3' UTR elements enhance expression of Pgs28, an ookinete protein of Plasmodium gallinaceum.
In Plasmodium parasites the fusion of gametes to form a fertilized zygote and morphogenesis into the motile ookinete are critical developmental stages in the parasite's complex life cycle. In analogous developmental stages of metazoan organisms 3' gene flanking regions are critical in the regulation of gene expression. To determine whether these mechanisms are conserved in the protozoan parasite we studied the 3' gene flanking elements necessary for the expression of Pgs28, the major surface protein of mature zygotes and ookinetes of the chicken malaria Plasmodium gallinaceum. The DNA sequence of the pgs28 3' gene flanking region contains 7 eukaryotic polyadenylation consensus signals (AATAAA/ATTAAA). An unusual 82% T-rich region is located 55 nucleotides upstream of the fifth polyadenylation signal (ATTAAA). The pgs28 mRNA terminates approximately 20 nucleotides from the polyadenylation signal in a poly (A) tail. To determine whether the T-rich region and polyadenylation signals were necessary for Pgs28 protein expression, sexual stage parasites were transfected with plasmids containing deletions of these elements utilizing firefly luciferase (LUC) and beta-glucuronidase (GUS) as markers of transient gene transfection. The parasites were allowed to develop in vitro to the ookinete stage and assayed for enzymatic activity. Cells transfected with plasmids containing deletions of the T-rich region or fifth eukaryotic polyadenylation consensus signal expressed 89 and 92%, less enzymatic activity respectively than those transfected with the full length pgs28 3' gene flanking region. The U-rich element and fifth eukaryotic polyadenylation consensus sequence within the pgs28 3' UTR are therefore necessary for Pgs28 protein expression.
Golightly
L M
LM
Department of Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA. lgolight@mail.med.cornell.edu
Mbacham
W
W
Daily
J
J
Wirth
D F
DF
D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D000953:Antigens, Protozoan; D001483:Base Sequence; D015152:Blotting, Northern; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D005966:Glucuronidase / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D010964:Plasmodium gallinaceum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014162:Transfection
D020413:3' Untranslated Regions; D000953:Antigens, Protozoan; C079421:Pgs28 protein, Plasmodium gallinaceum; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011061:Poly A; D008156:Luciferases; D005966:Glucuronidase