Nucleoside diphosphate kinase from the parasitic nematode Brugia malayi.
Using a reverse transcription-polymerase chain reaction (RT-PCR) procedure that exploited the presence of a conserved 22-nucleotide spliced leader (SL) sequence that is trans-spliced to the 5' end of nematode transcripts, a novel Brugia malayi (Bm) infective-stage SL cDNA expression library was constructed and characterized. The library was immunoscreened with rabbit anti-infective-stage antibodies (Ab) and an immunodominant clone, BmG4-7, was identified and characterized. BmG4-7 contained a full-length cDNA that had significant sequence similarity to nucleoside diphosphate kinase (NDK)-encoding sequences reported from a number of species, including Drosophila melanogaster and humans. BmNDK was found to be constitutively transcribed during all stages of parasite development. An anti-BmNDK Ab was used to immunostain a Western blot of extracts from adult and larval parasites. The Ab specifically recognized a 17.5-kDa molecule in all of the parasite extracts. Molecular modeling of the BmNDK showed several regions surrounding the conserved catalytic site that may be important in the design of drugs specific for the disruption of NTP synthesis in filarial parasites.
Ghosh
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I
Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
Raghavan
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N
FitzGerald
P C
PC
Scott
A L
AL
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017931:DNA Primers; D018076:DNA, Complementary; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D017238:Genes, Helminth; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D012326:RNA Splicing; D018510:RNA, Helminth / Q000378:metabolism; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014158:Transcription, Genetic
D000906:Antibodies; D017931:DNA Primers; D018076:DNA, Complementary; D018510:RNA, Helminth; D011994:Recombinant Proteins; D009701:Nucleoside-Diphosphate Kinase