Cytosolic tail sequences and subunit interactions are critical for synaptic localization of glutamate receptors.
J Cell Sci. 118:1945-56. Abstract
AMPA-type glutamate receptors mediate excitatory synaptic transmission in the nervous system. The receptor subunit composition and subcellular localization play an important role in regulating synaptic strength. GLR-1 and GLR-2 are the Caenorhabditis elegans subunits most closely related to the mammalian AMPA-type receptors. These subunits are expressed in overlapping sets of interneurons, and contain type-I PDZ binding motifs in their carboxy-terminal cytosolic tail sequences. We report that GLR-1 and GLR-2 may form a heteromeric complex, the localization of which depends on either GLR-1 or GLR-2 tail sequences. Subunit interactions alone can mediate synaptic localization as endogenous GLR-1, or GLR-2 subunits can rescue the localization defects of subunits lacking tail sequences. Moreover, GLR-2 cytosolic tail sequences are sufficient to confer synaptic localization on a heterologous reporter containing a single-transmembrane domain. The localization of this GLR-2 reporter requires both a PDZ-binding motif in the GLR-2 tail sequence, and sequences outside of this motif. The PDZ protein LIN-10 regulates the localization of the reporter through the sequences outside of the PDZ-binding motif. Our results suggest that multiple synaptic localization signals reside in the cytosolic tail sequence of the receptor subunits, and that channel assembly can rescue the synaptic localization defects of individual mutant subunits as long as there are also wild-type subunits in the receptor complex.
Cytological analysis of meiosis in fixed Drosophila ovaries.
Methods Mol Biol. 558:197-216. Abstract
Methods are described to analyze two different parts of the Drosophila ovary, which correspond to early stages (pachytene) and late stages (metaphase I and beyond) of meiosis. In addition to taking into account morphology, the techniques differ by fixation conditions and the method to isolate the tissue. Most of these methods are whole mounts, which preserve the three-dimensional structure.
Crystallographic Studies of a Novel DNA-binding Domain from the Yeast Transcriptional Activator Ndt80.
Acta Crystallogr D Biol Crystallogr. 58:2127-2130. Abstract
The Ndt80 protein is a transcriptional activator that plays a key role in the progression of the meiotic divisions in the yeast Saccharomyces cerevisiae. Ndt80 is strongly induced during the middle stages of the sporulation pathway and binds specifically to a promoter element called the MSE to activate transcription of genes required for the meiotic divisions. Here, the preliminary structural and functional studies to characterize the DNA-binding activity of this protein are reported. Through deletion analysis and limited proteolysis studies of Ndt80, a novel 32 kDa DNA-binding domain that is sufficient for DNA-binding in vitro has been defined. Crystals of the DNA-binding domain of Ndt80 in two distinct lattices have been obtained, for which diffraction data extend to 2.3 A resolution.
Crystal Structure of the MATa1/MATalpha2 Homeodomain Heterodimer in Complex with DNa Containing an A-tract.
Nucleic Acids Res. 26:5707-5718. Abstract
The crystal structure of the heterodimer formed by the DNA binding domains of the yeast mating type transcription factors, MATa1 and MATalpha2, bound to a 21 bp DNA fragment has been determined at 2.5 A resolution. The DNA fragment in the present study differs at four central base pairs from the DNA sequence used in the previously studied ternary complex. These base pair changes give rise to a (dA5).(dT5) tract without changing the overall base composition of the DNA. The resulting A-tract occurs near the center of the overall 60 degrees bend in the DNA. Comparison of the two structures shows that the structural details of the DNA bend are maintained despite the DNA sequence changes. Analysis of the A5-tract DNA subfragment shows that it contains a bend toward the minor groove centered at one end of the A-tract. The observed bend is larger than that observed in the crystal structures of A-tracts embedded in uncomplexed DNA, which are straight and have been presumed to be quite rigid. Variation of the central DNA base sequence reverses the two AT base pairs contacted in the minor groove by Arg7 of the alpha2 N-terminal arm without significantly altering the DNA binding affinity of the a1/alpha2 heterodimer. The Arg7 side chain accommodates the sequence change by forming alternate H bond interactions, in agreement with the proposal that minor groove base pair recognition is insensitive to base pair reversal. Furthermore, the minor groove spine of hydration, which stabilizes the narrowed minor groove caused by DNA bending, is conserved in both structures. We also find that many of the water-mediated hydrogen bonds between the a1 and alpha2 homeodomains and the DNA are highly conserved, indicating an important role for water in stabilization of the a1/alpha2-DNA complex.
Crystal Structure of the DNA-binding Domain from Ndt80, a Transcriptional Activator Required for Meiosis in Yeast.
Proc Natl Acad Sci U S A. 99:14041-14046. Abstract
Ndt80 is a transcriptional activator required for meiosis in the yeast Saccharomyces cerevisiae. Here, we report the crystal structure at 2.3 A resolution of the DNA-binding domain of Ndt80 experimentally phased by using the anomalous and isomorphous signal from a single ordered Se atom per molecule of 272-aa residues. The structure reveals a single approximately 32-kDa domain with a distinct fold comprising a beta-sandwich core elaborated with seven additional beta-sheets and three short alpha-helices. Inspired by the structure, we have performed a mutational analysis and defined a DNA-binding motif in this domain. The DNA-binding domain of Ndt80 is homologous to a number of proteins from higher eukaryotes, and the residues that we have shown are required for DNA binding by Ndt80 are highly conserved among this group of proteins. These results suggest that Ndt80 is the defining member of a previously uncharacterized family of transcription factors, including the human protein (C11orf9), which has been shown to be highly expressed in invasive or metastatic tumor cells.
Cross regulation of decapentaplegic and Ultrabithorax transcription in the embryonic visceral mesoderm of Drosophila.
Development (Cambridge, England). 117:1211-22. Abstract
The Drosophila decapentaplegic gene (dpp) encodes a TGF-beta family member involved in signal transduction during embryonic midgut formation. The shortvein (shv) class of cis-regulatory dpp mutants disrupt expression in parasegments 4 and 7 (ps4 and ps7) of the embryonic visceral mesoderm (VM) surrounding the gut and cause abnormalities in gut morphogenesis. We demonstrate that cis-regulatory elements directing expression in ps4 and ps7 are separable and identify DNA fragments that generate ps4 and ps7 expression patterns using reporter gene constructs. dpp reporter gene expression in both ps4 and ps7 is autoregulated as it requires endogenous dpp+ activity. Reporter gene ps7 expression requires the wild-type action of Ultra-bithorax (Ubx), and abdominal-A. Furthermore, the expression of certain Ubx reporter genes is coincident with dpp in the VM. Both the mis-expression of Ubx reporter genes in the developing gastric caecae at ps4 and its normal expression in ps7 are dependent upon endogenous dpp+ activity. We conclude that dpp both responds to and regulates Ubx in ps7 of the visceral mesoderm and that Ubx autoregulation within this tissue may be indirect as it requires more components than have previously been thought.
Corrected nucleotide sequence of M13mp18 gene III..
Gene. 114(1):81-3. Abstract
There are seven differences between the actual nucleotide (nt) sequence of bacteriophage M13mp18 gene III and the previously reported nt sequence (which had been compiled based on the nt sequence of wild-type bacteriophage M13 gene III).
Cooperative Regulation of Growth by Yorkie and Mad through bantam.
Developmental Cell. 20:109-22. Abstract
The Dpp and Fat-Hippo signaling pathways both regulate growth in Drosophila. Dpp is a BMP family ligand and acts via a Smad family DNA-binding transcription factor, Mad. Fat-Hippo signaling acts via a non-DNA-binding transcriptional coactivator protein, Yorkie. Here, we show that these pathways are directly interlinked. They act synergistically to promote growth, in part via regulation of the microRNA gene bantam, and their ability to promote growth is mutually dependent. Yorkie and Mad physically bind each other, and we identify a 410 bp minimal enhancer of bantam that responds to Yorkie:Mad in vivo and in cultured cells, and show that both Yorkie and Mad associate with this enhancer in vivo. Our results indicate that in promoting the growth of Drosophila tissues, Fat-Hippo and Dpp signaling contribute distinct subunits of a shared transcriptional activation complex, Yorkie:Mad.
Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia.
Nat Cell Biol. 3:983-91. Abstract
The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (alpha-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans.
Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent..
Proceedings of the National Academy of Sciences of the United States of America. 87(8):2882-6. Abstract
Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. [(N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with Kobs = 1 x 10(8) M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.
The Control of Axillary Meristem fate in the Maize Ramosa Pathway.
Development. 137:2849-2856. Abstract
Plant axillary meristems are composed of highly organized, self-renewing stem cells that produce indeterminate branches or terminate in differentiated structures, such as the flowers. These opposite fates, dictated by both genetic and environmental factors, determine interspecific differences in the architecture of plants. The Cys(2)-His(2) zinc-finger transcription factor RAMOSA1 (RA1) regulates the fate of most axillary meristems during the early development of maize inflorescences, the tassel and the ear, and has been implicated in the evolution of grass architecture. Mutations in RA1 or any other known members of the ramosa pathway, RAMOSA2 and RAMOSA3, generate highly branched inflorescences. Here, we report a genetic screen for the enhancement of maize inflorescence branching and the discovery of a new regulator of meristem fate: the RAMOSA1 ENHANCER LOCUS2 (REL2) gene. rel2 mutants dramatically increase the formation of long branches in ears of both ra1 and ra2 mutants. REL2 encodes a transcriptional co-repressor similar to the TOPLESS protein of Arabidopsis, which is known to maintain apical-basal polarity during embryogenesis. REL2 is capable of rescuing the embryonic defects of the Arabidopsis topless-1 mutant, suggesting that REL2 also functions as a transcriptional co-repressor throughout development. We show by genetic and molecular analyses that REL2 physically interacts with RA1, indicating that the REL2/RA1 transcriptional repressor complex antagonizes the formation of indeterminate branches during maize inflorescence development. Our results reveal a novel mechanism for the control of meristem fate and the architecture of plants.
Contributions of chaperone and glycosyltransferase activities of O-fucosyltransferase 1 to Notch signaling.
BMC Biology. 6:1. Abstract
BACKGROUND: O-fucosyltransferase1 (OFUT1) is a conserved ER protein essential for Notch signaling. OFUT1 glycosylates EGF domains, which can then be further modified by the N-acetylglucosaminyltransferase Fringe. OFUT1 also possesses a chaperone activity that promotes the folding and secretion of Notch. Here, we investigate the respective contributions of these activities to Notch signaling in Drosophila. RESULTS: We show that expression of an isoform lacking fucosyltransferase activity, Ofut1R245A, rescues the requirement for Ofut1 in embryonic neurogenesis. Lack of requirement for O-fucosylation is further supported by the absence of embryonic phenotypes in Gmd mutants, which lack all forms of fucosylation. Requirements for O-fucose during imaginal development were evaluated by characterizing clones of cells expressing only Ofut1R245A. These clones phenocopy fringe mutant clones, indicating that the absence of O-fucose is functionally equivalent to the absence of elongated O-fucose. CONCLUSION: Our results establish that Notch does not need to be O-fucosylated for fringe-independent Notch signaling in Drosophila; the chaperone activity of OFUT1 is sufficient for the generation of functional Notch.
Contiguous genomic DNA sequence comprising the 19-kD zein gene family from maize.
Plant physiology. 130:1626-35. Abstract
A new approach has been undertaken to analyze the sequences and linear organization of the 19-kD zein genes in maize (Zea mays). A high-coverage, large-insert genomic library of the inbred line B73 based on bacterial artificial chromosomes was used to isolate a redundant set of clones containing members of the 19-kD zein gene family, which previously had been estimated to consist of 50 members. The redundant set of clones was used to create bins of overlapping clones that represented five distinct genomic regions. Representative clones containing the entire set of 19-kD zein genes were chosen from each region and sequenced. Seven bacterial artificial chromosome clones yielded 1,160 kb of genomic DNA. Three of them formed a contiguous sequence of 478 kb, the longest contiguous sequenced region of the maize genome. Altogether, these DNA sequences provide the linear organization of 25 19-kD zein genes, one-half the number previously estimated. It is suggested that the difference is because of haplotypes exhibiting different degrees of gene amplification in the zein multigene family. About one-half the genes present in B73 appear to be expressed. Because some active genes have only been duplicated recently, they are so conserved in their sequence that previous cDNA sequence analysis resulted in "unigenes" that were actually derived from different gene copies. This analysis also shows that the 22- and 19-kD zein gene families shared a common ancestor. Although both ancestral genes had the same incremental gene amplification, the 19-kD zein branch exhibited a greater degree of far-distance gene translocations than the 22-kD zein gene family.
Construction of marker-free transplastomic plants.
Current Opinion in Biotechnology. 18:107-14. Abstract
Because of its prokaryotic-type gene expression machinery, maternal inheritance and the opportunity to express proteins at a high level, the plastid genome (plastome or ptDNA) is an increasingly popular target for engineering. The ptDNA is present as up to 10,000 copies per cell, making selection for marker genes essential to obtain plants with uniformly transformed ptDNA. However, the marker gene is no longer desirable when homoplastomic plants are obtained. Marker-free transplastomic plants can now be obtained with four recently developed protocols: homology-based excision via directly repeated sequences, excision by phage site-specific recombinanses, transient cointegration of the marker gene, and the cotransformation-segregation approach. Marker excision technology will benefit applications in agriculture and in molecular farming.
Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis.
Gene. 26:101-6. Abstract
The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M13mp10 and M13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.
Consensus DNA site for the Escherichia coli catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. coli lac DNA site..
Nucleic acids research. 17(24):10295-305. Abstract
We have synthesized two 40 base pair DNA fragments; one fragment contains the consensus DNA site for CAP (fragment 'ICAP'); the other fragment contains the E. coli lac promoter DNA site for CAP (fragment 'LCAP'). We have investigated the binding of CAP to the two DNA fragments using the nitrocellulose filter binding assay. Under standard conditions [( NaCl] = 200 mM, pH = 7.3), CAP exhibits a 450-fold higher affinity for ICAP than for LCAP. The salt dependence of the binding equilibrium indicates that CAP makes eight ion pairs with ICAP, but only six ion pairs with LCAP. Approximately half of the difference in binding free energy for interaction of CAP with ICAP vs. LCAP is attributable to this difference in ion-pair formation. The pH dependence of the binding equilibrium indicates that the eight CAP-ICAP ion pairs and the six CAP-LCAP ion pairs do not involve His residues of CAP.
Computational prediction and molecular confirmation of Helitron transposons in the maize genome.
BMC Genomics. 9:51. Abstract
Helitrons represent a new class of transposable elements recently uncovered in plants and animals. One remarkable feature of Helitrons is their ability to capture gene sequences, which makes them of considerable potential evolutionary importance. However, because Helitrons lack the typical structural features of other DNA transposable elements, identifying them is a challenge. Currently, most researchers identify Helitrons manually by comparing sequences. With the maize whole genome sequencing project underway, an automated computational Helitron searching tool is needed. The characterization of Helitron activities in maize needs to be addressed in order to better understand the impact of Helitrons on the organization of the genome.\\ We developed and implemented a heuristic searching algorithm in PERL for identifying Helitrons. Our HelitronFinder program will (i) take FASTA-formatted DNA sequences as input and identify the hairpin looping patterns, and (ii) exploit the consensus 5' and 3' end sequences of known Helitrons to identify putative ends. We randomly selected five predicted Helitrons from the program's high quality output for molecular verification. Four out of the five predicted Helitrons were confirmed by PCR assays and DNA sequencing in different maize inbred lines. The HelitronFinder program identified two head-to-head dissimilar Helitrons in a maize BAC sequence.\\ We have identified 140 new Helitron candidates in maize with our computational tool HelitronFinder by searching maize DNA sequences currently available in GenBank. Four out of five candidates were confirmed to be real by empirical methods, thus validating the predictions of HelitronFinder. Additional points to emerge from our study are that Helitrons do not always insert at an AT dinucleotide in the host sequences, that they can insert immediately adjacent to an existing Helitron, and that their movement may cause changes in the flanking region, such as deletions.
The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing.
Nucleic acids research. 9:2871-88. Abstract
We have determined the complete primary structure (8031 base pairs) of an infectious clone of cauliflower mosaic virus strain CM1841. The sequence was obtained using the strategy of cloning shotgun restriction fragments in the sequencing vector M13mp7. Comparison of the CM1841 sequence with that published for another caMV strain (Strasbourg) reveals 4.4% changes, mostly nucleotide substitutions with a few small insertions and deletions. The six open reading frames in the sequence of the Strasbourg isolate are also present in CM1841.
The complete Ac/Ds transposon family of maize..
BMC Genomics. 12:588 :doi:10.1186/1471-2164-12-588. Abstract
Background: The nonautonomous maize Ds transposons can only move in the presence of the autonomous
element Ac. They comprise a heterogeneous group that share 11-bp terminal inverted repeats (TIRs) and some
subterminal repeats, but vary greatly in size and composition. Three classes of Ds elements can cause mutations:
Ds-del, internal deletions of the 4.6-kb Ac element; Ds1, ~400-bp in size and sharing little homology with Ac, and
Ds2, variably-sized elements containing about 0.5 kb from the Ac termini and unrelated internal sequences. Here,
we analyze the entire complement of Ds-related sequences in the genome of the inbred B73 and ask whether
additional classes of Ds-like (Ds-l) elements, not uncovered genetically, are mobilized by Ac. We also compare the
makeup of Ds-related sequences in two maize inbreds of different origin.
Results: We found 903 elements with 11-bp Ac/Ds TIRs flanked by 8-bp target site duplications. Three resemble Ac,
but carry small rearrangements. The others are much shorter, once extraneous insertions are removed. There are
331 Ds1 and 39 Ds2 elements, many of which are likely mobilized by Ac, and two novel classes of Ds-l elements.
Ds-l3 elements lack subterminal homology with Ac, but carry transposase gene fragments, and represent decaying
Ac elements. There are 44 such elements in B73. Ds-l4 elements share little similarity with Ac outside of the 11-bp
TIR, have a modal length of ~1 kb, and carry filler DNA which, in a few cases, could be matched to gene
fragments. Most Ds-related elements in B73 (486/903) fall in this class. None of the Ds-l elements tested responded
to Ac. Only half of Ds insertion sites examined are shared between the inbreds B73 and W22.
Conclusions: The majority of Ds-related sequences in maize correspond to Ds-l elements that do not transpose in
the presence of Ac. Unlike actively transposing elements, many Ds-l elements are inserted in repetitive DNA, where
they probably become methylated and begin to decay. The filler DNA present in most elements is occasionally
captured from genes, a rare feature in transposons of the hAT superfamily to which Ds belongs. Maize inbreds of
different origin are highly polymorphic in their DNA transposon makeup.
Combined Analysis of Expression data and Transcription Factor Binding Sites in the Yeast Genome.
BMC Genomics. 5:59-59. Abstract
The analysis of gene expression using DNA microarrays provides genome wide profiles of the genes controlled by the presence or absence of a specific transcription factor. However, the question arises of whether a change in the level of transcription of a specific gene is caused by the transcription factor acting directly at the promoter of the gene or through regulation of other transcription factors working at the promoter.