Mutant/mutation
In the 'promoter-swap' mutant the promoter of fact-l is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060). Transcription of fact-l in  mutant gametocytes was down-regulated compared with wild-type gametocytes.

Protein (function)
FACT is a histone chaperone that has received considerable attention for its function in facilitating RNA transcription and DNA replication by dynamically altering chromatin through modifications of nucleosome structure. FACT is an evolutionarily conserved, abundant nuclear heterodimer/trimer. The large subunit FACT-L, in humans p140h/SPT16, is a homologue of the yeast Spt16/Cdc68 protein (suppressor of Ty). The small subunit of FACT (FACT-S) in human structure specific recognition protein 1 (SSRP1), possesses a C-terminal high-mobility group (HMG) box with an N-terminus homologous to the yeast protein Pob3 (DNA polymerase one binding). In yeast SSRP1 activity is represented by two distinct proteins, Pob3 and Nhp6A (or B) which encodes the missing HMG box. Functionally, both yeast and human FACT act during transcription initiation and displace histones H2A/H2B to effect nucleosome disassembly and facilitate transcription elongation.

BLAST-based homology searches with PBANKA_123220 identified clear homology to the eukaryotic SPT16 protein (e.g. 30% identity, 41% similarity to SPT16 of  Saccharomyces cerevisiae) with an identical arrangement of the Rttp106-like, SPT16 and M24 peptidase domains.

Attempts to disrupt the fact-l gene in P. berghei were unsuccessful (see RMgm-255), indicating an essential role of FACT-L for blood stage development/multiplication

Phenotype
Attempts to disrupt the fact-l gene in P. berghei were unsuccessful (see RMgm-255), indicating an essential role of FACT-L for blood stage development/multiplication.

In the 'promoter-swap' mutant the promoter of fact-l is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060). Transcription of fact-l in  mutant gametocytes was down-regulated compared with wild-type gametocytes.

Phenotype analyses of the promoter-swap mutant indicate that FACT-L plays an important role in the production of fertile male gametes.

The promoter-swap mutant produced normal numbers of female and male gametocytes. Fertility of female gametes is comparable to wild type female gametes as shown by crossing with wild type male gametes. Male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility is strongly reduced, resulting in strongly reduced ookinete formation. Residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony.

Additional information
Similar promoter-swap mutants have been generated in in the P. berghei reference line cl15cy1 (RMgm-652) and in a  reporter reference line of P. berghei that  express GFP constitutively (RMgm-653).

In the same paper a successful 'promoter-swap' mutants are described in which the promoter of fact-l is replaced by the 'asexual blood stage specific’ promoter of PBANKA_091420 that is silent in gametocytes. These mutants are: Δfact-lgam1a, 1044 in cl15cy1; Δfactlgam 1b, 1076cl1, cl3 in 507cl2; Δfact-lgam 1c, 1134cl1,cl2 in 820cl1m1cl1.

Phenotype analyses of a mutant expressing c-myc-tagged FACT-L (RMgm-655) showed that FACT-L is located predominantly in the parasite nucleus with evidence that a small proportion of FACT-L is located in the cytoplasm. Expression was detected in all blood stages with high levels of FACT-L in the nuclei of male gametocytes.

Other mutants
RMgm-255: Unsuccessful attempts to disrupt fact-l
RMgm-652:  An independent promoter-swap mutant in which the promoter of fact-l is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060). This mutant is made in the P. berghei reference line cl15cy1.
RMgm-653: An independent promoter-swap mutant in which the promoter of fact-l is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060). This mutant constitutively expresses GFP.