Molecular cloning of G1 phase mRNAs from a subtractive G1 phase cDNA library

1993 ◽  
Vol 71 (7-8) ◽  
pp. 372-380 ◽  
Author(s):  
Gin Wu ◽  
Shiawhwa Su ◽  
Tzyy-Yun Tzeng Kung ◽  
R. Curtis Bird
Keyword(s):  
Cell Cycle ◽  
Cdna Library ◽  
Light Chain ◽  
Hela Cells ◽  
Subtractive Hybridization ◽  
S Phase ◽  
G1 Phase ◽  
Chain Gene ◽  
Centrifugal Elutriation ◽  
Light Chain Gene

Many G1-phase-specific mRNAs have been identified from various normal or transformed cells based on serum induction and re-entry into the cell cycle from quiescence. However, these mRNAs may not represent some important genes expressed during G1 phase in continuously cycling cells. The eukaryotic cell cycle possesses two cdk (cyclin-dependent kinase) dependent regulatory gates through which cells pass during late G1 phase and G2 phase of each cycle. Subtractive hybridization was employed to synthesize a high R0t fraction cDNA library enriched in sequences expressed during G1 phase prior to passage through the G1-phase gate. To prepare G1-phase cells from continuously cycling cell populations, G1-phase HeLa cells were collected by centrifugal elutriation and highly synchronous S phase cells were obtained by double thymidine block followed by centrifugal elutriation. A G1-phase subtractive cDNA library was prepared by subtracting G1-phase cDNA with a 10-fold excess of S-phase mRNA. Single-stranded, G1-phase cDNAs were isolated by oligo(dA) chromatography. The library was screened with a high R0t fraction subtractive probe population. Following two rounds of screening, 20 positive clones were obtained. Northern blot analysis indicated that six of these clones were enhanced in expression level during G1 phase when compared with S phase. Nucleotide sequence comparison of each clone with the GenBank data base revealed that hG1.11 was highly homologous (99%) to the apoferritin light chain gene and clones hG1.6, hG1.10, hG1.17, and hG1.18 represented new G1-phase-enriched members of four human ribosomal protein gene families (71–95% homology). The last clone, hG1.1, encoded a highly charged polypeptide not previously identified. Additional study of these G1-phase-enriched mRNAs will be required to determine their role in cell cycle progression and the G1-phase gateway through which cells transit as they proceed through the cell cycle.Key words: cell cycle, G1 phase, subtractive hybridization, cDNA cloning, ribosomal proteins, apoferritin light chain, HeLa cells.

scholarly journals Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

1997 ◽  
Vol 139 (5) ◽  
pp. 1077-1087 ◽  
Author(s):  
Li Yang ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Nuclear Lamina ◽  
Normal Function ◽  
S Phase ◽  
Nuclear Volume ◽  
G1 Phase ◽  
Binding Region ◽  
Recombinant Polypeptide ◽  
Volume Increase

Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane that binds to both lamin B and chromatin and has a putative role in nuclear envelope (NE) organization. We found that microinjection of a recombinant polypeptide comprising the nucleoplasmic domain of rat LAP2 (residues 1–398) into metaphase HeLa cells does not affect the reassembly of transport-competent nuclei containing NEs and lamina, but strongly inhibits nuclear volume increase. This effect appears to be specifically due to lamin binding, because it also is caused by microinjection of the minimal lamin-binding region of LAP2 (residues 298–373) but not by the chromatin-binding domain (residues 1–88). Injection of the lamin-binding region of rat LAP2 into early G1 phase HeLa cells also strongly affects nuclear growth; it almost completely prevents the threefold nuclear volume increase that normally occurs during the ensuing 10 h. Moreover, injection of the fragment during early G1 phase strongly inhibits entry of cells into S phase, whereas injection during S phase has no apparent effect on ongoing DNA replication. Since the lamin-binding fragment of LAP2 most likely acts by inhibiting dynamics of the nuclear lamina, our results suggest that a normal function of LAP2 involves regulation of nuclear lamina growth. These data also suggest that lamina dynamics are required for growth of the NE and for nuclear volume increase during the cell cycle, and that progression into S phase is dependent on the acquisition of a certain nuclear volume.

scholarly journals Fluctuation in radioresponse of HeLa cells during the cell cycle evaluated based on micronucleus frequency

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hiroaki Shimono ◽  
Atsushi Kaida ◽  
Hisao Homma ◽  
Hitomi Nojima ◽  
Yusuke Onozato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Time Lapse ◽  
S Phase ◽  
G1 Phase ◽  
M Phase ◽  
G2 Phase ◽  
The Difference ◽  
Time Lapse Imaging ◽  
First Time

AbstractIn this study, we examined the fluctuation in radioresponse of HeLa cells during the cell cycle. For this purpose, we used HeLa cells expressing two types of fluorescent ubiquitination-based cell cycle indicators (Fucci), HeLa-Fucci (CA)2 and HeLa-Fucci (SA), and combined this approach with the micronucleus (MN) assay to assess radioresponse. The Fucci system distinguishes cell cycle phases based on the colour of fluorescence and cell morphology under live conditions. Time-lapse imaging allowed us to further identify sub-positions within the G1 and S phases at the time of irradiation by two independent means, and to quantitate the number of MNs by following each cell through M phase until the next G1 phase. Notably, we found that radioresponse was low in late G1 phase, but rapidly increased in early S phase. It then decreased until late S phase and increased in G2 phase. For the first time, we demonstrated the unique fluctuation of radioresponse by the MN assay during the cell cycle in HeLa cells. We discuss the difference between previous clonogenic experiments using M phase-synchronised cell populations and ours, as well as the clinical implications of the present findings.

scholarly journals hPOC5 is a centrin-binding protein required for assembly of full-length centrioles

2009 ◽  
Vol 185 (1) ◽  
pp. 101-114 ◽  
Author(s):  
Juliette Azimzadeh ◽  
Polla Hergert ◽  
Annie Delouvée ◽  
Ursula Euteneuer ◽  
Etienne Formstecher ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Drosophila Melanogaster ◽  
Hela Cells ◽  
Binding Protein ◽  
Distal Portion ◽  
S Phase ◽  
G1 Phase ◽  
Evolutionary Divergence ◽  
Distal Half

Centrin has been shown to be involved in centrosome biogenesis in a variety of eukaryotes. In this study, we characterize hPOC5, a conserved centrin-binding protein that contains Sfi1p-like repeats. hPOC5 is localized, like centrin, in the distal portion of human centrioles. hPOC5 recruitment to procentrioles occurs during G2/M, a process that continues up to the full maturation of the centriole during the next cell cycle and is correlated with hyperphosphorylation of the protein. In the absence of hPOC5, RPE1 cells arrest in G1 phase, whereas HeLa cells show an extended S phase followed by cell death. We show that hPOC5 is not required for the initiation of procentriole assembly but is essential for building the distal half of centrioles. Interestingly, the hPOC5 family reveals an evolutionary divergence between vertebrates and organisms like Drosophila melanogaster or Caenorhabditis elegans, in which the loss of hPOC5 may correlate with the conspicuous differences in centriolar structure.

scholarly journals Structure and sequence of the myosin alkali light chain gene expressed in adult cardiac atria and fetal striated muscle.

1988 ◽  
Vol 263 (25) ◽  
pp. 12669-12676 ◽  
Author(s):  
P J Barton ◽  
B Robert ◽  
A Cohen ◽  
I Garner ◽  
D Sassoon ◽  
...  
Keyword(s):  
Light Chain ◽  
Striated Muscle ◽  
Chain Gene ◽  
Light Chain Gene

Structure of extrachromosomal circular DNAs generated by immunoglobulin light chain gene rearrangements

1991 ◽  
Vol 27 (1) ◽  
pp. 19-23 ◽  
Author(s):  
Toshiyasu Hirama ◽  
Sunao Takeshita ◽  
Yataroh Yoshida ◽  
Hideo Yamagishi
Keyword(s):  
Light Chain ◽  
Chain Gene ◽  
Gene Rearrangements ◽  
Immunoglobulin Light Chain ◽  
Light Chain Gene ◽  
Circular Dnas

NEUROFERRITINOPATHY IN A JAPANESE FAMILY WITH A DUPLICATION IN THE FERRITIN LIGHT CHAIN GENE

Neurology ◽  
2008 ◽  
Vol 70 (Issue 16, Part 2) ◽  
pp. 1493-1494 ◽  
Author(s):  
E. Ohta ◽  
T. Nagasaka ◽  
K. Shindo ◽  
S. Toma ◽  
K. Nagasaka ◽  
...  
Keyword(s):  
Light Chain ◽  
Chain Gene ◽  
Japanese Family ◽  
Light Chain Gene
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