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Summer Colloquium 2002 Abstracts

Generation of Mitchondrial Hsp70 Transgenic Mice

Radhika Puttagunta

Emerging research indicates that mitochondria play a central role in the aging process. The epicenter of oxidative damage is the mitochondrion, where radical oxygen species (ROS) are created as byproducts of energy metabolism. ROS can damage proteins, lipids and DNA. Severe damage can lead to mitochondrial dysfunction, such as alterations in mitochondria structure, membrane potential, release of cytochrome c from the inner membrane matrix and induction of the apoptotic pathway. Cytosolic inducible HSP70 (heat shock protein of 70kDa) has shown a protective role against protein damage, oxidative stress, ischemia, apoptosis and mitochondrial dysfunction in cell lines, drosophila, and/or mice. The HSP70 family is large and highly conserved throughout evolution. First discovered as a response to heat shock, HSP70 is now known to have multiple functions. Of the human HSP70 family, mitochondrial HSP70 (MTHSP75) is most homologous to bacterial Hsp70 (DnaK). MTHSP75 is constitutively transcribed in the nucleus but localized to the inner membrane of the mitochondria, where it serves as an ATPase transporter of proteins into the mitochondria.

Once proteins arrive inside the mitochondria, MTHSP75 is able to fold them into their proper conformation. It also serves as a chaperone of proteins synthesized on mitochondrial ribosomes and can help degrade misfolded proteins within the mitochondrion. Under stress, MTHSP75 refolds denatured proteins properly and prevents aggregation. MTHSP75 is also known as glucose regulated protein of 75kD (GRP75). Another grp family member, GRP78, is a resident endoplasmic reticulum HSP70 family member involved in Ca++ homeostasis. GRP75 (MTHSP75) may contain a calcium binding domain and also be involved in mitochondrial Ca++ homeostasis. By creating a transgenic mouse overexpressing human MTHSP75 we are studying the role of this protein in protection against protein oxidative damage/aging and maintaining the integrity of the mitochondrion. The human MTHSP75 cDNA was cloned into the pCAGGS vector. Expression is driven at high levels in the heart and muscle, and at lower levels in the brain, by the chicken Beta-actin promoter and CMV enhancer. Five transgenic mice were generated, two of which died after birth prior to weaning. A high copy number of MTHSP75 leads to smaller animals and in some cases an early death. A low copy number founder of MTHSP75 has thus far shown no phenotype varying from wildtype C57Bl/6 mice. Northern analysis shows expression in muscle, heart and brain. Western analysis and immunohistochemistry confirm these results. Electron microscopy has shown that the protein is localizing to the mitochondria. These mice will be further analyzed for resistance to protein oxidative damage, retarding of aging and/or resistance to mitochondrial dysfunction under stressful conditions.

Conserved and novel roles of maize FLORICAULA/LEAFY homologs in reproductive development

Kirsten Bomblies

Genes involved in flower patterning and organ identity are conserved between widely divergent moncot and dicot flowering plants. Much is known about the upstream regulation that establishes intial expression of these genes in dicots, especially Arabidopsis, but the degree of conservation in moncots is unclear. The homologous transcription factors FLORICAULA of Antirrhinum and LEAFY of Arabidopsis share conserved roles in establishing flower meristem patterning in numerous dicots, but little is known about the function of these meristem identity genes in monocots. We used reverse genetics to examine the role of two duplicate FLORICAULA/LEAFY homologs in maize (Zea mays ssp mays) - a monocot species with dramatically different flower and inflorescence morphology from that of model dicot species such as Arabidopsis and Antirrhinum. Transposon insertions into the maize genes, ZFL1 and ZFL2, led to disruption of flower organ identity and patterning, as well as to defects in inflorescence architecture and the reproductive transition. Our results demonstrate that these genes share conserved roles with their dicot counterparts in flower and inflorescence patterning. Our data also suggest that ZFL function may play a novel role in controlling quantitative aspects of inflorescence phyllotaxy in maize. This observation supports candidacy of these genes for QTL that control morphological traits of inflorescence structure selected during maize domestication.

Cloning and Characterization of smg-6, a Gene Involved in mRNA Surveillance in C.elegans

Hongyu Shang

Eukaryotic transcripts that contain premature stop codons are less stable than their wild-type counterparts, a phenomenon termed nonsense-mediated mRNA decay (NMD) or mRNA surveillance. NMD in C.elegans requires functions of the seven smg genes, smg-1 through smg-7.

We mapped smg-6 relative to genetic and physical markers on chromosome III. Using cosmids from the region as hybridization probes, we detected a 1.8 kb deletion in smg-6(r1217). DNA fragments that cover the region deleted in smg-6(r1217) rescue smg-6 mutants, demonstrating that smg-6 derives from the deleted region. All potential open reading frames in the rescuing DNA encode novel proteins.

Sequence analysis of the smg-6 gene and its mRNAs is nearly complete. The smg-6 region encodes four main alternatively spliced mRNAs (transcripts I, II, III and IV). Transcript III is a fusion of transcript I and II, in which a part of the final exon of transcript I is spliced in frame to the first exon of transcript II. Both transcripts II and III are affected by smg-6(r1217). A cDNA clone of transcript II is sufficient to rescue smg-6(r1217). Transcript IV is an alternative-spliced mRNA related to transcript I, and its abundance is elevated in all tested smg(-) mutants. The protein products of transcripts I, II and III are able to be detected by western analysis. Protein I is not affected in all tested mg-6 mutants on western blots, whereas protein II and III are affected in most alleles.

SPD-1, a putative microtubule associated protein required for the formation of the spindle midzone and involved in the completion of cytokinesis

Koen Verbrugghe

Several mutants that disrupt the spindle midzone, including zen-4 and air-2, indicate that this structure is required for the completion of cytokinesis in C. elegans. The midzone may be required for regulating the various events required for sealing the two daughter cells, including the removal of the acto-myosin ring and the trafficking and fusion of membranes. We are studying a temperature sensitive mutant spd-1(oj5) in which the spindle midzone is disrupted in all cell cycles but cytokinesis fails only in EMS.

We have cloned spd-1 and identified it as the homolog of human PRC1(1) and tobacco MAP-65(2) and S. cerevisiae Ase1p(3). These proteins localize to the spindle midzone, or its equivalent, and are involved in cytokinesis in HeLa cells and spindle elongation in yeast. SPD-1 antibodies also recognize the spindle midzone in C. elegans embryos.

Live imaging of b-tubulin::GFP in spd-1 mutant embryos showed that the bundled microtubules of the spindle midzone are absent or much reduced during anaphase in all cell divisions. However, preliminary results indicate that ZEN-4 appears to localize correctly to the midzone of the one cell embryo. We are in the process of determining whether ZEN-4 and other known spindle midzone proteins localize correctly at all cell divisions of spd-1(oj5) embryos. We are also trying to determine why the EMS cell fails by determining the involvement of the wnt signalling pathway and the early polarity determining pathway.

  1. W. Jiang et al., Mol Cell 2, 877-85. (1998).
  2. A. Smertenko et. al. Nature Cell Biology 2, 750-753. (2000).
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Export of the yeast TRP channel YVC1 from the ER requires specific function of SEC12

Junyu Lin

Majority of membrane proteins are synthesized on the endoplasmic reticulum (ER) membrane and transported to their destination through vesicular traffic within a cell. ER export is the first step in membrane protein transport and is meditated by vesicles coated with COPII protein complex. YVC1, the yeast TRP channel homolog on the vacuolar membrane, is one of membrane cargo proteins transported out of the ER. Several YVC1 mutants are identified by their ability to inhibit cell growth in a random mutagenesis screening. Overproduction of mutant yvc1 affects normal functions of the ER. These YVC1 mutants can be suppressed by mutations in SEC12, a guanine nucleotide exchange factor that recruits SAR1 and initiates COPII coat formation on the ER membrane. Furthermore, overproduction of either SEC12 or SAR1 facilitates the ER export of wild type YVC1. These results indicate that interaction between SEC12 and YVC1 is essential for export of YVC1 from the ER.

Conserved and novel roles of maize FLORICAULA/LEAFY homologs in reproductive development.

Kati Geszvain

Bacterial RNA polymerase (RNAP) contains a flexible protein domain called the flap. The flap is composed of beta subunit residues 835-930 in E. coli and is formed by a long, narrow loop of protein that protrudes from the enzyme with a short segment of alpha helix at its tip. An interaction of the flap-tip helix with sigma70 region 4 appears to be essential for recognition of -35 element-dependent promoters. Region 4 makes specific contacts to bases in the -35 element, but is dispensable for transcription from -35 independent promoters (1). In the holoenzyme crystal structures from T. aquaticus and T. thermophilus, the flap-tip helix resides in a hydrophobic pocket on sigma70 region 4 opposite its DNA binding surface (2,3). A deletion of a portion of the flap that includes the flap-tip helix has been shown to be defective in initiation from -35 dependent, but not -35 independent, promoters (4). We screened the flap region for substitutions that produce a dominant growth defect when over-expressed in a wild-type background. These substitutions define a hydrophobic patch on the flap-tip helix. By systematically altering residues in this patch to alanine, or charged amino acids, we found that the interaction between the flap-tip and sigma70 region 4 is mediated by contacts between the hydrophobic surfaces on RNAP and sigma70, rather than by specific side-chain interactions.

Using trypsin protection assays, we demonstrated that disruption of the flap-tip hydrophobic patch disrupts the interaction between the flap and sigma70 and that protection from trypsin cleavage is due exclusively to region 4 of sigma70. Region 1.1 had been suggested to bind to the flap; however, a substitution in region 1.1 that severally impairs initiation (I53A) had no effect on trypsin protection. Substitutions in the flap-tip helix exhibit an initiation defect similar to that of the flap deletion, suggesting the interaction between the flap-tip helix hydrophobic patch and sigma70 region 4 is necessary for recognition of -35 dependent promoters.

  1. Kumar et al, 1993. JMB 232, 406-418.
  2. Vassylyev et al, 2002. Nature advance online publication, 8 May 2002 (DOI 10.1038/nature752).
  3. Murakami et al, 2002. Science 296, 1280-1284.
  4. Kuznedelov et al, 2002. Science 295, 855-857.

Arabidopsis Mutant with Defective Root Growth Response to Surface-derived Stimuli

Kai (Billy) Hung

Plant root is a dynamic organ that detects and adapts to the changing environment through regulated growth. A mutant with defects in root growth response to tilted agar surface, wvc16, has been identified using the heterologous Ac/Ds transposable element system in Arabidopsis. Mutant roots make a compressed waving pattern on tilted agar surface compared to the wild-type parents. In addition, this mutation also causes leaves, stems, pedicles, petals, and siliques to curl. Root growth rate assay using exogenous auxin sources revealed no significant difference between mutant and wild type plants. Current data suggest that wvc16 is at least semi-dominant and that the mutation is tightly linked to the Ds element. Further, preliminary results suggest that the phenotypes may be caused by miss-expression due to a divergent Cauliflower Mosaic virus 35S promoter present at the border of the Ds element. Genetic and molecular analyses are being done to further elucidate the nature of wvc16 mutation as well as the function of the wild-type allele.

The Role of Dynactin Complex in Spindle Alignment in C. elegans Early Embryo

Haining Zhang

Positioning of the mitotic spindle within dividing polarized cells is crucial for the proper inheritance of segregated cytoplasmic determinants to daughter cells. The orientation of the spindle determines the cell division plane. The cleavage furrow bisects the spindle during telophase, partitioning localized components to specific daughter cells. In C. elegans, the nucleus-centrosome complex rotates in germline lineage to ensue the proper segregation of P granules. Studies by Skop and White (Curr Biol. 1998), Gonczy et al. (J Cell Biol. 1999) have shown that knockout of dynein and dynactin function by RNAi abolishes spindle rotation in both P0 and P1 cells. Immunoflorescent staining with p150glued, the largest subunit of dynactin, revealed the accumulation of dynactin to the cell division remnant. It is hypothesized that this cortex spot may serve to tether the plus-ends of astral microtubules from one spindle pole and rotate the mitotic spindle towards this spot.

In order to study in more detail the dynamic distribution of dynactin in C. elegans early embryo, stable transgenic GFP fusion lines with p150 glued, dynamitin and arp-1 were constructed. Two-photon microscopy showed that these three dynactin components co-localized to meiotic and mitotic spindles, centrosomes, nuclear envelope and adjacent membranes between cells. Arp-1, which is also called centractin, has a prominent localization to centrosome compared to the other two components. Although the accumulation at the cell division remnant is not quite obvious in the living cell images, antibody staining of GFP in these transgenic lines confirmed its presence. We are now working on improving the sensitivity of two-photon imaging.

Mutants that have altered spindle alignment in either AB, P1 or both, such as par-3, par-2, gpb-1, ooc-3, ooc-5 and let-99 are being tested to see whether or not the dynactin complex localizes to cortical sites in cells that rotate their spindle and are missing in those that do not. In par-3 RNAi embryos, where spindles in both AB and P1 rotate, dynactin accumulates at the cell division remnant. While the accumulation of dynactin at the cell division remnant is not present in par-2 RNAi embryos, where neither the AB nor the P1 spindle rotates. These results indicate that accumulation of dynactin to the cortex is either required for or is the consequence of spindle rotation. We are now looking at dynactin localization in other mutants and have initiated a yeast-two hybrid screen to check for possible interactions between dynactin and these proteins.

High-throughput Systematic Mutagenesis in E.coli

Yisheng Kang

I have been working towards the construction of a set of E. coli strains harboring mutations in each of the predicted open reading frames in the genome. A complete set of PCR primers for the 4290 predicted ORFs is used to amplify each gene in a separate reaction. To create mutant alleles I introduce a kanamycin resistance cassette into each PCR fragment using an in vitro transposition reaction. I take advantage of the high efficiency of homologous recombination of linear fragments by lambda-red containing E. coli to introduce the mutation-containing fragment onto the chromosome following electro-transformation. Kanamycin resistant transformants are then screened by amplifying the target locus by PCR and confirming the presence and approximate location of the transposon insertion by restriction digestion and or sequencing. Each kanamycin resistant colony is likely an independent allele. To date I have constructed mutations in 1290 distinct genes and anticipate rapid progress on a large number of additional ORFs. Mutants are subjected to further phenotypic testing such as growth curves, Biolog phenotypic microarray analysis and global gene expression profiling.

Role of dlg-1 in formation and polarization of epithelial junctions.

Christopher Lockwood

We are interested in the genes involved with formation and function of epithelial junctions. Recently two proteins have been identified which localize and act at the C. elegans septate like junction. AJM-1, a novel coiled coil protein, and DLG-1, a homologue of Drosophila Discs large, have been shown to colocalize to a domain of apical junctions distinct from the catenin-cadherin complex. Loss of function studies with these genes demonstrate their requirement for proper junctional integrity. Individuals deficient for either protein arrest during elongation and have disrupted apical junctions at the level of electron microscopy. In addition, these two genes have been shown to interact both genetically and physically. A genetic interaction is seen by disruption of AJM-1 localization along the apical junction in the absence of DLG-1. A physical interaction is seen by the binding of AJM-1 to the N-terminus of DLG-1 in yeast 2-hybrid and GST pull down experiments.

In order to further characterize the formation and function of the DLG-1/AJM-1 domain we are undertaking a series of structure function experiments. A dlg-1 deletion mutant provided by the C. elegans knockout consortium confers a loss of function background in which rescue with constructs deleted for domains of dlg-1 can be performed. Constructs are being tested for their ability to rescue dlg-1 mutant phenotypes with respect to lethality and AJM-1 localization. In parallel we are performing directed 2-hybrid experiments with deletion constructs of dlg-1 and ajm-1 to further map the physical interaction domain.

Dishevelled Worms

Timothy Walston

Wnt signaling has been shown to have many roles during development in C. elegans. Mutations and RNAi of the three dishevelleds found in C. elegans have identified a new role for Wnt signaling during morphogenesis and further clarified the role of Wnt signaling in spindle orientation during early embryogenesis. A deletion of dsh-2 (or302), one of three dishevelleds in C. elegans, results in defects in dorsal intercalation and ventral enclosure during morphogenesis. The other two dishevelleds, dsh-1 and mig-5, when mutant, do not have defects in these processes. Dishevelleds signal through both the canonical Wg pathway and the planar cell polarity pathway. Constructs lacking certain domains of dsh-2 will be used to identify the pathway(s) involved through rescue of the morphogenetic defects in dsh-2(or302). Wnt signaling has been implicated in the spindle alignment of the cell ABar. Mutations and RNAi of the dishevelleds result in defects in ABar spindle alignment. We are currently investigating other members of Wnt signaling that may play a role in ABar spindle alignment.

Signal transduction in C. elegans male mating behaviors

Tiewen Liu

The nervous system of the adult C. elegans male possesses 381 neurons to the hermaphrodites's 302. These additional sex-specific neurons are required for male mating behaviors. We are specifically studying the molecular basis of male sensory behaviors. We ask the questions: what are the receptors of the sensory neurons that initiate the signal cascade? What molecules act during synaptic transmission? G-protein coupled receptors (GPCR) are a large family of seven-transmembrane spanning proteins involved in signal transduction in response to hormones, neurotransmitters, light, and chemicals. The C. elegans genome possesses over 1000 GPCRs with expression of 10 candidate GPCRs being highly enriched in adult males. Characterization of these sex-regulated GPCR genes is essential in determining a role in sex-specific sensory behaviors. To characterize the expression pattern and subcellular localization of candidate GPCRs in male nervous system, GPCR-X::GFP reporters will be constructed and expression patterns analyzed. To characterize the role(s) of candidate GPCRs that are expressed in male sensory neurons, we will attempt to demonstrate that GPCR candidates encode functional chemoreceptors. We will isolate deletions in candidate genes and perform behavioral and phenotypic analysis on mutants. Neuropeptides are non-classical neurotransmitters and play critical roles in synaptic signaling in the nervous system. 21 putative neuropeptide like protein (nlp) genes and 23 FMRFamide-like peptide (flp) genes (personal communication) have been identified in C. elegans. We are characterizing their expression patterns in adult males and analyzing the mutant phenotypes with respect to male mating behaviors.

Identifying the nuclear export mechanism for Upf3p, a factor required for the NMD pathway in yeast

Qioning Guan

Eukaryotic cells decay mRNAs that contain a premature stop codon at an accelerated rate relative to their wild-type counterparts, a phenomenon called nonsense-mediated mRNA decay (NMD). Three trans-acting factors Upf1p, Upf2p, and Upf3p are required for NMD in S. cerevisiae. The 45 KDa protein Upf3p contains three bipartite nuclear localization signal (NLS) sequences, and two leucine-rich nuclear export signal (NES) sequences. Our lab has demonstrated that a Upf3-Triple mutation, which contains three alanine substitutions in the NES-A element, changes the distribution of Upf3p from a primarily cytoplasmic to a nucleolar localization, and confers an Nmd- phenotype. When a functional NES from HIV-1 Rev was inserted near the C-terminus of the Upf3-Triple mutant, the nuclear export and NMD functions were partially restored. These results suggest that NES-A mediates the export of Upf3p from the nucleus to the cytoplasm, a process necessary for Upf3p to function in NMD. However, the export of Upf3p occurs independently of Crm1p, the major exportin for leucine-rich NES-containing proteins.

To identify proteins involved in the export of Upf3p, we performed a genetic screen for high-copy suppressors of the Upf3-Triple mutant. We screened more than ten genomic equivalents using a yeast 2m genomic DNA library for genes that, when over-expressed, would suppress the abnormal growth phenotypes of the Upf3-Triple mutant, and restore the NMD function. Plasmids containing two genomic fragments were identified multiple times. One set of plasmids contains one ORF (YGR125W) as the only common open reading frame. The other contains four open reading frames. Genetic and molecular studies are being carried out to further establish which ORFs code for products that cause suppression when over-expressed and what role the proteins might play in the NMD pathway.

Disruption of epithelial morphogenesis by perturbation of molecules affecting actin dynamics

Mark Sheffield

During C. elegans embryogenesis, the hypodermis undergoes three major morphogenetic processes that lead to formation of the worm's tubular shape: dorsal intercalation, ventral enclosure, and elongation. It has previously been demonstrated that proper completion of all three of these events requires an intact actin cytoskeleton in the hypodermis, but the mechanisms involved in regulation of actin during epithelial morphogenesis are not yet entirely clear. We have taken a candidate gene approach to examine whether molecules known to be involved in actin dynamics have roles in epithelial morphogenesis.

Members of the WASP family have been shown in vitro to act downstream of CDC-42 as activators of the Arp2/3 complex to promote actin branching during cell locomotion. Surprisingly, animals null for was-1, a C. elegans homolog of the WASP family, undergo normal embryogenesis. Drosophila Enabled is known to affect epithelial morphogenesis and the vertebrate Ena family members have been implicated in actin remodeling. unc-34 encodes an ortholog of Ena and has been shown to function in cell migration in C. elegans. As with was-1, a null allele of unc-34 results in virtually no defects in embryogenesis. However, RNAi of was-1 in unc-34(gm104) animals yields embryonic lethality. These embryos appear to be specifically defective for certain hypodermal cell migrations that occur during ventral enclosure, but not those during dorsal intercalation. In addition to its synergy with was-1, unc-34 has other morphogenetic roles. RNAi for a homolog of the WASP relative SCAR/WAVE generates hypodermal defects and lethality, and performing this experiment in an unc-34(gm104) background enhances this phenotype to yield embryos that are completely defective in morphogenesis. Finally, unc-34 enhances morphogenetic phenotypes associated with mutations in the cadherin/catenin complex similar to those seen in Drosophila. Experiments are underway to establish the localization of UNC-34 throughout embryogenesis, the specific causes of the various unc-34 synthetic phenotypes, and to establish which additional molecules are involved in these processes.

Cloning spd-3: A Gene Required for Spindle Alignment

Maria Vidal

In order to develop into a complex organism, cells must have the ability to divide asymmetrically to produce two daughter cells with different developmental potentials. A cell accomplishes this by segregating cytoplasmic components to opposite ends and positioning the cleavage plane to bisect the polarized cell. The cleavage plane is specified by the alignment of the mitotic spindle. To understand the molecular mechanism of spindle alignment we are studying a temperature sensitive, maternal effect, lethal mutation in the spd-3 gene of C. elegans. Cytological analysis reveals that the spd-3(oj35) mutant is defective in spindle positioning. During the first mitosis, the spindle fails to align along the anterior/posterior axis leading to abnormal cleavage configurations. Similar defects in spindle alignment are seen in dynactin complex mutants suggesting that SPD-3 may be involved with this complex. Localization of dynactin components is disrupted in spd-3(oj35) embryos further supporting this hypothesis. 3-point mapping has placed spd-3 between unc-44 (2.91) and unc-24 (3.54) on chromosome IV. This region contains 2 Mb of DNA and over 400 predicted open reading frames. To further narrow the region, I am utilizing the technique of Single Nucleotide Polymorphism (SNP) mapping. Currently, the position of spd-3 is narrowed to the region between SNPs C49A9 (3.0) and C48A7 (3.38), a region containing approximately 250 predicted ORFs. I am concurrently using a candidate gene approach which has uncovered an actin-like gene (alp-1) whose loss of function phenocopies spd-3(oj35). The spd-3(oj35) strain has no mutation in the alp-1 gene, and subsequent mapping eliminated alp-1 as a spd-3 candidate. However, because alp-1 is also required for spindle alignment and I will continue analysis of this gene.

Analyses of the involvement of ARG1, a DnaJ-like protein, in the gravitropic process of Arabidopsis thaliana

Kanok Boonsirichai

Gravitropism in plants involves the ability of organs to perceive the gravitational force, to transduce that information into physiological signals, to transmit such signals to the responding tissues, and for these tissues to receive and respond to the signals. ARG1 (Altered Response to Gravity) encodes a DnaJ-like protein that lacks the glycine/phenylalanine-rich region and the zinc-finger motifs common to many members of the DnaJ family. Mutations in ARG1 result in seedlings that show a slower kinetics in the bending response of the primary root and the hypocotyl upon gravistimulation. Expression analyses in the root tissues using plants expressing a GFP-ARG1 translational fusion and a cMYC-tagged ARG1 protein indicate that ARG1 can be found in the root cap and the elongation zones, where gravitropic processes occur in roots. Also, expression of ARG1under the control of the endodermis-specific SCR promoter and of the root-cap-specific RCP1 promoter can rescue the mutant phenotype of arg1-2 in hypocotyls and roots respectively, suggesting the involvement of ARG1 in early phases of gravitropism. ARG1 protein is present predominantly in the microsomal membrane pellet. Its association with the pellet can be disrupted in the presence of a high molarity of urea but not triton X-100, a property that is common among cytoskeletal and cytoskeleton-associated proteins. Together with the presence of a C-terminal coiled-coil region in ARG1 showing sequence similarity to proteins known to interact with cytoskeletons, this observation suggests that ARG1 may interact with cytoskeletal elements. Products of two other genes in the Arabidopsis genome share a high level of sequence similarity to ARG1. One of these genes, ARL2 (ARG1-Like 2), when mutated, results in seedlings with a similar alteration in the kinetics of root-bending response with that of arg1 mutants. arg1; arl2 double mutant seedlings also show a similar phenotype, suggesting that ARG1 and ARL2 may be involved in the same pathway or may interact with each other to facilitate the gravitropic response. In addition, genetic modifiers of ARG1 have been identified through an EMS-mutagenesis screen. Two of these show an enhanced randomization of their root growth behavior, resulting in seedlings with roots growing in the opposite direction to the gravity vector. The mutations responsible for these modifiers have been mapped to chromosome II and chromosome III.

Identification of Times During a Bacterial Growth Cycle When Changing NTP and ppGpp Concentrations Control rRNA Expression

Heath Murray

Escherichia coli rapidly modulates ribosome synthesis when nutritional conditions dictate a change in the level of protein synthesis, primarily by altering transcription initiation from rRNA promoters. For many years it has been known that the guanine nucleotide ppGpp is a negative regulator of rrn promoter activity, and more recent work has demonstrated that rrn promoters are sensitive to the concentration of their initiating nucleoside triphosphate (NTP). To determine when ppGpp and NTPs control rRNA expression throughout a growth cycle, we have measured the rate of rrn transcription initiation (by primer extension) and correlated this activity with changes in the concentrations of NTPs and ppGpp. Within seconds after addition of fresh media to stationary phase cells ("outgrowth"), rrn P1 promoter activity and NTP concentrations rise dramatically. Rather than decreasing (as would be predicted for a negative regulator of rRNA transcription), ppGpp concentrations increase slightly. We conclude that the increase in rRNA transcription initiation during outgrowth from stationary phase results from the rapid increase in the NTP pool. In contrast, during nutritional shifts from steady-state growth ("upshifts" and "downshifts"), changes in the level of ppGpp account for the regulation of the rrn promoters. Finally, during entry into stationary phase ("shut-off"), an increase in ppGpp and a decrease in NTPs act in concert to reduce rrn promoter activity. Later in stationary phase, low NTP concentrations persist, but elevated ppGpp concentrations do not, consistent with the model that the inactivity of rRNA promoters in deep stationary phase results at least in part from low initiating NTP concentrations. Our findings suggest that changes in the concentrations of NTPs and ppGpp account, at least in part, for the regulation of rrn promoters during changes in nutrient availability, and that depending on the physiological state of the cell, these molecules can either act independently or together to control rRNA transcription.

Regulation of axis induction in zebrafish embryos by canonical and non-canonical Wnt signaling pathways

Jamie Lyman

The hecate mutation causes a defect in dorsal axis induction. Mutant embryos are characterized by a lack of dorsal anterior structures, and the majority are radially symmetric posterio-ventralized embryos. In situ hybridizations with even skipped, goosecoid, and nieuwkoid exhibit staining patterns consistent with the ventralization of the embryos at the early gastrula stages. The wnt/?-catenin, pathway has been shown to play a role in the induction of the dorsal organizer. We hypothesize that the block caused by the hecate mutation may be in the wnt/?-catenin pathway or in a pathway that feeds into the wnt/?-catenin pathway. Injection of ?-catenin mRNA into hecate mutants can reverse the ventralization phenotype of the embryos as shown by the expression pattern of the dorsal marker, goosecoid. Similarly, injection of RNA coding for GSK3-binding protein (GBP) or a dominant negative GSK-3 product also results in the rescue of the ventralized phenotype. A second, non-canonical, Wnt signaling pathway involves intracellular Ca2+ release. Initial experiments indicate that the wild-type hecate gene product is a regulator of a step in the wnt/Ca2+ pathway, which in turn regulates activation of the canonical Wnt pathway an d axis induction.

WVD2 and WDL1 Modulate Helical Organ Growth and Anisotropic Cell Expansion in Arabidopsis thaliana

Chris Yuen

Wild-type A. thaliana seedlings exhibit a wavy growth pattern and slant to the right of the gravity vector on tilted agar surfaces. The root waving and slanting phenotypes are believed to derive from a combination of growth behaviors in response to displacement from the gravity vector and touch stimulation, as well as a surface-triggered circumnutation-like process in roots. We have identified two novel genes, WVD2 and WDL1, which suppress root waving and cause opposite (leftward) root skewing when activated by the CaMV 35S promoter, which has a strong expression pattern in many plant tissues. Transgenic 35S::WVD2 seedlings exhibit constitutive right-handed epidermal cell file rotation in both roots and etiolated hypocotyls. Moreover, the anisotropic expansion of cells is impaired, resulting in the formation of shorter and stockier organs throughout these plants. In roots, these phenotypes are accompanied by alterations in the arrangement of cortical microtubules. WVD2 and WDL1 transcripts were detected in multiple organs of wild-type plants. The respective products of these genes contain a novel conserved region, which we have termed the KLEEK domain, that is predicted to form coiled-coils. The KLEEK domain appears to be specific to plant proteins. Simultaneous co-suppression of both WVD2 and WDL1 causes a modest increase in rightward root slanting, relative to wild-type. Our observations suggest that both genes may be involved in modulating rotational polarity and anisotropic cell expansion during organ growth.

Age-Related Impairment of the Transcriptional Response to Stress in the Heart

Michael Edwards

We examined the gene expression profile of the response to oxidative stress in the hearts of 5 month old (young) and 25 month old (old) mice treated with a single interperitoneal injection of paraquat (50 mg/kg). Paraquat is a redox-cycler that reacts with molecular oxygen in vivo to generate reactive oxygen species in the tissues of the treated animal. The mice were sacrificed 1, 3, 5 and 7 hours after injected with paraquat and cardiac RNA was then isolated, labeled and hybridized to high-density oligonucleotide microarrays. Out of 9,977 genes probed on the microarray, 252 transcripts in the young mice and 260 transcripts in the old mice displayed a significant change in expression levels due to the paraquat treatment (ANOVA, P<0.01). Among these induced genes, a total of 53 transcripts were common to both age groups. A closer examination of these common genes revealed age-associated differences in expression patterns during paraquat exposure. The old mice had a greater average expression of commonly induced paraquat-responsive genes than the young animals for the majority of the time course. Only the young mice showed a significant increase in expression of all three isoforms of GADD45, a DNA damage-responsive protein. Furthermore, the number of immediate early genes (IEG) found to be upregulated by paraquat were 3 times greater in the young hearts than in the old and a subset of these genes, which are dependent on protein kinase C and MEK (MAPKK) signaling for transcription, had expression levels significantly higher in the young mice than in the old. These results demonstrate that there are observable differences in the transcriptional response to acute oxidative stress as a result of the aging process.

The minimal ORC Interaction Domain of the yeast heterochromatin protein Sir1p.

Melissa Bose

Silencing of the HM loci requires a Sir1p-Origin Recognition Complex (ORC) interaction. A small region of Sir1p, called the Silencer Recognition 'Domain' (SRD) (amino acids 489-505), is necessary for a Sir1p-ORC interaction. In this work we define the region of Sir1p sufficient to interact with Orc1p by the 2-hybrid assay and ORC by an in vitro interaction assay. We call this region (amino acids 489-611) the ORC Interaction Region (OIR). The OIR contains the SRD region (489-505) at its N-terminus that, on its own, is insufficient to interact specifically with ORC. Amino acid substitutions within the SRD abolish an OIR-ORC interaction but allow for a Sir1p-Sir4p interaction and HMR silencing by the Gal4-Sir1p-tethered mechanism. In contrast, several different amino acid substitutions in the OIR that lie outside of the SRD abolish Sir1p-ORC and Sir1p-Sir4p interactions and both natural and Gal4-Sir1p-tethered silencing. We propose that the OIR forms a discrete domain whose structure is necessary to present the SRD of Sir1p for binding by ORC. Consistent with this, the OIR shows similarity to a region of mouse Ku80 that is predicted to form an alpha-helical globular domain. Moreover, the OIR is resistant to partial proteolysis, suggesting that it is a stable structural domain. Mutations in the OIR are being analyzed for their effects on the structure of the OIR and interactions with ORC using a variety of assays.

Searching for G-protein coupled receptors in Arabidopsis thaliana

David Nelson

G-protein coupled receptors (GPCRs) compose a large superfamily of seven transmembrane domain proteins that are involved in the perception of a wide variety of signals including light, hormones, peptides, and other small molecules. While the members of this family are as divergent as their ligands, they share a common mode of signal transduction. Specific shifts in cytoplasmic loop conformations of the receptor cause activation of the associated heterotrimeric G-protein complex through an induced GDP/GTP exchange in the G-alpha subunit. The subsequent dissociation of the heterotrimeric complex permits downstream signaling to be effected.

In Arabidopsis, G-protein mediated signaling mechanisms have been implicated in a wide variety of processes including blue light perception, stomatal closure, seed germination, and cell division. With the recent completion of the Arabidopsis genome sequencing project, candidate genes for all the components of the classic heterotrimeric G-protein complex have been identified. However, there are far fewer genes encoding the subunits than are found in other non-plant eukaryotic systems. In addition, only one candidate GPCR has been identified and studied to date.

Altogether, this suggests that plants have undergone sufficient divergence as to make identification of GPCR system components through amino acid sequence comparison less obvious. To challenge the assumed lack of receptors, we are using a reverse genetics approach to investigate the function of a number of membrane proteins with potential roles as GPCRs. We are identifying knockout mutations in 12 genes from 3 different families, and have begun gauntlet screening for mutant phenotypes. In addition, we have recently undertaken a biochemical approach to identify plant membrane proteins that interact with the G-alpha subunit.

Global gene expression profiling of Escherichia coli K12: transition into stationary phase

Mingzhu Liu

Bacteria can survive for long time after entering into stationary phase and the stationary-phase response is tightly regulated in a temporal manner. We examined E. coli global gene expression profiles at 8 different time points during log phase and transition into stationary phase using DNA microarrays. An interesting observation is that cells had a resume of growth after 3 hours into stationary phase and then returned to non-growing state. Micoarray data showed there was acetate utilization during first hour entering into stationary phase and then propionate utilization 2 hours later. Stationary phase-induced genes seem mostly growth rate-dependant, being up-regulated after cells completely stop growing. Only a few genes showed transcripts difference in late log phase, which are involved in DNA replication inhibition and ribosome dimerization as well as acid response and possibly metal homeostasis. Most dramatic changes happened during the first hour after onset of stationary phase. They are the genes involved in central metabolism, stress response and transcription/translation apparatus. Sigma S transcript had a transient increase during this period while sigma E transcript showed a steady increase through 10 hours into stationary phase. Also, motifs searching were performed in up-stream regions of genes with similar expression patterns.