scholarly journals The Promoter of the Yeast INO4Regulatory Gene: a Model of the Simplest Yeast Promoter

2000 ◽  
Vol 182 (10) ◽  
pp. 2746-2752 ◽  
Author(s):  
Kelly A. Robinson ◽  
John M. Lopes
Keyword(s):  
Promoter Activity ◽  
Small Region ◽  
Transcription Start ◽  
Additional Element ◽  
S1 Nuclease ◽  
Activator Proteins ◽  
Transcription Start Sites ◽  
S1 Nuclease Mapping ◽  
Nuclease Mapping ◽  
Yeast Genes

ABSTRACT In Saccharomyces cerevisiae, the phospholipid biosynthetic genes are transcriptionally regulated in response to inositol and choline. This regulation requires the transcriptional activator proteins Ino4p and Ino2p, which form a heterodimer that binds to the UAS INO element. We have previously shown that the promoters of the INO4 and INO2 genes are among the weakest promoters characterized in yeast. Because little is known about the promoters of weakly expressed yeast genes, we report here the analysis of the constitutive INO4 promoter. Promoter deletion constructs scanning 1,000 bp upstream of theINO4 gene identified a small region (−58 to −46) that is absolutely required for expression. S1 nuclease mapping shows that this region contains the transcription start sites for the INO4gene. An additional element (−114 to −86) modestly enhancesINO4 promoter activity (fivefold). Thus, the region required for INO4 transcription is limited to 68 bp. These studies also found that INO4 gene expression is not autoregulated by Ino2p and Ino4p, despite the presence of a putative UAS INO element in the INO4promoter. We further report that the INO4 steady-state transcript levels and Ino4p levels are regulated twofold in response to inositol and choline, suggesting a posttranscriptional mechanism of regulation.

Transcription and processing of RNA from mouse ribosomal DNA transfected into hamster cells

1989 ◽  
Vol 9 (4) ◽  
pp. 1667-1671
Author(s):  
Raziuddin ◽  
R D Little ◽  
T Labella ◽  
D Schlessinger
Keyword(s):  
Gene Segment ◽  
State Level ◽  
Rrna Synthesis ◽  
Transcription Start ◽  
Base Pairs ◽  
S1 Nuclease ◽  
External Transcribed Spacer ◽  
S1 Nuclease Mapping ◽  
Nuclease Mapping ◽  
Entire Gene

Transcription of mouse genes coding for rRNA in CHO cells was promoter dependent at levels 3 to 10% of that of endogenous rRNA synthesis. Northern (RNA) and S1 nuclease mapping analyses demonstrated that transcription proceeds through the entire gene segment coding for rRNA in transfected constructs and continues, at least in some cases, into the adjoining plasmid sequences. S1 nuclease mapping also detected some processing cleavages in the transcripts, including those at the 3' terminus of 18S rRNA, those at the rapidly cleaved site at +650 in the external transcribed spacer, and those at a previously uncharacterized, rapidly cleaved site in the internal transcribed spacer. Deletion of sequences upstream or downstream from the promoter generally had no measurable effect on the level of transcription, but deletion of a 300-base-pair XhoI-XhoI fragment starting 1,287 base pairs from the transcription start site sharply increased the steady-state level of rRNA. Effects on processing were harder to test, because many intermediates are too unstable to detect even by S1 nuclease mapping; however, the data suggest that RNAs with deletions in the external transcribed spacer are processed poorly at distal sites. Processing at some sites may thus depend on interactions involving distant segments of rRNA.

A cis-acting element in the promoter region of the murine c-myc gene is necessary for transcriptional block

1989 ◽  
Vol 9 (12) ◽  
pp. 5340-5349
Author(s):  
H Miller ◽  
C Asselin ◽  
D Dufort ◽  
J Q Yang ◽  
K Gupta ◽  
...  
Keyword(s):  
Physiological State ◽  
S1 Nuclease ◽  
Cis Acting ◽  
Transcription Start Sites ◽  
Mapping Analysis ◽  
Exon 1 ◽  
Myc Gene ◽  
S1 Nuclease Mapping ◽  
Nuclease Mapping ◽  
In Cis

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

scholarly journals Transcription and processing of RNA from mouse ribosomal DNA transfected into hamster cells.

1989 ◽  
Vol 9 (4) ◽  
pp. 1667-1671 ◽  
Author(s):  
Raziuddin ◽  
R D Little ◽  
T Labella ◽  
D Schlessinger
Keyword(s):  
Gene Segment ◽  
State Level ◽  
Rrna Synthesis ◽  
Transcription Start ◽  
Base Pairs ◽  
S1 Nuclease ◽  
External Transcribed Spacer ◽  
S1 Nuclease Mapping ◽  
Nuclease Mapping ◽  
Entire Gene

Transcription of mouse genes coding for rRNA in CHO cells was promoter dependent at levels 3 to 10% of that of endogenous rRNA synthesis. Northern (RNA) and S1 nuclease mapping analyses demonstrated that transcription proceeds through the entire gene segment coding for rRNA in transfected constructs and continues, at least in some cases, into the adjoining plasmid sequences. S1 nuclease mapping also detected some processing cleavages in the transcripts, including those at the 3' terminus of 18S rRNA, those at the rapidly cleaved site at +650 in the external transcribed spacer, and those at a previously uncharacterized, rapidly cleaved site in the internal transcribed spacer. Deletion of sequences upstream or downstream from the promoter generally had no measurable effect on the level of transcription, but deletion of a 300-base-pair XhoI-XhoI fragment starting 1,287 base pairs from the transcription start site sharply increased the steady-state level of rRNA. Effects on processing were harder to test, because many intermediates are too unstable to detect even by S1 nuclease mapping; however, the data suggest that RNAs with deletions in the external transcribed spacer are processed poorly at distal sites. Processing at some sites may thus depend on interactions involving distant segments of rRNA.

scholarly journals A cis-acting element in the promoter region of the murine c-myc gene is necessary for transcriptional block.

1989 ◽  
Vol 9 (12) ◽  
pp. 5340-5349 ◽  
Author(s):  
H Miller ◽  
C Asselin ◽  
D Dufort ◽  
J Q Yang ◽  
K Gupta ◽  
...  
Keyword(s):  
Physiological State ◽  
S1 Nuclease ◽  
Cis Acting ◽  
Transcription Start Sites ◽  
Mapping Analysis ◽  
Exon 1 ◽  
Myc Gene ◽  
S1 Nuclease Mapping ◽  
Nuclease Mapping ◽  
In Cis

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

scholarly journals Location of the initial cleavage sites in mouse pre-rRNA.

1983 ◽  
Vol 3 (8) ◽  
pp. 1501-1510 ◽  
Author(s):  
L H Bowman ◽  
W E Goldman ◽  
G I Goldberg ◽  
M B Hebert ◽  
D Schlessinger
Keyword(s):  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
28S Rrna ◽  
Cleavage Sites ◽  
S1 Nuclease ◽  
5.8S Rrna ◽  
Initial Cleavage ◽  
S1 Nuclease Mapping ◽  
Mapping Techniques ◽  
Nuclease Mapping

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).

RAD3 gene of Saccharomyces cerevisiae: nucleotide sequence of wild-type and mutant alleles, transcript mapping, and aspects of gene regulation

1985 ◽  
Vol 5 (1) ◽  
pp. 17-26
Author(s):  
L Naumovski ◽  
G Chu ◽  
P Berg ◽  
E C Friedberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Coding Region ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Calculated Molecular Weight ◽  
Base Pair Deletion ◽  
S1 Nuclease Mapping ◽  
Essential Function ◽  
Nuclease Mapping

We determined the complete nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae. The coding region of the gene contained 2,334 base pairs that could encode a protein with a calculated molecular weight of 89,796. Analysis of RAD3 mRNA by Northern blots and by S1 nuclease mapping indicated that the transcript was approximately 2.5 kilobases and did not contain intervening sequences. Fusions between the RAD3 gene and the lac'Z gene of Escherichia coli were constructed and used to demonstrate that the RAD3 gene was not inducible by DNA damage caused by UV radiation or 4-nitroquinoline-1-oxide. Two UV-sensitive chromosomal mutant alleles of RAD3, rad3-1 and rad3-2, were rescued by gap repair of a centromeric plasmid, and their sequences were determined. The rad3-1 mutation changed a glutamic acid to lysine, and the rad3-2 mutation changed a glycine to arginine. Previous studies have shown that disruption of the RAD3 gene results in loss of an essential function and is associated with inviability of haploid cells. In the present experiments, plasmids carrying the rad3-1 and rad3-2 mutations were introduced into haploid cells containing a disrupted RAD3 gene. These plasmids expressed the essential function of RAD3 but not its DNA repair function. A 74-base-pair deletion at the 3' end of the RAD3 coding region or a fusion of this deletion to the E. coli lac'Z gene did not affect either function of RAD3.

Location of the initial cleavage sites in mouse pre-rRNA

1983 ◽  
Vol 3 (8) ◽  
pp. 1501-1510
Author(s):  
L H Bowman ◽  
W E Goldman ◽  
G I Goldberg ◽  
M B Hebert ◽  
D Schlessinger
Keyword(s):  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
28S Rrna ◽  
Cleavage Sites ◽  
S1 Nuclease ◽  
5.8S Rrna ◽  
Initial Cleavage ◽  
S1 Nuclease Mapping ◽  
Mapping Techniques ◽  
Nuclease Mapping

The locations of three cleavages that can occur in mouse 45S pre-rRNA were determined by Northern blot hybridization and S1 nuclease mapping techniques. These experiments indicate that an initial cleavage of 45S pre-rRNA can directly generate the mature 5' terminus of 18S rRNA. Initial cleavage of 45S pre-rRNA can also generate the mature 5' terminus of 5.8S rRNA, but in this case cleavage can occur at two different locations, one at the known 5' terminus of 5.8S rRNA and another 6 or 7 nucleotides upstream. This pattern of cleavage results in the formation of cytoplasmic 5.8S rRNA with heterogeneous 5' termini. Further, our results indicate that one pathway for the formation of the mature 5' terminus of 28S rRNA involves initial cleavages within spacer sequences followed by cleavages which generate the mature 5' terminus of 28S rRNA. Comparison of these different patterns of cleavage for mouse pre-rRNA with that for Escherichia coli pre-rRNA implies that there are fundamental differences in the two processing mechanisms. Further, several possible cleavage signals have been identified by comparing the cleavage sites with the primary and secondary structure of mouse rRNA (see W. E. Goldman, G. Goldberg, L. H. Bowman, D. Steinmetz, and D. Schlessinger, Mol. Cell. Biol. 3:1488-1500, 1983).

Oxalurate induction of multiple URA3 transcripts in Saccharomyces cerevisiae

1983 ◽  
Vol 3 (11) ◽  
pp. 1889-1897
Author(s):  
R G Buckholz ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hybridization Probe ◽  
S1 Nuclease ◽  
Ura3 Gene ◽  
Degradative Enzymes ◽  
S1 Nuclease Mapping ◽  
Dna Fragment ◽  
High Level ◽  
Nuclease Mapping ◽  
Gene Overlap

The URA3 gene from Saccharomyces cerevisiae is localized on a 1.1-kilobase (kb) DNA fragment. By using this fragment as a hybridization probe, we found that oxalurate, a gratuitous inducer of the allantoin degradative system, also serves to induce URA3 specific RNA. This response is restricted to oxalurate; other conditions which bring about high-level synthesis of the allantoin degradative enzymes did not produce the effect. Two classes of RNA (1.0 and 1.5 kb) were found to be oxalurate induced. Both classes are encoded by the URA3 gene, overlap, and probably do not significantly differ at their 5' termini. Northern blot mapping of the transcripts indicated that the 1.5-kb transcript was likely encoded by sequences extending up to 0.5 kb downstream from the 3' terminus of the 1.0-kb transcript. Analysis of the endpoints of the major 1.0-kb URA3 transcript by S1 nuclease mapping revealed the existence of two 5' termini, separated by 5 to 10 nucleotides, and seven 3' termini, separated by 5 to 20 nucleotides each, over a range of about 70 bases.

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