Sequences controlling transcription of the Chlamydomonas reinhardtii beta 2-tubulin gene after deflagellation and during the cell cycle

1994 ◽  
Vol 14 (8) ◽  
pp. 5165-5174
Author(s):  
J P Davies ◽  
A R Grossman
Keyword(s):  
Cell Cycle ◽  
Chlamydomonas Reinhardtii ◽  
Transcription Initiation ◽  
Chimeric Gene ◽  
Transcriptional Level ◽  
Sequence Motif ◽  
Tubulin Gene ◽  
Rich Region ◽  
Beta 2 ◽  
Encoding Genes

In Chlamydomonas reinhardtii, transcripts from the beta 2-tubulin gene (tubB2), as well as those from other tubulin-encoding genes, accumulate immediately after flagellar excision as well as at a specific time in the cell cycle. Control of tubB2 transcript accumulation following deflagellation is regulated, at least partially, at the transcriptional level. We have fused the tubB2 promoter to the arylsulfatase (ars) reporter gene, introduced this construct into C. reinhardtii, and compared expression of the chimeric gene with that of the endogenous tubB2 gene. After flagellar excision, transcripts from the tubB2/ars chimeric gene accumulate with kinetics similar to those of transcripts from the endogenous tubB2 gene. The tubB2/ars transcripts also accumulate in a cell cycle-specific manner; however, chimeric transcripts are more abundant earlier in the cell cycle than the endogenous tubB2 transcripts. To elucidate transcriptional control of tubB2, we have mutated or removed sequences in the tubB2 promoter and examined the effect on transcription. The tubB2 promoter shares features with the promoters of other tubulin-encoding genes; these include a GC-rich region between the TATA box and the transcription initiation site and multiple copies of a 10-bp sequence motif that we call the tub box. The tubB2 gene contains seven tub box motifs. Changing the GC-rich region to an AT-rich region or removing three of the seven tub box motifs did not significantly affect transcription of the chimeric gene. However, removing four or five tub box motifs prevented increased transcription following deflagellation and diminished cell cycle-regulated transcription from the tubB2 promoter.

scholarly journals Sequences controlling transcription of the Chlamydomonas reinhardtii beta 2-tubulin gene after deflagellation and during the cell cycle.

1994 ◽  
Vol 14 (8) ◽  
pp. 5165-5174 ◽  
Author(s):  
J P Davies ◽  
A R Grossman
Keyword(s):  
Cell Cycle ◽  
Chlamydomonas Reinhardtii ◽  
Transcription Initiation ◽  
Chimeric Gene ◽  
Transcriptional Level ◽  
Sequence Motif ◽  
Tubulin Gene ◽  
Rich Region ◽  
Beta 2 ◽  
Encoding Genes

In Chlamydomonas reinhardtii, transcripts from the beta 2-tubulin gene (tubB2), as well as those from other tubulin-encoding genes, accumulate immediately after flagellar excision as well as at a specific time in the cell cycle. Control of tubB2 transcript accumulation following deflagellation is regulated, at least partially, at the transcriptional level. We have fused the tubB2 promoter to the arylsulfatase (ars) reporter gene, introduced this construct into C. reinhardtii, and compared expression of the chimeric gene with that of the endogenous tubB2 gene. After flagellar excision, transcripts from the tubB2/ars chimeric gene accumulate with kinetics similar to those of transcripts from the endogenous tubB2 gene. The tubB2/ars transcripts also accumulate in a cell cycle-specific manner; however, chimeric transcripts are more abundant earlier in the cell cycle than the endogenous tubB2 transcripts. To elucidate transcriptional control of tubB2, we have mutated or removed sequences in the tubB2 promoter and examined the effect on transcription. The tubB2 promoter shares features with the promoters of other tubulin-encoding genes; these include a GC-rich region between the TATA box and the transcription initiation site and multiple copies of a 10-bp sequence motif that we call the tub box. The tubB2 gene contains seven tub box motifs. Changing the GC-rich region to an AT-rich region or removing three of the seven tub box motifs did not significantly affect transcription of the chimeric gene. However, removing four or five tub box motifs prevented increased transcription following deflagellation and diminished cell cycle-regulated transcription from the tubB2 promoter.

scholarly journals Repeated consensus sequence and pseudopromoters in the four coordinately regulated tubulin genes of Chlamydomonas reinhardi.

1984 ◽  
Vol 4 (6) ◽  
pp. 1115-1124 ◽  
Author(s):  
K J Brunke ◽  
J G Anthony ◽  
E J Sternberg ◽  
D P Weeks
Keyword(s):  
Transcription Initiation ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Amino Acid Sequences ◽  
Sequencing Data ◽  
Base Pairs ◽  
Tubulin Gene ◽  
Gene Set ◽  
Tubulin Genes ◽  
Beta 2

The 5' coding and promoter regions of the four coordinately regulated tubulin genes of Chlamydomonas reinhardi have been mapped and sequenced. DNA sequencing data shows that the predicted N-terminal amino acid sequences of Chlamydomonas alpha- and beta-tubulins closely match that of tubulins of other eucaryotes. Within the alpha 1- and alpha 2-tubulin gene set and the beta 1- and beta 2-tubulin gene set, both nucleotide sequence and intron placement are highly conserved. Transcription initiation sites have been located by primer extension analysis at 140, 141, 159, and 132 base pairs upstream of the translation initiator codon for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin genes, respectively. Among the structures with potential regulatory significance, the most striking is a 16-base-pair consensus sequence [GCTC(G/C)AAGGC(G/T)(G/C)--(C/A)(C/A)G] which is found in multiple copies immediately upstream of the TATA box in each of the four genes. An unexpected discovery is the presence of pseudopromoter regions in two of the transcribed tubulin genes. One pseudopromoter region is located 400 base pairs upstream of the authentic alpha 2-tubulin gene promoter, whereas the other is located within the transcribed 5' noncoding region of the beta 1-tubulin gene.

Tubulin gene expression in the Chlamydomonas reinhardtii cell cycle: elimination of environmentally induced artifacts and the measurement of tubulin mRNA levels

1988 ◽  
Vol 89 (3) ◽  
pp. 397-403
Author(s):  
D.S. Nicholl ◽  
J.A. Schloss ◽  
P.C. John
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Division ◽  
Chlamydomonas Reinhardtii ◽  
Environmental Conditions ◽  
Mrna Levels ◽  
Tubulin Gene ◽  
Mrna Accumulation ◽  
Tubulin Mrna ◽  
Flagellar Proteins

To investigate the involvement of tubulin gene expression in controlling cell division events in Chlamydomonas reinhardtii we have measured tubulin mRNA levels during the cell cycle under different environmental conditions. In C. reinhardtii cells grown under the synchronizing conditions of 14 h of light followed by 10 h of darkness, mRNAs for tubulin and associated flagellar proteins were found to accumulate periodically with a peak just prior to cell division. This was not seen when previously synchronized cells were transferred to constant environmental conditions in a turbidostat, suggesting that dramatic changes in tubulin mRNA levels are not required for successful completion of the cell cycle. A hypothesis to explain the patterns of tubulin mRNA accumulation found under different environmental conditions is presented.

scholarly journals Repeated consensus sequence and pseudopromoters in the four coordinately regulated tubulin genes of Chlamydomonas reinhardi

1984 ◽  
Vol 4 (6) ◽  
pp. 1115-1124
Author(s):  
K J Brunke ◽  
J G Anthony ◽  
E J Sternberg ◽  
D P Weeks
Keyword(s):  
Transcription Initiation ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Amino Acid Sequences ◽  
Sequencing Data ◽  
Base Pairs ◽  
Tubulin Gene ◽  
Gene Set ◽  
Tubulin Genes ◽  
Beta 2

The 5' coding and promoter regions of the four coordinately regulated tubulin genes of Chlamydomonas reinhardi have been mapped and sequenced. DNA sequencing data shows that the predicted N-terminal amino acid sequences of Chlamydomonas alpha- and beta-tubulins closely match that of tubulins of other eucaryotes. Within the alpha 1- and alpha 2-tubulin gene set and the beta 1- and beta 2-tubulin gene set, both nucleotide sequence and intron placement are highly conserved. Transcription initiation sites have been located by primer extension analysis at 140, 141, 159, and 132 base pairs upstream of the translation initiator codon for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin genes, respectively. Among the structures with potential regulatory significance, the most striking is a 16-base-pair consensus sequence [GCTC(G/C)AAGGC(G/T)(G/C)--(C/A)(C/A)G] which is found in multiple copies immediately upstream of the TATA box in each of the four genes. An unexpected discovery is the presence of pseudopromoter regions in two of the transcribed tubulin genes. One pseudopromoter region is located 400 base pairs upstream of the authentic alpha 2-tubulin gene promoter, whereas the other is located within the transcribed 5' noncoding region of the beta 1-tubulin gene.

scholarly journals To Divide or Not to Divide? How Deuterium Affects Growth and Division of Chlamydomonas reinhardtii

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 861
Author(s):  
Veronika Kselíková ◽  
Vilém Zachleder ◽  
Kateřina Bišová
Keyword(s):  
Cell Cycle ◽  
Chlamydomonas Reinhardtii ◽  
Cell Cycle Progression ◽  
Model Organism ◽  
Cycle Progression ◽  
Synchronous Cultures ◽  
Multiple Fission ◽  
First Choice ◽  
High Doses

Extensive in vivo replacement of hydrogen by deuterium, a stable isotope of hydrogen, induces a distinct stress response, reduces cell growth and impairs cell division in various organisms. Microalgae, including Chlamydomonas reinhardtii, a well-established model organism in cell cycle studies, are no exception. Chlamydomonas reinhardtii, a green unicellular alga of the Chlorophyceae class, divides by multiple fission, grows autotrophically and can be synchronized by alternating light/dark regimes; this makes it a model of first choice to discriminate the effect of deuterium on growth and/or division. Here, we investigate the effects of high doses of deuterium on cell cycle progression in C. reinhardtii. Synchronous cultures of C. reinhardtii were cultivated in growth medium containing 70 or 90% D2O. We characterize specific deuterium-induced shifts in attainment of commitment points during growth and/or division of C. reinhardtii, contradicting the role of the “sizer” in regulating the cell cycle. Consequently, impaired cell cycle progression in deuterated cultures causes (over)accumulation of starch and lipids, suggesting a promising potential for microalgae to produce deuterated organic compounds.

scholarly journals A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

1993 ◽  
Vol 13 (3) ◽  
pp. 1610-1618 ◽  
Author(s):  
J E Slansky ◽  
Y Li ◽  
W G Kaelin ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Phase Boundary ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Protein S ◽  
S Phase ◽  
Dependent Manner

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

The human transforming growth factor alpha promoter directs transcription initiation from a single site in the absence of a TATA sequence

1988 ◽  
Vol 8 (12) ◽  
pp. 5549-5554
Author(s):  
E B Jakobovits ◽  
U Schlokat ◽  
J L Vannice ◽  
R Derynck ◽  
A D Levinson
Keyword(s):  
Growth Factor ◽  
Transcription Initiation ◽  
Transforming Growth Factor ◽  
Promoter Sequence ◽  
Transforming Growth Factor Alpha ◽  
Sequence Motif ◽  
Factor Alpha ◽  
Mammalian Genes ◽  
Tata Sequence ◽  
The Brain

Transforming growth factor alpha (TGF-alpha) is a transformation-responsive mitogenic polypeptide that is expressed in the brain, epithelial cells, and activated macrophages. We isolated and characterized the TGF-alpha promoter and localized the 5' end of the TGF-alpha transcript to a unique position. Surprisingly, no apparent TATA box was present in the promoter sequence, suggesting that transcription from mammalian genes can initiate at unique and specific positions from promoters lacking this sequence motif.

A variant octamer motif in a Xenopus H2B histone gene promoter is not required for transcription in frog oocytes

1991 ◽  
Vol 11 (2) ◽  
pp. 641-654
Author(s):  
C Hinkley ◽  
M Perry
Keyword(s):  
Cell Cycle ◽  
Transcription Initiation ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Histone Gene ◽  
Site Directed Mutagenesis ◽  
Regulatory Sequences ◽  
Chromosomal Dna ◽  
Transcriptional Regulatory ◽  
Octamer Motif

Xenopus oocytes, arrested in G2 before the first meiotic division, accumulate histone mRNA and protein in the absence of chromosomal DNA replication and therefore represent an attractive biological system in which to examine histone gene expression uncoupled from the cell cycle. Previous studies have shown that sequences necessary for maximal levels of transcription in oocytes are present within 200 bp at the 5' end of the transcription initiation site for genes encoding each of the five major Xenopus histone classes. We have defined by site-directed mutagenesis individual regulatory sequences and characterized DNA-binding proteins required for histone H2B gene transcription in injected oocytes. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CBP, and ATF/CREB binding sites, required for maximal transcription. A sequence (CTTTACAT) in the H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is not required for transcription in oocytes. Nonetheless, substitution of a consensus octamer motif for the variant octamer element activates H2B transcription. Oocyte factors, presumably including the ubiquitous Oct-1 factor, specifically bind to the consensus octamer motif but not to the variant sequence. Our results demonstrate that a transcriptional regulatory element involved in lymphoid-specific expression of immunoglobulin genes and in S-phase-specific activation of mammalian H2B histone genes can activate transcription in nondividing amphibian oocytes.

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii confers resistance to anti-microtubule herbicides

1993 ◽  
Vol 106 (1) ◽  
pp. 209-218 ◽  
Author(s):  
S.W. James ◽  
C.D. Silflow ◽  
P. Stroom ◽  
P.A. Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Resistance Mutation ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Two Dimensional ◽  
Wild Type ◽  
Tubulin Gene ◽  
Alpha Tubulin ◽  
Tubulin Genes

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii was isolated by using the amiprophos-methyl-resistant mutation apm1-18 as a background to select new mutants that showed increased resistance to the drug. The upA12 mutation caused twofold resistance to amiprophos-methyl and oryzalin, and twofold hypersensitivity to the microtubule-stabilizing drug taxol, suggesting that the mutation enhanced microtubule stability. The resistance mutation was semi-dominant and mapped to the same interval on linkage group III as the alpha 1-tubulin gene. Two-dimensional gel immunoblots of proteins in the mutant cells revealed two electrophoretically altered alpha-tubulin isoforms, one of which was acetylated and incorporated into microtubules in the axoneme. The mutant isoforms co-segregated with the drug-resistance phenotypes when mutant upA12 was backcrossed to wild-type cells. Two-dimensional gel analysis of in vitro translation products showed that the non-acetylated variant alpha-tubulin was a primary gene product. DNA sequence analysis of the alpha 1-tubulin genes from mutant and wild-type cells revealed a single missense mutation, which predicted a change in codon 24 from tyrosine in wild type to histidine in mutant upA12. This alteration in the predicted amino acid sequence corroborated the approximately +1 basic charge shift observed for the variant alpha-tubulins. The mutant allele of the alpha 1-tubulin gene was designated tua1-1.

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