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V Lauermann

Publications and source records attributed to V Lauermann.

8 recordsLinked to original sources
PubMed1997

Plus-strand strong-stop DNA transfer in yeast Ty retrotransposons.

The yeast Ty1 LTR retrotransposon replicates by reverse transcription and integration; the process shows many similarities to the retroviral life cycle. However, we show that plus strand strong-stop DNA transfer in yeast Ty1 elements differs from the analogous retroviral process. By analysis of the native structure of the Ty1 primer binding site and by a series of manipulations of this region and assessment of the effects on retrotransposition, we show that primer binding site inheritance is not from the tRNA primer, which is inconsistent with classical retroviral models. This unusual inheritance pattern holds even when the Ty1 primer binding site is lengthened in order to be more retrovirus-like. Finally, the distantly related Ty3 element has an inheritance pattern like Ty1, indicating evolutionary conservation of the alternative pathway used by Ty1. Based on these results we arrive at a plus strand primer recycling model that explains Ty1 plus strand strong-stop DNA transfer and inheritance patterns in the primer binding site.

Binding Sites↗
PubMed1997

Maintenance of an unusual polypurine tract in HIV-2: stability to passage in culture.

A stretch of purine residues, the polypurine tract (PPT), is found in all retroviruses and is used to initiate plus-strand DNA synthesis. While the PPT of most lentiviruses is a homogeneous sequence of purine residues, the PPT of some isolates of the human and simian immunodeficiency viruses is interrupted with a single pyrimidine residue. The ROD strain of human immunodeficiency virus type 2 (HIV-2) has such a pyrimidine-containing variant PPT. Virus generated from an infectious molecular clone, pROD10, was used to infect two CD4-positive T-cell lines, H9 and CEM. The sequence of the PPT was determined after two passages. From both cell lines, the variant PPT was retained, demonstrating that the presence of a pyrimidine in the PPT was fully functional and that there was no strong selection for an all-purine PPT.

Amino Acid Sequence↗
PubMed1997

Increased length of long terminal repeats inhibits Ty1 transposition and leads to the formation of tandem multimers.

The Ty1 retrotransposon of Saccharomyces cerevisiae is bounded by long-terminal repeats (LTRs). We have constructed a variety of Ty1 elements in which the LTR length has been increased from the normal length of 334 bp to > 2 kb. Although small insertions in the LTR have minimal effects on transposition frequency, larger insertions dramatically reduce it. Nevertheless, elements with long LTRs are incorporated into the genome at a low frequency. Most of these rare insertion events represent Ty1 tandem (head to tail) multimers.

DNA, Fungal↗
PubMed1995

Plus-strand strong-stop DNA synthesis in retrotransposon Ty1.

Reverse transcription in the yeast retrotransposon Ty1 follows the general "rules" of retroviral replication overall. However, some details of the retroviral and Ty1 reverse transcription processes are different. We have identified and determined the structure of plus-strand strong-stop DNA and examined the effect of polypurine tract deletion mutations on its synthesis. Furthermore, we have defined the stop signal for plus-strand strong-stop DNA synthesis as an unusual 2'-O-ribosylated nucleotide in the primer tRNA. Full-length plus-strand strong-stop DNA, following strand transfer, would have a terminal 2-base mismatch with minus-strand DNA. These findings indicate that the mechanism of plus-strand strong-stop DNA transfer in Ty1 differs from that of the retroviral transfer and suggest that full-length plus-strand strong-stop DNA is not a direct intermediate in Ty1 retrotransposition.

Base Sequence↗
PubMed1995

Multiple molecular determinants for retrotransposition in a primer tRNA.

Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA(iMet), the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T psi C loop and the acceptor stem regions of the tRNA(iMet) affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.

Base Sequence↗
PubMed1994

The primer tRNA sequence is not inherited during Ty1 retrotransposition.

Yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism resembling retroviral replication. Long terminal repeat retroelements require primers for initiation of reverse transcription. The primer for minus-strand DNA synthesis is the 3' end of a cellular tRNA that base pairs to the complementary region of genomic RNA (the primer binding site). The genomic RNA of retroviruses and retrotransposons is shorter than its proviral DNA counterpart, lacking complete long terminal repeats. A variety of models have been proposed to describe how complete long terminal repeats are regenerated during reverse transcription. A common feature of these models is the requirement that the 3' portion of the primer tRNA be reverse-transcribed and then utilized in a strand-transfer reaction. We introduced a silent mutation into the Ty1 primer binding site and followed its fate during a single cycle of reverse transcription to directly test this aspect of the reverse transcription model. We demonstrate that the tRNA sequence is not inherited by progeny Ty1 elements during reverse transcription.

Base Sequence↗
PubMed1991

Ethanol improves the transformation efficiency of intact yeast cells.

A technique is described in which ethanol is used to improve the genetic transformation of intact yeast (Saccharomyces cerevisiae) cells pretreated with LiAc and PEG. Transformation efficiency was increased with increasing concentrations of ethanol with a peak at 10% concentration. The effect varies with different yeast strains and plasmids and up to a maximum of a 15-fold increase was observed.

DNA↗