Jean Beggs - Academia.edu (original) (raw)

Papers by Jean Beggs

Journal of Visualized Experiments, 2019

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into R... more The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Background RNA levels detected at steady state are the consequence of multiple dynamic processes ... more Background RNA levels detected at steady state are the consequence of multiple dynamic processes within the cell. In addition to synthesis and decay, transcripts undergo processing. Metabolic tagging with a nucleotide analog is one way of determining the relative contributions of synthesis, decay and conversion processes globally. Results By improving 4-thiouracil labeling of RNA in Saccharomyces cerevisiae we were able to isolate RNA produced during as little as 1Â minute, allowing the detection of nascent pervasive transcription. Nascent RNA labeled for 1.5, 2.5 or 5Â minutes was isolated and analyzed by reverse transcriptase-quantitative polymerase chain reaction and RNA sequencing. High kinetic resolution enabled detection and analysis of short-lived non-coding RNAs as well as intron-containing pre-mRNAs in wild-type yeast. From these data we measured the relative stability of pre-mRNA species with different high turnover rates and investigated potential correlations with sequen...

RNA, 2020

We reported previously that, in budding yeast, transcription rate affects both the efficiency and... more We reported previously that, in budding yeast, transcription rate affects both the efficiency and fidelity of pre-mRNA splicing, especially of ribosomal protein transcripts. Here, we report that the majority of ribosomal protein transcripts with non-consensus 5' splice sites are spliced less efficiently when transcription is faster, and more efficiently with slower transcription. These results support the "window of opportunity" model, and we suggest a possible mechanism to explain these findings.

Genome Research, 2017

The functional consequences of alternative splicing on altering the transcription rate have been ... more The functional consequences of alternative splicing on altering the transcription rate have been the subject of intensive study in mammalian cells but less is known about effects of splicing on changing the transcription rate in yeast. We present several lines of evidence showing that slow RNA polymerase II elongation increases both cotranscriptional splicing and splicing efficiency and that faster elongation reduces cotranscriptional splicing and splicing efficiency in budding yeast, suggesting that splicing is more efficient when cotranscriptional. Moreover, we demonstrate that altering the RNA polymerase II elongation rate in either direction compromises splicing fidelity, and we reveal that splicing fidelity depends largely on intron length together with secondary structure and splice site score. These effects are notably stronger for the highly expressed ribosomal protein coding transcripts. We propose that transcription by RNA polymerase II is tuned to optimize the efficiency ...

RNA, 2010

We describe methods for obtaining a quantitative description of RNA processing at high resolution... more We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3′-end cleav...

Recently, we reported that changes in transcription elongation rate affect the efficiency and fid... more Recently, we reported that changes in transcription elongation rate affect the efficiency and fidelity of precursor mRNA (pre-mRNA) splicing, especially of ribosomal protein (RP) transcripts. Here, we analyse these results in more detail, finding that the majority of RP transcripts with non-consensus 5′ splice sites have reduced splicing efficiency with faster transcription elongation, and improved efficiency with slower elongation, as might be predicted by the window of opportunity model for co-transcriptional splicing. In contrast, both faster and slower elongation reduce splicing fidelity, often for the same splicing events, and both faster and slower transcription increase fidelity with a different set of splicing events. We propose that certain non-consensus 5′ splice sites in ribosomal protein transcripts confer a stronger effect of transcription elongation rate on splicing efficiency, possibly by causing a rate-limiting step that delays activation of spliceosomes. The effects...

Yeast (Chichester, England), Jan 29, 2018

The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in... more The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in non-plant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin-binding receptor, TIR1, to target the AID-tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein but, as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required we regulated the expression of TIR1 using the ß-estradiol expression system. This is a fast-acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and ß-estradiol systems results in a tightly-controlled and fast auxin-induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the ...

eLife, Oct 13, 2017

Numerous links exist between co-transcriptional RNA processing and the transcribing RNAPII. In pa... more Numerous links exist between co-transcriptional RNA processing and the transcribing RNAPII. In particular, pre-mRNA splicing was reported to be associated with slowed RNAPII elongation. Here, we identify a site of ubiquitination (K1246) in the catalytic subunit of RNAPII close to the DNA entry path. Ubiquitination was increased in the absence of the Bre5-Ubp3 ubiquitin protease complex. Bre5 binds RNA in vivo, with a preference for exon 2 regions of intron-containing pre-mRNAs and poly(A) proximal sites. Ubiquitinated RNAPII showed similar enrichment. The absence of Bre5 led to impaired splicing and defects in RNAPII elongation in vivo on a splicing reporter construct. Strains expressing RNAPII with a K1246R mutation showed reduced co-transcriptional splicing. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.

RNA splicing, an essential part of eukaryotic pre-messenger RNA processing, can be simultaneous w... more RNA splicing, an essential part of eukaryotic pre-messenger RNA processing, can be simultaneous with transcription by RNA polymerase II. Here, we compare and review independent next-generation sequencing methods that quantify co-transcriptional splicing in budding yeast. Splicing in yeast is fast, taking place within seconds of intron transcription, while polymerase is within a few dozens of nucleotides of the 3' splice site. Ribosomal protein mRNAs are spliced particularly fast and co-transcriptionally. Intron-mediated regulation of some genes is also likely to be co-transcriptional. We suggest that intermediates of the splicing reaction, missing from current datasets, may hold key information about splicing kinetics.

Nucleic Acids Research, Dec 1, 1995

The protein sequence derived from a cloned yeast gene and partial cDNA has high sequence identity... more The protein sequence derived from a cloned yeast gene and partial cDNA has high sequence identity to 40S ribosomal subunit S5 proteins of higher eukaryotic origin. The open reading frame of the gene is flanked by consensus sequence motifs characteristic of ribosomal protein genes and the pattem of transcription of the gene in yeast cells subjected to nutritional shift or temperature shock is also typical of a ribosomal protein gene. The gene is single copy and essential for viability. The predicted sequence of the N-terminus of the protein identifies it as a phosphorylated ribosomal protein variously known as rpl 4, S2 or YS8, the least basic of the non-acidic ribosomal proteins of Saccharomyces cerevisiae.

Abnormal expression of chromosomal rabbit �-globin gene in Saccharomyces cerevisiae

Nature, 1980

A map of the restriction targets in yeast 2 micron plasmid DNA cloned on bacteriophage lambda

Mol Gen Genet, 1976

The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage lambda. The two for... more The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage lambda. The two forms of circular DNA which exist in equilibrium due to recombination between inverted repeat sequences were separated as stable clones, and a map of targets for restriction endonucleases EcoRI, HindIII and HpaI was constructed. The circular DNAs isolated from a particular oligomycin resistant strain and its parent oligomycin snesitive strain were compared by restriction endonuclease analysis, and no difference was detected. The potential uses of cloned 2 micron DNA in determining the possible biological role of these plasmids are considered.

Rna, Nov 1, 1999

Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitatio... more Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 39 end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 39 half of U6, which contains the 39 stem-loop and uridine-rich 39 end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs.

Synthesis of Rabbit Β-Globin-Specific Rna in Mouse L Cells and Yeast Transformed with Cloned Rabbit Chromosomal Β-Globin Dna

Eucaryotic Gene Regulation, 1979

Theory and Protocols, 2012

Gene 1 knockout Gene 2 knockout Transform double knockout strain with LEU2 and HIS3 plasmids with... more Gene 1 knockout Gene 2 knockout Transform double knockout strain with LEU2 and HIS3 plasmids with gene mutations (Protocol 1) Outcome: System to analyze genetic interactions between essential splicing factors. Question answered: Do two splicing factors interact genetically? Can one mutation suppress the phenotype of another mutation? Can the mechanisms of core splicing factor function be revealed?

Yeast Splicing Factors and Genetic Strategies for Their Analysis

Molecular Biology Intelligence Unit, 1995

Nature, Jan 14, 1978

Chimaeric plasmids have been constructed containing a yeast plasmid and fragments of yeast nuclea... more Chimaeric plasmids have been constructed containing a yeast plasmid and fragments of yeast nuclear DNA linked to pMB9, a derivative of the ColEl plasmid from E. coli. Two plasmids were isolated which complement leuB mutations in E. coli. These plasmids have been used to develop a method for transforming a leu2 strain of S. cerevisiae to Leu+ with high frequency. The yeast transformants contained multiple plasmid copies which were recovered by transformation in E. coli. The yeast plasmid sequence recombined intramolecularly during propagation in yeast.

Journal of general microbiology, 1976

Formation of benzoate and catechol during oxidation of benzyl alcohol by washed suspensions of Ac... more Formation of benzoate and catechol during oxidation of benzyl alcohol by washed suspensions of Acinetobacter calcoaceticus NCIB8250 confirmed earlier results indicating that this organism metabolizes benzyl alcohol via benzaldehyde, benzoate, and the 3-oxoadipate pathway. There was no evidence for feedback inhibition of benzyl alcohol dehydrogenase or benzaldehyde dehydrogenase II. Examination of growth curves and patterns of substrate utilization, as well as measurement of enzyme activities, showed that benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II are repressed when A. calcoaceticus utilizes L-mandelate or phenylglyoxylate. Growth of bacteria on L-mandelate prior to their inoculation into benzyl alcohol/salts medium leads to an exceptionally long lag period before benzyl alcohol is used at the maximum rate. Benzyl alcohol metabolism is also suppressed during growth on benzoate.

Journal of general microbiology, 1977

Specific activity of benzyl alcohol dehydrogenase in carbon-limited continuous cultures was at a ... more Specific activity of benzyl alcohol dehydrogenase in carbon-limited continuous cultures was at a maximum at a specific growth rate of 0.2 h-1, but fell off at lower and higher growth rates. The specific activity in nitrogen-limited cultures was always lower and was inversely proportional to growth rate. There was severe repression of benzyl alcohol dehydrogenase during metabolism of L(+)-mandelate or phenylglyoxylate in batch cultures. Synthesis of benzyl alcohol dehydrogenase was followed in experiments where various compounds, including a gratuitous inducer and an anti-inducer of the mandelate enzymes, were added to uninduced or pre-induced cultures and to constitutive and blocked mutants. The results led to the conclusion that there were at least two types of repression. One was caused by phenylglyoxylate carbon-lyase (or a compound synthesized co-ordinately with it), but not by the other mandelate enzymes or by L(+)-mandelate, phenylglyoxylate, benzaldehyde or benzoate. A second...

Journal of Visualized Experiments, 2019

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into R... more The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Background RNA levels detected at steady state are the consequence of multiple dynamic processes ... more Background RNA levels detected at steady state are the consequence of multiple dynamic processes within the cell. In addition to synthesis and decay, transcripts undergo processing. Metabolic tagging with a nucleotide analog is one way of determining the relative contributions of synthesis, decay and conversion processes globally. Results By improving 4-thiouracil labeling of RNA in Saccharomyces cerevisiae we were able to isolate RNA produced during as little as 1Â minute, allowing the detection of nascent pervasive transcription. Nascent RNA labeled for 1.5, 2.5 or 5Â minutes was isolated and analyzed by reverse transcriptase-quantitative polymerase chain reaction and RNA sequencing. High kinetic resolution enabled detection and analysis of short-lived non-coding RNAs as well as intron-containing pre-mRNAs in wild-type yeast. From these data we measured the relative stability of pre-mRNA species with different high turnover rates and investigated potential correlations with sequen...

RNA, 2020

We reported previously that, in budding yeast, transcription rate affects both the efficiency and... more We reported previously that, in budding yeast, transcription rate affects both the efficiency and fidelity of pre-mRNA splicing, especially of ribosomal protein transcripts. Here, we report that the majority of ribosomal protein transcripts with non-consensus 5' splice sites are spliced less efficiently when transcription is faster, and more efficiently with slower transcription. These results support the "window of opportunity" model, and we suggest a possible mechanism to explain these findings.

Genome Research, 2017

The functional consequences of alternative splicing on altering the transcription rate have been ... more The functional consequences of alternative splicing on altering the transcription rate have been the subject of intensive study in mammalian cells but less is known about effects of splicing on changing the transcription rate in yeast. We present several lines of evidence showing that slow RNA polymerase II elongation increases both cotranscriptional splicing and splicing efficiency and that faster elongation reduces cotranscriptional splicing and splicing efficiency in budding yeast, suggesting that splicing is more efficient when cotranscriptional. Moreover, we demonstrate that altering the RNA polymerase II elongation rate in either direction compromises splicing fidelity, and we reveal that splicing fidelity depends largely on intron length together with secondary structure and splice site score. These effects are notably stronger for the highly expressed ribosomal protein coding transcripts. We propose that transcription by RNA polymerase II is tuned to optimize the efficiency ...

RNA, 2010

We describe methods for obtaining a quantitative description of RNA processing at high resolution... more We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3′-end cleav...

Recently, we reported that changes in transcription elongation rate affect the efficiency and fid... more Recently, we reported that changes in transcription elongation rate affect the efficiency and fidelity of precursor mRNA (pre-mRNA) splicing, especially of ribosomal protein (RP) transcripts. Here, we analyse these results in more detail, finding that the majority of RP transcripts with non-consensus 5′ splice sites have reduced splicing efficiency with faster transcription elongation, and improved efficiency with slower elongation, as might be predicted by the window of opportunity model for co-transcriptional splicing. In contrast, both faster and slower elongation reduce splicing fidelity, often for the same splicing events, and both faster and slower transcription increase fidelity with a different set of splicing events. We propose that certain non-consensus 5′ splice sites in ribosomal protein transcripts confer a stronger effect of transcription elongation rate on splicing efficiency, possibly by causing a rate-limiting step that delays activation of spliceosomes. The effects...

Yeast (Chichester, England), Jan 29, 2018

The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in... more The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in non-plant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin-binding receptor, TIR1, to target the AID-tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein but, as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required we regulated the expression of TIR1 using the ß-estradiol expression system. This is a fast-acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and ß-estradiol systems results in a tightly-controlled and fast auxin-induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the ...

eLife, Oct 13, 2017

Numerous links exist between co-transcriptional RNA processing and the transcribing RNAPII. In pa... more Numerous links exist between co-transcriptional RNA processing and the transcribing RNAPII. In particular, pre-mRNA splicing was reported to be associated with slowed RNAPII elongation. Here, we identify a site of ubiquitination (K1246) in the catalytic subunit of RNAPII close to the DNA entry path. Ubiquitination was increased in the absence of the Bre5-Ubp3 ubiquitin protease complex. Bre5 binds RNA in vivo, with a preference for exon 2 regions of intron-containing pre-mRNAs and poly(A) proximal sites. Ubiquitinated RNAPII showed similar enrichment. The absence of Bre5 led to impaired splicing and defects in RNAPII elongation in vivo on a splicing reporter construct. Strains expressing RNAPII with a K1246R mutation showed reduced co-transcriptional splicing. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.

RNA splicing, an essential part of eukaryotic pre-messenger RNA processing, can be simultaneous w... more RNA splicing, an essential part of eukaryotic pre-messenger RNA processing, can be simultaneous with transcription by RNA polymerase II. Here, we compare and review independent next-generation sequencing methods that quantify co-transcriptional splicing in budding yeast. Splicing in yeast is fast, taking place within seconds of intron transcription, while polymerase is within a few dozens of nucleotides of the 3' splice site. Ribosomal protein mRNAs are spliced particularly fast and co-transcriptionally. Intron-mediated regulation of some genes is also likely to be co-transcriptional. We suggest that intermediates of the splicing reaction, missing from current datasets, may hold key information about splicing kinetics.

Nucleic Acids Research, Dec 1, 1995

The protein sequence derived from a cloned yeast gene and partial cDNA has high sequence identity... more The protein sequence derived from a cloned yeast gene and partial cDNA has high sequence identity to 40S ribosomal subunit S5 proteins of higher eukaryotic origin. The open reading frame of the gene is flanked by consensus sequence motifs characteristic of ribosomal protein genes and the pattem of transcription of the gene in yeast cells subjected to nutritional shift or temperature shock is also typical of a ribosomal protein gene. The gene is single copy and essential for viability. The predicted sequence of the N-terminus of the protein identifies it as a phosphorylated ribosomal protein variously known as rpl 4, S2 or YS8, the least basic of the non-acidic ribosomal proteins of Saccharomyces cerevisiae.

Abnormal expression of chromosomal rabbit �-globin gene in Saccharomyces cerevisiae

Nature, 1980

A map of the restriction targets in yeast 2 micron plasmid DNA cloned on bacteriophage lambda

Mol Gen Genet, 1976

The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage lambda. The two for... more The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage lambda. The two forms of circular DNA which exist in equilibrium due to recombination between inverted repeat sequences were separated as stable clones, and a map of targets for restriction endonucleases EcoRI, HindIII and HpaI was constructed. The circular DNAs isolated from a particular oligomycin resistant strain and its parent oligomycin snesitive strain were compared by restriction endonuclease analysis, and no difference was detected. The potential uses of cloned 2 micron DNA in determining the possible biological role of these plasmids are considered.

Rna, Nov 1, 1999

Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitatio... more Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 39 end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 39 half of U6, which contains the 39 stem-loop and uridine-rich 39 end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs.

Synthesis of Rabbit Β-Globin-Specific Rna in Mouse L Cells and Yeast Transformed with Cloned Rabbit Chromosomal Β-Globin Dna

Eucaryotic Gene Regulation, 1979

Theory and Protocols, 2012

Gene 1 knockout Gene 2 knockout Transform double knockout strain with LEU2 and HIS3 plasmids with... more Gene 1 knockout Gene 2 knockout Transform double knockout strain with LEU2 and HIS3 plasmids with gene mutations (Protocol 1) Outcome: System to analyze genetic interactions between essential splicing factors. Question answered: Do two splicing factors interact genetically? Can one mutation suppress the phenotype of another mutation? Can the mechanisms of core splicing factor function be revealed?

Yeast Splicing Factors and Genetic Strategies for Their Analysis

Molecular Biology Intelligence Unit, 1995

Nature, Jan 14, 1978

Chimaeric plasmids have been constructed containing a yeast plasmid and fragments of yeast nuclea... more Chimaeric plasmids have been constructed containing a yeast plasmid and fragments of yeast nuclear DNA linked to pMB9, a derivative of the ColEl plasmid from E. coli. Two plasmids were isolated which complement leuB mutations in E. coli. These plasmids have been used to develop a method for transforming a leu2 strain of S. cerevisiae to Leu+ with high frequency. The yeast transformants contained multiple plasmid copies which were recovered by transformation in E. coli. The yeast plasmid sequence recombined intramolecularly during propagation in yeast.

Journal of general microbiology, 1976

Formation of benzoate and catechol during oxidation of benzyl alcohol by washed suspensions of Ac... more Formation of benzoate and catechol during oxidation of benzyl alcohol by washed suspensions of Acinetobacter calcoaceticus NCIB8250 confirmed earlier results indicating that this organism metabolizes benzyl alcohol via benzaldehyde, benzoate, and the 3-oxoadipate pathway. There was no evidence for feedback inhibition of benzyl alcohol dehydrogenase or benzaldehyde dehydrogenase II. Examination of growth curves and patterns of substrate utilization, as well as measurement of enzyme activities, showed that benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II are repressed when A. calcoaceticus utilizes L-mandelate or phenylglyoxylate. Growth of bacteria on L-mandelate prior to their inoculation into benzyl alcohol/salts medium leads to an exceptionally long lag period before benzyl alcohol is used at the maximum rate. Benzyl alcohol metabolism is also suppressed during growth on benzoate.

Journal of general microbiology, 1977

Specific activity of benzyl alcohol dehydrogenase in carbon-limited continuous cultures was at a ... more Specific activity of benzyl alcohol dehydrogenase in carbon-limited continuous cultures was at a maximum at a specific growth rate of 0.2 h-1, but fell off at lower and higher growth rates. The specific activity in nitrogen-limited cultures was always lower and was inversely proportional to growth rate. There was severe repression of benzyl alcohol dehydrogenase during metabolism of L(+)-mandelate or phenylglyoxylate in batch cultures. Synthesis of benzyl alcohol dehydrogenase was followed in experiments where various compounds, including a gratuitous inducer and an anti-inducer of the mandelate enzymes, were added to uninduced or pre-induced cultures and to constitutive and blocked mutants. The results led to the conclusion that there were at least two types of repression. One was caused by phenylglyoxylate carbon-lyase (or a compound synthesized co-ordinately with it), but not by the other mandelate enzymes or by L(+)-mandelate, phenylglyoxylate, benzaldehyde or benzoate. A second...