scholarly journals Two distant and precisely positioned domains promote transcription of Xenopus laevis rRNA genes: analysis with linker-scanning mutants.

1986 ◽  
Vol 6 (12) ◽  
pp. 4585-4593 ◽  
Author(s):  
J J Windle ◽  
B Sollner-Webb
Keyword(s):  
Xenopus Laevis ◽  
Promoter Activity ◽  
Promoter Sequence ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Unexpected Result ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Transcriptional Initiation ◽  
Upstream Promoter

To examine the internal organization of the promoter of the Xenopus laevis rRNA gene, we constructed a series of linker-scanning mutants that traverse the rDNA initiation region. The mutant genes, which have 3 to 11 clustered base substitutions set within an otherwise unaltered rDNA promoter sequence, were injected into Xenopus oocyte nuclei, and their transcriptional capacity was assessed by S1 nuclease analysis of the resultant RNA. The data demonstrate that there are two essential promoter domains, the distal boundaries of which coincide with the promoter boundaries established previously by analysis of 5' and 3' deletion mutants. The upstream promoter domain is relatively small and extends from residues ca. -140 to -128. The downstream domain is considerably larger, encompassing residues ca. -36 to +10, and exactly corresponds in both size and position to the mammalian minimal promoter region. The Xenopus rDNA sequence between these two essential domains has a much smaller effect on the level of transcriptional initiation. In light of the fact that a large portion of this intervening region consists of a segment (residues -114 to -72) that is duplicated many times in the upstream spacer to form an rDNA enhancer sequence, it is noteworthy that a "-115/-77 linker scanner," in which virtually this entire segment is replaced by a polylinker sequence, has full promoter activity in the injected Xenopus borealis oocytes. Analysis of a parallel series of spacing change linker-scanning mutants revealed the unexpected result that the relative positions of the upstream and downstream promoter domains are very critical: all spacing alterations of more than 2 base pairs within this 100-base-pair region virtually abolish promoter activity. We conclude that the factors that bind to these two distant promoter domains must interact in a very precise stereospecific manner.

Two distant and precisely positioned domains promote transcription of Xenopus laevis rRNA genes: analysis with linker-scanning mutants

1986 ◽  
Vol 6 (12) ◽  
pp. 4585-4593
Author(s):  
J J Windle ◽  
B Sollner-Webb
Keyword(s):  
Xenopus Laevis ◽  
Promoter Activity ◽  
Promoter Sequence ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Unexpected Result ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Transcriptional Initiation ◽  
Upstream Promoter

To examine the internal organization of the promoter of the Xenopus laevis rRNA gene, we constructed a series of linker-scanning mutants that traverse the rDNA initiation region. The mutant genes, which have 3 to 11 clustered base substitutions set within an otherwise unaltered rDNA promoter sequence, were injected into Xenopus oocyte nuclei, and their transcriptional capacity was assessed by S1 nuclease analysis of the resultant RNA. The data demonstrate that there are two essential promoter domains, the distal boundaries of which coincide with the promoter boundaries established previously by analysis of 5' and 3' deletion mutants. The upstream promoter domain is relatively small and extends from residues ca. -140 to -128. The downstream domain is considerably larger, encompassing residues ca. -36 to +10, and exactly corresponds in both size and position to the mammalian minimal promoter region. The Xenopus rDNA sequence between these two essential domains has a much smaller effect on the level of transcriptional initiation. In light of the fact that a large portion of this intervening region consists of a segment (residues -114 to -72) that is duplicated many times in the upstream spacer to form an rDNA enhancer sequence, it is noteworthy that a "-115/-77 linker scanner," in which virtually this entire segment is replaced by a polylinker sequence, has full promoter activity in the injected Xenopus borealis oocytes. Analysis of a parallel series of spacing change linker-scanning mutants revealed the unexpected result that the relative positions of the upstream and downstream promoter domains are very critical: all spacing alterations of more than 2 base pairs within this 100-base-pair region virtually abolish promoter activity. We conclude that the factors that bind to these two distant promoter domains must interact in a very precise stereospecific manner.

The Xenopus laevis ribosomal gene terminator contains sequences that both enhance and repress ribosomal transcription

1989 ◽  
Vol 9 (9) ◽  
pp. 3777-3784
Author(s):  
S Firek ◽  
C Read ◽  
D R Smith ◽  
T Moss
Keyword(s):  
Xenopus Laevis ◽  
Point Mutation ◽  
Ribosomal Dna ◽  
Ribosomal Gene ◽  
Promoter Element ◽  
Base Pairs ◽  
Transcription Terminator ◽  
Upstream Promoter

A DNA segment approximately 200 base pairs upstream of the Xenopus laevis ribosomal promoter acts both as an upstream promoter element that augments transcription and as a transcription terminator. It is, however, unclear to what extent these two activities are related. A segment of the X. laevis ribosomal DNA, containing the terminator and the upstream promoter element, was subjected to point mutation, and the effects of the resulting mutations were investigated by oocyte microinjection. Analysis of 26 point mutants revealed not only sequences that augment 40S transcription but also those that repress it. The sequences that augmented transcription lay within the T3 homology box and also near the site of 3'-end formation. These sequences also played a role in termination. The sequences that repressed transcription lay within the G+C-rich DNA flanking the T3 box. It can be concluded that termination is probably essential but may not be sufficient for the activity of the upstream promoter element.

scholarly journals Upstream domains of the Xenopus laevis rDNA promoter are revealed in microinjected oocytes.

1986 ◽  
Vol 6 (4) ◽  
pp. 1228-1234 ◽  
Author(s):  
J Windle ◽  
B Sollner-Webb
Keyword(s):  
Xenopus Laevis ◽  
Dna Sequences ◽  
Promoter Region ◽  
Core Promoter ◽  
Rrna Genes ◽  
Deletion Mutants ◽  
Low Concentrations ◽  
Upstream Promoter ◽  
Polymerase I ◽  
Vector Dna

The DNA sequences involved in promoting transcription of the Xenopus laevis rRNA genes were determined by microinjecting a series of deletion mutants into oocyte nuclei. A very small promoter region is sufficient to direct efficient transcription when templates are microinjected at high rDNA concentration, since 5'delta- 9 and 3'delta +6 templates are fully active. However, as the concentration of injected template is decreased, an increasing requirement for upstream domains, extending to nucleotide approximately -170, is observed. The major downstream border of the required region does not change. This apparently expanding 5' promoter border results from the fact that, as the rDNA concentration is decreased, transcription from templates lacking the upstream promoter domain falls off much more sharply than does transcription from a complete promoter. In fact, the deleted promoters are virtually inactive below a threshold rDNA concentration. It is indeed the rDNA concentration that is important, for coinjected vector DNA does not increase the level of transcription obtained from low concentrations of the 5' deletions. From these data we conclude that polymerase I transcription factors can recognize and initiate transcription from a small core promoter domain, but that sequences extending upstream to nucleotide approximately -170 increase the efficiency of initiation. A model is presented that could account for these results.

scholarly journals Localization and structure of endonuclease cleavage sites involved in the processing of the rat 32S precursor to ribosomal RNA

1984 ◽  
Vol 220 (1) ◽  
pp. 105-116 ◽  
Author(s):  
K V Hadjiolova ◽  
O I Georgiev ◽  
V V Nosikov ◽  
A A Hadjiolov
Keyword(s):  
Secondary Structure ◽  
Ribosome Biogenesis ◽  
Rrna Gene ◽  
Computer Search ◽  
Cleavage Sites ◽  
Base Pairing ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Evolutionarily Conserved ◽  
Domain I

The initial endonuclease cleavage site in 32 S pre-rRNA (precursor to rRNA) is located within the rate rDNA sequence by S1-nuclease protection mapping of purified nucleolar 28 S rRNA and 12 S pre-rRNA. The heterogeneous 5′- and 3′-termini of these rRNA abut and map within two CTC motifs in tSi2 (internal transcribed spacer 2) located at 50-65 and 4-20 base-pairs upstream from the homogeneous 5′-end of the 28 S rRNA gene. These results show that multiple endonuclease cleavages occur at CUC sites in tSi2 to generate 28 S rRNA and 12 S pre-rRNA with heterogeneous 5′- and 3′-termini, respectively. These molecules have to be processed further to yield mature 28 S and 5.8 S rRNA. Thermal-denaturation studies revealed that the base-pairing association in the 12 S pre-rRNA:28 S rRNA complex is markedly stronger than that in the 5.8 S:28 S rRNA complex. The sequence of about one-quarter (1322 base-pairs) of the 5′-part of the rat 28 S rDNA was determined. A computer search reveals the possibility that the cleavage sites in the CUC motifs are single-stranded, flanked by strongly base-paired GC tracts, involving tSi2 and 28 S rRNA sequences. The subsequent nuclease cleavages, generating the termini of mature rRNA, seem to be directed by secondary-structure interactions between 5.8 S and 28 S rRNA segments in pre-rRNA. An analysis for base-pairing among evolutionarily conserved sequences in 32 S pre-rRNA suggests that the cleavages yielding mature 5.8 S and 28 S rRNA are directed by base-pairing between (i) the 3′-terminus of 5.8 S rRNA and the 5′-terminus of 28 S rRNA and (ii) the 5′-terminus of 5.8 S rRNA and internal sequences in domain I of 28 S rRNA. A general model for primary- and secondary-structure interactions in pre-rRNA processing is proposed, and its implications for ribosome biogenesis in eukaryotes are briefly discussed.

scholarly journals Selective nucleolus organizer inactivation in Arabidopsis is a chromosome position-effect phenomenon

2016 ◽  
Vol 113 (47) ◽  
pp. 13426-13431 ◽  
Author(s):  
Gireesha Mohannath ◽  
Frederic Pontvianne ◽  
Craig S. Pikaard
Keyword(s):  
Position Effect ◽  
Nucleolus Organizer ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Chromosome 2 ◽  
Chromosome 4 ◽  
Base Pairs ◽  
Genomic Change ◽  
Nucleolus Organizer Regions ◽  
Chromosome Position

Nucleolus organizer regions (NORs) are chromosomal loci where hundreds of rRNA genes are clustered. Despite being nearly identical in sequence, specific rRNA genes are selected for silencing during development via choice mechanism(s) that remain unclear. In Arabidopsis thaliana, rRNA gene subtypes that are silenced during development were recently mapped to the NOR on chromosome 2, NOR2, whereas active rRNA genes map to NOR4, on chromosome 4. In a mutant line deficient for ATXR5 or ATXR6-dependent histone H3 lysine 27 (H3K27) monomethylation, we show that millions of base pairs of chromosome 4, including the telomere, TEL4N, and much of NOR4, have been converted to the corresponding sequences of chromosome 2. This genomic change places rRNA genes of NOR2, which are normally silenced, at the position on chromosome 4 where active rRNA genes are normally located. At their new location, NOR2-derived rRNA genes escape silencing, independent of the atxr mutations, indicating that selective rRNA gene silencing is chromosome 2-specific. The chromosome 2 position effect is not explained by the NOR2-associated telomere, TEL2N, which remains linked to the translocated NOR, implicating centromere-proximal sequences in silencing.

scholarly journals The Xenopus laevis ribosomal gene terminator contains sequences that both enhance and repress ribosomal transcription.

1989 ◽  
Vol 9 (9) ◽  
pp. 3777-3784 ◽  
Author(s):  
S Firek ◽  
C Read ◽  
D R Smith ◽  
T Moss
Keyword(s):  
Xenopus Laevis ◽  
Point Mutation ◽  
Ribosomal Dna ◽  
Ribosomal Gene ◽  
Promoter Element ◽  
Base Pairs ◽  
Transcription Terminator ◽  
Upstream Promoter

A DNA segment approximately 200 base pairs upstream of the Xenopus laevis ribosomal promoter acts both as an upstream promoter element that augments transcription and as a transcription terminator. It is, however, unclear to what extent these two activities are related. A segment of the X. laevis ribosomal DNA, containing the terminator and the upstream promoter element, was subjected to point mutation, and the effects of the resulting mutations were investigated by oocyte microinjection. Analysis of 26 point mutants revealed not only sequences that augment 40S transcription but also those that repress it. The sequences that augmented transcription lay within the T3 homology box and also near the site of 3'-end formation. These sequences also played a role in termination. The sequences that repressed transcription lay within the G+C-rich DNA flanking the T3 box. It can be concluded that termination is probably essential but may not be sufficient for the activity of the upstream promoter element.

scholarly journals Isolation of tobacco DNA segments with plant promoter activity.

1986 ◽  
Vol 6 (12) ◽  
pp. 4486-4492 ◽  
Author(s):  
L M Herman ◽  
M C Van Montagu ◽  
A G Depicker
Keyword(s):  
Cell Lines ◽  
Promoter Activity ◽  
Nuclear Dna ◽  
Tobacco Plant ◽  
Promoter Sequence ◽  
Base Pairs ◽  
Kanamycin Selection ◽  
Promoter Probe ◽  
Npt Ii ◽  
Promoter Probe Vector

We constructed a promoter probe vector, pGVL120, to isolate plant DNA segments with promoter activity in tobacco. Plant nuclear DNA Sau3A fragments were inserted in front of the npt-II sequence, and a mixture of recombinant plasmids was mobilized to Agrobacterium sp. and used to transform tobacco protoplasts. By kanamycin selection, transformed plant cell lines containing NPT-II T-DNAs were isolated. Eight of these cell lines were regenerated and analyzed for the levels of NPT-II activity in stem, root, midrib, and leaf. These levels demonstrated novel regulation patterns in each isolate. One cell line, T20, was analyzed in detail and found to contain four different T-DNAs. One of the recloned T-DNAs, T20-2, contains an insert of 401 base pairs in front of the NPT-II sequence, and by reintroducing this T-DNA into plant cells we could demonstrate that this insert provides a promoter sequence. The NPT-II enzyme activity under the control of the P20 promoter is especially high in stem and root, but low in leaf and callus, both in the originally isolated T20 plant and in independently isolated transformants with the T20-2 T-DNA.

Isolation of tobacco DNA segments with plant promoter activity

1986 ◽  
Vol 6 (12) ◽  
pp. 4486-4492
Author(s):  
L M Herman ◽  
M C Van Montagu ◽  
A G Depicker
Keyword(s):  
Cell Lines ◽  
Promoter Activity ◽  
Nuclear Dna ◽  
Tobacco Plant ◽  
Promoter Sequence ◽  
Base Pairs ◽  
Kanamycin Selection ◽  
Promoter Probe ◽  
Npt Ii ◽  
Promoter Probe Vector

We constructed a promoter probe vector, pGVL120, to isolate plant DNA segments with promoter activity in tobacco. Plant nuclear DNA Sau3A fragments were inserted in front of the npt-II sequence, and a mixture of recombinant plasmids was mobilized to Agrobacterium sp. and used to transform tobacco protoplasts. By kanamycin selection, transformed plant cell lines containing NPT-II T-DNAs were isolated. Eight of these cell lines were regenerated and analyzed for the levels of NPT-II activity in stem, root, midrib, and leaf. These levels demonstrated novel regulation patterns in each isolate. One cell line, T20, was analyzed in detail and found to contain four different T-DNAs. One of the recloned T-DNAs, T20-2, contains an insert of 401 base pairs in front of the NPT-II sequence, and by reintroducing this T-DNA into plant cells we could demonstrate that this insert provides a promoter sequence. The NPT-II enzyme activity under the control of the P20 promoter is especially high in stem and root, but low in leaf and callus, both in the originally isolated T20 plant and in independently isolated transformants with the T20-2 T-DNA.

Upstream domains of the Xenopus laevis rDNA promoter are revealed in microinjected oocytes

1986 ◽  
Vol 6 (4) ◽  
pp. 1228-1234
Author(s):  
J Windle ◽  
B Sollner-Webb
Keyword(s):  
Xenopus Laevis ◽  
Dna Sequences ◽  
Promoter Region ◽  
Core Promoter ◽  
Rrna Genes ◽  
Deletion Mutants ◽  
Low Concentrations ◽  
Upstream Promoter ◽  
Polymerase I ◽  
Vector Dna

The DNA sequences involved in promoting transcription of the Xenopus laevis rRNA genes were determined by microinjecting a series of deletion mutants into oocyte nuclei. A very small promoter region is sufficient to direct efficient transcription when templates are microinjected at high rDNA concentration, since 5'delta- 9 and 3'delta +6 templates are fully active. However, as the concentration of injected template is decreased, an increasing requirement for upstream domains, extending to nucleotide approximately -170, is observed. The major downstream border of the required region does not change. This apparently expanding 5' promoter border results from the fact that, as the rDNA concentration is decreased, transcription from templates lacking the upstream promoter domain falls off much more sharply than does transcription from a complete promoter. In fact, the deleted promoters are virtually inactive below a threshold rDNA concentration. It is indeed the rDNA concentration that is important, for coinjected vector DNA does not increase the level of transcription obtained from low concentrations of the 5' deletions. From these data we conclude that polymerase I transcription factors can recognize and initiate transcription from a small core promoter domain, but that sequences extending upstream to nucleotide approximately -170 increase the efficiency of initiation. A model is presented that could account for these results.

scholarly journals Devosia ginsengisoli sp. nov., isolated from ginseng cultivation soil

2020 ◽  
Vol 70 (3) ◽  
pp. 1489-1495 ◽  
Author(s):  
Xiao-Tian Quan ◽  
Muhammad Zubair Siddiqi ◽  
Qing-Zhen Liu ◽  
Sang-Mi Lee ◽  
Wan-Taek Im
Keyword(s):  
Type Species ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Strictly Aerobic ◽  
The Novel ◽  
Base Pairs ◽  
Circular Chromosome ◽  
Content Type ◽  
Link Type ◽  
Ginseng Cultivation

A Gram-stain-negative, strictly aerobic, motile, ivory-coloured and rod-shaped bacterium (designated Gsoil 520T) isolated from ginseng cultivation soil was characterized by using a polyphasic approach to clarify its taxonomic position. Strain Gsoil 520T was observed to grow optimally at 30 °C and pH 7.0 on Reasoner's 2A agar medium. The results of phylogenetic analysis, based on 16S rRNA gene sequence similarities, indicated that Gsoil 520T belongs to the genus Devosia of the family Hyphomicrobiaceae and was most closely related to Devosia epidermidihirudinis E84T (98.0 %), Devosia yakushimensis Yak96BT (97.7 %), Devosia neptuniae J1T (97.7 %) and Devosia chinhatensis IPL18T (96.8 %). The complete genome of strain Gsoil 520T is a presumptive circular chromosome of 4 480 314 base pairs having G+C content of 63.7 mol%. A total of 4 354 genes, 4 303 CDS and 43 rRNA genes were assigned a putative function. The major isoprenoid quinone was Q-10. The main polar lipids were phosphatidylglycerol, diphosphatidylglycerol and two unidentified aminolipids (AL1 and AL3). The predominant fatty acids of strain Gsoil 520T were C18 : 1ω7c 11-methyl, C16 : 0 and C18 : 1ω7c/C18 : 1ω6c (summed feature 8) supporting the affiliation of strain Gsoil 520T to the genus Devosia . The low values of DNA–DNA hybridization distinguished strain Gsoil 520T from the recognized species of the genus Devosia . Thus, the novel isolate represents a novel species of the genus Devosia , for which the name Devosia ginsengisoli sp. nov. is proposed, with the type strain Gsoil 520T (=KACC 19440T=LMG 30329T).

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