Preparation of DNA and RNA from Trypanosoma brucei
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Last Update: 2021-02-12
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Source: Internet
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Author: User
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The protozoan
Trypanosoma brucei
is the most destructive parasite of domestic livestock in sub-Saharan Africa. The parasite lives extracellularly in its two hosts; alternately in the bloodstream of a mammal, and in the midgut and subsequently the salivary glands of the tsetse fly (
Glossina
spp.) vector. Most laboratory work has concentrated on two of these life cycle stages; rodent-adapted blood- stream forms propagated by syringe passage, and cultured procyclic forms representing the form found in the tsetse fly midgut. The blood- stream form has been intensively studied in order to describe and elucidate the control of antigenic variation that occurs through successive use of a series of genes, each encoding an antigenically distinct variant specific glycoprotein (VSG) (
1
,
2
). In this context VSGs were the first parasite antigens to be characterized at the level of c
DNA
and genomic clones (
3
–
5
). Such a characterization is, of course, dependent on the ability to obtain undegraded RNA of high quality. The use of VSG
cDNA
s to probe Southern blots of genomic DNA provided the demonstration that in most cases antigenic variation is the result of the duplicative transposition of a VSG gene to an expression site (
6
–
8
). Subsequently Northern blotting (
9
), primer extension (
9
–
11
), and S1 mapping (
11
) of RNA were used to demonstrate the presence of a mini-exon at the 5′ end of all mRNAs, leading to the discovery of trans-splicing.
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