Stage specific expression of poly(malic acid)‐affiliated genes in the life cycle of Physarum polycephalum

Abstract

Polymalic acid is receiving interest as a unique biopolymer of the plasmodia of mycetozoa and recently as a biogenic matrix for the synthesis of devices for drug delivery. The acellular slime mold Physarum polycephalum is characterized by two distinctive growth phases: uninucleated amoebae and multinucleated plasmodia. In adverse conditions, plasmodia reversibly transform into spherules. Only plasmodia synthesize poly(malic acid) (PMLA) and PMLA‐hydrolase (polymalatase). We have performed suppression subtractive hybridization (SSH) of cDNA from amoebae and plasmodia to identify plasmodium‐specific genes involved in PMLA metabolism. We found cDNA encoding a plasmodium‐specific, spherulin 3a‐like polypeptide, NKA48 (spherulin 3b), but no evidence for a PMLA‐synthetase encoding transcript. Inhibitory RNA (RNAi)‐induced knockdown of NKA48‐cDNA generated a severe reduction in the level of PMLA suggesting that spherulin 3b functioned in regulating the level of PMLA. Unexpectedly, cDNA of polymalatase was not SSH‐selected, suggesting its presence also in amoebae. Quantitative PCR then revealed low levels of mRNA in amoebae, high levels in plasmodia, and also low levels in spherules, in agreement with the expression under transcriptional regulation in these cells.

DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial nucleoid of Physarum polycephalum

“…Since plasmodium is a single multinucleate cell, materials injected into a part of it can spread to the entire plasmodium. We first tried an RNAi method by injecting siRNA, as described previously, in P. polycephalum (Haindl and Holler, 2005;Pinchai et al, 2006); however, expression of the targeted genes did not sufficiently decrease in our system. Therefore, we used MOs, which are nucleic acid analogs resistant to nucleases that are able to sustainably inhibit the expression of targeted genes by blocking the access of other molecules for translation and splicing in many organisms (Karkare and Bhatnagar, 2006).…”

Polymalic acid for translational nanomedicine

“…A temporary leakage in the plasmodial membrane may disrupt PMLA synthesis to some extent, but cell lysates of P. polycephalum plasmodia completely block the synthetic activity of malyl polymerase (but not malyl ligase), suggesting a tyrosine kinase–dependent cell injury signaling in deactivating the unidentified PMLA synthetase[ 39 ]. In addition, a plasmodium-specific polypeptide spherulin 3b (11.3 kDa, also named NKA48) assists in the PMLA synthesis, as the knockdown of NKA48 mRNA significantly lowers PMLA concentration [ 45 ]. Nonetheless, the identification of PMLA synthetase in P. polycephalum has been unsuccessful, partially because cell extracts are void of synthetase activity, and purified PMLA from P. polycephalum usually has a M w of 50–300 kDa [ 8 ].…”