Structural and functional characterizations of mung bean mitochondrial nucleoids

Abstract

Mitochondrial nucleoids isolated from mung bean seedlings exhibited a chromatin-like structure associated with a membrane component. A similar structure, which underwent discrete changes during cotyledon development, was identified in situ. Isolated nucleoids consisted of essentially the same phospholipids, including cardiolipin, as whole mitochondria and proteins of inner- and outer-mitochondrial-membrane origin. Actin was consistently found with mitochondrial nucleoids prepared with different detergent concentrations. Formaldehyde cross-linking of cytochalasin B- and proteinase K-treated mitochondria further revealed that actin was associated with DNA in nucleoids. Mitochondrial nucleoids were self-sufficient in directing DNA synthesis in vitro in a pattern mimicking mtDNA synthesis in isolated mitochondria. In pulse-field gel electrophoresis, newly synthesized mtDNA separated into two major components, well-bound and fast-moving forms. Nucleoids DNA synthesis was resistant to aphidicolin but sensitive to N-ethylmaleimide, which indicates that a γ-type DNA polymerase was responsible for this activity. Mitochondrial nucleoids were capable of self-directed RNA transcription in a non-random fashion in vitro. Consistent with and complementary to results from fungi and human cells done mostly in situ, our present work helps to establish the important paradigm that mitochondrial nucleoids in eukaryotes are more than mere mtDNA compaction and segregation entities but are centers of mtDNA maintenance and expression.

Characterization of the Structure and DNA Complexity of Mung Bean Mitochondrial Nucleoids

“…5) as described (Dai et al, 1991). Mitochondrial nucleoids were then purified exactly as described in our previous report (Dai et al, 2005).…”

“…Pulsed-field gel electrophoresis PFGE analyses of mitochondria and mitochondrial nucleoids were conducted exactly as described in our previous reports (Dai et al, 2005). The amounts of mitochondria and mitochondrial nucleoids used for PFGE were 200 μg and 20 μg, respectively.…”

“…Initially, nucleoids were defined morphologically as DNA-containing foci visualized inside mitochondria (Kuroiwa, 1982;Nass et al, 1965;Stevens, 1982) or chloroplasts (Eva and Chiang, 1984;Kuroiwa et al, 1982). Later, mitochondrial nucleoids were isolated at a defined density in gradient centrifugation and appeared to retain their morphological structure in vitro (Dai et al, 2005;Miyakawa et al, 1987;Newman et al, 1996;Suzuki et al, 1982). Spelbrink et al (2001) showed that nucleoids in mammalian cells are stable assemblies of multiple mitochondrial proteins with mtDNA.…”

“…Spelbrink et al (2001) showed that nucleoids in mammalian cells are stable assemblies of multiple mitochondrial proteins with mtDNA. In so far as DNA compaction, replication, and RNA transcription are concerned, mitochondrial nucleoids are roughly analogous to cell nuclei (Dai et al, 2005;Kuroiwa, 1982;Miyakawa et al, 1996;Sakai et al, 2004).…”

“…Regardless of its genomic size or its restriction-endonuclease-generated genomic map (circular or linear), when fractionated by pulsed-field gel electrophoresis (PFGE) most plant mtDNA migrates as a smear zone corresponding to 50 to 200 kb linear DNA molecules. However, much of the mtDNA from all plants remains in the well at the origin of electrophoresis (André and Walbot, 1995;Backert et al, 1997;Bendich, 1996;Dai et al, 2005;Maleszka et al, 1991;Narayanan et al, 1993;Oldenburg and Bendich, 1996;Sakai et al, 1999).…”