Biomedical subjects

D J Eichinger

Publications and source records attributed to D J Eichinger.

5 recordsLinked to original sources

PubMed1994

In-frame linker insertion mutagenesis of yeast transposon Ty1: phenotypic analysis.

A plasmid bearing a transpositionally functional GAL1::Ty1 fusion was mutagenized by insertion of four or five codons semirandomly throughout the plasmid. This collection of mutant plasmids was introduced into yeast cells and studied with regard to the properties of the mutant Ty1-encoded proteins and the transposition phenotypes observed. All of the transposition-inactivating mutations were previously found to be recessive with the exception of a single mutation in TYA. In this mutant, TYA protein of normal abundance is produced, but the virus-like particles containing this protein are unstable and have aberrant behavior. The effects of mutations in noncoding regions, as well as the capsid protein coding region TYA, and the regions encoding the protease, integrase and reverse transcriptase proteins are described. Effects on gene expression, types of proteins produced, proteolysis of precursor proteins, virus-like particle structure, and biochemical activities of the encoded proteins are summarized. In addition, we show that one of the mutations in the 3' LTR represents a new nonessential site into which foreign marker DNA can be inserted without compromising transposition.

Codon↗

PubMed1991

Doubling Ty1 element copy number in Saccharomyces cerevisiae: host genome stability and phenotypic effects.

Haploid yeast strains bearing approximately double the normal number of Ty1 elements have been constructed using marked GAL/Ty1 fusion plasmids. The strains maintain their high transposon copy number and overall genome structure in the absence of selection. The strains bearing extra Ty1 copies are surprisingly similar phenotypically to the parental strain. The results suggest that the limit to transposon copy number, if any, has not been reached. When these strains are crossed by wild-type strains (i.e., bearing the normal complement of Ty1 elements) or by strains of opposite mating type also bearing excess Ty1 elements, normal to very slightly reduced spore viability is observed, indicating that increasing the extent of transposon homology scattered around the genome does not result in significant increases in frequency of ectopic reciprocal recombination. The results suggest that yeast cells have evolved mechanisms for coping with excess transposon copies in the genome.

Blotting, Southern↗

PubMed1990

A specific terminal structure is required for Ty1 transposition.

Yeast retrotransposon Ty1 directs the synthesis of virus-like particles (VLPs) consisting of Ty1-encoded proteins, RNA, and reverse transcripts. Ty1 reverse transcripts, tagged with a selectable marker and found within VLPs, are capable of transposing into naked target DNA in vitro. Cassettes consisting of a Ty long terminal repeat (LTR), or delta, marked with supF, and flanked by appropriate restriction sites were constructed. These artificial substrates, whose termini resemble those of linear, full-length Ty1 reverse transcripts, can be coincubated with VLPs (containing unmarked reverse transcripts), resulting in the very efficient integration of the artificial substrate. The results suggest that Ty DNA is limiting for transposition in vivo, suggesting that inefficient reverse transcription regulates Ty1 transposition. Analysis of the transposition of these model substrates, which resemble in vivo Ty1 transposition intermediates or differ from them in subtle ways, shows that Ty transposition proceeds by the linkage of the 3' hydroxyl residue of the reverse transcript to target DNA.

Base Sequence↗

PubMed1988

The DNA intermediate in yeast Ty1 element transposition copurifies with virus-like particles: cell-free Ty1 transposition.

Yeast Ty1 elements are retrotransposons that transpose via an RNA intermediate found in a virus-like particle (Ty-VLP). A Ty-encoded reverse transcriptase activity found inside the particles is capable of giving rise to full-length reverse transcripts. The predominant form of these reverse transcripts is a full-length linear duplex DNA. We have developed a cell-free system for transposition of Ty1 DNA molecules into a bacteriophage lambda target. Purified Ty-VLPs and target DNA are the only macromolecular components required for the transposition reaction. A TYB-encoded protein, p90-TYB, contains amino acid sequences that are similar to those of retroviral integrase proteins. Mutations in the integrase coding region abolish transposition both in vivo and in vitro.

Antibodies, Monoclonal↗

PubMed1986

Circumsporozoite protein of Plasmodium berghei: gene cloning and identification of the immunodominant epitopes.

The gene encoding the circumsporozoite (CS) protein of the rodent malaria parasite Plasmodium berghei was cloned and characterized. A cDNA library made from P. berghei sporozoite RNA was screened with a monoclonal antibody for expression of CS protein epitopes. The resulting cDNA clone was used to isolate the CS protein gene from a lambda library containing parasite blood-stage DNA. The CS protein gene contains a central region encoding two types of tandemly repeated amino acid units, flanked by nonrepeated regions encoding amino- and carboxy-terminal signal and anchorlike sequences, respectively. One of the central repeated amino acid unit types contains the immunodominant epitopes.

Amino Acid Sequence↗