Related Experiment Videos
Expression of mRNAs encoding mammalian chromosomal proteins HMG-I and HMG-Y during cellular proliferation
K R Johnson1, J E Disney, C R Wyatt
1Program in Genetics and Cell Biology, Washington State University, Pullman 99164.
Abstract:
The high mobility group chromosomal proteins HMG-I and HMG-Y are closely related isoforms that are expressed at high levels in rapidly dividing, undifferentiated mammalian cells. We analyzed HMG-I/Y mRNA levels at various cell cycle stages in murine NIH/3T3 fibroblasts partially synchronized by seeding from quiescent, contact-inhibited cultures. Flow microfluorometric analysis of DNA content demonstrated a comparable degree of synchronization in such seeded NIH/3T3 cell populations as is obtained by serum deprivation or other means and has the added advantage of avoiding the use of possibly detrimental inhibitors or metabolic starvation to induce such synchrony. We show that HMG-I/Y mRNA levels gradually increase in NIH/3T3 cells during the first 16 h after seeding (G0/G1 to late S phase), but thereafter remain constant, in contrast to the cell cycle-regulated expression of the histone H3 gene. Although there is a 6-fold increase in HMG-I/Y expression during the transition from quiescent to proliferating NIH/3T3 cells, there is a much greater difference in expression (15- to 50-fold) among different cell types, possibly related to their state of differentiation. The HMG-I/Y mRNAs appear to be very stable; there was no decrease in their levels 6 h after actinomycin D transcription termination. The proportion of HMG-I to HMG-Y mRNAs was greater in the human than in the murine cells examined, appeared to be greater in proliferating than in quiescent cells, and did not always correspond with the HMG-I to HMG-Y protein ratio.
Insights
High mobility group chromosomal proteins HMG-I and HMG-Y (HMG-I/Y) mRNA levels increase during cell division in NIH/3T3 fibroblasts. These HMG-I/Y mRNAs are stable and show varied expression across cell types, correlating with differentiation states.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- High mobility group chromosomal proteins HMG-I and HMG-Y are isoforms crucial for rapidly dividing cells.
- Their expression patterns in relation to the cell cycle and differentiation are not fully understood.
Purpose of the Study:
- To analyze HMG-I/Y mRNA levels during different cell cycle stages in synchronized murine NIH/3T3 fibroblasts.
- To investigate the stability of HMG-I/Y mRNAs and compare their expression across various cell types and differentiation states.
Main Methods:
- Partial synchronization of NIH/3T3 fibroblasts by seeding from quiescent cultures.
- Flow microfluorometric analysis of DNA content for cell cycle staging.
- Measurement of HMG-I/Y mRNA levels using quantitative techniques.
- Assessment of mRNA stability via actinomycin D treatment.
Main Results:
- HMG-I/Y mRNA levels gradually increased from G0/G1 to late S phase in synchronized NIH/3T3 cells.
- Expression showed a 6-fold increase from quiescent to proliferating states, with greater differences (15- to 50-fold) among distinct cell types.
- HMG-I/Y mRNAs demonstrated high stability, with no significant decrease after actinomycin D treatment.
- The ratio of HMG-I to HMG-Y mRNA was higher in human than murine cells and in proliferating versus quiescent cells.
Conclusions:
- HMG-I/Y mRNA expression is regulated during the cell cycle and significantly influenced by the differentiation state of mammalian cells.
- The stability of HMG-I/Y mRNAs suggests a role in maintaining cellular function during proliferation and differentiation.