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J Colicelli

Publications and source records attributed to J Colicelli.

34 records · Page 2Linked to original sources
PubMed1990

Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein.

Adenylyl cyclase from S. cerevisiae contains at least two subunits, a 200 kd catalytic subunit and a subunit with an apparent molecular size of 70 kd, which we now call CAP (cyclase-associated protein). We cloned a cDNA encoding CAP by screening a yeast cDNA expression library in E. coli with antisera raised against the purified protein. The cDNA contained an open reading frame capable of encoding a 526 amino acid protein that is not homologous to any sequences in the current data bases. Adenylyl cyclase activity in membranes from cells that lacked CAP was not stimulated by RAS2 proteins in vitro. These results suggest that CAP is required for at least some aspects of the RAS-responsive signaling system. Mutants lacking CAP had four additional phenotypes that appear to be unrelated to effects of the RAS/adenylyl cyclase pathway: the inability to grow on rich medium (YPD), temperature sensitivity on minimal medium, sensitivity to nitrogen starvation, and a swollen cell morphology.

Adenylyl Cyclases↗
PubMed1990

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.

Adenylyl Cyclases↗
PubMed1990

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.

Adenylyl Cyclases↗
PubMed1989

Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase.

A rat brain cDNA library has been constructed in a Saccharomyces cerevisiae expression vector and used to isolate genes that can function in yeast to suppress the phenotypic effects of RAS2val19, a mutant form of the RAS2 gene analogous to an oncogenic mutant of the human HRAS gene. One cDNA, DPD, was cloned and its genetic and biochemical properties were characterized. A DPD product would share 80% amino acid sequence identity with the Drosophila melanogaster dunce-encoded protein over an extended region. We have shown that the DPD protein is a high-affinity cAMP-specific phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

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.

Base Sequence↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1986

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.

Animals↗
PubMed1986

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.

Cloning, Molecular↗
PubMed1985

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.

Animals↗
PubMed1985

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.

Animals↗
PubMed1985

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.

Animals↗
PubMed1984

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.

Chromosome Deletion↗
PubMed1984

Mutations in the gag gene of Moloney murine leukemia virus: effects on production of virions and reverse transcriptase.

We have constructed a series of deletion mutations in the p30 and p10 domains of the gag gene of Moloney murine leukemia virus. Mutants with deletions in P30 were completely defective in virion particle production even though an altered gag precursor protein is synthesized. This domain is apparently critical for particle formation. A mutant in P10 was able to release virion particles into the medium, and low levels of reverse transcriptase activity could be detected in these virions. To explore the effects of these mutations on the utilization of the gag-pol precursor, we have introduced these mutants into cells already releasing defective particles from an endogenous provirus which directs the synthesis of gag gene products and not pol gene products. The P10 mutant was capable of providing pol function as judged by the incorporation of high levels of reverse transcriptase into the particles and complete complementation for XC plaque formation. In contrast, the mutants in P30 were negative in this complementation test. Thus, those gag mutants which were unable on their own to assemble virion particles were also unable to contribute the gag-pol precursor to these particles. These mutations are the first to be mapped to the gag region which affect pol function, suggesting that the gag-pol precursor must be assembled before pol is functionally separated from the gag domain. The concordance of the effects of different mutations on both particle formation and gag-pol utilization suggests that similar domains of gag (namely, domains in the P30 region) are needed for these two processes.

Animals↗
PubMed1983

Deletion mutants of Moloney murine leukemia virus which lack glycosylated gag protein are replication competent.

A series of deletion mutations localized near the 5' end of the Moloney murine leukemia virus genome was generated by site-specific mutagenesis of cloned viral DNA. The mutants recovered from such deleted DNAs failed to synthesize the normal glycosylated gag protein gPr80gag. Two of the mutants made no detectable protein, and a third mutant, containing a 66-base pair deletion, synthesized an altered gag protein which was not glycosylated. All the mutants made normal amounts of the internal Pr65gag protein. The viruses were XC positive and replicated normally in NIH/3T3 cells as well as in lymphoid cell lines. These results indicate that the additional peptides of the glycosylated gag protein are encoded near the 5' end, that the glycosylated and internal gag proteins are synthesized independently, and that the glycosylated gag protein is not required during the normal replication cycle. In addition, the region deleted in these mutants apparently encodes no cis-acting function needed for replication. Thus, all essential sequences, including those for packaging viral RNA, must lie outside this area.

Animals↗