Analysis of Yeast Retrotransposon Ty Insertions at the Can1 Locus

The target site distribution for 55 independent Ty insertions that inactivate the function of the Saccharomyces cerevisiae CAN1 gene is reported. Under some selection conditions Ty elements inserted preferentially into the promoter and exhibited an orientation bias. In contrast, under other conditions no insertions were detected in the promoter region and transposition appeared to occur randomly throughout the CAN1 coding sequence. These results show that the target site distribution for Ty insertions may be a function of the selection conditions.     

Mutations in RAD6, a yeast gene encoding a ubiquitin-conjugating enzyme, stimulate retrotransposition.

The Saccharomyces cerevisiae DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme which polyubiquitinates histones in vitro. Here we show that mutations in rad6 increase the frequency of transposition of the retrotransposon Ty into the CAN1 and URA3 loci. Using isogenic RAD6 and rad6 strains, we measured a more than 100-fold increase in the spontaneous rate of retrotransposition due to rad6, although there was no increase in the Ty message level. This is the first time that a mutation in a host gene has been shown to result in an increased rate of retrotransposition.     

Ty4, a novel low-copy number element in Saccharomyces cerevisiae: one copy is located in a cluster of Ty elements and tRNA genes.

We have identified a composite element, Ty4, in S. cerevisiae that is ca 6.3 kb in length and contains two tau sequences as long terminal repeats. According to hybridization analyses, Ty4 occurs in low but varying copy number (one to four copies) in different yeast strains. By several criteria, Ty4 is a novel type of retroelement which is similar but not related to the other Ty elements in yeast. Two cosmid clones from strain C836 (c90 and c476) carrying individual copies of Ty4 were isolated. By restriction analysis and nucleotide sequence we show that c476 derives from the 'transposition right arm hot spot' of chromosome III [1]. The analysis of c476 revealed that an initiator tRNA(Met) gene is present at this locus and that an unusual concentration of different Ty elements has occurred: in addition to the Ty4, a Ty1 and a Ty2 element were detected in this region, confirming its highly polymorphic character.     

Localization of sequences required in cis for yeast Ty1 element transposition near the long terminal repeats: analysis of mini-Ty1 elements.

In order to identify and characterize sequences within Ty1 elements which are required in cis for transposition, a series of mini-Ty1 plasmids were constructed and tested for transposition. Mini-Ty1s are deletion mutants of the Ty1-H3 element; Ty1 gene products required for transposition are supplied in trans from a helper Ty1 which has intact open reading frames but lacks a 3' long terminal repeat (LTR) and therefore cannot transpose itself. Up to 5 kilobase pairs of internal sequences of the 6-kilobase-pair-long Ty1 element can be deleted without a significant effect on transposition. The smallest mini-Ty1 element capable of transposition contains the 3' LTR and the transcribed portion of the 5' LTR, 285 base pairs (bp) of internal sequence 3' to the 5' LTR, and 23 bp of internal sequence 5' to the 3' LTR. We conclude that Ty1-encoded proteins can act in trans and that cis-acting sequences in Ty1-H3 are all within or near the LTRs. Further deletion of the 285-bp internal sequence adjacent to the 5' LTR significantly reduced transposition frequency, and the mini-Ty1 RNA produced failed to be packaged into the viruslike particles efficiently. Surprisingly, several nonhomologous cellular mRNAs were also associated with viruslike particles.     

Ty1 copy number dynamics in Saccharomyces

To understand long terminal repeat (LTR)-retrotransposon copy number dynamics, Ty1 elements were reintroduced into a "Ty-less" Saccharomyces strain where elements had been lost by LTR-LTR recombination. Repopulated strains exhibited alterations in chromosome size that were associated with Ty1 insertions, but did not become genetically isolated. The rates of element gain and loss under genetic and environmental conditions known to affect Ty1 retrotransposition were determined using genetically tagged reference elements. The results show that Ty1 retrotransposition varies with copy number, temperature, and cell type. In contrast to retrotransposition, Ty1 loss by LTR-LTR recombination was more constant and not markedly influenced by copy number. Endogenous Ty1 cDNA was poorly utilized for recombination when compared with LTR-LTR recombination or ectopic gene conversion. Ty1 elements also appear to be more susceptible to copy number fluctuation in haploid cells. Ty1 gain/loss ratios obtained under different conditions suggest that copy number oscillates over time by altering the rate of retrotransposition, resulting in the diverse copy numbers observed in Saccharomyces.     

The RNase Z homologue encoded by Escherichia coli elaC gene is RNase BN

In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.     

Relationships among the rfb regions of Salmonella serovars A, B, and D.

The O antigens of Salmonella serogroups A, B, and D differ structurally in their side chain sugar residues. The genes encoding O-antigen biosynthesis are clustered in the rfb operon. The gene rfbJ in strain LT2 (serovar typhimurium, group B) and the genes rfbS and rfbE in strain Ty2 (serovar typhi, group D) account for the known differences in the rfb gene clusters used for determination of group specificity. In this paper, we report the nucleotide sequence of 2.9 kb of DNA from the rfb gene cluster of strain Ty2 and the finding of two open reading frames which have limited similarity with the corresponding open reading frames of strain LT2. These two genes complete the sequence of the rfb region of group D strain Ty2 if we use strain LT2 sequence where restriction site data show it to be extremely similar to the strain Ty2 sequence. The restriction map of the rfb gene cluster in group A strain IMVS1316 (serovar paratyphi) is identical to that of the cluster in strain Ty2 except for a frameshift mutation in rfbE and a triplicated region. The rfb gene clusters of these three strains are compared, and the evolutionary origin of these genes is discussed.     

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.