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.

scholarly journals 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.

Characterization of transcripts expressed from nitrogenase-3 structural genes of Azotobacter vinelandii

1992 ◽  
Vol 38 (9) ◽  
pp. 929-936 ◽  
Author(s):  
R. Premakumar ◽  
Marty R. Jacobson ◽  
Telisa M. Loveless ◽  
Paul E. Bishop
Keyword(s):  
Azotobacter Vinelandii ◽  
Coding Region ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Mrna Species ◽  
Mutant Strains ◽  
S1 Nuclease Mapping ◽  
Alternative Nitrogenase ◽  
Nuclease Mapping

Five major anfH-hybridizing mRNA species accumulated in Azotobacter vinelandii cells derepressed for nitrogenase-3 (an alternative nitrogenase, which appears to lack Mo and V). Using anfH-, anfD-, anfG-, anfK-, and orf1orf2-specific probes and mutant strains of A. vinelandii these mRNA species have been identified as encoding anfHDGKorf1orf2 (6.0 kb), anfHDGK (4.3 kb), anfHD (2.6 kb), vnfHorfFd (1.3 kb), and vnfH and (or) anfH(1.0 kb). A 0.6-kb mRNA species, which hybridized only to the orf1orf2-specific probe, and a 3.5-kb mRNA species, which hybridized to anfD or anfK, also accumulated under these conditions. Northern blot analysis and S1 nuclease mapping indicate that transcription of the anf structural gene cluster initiates at a unique nif consensus promoter situated 127 base pairs upstream from the anfH coding region. Observation of anfH-hybridizing mRNA species that accumulate in strains derepressed for nitrogen fixation demonstrates that transcription of the anfHDGKorf1orf2 cluster is normally repressed by Mo, V, and NH4+, whereas transcription of the vnfHorfFd cluster does not require the presence of V and is repressed only by Mo, but not NH4+. Analysis of the accumulation of mRNAs in a tungsten-tolerant strain revealed that Mo and V repression of anf transcription must occur by different mechanisms. Key words: Azotobacter vinelandii, nitrogenase-3, transcripts, regulation, molybdenum, vanadium.

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.

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 Upstream activation of ribosomal RNA biosynthesis in Saccharomyces cerevisiae

1985 ◽  
Vol 232 (1) ◽  
pp. 205-209 ◽  
Author(s):  
R V Quincey ◽  
R E Godfrey
Keyword(s):  
Shuttle Vector ◽  
Initiation Site ◽  
Rrna Gene ◽  
Coding Region ◽  
S1 Nuclease ◽  
Mapping Procedure ◽  
Nontranscribed Spacer ◽  
S1 Nuclease Mapping ◽  
Yeast Transformants ◽  
Nuclease Mapping

Yeast was transformed with eight recombinants that contained an rRNA minigene and upstream elements of rDNA in different orientations in the multi-copy yeast-Escherichia coli shuttle vector, pJDB207. The effect of these elements of upstream rDNA on the initiation of transcription of the minigene at the site for rRNA biosynthesis was determined by using an S1 nuclease mapping procedure to measure the abundance of the minigene transcript in RNA from the yeast transformants. Transcription of the minigene was enhanced 3-fold by DNA within a 2.2 kb element more than 1.5 kb upstream from the initiation site. Inversion of the 2.2 kb element decreased expression of the minigene by 40%. This 2.2 kb element contained approx. 500 bp from the 25S rRNA coding region at the 3′ end of the preceding rRNA gene and 1 kb of adjacent nontranscribed spacer rDNA. The enhancing activity was independent of interference from readthrough that might have contributed to the 7-fold decrease in minigene expression caused by removing all rDNA upstream from −209 bp.

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 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.

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).

scholarly journals DNA damage and heat shock dually regulate genes in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (1) ◽  
pp. 90-96 ◽  
Author(s):  
T McClanahan ◽  
K McEntee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Heat Shock ◽  
Shock Treatment ◽  
Northern Hybridization ◽  
Heat Shock Treatment ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Hsp70 Family ◽  
Transcriptional Start Sites

Two Saccharomyces cerevisiae genes isolated in a differential hybridization screening for DNA damage regulation (DDR genes) were also transcriptionally regulated by heat shock treatment. A 0.45-kilobase transcript homologous to the DDRA2 gene and a 1.25-kilobase transcript homologous to the DDR48 gene accumulated after exposure of cells to 4-nitroquinoline-1-oxide (NQO; 1 to 1.5 microgram/ml) or brief heat shock (20 min at 37 degrees C). The DDRA2 transcript, which was undetectable in untreated cells, was induced to high levels by these treatments, and the DDR48 transcript increased more than 10-fold as demonstrated by Northern hybridization analysis. Two findings argue that dual regulation of stress-responsive genes is not common in S. cerevisiae. First, two members of the heat shock-inducible hsp70 family of S. cerevisiae, YG100 and YG102, were not induced by exposure to NQO. Second, at least one other DNA-damage-inducible gene, DIN1, was not regulated by heat shock treatment. We examined the structure of the induced RNA homologous to DDRA2 after heat shock and NQO treatments by S1 nuclease protection experiments. Our results demonstrated that the DDRA2 transcript initiates equally frequently at two sites separated by 5 base pairs. Both transcriptional start sites were utilized when cells were exposed to either NQO or heat shock treatment. These results indicate that DDRA2 and DDR48 are members of a unique dually regulated stress-responsive family of genes in S. cerevisiae.

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