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I Grummt

Publications and source records attributed to I Grummt.

At least 91 records · Page 5Linked to original sources
PubMed1986

The core promoter of mouse rDNA consists of two functionally distinct domains.

We have determined the sequences constituting the minimal promoter of mouse rDNA. A very small region immediately upstream of the transcription start site (from -1 to -39) is sufficient to direct correct transcription initiation. Sequences immediately downstream of the transcription start site (+1 to +11) increase the efficiency of transcription initiation. Point mutations within the core promoter have been generated and assayed for their effects on template activity and on interaction with the pol I specific transcription factor TIF-IB. The core promoter element appears to consist of two functionally different domains. The distal sequence motif from position -22 to -16 is recognized by factor TIF-IB. Mutations within this region lead to similar changes of both template activity and binding of TIF-IB. Two point mutations within the proximal sequence motif from -15 to -1 do not affect TIF-IB binding although they severely impair transcription initiation. It is suggested, that this proximal region plays a role in the assembly of functional transcription initiation complexes rather than in the primary binding of TIF-IB.

Animals↗
PubMed1986

A repeated 18 bp sequence motif in the mouse rDNA spacer mediates binding of a nuclear factor and transcription termination.

DNA sequences and protein factors directing termination of mouse rDNA transcription in a nuclear extract system were examined. Termination is specific and requires a sequence element AGGTCGACCAGATTANTCCG (the Sall box) that is present eight times in the spacer region downstream of the 3' end of pre-rRNA. Exonuclease III protection experiments reveal the binding of a nuclear protein to the Sall box. Deletions, insertions, and point mutations in the Sall box reduce or abolish the interaction with the nuclear factor and disrupt transcription termination. A synthetic oligonucleotide corresponding to the Sall box consensus sequence governs transcription termination in vitro, although with reduced activity. Therefore, other sequences normally surrounding the Sall box appear to contribute to the accuracy and efficiency of termination.

Animals↗
PubMed1986

A purified transcription factor (TIF-IB) binds to essential sequences of the mouse rDNA promoter.

A transcription factor that is specific for mouse rDNA has been partially purified from Ehrlich ascites cells. This factor [designated transcription initiation factor (TIF)-IB] is required for accurate in vitro synthesis of mouse rRNA in addition to RNA polymerase I and another regulatory factor, TIF-IA. TIF-IB activity is present in extracts both from growing and nongrowing cells in comparable amounts. Prebinding competition experiments with wild-type and mutant templates suggest that TIF-IB interacts with the core control element of the rDNA promoter, which is located immediately upstream of the initiation site. The specific binding of TIF-IB to the RNA polymerase I promoter is demonstrated by exonuclease III protection experiments. The 3' border of the sequences protected by TIF-IB is shown to be on the coding strand at position -21 and on the noncoding strand at position -7. The results suggest that direct binding of TIF-IB to sequences in the core promoter element is the mechanism by which this factor imparts promoter selectivity to RNA polymerase I.

Animals↗
PubMed1985

Growth-dependent regulation of rRNA synthesis is mediated by a transcription initiation factor (TIF-IA).

Mouse RNA polymerase I requires at least two chromatographically distinct transcription factors (designated TIF-IA and TIF-IB) to initiate transcription accurately and efficiently in vitro. In this paper we describe the partial purification of TIF-IA by a four-step fractionation procedure. The amount or activity of TIF-IA fluctuates in response to the physiological state of the cells. Extracts from quiescent cells are incapable of specific transcription and do not contain detectable levels of TIF-IA. Transcriptionally inactive extracts can be restored by the addition of TIF-IA preparations that have been highly purified from exponentially growing cells. During the fractionating procedure TIF-IA co-purifies with RNA polymerase I, suggesting that it is functionally associated with the transcribing enzyme. We suggest that only those enzyme molecules that are associated with TIF-IA are capable to interact with TIF-IB and to initiate transcription.

Animals↗
PubMed1985

Spacer sequences downstream of the 28S RNA coding region are part of the mouse rDNA transcription unit.

Evidence is presented that more than 300 bp of spacer sequences downstream of the 28S RNA coding sequence are part of the mouse rDNA transcription unit. Studies in two cell-free transcription systems as well as analysis of cellular RNA indicate that RNA polymerase I does not terminate within the 334 bp 3' terminal spacer sequences contained in the rDNA clone used. Quantitative hybridization data, S1 mapping experiments and Northern analysis of nuclear RNA showed that the 14 kb pre-rRNA molecules hybridize with the same efficiency to both the 28S and the 3' NTS specific DNA probe. This indicates that the rRNA precursor contains both at the 5' and 3' end several hundreds bases of external transcribed spacer sequences which are eliminated in subsequent processing reactions.

Animals↗
PubMed1985

Functional RNA polymerase II promoters in solitary retroviral long terminal repeats (LTR-IS elements).

LTR-IS elements are middle repetitive sequences in the mouse genome with structural features of solitary retroviral LTRs. In order to get some insight in the possible functional role of these sequences the promotor activity of two LTR-IS representatives differing by 105 bp in their U3 region was investigated. Gene fusions between LTR-IS sequences and the bacterial gene coding for chloramphenicol acetyl transferase (CAT) were transfected into mouse 3T6 cells and the expression of CAT was measured. It is shown that the LTR-IS sequences represent weak RNA polymerase II promoters which require enhancement by cis-or trans-activating factors.

Acetyltransferases↗
PubMed1985

Transcription of mouse rDNA terminates downstream of the 3' end of 28S RNA and involves interaction of factors with repeated sequences in the 3' spacer.

RNA polymerase I terminates transcription of mouse rDNA 565 bp downstream of the 3' end of mature 28S rRNA. This specific termination event can be duplicated in a nuclear extract system. RNA molecules with authentic 3' ends are transcribed from ribosomal minigene constructs provided the templates retain a minimal length of downstream spacer sequences. The nucleotide sequence of the region of transcription termination contains a set of repetitive structural elements consisting of 18 bp conserved nucleotides surrounded by stretches of pyrimidines. Termination in vivo occurs within the first element. This site is preferentially used in vitro at low template concentrations. At increasing DNA concentrations a termination site within the second repetitive element is used. Competition experiments with defined 3'-terminal fragments suggest that transcription termination by RNA polymerase I requires interaction of some factor (or factors) with the repetitive structural elements in the 3' nontranscribed spacer.

Animals↗
PubMed1985

Efficient transcription of a protein-coding gene from the RNA polymerase I promoter in transfected cells.

The activity of the mouse ribosomal promoter was examined after fusion to the gene coding for chloramphenicol acetyltransferase (CAT) and transfection into mouse cells. Very little CAT enzyme but high levels of CAT-specific RNA correctly initiated at the ribosomal DNA start site were synthesized. The amount of specific transcripts was neither influenced by long stretches of upstream spacer sequences nor by the insertion of the Moloney murine sarcoma virus enhancer. The deletion mutant pMr delta-39, which has been shown to be fully active in vitro, exhibited a 90% decrease in template activity in vivo. A mutant in which 22 base pairs of ribosomal DNA (between positions -35 and -14) were substituted by foreign DNA sequences proved transcriptionally inactive. The fusion genes were only transcribed in mouse cells, indicating that species-specific transcription factors are involved in ribosomal promoter recognition.

Acetyltransferases↗
PubMed1984

Drastic rise of intracellular adenosine(5')tetraphospho(5')adenosine correlates with onset of DNA synthesis in eukaryotic cells.

An assay of adenosine(5')tetraphospho(5')adenosine (Ap4A), based on the luciferin/luciferase method for ATP measurement, was developed, which allows one to determine picomolar amounts of unlabeled Ap4A in cellular extracts. In eukaryotic cells this method yielded levels of Ap4A varying from 0.01 microM to 13 microM depending on the growth, cell cycle, transformation, and differentiation state of cells. After mitogenic stimulation of G1-arrested mouse 3T3 and baby hamster kidney fibroblasts the Ap4A pools gradually increased 1000-fold during progression through the G1 phase reaching maximum Ap4A concentrations of about 10 microM in the S phase. Quiescent 3T3 cells reach a high level of Ap4A (1 microM) in a 'committed' but prereplicative state if exposed to an external mitogenic stimulant (excess of serum) and simultaneously to a synchronizer which inhibits entry into the S phase (hydroxyurea). When the block for DNA replication was removed at varying times after removal of the stimulant decay of commitment to DNA synthesis was found correlated with a shrinkage of the Ap4A pool. Cells lacking a defined G1 phase (V79 lung fibroblasts, Physarum) possess a constitutively high base level of Ap4A (about 0.3 microM) even during mitosis. From this high level, Ap4A concentration increases only about tenfold during the S phase. Temperature-down-shift experiments, using chick embryo cells infected with transformation-defective temperature-sensitive viral mutants(td-ts), have shown that the expression of the transformed state at 35 degrees C is accompanied by a tenfold increase of the cellular Ap4A pool. Treatment of exponentially growing human cells with interferon leads, concomitantly with an inhibition of DNA syntheses, to a tenfold decrease in intracellular Ap4A levels within 20 h. The possibility of Ap4A being a 'second messenger' of cell cycle and proliferation control is discussed in the light of these results and those reported previously demonstrating that Ap4A is a ligand of mammalian DNA polymerase alpha, triggers DNA replication in quiescent mammalian cells and is active in priming DNA synthesis.

Adenine Nucleotides↗
PubMed1984

In vitro mutagenesis and transcriptional analysis of a mouse ribosomal promoter element.

An RNA polymerase I control region essential for initiation of pre-rRNA transcription has been identified by mutagenesis in vitro of mouse rDNA (ribosomal RNA genes) and transcription in a cell-free system derived from Ehrlich ascites cells. Substitution of nucleotides between -35 and -14 by foreign DNA sequences caused a loss of template activity, which indicates that an important promoter element is located within this region. To identify the nucleotides essential for RNA polymerase I function, single and multiple point mutations within this control region were generated and the modified DNAs were assayed for template activity. The phenotypes of mutants in which C-to-T transitions have been introduced at positions -36, -31, -27, -22, -21, and -13 were identical to the wild type. Conversion of G to A at position -15 resulted in a 20% increase of promoter activity, whereas a G-to-A transition at -16 decreased transcription by 95%. Competition experiments between mutant and wild-type DNAs suggest that the guanine at -16, which is evolutionarily highly conserved, interacts with essential components of the transcription apparatus.

Animals↗
PubMed1983

Formation of stable preinitiation complexes is a prerequisite for ribosomal DNA transcription in vitro.

Cytoplasmic extracts from cultured mouse cells contain the factor(s) required for specific transcription initiation of rDNA by RNA polymerase I. Prior to transcription the essential proteins bind to the ribosomal gene and remain bound to the template for several rounds of transcription. The assembly of these preinitiation complexes in vitro has been demonstrated by kinetic analysis of the transcription reaction and by competition experiments. Complex formation involves an initial, rapid binding of transcription factor(s) to rDNA sequences followed by additional events which arrange the DNA-protein complex into a transcriptionally active state. Once the complexes have formed they persist for at least 2 hours in vitro and are resistant to elevated salt concentrations. The assembly of the complexes was inhibited when the template DNA was incubated with histones prior to the addition of S-100 extract. If, however, preinitiation complex formation was allowed to occur before the addition of histones, the interference of histones with specific transcription was much less pronounced.

Animals↗
PubMed1983

Expression of an mRNA coding gene under the control of an RNA polymerase I promoter.

We have placed a 225-bp fragment from the 5' end of the mouse rDNA transcription unit (from -169 to +56) in front of the SV40 tumor antigen coding sequence. After microinjection of this chimeric plasmid into nuclei of mouse L-cells expression of SV40 large T antigen has been observed. The expression of T antigen was dependent on the correct orientation of the rDNA fragment relative to the T antigen-coding region and was seen only in mouse cells. This indicates that the 225-bp rDNA fragment contains the sequence information required for pre-rRNA transcription and demonstrates for the first time that a protein-coding gene can be transcribed and expressed under the control of an RNA polymerase I promoter.

Animals↗
PubMed1982

Nucleotide sequence requirements for specific initiation of transcription by RNA polymerase I.

The nucleotide sequence(s) specifying RNA polymerase I initiation has been investigated by studying the transcription of deleted and nondeleted mouse ribosomal RNA gene (rDNA) templates in vitro. The deletion of 5'-flanking sequences upstream from position -- 39 did not affect transcriptional activity, but removal of sequences between positions -- 39 and -- 34 resulted in a 90% decrease of rDNA transcription. The template activity was completely eliminated by the further deletion of nucleotides -- 33 to -- 13. It is concluded that sequences between -- 34 and -- 12, upstream from the transcribed region, represent an essential control region for the initiation of transcription in vitro. Therefore, this region may be functionally analogous to the T-A-T-A box of RNA polymerase II promoters. In addition to this control region, sequences located further upstream (between positions -- 45 and -- 169) may also exert some function in efficient transcription initiation as revealed by competition experiments between wildtype and mutant rDNA templates.

Animals↗
PubMed1981

Mapping of a mouse ribosomal DNA promoter by in vitro transcription.

An in vitro transcription system that provides proper initiation of RNA polymerase I on cloned rDNA has been used to identify the start site for rDNA transcription. Different subclones that span defined regions of the 5' terminal region of the ribosomal gene have been constructed and assayed in the cell-free system for their ability to promote specific initiation of pre-rRNA synthesis. It is shown that rapid processing at the 5' end of the primary transcript occurs both in vivo and in vitro which in former studies has led to a wrong interpretation of the S1 nuclease mapping data (1 - 3). RNA polymerase I starts in vitro at a unique point on the rDNA yielding run-off transcripts that have a triphosphorylated 5' end pppApC. If multiple copies of the promoter-containing rDNA fragment were placed in head-to-tail orientation in front of the transcribed region distinct RNA products were synthesized that have been started at the tandem initiation sites. Removal of sequences upstream the initiation site indicates that 5' flanking regions are essential for specific transcription.

Animals↗
PubMed1981

The nucleotide sequence of the initiation region of the ribosomal transcription unit from mouse.

The 5' end of 45S pre-rRNA has been located on a cloned rDNA fragment from mouse by r-loop mapping and the nuclease S1 protection technique. 45S pre-rRNA could be shown to represent the primary transcript of the ribosomal genes because 5' polyphosphate termini have been detected by an enzymatic assay. The sequence of about 1100 nucleotides surrounding the initiation site for ribosomal RNA transcription has been determined. Features of this region of the ribosomal DNA will be discussed. A comparison of the nucleotide sequence with corresponding areas of ribosomal genes from other eukaryotes does not reveal significant homology in the region of transcription initiation.

Animals↗
PubMed1981

Specific transcription of mouse ribosomal DNA in a cell-free system that mimics control in vivo.

Cloned ribosomal DNA (rDNA) from mouse, which contains the initiation site of 45S pre-rRNA transcription and 5' flanking sequences, has been used as the template in an in vitro transcription system. In the presence of extracts from rapidly growing Ehrlich ascites cells, RNA polymerase I initiates specifically in that region of purified rDNA where the 5' end of 45S rRNA has been mapped. This is shown by electrophoretic analysis of the length of run-off transcripts synthesized from truncated templates, by S1 nuclease mapping, and by hybridization analysis of the in vitro products. The ability of the crude extracts to promote faithful transcription of mouse rDNA correlates with the proliferation rate of the cells. Only extracts prepared from exponentially growing mouse cells contain the factor(s) required for the faithful transcription of mouse ribosomal genes. Extracts from nongrowing or slowly growing mouse cells show very little activity. Thus, the cell-free system somehow reflects the rRNA synthetic activity of the cell and will prove valuable for the identification and purification of the various factors that are involved in the specific read-out of rDNA and may play a central role in the regulation of transcription of the ribosomal genes.

Animals↗
PubMed1980

Structural organization of mouse rDNA: comparison of transcribed and non-transcribed regions.

The DNA of the recombinant phage lambda gtWES Mr974 (GRUMMT et al., 1979) which contains the 18S region and adjacent spacer sequences of the ribosomal genes from mouse has been digested with the restriction endonuclease SalI. Fragments corresponding to the non-transcribed spacer (A and D) and the external transcribed spacer (B) have been prepared and their nucleotide composition and sequence organization has been determined. The data indicate that the part of the non-transcribed spacer contained in Mr974 consists of at least two structural domains of distinct sequence characteristics. Fragment A contains 49% G + C and exhibits a high sequence complexity. Fragment D, the spacer fragment flanking the coding region, is very rich in G + C and is obviously composed of an internally repetitive sequence which is cut by several restriction enzymes into a similar set of repetitive fragments. Most of the fragments have sizes that are multiples of 60 and 80 or 140 base pairs, respectively, suggesting an alternating 60/80bp arrangement. This regular sequence in fragment D accounts both for the observed instability and length heterogeneity of the rDNA insert in several clones and probably for the heterogeneity in the structure of the ribosomal repeats in the genomic DNA.

Animals↗