Linda Margossian

Mutations in FIE, a WD Polycomb Group Gene, Allow Endosperm Development without Fertilization

A fundamental problem in biology is to understand how fertilization initiates reproductive development. Higher plant reproduction is unique because two fertilization events are required for sexual reproduction. First, a sperm must fuse with the egg to form an embryo. A second sperm must then fuse with the adjacent central cell nucleus that replicates to form an endosperm, which is the support tissue required for embryo and/or seedling development. Here, we report cloning of the Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) gene. The FIE protein is a homolog of the WD motif–containing Polycomb proteins from Drosophila and mammals. These proteins function as repressors of homeotic genes. A female gametophyte with a loss-of-function allele of fie undergoes replication of the central cell nucleus and initiates endosperm development without fertilization. These results suggest that the FIE Polycomb protein functions to suppress a critical aspect of early plant reproduction, namely, endosperm development, until fertilization occurs.

The BELL1 gene encodes a homeodomain protein involved in pattern formation in the Arabidopsis ovule primordium

Ovule development in Arabidopsis involves the formation of three morphologically defined proximal-distal pattern elements. Integuments arise from the central pattern element. Analysis of Bell 1 (Bel 1) mutant ovules indicated that BEL1 was required for integument development. Cloning of the BEL1 locus reveals that it encodes a homeodomain transcription factor. Prior to integument initiation, BEL1 RNA localizes to the central domain, providing molecular evidence for a central pattern element. Therefore, proximal-distal patterning of the ovule involves the regulated expression of the BEL1 gene that controls integument morphogenesis. A model for BEL1 function is evaluated with regard to new data showing the expression pattern of the floral homeotic gene AGAMOUS (AG) early in wild-type and BEL1 ovule development.

Imprinting of the MEA Polycomb Gene Is Controlled by Antagonism between MET1 Methyltransferase and DME Glycosylase

The MEA Polycomb gene is imprinted in the Arabidopsis endosperm. DME DNA glycosylase activates maternal MEA allele expression in the central cell of the female gametophyte, the progenitor of the endosperm. Maternal mutant dme or mea alleles result in seed abortion. We identified mutations that suppress dme seed abortion and found that they reside in the MET1 methyltransferase gene, which maintains cytosine methylation. Seeds with maternal dme and met1 alleles survive, indicating that suppression occurs in the female gametophyte. Suppression requires a maternal wild-type MEA allele, suggesting that MET1 functions upstream of, or at, MEA. DME activates whereas MET1 suppresses maternal MEA::GFP allele expression in the central cell. MET1 is required for DNA methylation of three regions in the MEA promoter in seeds. Our data suggest that imprinting is controlled in the female gametophyte by antagonism between the two DNA-modifying enzymes, MET1 methyltransferase and DME DNA glycosylase.

Interaction of a developmentally regulated DNA-binding factor with sites flanking two different fruit-ripening genes from tomato.

To investigate mechanisms that control fruit development, we have begun experiments to identify proteins that control gene expression during tomato fruit ripening. We focused on the regulation of two different genes, E4 and E8, whose transcription is coordinately activated at the onset of fruit ripening. We report here that a DNA-binding protein specifically reacts with similar sequences flanking the E4 and E8 genes. The E4 binding site is at position -34 to -18 and, therefore, overlaps the region (TATA box) that in many eukaryotic genes serves to determine the efficiency and initiation site of transcription. In contrast, the E8 binding site is distal, located at -936 to -920 relative to the start of E8 gene transcription. Gel electrophoresis mobility retardation experiments indicate that the DNA binding activity that interacts with these two sites increases at the onset of fruit ripening. Taken together, these results suggest that this DNA-binding protein may function to coordinate E4 and E8 gene expression during fruit ripening.

An Antisense Gene Stimulates Ethylene Hormone Production during Tomato Fruit Ripening.

The ripening of many fruits is controlled by an increase in ethylene hormone concentration. E8 is a fruit ripening protein that is related to the enzyme that catalyzes the last step in the ethylene biosynthesis pathway, 1-aminocyclopropane-1-carboxylic (ACC) oxidase. To determine the function of E8, we have transformed tomato plants with an E8 antisense gene. We show here that the antisense gene inhibits the accumulation of E8 protein during ripening. Whereas others have shown that reduction of ACC oxidase results in reduced levels of ethylene biosynthesis, we find that reduction of the related E8 protein produces the opposite effect, an increase in ethylene evolution specifically during the ripening of detached fruit. Thus, E8 has a negative effect on ethylene production in fruit.

W-reactivation of phage lambda in recF, recL, uvrA, and uvrB mutants of E. coli K-12

SummaryW-reactivation is reduced by recF143 and recF144 mutations and is undetectable if a second mutation at either the uvrA or uvrB locus is combined with recF143. The uvrA and uvrB mutations alone block W-reactivation partially. A recL152 mutation also partially blocks W-reactivation by itself. In combination with a uvrB5 mutation, recL125 blocks W-reactivation completely but in combination with recF143, significant residual W-reactivation ability remains. We suggest that the phenomenon of W-reactivation is the result of at least two modes or pathways. The observation that recF143 uvrB5 and recF143 uvrA6 strains permit normal levels of mutagenesis (Kato et al., 1977) but completely block all W-reactivation leads us to suggest further that the mechanism(s) of W-reactivation is at least partly different from that of UV mutagenesis.

Genetic and physical mapping of recF in Escherichia coli K-12.

SummaryTwo factor transductional crosses place recF at approximately 82 min on the E. coli chromosome; recF is highly cotransducible with dnaA and gyrB (cou). Transductional analysis with a series of λtna specialized transducing phages carrying chromosomal DNA from the tnaA region place recF between dnaA and gyrB. This analysis also indicates that a gene lying in the same region and producing an easily detectable protein (estimated MW of 45 kD) is dnaN and not recF.

Effects of a recA operator mutation on mutant phenotypes conferred by lexA and recF mutations

Derepression of recA by an operator mutation (recAo281) produces effects opposite to those obtained from its derepression following DNA damage. Inducible reactivation of lambda vir and S13 phages is decreased and inducible UV mutagenesis of a phi X174 amber mutant is lessened in a recAo281 strain compared to a recAo+ strain. The decreases could not be accounted for by increases in constitutive levels of these processes. Consistent with these results the UV resistance of a recAo281 strain is less than that of a recAo+ strain. This may indicate that too much recA protein immediately after irradiation interferes with derepression of the lexA regulon or functioning of its products. Effects of increasing the recAo+ and recA+ copy number on a Co1E1 plasmid are compared with the effects of recAo281. recAo281 partially suppresses UV sensitivity due to lexA102 and lexA3 in E. coli K-12. This increase in resistance is not correlated with an increase in constitutive or inducible reactivation of UV-irradiated lambda vir or S13. This is consistent with the previous suggestion that the UV resistance stems from a decrease in DNA degradation allowing an increase in DNA repair. lexA3 blocks UV mutagenesis of phi X174 as measured by reversion of amber mutations and this was not suppressed by recAo281. recF143 blocks UV mutagenesis of phi X174. recAo281 suppresses neither this effect nor the decrease in bacterial UV resistance caused by recF143.