We have similarly observed a major reduction in SP600125 SICAvar transcript signals in global microarray profiles from parasites grown in splenectomized animals or in vitro culture. The absence of full-length SICAvar signals on northern blots with SICA ring-stage samples is likely to be attributed to some degree of transcriptional repression in the SICA parasites, and may involve epigenetic mechanisms comparable to those being discovered with regards to var gene regulation in P. falciparum. Importantly, our data supports the proposition that the NSC-718781 spleen is critical for SICAvar transcription, and, we predict, the accumulation and maintenance of full-length SICAvar transcripts targeted for translation. qRT-PCR data confirming the presence of PCR-targeted transcript segments, suggests multifactorial synergistic and well-honed molecular processes. We predict that transcriptional control mechanisms as well as posttranscriptional gene-silencing tactics may result in the stagespecific maintenance of the low level of SICAvar transcript sequences that are detectable by qRT-PCR in the absence of the spleen. The natural turnover of RNA observed in the P. knowlesi intra-erythrocytic development cycle from SICA parasites does not simply result in degraded RNA. This point is illustrated by the presence of as many as 22 SICAvar transcripts represented in a Pk11+ cDNA library produced with RNA from late trophozoite and early schizont-stage parasites. and. It is noteworthy that this set of cDNAs did not have a predominant sequence, and in fact, only four corresponded to the 205 SICAvar allele known to produce protein in the Pk11+ parasites. Furthermore, the putative products encoded by the majority of these cDNAs were not identified via gene ID hits in the LC-MS/MS experiments reported here in Figure 5. These SICAvar gene transcripts clearly were not destined to be translated. Hints of translational repression have also been revealed in principle by evidence that SICAvar transcripts detected by microarrays are not 
necessarily expressed as protein. As postulated earlier for SICA parasites, it appears that some form of SICAvar RNA posttranscriptional gene silencing mechanism may be operating in both SICA and SICA parasites to achieve translational repression. Additional research is warranted in this area. Translational repression has been described for the P. falciparum var2csa gene and in Plasmodium sexual stage biology. This field remains a relatively new area of exploration in Plasmodium parasites and malaria infections. A high degree of mRNA turnover in P. falciparum has also been noted on a genome-wide scale, especially in the early intra-erythrocytic cycle. We suspect that both processed SICAvar mRNA and putative SICAvar non-coding transcript sequences are being detected in our RT-PCR experiments and on northern blots. Partial var gene sense and antisense non-coding RNAs have been identified in P. falciparum and confirmed to be products of a bi-directional promoter in the var gene intron. Whether a functional promoter exists in SICAvar genes within their comparable ��final�� intron or within other introns, remains to be determined, reviewed in 1. It is worth noting in this regard that the final intron has conserved motifs within the SICAvar gene family. Sense and antisense non-coding RNAs are now known to be common in P. falciparum. Their functions remain largely unexplored but they are predicted to be important for gene regulation. Importantly, our data raise new questions regarding the molecular processes relating to variant antigen gene expression in a living host compared to in vitro culture environments. Some splenic and potentially other host factors may be synergistically important for the natural in vivo regulation of the SICAvar gene family. Similar factors may also apply for var gene expression.