Biomedical subjects

J F Lawler

Publications and source records attributed to J F Lawler.

7 recordsLinked to original sources

PubMed2001

Frameshift signal transplantation and the unambiguous analysis of mutations in the yeast retrotransposon Ty1 Gag-Pol overlap region.

The yeast retrotransposon Ty1 encodes a 7-nucleotide RNA sequence that directs a programmed, +1 ribosomal frameshifting event required for Gag-Pol translation and retrotransposition. We report mutations that block frameshifting, which can be suppressed in cis by "transplanting" the frameshift signal to a position upstream of its native location. These "frameshift transplant" mutants transpose with only a modest decrease in efficiency, suggesting that the location of the frameshift signal in a functional Ty1 element may vary. The genomic architecture of Ty1 is such that Gag, Ty1 PR (PR), and the Gag-derived p4 peptide share a common sequence. The functional independence of the movement of the frameshift signal to a new location within the Ty1 element is used to unambiguously attribute the effect of mutations deleterious to transposition in this region of overlapping coding sequences to effects on the Ty1 (PR). This work defines the amino terminus of the Ty1 PR and introduces a new technique for studying viral genome organization.

Amino Acid Sequence↗

PubMed2001

Ty1 proteolytic cleavage sites are required for transposition: all sites are not created equal.

The retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation. Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner. To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized. Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes. Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT. In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites. Retrotransposons with mutations in each of these sites have transposition defects. Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in trans by endogenous Ty1 copies. Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa. Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first. Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway.

Binding Sites↗

PubMed2001

Human L1 retrotransposition: cis preference versus trans complementation.

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the approximately 400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.

Base Sequence↗

PubMed1999

Viral protease assay based on GAL4 inactivation is applicable to high-throughput screening in mammalian cells.

We present an assay for viral proteases that relies on the proteolytic cleavage of substrate leading to the dissociation of the yeast transcription factor GAL4. A consensus substrate for the cytomegalovirus protease is fused between the DNA binding and transactivating domains of GAL4. Proteolysis inactivates the transcription factor which drives a luciferase reporter system. The assay is performed in mammalian cells, has a robust signal-to-noise ratio, and assesses proteolysis in a physiologic context. A unique feature of the assay is its ability to detect inhibitors of viral replication that act on viral targets other than the protease.

Amino Acid Sequence↗

PubMed1999

Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats coding for glutamine in the HD gene product huntingtin. Although HD symptoms reflect preferential neuronal death in specific brain regions, huntingtin is expressed in almost all tissues, so abnormalities outside the brain might be expected. Although involvement of nuclei and mitochondria in HD pathophysiology has been suggested, specific intracellular defects that might elicit cell death have been unclear. Mitochondria dysfunction is reported in HD brains; mitochondria are organelles that regulates apoptotic cell death. We now report that lymphoblasts derived from HD patients showed increased stress-induced apoptotic cell death associated with caspase-3 activation. When subjected to stress, HD lymphoblasts also manifested a considerable increase in mitochondrial depolarization correlated with increased glutamine repeats.

Adolescent↗

PubMed1999

The primer generator: a program that facilitates the selection of oligonucleotides for site-directed mutagenesis.

Site-directed mutagenesis is a powerful tool that has enabled molecular biologists to perform functional analysis of altered nucleic acids and proteins. Newer PCR-based mutagenesis techniques have reduced the process of mutagenesis to as little as one day. While each technique has its advantages, both require a strategy to isolate the desired clone from a population that contains mutagenized and wild-type genes. In this report, we describe a World Wide Web-based computer program that facilitates the design of mutagenic primers such that successfully mutagenized clones can be identified by the presence or absence of a unique restriction site.

Amino Acid Sequence↗

PubMed1998

Tumor necrosis factor-alpha stimulates the maturation of sterol regulatory element binding protein-1 in human hepatocytes through the action of neutral sphingomyelinase.

The mechanism by which genes involved in cholesterol biosynthesis and import are preferentially up-regulated in response to sterol depletion was elucidated with the cloning of sterol regulatory element binding protein-1 (SREBP-1). SREBP-1 is a transcription factor whose entry into the nucleus is gated by sterol-regulated proteolysis. We have investigated the role of tumor necrosis factor-alpha (TNF-alpha) as a mediator of SREBP-1 maturation in human hepatocytes. TNF-alpha is capable of inducing SREBP-1 maturation in a time- and dose-dependent manner that is consistent with the kinetics of TNF-alpha-mediated activation of neutral sphingomyelinase (N-SMase). Antibodies to N-SMase inhibit TNF-alpha-induced SREBP-1 maturation suggesting that N-SMase is a necessary component of this signal transduction pathway. Ceramide, a product of sphingomyelin hydrolysis, is also capable of inducing SREBP-1 maturation. The mature form of SREBP-1 generated by TNF-alpha, sphingomyelinase or ceramide treatment translocates to the nucleus and binds the sterol regulatory element. This promotes transcription of the gene upstream of the sterol regulatory element. A unique finding of our studies is that ceramide stimulated SREBP-1 maturation even in the presence of cholesterol and 25-hydroxycholesterol both of which are known suppressors of SREBP-1 maturation. Our findings indicate that ceramide-mediated maturation of SREBP-1 maturation is a novel sterol-independent mechanism by which cholesterol homeostasis may be regulated.

Antibodies↗