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 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.

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 Location and characterization of two widely separated glucocorticoid response elements in the phosphoenolpyruvate carboxykinase gene.

1988 ◽  
Vol 8 (1) ◽  
pp. 96-104 ◽  
Author(s):  
D D Petersen ◽  
M A Magnuson ◽  
D K Granner
Keyword(s):  
Transcription Initiation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Maximal Response ◽  
Flanking Sequence ◽  
Base Pairs ◽  
Chimeric Genes

Chimeric genes were constructed by fusion of various regions of the 5'-flanking sequence from the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene to the chloramphenicol acetyltransferase-coding sequence and to simian virus 40 splice and polyadenylation sequences. These were used to demonstrate that two glucocorticoid regulatory elements (GREs) combine to confer glucocorticoid responsiveness upon the PEPCK gene in H4IIE hepatoma cells. Both elements, a distal one whose 5' boundary is located between -1264 and -1111 base pairs and a proximal one located between -468 and -420 base pairs relative to the transcription initiation site, act independently, in various positions and orientations, and upon the heterologous thymidine kinase promoter. Each element accounts for half of the maximal response of the chimeric genes. Therefore, two widely separated enhancerlike elements contribute equally to the response of the PEPCK gene to glucocorticoid hormones. Neither of the PEPCK GREs contains the TGTTCT consensus sequence associated with most other GREs.

Location and characterization of two widely separated glucocorticoid response elements in the phosphoenolpyruvate carboxykinase gene

1988 ◽  
Vol 8 (1) ◽  
pp. 96-104
Author(s):  
D D Petersen ◽  
M A Magnuson ◽  
D K Granner
Keyword(s):  
Transcription Initiation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Maximal Response ◽  
Flanking Sequence ◽  
Base Pairs ◽  
Chimeric Genes

Chimeric genes were constructed by fusion of various regions of the 5'-flanking sequence from the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene to the chloramphenicol acetyltransferase-coding sequence and to simian virus 40 splice and polyadenylation sequences. These were used to demonstrate that two glucocorticoid regulatory elements (GREs) combine to confer glucocorticoid responsiveness upon the PEPCK gene in H4IIE hepatoma cells. Both elements, a distal one whose 5' boundary is located between -1264 and -1111 base pairs and a proximal one located between -468 and -420 base pairs relative to the transcription initiation site, act independently, in various positions and orientations, and upon the heterologous thymidine kinase promoter. Each element accounts for half of the maximal response of the chimeric genes. Therefore, two widely separated enhancerlike elements contribute equally to the response of the PEPCK gene to glucocorticoid hormones. Neither of the PEPCK GREs contains the TGTTCT consensus sequence associated with most other GREs.

scholarly journals Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences.

1986 ◽  
Vol 6 (1) ◽  
pp. 302-314 ◽  
Author(s):  
R D Andersen ◽  
B W Birren ◽  
S J Taplitz ◽  
H R Herschman
Keyword(s):  
Rna Polymerase Ii ◽  
Structural Gene ◽  
Transcription Initiation ◽  
Metal Ion ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Nuclease Mapping

As shown by Southern blot analysis, the metallothionein-1 (MT-1) genes in rats comprise a multigene family. We present the sequence of the MT-1 structural gene and compare its features with other metallothionein genes. Three MT-1 pseudogenes which we sequenced apparently arose by reverse transcription of processed mRNA transcripts. Two of these, MT-1 psi a and MT-1 psi c, are retrogenes which derive from the MT-1 mRNA, having diverged from the MT-1 gene 6.9 and 2.6 million years ago, respectively. The third, MT-1 psi b, differs from the MT-1 cDNA by only three nucleotide alterations. Surprisingly, MT-1 psi b also preserves sequence homology for 142 base pairs 5' to the transcription initiation site of the parent gene; it contains a promoter sequence sufficient for specifying metal ion induction. We identified, by S1 nuclease mapping, an RNA polymerase II initiation site 432 base pairs 5' of the MT-1 transcription initiation site of the MT-1 structural gene which could explain the formation of the mRNA precursor to this pseudogene. We were unable to detect MT-1 psi b transcripts, either in liver tissue or after transfection. We conclude that the absence of detectable transcripts from this pseudogene is due to either a reduced level of transcription or the formation of unstable transcripts as a consequence of the lack of a consensus sequence normally found 3' of transcription termination in the MT-1 structural gene.

scholarly journals Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells

Processes ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 2118
Author(s):  
Carina Bannach ◽  
Daniel Ruiz Buck ◽  
Genna Bobby ◽  
Leo P. Graves ◽  
Sainan Li ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Gene Promoter ◽  
Protein Product ◽  
Expression Vectors ◽  
Coding Region ◽  
Base Pairs ◽  
Autographa Californica Nucleopolyhedrovirus ◽  
Transfer Vectors

Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, then some products will comprise a chimeric molecule with part of the polyhedrin protein. In this study, we showed that if AUU is placed in the frame with a Strep tag and eGFP coding region, we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors.

Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences

1986 ◽  
Vol 6 (1) ◽  
pp. 302-314
Author(s):  
R D Andersen ◽  
B W Birren ◽  
S J Taplitz ◽  
H R Herschman
Keyword(s):  
Rna Polymerase Ii ◽  
Structural Gene ◽  
Transcription Initiation ◽  
Metal Ion ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Nuclease Mapping

As shown by Southern blot analysis, the metallothionein-1 (MT-1) genes in rats comprise a multigene family. We present the sequence of the MT-1 structural gene and compare its features with other metallothionein genes. Three MT-1 pseudogenes which we sequenced apparently arose by reverse transcription of processed mRNA transcripts. Two of these, MT-1 psi a and MT-1 psi c, are retrogenes which derive from the MT-1 mRNA, having diverged from the MT-1 gene 6.9 and 2.6 million years ago, respectively. The third, MT-1 psi b, differs from the MT-1 cDNA by only three nucleotide alterations. Surprisingly, MT-1 psi b also preserves sequence homology for 142 base pairs 5' to the transcription initiation site of the parent gene; it contains a promoter sequence sufficient for specifying metal ion induction. We identified, by S1 nuclease mapping, an RNA polymerase II initiation site 432 base pairs 5' of the MT-1 transcription initiation site of the MT-1 structural gene which could explain the formation of the mRNA precursor to this pseudogene. We were unable to detect MT-1 psi b transcripts, either in liver tissue or after transfection. We conclude that the absence of detectable transcripts from this pseudogene is due to either a reduced level of transcription or the formation of unstable transcripts as a consequence of the lack of a consensus sequence normally found 3' of transcription termination in the MT-1 structural gene.

scholarly journals The heat shock consensus sequence is not sufficient for hsp70 gene expression in Drosophila melanogaster.

1985 ◽  
Vol 5 (1) ◽  
pp. 197-203 ◽  
Author(s):  
J Amin ◽  
R Mestril ◽  
R Lawson ◽  
H Klapper ◽  
R Voellmy
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Cell Types ◽  
Hsp70 Gene ◽  
Base Pairs ◽  
Hybrid Gene ◽  
Induced Expression ◽  
African Green Monkey Kidney

A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed. Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D. melanogaster and COS-1 (African green monkey kidney) cells. Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types. Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D. melanogaster cells. Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D. melanogaster gene in homologous cells. A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D. melanogaster cells.

scholarly journals Chlamydomonas reinhardtii Tubulin-Gene Disruptants for Efficient Isolation of Strains Bearing Novel Tubulin Mutations

2020 ◽  
Author(s):  
Takako Kato-Minoura ◽  
Yutaro Ogiwara ◽  
Takashi Yamano ◽  
Hideya Fukuzawa ◽  
Ritsu Kamiya
Keyword(s):  
Drug Resistance ◽  
Chlamydomonas Reinhardtii ◽  
Structural Changes ◽  
Human Cancer ◽  
Amino Acid Sequences ◽  
Drug Binding ◽  
Tubulin Gene ◽  
Tubulin Genes ◽  
Tubulin Mutations ◽  
Β Tubulin

ABSTRACTThe single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 and tua2) and two β-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we used an aphVIII gene cassette insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. None of the disruptants exhibited apparent defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate novel tubulin-mutants resistant to the anti-tubulin agents propyzamide and oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 24 strains bearing 12 different β-tubulin mutations. Some of these mutations are known to confer drug resistance in human cancer cells. Thus, single-tubulin-gene disruptants are an efficient means of isolating novel C. reinhardtii tubulin mutants.IMPORTANCEChlamydomonas reinhardtii is a useful organism for the study of tubulin function; however, only five kinds of tubulin mutations have been reported to date. This scarcity is partly due to C. reinhardtii possessing two tubulin genes each for α- and β-tubulin. Here, we obtained several strains in which one of the α- or β-tubulin genes was disrupted, and then used those disruptants to isolate 32 strains bearing 19 mostly novel tubulin mutations that conferred differing degrees of resistance to two anti-tubulin compounds. The majority of the tubulin mutations were located outside of the drug-binding sites in the three-dimensional tubulin structure, suggesting that structural changes underlie the drug resistance conferred by these mutations. Thus, single-tubulin-gene disruptants are an efficient means of generating tubulin mutants for the study of the structure–function relationship of tubulin and for the development of novel therapies based on anti-tubulin agents.

The heat shock consensus sequence is not sufficient for hsp70 gene expression in Drosophila melanogaster

1985 ◽  
Vol 5 (1) ◽  
pp. 197-203
Author(s):  
J Amin ◽  
R Mestril ◽  
R Lawson ◽  
H Klapper ◽  
R Voellmy
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Cell Types ◽  
Hsp70 Gene ◽  
Base Pairs ◽  
Hybrid Gene ◽  
Induced Expression ◽  
African Green Monkey Kidney

A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed. Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D. melanogaster and COS-1 (African green monkey kidney) cells. Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types. Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D. melanogaster cells. Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D. melanogaster gene in homologous cells. A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D. melanogaster cells.

Sign in / Sign up

Export Citation Format

Share Document