ISOLATION AND MOLECULAR CHARACTERIZATION OF TRYPANOSOMATID BASIC NUCLEAR PROTEINS

Najib  M.A. SAYED

Trypanosomatids are causative agents of diseases of great public and international health significance. There is a vast literature on eukaryotic chromatin structure and regulation of gene expression, but little is known about the nuclear organization as well as transcription factors in these parasitic protozoa. When compared with higher eukaryotes, trypanosomatids present behavioral dissimilarities during cell division such as persistence of the nuclear membrane and lack of chromatin condensation. Their gene expression is regulated stage‑specifically, but it is not necessarily achieved by transcriptional control through individual promoters. Multigene transcription units are expressed constitutively throughout the developmental cycle, producing long primary transcripts which are processed by a transcripts which are processed by a trans‑splicing mechanism to yield capped and polyadenylated gene‑length transcripts. While a typical nucleosomic organization of chromatin has been demonstrated, the presence of histone H1 and High Mobility Group (HMG) proteins has not been shown unambiguously in these organisms. Studies on control of transcription through individual promoters have just begun. The role of histone Hl is central in the packaging of DNA into chromatin, while HMGs are thought to activate genes and/or maintain the transcriptionally active state of chromatin in higher eukaryotes.

In efforts to characterize HMG proteins and histone Hl from trypanosomatids, we identified four basic proteins from Leishmania tarentolae using perchloric acid (PCA) or trichloroacetic acid (TCA) extraction procedures. The acid soluble proteins obtained have apparent molecular weights of 10,14, 19 and 22 kDa based on SDS‑PAGE analysis. Amino acid composition and partial N‑terminal sequence data on two of these revealed them to be very Lysine and Alanine Rich proteins, VLAR14 and 22. Degenerate oligonucleotide primers were used to amplify a 108 bq fragment coding for the N‑terminal region of VLAR14. This fragment was used to screen an L.tarentolae genomic library. We isolated five clones encoding for VLAR14 and sequenced its gene. Homologues were identified in seven related trypanosomatids. FASTA searches with the deduced protein show considerable homology to the tail region of histone H1. Polyclonal mouse antisera raised against VLAR14, VLAR22 and a multiple antigenic peptide (MAP) derived from VLAR14 were used to localize both proteins to the nucleus and the flagellum of L.tarentolae.