scholarly journals Common factors direct transcription through the proximal sequence elements (PSEs) of the embryonic sea urchin U1, U2, and U6 genes despite minimal similarity among the PSEs.

1996 ◽  
Vol 16 (3) ◽  
pp. 1275-1281 ◽  
Author(s):  
J M Li ◽  
R P Haberman ◽  
W F Marzluff
Keyword(s):  
Sea Urchin ◽  
Affinity Column ◽  
Rrna Gene ◽  
Start Site ◽  
Small Nuclear Rna ◽  
Mobility Shift ◽  
Sequence Element ◽  
Nuclear Rna ◽  
U6 Gene

The proximal sequence element (PSE) for the sea urchin U6 small nuclear RNA gene has been defined. The most critical nucleotides for expression, located 61 to 64 nucleotides (nt) from the transcription start site, are 4 nt, AACT, at the 5' end of the PSE. Two nucleotide mutations in this region abolish transcription of the sea urchin U6 gene in vitro. The same two nucleotide mutations greatly reduce the binding of specific factors detected by an electrophoretic mobility shift assay. There is also a conserved AC dinucleotide 57 nt from the start site of the sea urchin U1 and U2 PSEs. The sea urchin U1 and U2 PSEs were substituted for the sea urchin U6 PSE, with the conserved AC sequences aligned with those of the U6 PSE. Both of these genes were expressed at levels higher than those observed with the wild-type U6 gene. Similar complexes are formed on the U1 and U2 PSEs, and formation of the complexes is inhibited efficiently by the U6 PSE. In addition, the E-box sequence present upstream of the PSE enhances U6 transcription from both the U1 and U2 PSEs. Finally, depletion of a nuclear extract with a DNA affinity column containing the U6 PSE sequence reduces expression of the U6 genes driven by the U6, U1, or U2 PSE but does not affect expression of the 5S rRNA gene. These data support the possibility that the same factor(s) interacts with the PSE sequences of the U1, U2, and U6 small nuclear RNA genes expressed in early sea urchin embryogenesis.

scholarly journals The Xenopus laevis U2 gene distal sequence element (enhancer) is composed of four subdomains that can act independently and are partly functionally redundant.

1989 ◽  
Vol 9 (4) ◽  
pp. 1682-1690 ◽  
Author(s):  
G Tebb ◽  
I W Mattaj
Keyword(s):  
Transcription Factor ◽  
Xenopus Oocytes ◽  
Small Nuclear Rna ◽  
Base Pairs ◽  
Sequence Element ◽  
Nuclear Factors ◽  
Nuclear Rna ◽  
The Individual ◽  
Mutant Genes

The sequences involved in enhancement of transcription of the Xenopus U2 small nuclear RNA gene by the distal sequence element (DSE) of its promoter were analyzed in detail by microinjection of mutant genes into Xenopus oocytes. The DSE was shown to be roughly 60 base pairs long. Within this region, four motifs were found to contribute to DSE function: an ATGCAAAT octamer sequence, an SpI binding site, and two additional motifs which, since they are related in sequence, may bind the same transcription factor. These motifs were named D2 (for DSE; U2). Both the octamer sequence and the SpI site bound nuclear factors in vitro, but no factor binding to the D2 motifs was detected. All four elements were independently capable of enhancing transcription of the U2 gene to some extent. Furthermore, when assayed under both competitive and noncompetitive conditions, the individual units of the DSE displayed functional redundancy.

scholarly journals Transcription of the sea urchin U6 gene in vitro requires a TATA-like box, a proximal sequence element, and sea urchin USF, which binds an essential E box.

1994 ◽  
Vol 14 (3) ◽  
pp. 2191-2200 ◽  
Author(s):  
J M Li ◽  
R A Parsons ◽  
W F Marzluff
Keyword(s):  
Sea Urchin ◽  
Rna Polymerase Iii ◽  
Nuclear Extract ◽  
Major Protein ◽  
In Vitro Mutagenesis ◽  
Flanking Sequence ◽  
Sequence Element ◽  
Proximal Sequence Element ◽  
U6 Gene

The tandemly repeated gene set encoding the sea urchin U6 gene has been cloned from the sea urchin Strongylocentrotus purpuratus. The U6 gene is transcribed by RNA polymerase III in a sea urchin nuclear extract. Like that of the vertebrate U6 genes, transcription of the sea urchin U6 gene does not require any internal sequences or 3' sequences but requires only 5' flanking sequences. Only 88 nucleotides of 5' flanking sequence are required for maximal expression in vitro. Mutagenesis experiments demonstrated the requirement for three elements, a CACGTG element at -80, a proximal sequence element at about -55, and the TATA-like box at -25. The major protein in sea urchin extracts that interacts with the CACGTG element is sea urchin USF, and immunodepletion of sea urchin USF greatly reduces transcription. The USF binding site in the U6 gene is highly homologous (11 of 13 nucleotides) with the USF binding sites found in the promoter of the S. purpuratus spec genes.

The Xenopus laevis U2 gene distal sequence element (enhancer) is composed of four subdomains that can act independently and are partly functionally redundant

1989 ◽  
Vol 9 (4) ◽  
pp. 1682-1690
Author(s):  
G Tebb ◽  
I W Mattaj
Keyword(s):  
Transcription Factor ◽  
Xenopus Oocytes ◽  
Small Nuclear Rna ◽  
Base Pairs ◽  
Sequence Element ◽  
Nuclear Factors ◽  
Nuclear Rna ◽  
The Individual ◽  
Mutant Genes

The sequences involved in enhancement of transcription of the Xenopus U2 small nuclear RNA gene by the distal sequence element (DSE) of its promoter were analyzed in detail by microinjection of mutant genes into Xenopus oocytes. The DSE was shown to be roughly 60 base pairs long. Within this region, four motifs were found to contribute to DSE function: an ATGCAAAT octamer sequence, an SpI binding site, and two additional motifs which, since they are related in sequence, may bind the same transcription factor. These motifs were named D2 (for DSE; U2). Both the octamer sequence and the SpI site bound nuclear factors in vitro, but no factor binding to the D2 motifs was detected. All four elements were independently capable of enhancing transcription of the U2 gene to some extent. Furthermore, when assayed under both competitive and noncompetitive conditions, the individual units of the DSE displayed functional redundancy.

In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements

1993 ◽  
Vol 13 (9) ◽  
pp. 5918-5927
Author(s):  
Z Zamrod ◽  
C M Tyree ◽  
Y Song ◽  
W E Stumph
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Tata Box ◽  
Start Site ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Nuclear Rna ◽  
Tata Sequence ◽  
Tata Box Binding Protein

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

scholarly journals Characterization of two developmentally regulated sea urchin U2 small nuclear RNA promoters: a common required TATA sequence and independent proximal and distal elements.

1992 ◽  
Vol 12 (2) ◽  
pp. 650-660 ◽  
Author(s):  
B Stefanovic ◽  
W F Marzluff
Keyword(s):  
Sea Urchin ◽  
Core Promoter ◽  
Sequence Similarity ◽  
Single Copy ◽  
Upstream Sequence ◽  
Small Nuclear Rna ◽  
Developmentally Regulated ◽  
Sequence Element ◽  
Nuclear Rna ◽  
Tata Sequence

The promoters of two U2 small nuclear RNA genes isolated from the sea urchin Lytechinus variegatus were mapped by microinjection of genes into sea urchin zygotes. One gene, LvU2E, is expressed only in oocytes and embryos and is found in a tandemly repeated gene set, while the other gene, LvU2L, is a single-copy gene and is expressed in embryos and somatic cells. The promoters each contain a TATA sequence at -25 which is required for expression, a proximal sequence element (PSE) centered at -55 required for expression, a sequence at -100 which couples the core promoter (PSE plus TATA box) to the upstream element, and an upstream sequence which stimulates expression fourfold. The PSE together with the TATA sequence is sufficient to determine the transcription start site. There is no sequence similarity between the -100 and PSE sequences of the two genes. The -100 sequences can be interchanged between the two genes. The LvU2E PSE functions in the context of the LvU2L gene, but the LvU2L PSE functions poorly in the context of the LvU2E gene.

scholarly journals Functional characterization of elements in a human U6 small nuclear RNA gene distal control region

1993 ◽  
Vol 13 (8) ◽  
pp. 4670-4678
Author(s):  
D A Danzeiser ◽  
O Urso ◽  
G R Kunkel
Keyword(s):  
Control Region ◽  
Functional Characterization ◽  
Proximal Promoter ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Related Sequence ◽  
Octamer Motif ◽  
Nuclear Rna ◽  
U6 Gene

The promoters of vertebrate U6 small nuclear RNA genes contain a distal control region whose presence results in at least an eightfold level of transcriptional activation in vivo. Previous transfection experiments have demonstrated that most of the distal control region of a human U6 gene resides in a restriction fragment located from -244 to -149 relative to the transcriptional start site. Three octamer-related motifs that bind recombinant Oct-1 transcription factor in vitro exist in this segment of DNA. However, transfection of human 293 cells with various plasmid templates in which these Oct-1 binding sites had been disrupted individually or in combination showed that only the consensus octamer motif located between positions -221 to -214 was functional. Even so, the consensus octamer motif mutant template was expressed at only a moderately reduced level relative to the wild-type promoter. When another octamer-related sequence located nearby, one that did not bind Oct-1 in vitro, was disrupted along with the perfect octamer site, expression was reduced fivefold in transfected cells. A factor that binds this functional, nonconsensus octamer site (NONOCT) was detected in crude cellular extracts. However, the NONOCT sequence was not essential for activation, since its disruption caused only a 40% reduction in U6 gene expression, and mutagenesis to convert the NONOCT sequence to a consensus octamer motif restored wild-type expression. Furthermore, in vitro transcription of a human U6 proximal promoter joined to a single copy of the octamer motif was stimulated by the addition of recombinant Oct-1 protein.

Characterization of two developmentally regulated sea urchin U2 small nuclear RNA promoters: a common required TATA sequence and independent proximal and distal elements

1992 ◽  
Vol 12 (2) ◽  
pp. 650-660
Author(s):  
B Stefanovic ◽  
W F Marzluff
Keyword(s):  
Sea Urchin ◽  
Core Promoter ◽  
Sequence Similarity ◽  
Single Copy ◽  
Upstream Sequence ◽  
Small Nuclear Rna ◽  
Developmentally Regulated ◽  
Sequence Element ◽  
Nuclear Rna ◽  
Tata Sequence

The promoters of two U2 small nuclear RNA genes isolated from the sea urchin Lytechinus variegatus were mapped by microinjection of genes into sea urchin zygotes. One gene, LvU2E, is expressed only in oocytes and embryos and is found in a tandemly repeated gene set, while the other gene, LvU2L, is a single-copy gene and is expressed in embryos and somatic cells. The promoters each contain a TATA sequence at -25 which is required for expression, a proximal sequence element (PSE) centered at -55 required for expression, a sequence at -100 which couples the core promoter (PSE plus TATA box) to the upstream element, and an upstream sequence which stimulates expression fourfold. The PSE together with the TATA sequence is sufficient to determine the transcription start site. There is no sequence similarity between the -100 and PSE sequences of the two genes. The -100 sequences can be interchanged between the two genes. The LvU2E PSE functions in the context of the LvU2L gene, but the LvU2L PSE functions poorly in the context of the LvU2E gene.

scholarly journals In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements.

1993 ◽  
Vol 13 (9) ◽  
pp. 5918-5927 ◽  
Author(s):  
Z Zamrod ◽  
C M Tyree ◽  
Y Song ◽  
W E Stumph
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Tata Box ◽  
Start Site ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Nuclear Rna ◽  
Tata Sequence ◽  
Tata Box Binding Protein

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

scholarly journals Transcription of the sea urchin U6 gene in vitro requires a TATA-like box, a proximal sequence element, and sea urchin USF, which binds an essential E box

1994 ◽  
Vol 14 (3) ◽  
pp. 2191-2200
Author(s):  
J M Li ◽  
R A Parsons ◽  
W F Marzluff
Keyword(s):  
Sea Urchin ◽  
Rna Polymerase Iii ◽  
Nuclear Extract ◽  
Major Protein ◽  
In Vitro Mutagenesis ◽  
Flanking Sequence ◽  
Sequence Element ◽  
Proximal Sequence Element ◽  
U6 Gene

The tandemly repeated gene set encoding the sea urchin U6 gene has been cloned from the sea urchin Strongylocentrotus purpuratus. The U6 gene is transcribed by RNA polymerase III in a sea urchin nuclear extract. Like that of the vertebrate U6 genes, transcription of the sea urchin U6 gene does not require any internal sequences or 3' sequences but requires only 5' flanking sequences. Only 88 nucleotides of 5' flanking sequence are required for maximal expression in vitro. Mutagenesis experiments demonstrated the requirement for three elements, a CACGTG element at -80, a proximal sequence element at about -55, and the TATA-like box at -25. The major protein in sea urchin extracts that interacts with the CACGTG element is sea urchin USF, and immunodepletion of sea urchin USF greatly reduces transcription. The USF binding site in the U6 gene is highly homologous (11 of 13 nucleotides) with the USF binding sites found in the promoter of the S. purpuratus spec genes.

scholarly journals Functional characterization of elements in a human U6 small nuclear RNA gene distal control region.

1993 ◽  
Vol 13 (8) ◽  
pp. 4670-4678 ◽  
Author(s):  
D A Danzeiser ◽  
O Urso ◽  
G R Kunkel
Keyword(s):  
Control Region ◽  
Functional Characterization ◽  
Proximal Promoter ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Related Sequence ◽  
Octamer Motif ◽  
Nuclear Rna ◽  
U6 Gene

The promoters of vertebrate U6 small nuclear RNA genes contain a distal control region whose presence results in at least an eightfold level of transcriptional activation in vivo. Previous transfection experiments have demonstrated that most of the distal control region of a human U6 gene resides in a restriction fragment located from -244 to -149 relative to the transcriptional start site. Three octamer-related motifs that bind recombinant Oct-1 transcription factor in vitro exist in this segment of DNA. However, transfection of human 293 cells with various plasmid templates in which these Oct-1 binding sites had been disrupted individually or in combination showed that only the consensus octamer motif located between positions -221 to -214 was functional. Even so, the consensus octamer motif mutant template was expressed at only a moderately reduced level relative to the wild-type promoter. When another octamer-related sequence located nearby, one that did not bind Oct-1 in vitro, was disrupted along with the perfect octamer site, expression was reduced fivefold in transfected cells. A factor that binds this functional, nonconsensus octamer site (NONOCT) was detected in crude cellular extracts. However, the NONOCT sequence was not essential for activation, since its disruption caused only a 40% reduction in U6 gene expression, and mutagenesis to convert the NONOCT sequence to a consensus octamer motif restored wild-type expression. Furthermore, in vitro transcription of a human U6 proximal promoter joined to a single copy of the octamer motif was stimulated by the addition of recombinant Oct-1 protein.

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