Characterization of the developmental mutant redA of Dictyostelium discoideum

Author(s)

Daniela Carvalho Gonzalez

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2002-11-25

Examining board members

Aline Maria da Silva; Pio Colepicolo Neto; Luís Eduardo Soares Netto

• Advisor: Aline Maria da Silva

Abstract

The Dictyostelium discoideum redA mutant, obtained by random gene inactivation, exhibits normal growth but has its developmental cycle impaired at tight mound stage. In this study we describe the characterization of this mutant whose defective gene encodes the enzyme NADPH cytochrome P450 reductase (NCPR). NCPR is known to play an essential role in the transfer of reducing equivalents from NADPH to various cytochrome P450 isoforms. We isolated a 2094 bp cDNA that encodes D. discoideum NCPR (DdNCPR) by screening a cDNA library using as probe the mutated gene fragment rescued from redA cells. Analysis of the deduced aminoacid sequence of DdNCPR cDNA shows that it encodes a 631 aminoacid protein with 31% of identity and 50% of similarity with human NCPR. Northern blot analysis showed that DdNCPR mRNA levels is maximum during growth phase and decreases at early stages of the development. After slug stage this mRNA is not detectable. D. discoideum has a single copy of NCPR gene and, as shown by analysis of other NCPR knockout mutants, inactivation of this gene is strongly correlated to the redA phenotype. However, redA, as well as the other NCPR knockout strains, do have growth alterations and in some circumstances they do not show the described developmental defects. Thus, it is possible that one or more proteins be able to compensate for the lack of NCPR in these mutants. Our results also suggest that the redA developmental phenotype might play a role of NCPR on the metabolism of DIF-1, a prime candidate for controlling prestalk and prespore cell differentiation during D. discoideum development. (AU)