7. Genomatix software: This is used for promoter analysis of Genomic DNA sequence
3.3 Discussion
The genes cmd, trm-9 and nca-2 encode for a CaM, a cation ATPases and a PMCA type Ca2+-ATPase that possesses conserved domains (Figures 3.3-3.7). I studied the CaM role in growth of N. crassa using its antagonists TFP and CPZ. The CaM antagonists TFP and CPZ affected growth, hyphae morphology, aerial hyphae development, carotenoids accumulation and sexual development in N. crassa (Figures 3.8-3.11; Figure 3.13; Tables 3.1-3.3). The crystal structure of mammalian CaM has been refined at 2.2 Å, which shows dumbbell shaped molecule connected by a seven-turn α-helix, with an overall length of 65 Å. CaM also contains four Ca2+-binding loops and two short, double- stranded antiparallel β-sheets between pairs of adjacent Ca2+-binding loops referred to as EF-hand structures. In addition, X-ray structure of CaM also shows a large hydrophobic cleft in each half of the molecule, which is responsible for interaction with many of the pharmacological agents known to bind to CaM (Babu et al. 1988). Various naturally occurring compounds isolated from a wide variety of natural sources, including many fungi and plants has CaM-inhibitory properties, belonging to different structural classes such as alkaloid and peptide (Martínez-Luis et al. 2007; Mata et al. 2014). Among the most potent anti-CaM substances, however, the first class of drugs shown to demonstrate this property was the phenothiazines (Levin and Weiss 1976). Inhibition of CaM function by a phenothiazines first demonstrated by Levin and Weiss, and the effect of TFP inhibition on CaM activated enzymes can also be overcome by excess CaM (Levin and Weiss 1976, 1977). These different pharmacological compounds share many common structural features and display a large hydrophobic region, consisting of two or three aromatic rings and a side-chain amino group that is at least four atoms removed from the ring structure (Weiss et al. 1982). Interactions of drugs with CaM appear to involve two kinds of attachments, a hydrophobic interaction between lipophilic portions of the drug and non-polar regions of CaM and an electrostatic interaction occurs between a positively charged amino group on the drug and a negatively charged acidic residue on CaM (Weiss et al. 1982). The mode of action of phenothiazines are not specific for CaM protein (Mori et al. 1980; Wise et al. 1982; Hait and Lee 1985).
The phenothiazine, TFP and CPZ, inhibited the CaM-induced activation of the
(Ca2++Mg2+)-ATPase of erythrocyte membranes (Raess and Vincenzi 1980; Roufogalis 1981). CPZ is about one-fourth less potent an inhibitor of CaM than TFP (Levin and Weiss 1976; Roufogalis 1981; Weiss et al. 1982). This could explain more potent effect of the TFP than CPZ on carotenoid accumulation in N. crassa (Figure 3.11; Table 3.3).
The effect of the CaM antagonists TFP and CPZ in N. crassa was further supported by the reduced expression of the cmd gene (Figure 3.12).
In N. crassa, nine ATPases including seven Ca2+-and cation-ATPases and two cation-ATPases have been identified (Benito et al. 2000). The trm-9 gene encodes a cation-ATPases, homolog of the yeast spf1 gene. The spf1 gene encodes a novel P-type ATPase of S. cerevisiae. Replacement of aspartic acid at 487 position to asparagine leads to compete loss of function of spf1 gene, which suggested that this residue plays an important role as an acceptor of the terminal phosphate of ATP during ATP hydrolysis and energy transduction. The pmr1 gene, which encodes a yeast golgi ion pump, is functionally related to spf1. The spf1 mutant shares many phenotypic features with the pmr1 mutant. Moreover, absence of spf1 gene causes lack of control of HMG-CoA reductase enzyme degradation in sterol production (Cronin et al. 2000; Suzuki 2001). In addition, the PMCA type Ca2+-ATPase, NCA-2 plays a major role in pumping Ca2+ out of the cell and the Δnca-2 knockout mutant shows sensitivity to high concentration of extracellular Ca2+, slow growth and female sterility phenotype (Bowman et al. 2011).
In this work, I generated the ∆trm-9∆nca-2 double mutant and studied its phenotype (Figures 3.14,-3.15). The ∆trm-9∆nca-2 double mutant showed novel colony morphology with less-branched hyphae (Figures 3.16-3.17). The trm-9nca-2 double mutant displayed reduced aerial hyphae, slow growth rate, reduced carotenoid accumulation, increased sensitivity to Ca2+ and UV stress and reduced viability in the acquisition of thermotolerance induced by heat shock temperature (Figures 3.18-3.21;
Figures 3.23-3.27; Table 3.5-3.10). However, the slow growth rate of the trm-9nca- 2 mutant strain was not due to a defect in ergosterol biosynthesis (Figure 3.22).
In N. crassa, growth is manifested by an increase in colony diameter and hyphal front, development of aerial hyphae and biomass accumulation. Hyphae extend at its tip, which is measured as apical growth and radial growth. Biomass is the amount of living matter in at a given time and measured in terms of the dried organic mass, since more than half is constituted by water. Hyphal branching is necessary for efficient colonization and utilization of the substrate upon which N. crassa grows. Aerial hyphae are hyphal
extensions which project above water-air interface and bear the asexual spores (conidia) that are hydrophobic in nature due to the presence of hydrophobin protein. In addition, the characteristic orange pigmentation of N. crassa is due to accumulation of xanthophyll neurosporaxanthin and variable amounts of carotenoids precursors (Harding et al. 1969; Harding and Turner 1981; Díaz-Sánchez et al. 2011). The albino (al) genes al-1, al-2 and al-3 loci found to be structural genes coding for a phytoene dehydrogenase, phytoenesynthetase and GGPP synthetase enzyme and regulated by the white collar-1 (wc-1) required for carotenoid biosynthesis in N. crassa (Harding and Turner 1981). I found here that the cmd, trm-9 and nca-2 play an important role in growth and pigmentation in N. crassa. UV irradiation damages DNA by inducing formation of cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts and cells possess a molecular mechanism to protect DNA from this damage (Sinha and Häder 2002; Cadet et al. 2005). Carotenoids also possess a protective role against oxidative stress by quenching reactive oxygen species (Vershinin 1999). There is an interrelation of heat shock, oxidative stress and thermotolerance in N. crassa. There is a substantial increase in free radicals. O2 in heat-shocked cells compared with non-shocked controls. Exposure of cells to .O2 generating agents results in the induction of proteins specifically required for defense against oxidative damage that leads to production of H2O2 catalyzed by the multiple isoforms of N. crassa superoxide dismutase. H2O2, a deleterious agent, also generates -OH, an even more potent species, via the Haber-Weiss reaction (Kapoor et al.
1990). I found that genetic interaction of trm-9 and nca-2 play an important role in cell survival under Ca2+ and UV stress and exposure to heat-shock temperature in N. crassa.
Thus, in this study, I have shown that cmd, trm-9 and nca-2 genes play an important role in growth, pigmentation and stress-tolerance in N. crassa.
A part of this chapter was published in Genomics and Applied Biology (Laxmi and Tamuli 2015) and presented in “4th Meeting of the Asian Forum of Chromosome and Chromatin Biology on Epigenetic Mechanisms in Development and Disease, 2012” held at Centre for Cellular and Molecular Biology, Hyderabad, India, “8th International Conference on Yeast Biology, 2013” held at Institute of Microbial Technology, Chandigarh, India and International Conference on Disease Biology and Therapeutics, 2014” held at Institute of Advanced Study in Science and Technology, Guwahati, India.
Additional functions of the cmd gene have been discussed in the next Chapter.