Tau, sigma, and delta. A family of repeated elements in yeast

We report here the isolation and structure of a new repeated DNA element, tau. This element, from Saccharomyces cerevisiae, is 371 base pairs long and is flanked on either end by the same invertedly repeated sequence found at the ends of some Ty and sigma elements in yeast, copia elements in Drosophila and spleen necrosis virus. The tau inverted repeats are themselves flanked by a 5-base pair directly repeated genomic sequence that is present only once in a cognate tau-allele. These structural characteristics, the presence of multiple copies of tau in the genome, and the isolation of tau+ and tau- allelic pairs suggest that tau may be capable of transposition either alone or in association with some larger element. Detailed sequence analysis of the tau, sigma, and delta elements revealed that all three contain significant regions of homology, suggesting that they are probably members of a single family derived from a common progenitor.     

Frameshift suppressor mutations outside the anticodon in yeast proline tRNAs containing an intervening sequence.

Extragenic suppressors of +1 frameshift mutations in proline codons map in genes encoding two major proline tRNA isoacceptors. We have shown previously that one isoacceptor encoded by the SUF2 gene (chromosome 3) contains no intervening sequence. SUF2 suppressor mutations result from the base insertion of a G within a 3'-GGA-5' anticodon, allowing the tRNA to read a 4-base code word. In this communication we describe suppressor mutations in genes encoding a second proline tRNA isoacceptor (wild-type anticodon 3'-GGU-5') that result in a novel mechanism for translation of a 4-base genetic code word. The genes that encode this isoacceptor include SUF7 (chromosome 13), SUF8 (chromosome 8), trn1 (chromosome 1), and at least two additional unmapped genes, all of which contain an intervening sequence. We show that suppressor mutations in the SUF7 and SUF8 genes result in G-to-U base substitutions at position 39 that disrupted the normal G . C base pairing in the last base pair of the anticodon stem adjacent to the anticodon loop. These anticodon stem mutations might alter the size of the anticodon loop and permit the use of a 3'-GGGU-5' sequence within the loop to read 4-base proline codons. Uncertainty regarding the exact structure of the mature suppressor tRNAs results from the possibility that anticodon stem mutations might affect sites of intervening sequence removal. The possible role of the intervening sequence in the generation of mature suppressor tRNA is discussed. Besides an analysis of suppressor tRNA genes, we have extended previous observations of the apparent relationship between tRNA genes and repetitive delta sequences found as solo elements or in association with the transposable element TY1. Hybridization studies and a computer analysis of the DNA sequence surrounding the SUF7 gene revealed two incomplete, inverted delta sequences that form a stem and loop structure located 165 base pairs from the 5' end of the tRNA gene. In addition, sequences beginning 164 base pairs from the 5' end of the trn1 gene also exhibit partial homology to delta. These observations provide further evidence for a nonrandom association between tRNA genes and delta sequences.     

The histidine tRNA genes of yeast

Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis. We have sequenced three of the histidine tRNA genes. The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis. None of the three yeast histidine tRNA genes has an intervening sequence. Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene. One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats. The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene. These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.     

The non-autonomous retrotransposon SVA is trans-mobilized by the human LINE-1 protein machinery

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.     

Transfer RNA Genes Are Genomic Targets for De Novo Transposition of the Yeast Retrotransposon Ty3

Insertions of the yeast element Ty3 resulting from induced retrotransposition were characterized in order to identify the genomic targets of transposition. The DNA sequences of the junctions between Ty3 and flanking DNA were determined for two insertions of an unmarked element. Each insertion was at position -17 from the 5' end of a tRNA-coding sequence. Ninety-one independent insertions of a marked Ty3 element were studied by Southern blot analysis. Pairs of independent insertions into seven genomic loci accounted for 14 of these insertions. The DNA sequence flanking the insertion site was determined for at least one member of each pair of integrated elements. In each case, insertion was at position -16 or -17 relative to the 5' end of one of seven different tRNA genes. This proportion of genomic loci used twice for Ty3 integration is consistent with that predicted by a Poisson distribution for a number of genomic targets roughly equivalent to the estimated number of yeast tRNA genes. In addition, insertions upstream of the same tRNA gene in one case were at different positions, but in all cases were in the same orientation. Thus, genomic insertions of Ty3 in a particular orientation are apparently specified by the target, while the actual position of the insertion relative to the tRNA-coding sequence can vary slightly.     

Macrotransposition and other complex chromosomal restructuring in maize by closely linked transposons in direct orientation

Several observations indicate that compatible ends of separate, yet closely linked, transposable elements (TEs) can interact in alternative transposition reactions. First, pairs of TEs cause chromosome breaks with frequencies inversely related to the intertransposon distance. Second, some combinations of two TEs produce complex rearrangements that often include DNA adjacent to one or both elements. In pairs of TEs in direct orientation, alternative reactions involving the external ends of the two TEs should lead to the transposition of a macrotransposon consisting of both elements plus the intervening chromosomal segment. Such macrotransposons have been hypothesized previously based on deletions, but no macrotransposon insertions have been recovered. To detect macrotransposition, we have analyzed heritable chromosomal rearrangements produced by a chromosome-breaking pair of Ac and Ds elements situated 6.5 kb apart in direct orientation in a part of the maize (Zea mays) genome dispensable for viability. Here, we show that the postulated macrotransposon can excise and reinsert elsewhere in the genome. In addition, this transposon pair produces other complex rearrangements, including deletions, inversions, and reshuffling of the intertransposon segment. Thus, closely linked TE pairs, a common transposition outcome in some superfamilies, are adept at restructuring chromosomes and may have been instrumental in reshaping plant genomes.     

Regulation of the rat alpha-fetoprotein gene expression in liver. Both the promoter region and an enhancer element are liver-specific and negatively modulated by dexamethasone.

Cis-acting elements involved in the control of rat alpha-fetoprotein gene expression in the liver and its modulation by glucocorticoid hormones were detected after transfection of chloramphenicol acetyltransferase constructs and their transient expression into two hepatoma cell lines. The proximal promoter region (-324 to -15) was found to contain all the information necessary for tissue-specific expression. It is also involved in the negative gene modulation by glucocorticoids and includes an activating regulatory domain allowing efficient expression in the HepG2 cells. Three regions within 7 kilobase pairs of the 5' extragenic sequences are capable of stimulating the chloramphenicol acetyltransferase activity driven by the alpha-fetoprotein promoter sequence. One of these regions, at about -2.5 kilobase pairs, contains a short indivisible 170-base pair DNA element that fulfills all the criteria of a tissue-specific enhancer, i.e. orientation and position independence, as well as cell-specific stimulation of gene expression driven by a homologous or heterologous promoter. The enhancing properties of this element are totally abolished by glucocorticoids. DNase I footprinting experiments indicate that several rat liver nuclear proteins interact with this enhancer element.     

Retrotransposon insertions in rice gene pairs associated with reduced conservation of gene pairs in grass genomes

Small-scale changes in gene order and orientation are common in plant genomes, even across relatively short evolutionary distances. We investigated the association of retrotransposons in and near rice gene pairs with gene pair conservation, inversion, rearrangement, and deletion in sorghum, maize, and Brachypodium. Copia and Gypsy LTR-retrotransposon insertions were found to be primarily associated with reduced frequency of gene pair conservation and an increase in both gene pair rearrangement and gene deletions. SINEs are associated with gene pair rearrangement, while LINEs are associated with gene deletions. Despite being more frequently associated with retrotransposons than convergent and tandem pairs, divergent gene pairs showed the least effects from that association. In contrast, convergent pairs were least frequently associated with retrotransposons yet showed the greatest effects. Insertions between genes were associated with the greatest effects on gene pair arrangement, while insertions flanking gene pairs had significant effects only on divergent pairs.