Characterization and properties of the spinach chloroplast transcriptionally active chromosome isolated at high ionic strength

Abstract

The transcriptionally active chromosome (TAC) of spinach (Spinacia oleracea L.) chloroplasts has been isolated at a high ionic strength, with low mechanical shearing, by glycerol gradient centrifugation. The properties of the TAC differ from those previously reported for the TAC isolated either from Euglena chloroplasts or from spinach using a low-ionic-strength solubilization medium and gel filtration. The high-salt-isolated TAC is homogenous in density but not in size and contains fewer weakly bound proteins than its lowsalt-isolated homologue. In vitro, it promotes elongation of the RNA chains previously initiated in vivo. Transcription is not limited to the ribosomal DNA. The transcriptional pattern is not strongly affected by the high-salt preparation. Ribonuclease pretreatment of the TAC, prior to the in-vitro transcription, leads to a more than tenfold increase of the transcription activity. These properties are discussed in relation to the structure of the spinach chloroplast chromosome.