[Activation of the ribosomal gene during blast transformation of human lymphocytes].
BACKGROUND
Natural lectin, phytohemagglutinin, initiates the transformation of normally quiescent T lymphocytes into proliferating lymphoblast-like cells. Recently we have shown that the transformation is accompanied by strong promotion of ribosomal RNA synthesis and by phosphorylation of its activator, initiating factor UBF, both culminating in a synthetic phase of the first cell division cycle. In contrast we have revealed that the UBF gene was activated and its transcription culminated in the early G1 phase. We examined three possible delaying mechanisms: the kinetics of unwinding of rDNA chromatin, the kinetics of transcription of genes coding for the second initiating factor, SL1 complex, and the kinetics of the translation of UBF protein product.


RESULTS
Up to 48 hrs following the addition of phytohemagglutinin to the growth medium, we monitored structural changes in the rDNA chromatin using indirect antiUBF immunofluorescence. The data indicated an increased number of separated transcriptional units during the G1 phase of the first cycle. In a time interval of up to 70 hrs we measured the mRNA levels of four constituents of SL1 complex: TAF110, TAF63, TAF48 and TBP using the RT-PCR method. We found a close correlation between the kinetics of the transcription of UBF and SL1 genes and the maximal rate in the early G1 phase. Using metabolic labelling with 35S methionine/cysteine we monitored the translation of UBF protein in PHA stimulated lymphocytes. The data suggested that UBF translation, starting in the S phase, paralleled chromosomal DNA replication.


CONCLUSIONS
During the transformation of normal T lymphocytes into proliferating blast-like cells, the multicopy rDNA gene unwinds in the G1 phase of the first cycle forming individual transcriptional units. Genes coding for factors which initiate synthesis of ribosomal precursors are activated in the early G1 phase. The G1 synthesis of ribosomal RNA is accelerated by phosphorylation of the hypophosphorylated UBF pool. As blastic transformation develops UBF translation is triggered in the S phase and neosynthesized UBF, activated by phosphorylation, pushes the synthesis of ribosomal precursors to maximal efficiency. The process of blastic transformation interferes throughout the entire prolonged G1 phase of the first cell division cycle.
Kalousek
I
I
Oddĕlení bunĕcné biochemie UHKT, Praha. ivan.kalousek@uhkt.cz
Hlavácová
M
M
Röselová
P
P
Otevrelová
P
P
D004275:DNA, Ribosomal / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects / Q000235:genetics*; D010835:Phytohemagglutinins / Q000494:pharmacology; D035463:Pol1 Transcription Initiation Complex Proteins / Q000235:genetics; D012335:RNA, Ribosomal / Q000096:biosynthesis / Q000235:genetics*
D004275:DNA, Ribosomal; D010835:Phytohemagglutinins; D035463:Pol1 Transcription Initiation Complex Proteins; D012335:RNA, Ribosomal; C063844:transcription factor UBF