A simple and rapid PCR-based method to isolate complete small macronuclear minichromosomes from hypotrich ciliates: 5S rDNA and S26 ribosomal protein gene of Oxytricha (Sterkiella) nova.
Hypotrich ciliates present a macronuclear genome consisting of gene-sized instead of chromosome-sized DNA molecules. Exploiting this unique eukaryotic genome feature, we introduce, for the first time in ciliates, a rapid and easy PCR method using telomeric primers to isolate small complete macronuclear DNA molecules or minichromosomes. Two presumably abundant macronuclear DNA molecules, containing ribosomal genes, were amplified from the Oxytricha (Sterkiella) nova complete genome after using this method, and then were cloned and sequenced. The 5S rDNA sequence of O. (S.) nova is the third one reported among hypotrich ciliates; its primary and secondary structure is compared with other eukaryotic 5S rRNAs. The ribosomal protein S26 gene is the first one reported among ciliates. This "End-End-PCR" method might be useful to obtain similar gene-sized macronuclear molecules from other hypotrich ciliates, and, therefore, to increase our knowledge on ribosomal genes in these eukaryotic microorganisms.
Departamento de Microbiología-III, Facultad de Biología, Universidad Complutense, Madrid, Spain.
Sergio
S
Callejas
Juan Carlos
JC
Gutiérrez
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000235:genetics; D002875:Chromosomes / Q000235:genetics*; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics* / Q000302:isolation & purification; D005796:Genes / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016813:Oxytricha / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012341:RNA, Ribosomal, 5S / Q000737:chemistry / Q000235:genetics*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics*; D013997:Time Factors
D016054:DNA, Protozoan; D012341:RNA, Ribosomal, 5S; D012269:Ribosomal Proteins