The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse

Urano, Yoshio; Kominami, Ryo; Mishima, Yukio; Muramatsu, Masami

doi:10.1093/nar/8.24.6043

Cited by 78 publications

(48 citation statements)

References 47 publications

(42 reference statements)

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“…9) reflects the smaller amount of rDNA in clone MOR-30-ra than in clone MOR-30 even though the dhfr gene had been o amplified in the former (Fig. 4A and B) (15,38). Our data rule out methylation at most, although not all, of these or other CpG sites.…”

Section: Methodsmentioning

confidence: 62%

Transcription of mouse rDNA and associated formation of the nucleolus organizer region after gene transfer and amplification in Chinese hamster cells.

Dhar¹,

Miller²,

Miller³

1985

Mol. Cell. Biol.

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Mouse rDNA can initiate transcription by using only Chinese hamster cell components, and this is associated with nucleolus organizer activity. To demonstrate this, we transferred a 3.2-kilobase segment of mouse rDNA containing the promoter, the transcription initiation site, and part of the external transcribed spacer to dihydrofolate reductase-deficient Chinese hamster cells by cotransformation with an abbreviated mouse dhfr gene. Stepwise selection for methotrexate resistance produced sublines in which the mouse rDNA was usualy coamplified with the donor dhfr DNA and occupied the same site or sites in the hamster genome, as shown by in situ hybridization. Transcription from mouse rDNA was demonstrated in two such lines, and S1 protection mapping indicated faithful initiation of the transcript. In some cells from both lines, the chromosome segments containing amplified mouse rDNA showed multiple silver-staining regions (i.e., active nucleolus organizers). Although the transferred mouse rDNA was able to use the rDNA transcriptional machinery of the Chinese hamster, the level of transcription was much lower than expected from the rDNA copy number, and a large fraction of each amplified region showed no silver staining. Since the absence of silver staining is generally correlated with the absence of transcription, many copies of the amplified mouse rDNA may have been in a chromatin conformation in which they could not be transcribed. This was not associated with the extensive methylation seen in other amplified, inactive rDNA sequences.rRNA genes (rDNA) in mammals occur as tandemly repetitive copies present in highly variable numbers at the nucleolus organizer regions (NORs) of chromosomes. These sites can be detected in metaphase chromosomes by in situ hybridization with labeled cDNA to 18S and 28S rRNA or rDNA probes or by a simple silver-staining reaction (11,17). The number of rRNA genes and the size of the silver-stained NORs (AgNORs) generally parallel the level of nucleolus organizer activity and, presumably, the rRNA transcriptional activity (24,40). Silver staining is restricted to potentially active NORs; it is absent from the nonexpressed rDNA clusters on chromosomes of one species in-rodent-human hybrid cells (7,27,32) and is also absent from homogeneously staining chromosomal regions containing greatly amplified and hypermethylated rDNA (28,37).Transcription of rDNA requires both RNA polymerase I and several protein cofactors; the cofactors from rodent cells do not support in vitro transcription from human rDNA and vice versa (13, 20, 23, 31 predominates (8,26,41,42), and it is unclear whether mouse and hamster rDNAs can use the same transcription cofactors.In this paper, we show that mouse rDNA can be transcribed by using only Chinese hamster components. We describe the transfer of a cloned mouse rRNA gene fragment into dihydrofolate reductase-deficient (dhfr-) hamster cells by DNA-mediated cotransformation with a constructed dhfr gene (9), coamplification of the two genes by stepwise selection of me...

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Section: Methodsmentioning

confidence: 62%

Transcription of mouse rDNA and associated formation of the nucleolus organizer region after gene transfer and amplification in Chinese hamster cells.

Dhar¹,

Miller²,

Miller³

1985

Mol. Cell. Biol.

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“…9B) is conserved in all species, but most secondary-structure models for eucaryotic large rRNAs predict that these 28S rRNA nucleotides are stably base paired with the 3' end of 5.8S rRNA (reviewed by Hadjiolov [24]). Two short complementarities between U3 and pre-rRNA sequences are found near site 2, which is a processing site several hundred nucleotides downstream of the transcription initiation site (12,39,62) (Fig. 9C and D), but conservation of this potential interaction cannot be judged, as this processing site has not been identified in any of the other species for which U3 sequences are available.…”

Section: Discussionmentioning

confidence: 99%

Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA.

1987

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The human U3 ribonucleoprotein (RNP) has been analyzed to determine its protein constituents, sites of protein-RNA interaction, and RNA secondary structure. By using anti-U3 RNP antibodies and extracts prepared from HeLa cells labeled in vivo, the RNP was found to contain four nonphosphorylated proteins of 36, 30, 13, and 12.5 kilodaltons and two phosphorylated proteins of 74 and 59 kilodaltons. U3 nucleotides 72-90, 106-121, 154-166, and 190-217 must contain sites that interact with proteins since these regions are immunoprecipitated after treatment of the RNP with RNase A or TI. The secondary structure was probed with specific nucleases and by chemical modification with single-strand-specific reagents that block subsequent reverse transcription. Regions that are single stranded (and therefore potentially able to interact with a substrate RNA) include an evolutionarily conserved sequence at nucleotides 104-112 and nonconserved sequences at nucleotides 65-74, 80-84, and 88-93. Nucleotides 159-168 do not appear to be highly accessible, thus making it unlikely that this U3 sequence base pairs with sequences near the 5.8S rRNA-internal transcribed spacer II junction, as previously proposed. Alternative functions of the U3 RNP are discussed, including the possibility that U3 may participate in a processing event near the 3' end of 28S rRNA. The nucleolar location of U3 (7,34,43,45,64; Reimer et al., in press) and the observations that it can be found associated with a >60S RNP particle (16) and with a 28 to 35S RNA (after deproteinization) (8,45,67) suggest an involvement in rRNA processing, ribosome assembly, or transport.Specific theories regarding the function of the U3 RNP (3, 13, 60) have been based primarily on assumptions concerning the identity of the 28 to 35S RNA with which it associates (8, 45, 67) and on complementarity between U3 and prerRNA sequences. In mammalian cells, a 47S precursor is processed in several steps to yield 18S, 5.8S, and 28S rRNAs (reviewed by Hadjiolov [24] Here we report structural studies and a derived model for the secondary structure of the RNA in the human U3 RNP. The protein constituents of the U3 RNP and protein-RNA interaction sites have also been analyzed. We conclude that U3 nucleotides 159 to 168 (human) are not highly accessible in the RNP and therefore probably do not base pair with ITS-II sequences. In contrast, a highly conserved sequence at nucleotides 104-112 is available for interacting with prerRNA. Complementarity between this region of U3 and conserved pre-rRNA sequences suggests that U3 may be involved in processing events occurring at or beyond the 3' end of 28S rRNA.

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“…comm.). Sequence homogenisation between the 240 unit and the Pol I initiation region Very little homology can be detected between the Pol I initiation regions of the rDNA of Xenopus (8), Drosophila (17), mouse (25) and man (26); or between the NTS's of D.melanogaster and D.hydei and D.virilis (19,20). There is no apparent recognition of the D.virilis Pol I promoter in a cell-free system of D.melanogaster (3).…”

Section: Construction Of Clonesmentioning

confidence: 99%

Multiple Pol I initiation sequences in rDNA spacers ofDrosophila melanogaster

1982

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The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse

Cited by 78 publications

References 47 publications

Transcription of mouse rDNA and associated formation of the nucleolus organizer region after gene transfer and amplification in Chinese hamster cells.

Transcription of mouse rDNA and associated formation of the nucleolus organizer region after gene transfer and amplification in Chinese hamster cells.

Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA.

Multiple Pol I initiation sequences in rDNA spacers ofDrosophila melanogaster

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