The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals.

Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.

APETALA1 and SEPALLATA3 interact to promote flower development.

In Arabidopsis, the closely related APETALA1 (AP1) and CAULIFLOWER (CAL) MADS-box genes share overlapping roles in promoting flower meristem identity. Later in flower development, the AP1 gene is required for normal development of sepals and petals. Studies of MADS-domain proteins in diverse species have shown that they often function as heterodimers or in larger ternary complexes, suggesting that additional proteins may interact with AP1 and CAL during flower development. To identify proteins that may interact with AP1 and CAL, we used the yeast two-hybrid assay. Among the five MADS-box genes identified in this screen, the SEPALLATA3 (SEP3) gene was chosen for further study. Mutations in the SEP3 gene, as well as SEP3 antisense plants that have a reduction in SEP3 RNA, display phenotypes that closely resemble intermediate alleles of AP1. Furthermore, the early flowering phenotype of plants constitutively expressing AP1 is significantly enhanced by constitutive SEP3 expression. Taken together, these studies suggest that SEP3 interacts with AP1 to promote normal flower development.

The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required for normal patterning and growth in the floral meristem.

Genetic and molecular studies have suggested that the UNUSUAL FLORAL ORGANS (UFO) gene, from Arabidopsis thaliana, is expressed in all shoot apical meristems, and is involved in the regulation of a complex set of developmental events during floral development, including floral meristem and floral organ identity. Results from in situ hybridization using genes expressed early in floral development as probes indicate that UFO controls growth of young floral primordia. Transgenic constructs were used to provide evidence that UFO regulates floral organ identity by activating or maintaining transcription of the class B organ-identity gene APETALA 3, but not PISTILLATA. In an attempt to understand the biochemical mode of action of the UFO gene product, we show here that UFO is an F-box protein that interacts with Arabidopsis SKP1-like proteins, both in the yeast two-hybrid system and in vitro. In yeast and other organisms both F-box proteins and SKP1 homologues are subunits of specific ubiquitin E3 enzyme complexes that target specific proteins for degradation. The protein selected for degradation by the complex is specified by the F-box proteins. It is therefore possible that the role of UFO is to target for degradation specific proteins controlling normal growth patterns in the floral primordia, as well as proteins that negatively regulate APETALA 3 transcription.

Divergence of function and regulation of class B floral organ identity genes.

Regulatory mechanisms controlling basic aspects of floral morphogenesis seem to be highly conserved among plant species. The class B organ identity genes, which are required to establish the identity of organs in the second (petals) and third (stamens) floral whorls, are a good example of such conservation. This work compares the function of two similar class B genes in the same genetic background. The DEFICIENS (DEF) gene from Antirrhinum, including its promoter, was transformed into Arabidopsis and compared in function and expression with the Arabidopsis class B genes APETALA3 (AP3) and PISTILLATA (PI). The DEF gene was expressed in the second, third, and fourth whorls, as was PI. Functionally, DEF could replace AP3 in making petals and stamens. The DEF gene's AP3-like function and PI-like expression caused transformation of fourth-whorl carpels to stamens. Like AP3, all aspects of DEF function in Arabidopsis required a functional PI protein. Surprisingly, DEF could not replace the AP3 protein in properly maintaining AP3 transcripts (autoregulation). Our data allow us to revise the current model for class B autoregulation and propose a hypothesis for the evolution of class B gene expression in dicotyledonous plants.

International Commission for Protection Against Environmental Mutagens and Carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability.

DNA precursor pool imbalances can elicit a variety of genetic effects and modulate the genotoxicity of certain DNA-damaging agents. These and other observations indicate that the control of DNA precursor concentrations is essential for the maintenance of genetic stability, and suggest that factors which offset this control may contribute to environmental mutagenesis and carcinogenesis. In this article, we review the biochemical and genetic mechanisms responsible for regulating the production and relative amounts of intracellular DNA precursors, describe the many outcomes of perturbations in DNA precursor levels, and discuss implications of such imbalances for sensitivity to DNA-damaging agents, population monitoring, and human diseases.

Elevated intracellular dCTP levels reduce the induction of GC-->AT transitions in yeast by ethyl methanesulfonate or N-methyl-N'-nitro-N- nitrosoguanidine but increase alkylation-induced GC-->CG transversions.

The effect of an increased intracellular dCTP:dTTP ratio on the specificities of ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis was examined in the yeast Saccharomyces cerevisiae. To do so, we used a dCMP deaminase-deficient (dcd1) strain having a dCTP:dTTP ratio > 77-fold larger than its isogenic wild-type parent under the treatment conditions employed. This DNA precursor imbalance lowered the frequencies of EMS- or MNNG-induced SUP4-o mutations by 75 or 45%, respectively, relative to the corresponding values for the wild-type strain. A total of 405 SUP4-o mutations produced by the alkylating agents in the dcd1 background were characterized by DNA sequencing and the mutational spectra were compared to those for 399 mutations induced in the wild-type parent and 207 mutations that arose spontaneously in the dcd1 strain. Unexpectedly, the frequencies of EMS- and MNNG-induced GC-->AT transitions in the dcd1 strain were found to be reduced by 93 and 68%, respectively, considerably more than the decreases for the overall SUP4-o mutation frequencies. The differences were due mainly to substantial increases in the frequencies of GC-->CG transversions. Although these events were the predominant type of spontaneous substitution in the dcd1 strain, they were more frequent after alkylation treatment and were distributed differently than the spontaneous GC-->CG transversions. Preferences for the EMS- or MNNG-induced GC-->AT transitions to occur at GC sites having the guanine located on the transcribed strand or preceded by a 5' purine, respectively, also were diminished in the dcd1 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutational specificity of thymine nucleotide depletion in yeast.

Relative to normal growth conditions for a wild-type strain of the yeast Saccharomyces cerevisiae, withholding thymidylate (dTMP) severely diminished the dTTP pool but elevated the dATP, dGTP and dCTP levels (120-, 8.5- and 3.6-fold, respectively) in an isogenic dTMP auxotroph. This treatment also increased the frequency of mutations in a tRNA gene (SUP4-o) by 15-fold. Single base-pair events accounted for 97% of the 89 SUP4-o mutations characterized by DNA sequencing and the ratio of transversions to transitions was 3-fold greater than that for spontaneous substitutions in the wild-type strain. This difference was due to decreases in the fractions of transitions and an increase in the proportion of A.T-->T.A transversions. The largest increases in mutation frequency were observed for transversions at A.T pairs, consistent with dATP and dGTP being incorporated in place of dTTP during DNA replication. Similarly, misinsertion of dATP and dGTP could have promoted the single base-pair deletions and insertion detected. Analysis of the distributions of substitutions indicated no preference for dATP misinsertion to occur at sites flanked by a specific 5' or 3' base or on the transcribed or nontranscribed strands. However, the presence of mutational hotspots and site-specific variations in the substitution frequencies implied a role for DNA sequence context in the mutational specificity of dTTP depletion.

DNA repair modifies the site and strand specificity of ethyl methanesulfonate mutagenesis in yeast.

The influence of DNA repair on the specificity of ethyl methanesulfonate (EMS) mutagenesis in a plasmid-borne copy of the Saccharomyces cerevisiae SUP4-o gene was investigated. Isogenic yeast strains that are repair-proficient (RAD) or defective for nucleotide excision (rad1), postreplication (rad18) or recombinational repair (rad52) were treated with EMS. Compared to the RAD wild-type, the maximum SUP4-o mutation frequency was 2-fold greater in the rad1 background whereas it was approximately 50% less in the rad18 and rad52 strains. The majority (779/788) of SUP4-o mutations characterized by DNA sequencing were single base pair changes, primarily (> 91%) G.C-->A.T transitions in the RAD, rad1 and rad18 strains. In the rad52 background, only 57% of the substitutions were G.C-->A.T transitions with transversions at G.C pairs accounting for almost all of the remaining changes. Comparisons of the distributions of single base pair substitutions in SUP4-o revealed that there was no excision repair-dependent bias for G.C-->A.T events to occur at sites flanked by a 5' or 3' A.T pair as observed previously for EMS mutagenesis of the lacIgene in Escherichia coli (Burns et al., 1986). These transitions also did not occur more often at sites where the guanine was flanked by a 5' purine than by a 5' pyrimidine. However, they exhibited a small preference for sites having the guanine on the transcribed strand in the RAD and rad52, but not rad1 or rad18, strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutational specificity of DNA precursor pool imbalances in yeast arising from deoxycytidylate deaminase deficiency or treatment with thymidylate.

Disruption of the dCMP deaminase (DCD1) gene, or provision of excess dTMP to a nucleotide-permeable strain, produced dramatic increases in the dCTP or dTTP pools, respectively, in growing cells of the yeast Saccharomyces cerevisiae. The mutation rate of the SUP4-o gene was enhanced 2-fold by the dCTP imbalance and 104-fold by the dTTP imbalance. 407 SUP4-o mutations that arose under these conditions, and 334 spontaneous mutations recovered in an isogenic strain having balanced DNA precursor levels, were characterized by DNA sequencing and the resulting mutational spectra were compared. Significantly more (greater than 98%) of the changes resulting from nucleotide pool imbalance were single base-pair events, the majority of which could have been due to misinsertion of the nucleotides present in excess. Unexpectedly, expanding the dCTP pool did not increase the fraction of A.T----G.C transitions relative to the spontaneous value nor did enlarging the dTTP pool enhance the proportion of G.C----A.T transitions. Instead, the elevated levels of dCTP or dTTP were associated primarily with increases in the fractions of G.C----C.G or A.T----T.A. transversions, respectively. Furthermore, T----C, and possibly A----C, events occurred preferentially in the dcd1 strain at sites where dCTP was to be inserted next. C----T and A----T events were induced most often by dTMP treatment at sites where the next correct nucleotide was dTTP or dGTP (dGTP levels were also elevated by dTMP treatment). Finally, misinsertion of dCTP or dTTP did not exhibit a strand bias. Collectively, our data suggest that increased levels of dCTP and dTTP induced mutations in yeast via nucleotide misinsertion and inhibition of proofreading but indicate that other factors must also be involved. We consider several possibilities, including potential roles for the regulation and specificity of proofreading and for mismatch correction.

Base-pair substitutions alter the site-specific mutagenicity of UV and MNNG in the SUP4-o gene of yeast.

Yeast strains carrying SUP4-o genes that have base-pair substitutions at hotspots for UV or MNNG mutagenesis were treated with these agents. In both cases, the induced mutation frequencies were substantially reduced. Furthermore, specific substitutions at positions in SUP4-o that had not been mutated by MNNG resulted in the recovery of MNNG-induced mutations at these sites. These results demonstrate that base-pair identity is an important factor determining the site-specific mutagenicity of UV and MNNG in yeast. For UV, our findings suggest that the type of lesion that is induced, but not flanking DNA sequences, plays a role in specifying mutability at the sites examined. In contrast, DNA sequence context seems to be an important factor for MNNG mutagenesis.

Disruption of the RAD52 gene alters the spectrum of spontaneous SUP4-o mutations in Saccharomyces cerevisiae.

Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced canavanine uptake is associated with nucleotide permeability in a thymidylate auxotroph of Saccharomyces cerevisiae.

The recovery of spontaneous canavanine-resistant mutants is reduced dramatically in a strain of Saccharomyces cerevisiae that carries a suppressed can 1-100 allele and is permeable to and auxotrophic for thymidylate. This effect does not occur in an isogenic strain that neither takes up nor requires the nucleotide. However, it is observed for another isogenic strain which is permeable to but not auxotrophic for thymidylate, indicating that the effect is related to thymidylate permeability. Apparently, increased sensitivity of the permeable cells to growth inhibition by canavanine accounts for the diminished mutant recovery. In turn, enhanced uptake of canavanine in these cells seems to be responsible for the increased sensitivity. The experimental findings suggest that the elevated transport of canavanine in the thymidylate auxotroph is unlikely to be due to enhanced suppression of the can 1-100 allele or to activation of the yeast general amino acid permease.

Role of neighbouring bases and assessment of strand specificity in ethylmethanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in the SUP4-o gene of Saccharomyces cerevisiae.

A total of 318 forward mutations induced by ethylmethanesulphonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the SUP4-o gene of the yeast Saccharomyces cerevisiae was characterized by DNA sequence analysis. Only base-pair substitutions were detected among the mutations examined and, for both agents, the majority (greater than 96%) were G.C to A.T. transitions. The remaining changes included A.T to G.C transitions and transversions at G.C sites. For EMS, two of the transversions were accompanied by nearby G.C to A.T transitions. There was considerable overlap of the sites within the SUP4-o gene that were mutated by EMS and MNNG and of the sites that each agent failed to mutate. However, EMS and MNNG mutagenesis differed with respect to the frequencies at which mutations were recovered at G.C pairs where the guanine is flanked (5') by a purine or pyrimidine. EMS exhibited no preference for either type of site, whereas a G.C site was 12-fold or fivefold more likely to be mutated by MNNG if preceded by a 5' adenine or guanine, respectively, than if flanked by a 5' pyrimidine. Finally, neither EMS nor MNNG mutagenesis showed a preference for G.C sites having the guanine on the non-transcribed strand.

DNA sequence analysis of spontaneous mutations in the SUP4-o gene of Saccharomyces cerevisiae.

A collection of 196 spontaneous mutations in the SUP4-o gene of the yeast Saccharomyces cerevisiae was analyzed by DNA sequencing. The classes of mutation identified included all possible types of base-pair substitution, deletions of various lengths, complex alterations involving multiple changes, and insertions of transposable elements. Our findings demonstrate that at least several different mechanisms are responsible for spontaneous mutagenesis in S. cerevisiae.