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Differential localization of nuclear-encoded tRNAs between the cytosol and mitochondrion in Leishmania tarentolae

Published online by Cambridge University Press:  11 January 2002

STEPHEN T. KAPUSHOC ,
JUAN D. ALFONZO  and
LARRY SIMPSON
STEPHEN T. KAPUSHOC
Affiliation:
Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California, 90095-1662, USA
JUAN D. ALFONZO
Affiliation:
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California, 90095-1662, USA
LARRY SIMPSON
Affiliation:
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California, 90095-1662, USA Howard Hughes Medical Institute, University of California, Los Angeles, California, 90095-1662, USA
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Abstract

All mitochondrial tRNAs of the kinetoplastid protozoan Leishmania tarentolae are encoded in the nucleus and are imported from the cytosol into the mitochondrion. We previously reported the partitioning of five tRNAs and found that all were shared between the two compartments to different extents. To increase our knowledge of the tRNAs of this organism, and to attempt to understand the signals involved in their subcellular localization, a method to RT-PCR amplify new tRNAs was developed. Various tRNAs were 3′ polyadenylated and reverse transcribed with a sequence-tagged primer. The cDNA was tagged by ligation to an anchor oligonucleotide, and the resulting double-tagged cDNA was amplified by PCR. Four new tRNAs were obtained, bringing to 20 the total number of L. tarentolae tRNAs identified to date. The subcellular localization of 17 tRNAs was quantitatively analyzed by two-dimensional gel electrophoresis and northern hybridization. In general, the previously suggested operational classification of tRNAs into three groups (mainly cytosolic, mainly mitochondrial, and shared between the two compartments) is still valid, but the relative abundance of each tRNA in the cytosol or mitochondrion varied greatly as did the level of expression.

Keywords

Leishmania tarentolaeoligonucleotide ligationRNA quantitationsubcellular localizationtRNA importation

Information

Type
Research Article
Information
RNA , Volume 8 , Issue 1 , January 2002 , pp. 57 - 68
Copyright
2002 RNA Society

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