Characterization of new transposable element sub-families from white clover (Trifolium repens) using PCR amplification.

Transposable elements (TEs) dominate the landscapes of most plant and animal genomes. Once considered junk DNA and genetic parasites, these interspersed, repetitive DNA elements are now known to play major roles in both genetic and epigenetic processes that sponsor genome variation and regulate gene expression. Knowledge of TE consensus sequences from elements in species whose genomes have not been sequenced is limited, and the individual TEs that are encountered in clones or short-reads rarely represent potentially canonical, let alone, functional representatives.

Computational and experimental analyses of retrotransposon-associated minisatellite DNAs in the soybean genome.

Background: Retrotransposons are mobile DNA elements that spread through genomes via the action of element-encoded reverse transcriptases. They are ubiquitous constituents of most eukaryotic genomes, especially those of higher plants. The pericentromeric regions of soybean (Glycine max) chromosomes contain >3,200 intact copies of the Gmr9/GmOgre retrotransposon.

Evolutionary conservation, diversity and specificity of LTR-retrotransposons in flowering plants: insights from genome-wide analysis and multi-specific comparison.

The availability of complete or nearly complete genome sequences from several plant species permits detailed discovery and cross-species comparison of transposable elements (TEs) at the whole genome level. We initially investigated 510 long terminal repeat-retrotransposon (LTR-RT) families comprising 32370 elements in soybean (Glycine max (L.) Merr.

A shotgun approach to discovering and reconstructing consensus retrotransposons ex novo from dense contigs of short sequences derived from Genbank Genome Survey Sequence database records.

Retrotransposons constitute the majority of pseudogenic protein coding regions of most eukaryotic genomes. Most genomes carry tens to thousands of retrotransposon copies derived from dozens of distinct families, but most if not all of these copies are non-functional and contain disabling mutations, including large numbers of indels. Until recently, most regions rich in these elements were virtually ignored in all but the most complete genome sequencing projects, and the full extent of their impact on the structure and function of the genomes of higher eukaryotes was under-appreciated.

Analysis of insertional sites of the SIRE1 retroelement family from Glycine max using GenBank BAC-end sequences.

SIRE1 is a 2000-copy member of the Ty1/copia retroelement family found in the soybean genome and is closely related to sireviruses found in the genomes of other legumes. Although these elements closely resemble typical plant members of the Ty1/copia family, they are unusual in that they possess an envelope-like coding region immediately downstream of the reverse transcriptase gene. Despite its copy number, very few members of the SIRE1 family are currently present in publicly available genomic assemblies or draft contigs.

Diaspora, a large family of Ty3-gypsy retrotransposons in Glycine max, is an envelope-less member of an endogenous plant retrovirus lineage.

Background: The chromosomes of higher plants are littered with retrotransposons that, in many cases, constitute as much as 80% of plant genomes. Long terminal repeat retrotransposons have been especially successful colonizers of the chromosomes of higher plants and examinations of their function, evolution, and dispersal are essential to understanding the evolution of eukaryotic genomes. In soybean, several families of retrotransposons have been identified, including at least two that, by virtue of the presence of an envelope-like gene, may constitute endogenous retroviruses.

SIRE1, an endogenous retrovirus family from Glycine max, is highly homogeneous and evolutionarily young.

SIRE1 is unusual among Ty1-copia retrotransposons in that it has an additional open reading frame with structural features similar to retroviral envelope proteins between pol and the 3' LTR. Here we report the characterization and comparison of eight different SIRE1 elements derived from a soybean genomic library, as well as SIRE1 reverse transcriptases from Glycine soja. The DNA sequences of the eight SIRE1 elements are highly homogeneous and share greater than 95% nucleotide identity.

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.

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.

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.

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.

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.

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.

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.

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.

Antisuppression of class I suppressors in an isopentenylated-transfer RNA deficient mutant of Saccharomyces cerevisiae.

The effect of a previously isolated antisuppressor mutation from bakers' yeast, that reduced the efficiency of the tyrosine-inserting ochre suppressor, SUP7-o, on other tyrosine-inserting ochre suppressors has been determined. As expected, the antisuppressor mutation, mod5-1, restricted the capacity of all eight tyrosine-inserting ochre suppressors to suppress nonsense mutations. Based on the suppression of five ochre alleles in the presence of mod5, the eight class I suppressors can be grouped into three subclasses.

Thiolated nucleotides in yeast transfer RNA.

By culturing Saccharomyces cerevisiae in growth medium containing Mg35SO4, we have determined the extent and variation of tRNA thiolation in this yeast. We find that 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U)1 is the major, if not only, thiolated derivative in S. cerevisiae tRNA.

Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae.

We have isolated a mutant of Saccharomyces cerevisiae that contains 1.5% of the normal tRNA complement of isopentenyladenosine (i6A). The mutant was characterized by the reduction in efficiency of a tyrosine inserting UAA nonsense suppressor.

Genetic analysis of a transposable suppressor gene in Saccharomyces cerevisiae.

We have demonstrated in Saccharomyces cerevisiae the transposition of a gene coding for an efficient ochre (UAA) suppressor from a centromere-linked site on chromosome III to two new sites in the yeast genome. One site is on chromosome VI, very close to, if not allelic with, SUP11, one of eight genes coding for a tyrosine-inserting suppressor. The second site is on chromosome III, unlinked to the centromere and distal to the mating type locus.