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We previously constructed a collection of fission yeast strains that express various mammalian cyclic nucleotide phosphodiesterases (PDEs) and developed a cell-based high throughput screen (HTS) for small molecule PDE inhibitors. Here we describe a compound, BC54, that is a selective inhibitor of enzymes from the cAMP-specific PDE4 and PDE7 families. Consistent with the biological effect of other PDE4 and PDE7 inhibitors, BC54 displays potent anti-inflammatory properties and is superior to a combination of rolipram (a PDE4 inhibitor) and BRL50481 (a PDE7A inhibitor) for inducing apoptosis in chronic lymphocytic leukemia (CLL) cells. We further exploited PKA-regulated growth phenotypes in fission yeast to isolate two mutant alleles of the human PDE4B2 gene that encode enzymes possessing single amino acid changes that confer partial resistance to BC54. We confirm this resistance to both BC54 and rolipram via yeast-based assays and, for PDE4B2T407A, in vitro enzyme assays. Thus, we are able to use this system for both chemical screens to identify biologically-active PDE inhibitors and molecular genetic studies to characterize the interaction of these molecules with their target enzymes. Based on its potency, selectivity, and effectiveness in cell culture, BC54 should be a useful tool to study biological processes regulated by PDE4 and PDE7 enzymes.

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 PDE7 inhibitory activity (BC12-4) displays similar biological activity, suggesting that BC12 does not act via PDE7 inhibition. To investigate the mechanism of inhibition of IL-2 production by BC12, we performed microarray analyses using unstimulated and stimulated Jurkat T cells in the presence or absence of BC12 or BC12-4. Our studies show these compounds affect the transcriptional response to stimulation and act via one or more shared targets to produce both anti-inflammatory and pro-stress effects. These results demonstrate potent immunomodulatory activity for BC12 and BC12-4 in T lymphocytes and suggest a potential clinical use as an immunotherapeutic to treat T lymphocyte-mediated diseases.

Studies of the phosphodiesterase PDE7 family are impeded by there being only one commercially available PDE7 inhibitor, BRL50481. The authors have employed a high-throughput screen of commercial chemical libraries, using a fission yeast-based assay, to identify PDE7 inhibitors that include steroids, podocarpanes, and an unusual heterocyclic compound, BC30. In vitro enzyme assays measuring the potency of BC30 and 2 podocarpanes, in comparison with BRL50481, produce data consistent with those from yeast-based assays. In other enzyme assays, BC30 stimulates the PDE4D catalytic domain but not full-length PDE4D2, suggesting an allosteric site of action. BC30 significantly enhances the anti-inflammatory effect of the PDE4 inhibitor rolipram as measured by release of tumor necrosis factor alpha from activated monocytes. These studies introduce several new PDE7 inhibitors that may be excellent candidates for medicinal chemistry because of the requirements for drug-like characteristics placed on them by the nature of the yeast-based screen.

A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC50 value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems.

Fission yeast strains have been engineered so that their growth behavior reflects the activity of heterologous cyclic nucleotide phosphodiesterases (PDEs). These strains can be used in High Throughput Screens (HTSs) for PDE inhibitors that possess ‘drug-like’ characteristics, displaying activity in a growth stimulation assay over a 48-hr period. Through three generations of development, a collection of strains expressing 10 of the 11 mammalian PDE families that is appropriate for small molecule inhibitor screening has been generated in our laboratory. Strains unable to synthesize cyclic nucleotides allow characterization of PDE activity in that the enzyme’s potency is reflected in the amount of either cAMP or cGMP that must be added to the growth medium in order to stimulate cell growth. In the future, this system could be used to screen cDNA libraries for biological regulators of target PDEs and for the construction of strains that co-express PDEs and associated regulatory proteins to facilitate molecular and genetic studies of their functions and, in particular, to identify if different PDE-partner protein complexes show distinct patterns of inhibitor sensitivity.

The Schizosaccharomyces pombe fbp1 gene is transcriptionally repressed by protein kinase A (PKA) that is activated by extracellular glucose via a cAMP-signaling pathway. We previously used an fbp1–ura4 reporter that places uracil biosynthesis under the control of the glucose-sensing pathway to identify mutations in genes of the cAMP pathway. More recently, this reporter has been used in high throughput screens for small molecule inhibitors of heterologously-expressed cyclic nucleotide phosphodiesterases (PDEs) that hydrolyse cAMP to 5′ AMP. Here we show that strains lacking the adenylyl cyclase gene respond to either exogenous cAMP or cGMP to activate PKA, thus regulating fbp1–ura4 expression and other PKA-regulated processes such as conjugation and the nuclear export of an Rst2–GFP fusion protein. Expression of cGMP-specific PDEs or ones that hydrolyse both cAMP and cGMP increases the amount of exogenous cGMP required to activate PKA in order to repress fbp1–ura4 expression, creating conditions that allow detection of inhibitors of these PDEs. As proof of this concept, we screened a collection of compounds previously identified as inhibitors of cAMP-specific PDE4 or PDE7 enzymes for their ability to inhibit the mammalian cGMP-specific PDE5A enzyme. We identified compound BC76, which inhibits PDE5A in an in vitro enzyme assay with an IC 50 of 232 nM. Further yeast-based assays show that BC76 inhibits PDE1, PDE4, PDE5, PDE8, PDE10 and PDE11, thus demonstrating the utility of this system for detecting and characterising inhibitors of either cAMP- or cGMP-metabolising PDEs.

Use of a 4.5-kilobase-pair (kb) segment of single-copy DNA from a human genomic library as a hybridization probe of genomic human DNAs revealed allelic Taq I restriction fragments 10.6, 11.8, and 14.6 kb long. Among 12 unrelated individuals, all 6 males exhibited the 14.6-kb fragment in addition to one of the other fragments. Three of the females displayed 10.6- and 11.8-kb fragments, and the other three displayed only one fragment length; none had the 14.6-kb fragment. Hybridization of this probe to Taq I-digested DNAs from human-rodent hybrid cell lines (which have partial complements of human chromosomes) demonstrated segregation of the 14.6-kb fragment with the human Y chromosome and segregation of the 10.6- and 11.8-kb fragments with the human X chromosome. Furthermore, hybridization of this probe to Taq I-digested DNAs from 48 members of a single kindred revealed Y-linked inheritance of the 14.6-kb fragment and X-linked inheritance of the 10.6- and 11.8-kb fragments. These experiments demonstrate homology between single-copy sequences on the human X and Y chromosomes.

The non-transcribed spacer regions in the ribosomal DNA cistrons of Calliphora erythrocephala vary in length. This length variation is shown to be due to variable numbers of small repeated units found in certain areas of the NTS † regions, as has been found in several other systems. In contrast to the other organisms, however, the length variation in C. erythrocephala leads to the formation of only two major size classes with a length difference of about 1 kb. We have investigated the nature of this length difference by means of electron microscope heteroduplex and restriction enzyme digestion analyses. We demonstrate that the length variation in C. erythrocephala is due to a variation in the number of 350 bp repeating units defined by the presence of sites for three restriction enzymes, Hha I, Sau 3A and Alu I. Furthermore, the existence of an Xba I site within one 350 bp unit and the lack of a Hha I restriction site within another 350 bp unit reveals that at least some sequence differences exist among the repeating units. These restriction site differences can be viewed as genetic markers and lead to the hypothesis that the longer NTS class originated from the shorter NTS class by an unequal crossover event that served to duplicate the region containing repetitive units. An interesting feature of our observation is that not all NTS length classes are equally represented, suggesting special rules for the unequal recombination events often hypothesized as the basis for such variation.

The eggshell of Drosophila melanogaster is composed of a set of proteins synthesized by the follicular epithelium during the last third of oogenesis and organized into an inner zone (vitelline membrane) and an outer zone (chorion). To study these proteins, the authors developed techniques for mass-isolating follicles of mixed stages, mature (stage 14) follicles, chorion from stage 14 follicles, and chorion and vitelline membrane from laid eggs. The eggshell is composed mainly of protein and is unusually rich in proline and alanine. Six proteins of the chorion have been identified on polyacrylamide gels. The program of synthesis of these proteins was studied by incubating follicles of different developmental stages in culture with 3 H-labeled amino acids and displaying the labeled proteins on gels with the aid of autofluorography. The proteins are synthesized in a specific overlapping sequence during stages 10–14, a period when chorion deposition is known to occur. In addition, putative vitelline membrane proteins have been identified by their preferential incorporation of [ 3 H]proline and [ 3 H]alanine during stages of active vitelline membrane synthesis.

The growth of clones of human genomic DNA fragments in a bacteriophage lambda vector has been examined in a number of different Escherichia coli hosts. A large proportion (8.9%) of the phages carrying different fragments of the human genome fail to grow on standard rec+ hosts but will grow on hosts carrying mutations in the recB, recC, and sbcB genes. Heteroduplex analysis in the electron microscope of DNA from four of these phages revealed substantial secondary structure, including snap-back regions 200-500 base pairs in length. Such structures were not found in phages from the same DNA library that grow in rec+ hosts. These results are interpreted in the light of prior observations [Leach, D.R.F. & Stahl, F. (1983) Nature (London) 305, 448-451] showing that inverted repetitions cloned in phage lambda can be propagated in recB recC sbcB hosts but not in rec+ hosts.

A class of recombinant phage lambda clones are recovered from human genomic libraries on Escherichia coli recB21 recC22 sbcB15 cells, which fail to form plaques on wild-type cells. We report experiments which address the mechanism of this inhibition. The introduction of the recombination-stimulating sequence chi into one such clone allows growth of this phage on Rec+ cells. In addition, the insertion of lambda gam+ gene into a rec+-inhibited clone results in the ability of the phage to form plaques on wild-type cells. Since lambda Gam protein is an inhibitor of host RecBC enzyme, we tested a collection of such phage for growth on a variety of hosts altered in RecBC function. Host permissiveness correlated with the inactivation of the RecBC nucleolytic activities and not with the recombinational activities. These observations suggest that the inserted DNA sequences of these phage limit the production of packageable chromosomes. This conclusion is easily reconciled with our current knowledge of the interaction of the host recombination systems with lambda replication and encapsidation. Based on these experiments we have constructed strains, both recombination-proficient and recombination-deficient, which serve as improved hosts for the recovery of genomic sequences which are otherwise inhibitory to the growth of phage lambda.

Genomic segments which contain inverted repetitions longer than 300 bp are frequently lost from recombinant libraries grown on rec+ hosts. We have found that 9% of phage lambda clones that contain 15-20-kb insertions of human or Drosophila DNA are inhibited on rec+ hosts and as a result will become under-represented in amplified genomic libraries. We have therefore examined several factors of both host and vector origin which affect the fidelity of representation of genomic sequences in recombinant DNA libraries constructed in bacteriophage lambda vectors. This loss may be diminished if the vector carries either a chi element or a functional gam gene. The most successful approach, however, involves using a host with mutations in recB, recC, and sbcB, or in recD. We have shown that recombinant clones which require such mutant hosts for growth are somewhat more likely to contain DNA derived from loci in the genome which are polymorphic than are clones recovered on conventional hosts.

A locus in the human genome, not associated with any specific gene, has been found to be a site of restriction fragment length polymorphism. The polymorphism was found by hybridizing a 16-kilobase-pair segment of single-copy human DNA, selected from the human genome library cloned in phage lambda CH4A, to a Southern transfer of total human DNA digested with EcoRI. DNAs from a number of individuals from within Mormon pedigrees as well as random individuals have been examined. The locus is highly variable, with at least eight alleles present, homozygotes accounting for less than 25% of the individuals examined. The polymorphism appears to be the result of DNA rearrangements rather than base-pair substitutions or modifications. Examination of the DNA from seven members of a family revealed fragment lengths that are consistent with their inheritance as Mendelian alleles through three generations.

710 ASSIGNMENT OF THE FIRST HIGHLY POLYMORPHIC DNA MARKER LOCUS TO A HUMAN CHROMOSOME REGION