Ethan R. Signer

Epigenetic repeat-induced gene silencing (RIGS) inArabidopsis

In several plant systems expression of structurally intact genes may be silenced epigenetically when a transgenic construct increases the copy number of DNA sequences. Here we report epigenetic silencing inArabidopsis lines containing transgenic inserts of defined genetic structure, all at the same genomic locus. These comprise an allelic series that includes a single copy of the primary insert, which carries repeated drug resistance transgenes, and a set of its derivatives, which as a result of recombination within the insert carry different numbers and alleles of resistance genes. Although the drug resistance genes remained intact, both the primary and some recombinant lines nevertheless segregated many progeny that were partly or fully drug-sensitive because of silencing. As in other systems silencing was reversible, and correlated with decreased steady-state mRNA and increased DNA methylation. Each different number and combination of genes, on the same or different (i.e., homologous) chromosomes, conditioned its own idiosyncratic segregation pattern. Strikingly, lines with a single gene segregated only a few slightly drug-sensitive progeny whereas multi-gene lines segregated many highly sensitive progeny, indicating dependence of silencing at this locus on repeated sequences. This argues strongly against explanations based on antisense RNA, but is consistent with explanations based on ectopic DNA pairing. One possibility is that silencing reflects the interaction of paired homologous DNA with flanking heterologous DNA, which induces condensation of chromatin into a non-transcribable state.

The Role of Recombination in Growth of Bacteriophage Lambda I. The Gamma Gene

Although recombination in microorganisms has been studied extensively, the role of recombination in growth is not entirely understood. For example, it is not yet clear to what extent recombination proteins are involved in events such as repair or synthesis of DNA. One way to approach the problem is to study the effect on growth of a deficiency in recombination. Recombination-deficient mutants have been isolated in several systems. In E. coli (Howard-Flanders and Theriot, 1966; Clark, 1967) they define three cistrons recA, B, and C, of which B and C determine an exonuclease (Barbour and Clark, 1970). None of the three cistrons appears to be essential for cell growth, since the mutants are viable. (However, since the selection procedure scored only viable cells, these mutants might retain a low but essential level of the gene products.) Recombination-deficient mutants have also been isolated in several bacteriophages. In phage P22, these mutants ( erf − ) grow in rec + but not in rec − hosts, suggesting that recombination proteins are essential for growth in P22 (Botstein and Matz, 1970). In phage T4, mutations in several essential cistrons seem to affect recombination (Bernstein, 1968). In phage λ, recombination-deficient mutants ( red − , Signer and Weil, 1968; Echols and Gingery, 1968) define two genes (Shulman et al., 1970), namely exo (exonuclease, Korn and Weissbach, 1963) and β (beta protein, Radding and Schreffler, 1966). In this phage neither gene appears to be essential for growth, since both exo − mutants (including deletion mutants) and β − mutants (including amber mutants) make plaques on rec − ...

Properties of recombination-deficient mutants of bacteriophage lambda☆

Abstract We have studied the complementation behavior, genetic mapping and exonuclease activity of various recombination-deficient (red−) mutants of bacteriophage λ. In collaboration with C. Radding (Radding, 1970) , we have observed that the red− mutants tested often affect one or both of two gene products—exonuclease and β-protein—indicating that these proteins are involved in genetic recombination.

Cauliflower mosaic virus P35S promoter activity in Escherichia coli.

SummaryWe present evidence that the cauliflower mosaic virus promoter P35S can direct expression of the bacterial neomycin phosphotransferase II (NPTll) gene in . Transcription is initiated at several sites, the major one being located approximately 315 bases upstream of the plant start site. The nucleotide sequence directly preceding this start site is strongly homologous to the prokaryotic promoter consensus sequence. Thus constructs designed for introduction into plants can be expressed in E. coli.

The Role of Recombination in Growth of Bacteriophage Lambda II. Inhibition of Growth by Prophage P2

In this series we are investigating the role of recombination in the growth of bacteriophage λ . In the preceding paper we described a new λ gene called γ (gamma). γ − mutants are slightly deficient in repair of UV damage (in recA − hosts) and also in recombination. Like recombination-deficient ( red − ) mutants, γ − mutants do not form plaques on certain strains of Escherichia coli (among which are E. coli mutants deficient in DNA polymerase). Phages that are both red − and γ − do not form plaques on recA − strains of E. coli, possibly because of cumulative adverse effects of the two mutations on growth. Failure to form plaques on recA − is called the fec − phenotype (Manly et al., 1969). Wild-type λ does not form plaques on lysogens carrying the unrelated phage P2 (Lederberg, 1957), and is therefore said to be spi + (sensitive to P2 interference). Lindahl et al. (1970), who studied this phenomenon, described spi − mutants of λ that can form plaques on P2 lysogens. They concluded that the spi − phenotype required inactivation of at least two λ genes: one gene maps under the bio 72 deletion; another maps between the ends of the bio 72 and bio 1 deletions. They also found that the spi − mutants they studied are fec − . We show here that the combination red − γ − , which is sufficient for the fec − phenotype, gives only a partial spi − phenotype. The full spi − phenotype requires in addition, the mutation of a new gene called δ (delta), located between int and exo . The δ − mutation does...

Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives

SummaryTwo derivatives of the prokaryotic transposon Tn5 were constructed in vitro. In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin. In Tn5-235, the Escherichia coli β-galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter. Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti. When a Tn5-derivative is introduced into an R. meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons. In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations. We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage ϕ M12. In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac+ phenotype. Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative.

Cytosine deaminase as a negative selective marker for Arabidopsis

Cytosine deaminase (CD), produced by prokaryotes but not by higher eukaryotes including plants, deaminates cytosine to uracil. The enzyme likewise converts 5-fluorocytosine (5FC), which by itself is not toxic, to 5-fluorouracil (5FU), which is toxic. The Escherichia coli codA-coding sequence encoding CD, together with appropriate regulatory elements, was introduced into Arabidopsis. Neither untransformed controls, nor transgenic plants expressing no CD mRNA, were sensitive to 5FC. Conversely, for most transgenic plants expressing CD mRNA, in the presence of 5FC calli and seedlings failed to proliferate, and seeds failed to germinate. A few transgenic plants with many codA copies expressed less CD mRNA and remained insensitive to 5FC, which likely reflected epigenetic repeat-induced gene silencing. Thus 5FC, presumably through conversion by the enzyme to 5FU, can be used to select against plants that express CD.

Activity of the yeast FLP recombinase in Arabidopsis

The coding sequence for FLP recombinase, originally from the 2 μ plasmid of Saccharomyces cerevisiae, was introduced into Arabidopsis behind the cauliflower mosaic virus 35S promoter. FLP activity was monitored by the glucuronidase activity resulting from inversion of an antisense-oriented GUS reporter gene flanked by a pair of FRT target sites in inverted repeat. FLP-dependent Gus activity was observed in both transient assays and transgenic plants. The FLP system will be useful for a variety of in planta genetic manipulations.

On the control of lysogeny in phage λ

Abstract The int (prophage integration) gene of phage λ requires the product of gene N for expression. However, in the presence of the immunity repressor the N product of a heteroimmune phage is not sufficient to induce a high level of int expression, although there is a low level of int expression under these conditions. The relevance of these findings to lysogeny and control of gene expression is discussed.

Positive-negative selection for homologous recombination in Arabidopsis.

In plants gene knock-outs and targeted mutational analyses are hampered by the inefficiency of homologous recombination. We have developed a strategy to enrich for rare events of homologous recombination in Arabidopsis using combined positive and negative selection. The T-DNA targeting construct contained two flanking regions of the target alcohol dehydrogenase gene as homologous sequences, and neomycin phosphotransferase and cytosine deaminase as positive and negative markers, respectively. A root explant transformation procedure was used to obtain transgenic calli. Among 6250 transformants isolated by positive selection, 39 were found to be resistant to negative selection as well. Of these 39, at least one had undergone homologous recombination correlated with a unidirectional transfer of information. Although the ADH locus was not changed, our data demonstrate that a homologous recombination event can be selected by positive negative selection in plants.