4324c
155.0
1 . 84-1
058.0
<1.7
4-2
4
133.3
3.0
4-3
093.0
< 1.1
4-5
073.0
<1.4
4-9
14
69.5
2 0 . 14-10
079.0
<1.3
4-11
05.9
<16.9
4-17
073.3
<1.4
4-18
0121.5
<0.8
4—20
126.8
3.7
4-21
043.5
<2.3
4-22
077.0
<1.3
Spontaneous reversion frequencies of the different GOGAT mutants. Aliquots of glutamate grown stationary phase cultures of each mutant were plated on to MM + NH4+. The plates were incubated at 190C for 6 days before being screened. The reversion frequency of the gdhl-hocus was determined by plating 4324c onto MM + NH4+ + D-histidine.
83
The data shows that gdhl-1 reverts at a frequency of 1.8x10”8. Revertants were obtained for only 3/12
of the mutants tested. The reversion frequencies of 4-2 and 4-20 were comparable to that of the parental strain but 4-9 reverted eleven-times more frequently. The reversion frequencies of the majority of the other strains tested were comparable to or less than that of gdhl-1. Although no revertants were obtained from these strains, the number of cells screened was similar, or, for the majority of the strains examined, greater than the number for 4324c. The maximal reveision frequency for 4-11 was eight-fold higher than that for 4324c, but ten-fold fewer cells had been examined.
It would be futile to speculate about whether any of the mutations are non-revertable because insufficient cell numbers have been examined. The reversion frequencies are low enough, for all the strains examined, so that reversion of the mutation causing loss of GOGAT activity is not a problem in cloning or growth experiments. It is unclear why some mutants carrying two mutations, whose reversion would independently cause loss of glutamate auxotrophy, should revert less frequently than the parental strain.
5.4.1 There are several explanations of why the GLU” mutants described above have reduced GOGAT activity. Firstly, the mutation could lie within the coding sequence of the GOGAT genes. These sequence alterations could result in changes in the primary structure of the polypeptide(s) and subsequent loss/decrease in catalytic activity. Secondly, lesions could lie in regulatory regions which modulate gene expression either at the level of transcription or translation. Such mutations would cause a decrease in the number of enzyme molecules but not in the activity of each molecule. Thirdly, a non specific mutation results in altered levels of an effector e.g. a TCA cycle intermediate which modulates the activity of the enzyme, although the number of enzyme molecules and the activity per se per molecule remains unaltered. Additional explanations of the reduced GOGAT activities are possible. To determine if any of the GLU” mutants isolated carry mutations which lie within the GOGAT coding regions, physiochemical properties (e.g. stability to heal or pH, sensitivity to inhibitor) of GOGAT from the mutant strains was examined.
5.4.2.1 The approach first tried was an investigation of the thermolability of the mutant GOGAT enzymes. The mutants were initially screened for a fs-phenotype. The mutants were inoculated into MM + glutamate and incubated at 19^0 for 5 days. Appropriately diluted aliquots of each culture (to give about 100 colonies per plate) were spread onto MM + NH4+ at 2 or 20mM, and incubated at 19 or 30OC for up to 6 days.
Single colonies of Z.1278b and 4324c were observed after about 40h growth at 3QOC, but at 19^0 colonies appeared at 3-4 days. It was found that only strain 4-25 grew on MM + NH4+ under these conditions. At 190C single colonies were visible after about 4 days incubation, but at 30°C no growth
84
was apparent even after 6 days incubation. As has been slated above, the ability to grow on MM + NH4+ could be due to reversion of tlie gdhl-1 allele. This possibility was excluded by plating the cells onto MM + NH4 + + D-histidine. No growth of strain 4-25 on tiiis lest medium was apparent, even after 6 days incubation at 190C. It would appear that, on the basis of these plate tests, the mutation causing tlie loss of GOGAT in 4-25 confers a /^-phenotype on this strain. This is shown in Fig. 17. The same experiment using strain 4-9 is shown in Fig. 18. Strain 4-9 did not exhibit a rj-phenolype. The putative rj:-mutant was taken for further study.
5.4.2.2 The approach used to identify whether tlie mutation in 4-25 lies witliin the GOGAT structural gene exploits the r^-phenotype of tlie mutant. If a particular gene specifies a polypeptide which is structurally part of the GOGAT molecule (tlie S. cerevisiae enzyme is a heteromeric dimer), ts-
mutations at such a locus should make functional enzyme when the cells are grown at the permissive temperature (19^0). However, this enzyme should be more heat labile than the wild type enzyme. If the gene in question specifies a product not structurally part of the GOGAT molecule, the GOGAT moiety in such a mutant should have the same thermostability as the wild type enzyme.
To test this possibility, 4-25 was grown at both permissive and restrictive (30OC) temperatures. GOGAT was assayed in extracts prepared from each culture: cells grown at die permissive temperature were assayed at 190C, and ones grown at the restrictive temperature at 30^C. The results are presented in Table 15.
The data show that GOGAT activity in 4-25 grown at 30OC is 46% of the activity measured in 4324c. At 190c , the GOGAT level in 4-25 is 86% of the parental activity. This shows that GOGAT in strain 4-25 is more stable at 19 tlian 3QOC, as would be expected of a rs-mutant. The 4-25 enzyme has an altered thermolability relative to the wild type. Therefore it suggests tliat the mutation in 4-25 lies within the GOGAT structural genes.
Although the percentage figure for 4-9 increases from 2 to 8, the measured activities increased from 1 to 3mU/mg. The increase in absolute activity is not marked and probably reflects sampling errors. The decrease in parental activity increases this variation. The 30OC figure is tlie mean of four independent experiments while the 190C value was obtained from a single experiment. It is therefore improbable that GOGAT in 4-9 has an altered thermolability relative to the wild type.
5.4.2.3 The data presented in Figs. 17 and 18 suggest that strain 4-25 has a r^-phenotype and that 4-9 does not. To investigate this further, growth curves of both these strains were made at the permissive and restrictive temperatures.
F ig . 17
IO
imM
2 m M T he t s - n a t u r e o f t h e m u t a t i o n c o n f e r r i n g t h e g l u p h e n o t y p e o n s t r a i n 4-25 i s d e m o n s t r a t e d by t h e a b i l i t y o f t h i s s t r a i n t o g ro w o n + NH^ a t 19°C b u t n o t a t 5 0 ° C . 4 - 2 5 w as s t r e a k e d o n t o MK a n d i n c u b a t e d a t 19°C f o r 4 d a y s o r 50°C f o r 6 d a y s .F i g . 18a
l O m M
a O m M
2 Jh NIW
L ,
The n o n -t8 -n a tu re of the m utation c a r r ie d by s t r a i n 4-9 was shown
by the i n a b i l i t y of t h is s t r a i n to grow on MM +
a t both 19°C
(a) and 30°C (b ). 4-9 was stre a k e d onto MM and in cubated a t the
a p p ro p ria te tem perature f o r 6 days.
F i g . 18b
% O m M