HAT selection

Centrifuge for 3 min at 400g, and then resuspend the cells in 200 mL HAT selection medium, and add irradiated fibroblast or thymocyte feeder cells. Plate 2-mL aliquots into four 24-well plates or, if necessary, five 96-well plates (fusions with SP2/0 myeloma) (see Note 6). 

By growing cells in the presence of increasing concentrations of aminopterin a number of resistant cells lines have been isolated (Hakala and Ishihara, 1962 Littlefield, 1969). These have been characterised as having either an altered permeability to the drug or an altered folate reductase or an increased rate of synthesis and hence increased amounts of the enzyme (Alt et al., 1976), resulting, at least in part, from a selective amplification of the dihydrofolate reductase gene (Alt et al., 1978 Schimke et al., 1988). The problem is considered in more detail in 11.8.1. The importance of the antifolates lies in their role in the HAT selection technique ( 13.5) devised by Szybalski (1962) (see also Szybalski et al., 1962 and Littlefield, 1964) for the isolation of hybrids between mutant cells defective on the one hand in thymidine kinase and on the other hand... 

The ability of cells to uptake thymidine and phosphorylate it is exploited in the so-called HAT selection medium (see here), which selects for cells that have functional nucleotide salvage pathways. 

HAT Selection - The compounds hypoxanthine, aminopterin (see here), and thymidine (H,A, and T, respectively) can be used to select for cells having functional salvage pathways. Aminopterin inhibits dihydrofolate reductase, which blocks de novo purine and thymidine synthesis. Only cells which can utilize thymidine (pyrimidine salvage) and hypoxanthine (purine salvage) can grow in this medium. 

Figure 3 Schematic representation of HAT selection. HAT medium contains aminopterin to block the de novo pathway and hypoxanthine and thymidine to allow growth by the salvage pathway. Cells that lack either HGPRT or TK will die in HAT medium, because they lack the ability to use the salvage pathway to synthesize nucleic acids.

A number of protocols have been described for the generation of mouse hybrid-omas and workers have their own particular protocol. We have used a standard procedure for the fusion of mouse and rat myelomas with considerable success over the last twenty years and this is described in Protocol 7. Basically, 10 lymphocytes are mixed with 2 x 10 mouse myeloma cells or 5 X 10 Y3 cells in a round-bottomed tube and pelleted by centrifugation. Then the ceUs are fused by the addition of 1 ml of 50% PEG 1500 and plated into HAT selection medium to allow the growth of the hybridomas generated. 

Centrifuge for 3 min at 400 g, then resuspend the cells in 200 ml of HAT selection medium (containing feeder cells where necessary), and plate 2 ml aliquots into four 24-well plates or 200 pi aliquots into ten 96-well plates. Incubate at 37 °C in 5% C02-... 

Most of what has been described so far for stiffener design involves shape and size of the stiffener. Those issues involve selection of the type of stiffener, H-shaped cross section, blade, hat-shaped, etc. as well as the specific dimensions and material makeup of each stiffener element. Other obvious factors in the design of a stiffener include how far apart we space them, at what orientation we place them, and, perhaps most obviously in connection with what we addressed in Section 7.3, out of what material we make the elements. As you saw in some of the previous sketches for stiffeners, we are able with a composite stiffener to use different materials in different places very easily and to essentially optimize our materials usage so that the stiffening comes out to be as good as we can possibly make it. 

The multiple conformational restriction of dermorphin-related tet-rapeptide analogues that was performed represents a rational design of opioid peptidomimetics characterized by a high degree of structural rigid-ification. This is indicated by the fact that the p-selective agonist H-Hat-D-Orn-Aic-Glu-NH2 contains only two freely rotatable bonds, whereas there are 14 freely rotatable bonds in [Leu5]enkephalin. 

Table 3. Selected list of HAT and HMT disruptions in human diseases...

Lachner M, O Carroll D, Rea S, Mechtler K Jenuwein T (2001) Methylation of histone H3 lysine 9 creates a binding site for HPl proteins. Nature 410 116-120 Lau OD, Kundu TK, Soccio RE, Ait-Si-Ali S, KhaUl EM, Vassilev A, Wolffe AP, Nakatani Y, Roeder RG, Cole PA (2000) HATs off selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF. Mol Cell 5 589-595... 

The first reported selective inhibitors of the HATs p300 and PCAF were peptides representing structurally simple bi-substrate analogs of H3 and acetyl-CoA [10, 11]. Hence, p300 was inhibited by Lys-CoA 1 and the more specific PCAF by the 20-amino-acid peptide H3-CoA-20 2 (Figure 11.2). Unfortunately, these inhibitors showed low cell permeability and high metabolic instability, which decreases their suitability for investigations in vivo [12]. 

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