is the most obscure of all the proteases, and there are no simple chemical models for guidance. 

Although technically simple, the use of retroviruses and retroviral vectors has not found widespread application. This is mainly due to the size limitations for transfected DNA (about 10 kb), as well as to the unresolved problems of reproducibly expressing a virally transfected eukaryotic gene. However, the methods of pronuclear microinjection and the use of embryonic stem cells are being successfully applied by a growing number of research groups. 

Although the existence of this enzyme may not be surprising, the mechanism by which it acts is remarkable and unprecedented. Telomerase, like some other enzymes described in this chapter, contains both RNA and protein components. The RNA component is about 150 nucleotides long and contains about 1.5 copies of the appropriate CyKx telomere repeat. This region of the RNA acts as a template for synthesis of the T -G strand of the telomere. Telomerase thereby acts as a cellular reverse transcriptase that provides the active site for RNA-dependent DNA synthesis. Unlike retroviral reverse transcriptases, telomerase copies only a small segment of RNA that it carries within itself. Telomere synthesis requires the 3 end of a chromosome as primer and proceeds in the usual 5 —>3 direction. Having syn-... 

At present, we can induce CML in mice with high efficiency, shown by 100% induction of CML in mice (14). The same CML disease could be induced in most of the inbred mouse strain including C57BL/6, BALB/c, and viable gene knockout mice strains (15). Because all recipients develop CML with a short latency (about 3 weeks), this provides an excellent model for evaluating therapeutic agents for CML treatment (15). As CML is derived from the hematopoietic stem cells which harbor BCR-ABL oncogene, CML leukemia stem cells can also be studied in this model (15). In conclusion, this retroviral model system pro-... 

Manganese(II) ions may also be employed in the hydrolases that compose the ribonuclease H domain of reverse transcriptases (3, 4). Many of these enzymes, which may employ either Mn" or Mg11, are found in a variety of organisms where they may or may not be essential (e.g., Escherichia coli) (3, 4). This class of enzymes catalyzes the hydrolysis of DNA-RNA hybrids. However, retroviral reverse transcriptases are critical for the replication of retroviruses, and Mn11 may be the required cofactor for these ribonuclease hydrolases to function (3, 4). One example of this family of hydrolase enzymes is the RNase H enzyme from HIV-I. This enzyme has been crystallographically characterized (11). In the crystal structure at 2.4-A resolution, the two Mn 1 ions are separated by a distance of about 4 A. They are bound to carboxylate residues that are located near the surface of the enzyme. One of these carboxylates bridges the two manganese... 

To overcome this problem, two approaches have been taken by using gene manipulation in mice. One approach involves the overexpression of LIF (Metcalf and Gearing, 1989). Cells of a murine hematopoietic cell line which are multiply infected with a retroviral construct containing LIF cDNA, secrete high levels of LIF. Injection of these cells into mice causes them to exhibit fatal syndromes within 12-70 days of injection. Irradiated animals injected with FD/LIF develop these fatal syndromes as early as 10 days after injection, whereas non-irradiated animals injected with FD/LIF develop the syndromes only about 30 or more days after injection. In both cases, the increased level of LIF causes serious abnormalities (Table 2). 

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