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Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial genome 总被引:22,自引:8,他引:14
Macey JR; Larson A; Ananjeva NB; Fang Z; Papenfuss TJ 《Molecular biology and evolution》1997,14(1):91-104
Two novel mitochondrial gene arrangements are identified in an agamid
lizard and a ranid frog. Statistical tests incorporating phylogeny indicate
a link between novel vertebrate mitochondrial gene orders and movement of
the origin of light-strand replication. A mechanism involving errors in
light-strand replication and tandem duplication of genes is proposed for
rearrangement of vertebrate mitochondrial genes. A second mechanism
involving small direct repeats also is identified. These mechanisms
implicate gene order as a reliable phylogenetic character. Shifts in gene
order define major lineages without evidence of parallelism or reversal.
The loss of the origin of light-strand replication from its typical
vertebrate position evolves in parallel and, therefore, is a less reliable
phylogenetic character. Gene junctions also evolve in parallel. Sequencing
across multigenic regions, in particular transfer RNA genes, should be a
major focus of future systematic studies to locate novel gene orders and to
provide a better understanding of the evolution of the vertebrate
mitochondrial genome.
相似文献
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Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex 总被引:1,自引:0,他引:1
A novel form of mitochondrial DNA (mtDNA) inheritance has previously been
documented for the blue mussel (Mytilus edulis). Female mussels inherit
their mtDNA solely from their mother while males inherit mtDNA from both
their mother and their father. In males, the paternal mtDNA is
preferentially amplified so that the male gonad is highly enriched for the
paternal mtDNA that is then transmitted from fathers to sons. We
demonstrate that this mode of mtDNA inheritance also operates in the
closely related species M. galloprovincialis and M. trossulus. The
evolutionary relationship between the male and female mtDNA lineages is
estimated by phylogenetic analysis of 455 nucleotides from the large
subunit ribosomal RNA gene. We have found that the male and female lineages
are highly divergent; the divergence of these lineages began prior to the
speciation of the three species of blue mussels. Further, the separation
between the male and female lineages is estimated to have occurred between
5.3 and 5.7 MYA.
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Design and characterization of a hyperstable p16INK4a that restores Cdk4 binding activity when combined with oncogenic mutations 总被引:3,自引:0,他引:3
Cyclin-dependent kinase inhibitor p16(INK4a) is the founding member of the INK4 family of tumor suppressors capable of arresting mammalian cell division. Missense mutations in the p16(INK4a) gene (INK4a/CDKN2A/MTS1) are strongly linked to several types of human cancer. These mutations are evenly distributed throughout this small, ankyrin repeat protein and the majority of them disrupt the native secondary and/or tertiary structure, leading to protein unfolding, aggregation and loss of function. We report here the use of multiple stabilizing substitutions to increase the stability of p16(INK4a) and furthermore, to restore Cdk4 binding activity of several defective, cancer-related mutant proteins. Stabilizing substitutions were predicted using four different techniques. The three most effective substitutions were combined to create a hyperstable p16(INK4a) variant that is 1.4 kcal/mol more stable than wild-type. This engineered construct is monomeric in solution with wild-type-like secondary and tertiary structure and cyclin-dependent kinase 4 binding activity. Interestingly, these hyperstable substitutions, when combined with oncogenic mutations R24P, P81L or V126D, can significantly restore Cdk4 binding activity, despite the divergent features of each destabilizing mutation. Extensive biophysical studies indicate that the hyperstable substitutions enhance the binding activity of mutant p16 through several different mechanisms, including an increased amount of secondary structure and thermostability, reduction in exposed hydrophobic surface(s) and/or a reduced tendency to aggregate. This apparent global suppressor effect suggests that increasing the thermodynamic stability of p16 can be used as a general strategy to restore the biological activity to defective mutants of this important tumor suppressor protein. 相似文献
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Braunstein M Griffin TJ IV Kriakov JI Friedman ST Grindley ND Jacobs WR 《Journal of bacteriology》2000,182(10):2732-2740
Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552'phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552'phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms. 相似文献
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Michael J Gramer Ewald TJ van den Bremer Muriel D van Kampen Amitava Kundu Peter Kopfmann Eric Etter David Stinehelfer Justin Long Tom Lannom Esther H Noordergraaf Jolanda Gerritsen Aran F Labrijn Janine Schuurman Patrick HC van Berkel Paul WHI Parren 《MABS-AUSTIN》2013,5(6):962-973
The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production. The bispecific antibody was generated by mixing the two parental molecules under controlled reducing conditions, resulting in efficient Fab-arm exchange of >95% at kg scale. The reductant was removed via diafiltration, resulting in spontaneous reoxidation of interchain disulfide bonds. Aside from the bispecific nature of the molecule, extensive characterization demonstrated that the IgG1 structural integrity was maintained, including function and stability. These results demonstrate the suitability of this bispecific IgG1 format for commercial-scale manufacturing using standard antibody manufacturing techniques. 相似文献