EFFECTS OF CELL CYCLE REGULATORY GENES ON THE UTILIZATION OF HISTONE H3/H4 PROMOTERS IN ALFALFA AND MAIZE PROTOPLASTS

Setenci, Fahriye
M.Sc., Department of Biotechnology
Supervisor: Prof. Dr. Meral YUCEL
Co-supervisor: Assoc. Prof. Dr. Huseyin A. Oktem
January 1997, 107 pages

Different from eukaryotes, plants have the capacity to reactivate the cell division cycle at fully differentiated histological stage. Physiological factors that initiate the activation of cell division can be experimentally monitored in in vitro cultured plant cell upon the application of mitosis.

Replication-dependent histone genes are the most predominant class of histone proteins and their expression increases during the S-phase of cell cycle. Several histone genes and their corresponding sequences have been isolated and characterized from different plant species.

In this study, we describe the use of a histone-GUS expression casette as indicative of S-phase activation upon co-transformation using a plasmid carrying cell cycle regulatory genes. Alfalfa (Medicago sativa) cyclins and cdc2 genes were introduced into alfalfa and maize protoplasts. The protoplasts were isolated from alfalfa A2 suspension culture and co-transformed using plasmids carrying the alfalfa histone H3.1 promoter-GUS and cDNAs of the cell cycle genes Mscyc1 and Mscdc2 which are under the control of the CaMV 35S promoter by PEG-mediated direct DNA uptake method. Similarly a maize suspension culture of HE/89 protoplasts were transformed using a wheat H4-GUS sequence which was fused to one of the expression cassettes of cell cycle genes. Samples were taken 2 and 3 days after transformation; the progression of S-phase activation was monitored using GUS enzyme (fluorometric) activity assay.

The preliminary data suggest that the replication-dependent histone gene promoters when fused to marker genes, can be used to monitor the effects of cell cycle genes which are overexpressed.

Key words: Cell cycle, cyclin, cdc2, MPF, histones, GUS, alfalfa, maize.