Entamoeba histolytica: cloning and expression of the poly(A) polymerase EhPAP.
In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability, and translation. Here we identified and cloned a gene codifying for a putative nuclear poly(A) polymerase (EhPAP) in Entamoeba histolytica. Protein sequence alignments with eukaryotic PAPs showed that EhPAP has the RNA-binding region and the PAP central domain with the catalytic nucleotidyl transferase domain described for other nuclear PAPs. Recombinant EhPAP expressed in bacteria was used to generate specific antibodies, which recognized two EhPAP isoforms of 60 and 63kDa in nuclear and cytoplasmic extracts by Western blot assays. RT-PCR assays showed that EhPap mRNA expression varies in multidrug-resistant trophozoites growing in different emetine concentrations. Moreover, EhPap mRNA expression is about 10- and 7-fold increased in G1 and S phase, respectively, through cell cycle progression. These results suggest the existence of a link between EhPAP expression and MDR and cell cycle regulation, respectively.
García-Vivas
Jessica
J
Programa Institucional de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía del IPN, Guillermo Massieu Helguera #239, Fracc. La Escalera, Ticomán, Mexico D.F. 07300, Mexico.
López-Camarillo
César
C
Azuara-Liceaga
Elisa
E
Orozco
Esther
E
Marchat
Laurence A
LA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry; D018432:Drug Resistance, Multiple / Q000502:physiology; D004748:Entamoeba histolytica / Q000145:classification / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
D016054:DNA, Protozoan; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011062:Polynucleotide Adenylyltransferase