Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae.

The interaction between RAS proteins and adenylyl cyclase was studied by using dominant interfering mutations of adenylyl cyclase from the yeast Saccharomyces cerevisiae. RAS proteins activate adenylyl cyclase in this organism. A plasmid expressing a catalytically inactive adenylyl cyclase was found to interfere dominantly with this activation. The interfering region mapped to the leucine-rich repeat region of adenylyl cyclase, which is homologous to domains present in several other proteins and is thought to participate in protein-protein interactions.

A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1.

The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.

Mutational mapping of RAS-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase.

Large deletion and small insertion mutations in the adenylyl cyclase gene of Saccharomyces cerevisiae were used to map regions required for activation by RAS protein in vitro. The amino-terminal 605 amino acids were found to be dispensable for responsiveness to RAS protein. All other deletions in adenylyl cyclase destroyed its ability to respond to RAS. Small insertion mutations within the leucine-rich repeat region also prevented RAS responsiveness, while other insertions did not.

Two human cDNAs, including a homolog of Arabidopsis FUS6 (COP11), suppress G-protein- and mitogen-activated protein kinase-mediated signal transduction in yeast and mammalian cells.

We have isolated two novel human cDNAs, gps1-1 and gps2, that suppress lethal G-protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Suppression of other pathway-activating events was examined. In wild-type cells, expression of either gps1-1 or gps2 led to enhanced recovery from cell cycle arrest induced by pheromone. Sequence analysis indicated that gps1-1 contains only the carboxy-terminal half of the gps1 coding sequence. The predicted gene product of gps1 has striking similarity to the protein encoded by the Arabidopsis FUS6 (COP11) gene, a negative regulator of light-mediated signal transduction that is known to be essential for normal development. A chimeric construct containing gps1 and FUS6 sequences also suppressed the yeast pheromone pathway, indicating functional conservation between these human and plant genes. In addition, when overexpressed in mammalian cells, gps1 or gps2 potently suppressed a RAS- and mitogen-activated protein kinase-mediated signal and interfered with JNK activity, suggesting that signal repression is part of their normal function. For gps1, these results are consistent with the proposed function of FUS6 (COP11) as a signal transduction repressor in plants.

Sequence and spacing requirements of a retrovirus integration site.

Following infection, retroviruses insert a DNA copy of their RNA genome into the host cell genome. This integrative recombination reaction occurs at specific sites on the viral DNA: inverted repeat sequences near the termini of the linear DNA form of the viral genome. We have described elsewhere the generation and analysis of deletion mutations at one of the inverted repeat sequences in Moloney murine leukemia virus. We describe here the effects of insertion mutations made at this locus. Our results show that substantial sequence changes at the site of recombination can be tolerated, and that the spacing between the cleavage sites on the viral DNA can be expanded as well as contracted while still allowing efficient viral integration. After several rounds of virus replication, each of the insertion mutants gave rise to pseudorevertants with new alterations at the integration site.

Cloning of a rat cDNA encoding dihydroxypolyprenylbenzoate methyltransferase by functional complementation of a Saccharomyces cerevisiae mutant deficient in ubiquinone biosynthesis.

3,4-Dihydroxy-5-hexaprenylbenzoate methyltransferase (DHHB-MTase) is the product of the COQ3 gene in Saccharomyces cerevisiae and catalyses the fourth step in the biosynthesis of ubiquinone (coenzyme Q) from p-hydroxybenzoic acid. A full-length cDNA encoding a mammalian homologue of DHHB-MTase was isolated from a newly constructed rat testis cDNA library by functional complementation of a coq3 deletion mutant of S. cerevisiae. The complementing clone contained a 1.1-kb poly(A)(+)-tailed insert with a 858-bp open reading frame and presumably encodes 3,4-dihydroxy-5-polyprenylbenzoate-MTase. The deduced rat amino acid (aa) sequence has a 39% identity over 138 aa with the yeast DHHB-MTase and a 37% identity over this same region with an Escherichia coli protein encoded by the ubiG gene, a MTase that catalyses the terminal step of ubiquinone biosynthesis. The rescue of the yeast coq3 mutant by the rat homologue suggests that yeast and rat synthesize ubiquinone via the same early steps in this pathway.

ABL fusion oncogene transformation and inhibitor sensitivity are mediated by the cellular regulator RIN1.

ABL gene translocations create constitutively active tyrosine kinases that are causative in chronic myeloid leukemia, acute lymphocytic leukemia and other hematopoietic malignancies. Consistent retention of ABL SH3/SH2 autoinhibitory domains, however, suggests that these leukemogenic tyrosine kinase fusion proteins remain subject to regulation. We resolve this paradox, demonstrating that BCR-ABL1 kinase activity is regulated by RIN1, an ABL SH3/SH2 binding protein. BCR-ABL1 activity was increased by RIN1 overexpression and decreased by RIN1 silencing. Moreover, Rin1(-/-) bone marrow cells were not transformed by BCR-ABL1, ETV6-ABL1 or BCR-ABL1(T315I), a patient-derived drug-resistant mutant, as judged by growth factor independence. Rescue by ectopic RIN1 verified a cell autonomous mechanism of collaboration with BCR-ABL1 during transformation. Sensitivity to the ABL kinase inhibitor imatinib was increased by RIN1 silencing, consistent with RIN1 stabilization of an activated BCR-ABL1 conformation having reduced drug affinity. The dependence on activation by RIN1 to unleash full catalytic and cell transformation potential reveals a previously unknown vulnerability that could be exploited for treatment of leukemic cases driven by ABL translocations. The findings suggest that RIN1 targeting could be efficacious for imatinib-resistant disease and might complement ABL kinase inhibitors in first-line therapy.

Isolation of an integrated provirus of Moloney murine leukemia virus with long terminal repeats in inverted orientation: integration utilizing two U3 sequences.

We have previously described the construction of a mutant of Moloney murine leukemia virus, in594-2, which carries a 2-base-pair insertion in the U5 region of the genome and is partially defective in forming the integrated proviral DNA. We have now recovered a cloned copy of an unusual provirus from rat cells infected with this mutant. The viral genome is flanked by long terminal repeats in inverted orientation, with U3 sequences joined to cellular DNA at both of the outer edges. In addition, the provirus is a recombinant, containing a segment of a VL30 element in inverted orientation in place of the Moloney murine leukemia virus env region. The recovery of this provirus indicates that two U3 regions can be used for viral integration and suggests that there may be no absolute requirement in the reaction for those U5 sequences outside the 13-base-pair inverted repeats.

Yeast model system for study of mammalian phosphodiesterases.

Facile manipulation and rapid regeneration have helped to establish the yeast Saccharomyces cerevisiae as a genetic workhorse. More recently, these simple eukaryotes have been used for the biochemical analysis of mammalian proteins. This article describes the use of a yeast expression system for both in vitro and in vivo assays of mammalian phosphodiesterase (PDE) activity using yeast cells devoid of endogenous PDEs. It also presents simple methodologies for the analysis of the pharmacological properties of mammalian PDEs and describes the use of a powerful genetic selection for mutant forms of PDEs with altered biochemical and pharmacological characteristics.

Isolation of a recombinant murine leukemia virus utilizing a new primer tRNA.

We have previously described the construction of a mutant of Moloney murine leukemia virus bearing a deletion at the normal site of integration of the viral DNA. We have now recovered a revertant of the virus after abortive infection of mouse cells and have determined the structure of the new virus. The revertant is a recombinant virus containing a 500-base-pair patch of new sequences derived from the mouse genome. The integration site was perfectly restored to the wild-type sequence, although the patch of DNA was overall only 80% homologous to Moloney murine leukemia virus. Surprisingly, the tRNA primer binding site was no longer homologous to the usual proline tRNAs, but was a perfect match for glutamine tRNA. This result suggests that the Moloney murine leukemia virus reverse transcriptase is not specific to one tRNA, but can utilize different tRNAs to prime the synthesis of viral DNA. Comparisons with published reports allowed the identification of sequences that are 94% homologous to the patch sequence, present in one of the endogenous retroviral sequences of the mouse. No replication-competent members of this family, utilizing the glutamine tRNA primer, have been previously isolated.

Identification of endogenous retroviral sequences as potential donors for recombinational repair of mutant retroviruses: positions of crossover points.

Mutants of Moloney murine leukemia virus carrying deletions in essential regions of the genome can revert after infection of mouse cells by recombination with endogenous retroviral sequences. We have identified cloned DNAs containing potential donor sequences for two such recombination events and determined the nucleotide sequences in the relevant regions. Comparison of these sequences with that of the original mutants and the revertant viruses allowed a determination of the crossover points that were used in formation of the revertants. Each crossover occurred in short stretches (17-24 bp) of perfect homology between the two parent sequences.

Mutants and pseudorevertants of Moloney murine leukemia virus with alterations at the integration site.

Soon after infection, retroviruses synthesize a DNA copy of the genomic RNA and insert that DNA into the cellular genome by recombination at inverted repeat sequences at the termini of the viral genome. We have generated mutations that alter one terminus of the genome of Moloney murine leukemia virus (M-MuLV). Some mutations did not prevent integration of the viral DNA even though the very terminal bases were disrupted. Other mutations had dramatic effects on the efficiency of infection; in these cases the formation of preintegrative DNA was normal but the establishment of the productive provirus was prevented. One of these defective mutants gave rise to a pseudorevertant which differed from the wild type but displayed normal infectivity. An unusual number of bases of viral DNA were removed during the integration reaction carried out by this virus.

Structure of a cloned circular retroviral DNA containing a tRNA sequence between the terminal repeats.

In the course of analyzing a series of cloned circular retroviral DNAs, we recovered an unusual clone. The molecule consisted of a complete viral genome containing two copies of the long terminal repeat with extra sequences between the repeats. These extra bases proved to be a nearly complete DNA copy of a glycine tRNA, including bases that corresponded to modified and nonpairing bases of the mature tRNA. A model is proposed to account for the formation of the aberrant clone.

Recombination between a defective retrovirus and homologous sequences in host DNA: reversion by patch repair.

The genomes of mammalian species contain multiple copies of sequences homologous to exogenous retroviruses. When a mutant retrovirus carrying a lethal deletion in an essential viral gene was introduced into mammalian cells, revertant viruses appeared and spread throughout the culture. Analysis of one such revertant showed that the mutation had been repaired by homologous recombination with endogenous sequences. Our results suggest that defective retroviruses can draw upon the genetic complement of the host cell to repair lesions in viral genes.

Use of a yeast expression system for the isolation and analysis of drug-resistant mutants of a mammalian phosphodiesterase.

Saccharomyces cerevisiae strain PP5 has a phosphodiesterase (PDE) deficiency that results in heat-shock sensitivity due to the intracellular accumulation of cAMP. This strain also carries the cam mutation, which confers permeability to cAMP and, as shown here, to other compounds. Expression of rat type IV PDE in these cells caused them to revert to heat-shock resistance. Treatment of the transformed PP5 cells with rolipram, an antidepressant in humans and a potent inhibitor of type IV PDEs, reinstated sensitivity to heat shock. The biochemical properties of deletion mutants of this PDE were determined, and an active enzyme of minimum length was created. Reversion to heat-shock resistance was then used to select for PDE mutants refractory to the inhibitory effects of rolipram. Four mutants (A1, A2, A3, and A5) were isolated. Each carries a single point mutation; two have mutations in the same codon. Each mutant showed distinct properties, based on analysis of their substrate kinetics and IC50 values for a variety of inhibitors. Mutant A5 had a reduced activity for substrate, mutants A1 and A3 showed no change in substrate kinetics, and mutant A2 displayed an increase in activity. For most mutants, the drug resistance was confined to the class of drug used in the selection. This study shows that it is possible to recreate in yeast cells the susceptibility of mammalian enzymes to pharmacological agents. Our study also demonstrates that such systems can be used to select rare mutants useful in the analysis of drug-protein interactions.

Analysis of mutations in the envelope gene of Moloney murine leukemia virus: separation of infectivity from superinfection resistance.

Six deletion mutations and an insertion were generated in the env gene of cloned copies of Moloney murine leukemia virus DNA. All seven mutants were replication-defective as tested by transformation of NIH/3T3 cells. The mutant DNAs were introduced into NIH/3T3 cells to generate stable producer lines; all released virion particles into the medium, suggesting that none of the mutations affected overall viral gene expression, gag and pol gene expression, gag and pol gene functions, or virion budding. Several of the mutations reduced the lifetime of the env protein or blocked its export to the cell surface. One mutation altering the membrane-spanning region and the cytoplasmic tail of the TM protein had no effect on export of the protein, proteolytic processing, or incorporation into virion particles, but still blocked the infectivity of the resulting virus. The results suggest that alterations in the transmembrane region can affect early steps of infection, such as the fusion of virion and host membranes. Cells expressing this mutant env protein were fully resistant to superinfection by wild-type virus. Thus, induction of virus resistance, presumably reflecting blocking the virus receptor, can be separated from virus infectivity.

A temperature-sensitive mutation constructed by "linker insertion" mutagenesis.

The in vitro mutagenesis of cloned DNAs allows the formation of virtually any specific mutation, but no method has been found which might routinely lead to the important phenotype of temperature sensitivity. We have studied three linker insertion mutations in the envelope gene of Moloney murine leukemia virus (M-MuLV), and found that one was exquisitely temperature-sensitive for plaque formation. We suggest that the construction of short insertion mutations may be a fruitful approach for the generation of temperature-sensitive phenotypes in cloned genes.

Construction and analysis of deletion mutations in the pol gene of Moloney murine leukemia virus: a new viral function required for productive infection.

We have used in vitro mutagenesis to explore the functions of the gene products encoded by the pol gene of Moloney murine leukemia virus (M-MuLV). Deletions were constructed at a variety of positions in the gene, and the altered DNA copies of the viral genome were introduced into mouse cells by cotransformation. The mutants could be divided into two classes depending on the phenotype and map position of the deletion within the pol gene. Mutants with deletions mapping in the 5' portion of the gene were found to be completely deficient in reverse transcriptase activity. Mutants mapping in the 3' portion of the gene, however, assembled and released virions with normal levels of reverse transcriptase and RNAase H activities. When applied to permissive cells, these virions directed the synthesis of all three forms of unintegrated viral DNA: full-length, double-stranded linear DNA and the two circular forms with one and two copies of the long terminal repeat sequences. The infection was arrested at this point and the infected cells did not become producers of virus. Thus the 3' portion of the pol gene encodes a polypeptide with a function distinct from that of reverse transcriptase, which is not required for synthesis of viral DNA but is essential for establishment of that DNA in a stable, active form in the infected cell. We suggest that this function may be the integration of the proviral DNA.