Genetic and molecular analysis of the Spm-dependent a-m2 alleles of the maize a locus

The Suppressor-mutator (Spm) transposable element family of maize consists of the fully functional standard Spm (Spm-s) and many mutant elements. Insertion of an Spm element in or near a gene can markedly alter its expression, in some cases bringing the gene under the control of the mechanisms that regulate expression of the element. To gain insight into such mechanisms, as well as to enlarge our understanding of the Spm element's genetic organization, we have analyzed derivatives of a unique Spm insertion at the maize a locus in which the gene is co-expressed and co-regulated with the element. We describe the genetic properties and the structure of the a locus and Spm element in 9 strains (collectively designated the a-m2 alleles) selected by McClintock from the original a-m2 allele for heritable changes affecting either the Spm element or expression of the a gene. Most of the mutations are intra-element deletions within the 8.3-kb Spm element; many alter both Spm function and expression of the gene. Spm controls a gene expression in alleles with internally deleted, transposition-defective Spm elements and element ends contain the target sequences that mediate Spm's ability to activate expression of the gene. We argue that the properties of the a-m2 alleles reflect the operation of an element-encoded positive regulatory mechanism, as well as a negative regulatory mechanism that affects expression of the element, but appears not to be mediated by an element-encoded gene product.     

Host versus in vitro signals and intrastrain allelic differences in the expression of a Candida albicans virulence gene

The yeast Candida albicans is a harmless colonizer of mucosal surfaces in healthy people but can become a serious pathogen in immunocompromised patients, causing superficial as well as systemic infections. The evolution of gene families encoding pathogenicity-related functions, like adhesins and secreted aspartic proteinases (Saps), which are differentially induced by host signals at various stages of colonization and infection, may have allowed C. albicans an optimal adaptation to many different host niches. We found that even the two alleles of a single gene can be differentially regulated in the diploid C. albicans. In the model strain SC5314, the in vitro expression of one of the two SAP2 alleles, SAP2-1, depended on the presence of a functional SAP2-2 allele. In contrast, inactivation of SAP2-1 did not in-fluence the expression of SAP2-2. The proteinase encoded by the SAP2-2 allele serves as a signal sensor and amplifier to enhance its own expression as well as to induce the SAP2-1 allele to achieve maximal proteolytic activity under appropriate conditions. Using in vivo expression technology, we could demonstrate that the SAP2-1 allele is significantly activated only in the late stages of systemic candidiasis in mice, whereas the SAP2-2 allele is induced much earlier. The differential regulation of the two SAP2 alleles was due to differences in their pro-moters, which contained a variable number of two pentameric nucleotide repeats. Mutations that reduced or increased the copy number of these repeats diminished the inducibility of the SAP2 promoter during infection but not in vitro, suggesting that the mutations affected interactions of regulatory factors that are necessary for SAP2 activation in vivo but dispensable for its induction in vitro. Therefore, the signals and signal transduction pathways that mediate SAP2 expression within certain host niches may differ from those that activate the gene in vitro. In addition to the generation of gene families whose members exhibit functional and regulatory diversification, C. albicans seems to use its diploid genome to create further variability and host adaptation by differential evolution of even the two alleles of a single gene.     

Comparison of Waxy gene regulation in the endosperm and pollen in Oryza sativa L

The Waxy (Wx) gene controls amylose synthesis in rice (Oryza sativa) and its expression is regulated organ-specifically. The Wx gene is expressed in the endosperm and pollen but not in other organs. In order to know whether Wx gene regulation is the same in the endosperm and pollen, we compared expression patterns of the rice Wx gene in these two organs by immunoblot analysis. We focused on the allelic differences (Wxa and Wxb), cool temperature response and effects of the mutation at the du loci. The results obtained are as follows. First, the quantitative regulation depending on two alleles, Wxa and Wxb, was common to both organs; Wx protein levels from the Wxa allele were about 10-fold higher than those from the Wxb allele in the pollen as well as in the endosperm. Second, in both the endosperm and pollen, expression of the Wxb gene, but not the Wxa gene, was enhanced in response to cool temperature. In contrast to these two types of regulation, analysis of two du mutants, 2035 (du1) and 76-3 (du2), revealed that the pattern of reduction in Wx protein levels in the pollen was distinct from that in the endosperm, suggesting that functions of the two du+ genes differ in these two organs.     

Enhancement of allele discrimination by introduction of nucleotide mismatches into siRNA in allele-specific gene silencing by RNAi

Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs) against mutant alleles of the human Prion Protein (PRNP) gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs), of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense-strand siRNA elements, which possibly increase the assembly of antisense-strand (guide) siRNAs into RNA-induced silencing complexes (RISCs), may enhance ASP-RNAi in the case of inert siRNA duplexes. Therefore, the data presented here suggest that structural modification of functional portions of an siRNA duplex by base substitution could greatly influence allele discrimination and gene silencing, thereby contributing to enhancement of ASP-RNAi.     

Enhancer-promoter communication at the yellow gene of Drosophila melanogaster: diverse promoters participate in and regulate trans interactions

The many reports of trans interactions between homologous as well as nonhomologous loci in a wide variety of organisms argue that such interactions play an important role in gene regulation. The yellow locus of Drosophila is especially useful for investigating the mechanisms of trans interactions due to its ability to support transvection and the relative ease with which it can be altered by targeted gene replacement. In this study, we exploit these aspects of yellow to further our understanding of cis as well as trans forms of enhancer-promoter communication. Through the analysis of yellow alleles whose promoters have been replaced with wild-type or altered promoters from other genes, we show that mutation of single core promoter elements of two of the three heterologous promoters tested can influence whether yellow enhancers act in cis or in trans. This finding parallels observations of the yellow promoter, suggesting that the manner in which trans interactions are controlled by core promoter elements describes a general mechanism. We further demonstrate that heterologous promoters themselves can be activated in trans as well as participate in pairing-mediated insulator bypass. These results highlight the potential of diverse promoters to partake in many forms of trans interactions.     

Perspective: maternal kin groups and the origins of asymmetric genetic systems-genomic imprinting, haplodiploidy, and parthenogenesis

The genetic systems of animals and plants are typically eumendelian. That is, an equal complement of autosomes is inherited from each of two parents, and at each locus, each parent's allele is equally likely to be expressed and equally likely to be transmitted. Genetic systems that violate any of these eumendelian symmetries are termed asymmetric and include parent-specific gene expression (PSGE), haplodiploidy, thelytoky, and related systems. Asymmetric genetic systems typically arise in lineages with close associations between kin (gregarious siblings, brooding, or viviparity). To date, different explanatory frameworks have been proposed to account for each of the different asymmetric genetic systems. Haig's kinship theory of genomic imprinting argues that PSGE arises when kinship asymmetries between interacting kin create conflicts between maternally and paternally derived alleles. Greater maternal than paternal relatedness within groups selects for more "abstemious" expression of maternally derived alleles and more "greedy" expression of paternally derived alleles. Here, I argue that this process may also underlie origins of haplodiploidy and many origins of thelytoky. The tendency for paternal alleles to be more "greedy" in maternal kin groups means that maternal-paternal conflict is not a zero-sum game: the maternal optimum will more closely correspond to the optimum for family groups and demes and for associated entities such as symbionts. Often in these circumstances, partial or complete suppression of paternal gene expression will evolve (haplodiploidy, thelytoky), or other features of the life cycle will evolve to minimize the conflict (monogamy, inbreeding). Maternally transmitted cytoplasmic elements and maternally imprinted nuclear alleles have a shared interest in minimizing agonistic interactions between female siblings and may cooperate to exclude the paternal genome. Eusociality is the most dramatic expression of the conflict-reducing effects of haplodiploidy, but its original and more widespread function may be suppression of intrafamilial cannibalism. In rare circumstances in which paternal gene products gain access to maternal physiology via a placenta, PSGE with greedy paternal gene expression can persist (e.g., in mammals).     

Menin and JunD regulate gastrin gene expression through proximal DNA elements

Mutations in the MEN1 gene correlate with multiple endocrine neoplasia I (MEN1). Gastrinomas are the most malignant of the neuroendocrine tumors associated with MEN1. Because menin and JunD proteins interact, we examined whether JunD binds to and regulates the gastrin gene promoter. Both menin and JunD are ubiquitous nuclear proteins that we showed colocalize in the gastrin-expressing G cells of the mouse antrum. Transfection with a JunD expression vector alone induced endogenous gastrin mRNA in AGS human gastric cells, and the induction was blocked by menin overexpression. We mapped repression by menin to both a nonconsensus AP-1 site and proximal GC-rich elements within the human gastrin promoter. Chromatin immunoprecipitation assays, EMSAs, and DNA affinity precipitation assays documented that JunD and Sp1 proteins bind these two elements and are both targets for menin regulation. Consistent with menin forming a complex with histone deacetylases, we found that repression of gastrin gene expression by menin was reversed by trichostatin A. In conclusion, proximal DNA elements within the human gastrin gene promoter mediate interactions between JunD, which induces gastrin gene expression and menin, which suppresses JunD-mediated activation.     

Independent integration of rodent identifier (ID) elements into orthologous sites of some RT6 alleles of Rattus norvegicus and Rattus rattus

The two alleles of the rat T-cell differentiation alloantigen RT6 are highly divergent and their expression is distinctively regulated. While the majority of T cells of RT6a/RT6b heterozygous laboratory rats (Rattus norvegicus) expresses both alleles, a subpopulation expresses only RT6b. To identify cis-regulatory elements that potentially control monoallelic expression, we compared the sequences of both alleles. A striking difference is the presence or absence of a rodent identifier (ID) sequence in intron 7. All investigated inbred RT6a rat strains (n=7) had this integration, while it was absent in all investigated RT6b rats (n=9). An ID element was also identified at precisely the same integration site in one RT6 allele of the closely related species Rattus rattus. The ID elements of both species showed nucleotide substitutions characteristic of different subfamilies, and their flanking repeats differed in length, indicating that two independent integration events had occurred into the same site adjacent to a mammalianwide interspersed repeat. Analysis of the surrounding sequences did not disclose any motifs to explain preferential integration into one allele. Our data indicate that the RT6 alleles diverged about 1 myr ago and that the ID element integrated into the RT6a locus soon after this. We have previously shown that DNA methylation plays an important role in regulating monoallelic RT6 expression. The possibility that the ID element in the RT6a allele interferes with the required demethylation process and thus accounts for monoallelic expression is discussed.     

Identification of New Genes Involved in the Regulation of Yeast Alcohol Dehydrogenase II

Recessive mutations in two negative control elements, CRE1 and CRE2, have been obtained that allow the glucose-repressible alcohol dehydrogenase (ADHII) of yeast to escape repression by glucose. Both the cre1 and cre2 alleles affected ADHII synthesis irrespective of the allele of the positive effector, ADR1. However, for complete derepression of ADHII synthesis, a wild-type ADR1 gene was required. Neither the cre1 nor cre2 alleles affected the expression of several other glucose-repressible enzymes. A third locus, CCR4, was identified by recessive mutations that suppressed the cre1 and cre2 phenotypes. The ccr4 allele blocked the derepression of ADHII and several other glucose-repressible enzymes, indicating that the CCR4 gene is a positive control element. The ccr4 allele had no effect on the repression of ADHII when it was combined with the ADR1-5c allele, whereas the phenotypically similar ccr1 allele, which partially suppresses ADR1-5c, did not suppress the cre1 or cre2 phenotype. Complementation studies also indicated that ccr1 and snf1 are allelic. A model of ADHII regulation is proposed in which both ADR1 and CCR4 are required for ADHII expression. CRE1 and CRE2 negatively control CCR4, whereas CCR1 is required for ADR1 function.