Phylogenetic evidence for Ty1-copia-like endogenous retroviruses in plant genomes.

SIRE-1 is a multi-copy, Ty1-copia-like retroelement family found in the genome of Glycine max. A sequenced SIRE-1 genomic copy has an uninterrupted ORF that can be translated into a gag-pol polyprotein, followed by an unprecedented second ORF whose conceptual translation yielded a theoretical protein predicted to possess many of the same secondary structural elements found in mammalian retroviral envelope proteins. Similar, but clearly pseudogenic, envelope-like sequences were recovered from conceptual translations of 10 Arabidopsis GenBank accessions. All were associated with identifiable Ty1-copia-like retroelements. Phylogenetic analysis of the adjacent ribonuclease H regions from these sequences and three similarly endowed elements, two from maize and one from tomato, indicate that the 14 elements constitute a monophyletic group distinct from several closely related plant Ty1-copia-like elements in which pol is immediately followed by a downstream LTR. The conservation of identifiable env-like gene features suggests that these plant elements are endogenous retroviruses whose ancestors were acquired from animal vectors. The finding that the env and env-less retroelements identified in this study form distinct lineages does not support the hypothesis that horizontal transmission of retrotransposons is sponsored by ancestral infectious retroviruses that subsequently lost all traces of env genes.

SIRE-1, a copia/Ty1-like retroelement from soybean, encodes a retroviral envelope-like protein.

The soybean genome hosts a family of several hundred, relatively homogeneous copies of a large, copia/Ty1-like retroelement designated SIRE-1. A copy of this element has been recovered from a Glycine max genomic library. DNA sequence analysis of two SIRE-1 subclones revealed that SIRE-1 contains a long, uninterrupted, ORF between the 3' end of the pol ORF and the 3' long terminal repeat (LTR), a region that harbors the env gene in retroviral genomes. Conceptual translation of this second ORF produces a 70-kDa protein. Computer analyses of the amino acid sequence predicted patterns of transmembrane domains, alpha-helices, and coiled coils strikingly similar to those found in mammalian retroviral envelope proteins. In addition, a 65-residue, proline-rich domain is characterized by a strong amino acid compositional bias virtually identical to that of the 60-amino acid, proline-rich neutralization domain of the feline leukemia virus surface protein. The assignment of SIRE-1 to the copia/Ty1 family was confirmed by comparison of the conceptual translation of its reverse transcriptase-like domain with those of other retroelements. This finding suggests the presence of a proretrovirus in a plant genome and is the strongest evidence to date for the existence of a retrovirus-like genome closely related to copia/Ty1 retrotransposons.

Sequence analysis of a cDNA containing the gag and prot regions of the soybean retrovirus-like element, SIRE-1.

SIRE-1 is a family of several hundred dispersed copies of a very large DNA element from Glycine max that has features characteristic of retroviruses and retrotransposons. A 2.4 kb SIRE-1-specific fragment was recovered from a soybean cDNA library and sequenced. The sequence contains two ORFs. Theoretical translation of ORF1 produces a gag-prot-like polyprotein containing highly conserved motifs found in retroelement nucleocapsids (CX2CX4HX4C) and aspartic proteases (LDSG). The second ORF is foreshortened. The cDNA also contains nearly 200 bp of a putative 5' LTR just upstream of a tRNA primer-binding site.

Cytokinins affect spore formation but not cell division in the yeast Saccharomyces cerevisiae.

Cytokinins are N6-substituted adenine derivatives that function as essential growth hormones in higher plants. In experimental systems, cytokinins can influence cell growth and differentiation among both plant and non-plant tissues. The single-celled yeast, Saccharomyces cerevisiae, has served as an effective and useful model system for the study of a wide range of cellular phenomena generally associated with higher eukaryotes, including mammals. In an attempt to assess the efficacy of its use to dissect the molecular basis for plant hormone action, the effects of cytokinins on S. cerevisiae with respect to cell division rates and sporulation efficiencies were monitored. While none of the cytokinins tested influenced mitotic generation times, micromolar concentrations of kinetin enhanced the formation of yeast haploid ascospores and even lower concentrations of isopentenyladenine inhibited ascus formation.

SIRE-1, a long interspersed repetitive DNA element from soybean with weak sequence similarity to retrotransposons: initial characterization and partial sequence.

Gm776 is a 776-bp subregion of a member of an interspersed family of relatively homogeneous repetitive DNA elements from soybean (Glycine max). The fragment was originally amplified from soybean DNA by the polymerase chain reaction using a single 22-nucleotide primer, and consequently terminates in an inverted repeat. The elements defined by Gm776 are at least 10.6 kb in length and constitute a family of 500-800 members per haploid genome. The family has been designated SIRE-1 (soybean interspersed repetitive element 1). Overlapping regions of Gm776 exhibit suggestive DNA sequence similarity to Ta1 and Ty1, copia-like retrotransposons from Arabidopsis thaliana and Saccharomyces cerevisiae, respectively. However, there are no similarities at the amino acid level, and the regions of similarity are not functionally related.

Cytokinin Utilization by Adenine-Requiring Mutants of the Yeast Saccharomyces cerevisiae.

By monitoring the growth of several adenine auxotrophs of the yeast Saccharomyces cerevisiae on cytokinin-supplemented media, we have demonstrated that this organism can utilize some of these derivatives as a source of adenine. Growth of a mutant lacking adenylosuccinate synthetase suggests that the conversion of cytokinins to adenine does not involve a hypoxanthine intermediate and may be catalyzed by an enzyme analogous to cytokinin oxidase.

Adenosine accumulation in Saccharomyces cerevisiae cultured in medium containing low levels of adenine.

By monitoring the in vivo incorporation of low concentrations of radiolabeled adenine into acid-soluble compounds, we observed the unusual accumulation of two nucleosides in Saccharomyces cerevisiae that were previously considered products of nucleotide degradation. Under the culture conditions used in the present study, radiolabeled adenosine was the major acid-soluble intracellular derivative, and radiolabeled inosine was initially detected as the second most prevalent derivative in a mutant lacking adenine aminohydrolase. The use of yeast mutants defective in the conversion of adenine to hypoxanthine or to AMP renders very unlikely the possibility that the presence of adenosine and inosine is attributable to nucleotide degradation. These data can be explained by postulating the existence of two enzyme activities not previously reported in S. cerevisiae. The first of these activities transfers ribose to the purine ring and may be attributable to purine nucleoside phosphorylase (EC 2.4.2.1) or adenosine phosphorylase (EC 2.4.2.-). The second enzyme converts adenosine to inosine and in all likelihood is adenosine aminohydrolase (EC 3.5.4.4).

Presence and source of free isopentenyladenosine in yeasts.

Cytokinins are a class of naturally occurring compounds that regulate growth and differentiation in tissues of higher plants. Many cytokinins are isopentenylated derivatives of adenine and its riboside, adenosine. By virtue of the post-transcriptional isopentenylation of specific anticodon loop adenosine residues in certain tRNA sequences, cytokinins are nearly universal, but tRNA-independent (de novo) cytokinin synthesis has been demonstrated in a few species. Using a radioimmunoassay, we have demonstrated that haploid strains of Saccharomyces cerevisiae and Schizosaccharomyces pombe contain, respectively, 0.8 and 0.9 microgram of the free cytokinin, N6-(delta 2-isopentenyl)adenosine, per g of cells (wet weight). Strains of both species characterized by mutations that result in deficiencies of isopentenylated tRNAs have somewhat elevated levels of free N6-(delta 2-isopentenyl)adenosine. These findings lead to the conclusion that the major, if not exclusive, source of free cytokinins in these two yeasts is a synthetic pathway independent of isopentenylated RNA turnover.

An antisuppressor mutant of Saccharomyces cerevisiae deficient in isopentenylated tRNA has reduced delta 2-isopentenylpyrophosphate: tRNA-delta 2-isopentenyl transferase activity.

We have previously reported the isolation and initial characterization of a mutation in Saccharomyces cerevisiae, designated mod5-1, that reduces the capacity of altered tyrosine tRNAs to suppress ochre nonsense mutations. The mutation results in the virtual elimination of the modified tRNA nucleoside, N6-delta 2-(isopentenyl) adenosine, normally found adjacent to the anticodons of certain tRNA species. We demonstrate here that MOD5 codes for delta 2-isopentenylpyrophosphate: tRNA-delta 2-isopentenyl transferase, or a protein that regulates its synthesis.