Nucleotide sequences of nuclear tRNACys genes from Nicotiana and Arabidopsis and expression in HeLa cell extract

1996 ◽  
Vol 32 (3) ◽  
pp. 549-552 ◽  
Author(s):  
Carsten Urban ◽  
Kathleen N. Smith ◽  
Hildburg Beier
Keyword(s):  
Hela Cell ◽  
Cell Extract ◽  
Nucleotide Sequences ◽  
Hela Cell Extract

Transcription factor binding is limited by the 5'-flanking regions of a Drosophila tRNAHis gene and a tRNAHis pseudogene

1984 ◽  
Vol 4 (12) ◽  
pp. 2714-2722
Author(s):  
L Cooley ◽  
J Schaack ◽  
D J Burke ◽  
B Thomas ◽  
D Söll
Keyword(s):  
Complex Formation ◽  
Hela Cell ◽  
Cell Extract ◽  
Deletion Analysis ◽  
Stable Complex ◽  
Flanking Sequence ◽  
Factor Binding ◽  
Real Gene ◽  
Hela Cell Extract ◽  
Flanking Regions

We determined the sequence of a Drosophila tRNA gene cluster containing a tRNAHis gene and a tRNAHis pseudogene in close proximity on the same DNA strand. The pseudogene contains eight consecutive base pairs different from the region of the bona fide gene which codes for the 3' portion of the anticodon stem of tRNAHis. The tRNAHis gene is transcribed efficiently in Drosophila Kc cell extract, whereas the pseudogene is not. The pseudogene is also a much poorer competitor than the real gene in a stable transcription complex formation assay, even though the sequence alteration in the pseudogene does not affect the sequence or spacing of the putative internal transcription control regions. Recombinant clones were constructed in which the 5'-flanking regions are exchanged. The transcription efficiencies and competitive abilities of the recombinant clones resemble those of the genes from which the 5' flank was derived; for example, the tRNAHis pseudogene with the 5'-flanking sequence of the tRNAHis gene is now efficiently transcribed. Deletion analysis of the pseudogene 5' flank failed to uncover an inhibitory element. Deletion analysis of the real gene showed very high dependence on the presence of the wild-type 5'-flanking sequence for factor binding to the internal control regions and stable complex formation. The 5'-flanking sequence of a Drosophila tRNAArg gene active in the Drosophila Kc cell extract does not restore transcriptional activity or stable complex formation. The tRNAHis gene and pseudogene behave atypically in HeLa cell extract. Both genes compete for HeLa transcription factors, but neither of them is efficiently transcribed. Removal of the 5'-flanking sequences of each gene and replacement with various sequences, including the tRNAArg gene 5' flank, does not allow increased transcription in HeLa cell extract.

MPM-2 antibody-reactive phosphorylations can be created in detergent-extracted cells by kinetochore-bound and soluble kinases

1997 ◽  
Vol 110 (17) ◽  
pp. 2013-2025 ◽  
Author(s):  
L. Renzi ◽  
M.S. Gersch ◽  
M.S. Campbell ◽  
L. Wu ◽  
S.A. Osmani ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Hela Cell ◽  
Map Kinase ◽  
Kinase Inhibitors ◽  
Casein Kinase Ii ◽  
Cell Extract ◽  
Casein Kinase ◽  
Mitotic Chromosomes ◽  
Phosphatase Inhibitors ◽  
Hela Cell Extract

The MPM-2 antibody labels mitosis-specific and cell cycle-regulated phosphoproteins. The major phosphoproteins of mitotic chromosomes recognized by the MPM-2 antibody are DNA topoisomerase II (topoII) alpha and beta. In immunofluorescence studies of PtK1 cytoskeletons, prepared by detergent lysis in the presence of potent phosphatase inhibitors, the MPM-2 antibody labels phosphoproteins found at kinetochores, chromosome arms, midbody and spindle poles of mitotic cells. In cells extracted without phosphatase inhibitors, labeling of the MPM-2 antibodies at kinetochores is greatly diminished. However, in cytoskeletons this epitope can be regenerated through the action of kinases stably bound at the kinetochore. Various kinase inhibitors were tested in order to characterize the endogenous kinase responsible for these phosphorylations. We found that the MPM-2 epitope will not rephosphorylate in the presence of the broad specificity kinase inhibitors K-252a, staurosporine and 2-aminopurine. Several other inhibitors had no effect on the rephosphorylation indicating that the endogenous MPM-2 kinase at kinetochores is not p34cdc2, casein kinase II, MAP kinase, protein kinase A or protein kinase C. The addition of N-ethylmaleimide inactivated the endogenous kinetochore kinase; this allowed testing of several purified kinases in the kinetochore rephosphorylation assay. Active p34cdc2-cyclin B, casein kinase II and MAP kinase could not generate the MPM-2 phosphoepitope. However, bacterially expressed NIMA from Aspergillus and ultracentrifuged mitotic HeLa cell extract were able to catalyze the rephosphorylation of the MPM-2 epitope at kinetochores. Furthermore, fractionation of mitotic HeLa cell extract showed that kinases that create the MPM-2 epitope at kinetochores and chromosome arms are distinct. Our results suggest that multiple kinases (either soluble or kinetochore-bound), including a homolog of mammalian NIMA, can create the MPM-2 phosphoepitope. The kinetochore-bound kinase that catalyzes the formation of the MPM-2 phosphoepitope may play an important role in key events such as mitotic kinetochore assembly and sister chromatid separation at anaphase.

scholarly journals RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract

1983 ◽  
Vol 11 (5) ◽  
pp. 1405-1418 ◽  
Author(s):  
W. Filipowicz ◽  
M. Konarska ◽  
H. J. Gross ◽  
A. J. Shatkin
Keyword(s):  
Hela Cell ◽  
Cell Extract ◽  
Cyclase Activity ◽  
Hela Cell Extract ◽  
Rna Ligation

scholarly journals Zinc ions are differentially required for the transcription of ribosomal 5S RNA and tRNA in a HeLa-cell extract

1984 ◽  
Vol 12 (23) ◽  
pp. 8971-8985 ◽  
Author(s):  
Edgar Wingender ◽  
Dagmar Dilloo ◽  
Klaus H. Seifart
Keyword(s):  
Hela Cell ◽  
Cell Extract ◽  
Zinc Ions ◽  
5S Rna ◽  
Hela Cell Extract

scholarly journals Yeast tRNA3Leu gene transcribed and spliced in a HeLa cell extract.

1981 ◽  
Vol 78 (10) ◽  
pp. 5963-5967 ◽  
Author(s):  
D. N. Standring ◽  
A. Venegas ◽  
W. J. Rutter
Keyword(s):  
Hela Cell ◽  
Cell Extract ◽  
Hela Cell Extract

scholarly journals Transcription factor binding is limited by the 5'-flanking regions of a Drosophila tRNAHis gene and a tRNAHis pseudogene.

1984 ◽  
Vol 4 (12) ◽  
pp. 2714-2722 ◽  
Author(s):  
L Cooley ◽  
J Schaack ◽  
D J Burke ◽  
B Thomas ◽  
D Söll
Keyword(s):  
Complex Formation ◽  
Hela Cell ◽  
Cell Extract ◽  
Deletion Analysis ◽  
Stable Complex ◽  
Flanking Sequence ◽  
Factor Binding ◽  
Real Gene ◽  
Hela Cell Extract ◽  
Flanking Regions

We determined the sequence of a Drosophila tRNA gene cluster containing a tRNAHis gene and a tRNAHis pseudogene in close proximity on the same DNA strand. The pseudogene contains eight consecutive base pairs different from the region of the bona fide gene which codes for the 3' portion of the anticodon stem of tRNAHis. The tRNAHis gene is transcribed efficiently in Drosophila Kc cell extract, whereas the pseudogene is not. The pseudogene is also a much poorer competitor than the real gene in a stable transcription complex formation assay, even though the sequence alteration in the pseudogene does not affect the sequence or spacing of the putative internal transcription control regions. Recombinant clones were constructed in which the 5'-flanking regions are exchanged. The transcription efficiencies and competitive abilities of the recombinant clones resemble those of the genes from which the 5' flank was derived; for example, the tRNAHis pseudogene with the 5'-flanking sequence of the tRNAHis gene is now efficiently transcribed. Deletion analysis of the pseudogene 5' flank failed to uncover an inhibitory element. Deletion analysis of the real gene showed very high dependence on the presence of the wild-type 5'-flanking sequence for factor binding to the internal control regions and stable complex formation. The 5'-flanking sequence of a Drosophila tRNAArg gene active in the Drosophila Kc cell extract does not restore transcriptional activity or stable complex formation. The tRNAHis gene and pseudogene behave atypically in HeLa cell extract. Both genes compete for HeLa transcription factors, but neither of them is efficiently transcribed. Removal of the 5'-flanking sequences of each gene and replacement with various sequences, including the tRNAArg gene 5' flank, does not allow increased transcription in HeLa cell extract.

Promoter activity of the two chicken 5-crystallin genes in a Hela cell extract

1988 ◽  
Vol 7 (4) ◽  
pp. 331-340 ◽  
Author(s):  
Gokul C. Das ◽  
Joram Piatigorsky
Keyword(s):  
Hela Cell ◽  
Promoter Activity ◽  
Cell Extract ◽  
Hela Cell Extract

scholarly journals Accurate transcription of simian virus 40 chromatin in a HeLa cell extract.

1982 ◽  
Vol 44 (3) ◽  
pp. 772-781 ◽  
Author(s):  
J N Brady ◽  
M Radonovich ◽  
N P Salzman
Keyword(s):  
Hela Cell ◽  
Simian Virus 40 ◽  
Cell Extract ◽  
Simian Virus ◽  
Hela Cell Extract

scholarly journals Detection of isotype-specific autoantibodies to calpastatin in sera from patients with rheumatic diseases using heat-treated HeLa cell extract as an antigen source for immunoblotting.

1998 ◽  
Vol 21 (4) ◽  
pp. 150-158 ◽  
Author(s):  
Rie Takada ◽  
Maiko Matsumoto ◽  
Memi Yosida ◽  
Takaki Nojima ◽  
Michito Hirakata ◽  
...  
Keyword(s):  
Hela Cell ◽  
Rheumatic Diseases ◽  
Cell Extract ◽  
Hela Cell Extract ◽  
Heat Treated
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