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Characterization of a transpositionally active Ty3 element and identification of the Ty3 integrase protein.
Get PDFTy3 is a Saccharomyces cerevisiae retrotransposon associated with tRNA genes. Two Ty3 elements have been cloned and characterized. The complete nucleotide sequence for one element, Ty3-2, was reported previously (L. J. Hansen, D. L. Chalker, and S. B. Sandmeyer, Mol. Cell. Biol. 9:5245-5256, 1988). However, this element is incapable of autonomous transposition. The complete DNA sequence of a transpositionally competent Ty3 element, Ty3-1, is presented here. Its sequence translates into two overlapping open reading frames, TYA3-1 and TYB3-1, which encode proteins with homology to the proteins specified by the retroviral gag and pol genes, respectively. Comparison of the Ty3-1 nucleotide sequence to Ty3-2 suggests that the TYB3-2 open reading frame of Ty3-2 is truncated by the deletion of a single nucleotide, which causes a frameshift mutation. Restoration of the reading frame with insertion of a single adenine by site-directed mutagenesis converted Ty3-2 into a transpositionally active element, Ty3-2(+ A). Western blot analysis with antibodies made against synthetic peptides identified integrase (IN) proteins in viruslike particle preparations from cells expressing Ty3 elements. Cells expressing Ty3-1 and Ty3-2 (+A) produce antibody-reactive proteins with approximate molecular masses of 61 and 58 kilodaltons (kDa), while cells expressing Ty3-2 produce reactive proteins of approximately 52 and 49 kDa. Together, these data show that the 61- or 58-kDa protein, or both, provides the integrase function of Ty3
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Tye GAG3 and POL3 genes encode the components of intracellular particles
Get PDFTy3 is a Saccharomyces cerevisiae retrotransposon that integrates near the transcription initiation sites of polymerase III-transcribed genes. It is distinct from the copialike Ty1 and Ty2 retrotransposons of S. cerevisiae in both the sequences of encoded proteins and gene order. It is a member of the gypsylike family of retrotransposons which resemble animal retroviruses. This study was undertaken to investigate the nucleocapsid particle of a transpositionally active gypsylike retrotransposon. Characterization of extracts from cells in which Ty3 expression was induced showed the presence of Ty3 nucleoprotein complexes, or viruslike particles, that migrated on linear sucrose gradients with a size of 156S. These particles are composed of Ty3 RNA, full-length, linear DNA, and proteins. In this study, antibodies raised against peptides predicted from the Ty3 sequence were used to identify Ty3-encoded proteins. These include the capsid (26 kDa), nucleocapsid (9 kDa), and reverse transcriptase (55 kDa) proteins. Ty3 integrase proteins of 61 and 58 kDa were identified previously (L. J. Hansen and S. B. Sandmeyer, J. Virol. 64:2599-2607, 1990). Reverse transcriptase activity associated with the particles was measured by using exogenous and endogenous primer-templates. Immunofluorescence studies of cells overexpressing Ty3 revealed cytoplasmic clusters of immunoreactive proteins. Transmission electron microscopy showed that Ty3 viruslike particles are about 50 nm in diameter. Thus, despite the unusual position specificity of Ty3 upstream of tRNA-coding regions, aspects of the Ty3 life cycle are fundamentally similar to those of retroviruses
Selective-Area MOCVD Growth and Carrier-Transport-Type Control of InAs(Sb)/GaSb Core-Shell Nanowires
No full textWe
report the first selective-area growth of high quality InAs(Sb)/GaSb
core–shell nanowires on Si substrates using metal–organic
chemical vapor deposition (MOCVD) without foreign catalysts. Transmission
electron microscopy (TEM) analysis reveals that the overgrowth of
the GaSb shell is highly uniform and coherent with the InAs(Sb) core
without any misfit dislocations. To control the structural properties
and reduce the planar defect density in the self-catalyzed InAs core
nanowires, a trace amount of Sb was introduced during their growth.
As the Sb content increases from 0 to 9.4%, the crystal structure
of the nanowires changes from a mixed wurtzite (WZ)/zinc-blende (ZB)
structure to a perfect ZB phase. Electrical measurements reveal that
both the n-type InAsSb core and p-type GaSb shell can work as active
carrier transport channels, and the transport type of core–shell
nanowires can be tuned by the GaSb shell thickness and back-gate voltage.
This study furthers our understanding of the Sb-induced crystal-phase
control of nanowires. Furthermore, the high quality InAs(Sb)/GaSb
core–shell nanowire arrays obtained here pave the foundation
for the fabrication of the vertical nanowire-based devices on a large
scale and for the study of fundamental quantum physics
Origin of the low thermal conductivity of the thermoelectric material beta-Zn(4)Sb(3): An ab initio theoretical study
No full textBy modeling beta-Zn(4)Sb(3) material as a Zn(36)Sb(30) crystal with defects, the crystal structure and thermal properties of beta-Zn(4)Sb(3) are studied by ab initio method to explain its extremely low thermal conductivity at moderate temperature. The formation and migration energies of defects are calculated and used to explain the partial occupation of Zn at the lattice sites, the disordered local structures and the origin of the low thermal conductivity of beta-Zn(4)Sb(3). Our study also unravels the puzzling dependence of thermal conductivity on doping in beta-Zn(4)Sb(3). A doping strategy is proposed to improve the thermoelectric performance of beta-Zn(4)Sb(3). (C) 2011 American Institute of Physics. [doi:10.1063/1.3599483
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Sites of RNA polymerase III transcription initiation and Ty3 integration at the U6 gene are positioned by the TATA box.
Get PDFThe function of a TATA element in RNA polymerase (EC 2.7.7.6) III transcription of a naturally TATA-containing U6 snRNA gene and a naturally TATA-less tRNA gene was probed by transcription and Ty3 transposition analyses. Deletion of the TATA box from a U6 minigene did not abolish transcription and Ty3 integration but changed the positions of initiation and insertion. Insertion of the U6 TATA box at three positions upstream of the TATA-less SUP2 tRNA(Tyr) gene resulted in novel transcription initiation and Ty3 integration patterns that depended upon position of the insertion. Nevertheless, the predominant tRNA gene initiation sites were not affected by insertion of the TATA sequence and remained at a fixed distance from the internal box A promoter element. Insertions of the TATA box upstream of a SUP2 box A mutant affected the level of transcription and restricted the use of upstream start sites, but they neither enhanced the use of TATA-dependent initiation sites nor restored expression to the level of the wild-type gene. We conclude that (i) the U6 TATA box is essential in vivo for correct initiation but not for transcription, (ii) a TATA box does not compensate for a weak box A sequence and so cannot perform equivalently, and (iii) the TATA-binding protein, and probably components of transcription factor IIIB, are present on the target at the time of Ty3 integration
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Cellular stress inhibits transposition of the yeast retrovirus-like element Ty3 by a ubiquitin-dependent block of virus-like particle formation.
Get PDFMany stress proteins and their cognates function as molecular chaperones or as components of proteolytic systems. Viral infection can stimulate synthesis of stress proteins and particular associations of viral and stress proteins have been documented. However, demonstrations of functions for stress proteins in viral life cycles are few. We have initiated an investigation of the roles of stress proteins in eukaryotic viral life cycles using as a model the Ty3 retrovirus-like element of Saccharomyces cerevisiae. During stress, Ty3 transposition is inhibited; Ty3 DNA is not synthesized and, although precursor proteins are detected, mature Ty3 proteins and virus-like particles (VLPs) do not accumulate. The same phenotype is observed in the constitutively stressed ssa1 ssa2 mutant, which lacks two cytoplasmic members of the hsp70 family of chaperones. Ty3 VLPs preformed under nonstress conditions are degraded more rapidly if cells are shifted from 30 degrees C to 37 degrees C. These results suggest that Ty3 VLPs are destroyed by cellular stress proteins. Elevated expression of the yeast UBP3 gene, which encodes a protease that removes ubiquitin from proteins, allows mature Ty3 proteins and VLPs to accumulate in the ssa1 ssa2 mutant, suggesting that, at least under stress conditions, ubiquitination plays a role in regulating Ty3 transposition
Ty3 integrase mutants defective in reverse transcription or 3'-end processing of extrachromosomal Ty3 DNA.
No full textSites of RNA polymerase III transcription initiation and Ty3 integration at the U6 gene are positioned by the TATA box.
No full textTransfer RNA genes are genomic targets for de Novo transposition of the yeast retrotransposon Ty3.
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