Characterisation of a rice vacuolar invertase isoform, OsINV2, for growth and yield-related traits

OsINV2, a rice vacuolar invertase isoform, was assessed for its functional roles in plant growth and development with key focus on its agronomic traits such as grain weight, grain filling percentage, grain number and dry weights at various stages until harvest. Lack of differences between the wild-type and the mutants with respect to any of the aforementioned traits tested revealed a possibility of functional compensation of OsINV2 in the mutants conceivably by its isoform. This was confirmed by OsINV2 promoter::GUS studies, where its spatial and temporal expression in the panicle elongation stages showed that although OsINV2 expression was observed from the stage with young panicles ~1 cm in length to the flag leaf stage, significant differences with respect to panicle and spikelet phenotypes between the wild-type and the mutant were not present. However, complement lines displaying an overexpression phenotype of OsINV2 possessed a higher stem non-structural carbohydrate content under both monoculm and normal tillering conditions. A trade-off between the spikelet number and grain weight in the complement lines grown under monoculm conditions was also observed, pointing towards the necessity of OsINV2 regulation for grain yield-related traits.

Heat Stress-Induced Cup9-Dependent Transcriptional Regulation ofSIR2

The epigenetic writer Sir2 maintains the heterochromatin state of chromosome in three chromosomal regions, namely, the silent mating type loci, telomeres, and the ribosomal DNA (rDNA). In this study, we demonstrated the mechanism by which Sir2 is regulated under heat stress. Our study reveals that a transient heat shock causes a drastic reduction in theSIR2transcript which results in sustained failure to initiate silencing for as long as 90 generations. Hsp82 overexpression, which is the usual outcome of heat shock treatment, leads to a similar downregulation ofSIR2transcription. Using a series of genetic experiments, we have established that heat shock or Hsp82 overexpression causes upregulation ofCUP9that, in turn, repressesSIR2transcription by binding to its upstream activator sequence. We have mapped thecisregulatory element ofSIR2. Our study shows that the deletion ofcup9causes reversal of the Hsp82 overexpression phenotype and upregulation ofSIR2expression in heat-induced Hsp82-overexpressing cells. On the other hand, we found that Cup9 overexpression repressesSIR2transcription and leads to a failure in the establishment of heterochromatin. The results of our study highlight the mechanism by which environmental factors amend the epigenetic configuration of chromatin.

Massive Gene Duplication Event among Clinical Isolates of the Mycobacterium tuberculosis W/Beijing Family

ABSTRACT As part of our effort to uncover the molecular basis for the phenotypic variation among clinical Mycobacterium tuberculosis isolates, we have previously reported that isolates belonging to the W/Beijing lineage constitutively overexpress the DosR-regulated transcriptional program. While generating dosR knockouts in two independent W/Beijing sublineages, we were surprised to discover that they possess two copies of dosR. This dosR amplification is part of a massive genomic duplication spanning 350 kb and encompassing >300 genes. In total, this equates to 8% of the genome being present as two copies. The presence of IS6110 elements at both ends of the region of duplication, and in the novel junction region, suggests that it arose through unequal homologous recombination of sister chromatids at the IS6110 sequences. Analysis of isolates representing the major M. tuberculosis lineages has revealed that the 350-kb duplication is restricted to the most recently evolved sublineages of the W/Beijing family. Within these isolates, the duplication is partly responsible for the constitutive dosR overexpression phenotype. Although the nature of the selection event giving rise to the duplication remains unresolved, its evolution is almost certainly the result of specific selective pressure(s) encountered inside the host. A preliminary in vitro screen has failed to reveal a role of the duplication in conferring resistance to common antitubercular drugs, a trait frequently associated with W/Beijing isolates. Nevertheless, this first description of a genetic remodeling event of this nature for M. tuberculosis further highlights the potential for the evolution of diversity in this important global pathogen.

Mouse Disabled 1 Regulates the Nuclear Position of Neurons in a Drosophila Eye Model

ABSTRACT Nucleokinesis has recently been suggested as a critical regulator of neuronal migration. Here we show that Disabled 1 (Dab1), which is required for neuronal positioning in mammals, regulates the nuclear position of postmitotic neurons in a phosphorylation-site dependent manner. Dab1 expression in the Drosophila visual system partially rescues nuclear position defects caused by a mutation in the Dynactin subunit Glued. Furthermore, we observed that a loss-of-function allele of amyloid precursor protein (APP)-like, a kinesin cargo receptor, enhanced the severity of a Dab1 overexpression phenotype characterized by misplaced nuclei in the adult retina. In mammalian neurons, overexpression of APP reduced the ability of Reelin to induce Dab1 tyrosine phosphorylation, suggesting an antagonistic relationship between APP family members and Dab1 function. This is the first evidence that signaling which regulates Dab1 tyrosine phosphorylation determines nuclear positioning through Dab1-mediated influences on microtubule motor proteins in a subset of neurons.

The Acetyltransferase Activity of CBP Is Required for wingless Activation and H4 Acetylation in Drosophila melanogaster

ABSTRACT CBP is a critical coactivator of transcription, but little is understood about the importance of its intrinsic acetyltransferase (AT) activity in gene activation in vivo. We show that the intrinsic AT function of CBP in Drosophila melanogaster (dCBP) is necessary to maintain a dCBP overexpression phenotype in the eye, for the in vivo activation of a specific target gene, wingless, and for the global acetylation of histone H4. These findings indicate that a point mutation which alters the intrinsic AT activity of CBP (only one of many CBP functions) has profound effects on CBP-induced gene activation in a physiologically intact transcription system. Furthermore, the effects of CBP AT activity are not limited to a few specific promoters, but rather CBT AT activity may play a role in regulating global histone acetylation throughout the developing organism.

A Mutational Analysis of dishevelled in Drosophila Defines Novel Domains in the Dishevelled Protein as Well as Novel Suppressing Alleles of axin

Drosophila dishevelled (dsh) functions in two pathways: it is necessary to transduce Wingless (Wg) signaling and it is required in planar cell polarity. To learn more about how Dsh can discriminate between these functions, we performed genetic screens to isolate additional dsh alleles and we examined the potential role of protein phosphorylation by site-directed mutagenesis. We identified two alleles with point mutations in the Dsh DEP domain that specifically disrupt planar polarity signaling. When positioned in the structure of the DEP domain, these mutations are located close to each other and to a previously identified planar polarity mutation. In addition to the requirement for the DEP domain, we found that a cluster of potential phosphorylation sites in a binding domain for the protein kinase PAR-1 is also essential for planar polarity signaling. To identify regions of dsh that are necessary for Wg signaling, we screened for mutations that modified a GMR-GAL4;UAS-dsh overexpression phenotype in the eye. We recovered many alleles of the transgene containing missense mutations, including mutations in the DIX domain and in the DEP domain, the latter group mapping separately from the planar polarity mutations. In addition, several transgenes had mutations within a domain containing a consensus sequence for an SH3-binding protein. We also recovered second-site-suppressing mutations in axin, mapping at a region that may specifically interact with overexpressed Dsh.

A Genetic Screen for Modifiers of E2F in Drosophila melanogaster

The activity of the E2F transcription factor is regulated in part by pRB, the protein product of the retinoblastoma tumor suppressor gene. Studies of tumor cells show that the p16ink4a/cdk4/cyclin D/pRB pathway is mutated in most forms of cancer, suggesting that the deregulation of E2F, and hence the cell cycle, is a common event in tumorigenesis. Extragenic mutations that enhance or suppress E2F activity are likely to alter cell-cycle control and may play a role in tumorigenesis. We used an E2F overexpression phenotype in the Drosophila eye to screen for modifiers of E2F activity. Coexpression of dE2F and its heterodimeric partner dDP in the fly eye induces S phases and cell death. We isolated 33 enhancer mutations of this phenotype by EMS and X-ray mutagenesis and by screening a deficiency library collection. The majority of these mutations sorted into six complementation groups, five of which have been identified as alleles of brahma (brm), moira (mor) osa, pointed (pnt), and polycephalon (poc). osa, brm, and mor encode proteins with homology to SWI1, SWI2, and SWI3, respectively, suggesting that the activity of a SWI/SNF chromatin-remodeling complex has an important impact on E2F-dependent phenotypes. Mutations in poc also suppress phenotypes caused by p21CIP1 expression, indicating an important role for polycephalon in cell-cycle control.

Genetic and Molecular Analysis of fox-1, a Numerator Element Involved in Caenorhabditis elegans Primary Sex Determination

fox-1 was previously identified as a candidate numerator element based on its overexpression phenotype. FOX-1 is an RRM-type RNA-binding protein, which can bind RNAs in vitro. Western analysis detects FOX-1 throughout development. fox-1::lacZ comes on ubiquitously early during embryogenesis. Postembryonically, fox-1::lacZ is expressed sex specifically in a subset of cells in the head and tail. We describe a Tc1derived deletion allele [fox-1(Δ)] that removes the RRM domain. fox-1(Δ) confers no phenotype in XXs, but can rescue XO-specific lethality and feminization caused by duplications of the left end of the X. fox-1(Δ) synergizes with putative numerators, resulting in abnormal XX development. Genetic analysis indicated that fox-1(Δ) leads to a slight increase in xol-1 activity, while fox-1(gf) leads to partial loss of xol-1 activity, and xol-1 is epistatic to fox-1. RNase protection experiments revealed increased levels of the 2.2-kb xol-1 message in fox-1(Δ) animals, and reduced levels in fox-1(gf) animals. Additionally, fox-1(Δ) impairs male mating efficiency, which, we propose, represents another function of fox-1, independent of xol-1 and its role in sex determination.

Identification of MYO4, a second class V myosin gene in yeast

We have isolated a fourth myosin gene (MYO4) in yeast (Saccharomyces cerevisiae). MYO4 encodes a approximately 170 kDa (1471 amino acid) class V myosin, using the classification devised by Cheney et al. (1993a; Cell Motil. Cytoskel. 24, 215–223); the motor domain is followed by a neck region containing six putative calmodulin-binding sites and a tail with a short potential ‘coiled-coil’ domain. A comparison with other myosins in GenBank reveals that Myo4 protein is most closely related to the yeast Myo2 protein, another class V myosin. Deletion of MYO4 produces no detectable phenotype, either alone or in conjunction with mutations in myo2 or other myosin genes, the actin gene, or secretory genes. However, overexpression of MYO4 or MYO2 results in several morphological abnormalities, including the formation of short strings of unseparated cells in diploid strains, or clusters of cells in haploid strains. Alterations of MYO4 or MYO2 indicate that neither the motor domains nor tails of these myosins are required to confer the overexpression phenotype, whereas the neck region may be required. Although this phenotype is similar to that seen upon MYO1 deletion, we provide evidence that the overexpression of Myo4p or Myo2p is not simply interfering with Myo1p function.

Xwnt-11: a maternally expressed Xenopus wnt gene

We have isolated and characterized a novel Xenopus wnt gene, Xwnt-11, whose expression pattern and overexpression phenotype suggest that it may be important for dorsal-ventral axis formation. Xwnt-11 mRNA is present during oogenesis and embryonic development through swimming tadpole stages. Xwnt-11 mRNA is ubiquitous in early oocytes and is localized during mid-oogenesis. By late oocyte stages, Xwnt-11 mRNA is localized to the vegetal cortex, with some mRNA in the vegetal cytoplasm. After egg maturation, Xwnt-11 mRNA is released from the vegetal cortex and is found in the vegetal cytoplasm. This early pattern of Xwnt-11 mRNA localization is similar to another vegetally localized maternal mRNA, Vg1 (D. A. Melton (1987) Nature 328, 80–82). In the late blastula, Xwnt-11 mRNA is found at high levels in the dorsal marginal zone. As gastrulation proceeds, Xwnt-11 mRNA appears in the lateral and ventral marginal zone and, during tadpole stages, it is found in the somites and first branchial arch. Injection of Xwnt-11 mRNA into UV-ventralized embryos can substantially rescue the UV defect by inducing the formation of dorsal tissues. The rescued embryos develop somitic muscle and neural tube; however, they lack notochord and anterior head structures.