Charles Hoffman | Boston College (original) (raw)

Papers by Charles Hoffman

Cellular Signalling, Dec 1, 2017

Molecular and Cellular Biology, Dec 1, 1992

We have used degenerate oligonucleotide probes based on sequences conserved among known protein t... more We have used degenerate oligonucleotide probes based on sequences conserved among known protein tyrosine phosphatases (PITPases) to identify two Schizosaccharomyces pombe genes encoding PIPases. We previously described the cloning of pypl+ (S.

Proceedings of the National Academy of Sciences of the United States of America, Apr 15, 1991

Degenerate oligonucleotide probes encoding sequences conserved among mammalian protein-tyrosineph... more Degenerate oligonucleotide probes encoding sequences conserved among mammalian protein-tyrosinephosphatases (PTPases) were used to amplify DNA fragments from a Schizosaccharomyces pombe cDNA library by polymerase chain reaction (PCR) methods. A cloned PCR product predicted peptide sequences similar to those found in PTPases but not identical to any published sequences. A S. pombe gene,

Cellular Signalling, 2019

The fission yeast Schizosaccharomyces pombe uses a cAMP signaling pathway to link glucosesensing ... more The fission yeast Schizosaccharomyces pombe uses a cAMP signaling pathway to link glucosesensing to Protein Kinase A activity in order to regulate cell growth, sexual development, gluconeogenesis, and exit from stationary phase. We previously used a PKA-repressed fbp1-ura4 reporter to conduct high throughput screens (HTSs) for inhibitors of heterologously-expressed mammalian cyclic nucleotide phosphodiesterases (PDEs). Here, we describe the successful expression of all ten mammalian adenylyl cyclase (AC) genes, along with the human GNAS Gα s gene. By measuring expression of an fbp1-GFP reporter together with direct measurements of intracellular cAMP levels, we can detect both basal AC activity from all ten AC genes as well as GNAS-stimulated activity from eight of the nine transmembrane ACs (tmACs; AC2-AC9). The ability to use this platform to conduct HTS for novel chemical probes that reduce PKA activity was demonstrated by a pilot screen of the LOPAC® 1280 library, leading to the identification of diphenyleneiodonium chloride (DPI) as an inhibitor of basal AC activity. This screening technology could open the door to the development of therapeutic compounds that target GNAS or the ACs, an area in which there is significant unmet need.

Journal of Molecular Biology, 1985

Current Genetics, 2011

While the counterselectable Schizosaccharomyces pombe ura4 + gene can be used to prepare a site i... more While the counterselectable Schizosaccharomyces pombe ura4 + gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4 + confers 5FOA-resistant (5FOA R) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOA R mutants. Relative to the same approach using the homologous URA3 + gene in Saccharomyces cerevisiae, knock-ins in S. pombe are harder to identify due to a lower eYciency of homologous recombination and a relatively high background of spontaneous 5FOA R colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we Wrst determined that 5FOA R strains carry mutations in either of two genes; ura4 + and ura5 +. We then cloned the S. pombe ura5 + orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5 + together with the S. pombe lys7 + gene. Using this doubly marked cassette to disrupt the sck1 + kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4 ¡ and ura5 ¡ mutants by screening 5FOA R colonies for the loss of the lys7 + marker. The utility of this system, especially when the phenotype for the strain carrying the knock-in allele is indistinguishable from that of the disruption strain, is borne out by the fact that »95% of 5FOA R colonies in our studies arose from background ura4 ¡ and ura5 ¡ mutations. Keywords Schizosaccharomyces pombe • Fission yeast • ura5 + • lys7 + • Selectable marker • Gene knock-in Communicated by P. Sunnerhagen.

Current Genetics, 2006

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used ... more Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2 + gene, which is homologous to Saccharomyces cerevisiae LYS4 +. We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2 +-based cloning vector pRH3. In addition, we deleted the S. pombe his7 + gene with a lys2 +-marked polymerase chain reaction (PCR) product and the S. pombe lys2 + gene with a his7 +-marked PCR product. Strains carrying these deletions of lys2 + or his7 + serve as relatively efficient hosts for the deletion of the ade6 + gene by lys2 +-or his7 +-marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2 + as a selectable marker, along with improved strains for the use of his7 + to mark gene deletions.

Molecular and Cellular Biology, 1992

We have used degenerate oligonucleotide probes based on sequences conserved among known protein t... more We have used degenerate oligonucleotide probes based on sequences conserved among known protein tyrosine phosphatases (PTPases) to identify two Schizosaccharomyces pombe genes encoding PTPases. We previously described the cloning of pyp1+ (S. Ottilie, J. Chernoff, G. Hannig, C. S. Hoffman, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 88:3455-3459, 1991), and here we describe a second gene, called pyp2+. The C terminus of each protein contains sequences conserved in the apparent catalytic domains of all known PTPases. Disruption of pyp2+ results in viable cells, as was the case for pyp1+, whereas disruption of pyp2+ and pyp1+ results in synthetic lethality. Overexpression of either pyp1+ or pyp2+ in wild-type strains leads to a delay in mitosis but is suppressed by a wee1-50 mutation at 35 degrees C or a cdc2-1w mutation. A pyp1 disruption suppresses the temperature-sensitive lethality of a cdc25-22 mutation. Our data suggest that pyp1+ and pyp2+ act as negative regulators of mitosi...

Genetics, 1982

The usual sequence of forms in the Physarum polycephalum life cycle is plasmodium-spore-amoeba-pl... more The usual sequence of forms in the Physarum polycephalum life cycle is plasmodium-spore-amoeba-plasmodium. So-called "amoebaless life cycle" or alc mutants of this Myxomycete undergo a simplified plasmodium-spore-plasmodium life cycle. We have analyzed three independently isolated alc mutants and found in each case that the failure of the spores to give rise to amoebae is due to a recessive Mendelian allele. The three mutations are tightly linked to one another and belong to a single complementation group, alcA. The mutations are pleiotropic, not only interfering with the establishment of the amoebal form at spore germination, but also affecting the phenotype of alc amoebae, which occasionally arise from alc spores. The alc amoebae (1) grow more slowly than wild type, particularly at elevated temperatures; (2) tend to transform directly into plasmodia, circumventing the sexual fusion of amoebae that usually accompanies plasmodium formation; and (3) form plasmodia by the se...

microPublication Biology, 2021

The fission yeast Schizosaccharomyces pombe produces a cAMP signal in response to glucose detecti... more The fission yeast Schizosaccharomyces pombe produces a cAMP signal in response to glucose detection. Previous characterization of this signaling focused on intracellular levels of cAMP. Here, we find that the cAMP is secreted into the medium almost immediately. This is not due to PKA activation as might have been expected. In addition, a strain that is highly deficient in drug efflux shows only a modest reduction in the secretion of cAMP to the growth medium. These observations reveal a previously unappreciated aspect of cAMP metabolism in an important model organism, leading to new questions regarding the mechanism and benefit of cAMP export in S. pombe.

Genetics, 2001

The Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcrip... more The Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 t...

Genetics, 2003

Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. ... more Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1+ gene and recombination at the meiotic recombination hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ATF-type transcription factor Atf1•Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1+ transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1+ promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1+ was remodeled under derepressed conditions in concert with the robust activation of fbp1+ transcription. Strains with tup11Δ tup12Δ double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingl...

Genetics, 2001

Fission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrime... more Fission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrimeric G protein. The gpa2 Gα and git5 Gβ are both required for glucose-triggered cAMP signaling. The git5 Gβ is a unique member of the Gβ family in that it lacks an amino-terminal coiled-coil domain shown to be essential for mammalian Gβ folding and interaction with Gγ subunits. Using a git5 bait in a two-hybrid screen, we identified the git11 Gγ gene. Co-immunoprecipitation studies confirm the composition of this Gβγ dimer. Cells deleted for git11 are defective in glucose repression of both fbp1 transcription and sexual development, resembling cells lacking either the gpa2 Gα or the git5 Gβ. Overexpression of the gpa2 Gα partially suppresses loss of either the git5 Gβ or the git11 Gγ, while mutational activation of the Gα fully suppresses loss of either Gβ or Gγ. Deletion of gpa2 (Gα), git5 (Gβ), or git11 (Gγ) confer quantitatively distinct effects on fbp1 repression, indicating that the ...

International immunopharmacology, Jan 13, 2016

We have used a high throughput small molecule screen, using a fission yeast-based assay, to ident... more We have used a high throughput small molecule screen, using a fission yeast-based assay, to identify novel phosphodiesterase 7 (PDE7) inhibitors. One of the most effective hit compounds was BC12, a barbituric acid-based molecule that exhibits unusually potent immunosuppressive and immunomodulatory actions on T lymphocyte function, including inhibition of T cell proliferation and IL-2 cytokine production. BC12 treatment confers a >95% inhibition of IL-2 secretion in phytohaemagglutinin (PHA) plus phorbol-12-myristate-13-acetate (PMA) stimulated Jurkat T cells. The effect of BC12 on IL-2 secretion is not due to decreased cell viability; rather, BC12 blocks up-regulation of IL-2 transcription in activated T cells. BC12 also inhibits IL-2 secretion in human peripheral T lymphocytes stimulated in response to CD3/CD28 co-ligation or the combination of PMA and ionomycin, as well as the proliferation of primary murine T cells stimulated with PMA and ionomycin. A BC12 analog that lacks PD...

Genetics, 2015

The fission yeast Schizosaccharomyces pombe is an important model organism for the study of eukar... more The fission yeast Schizosaccharomyces pombe is an important model organism for the study of eukaryotic molecular and cellular biology. Studies of S. pombe, together with studies of its distant cousin, Saccharomyces cerevisiae, have led to the discovery of genes involved in fundamental mechanisms of transcription, translation, DNA replication, cell cycle control, and signal transduction, to name but a few processes. However, since the divergence of the two species approximately 350 million years ago, S. pombe appears to have evolved less rapidly than S. cerevisiae so that it retains more characteristics of the common ancient yeast ancestor, causing it to share more features with metazoan cells. This Primer introduces S. pombe by describing the yeast itself, providing a brief description of the origins of fission yeast research, and illustrating some genetic and bioinformatics tools used to study protein function in fission yeast. In addition, a section on some key differences between...

PKA (protein kinase A) in the fission yeast Schizosaccharomyces pombe controls transcription of g... more PKA (protein kinase A) in the fission yeast Schizosaccharomyces pombe controls transcription of genes involved in metabolism, cell growth and sexual development. In the present review, we discuss phenotypes associated with either high or low PKA activity in the context of how they can be used to carry out genetic or small-molecule screens that affect components of the PKA pathway. Although our recent research has focused on the study of heterologously expressed cyclic nucleotide PDEs (phosphodiesterases), these same methods can be used to target other S. pombe proteins or their functionally equivalent orthologues that act in the PKA pathway.

Cellular Signalling, Dec 1, 2017

Molecular and Cellular Biology, Dec 1, 1992

We have used degenerate oligonucleotide probes based on sequences conserved among known protein t... more We have used degenerate oligonucleotide probes based on sequences conserved among known protein tyrosine phosphatases (PITPases) to identify two Schizosaccharomyces pombe genes encoding PIPases. We previously described the cloning of pypl+ (S.

Proceedings of the National Academy of Sciences of the United States of America, Apr 15, 1991

Degenerate oligonucleotide probes encoding sequences conserved among mammalian protein-tyrosineph... more Degenerate oligonucleotide probes encoding sequences conserved among mammalian protein-tyrosinephosphatases (PTPases) were used to amplify DNA fragments from a Schizosaccharomyces pombe cDNA library by polymerase chain reaction (PCR) methods. A cloned PCR product predicted peptide sequences similar to those found in PTPases but not identical to any published sequences. A S. pombe gene,

Cellular Signalling, 2019

The fission yeast Schizosaccharomyces pombe uses a cAMP signaling pathway to link glucosesensing ... more The fission yeast Schizosaccharomyces pombe uses a cAMP signaling pathway to link glucosesensing to Protein Kinase A activity in order to regulate cell growth, sexual development, gluconeogenesis, and exit from stationary phase. We previously used a PKA-repressed fbp1-ura4 reporter to conduct high throughput screens (HTSs) for inhibitors of heterologously-expressed mammalian cyclic nucleotide phosphodiesterases (PDEs). Here, we describe the successful expression of all ten mammalian adenylyl cyclase (AC) genes, along with the human GNAS Gα s gene. By measuring expression of an fbp1-GFP reporter together with direct measurements of intracellular cAMP levels, we can detect both basal AC activity from all ten AC genes as well as GNAS-stimulated activity from eight of the nine transmembrane ACs (tmACs; AC2-AC9). The ability to use this platform to conduct HTS for novel chemical probes that reduce PKA activity was demonstrated by a pilot screen of the LOPAC® 1280 library, leading to the identification of diphenyleneiodonium chloride (DPI) as an inhibitor of basal AC activity. This screening technology could open the door to the development of therapeutic compounds that target GNAS or the ACs, an area in which there is significant unmet need.

Journal of Molecular Biology, 1985

Current Genetics, 2011

While the counterselectable Schizosaccharomyces pombe ura4 + gene can be used to prepare a site i... more While the counterselectable Schizosaccharomyces pombe ura4 + gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4 + confers 5FOA-resistant (5FOA R) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOA R mutants. Relative to the same approach using the homologous URA3 + gene in Saccharomyces cerevisiae, knock-ins in S. pombe are harder to identify due to a lower eYciency of homologous recombination and a relatively high background of spontaneous 5FOA R colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we Wrst determined that 5FOA R strains carry mutations in either of two genes; ura4 + and ura5 +. We then cloned the S. pombe ura5 + orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5 + together with the S. pombe lys7 + gene. Using this doubly marked cassette to disrupt the sck1 + kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4 ¡ and ura5 ¡ mutants by screening 5FOA R colonies for the loss of the lys7 + marker. The utility of this system, especially when the phenotype for the strain carrying the knock-in allele is indistinguishable from that of the disruption strain, is borne out by the fact that »95% of 5FOA R colonies in our studies arose from background ura4 ¡ and ura5 ¡ mutations. Keywords Schizosaccharomyces pombe • Fission yeast • ura5 + • lys7 + • Selectable marker • Gene knock-in Communicated by P. Sunnerhagen.

Current Genetics, 2006

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used ... more Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2 + gene, which is homologous to Saccharomyces cerevisiae LYS4 +. We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2 +-based cloning vector pRH3. In addition, we deleted the S. pombe his7 + gene with a lys2 +-marked polymerase chain reaction (PCR) product and the S. pombe lys2 + gene with a his7 +-marked PCR product. Strains carrying these deletions of lys2 + or his7 + serve as relatively efficient hosts for the deletion of the ade6 + gene by lys2 +-or his7 +-marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2 + as a selectable marker, along with improved strains for the use of his7 + to mark gene deletions.

Molecular and Cellular Biology, 1992

We have used degenerate oligonucleotide probes based on sequences conserved among known protein t... more We have used degenerate oligonucleotide probes based on sequences conserved among known protein tyrosine phosphatases (PTPases) to identify two Schizosaccharomyces pombe genes encoding PTPases. We previously described the cloning of pyp1+ (S. Ottilie, J. Chernoff, G. Hannig, C. S. Hoffman, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 88:3455-3459, 1991), and here we describe a second gene, called pyp2+. The C terminus of each protein contains sequences conserved in the apparent catalytic domains of all known PTPases. Disruption of pyp2+ results in viable cells, as was the case for pyp1+, whereas disruption of pyp2+ and pyp1+ results in synthetic lethality. Overexpression of either pyp1+ or pyp2+ in wild-type strains leads to a delay in mitosis but is suppressed by a wee1-50 mutation at 35 degrees C or a cdc2-1w mutation. A pyp1 disruption suppresses the temperature-sensitive lethality of a cdc25-22 mutation. Our data suggest that pyp1+ and pyp2+ act as negative regulators of mitosi...

Genetics, 1982

The usual sequence of forms in the Physarum polycephalum life cycle is plasmodium-spore-amoeba-pl... more The usual sequence of forms in the Physarum polycephalum life cycle is plasmodium-spore-amoeba-plasmodium. So-called "amoebaless life cycle" or alc mutants of this Myxomycete undergo a simplified plasmodium-spore-plasmodium life cycle. We have analyzed three independently isolated alc mutants and found in each case that the failure of the spores to give rise to amoebae is due to a recessive Mendelian allele. The three mutations are tightly linked to one another and belong to a single complementation group, alcA. The mutations are pleiotropic, not only interfering with the establishment of the amoebal form at spore germination, but also affecting the phenotype of alc amoebae, which occasionally arise from alc spores. The alc amoebae (1) grow more slowly than wild type, particularly at elevated temperatures; (2) tend to transform directly into plasmodia, circumventing the sexual fusion of amoebae that usually accompanies plasmodium formation; and (3) form plasmodia by the se...

microPublication Biology, 2021

The fission yeast Schizosaccharomyces pombe produces a cAMP signal in response to glucose detecti... more The fission yeast Schizosaccharomyces pombe produces a cAMP signal in response to glucose detection. Previous characterization of this signaling focused on intracellular levels of cAMP. Here, we find that the cAMP is secreted into the medium almost immediately. This is not due to PKA activation as might have been expected. In addition, a strain that is highly deficient in drug efflux shows only a modest reduction in the secretion of cAMP to the growth medium. These observations reveal a previously unappreciated aspect of cAMP metabolism in an important model organism, leading to new questions regarding the mechanism and benefit of cAMP export in S. pombe.

Genetics, 2001

The Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcrip... more The Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 t...

Genetics, 2003

Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. ... more Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1+ gene and recombination at the meiotic recombination hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ATF-type transcription factor Atf1•Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1+ transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1+ promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1+ was remodeled under derepressed conditions in concert with the robust activation of fbp1+ transcription. Strains with tup11Δ tup12Δ double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingl...

Genetics, 2001

Fission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrime... more Fission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrimeric G protein. The gpa2 Gα and git5 Gβ are both required for glucose-triggered cAMP signaling. The git5 Gβ is a unique member of the Gβ family in that it lacks an amino-terminal coiled-coil domain shown to be essential for mammalian Gβ folding and interaction with Gγ subunits. Using a git5 bait in a two-hybrid screen, we identified the git11 Gγ gene. Co-immunoprecipitation studies confirm the composition of this Gβγ dimer. Cells deleted for git11 are defective in glucose repression of both fbp1 transcription and sexual development, resembling cells lacking either the gpa2 Gα or the git5 Gβ. Overexpression of the gpa2 Gα partially suppresses loss of either the git5 Gβ or the git11 Gγ, while mutational activation of the Gα fully suppresses loss of either Gβ or Gγ. Deletion of gpa2 (Gα), git5 (Gβ), or git11 (Gγ) confer quantitatively distinct effects on fbp1 repression, indicating that the ...

International immunopharmacology, Jan 13, 2016

We have used a high throughput small molecule screen, using a fission yeast-based assay, to ident... more We have used a high throughput small molecule screen, using a fission yeast-based assay, to identify novel phosphodiesterase 7 (PDE7) inhibitors. One of the most effective hit compounds was BC12, a barbituric acid-based molecule that exhibits unusually potent immunosuppressive and immunomodulatory actions on T lymphocyte function, including inhibition of T cell proliferation and IL-2 cytokine production. BC12 treatment confers a >95% inhibition of IL-2 secretion in phytohaemagglutinin (PHA) plus phorbol-12-myristate-13-acetate (PMA) stimulated Jurkat T cells. The effect of BC12 on IL-2 secretion is not due to decreased cell viability; rather, BC12 blocks up-regulation of IL-2 transcription in activated T cells. BC12 also inhibits IL-2 secretion in human peripheral T lymphocytes stimulated in response to CD3/CD28 co-ligation or the combination of PMA and ionomycin, as well as the proliferation of primary murine T cells stimulated with PMA and ionomycin. A BC12 analog that lacks PD...

Genetics, 2015

The fission yeast Schizosaccharomyces pombe is an important model organism for the study of eukar... more The fission yeast Schizosaccharomyces pombe is an important model organism for the study of eukaryotic molecular and cellular biology. Studies of S. pombe, together with studies of its distant cousin, Saccharomyces cerevisiae, have led to the discovery of genes involved in fundamental mechanisms of transcription, translation, DNA replication, cell cycle control, and signal transduction, to name but a few processes. However, since the divergence of the two species approximately 350 million years ago, S. pombe appears to have evolved less rapidly than S. cerevisiae so that it retains more characteristics of the common ancient yeast ancestor, causing it to share more features with metazoan cells. This Primer introduces S. pombe by describing the yeast itself, providing a brief description of the origins of fission yeast research, and illustrating some genetic and bioinformatics tools used to study protein function in fission yeast. In addition, a section on some key differences between...

PKA (protein kinase A) in the fission yeast Schizosaccharomyces pombe controls transcription of g... more PKA (protein kinase A) in the fission yeast Schizosaccharomyces pombe controls transcription of genes involved in metabolism, cell growth and sexual development. In the present review, we discuss phenotypes associated with either high or low PKA activity in the context of how they can be used to carry out genetic or small-molecule screens that affect components of the PKA pathway. Although our recent research has focused on the study of heterologously expressed cyclic nucleotide PDEs (phosphodiesterases), these same methods can be used to target other S. pombe proteins or their functionally equivalent orthologues that act in the PKA pathway.