Additional remarks phenotype | Mutant/mutation
The mutant expresses a mutated form of HAP2/GCS1 lacking the trans-membrane (TM) domain and the entire C-terminal region downstream from the TM domain (amino acids 680-827).
Protein (function)
In Arabidopsis the male-specific sterility protein HAP2 has been identified by a genetic screen (HAP mutants: containing haploid-disrupting (hapless) mutations; Johnson M.A. et al., 2004, Genetics 168, 971-82) . A HAP2 family member called GCS1 (for generative cell-specific) was subsequently identified in a screen for lily genes whose transcripts were up-regulated in sperm (generative cells) . HAP2 is conserved and members were found in rice, Chlamydomonas, a red alga, a slime mold, Plasmodium falciparum, and Leishmania major.
Phenotype analyses of Plasmodium mutants lacking expression of HAP2/GCS1 (RMgm-155, RMgm-156) indicate a role of HAP/GCS1 in fertilisation. Female gametes are fertile, whereas male gametes are sterile. Male gametes are able to attach to female gametes and form tight prefusion membrane attachments but the membranes of the gametes do not merge or fuse. As a result no zygotes/ookinetes are formed.
Phenotype
The phenotype of the -TM/C KI mutant is comparable to mutants lacking expression of the complete HAP2/GCS1 protein (see RMgm-155, RMgm-156). Whereas female gametes are formed, no ookinetes are produced which indicate the absence of fertilization and the importance of the TM and C-terminal region (amino acids 680-827) for a functional HAP/GCS1 protein. Mutants which express HAP2/GCS1 lacking only the C-terminal region downstream from the TM domain (RMgm-606) showed normal ookinete formation, indicating the importance of the TM domain for a functional HAP2/GCS1 protein.
Additional information
The phenotype has not been analyzed in detail. No information is provided on production of gametocytes and gametes, the process of fertilization or gamete fusion. The effect of the mutation has only been analyzed by quantification of ookinete production.
A mutant has been generated in which the endogenous hap2/gsc1 gene was replaced by a wild type copy of hap2/gsc1 (mutant PbGCS1 Full KI). This mutant was used as a control to analyse the effect of the genetic modification of the locus on male fertility. The DNA construct used to generate the PbGCS1 Full KI line served as PCR template for preparing the DNA constructs used to generate the -HG (RMgm-605), -C (RMgm-606) and -TM/C mutant lines.
Other mutants
RMgm-155: An independent mutant lacking expression of HAP2/GCS1.
RMgm-156: A mutant lacking expression of HAP2/GCS1.
RMgm-157: A mutant expressing a GFP-tagged form of HAP2/GCS1
RMgm-167: A mutant expressing a GFP-tagged form of HAP2/GCS1
RMgm-158: A mutant expressing GFP under the control of the hap2/gcs1 promoter
RMgm-605: A mutant expressing a mutated form of HAP2/GCS1 lacking the HAP2-GCS1 (HG) domain
RMgm-606: A mutant expressing a mutated form of HAP2/GCS1 lacking the entire C-terminal region downstream from the TM domain
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