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Reverse transcription-polymerase chain reaction construction of plasmid-based, full-length cDNA libraries from Leishmania infantum for in vitro expression screening. 2003

Bernard Couvreur, and Alex Bollen, and Dominique Le Ray, and Jean-Claude Dujardin
Laboratory of Applied Genetics, IBMM, Free University of Brussels, Gosselies, Belgium.

We describe a streamlined reverse transcription-polymerase chain reaction methodology for constructing full-length cDNA libraries of trypanosomatids on the basis of conserved sequences located at the 5' and 3'ends of trans-spliced mRNAs. The amplified cDNA corresponded to full-length messengers and was amenable to in vitro expression. Fractionated libraries could be rapidly constructed in a plasmid vector by the TA cloning method (Invitrogen). We believe this is useful when there are concerns over the use of restriction enzymes and phage technology as well as in cases where expression of proteins in their native conformation is desired.

UI MeSH Term Description Entries
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012333 RNA, Messenger RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm. Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated
D015723 Gene Library A large collection of DNA fragments cloned (CLONING, MOLECULAR) from a given organism, tissue, organ, or cell type. It may contain complete genomic sequences (GENOMIC LIBRARY) or complementary DNA sequences, the latter being formed from messenger RNA and lacking intron sequences. DNA Library,cDNA Library,DNA Libraries,Gene Libraries,Libraries, DNA,Libraries, Gene,Libraries, cDNA,Library, DNA,Library, Gene,Library, cDNA,cDNA Libraries
D016053 RNA, Protozoan Ribonucleic acid in protozoa having regulatory and catalytic roles as well as involvement in protein synthesis. Protozoan RNA
D016054 DNA, Protozoan Deoxyribonucleic acid that makes up the genetic material of protozoa. Protozoan DNA
D017931 DNA Primers Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide
D018314 Leishmania infantum A parasitic hemoflagellate of the subgenus Leishmania leishmania that infects man and animals and causes visceral leishmaniasis (LEISHMANIASIS, VISCERAL). Human infections are confined almost entirely to children. This parasite is commonly seen in dogs, other Canidae, and porcupines with humans considered only an accidental host. Transmission is by Phlebotomus sandflies. Leishmania (Leishmania) infantum,Leishmania donovani chagasi,Leishmania donovani infantum,Leishmania (Leishmania) chagasi,Leishmania chagasi,Leishmania chagasi chagasi,Leishmania chagasi subsp. Chagasi,Leishmania infantum chagasi,Leishmania leishmania infantum,chagasi chagasi, Leishmania,chagasi, Leishmania,chagasi, Leishmania chagasi,chagasi, Leishmania infantum,donovani chagasi, Leishmania,donovani infantum, Leishmania,infantum chagasi, Leishmania,infantum, Leishmania donovani,infantum, Leishmania leishmania,leishmania infantum, Leishmania
D020133 Reverse Transcriptase Polymerase Chain Reaction A variation of the PCR technique in which cDNA is made from RNA via reverse transcription. The resultant cDNA is then amplified using standard PCR protocols. Polymerase Chain Reaction, Reverse Transcriptase,Reverse Transcriptase PCR,PCR, Reverse Transcriptase,Transcriptase PCR, Reverse
D022821 RNA Splice Sites Nucleotide sequences located at the ends of EXONS and recognized in pre-messenger RNA by SPLICEOSOMES. They are joined during the RNA SPLICING reaction, forming the junctions between exons. Splice Acceptor Site,Splice Donor Site,3' Splice Site,5' Splice Site,Alternative Splice Sites,Cryptic Splice Sites,3' Splice Sites,5' Splice Sites,Acceptor Site, Splice,Acceptor Sites, Splice,Alternative Splice Site,Cryptic Splice Site,Donor Site, Splice,Donor Sites, Splice,Splice Acceptor Sites,Splice Donor Sites,Splice Site, 3',Splice Site, 5',Splice Site, Alternative,Splice Site, Cryptic,Splice Sites, 3',Splice Sites, 5',Splice Sites, Alternative,Splice Sites, Cryptic

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