Ty1 mobilizes subtelomeric Y' elements in telomerase-negative Saccharomyces cerevisiae survivors.

When telomerase is inactivated in Saccharomyces cerevisiae, telomeric DNA shortens with every cell division, and cells stop dividing after approximately 100 generations. Survivors that form in these senescent populations and resume growing have variably amplified arrays of subtelomeric Y' elements. We marked a chromosomal Y' element with the his3AI retrotransposition indicator gene and found that Y'HIS3 cDNA was incorporated into the genome at approximately 10- to 1,000-fold-higher frequencies in survivors compared to telomerase-positive strains.

Ty1 defect in proteolysis at high temperature.

Retrotransposition of the Ty1 element of Saccharomyces cerevisiae is temperature sensitive. Transposition activity of Ty1 is abolished at temperatures above 34 degrees C. In this report, we show that the major block to transposition at high temperature is the inhibition of processing of the Gag-Pol-p199 polyprotein and the concomitant reduction of reverse transcriptase (RT) activity.

A nucleocapsid functionality contained within the amino terminus of the Ty1 protease that is distinct and separable from proteolytic activity.

Ty1 is the most successful of the five endogenous yeast retrotransposons. The life cycle of Ty1 dictates that a number of nucleocapsid (NC)-facilitated events occur although the protein(s) responsible for these events has not been identified. The positioning of the NC peptide is conserved at the carboxy terminus of the Gag protein among most long terminal repeat (LTR)-containing retroelements.

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.

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.

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.

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.

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.

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.

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.