Insertion of the Mu1 transposable element into the first intron of maize Adh1 interferes with transcript elongation but does not disrupt chromatin structure

Abstract

The presence of the Mu1 transposable element within the first intervening sequence of the maize Adh1 gene interfered with transcription through that gene. Insertion of the element did not have an apparent effect on transcription initiation or chromatin structure. In nuclei isolated from anaerobically induced roots, in which Adh1 is transcriptionally active, a subset of the Adh1 chromatin is arranged in a unique conformation characterized by a generalized sensitivity to nucleases, specific DNAase I sensitive sites and a nucleosome array distinct from the inactive configuration present in leaf nuclei. The chromatin organization of the Mu1-induced mutant alleles is indistinguishable from that of the progenitor Adh1-S allele and a point mutant allele that is null for ADH1 activity. The initiation of transcription also proved to be unaffected in these mutants. Nuclear runoff experiments indicated that Adh1 sequences upstream from the point of Mu1 insertion were transcribed normally, but sequences downstream to the insertion were drastically reduced relative to a reference gene expressed in anaerobic root nuclei. Thus, it was concluded that the defect in these Mu1-induced mutants does not reside at the level of gene accessibility or transcript initiation. Rather, Mu1 presents an impediment to the progress of the polymerase II complex during transcript elongation.

Insertion of Mu1 elements in the first intron of the Adh1-S gene of maize results in novel RNA processing events.

“…This conclusion is consistent with two previous reports. Nuclear run-off assays were used to analyze the Adhl-S3034 allele (Vayda and Freeling, 1986), and it was found that the Adhl-S transcript RNA did not effectively traverse the M u l element, suggesting that it was polyadenylated or terminated. Sequence analysis of seven cDNA clones (Ortiz and Strommer, 1990) derived from the Adhl-S3034 and Adh 1 -S4477 alleles under hypoxic stress demonstrated that polyadenylation was occurring within the AT-rich central region of Mul as well as in the TIR sequence.…”

“…Rowland and Strommer (1 985), using nuclear run-off assays, suggested that the Mul element affected transcription initiation but not RNA processing. In contrast, Vayda and Freeling (1986) concluded from their nuclear run-off assays that transcription initiation was unaffected but that transcription elongation was impeded in some way by the Mul sequences. In a recent study, Ortiz and Strommer (1990) concluded that RNA processing of the Mul -containing chimeric transcripts was altered by aberrant splicing and by cryptic polyadenylation during hypoxic stress; these conclusions were based on RNA gel blots and the sequences of severa1 cDNAs of Adhl-S3034 transcripts from hypoxic roots.…”

Hypoxic Stress Inhibits Multiple Aspects of the Potato Tuber Wound Response

“…SC) and alcohol dehydrogenase (Fig. SD) are associated with anaerobic metabolism in other plant species (10,14,26,30), and the protein products of these genes are detected by Western blot analysis (19) in tubers incubated under hypoxic conditions for 24 h (Fig. 5, E and F) prevents the accumulation of the wound-inducible class of RNA species.…”

“…RNA species were identified by formaldehyde agarose gel electrophoresis and Northern blot analyses using the methods described previously (20). In all analyses, equal amounts (30 …”

DNA sequences required for anaerobic expression of the maize alcohol dehydrogenase 1 gene

“…The absence of other homologies of substantial length (>10 base pairs) between the promoters ofthese two genes supports the suggestion that the conserved sequences may have a functional role. The ARE also corresponds to one of the nuclease-sensitive regions of Adhi in the chromatin of nuclei isolated from anaerobic roots (5,21). Increased nuclease sensitivity is often associated with genes in a transcriptionally active state (22) and would suggest that the ARE may be part ofa transcriptionally active region in the chromatin of anaerobic maize roots.…”