Cloning novel alfalfa cyclin sequences--a RACE-PCR approach.
Cyclins are a complex group of proteins involved in regulation of the eukaryotic cell division cycle via their interaction with cyclin dependent kinases (Cdks). Cyclin gene sequences have been cloned from a number of plant species, including alfalfa, but the diversity of these genes suggests that there are many plant cyclins which have yet to be characterized. A RACE-PCR strategy has been adopted for cloning cyclin gene sequences expressed during direct somatic embryogenesis in alfalfa. RT-PCR with nested degenerate primers was used to amplify the highly conserved "cyclin box" region of a novel A-like cyclin mRNA sequence expressed after induction of somatic embryogenesis. The sequence of this PCR product was used to design primers for 5'- and 3'-RACE protocols. 5'-RACE using a modified SLIC (single strand ligation to single stranded cDNA) procedure revealed considerable sequence heterogeneity in the N-terminal region of the coding sequence with several closely related sequences apparent. Conventional 3'-RACE generated a single cyclin sequence. The complete coding sequence of one member of this A-like cyclin subgroup has been obtained by this RACE strategy and confirmed by PCR amplification and sequencing of alfalfa genomic DNA.
Russinova
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Norman Borlaug Institute for Plant Science Research, De Montfort University, Scraptoft, Leicester, UK.
Slater
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A
Atanassov
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AI
Elliott
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MC
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016213:Cyclins / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D017343:Genes, Plant; D000455:Medicago sativa / Q000235:genetics*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017386:Sequence Homology, Amino Acid
D016213:Cyclins; D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins