Replacement folds

Figure 2.19. Potential secondary structure of the A-B fold of the ori sequence present in the repeat units of mitochondrial genomes from petites al/lR/1 and Z1 (ori ) and of the replacement folds that can be formed in the oril sequence present in the mitochondrial genomes of petites 14 and 26. In the case of petite 14, the residual nucleotides from the partially deleted p stretch (/ A) can generate a hairpin structure with nucleotides from the preceding repeat unit, but the stem, only formed by 13 A/T nucleotides, carries different terminal and side loops compared to the A-B fold. In the case of petite 26, the upper part of the stem and terminal loop are identical to those of the A-B fold, but the lower part is replaced by three A T pairs (three nucleotides are derived from the preceding repeat unit). The sequence involved in the structure shown (GC clusters A and B, sequences p and are those indicated in Fig. 2.13. (From Goursot et al., 1988).

In view of the presumed lower thermodynamical stability of replacement folds compared to the A-B fold, crosses of petites 14 and 26 with wild-type cells were also performed at both a higher (33°C) and a lower (23°C) temperature than the standard one, 28°C. The results of Table 2.2 indicate that such temperatures decreased and increased, respectively, the replicative ability of the two mutants, whereas that of the control orT petite Z1 did not show any significant change between these temperatures. The decrease of the replicative ability was stronger for petite 14 than for petite 26, as expected from the presumably lower thermodynamical stability of the replacement fold of the former. In the first case, the proportion of wild-type colonies obtained at 23°C and 33°C, respectively, was 10% and 55%, in the second 11% and 42%. The changes in replicative ability just described are immediate and reversible. 

The results presented indicate that an environmental factor, temperature, can reversibly affect the replicative ability of a genome by altering its secondary (and possibly, its tertiary) structure. Indeed, (i) these changes cannot be ascribed to enzymes involved in DNA replication, as in temperature-sensitive mutants, since the petites discussed here are iso-nuclear, and lack mitochondrial protein synthesis, like all petites (ii) the different effects of temperature on the replicative ability of petites Zl, 14 and 26 show an excellent correlation with those expected from the secondary structures of the postulated A-B fold and replacement folds (de Zamaroczy et al., 1981 1984), an effect on tertiary structures being also possible. 

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

We thus have here a case where a mutation on the surface of the globin fold, replacing a hydrophilic residue with a hydrophobic one, changes important properties of the molecule and produces a lethal disease. Why has the... 

FIGURE 5.31 Subdivision of the 100-fold uncertainty factor showing the relationship between the use of uncertainty factors (above the dashed line) and proposed subdivisions based on toxicokinetics and toxicodynamics. Actual data should be used to replace the default values if available, ... 

In this chapter, the experiment in which Craik and Rntter replaced Asp with Asn in trypsin (redncing activity 10,000-fold) was discnssed. 

Gas-fired water heaters use the same general method of construction, except that the elements are replaced with a burner beneath the tank. The combustion products from the burner are vented through a flue made out of the same thickness steel as the tank, that goes up through the center of the tank. To increase heat transfer from the hot flue gases to the inner wall of the flue, a baffle is inserted down the flue. This baffle is a twisted strip of sheet metal with folds and tabs on it. The folds and tabs are designed to... 

The subsequent advance was rather fortuitous and rested more with serendipity than with scientific logic. A search was made for cheaper more effective replacements for casein hydrolysate. Amongst the tested materials was com steep liquor (CSL). CSL is a by-product of the manufacture of starch from maize kemals. Whole maize is incubated in warm water, at 50°C acidified with SO2. Thermophilic bacteria hydrolyse proteins and other components of the kemals, thereby loosening the starch granules. These are removed, leaving behind the steep liquor which is used to treat further maize kemals. Ultimately, the liquor is too viscous to re-use and the liquor is concentrated and used as cattle feed. It was this material that was used for penicillin fermentation. Surprisingly, the yield of penicillin increased by a further 5-10 fold giving yields of 50-100 ig ml. 

If the experiment to which the calculation refers were actually attempted, several differences would appear. A lost important, the x-ray power would be expended over a vide spectrum. The intensity in Equation-4-10 would be the integrated intensity from the short-wavelength limit to the critical absorption wavelength. /Also, gmax and wka would need to be replaced by values that reflect the wavelength range of the integrated intensity. The net effect of all these differences would be to reduce /k below the value of Equation 4-16, perhaps by as much as ten-fold. 

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