bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of a Class III Myosin to Filopodia Tips in Transfected HeLa Cells Requires an Actin-binding Site in its Tail Domain
Authors: Les Erickson F. , Corsa Amoreena C. , Dosé Andréa C. , Burnside Beth
Published: July 2003

Bass Myo3A, a class III myosin, was expressed in HeLa cells as a GFP fusion in order to study its cellular localization. GFP-Myo3A localized to the cytoplasm and to the tips of F-actin bundles in filopodia, a localization that is consistent with the observed concentration toward the distal ends of F-actin bundles in photoreceptor cells. A mutation in the motor active site resulted in a loss of filopodia localization, suggesting that Myo3A motor activity is required for filopodial tip localization. Deletion analyses showed that the NH2-terminal kinase domain is not required but the CO2H-terminal 22 amino acids of the Myo3A tail are required for filopodial localization. Expression of this tail fragment alone produced fluorescence associated with F-actin throughout the cytoplasm and filopodia and a recombinant tail fragment bound to F-actin in vitro. An actin-binding motif was identified within this tail fragment, and a mutation within this motif abolished both filopodia localization by Myo3A and F-actin binding by the tail fragment alone. Calmodulin localized to filopodial tips when coexpressed with Myo3A but not in the absence of Myo3A, an observation consistent with the previous proposal that class III myosins bind calmodulin and thereby localize it in certain cell types.
Identification and Characterization of the Endocytic Transmembrane Glycoprotein Endo180 as a Novel Collagen Receptor
Authors: Wienke Dirk , MacFadyen John R. , Isacke Clare M.
Published: July 2003

Endo180, a member of the mannose receptor family, is constitutively recycled between clathrin-coated pits on the cell surface and intracellular endosomes. Its large extracellular domain contains an N-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. The second of these lectin-like domains has been shown to mediate Ca2+-dependent mannose binding. In addition, cross-linking studies have identified Endo180 as a urokinase plasminogen activator receptor–associated protein and this interaction can be blocked by collagen V. Here we demonstrate directly using in vitro assays, cell-based studies and tissue immunohistochemistry that Endo180 binds both to native and denatured collagens and provide evidence that this is mediated by the fibronectin type II domain. In cell culture systems, expression of Endo180 results in the rapid uptake of soluble collagens for delivery to lysosomal degradative compartments. Together with the observed restricted expression of Endo180 in both embryonic and adult tissue, we propose that Endo180 plays a physiological role in mediating collagen matrix remodelling during tissue development and homeostasis and that the observed receptor upregulation in pathological conditions may contribute to disease progression.
Lipid Raft Targeting of the TC10 Amino Terminal Domain Is Responsible for Disruption of Adipocyte Cortical Actin
Authors: Chunqiu Hou June , Pessin Jeffrey E.
Published: July 2003

Overexpression of the Rho family member TC10α, disrupts adipocyte cortical actin structure and inhibits insulin-stimulated GLUT4 translocation when targeted to lipid raft microdomains. This appears to be independent of effecter domain function because overexpression of the wild-type (TC10/WT), constitutively GTP-bound (TC10/Q75L), and constitutively GDP bound (TC10/T31N) all inhibit adipocyte cortical actin structure and GLUT4 translocation. To examine the structural determinants responsible for these effects, we generated a series of chimera proteins between TC10 with that of H-Ras and K-Ras. Chimera containing the 79 (TC10–79/H-Ras), 41 (TC10–41/H-Ras), or 16 (TC10–16/H-Ras) amino acids of the TC10 amino terminal extension fused to H-Ras disrupted cortical actin and inhibited insulin-stimulated GLUT4 translocation. In contrast, the same amino terminal TC10 extensions fused to K-Ras had no significant effect on either GLUT4 translocation or cortical actin structure. Similarly, expression of TC10β was without effect, whereas fusion of the amino terminal 8 amino acid of TC10α onto TC10β resulted in an inhibition of insulin-stimulated GLUT4 translocation. Within the amino terminal extension point mutation analysis demonstrated that both a GAG and GPG sequences when lipid raft targeted was essential for these effects. Furthermore, expression of the amino terminal TC10 deletions ΔNT-TC10/WT or ΔNT-TC10/T31N had no detectable effect on cortical actin organization and did not perturb insulin-stimulated GLUT4 translocation. Surprisingly, however, expression of ΔNT-TC10/Q75L remained fully capable of inhibiting insulin-stimulated GLUT4 translocation without affecting cortical actin. These data demonstrate that inhibitory effect of TC10 overexpression on adipocyte cortical actin organization is due to the specific lipid raft targeting of the unusual TC10 amino terminal extension.
Interaction with Tap42 Is Required for the Essential Function of Sit4 and Type 2A Phosphatases
Authors: Wang Huamin , Wang Xiaodong , Jiang Yu
Published: July 2003

In Saccharomyces cerevisiae, Pph21 and Pph22 are the two catalytic subunits of type 2A phosphatase (PP2Ac), and Sit4 is a major form of 2A-like phosphatase. The function of these phosphatases requires their association with different regulatory subunits. In addition to the conventional regulatory subunits, namely, the A and B subunits for Pph21/22 and the Sap proteins for Sit4, these phosphatases have been found to associate with a protein termed Tap42. In this study, we demonstrated that Sit4 and PP2Ac interact with Tap42 via an N-terminal domain that is conserved in all type 2A and 2A-like phosphatases. We found that the Sit4 phosphatase in the sit4-102 strain contains a reverse-of-charge amino acid substitution within its Tap42 binding domain and is defective for formation of the Tap42-Sit4 complex. Our results suggest that the interaction with Tap42 is required for the activity as well as for the essential function of Sit4 and PP2Ac. In addition, we showed that Tap42 is able to interact with two other 2A-like phosphatases, Pph3 and Ppg1.
Urmylation: A Ubiquitin-like Pathway that Functions during Invasive Growth and Budding in Yeast
Authors: Goehring April S. , Rivers David M. , Sprague George F.
Published: July 2003

Ubiquitin is a small modifier protein that is conjugated to substrates to target them for degradation. Recently, a surprising number of ubiquitin-like proteins have been identified that also can be attached to proteins. Herein, we identify two molecular functions for the posttranslational protein modifier from Saccharomyces cerevisiae, Urm1p. Simultaneous loss of Urm1p and Cla4p, a p21-activated kinase that functions in budding, is lethal. This result suggests a role for the urmylation pathway in budding. Furthermore, loss of the urmylation pathway causes defects in invasive growth and confers sensitivity to rapamycin. Our results indicate that the sensitivity to rapamycin is due to a genetic interaction with the TOR pathway, which is important for regulation of cell growth in response to nutrients. We have found that Urm1p can be attached to a number of proteins. Loss of five genes that are also essential in a cla4Δ strain, NCS2, NCS6, ELP2, ELP6, and URE2, affect the level of at least one Urm1p conjugate. Moreover, these five genes have a role in invasive growth and display genetic interactions with the TOR pathway. In summary, our results suggest the urmylation pathway is involved in nutrient sensing and budding.
Actin Filament Turnover Regulated by Cross-linking Accounts for the Size, Shape, Location, and Number of Actin Bundles inDrosophilaBristles
Authors: Tilney Lewis G. , Connelly Patricia S. , Ruggiero Linda , Vranich Kelly A. , Guild Gregory M.
Published: July 2003

Drosophila bristle cells are shaped during growth by longitudinal bundles of cross-linked actin filaments attached to the plasma membrane. We used confocal and electron microscopy to examine actin bundle structure and found that during bristle elongation, snarls of uncross-linked actin filaments and small internal bundles also form in the shaft cytoplasm only to disappear within 4 min. Thus, formation and later removal of actin filaments are prominent features of growing bristles. These transient snarls and internal bundles can be stabilized by culturing elongating bristles with jasplakinolide, a membrane-permeant inhibitor of actin filament depolymerization, resulting in enormous numbers of internal bundles and uncross-linked filaments. Examination of bundle disassembly in mutant bristles shows that plasma membrane association and cross-bridging adjacent actin filaments together inhibits depolymerization. Thus, highly cross-bridged and membrane-bound actin filaments turn over slowly and persist, whereas poorly cross-linked filaments turnover more rapidly. We argue that the selection of stable bundles relative to poorly cross-bridged filaments can account for the size, shape, number, and location of the longitudinal actin bundles in bristles. As a result, filament turnover plays an important role in regulating cytoskeleton assembly and consequently cell shape.
DNA Damage during the Spindle-Assembly Checkpoint Degrades CDC25A, Inhibits Cyclin–CDC2 Complexes, and Reverses Cells to Interphase
Authors: Chow Jeremy P.H. , Siu Wai Yi , Fung Tsz Kan , Chan Wan Mui , Lau Anita , Arooz Talha , Ng Chuen-Pei , Yamashita Katsumi , Poon Randy Y.C.
Published: July 2003

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.
Yeast Kinetochores Do Not Stabilize Stu2p-dependent Spindle Microtubule Dynamics
Authors: Pearson Chad G. , Maddox Paul S. , Zarzar Ted R. , Salmon E.D. , Bloom Kerry
Published: July 2003

The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule–kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.
Visualization of Protein Compartmentation within the Plasma Membrane of Living Yeast Cells
Authors: Malínská Katerina , Malínský Jan , Opekarová Miroslava , Tanner Widmar
Published: July 2003

Different distribution patterns of the arginine/H+symporter Can1p, the H+plasma membrane ATPase Pma1p, and the hexose transport facilitator Hxt1p within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using fluorescence protein tagging of these proteins. Although Hxt1p-GFP was evenly distributed through the whole cell surface, Can1p-GFP and Pma1p-GFP were confined to characteristic subregions in the plasma membrane. Pma1p is a well-documented raft protein. Evidence is presented that Can1p, but not Hxt1p, is exclusively associated with lipid rafts, too. Double labeling experiments with Can1p-GFP– and Pma1p-RFP–containing cells demonstrate that these proteins occupy two different nonoverlapping membrane microdomains. The size of Can1p-rich (Pma1p-poor) areas was estimated to 300 nm. These domains were shown to be stable in growing cells for >30 min. To our knowledge, this is the first observation of a cell polarization-independent lateral compartmentation in the plasma membrane of a living cell.
The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast
Authors: Caviston Juliane P. , Longtine Mark , Pringle John R. , Bi Erfei
Published: July 2003

The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36Gallele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42V36Gor GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth.
The Balance of Driving Forces During Genome Evolution in Prokaryotes
Authors: Kunin Victor , Ouzounis Christos A.
Published: July 2003

Genomes are shaped by evolutionary processes such as gene genesis,
horizontal gene transfer (HGT), and gene loss. To quantify the relative
contributions of these processes, we analyze the distribution of 12,762
protein families on a phylogenetic tree, derived from entire genomes of 41
Bacteria and 10 Archaea. We show that gene loss is the most important factor
in shaping genome content, being up to three times more frequent than HGT,
followed by gene genesis, which may contribute up to twice as many genes as
HGT. We suggest that gene gain and gene loss in prokaryotes are balanced;
thus, on average, prokaryotic genome size is kept constant. Despite the
importance of HGT, our results indicate that the majority of protein families
have only been transmitted by vertical inheritance. To test our method, we
present a study of strain-specific genes of Helicobacter pylori, and
demonstrate correct predictions of gene loss and HGT for at least 81% of
validated cases. This approach indicates that it is possible to trace genome
content history and quantify the factors that shape contemporary prokaryotic
genomes.
A Rapid Method of Genomic Array Analysis of Scaffold/Matrix Attachment Regions (S/MARs) Identifies a 2.5-Mb Region of Enhanced Scaffold/Matrix Attachment at a Human Neocentromere
Authors: Sumer Huseyin , Craig Jeffrey M. , Sibson Mandy , Choo K.H. Andy
Published: July 2003

Human neocentromeres are fully functional centromeres that arise at previously noncentromeric regions of the genome. We have tested a rapid procedure of genomic array analysis of chromosome scaffold/matrix attachment regions (S/MARs), involving the isolation of S/MAR DNA and hybridization of this DNA to a genomic BAC/PAC array. Using this procedure, we have defined a 2.5-Mb domain of S/MAR-enriched chromatin that fully encompasses a previously mapped centromere protein-A (CENP-A)-associated domain at a human neocentromere. We have independently verified this procedure using a previously established fluorescence in situ hybridization method on salt-treated metaphase chromosomes. In silico sequence analysis of the S/MAR-enriched and surrounding regions has revealed no outstanding sequence-related predisposition. This study defines the S/MAR-enriched domain of a higher eukaryotic centromere and provides a method that has broad application for the mapping of S/MAR attachment sites over large genomic regions or throughout a genome.
Genome Sequence of an M3 Strain of Streptococcus pyogenes
Reveals a Large-Scale Genomic Rearrangement in Invasive Strains and New Insights into Phage Evolution
Authors: Nakagawa Ichiro , Kurokawa Ken , Yamashita Atsushi , Nakata Masanobu , Tomiyasu Yusuke , Okahashi Nobuo , Kawabata Shigetada , Yamazaki Kiyoshi , Shiba Tadayoshi , Yasunaga Teruo , Hayashi Hideo , Hattori Masahira , Hamada Shigeyuki
Published: July 2003

Group Astreptococcus (GAS) is a gram-positive bacterial pathogen that causes various suppurative infections and nonsuppurative sequelae. Since the late 1980s, streptococcal toxic-shock like syndrome (STSS) and severe invasive GAS infections have been reported globally. Here we sequenced the genome of serotype M3 strain SSI-1, isolated from an STSS patient in Japan, and compared it with those of other GAS strains. The SSI-1 genome is composed of 1,884,275 bp, and 1.7 Mb of the sequence is highly conserved relative to strain SF370 (serotype M1) and MGAS8232 (serotype M18), and almost completely conserved relative to strain MGAS315 (serotype M3). However, a large genomic rearrangement has been shown to occur across the replication axis between the homologous rrn-comX1 regions and between two prophage-coding regions across the replication axis. Atotal of 1 Mb of chromosomal DNA is inverted across the replication axis. Interestingly, the recombinations between the prophage regions are within the phage genes, and the genes encoding superantigens and mitogenic factors are interchanged between two prophages. This genomic rearrangement occurs in 65% of clinical isolates (64/94) collected after 1990, whereas it is found in only 25% of clinical isolates (7/28) collected before 1985. These observations indicate that streptococcal phages represent important plasticity regions in the GAS chromosome where recombination between homologous phage genes can occur and result not only in new phage derivatives, but also in large chromosomal rearrangements.
Divergence in the Spatial Pattern of Gene Expression Between Human Duplicate Genes
Authors: Makova Kateryna D. , Li Wen-Hsiung
Published: July 2003

Microarray gene expression data provide a wealth of information for
elucidating the mode and tempo of molecular evolution. In the present study,we
analyze the spatial expression pattern of human duplicate gene pairs by using
oligonucleotide microarray data,and study the relationship between coding
sequence divergence and expression divergence. First,we find a strong positive
correlation between the proportion of duplicate gene pairs with divergent
expression (as presence or absence of expression in a tissue) and both
synonymous (KS) and nonsynonymous divergence
(KA). The divergence of gene expression between human
duplicate genes is rapid, probably faster than that between yeast duplicates
in terms of generations. Second,we compute the correlation coefficient
(R) between the expression levels of duplicate genes in different
tissues and find a significant negative correlation between R and
KS. There is also a negative correlation between
R and KA,when KA ≤ 0.2.
These results indicate that protein sequence divergence and divergence of
spatial expression pattern are initially coupled. Finally,we compare the
functions of those duplicate genes that show rapid divergence in spatial
expression pattern with the functions of those duplicate genes that show no or
little divergence in spatial expression.
Multimeric Threading-Based Prediction of Protein–Protein Interactions on a Genomic Scale: Application to the Saccharomyces cerevisiae Proteome
Authors: Lu Long , Arakaki Adrian K. , Lu Hui , Skolnick Jeffrey
Published: July 2003

MULTIPROSPECTOR, a multimeric threading algorithm for the prediction of protein–protein interactions, is applied to the genome of Saccharomyces cerevisiae. Each possible pairwise interaction among more than 6000 encoded proteins is evaluated against a dimer database of 768 complex structures by using a confidence estimate of the fold assignment and the magnitude of the statistical interfacial potentials. In total, 7321 interactions between pairs of different proteins are predicted, based on 304 complex structures. Quality estimation based on the coincidence of subcellular localizations and biological functions of the predicted interactors shows that our approach ranks third when compared with all other large-scale methods. Unlike other in silico methods, MULTIPROSPECTOR is able to identify the residues that participate directly in the interaction. Three hundred seventy-four of our predictions can be found by at least one of the other studies, which is compatible with the overlap between two different other methods. From the analysis of the mRNA abundance data, our method does not bias towards proteins with high abundance. Finally, several relevant predictions involved in various functions are presented. In summary, we provide a novel approach to predict protein–protein interactions on a genomic scale that is a useful complement to experimental methods.
An Evolutionarily Structured Universe of Protein Architecture
Authors: Caetano-Anollés Gustavo , Caetano-Anollés Derek
Published: July 2003

Protein structural diversity encompasses a finite set of architectural
designs. Embedded in these topologies are evolutionary histories that we here
uncover using cladistic principles and measurements of protein-fold usage and
sharing. The reconstructed phylogenies are inherently rooted and depict
histories of protein and proteome diversification. Proteome phylogenies showed
two monophyletic sister-groups delimiting Bacteria and Archaea, and a topology
rooted in Eucarya. This suggests three dramatic evolutionary events and a
common ancestor with a eukaryotic-like, gene-rich, and relatively modern
organization. Conversely, a general phylogeny of protein architectures showed
that structural classes of globular proteins appeared early in evolution and
in defined order, the α/β class being the first. Although most
ancestral folds shared a common architecture of barrels or interleaved
β-sheets and α-helices, many were clearly derived, such as
polyhedral folds in the all-α class and β-sandwiches,
β-propellers, and β-prisms in all-β proteins. We also describe
transformation pathways of architectures that are prevalently used in nature.
For example, β-barrels with increased curl and stagger were favored
evolutionary outcomes in the all-β class. Interestingly, we found cases
where structural change followed the α-to-β tendency uncovered in
the tree of architectures. Lastly, we traced the total number of enzymatic
functions associated with folds in the trees and show that there is a general
link between structure and enzymatic function.
Quantitative Assessment of Transcriptome Differences Between Brain Territories
Authors: de Chaldée Michel , Gaillard Marie-Claude , Bizat Nicolas , Buhler Jean-Marie , Manzoni Olivier , Bockaert Joël , Hantraye Philippe , Brouillet Emmanuel , Elalouf Jean-Marc
Published: July 2003

Transcriptome analysis of mammalian brain structures is a potentially
powerful approach in addressing the diversity of cerebral functions. Here, we
used a microassay for serial analysis of gene expression (SAGE) to generate
quantitative mRNA expression profiles of normal adult mouse striatum, nucleus
accumbens, and somatosensory cortex. Comparison of these profiles revealed 135
transcripts heterogeneously distributed in the brain. Among them, a majority
(78), although matching a registered sequence, are novel regional markers. To
improve the anatomical resolution of our analysis, we performed in situ
hybridization and observed unique expression patterns in discrete brain
regions for a number of candidates. We assessed the distribution of the new
markers in peripheral tissues using quantitative RT–PCR, Northern
hybridization, and published SAGE data. In most cases, expression was higher
in the brain than in peripheral tissues. Because the markers were selected
according to their expression level, without reference to prior knowledge, our
studies provide an unbiased, comprehensive molecular signature for various
mammalian brain structures that can be used to investigate their plasticity
under a variety of circumstances.
Intronic Sequences Flanking Alternatively Spliced Exons Are Conserved Between Human and Mouse
Authors: Sorek Rotem , Ast Gil
Published: July 2003

Comparison of the sequences of mouse and human genomes revealed a
surprising number of nonexonic, nonexpressed conserved sequences, for which no
function could be assigned. To study the possible correlation between these
conserved intronic sequences and alternative splicing regulation, we developed
a method to identify exons that are alternatively spliced in both human and
mouse. We compiled two exon sets: one of alternatively spliced conserved exons
and another of constitutively spliced conserved exons. We found that 77% of
the conserved alternatively spliced exons were flanked on both sides by long
conserved intronic sequences. In comparison, only 17% of the conserved
constitutively spliced exons were flanked by suchconserved intronic sequences.
The average length of the conserved intronic sequences was 103 bases in the
upstream intron and 94 bases in the downstream intron. The average identity
levels in the immediately flanking intronic sequences were 88% and 80% for the
upstream and downstream introns, respectively, higher than the conservation
levels of 77% that were measured in promoter regions. Our results suggest that
the function of many of the intronic sequence blocks that are conserved
between human and mouse is the regulation of alternative splicing.
Identification of Novel ErbB3-Interacting Factors Using the Split-Ubiquitin Membrane Yeast Two-Hybrid System
Authors: Thaminy Safia , Auerbach Daniel , Arnoldo Anthony , Stagljar Igor
Published: July 2003

Analysis of membrane protein interactions is difficult because of the
hydrophobic nature of these proteins, which often renders conventional
biochemical and genetic assays fruitless. This is a substantial problem
because proteins that are integral or associated with membranes represent
approximately one-third of all proteins in a typical eukaryotic cell. We have
shown previously that the modified split-ubiquitin system can be used as a
genetic assay for the in vivo detection of interactions between the two
characterized yeast transmembrane proteins, Ost1p and Wbp1p. This so-called
split-ubiquitin membrane yeast two-hybrid (YTH) system uses the
split-ubiquitin approach in which reconstitution of two ubiquitin halves is
mediated by a protein–protein interaction. Here we converted the
split-ubiquitin membrane YTH system into a generally applicable in vivo
screening approach to identify interacting partners of a particular mammalian
transmembrane protein. We have demonstrated the effectiveness of this approach
by using the mammalian ErbB3 receptor as bait and have identified three
previously unknown ErbB3-interacting proteins. In addition, we have confirmed
one of the newly found interactions between ErbB3 and the membrane-associated
RGS4 protein by coimmunoprecipitating the two proteins from human cells. We
expect the split-ubiquitin membrane YTH technology to be valuable for the
identification of potential interacting partners of integral membrane proteins
from many model organisms.
Comparative Complete Genome Sequence Analysis of the Amino Acid Replacements Responsible for the Thermostability ofCorynebacterium efficiens
Authors: Nishio Yousuke , Nakamura Yoji , Kawarabayasi Yutaka , Usuda Yoshihiro , Kimura Eiichiro , Sugimoto Shinichi , Matsui Kazuhiko , Yamagishi Akihiko , Kikuchi Hisashi , Ikeo Kazuho , Gojobori Takashi
Published: July 2003

Corynebacterium efficiensis the closest relative ofCorynebacterium glutamicum,a species widely used for the industrial production of amino acids.C. efficiensbut notC. glutamicumcan grow above 40°C. We sequenced the completeC. efficiensgenome to investigate the basis of its thermostability by comparing its genome with that ofC. glutamicum. The difference in GC content between the species was reflected in codon usage and nucleotide substitutions. Our comparative genomic study clearly showed that there was tremendous bias in amino acid substitutions in all orthologous ORFs. Analysis of the direction of the amino acid substitutions suggested that three substitutions are important for the stability of theC. efficiensproteins: from lysine to arginine, serine to alanine, and serine to threonine. Our results strongly suggest that the accumulation of these three types of amino acid substitutions correlates with the acquisition of thermostability and is responsible for the greater GC content ofC. efficiens.
Comparative Analysis of Gene-Expression Patterns in Human and African Great Ape Cultured Fibroblasts
Authors: Karaman Mazen W. , Houck Marlys L. , Chemnick Leona G. , Nagpal Shailender , Chawannakul Daniel , Sudano Dominick , Pike Brian L. , Ho Vincent V. , Ryder Oliver A. , Hacia Joseph G.
Published: July 2003

Although much is known about genetic variation in human and African great ape (chimpanzee, bonobo, and gorilla) genomes, substantially less is known about variation in gene-expression profiles within and among these species. This information is necessary for defining transcriptional regulatory networks that contribute to complex phenotypes unique to humans or the African great apes. We took a systematic approach to this problem by investigating gene-expression profiles in well-defined cell populations from humans, bonobos, and gorillas. By comparing these profiles from 18 human and 21 African great ape primary fibroblast cell lines, we found that gene-expression patterns could predict the species, but not the age, of the fibroblast donor. Several differentially expressed genes among human and African great ape fibroblasts involved the extracellular matrix, metabolic pathways, signal transduction, stress responses, as well as inherited overgrowth and neurological disorders. These gene-expression patterns could represent molecular adaptations that influenced the development of species-specific traits in humans and the African great apes.
The Pattern of Polymorphism on Human Chromosome 21
Authors: Innan Hideki , Padhukasahasram Badri , Nordborg Magnus
Published: July 2003

Polymorphism data from 20 partially resequenced copies of human chromosome 21—more than 20,000 polymorphic sites—were analyzed. The allele-frequency distribution shows no deviation from the simplest population genetic model with a constant population size (although we show that our analysis has no power to detect population growth). The average rate of recombination per site is estimated to be roughly one-half of the rate of mutation per site, again in agreement with simple model predictions. However, sliding-window analyses of the amount of polymorphism and the extent of linkage disequilibrium (LD) show significant deviations from standard models. This could be due to the history of selection or demographic change, but it is impossible to draw strong conclusions without much better knowledge of variation in the relationship between genetic and physical distance along the chromosome.
The Zebrafish Annexin Gene Family
Authors: Farber Steven A. , De Rose Robert A. , Olson Eric S. , Halpern Marnie E.
Published: July 2003

The Annexins (ANXs) are a family of calcium- and phospholipid-binding proteins that have been implicated in many cellular processes, including channel formation, membrane fusion, vesicle transport, and regulation of phospholipase A2 activity. As a first step toward understanding in vivo function, we have cloned 11 zebrafish anx genes. Four genes (anx1a, anx2a, anx5,and anx11a) were identified by screening a zebrafish cDNA library with a Xenopus anx2 fragment. For these genes, full-length cDNA sequences were used to cluster 212 EST sequences generated by the Zebrafish Genome Resources Project. The EST analysis revealed seven additional anx genes that were subsequently cloned. The genetic map positions of all 11 genes were determined by using a zebrafish radiation hybrid panel. Sequence and syntenic relationships between zebrafish and human genes indicate that the 11 genes represent orthologs of human anx1,2,4,5,6,11,13,and suggest that several zebrafish anx
genes resulted from duplications that arose after divergence of the zebrafish and mammalian genomes. Zebrafish anx genes are expressed in a wide range of tissues during embryonic and larval stages. Analysis of the expression patterns of duplicated genes revealed both redundancy and divergence, with the most similar genes having almost identical tissue-specific patterns of expression and with less similar duplicates showing no overlap. The differences in gene expression of recently duplicated anx genes could explain why highly related paralogs were maintained in the genome and did not rapidly become pseudogenes.
Customized Molecular Phenotyping by Quantitative Gene Expression and Pattern Recognition Analysis
Authors: Akilesh Shreeram , Shaffer Daniel J. , Roopenian Derry
Published: July 2003

Description of the molecular phenotypes of pathobiological processes in
vivo is a pressing need in genomic biology. We have implemented a
high-throughput real-time PCR strategy to establish quantitative expression
profiles of a customized set of target genes. It enables rapid, reproducible
data acquisition from limited quantities of RNA, permitting serial sampling of
mouse blood during disease progression. We developed an easy to use
statistical algorithm—Global Pattern Recognition—to readily
identify genes whose expression has changed significantly from healthy
baseline profiles. This approach provides unique molecular signatures for
rheumatoid arthritis, systemic lupus erythematosus, and graft versus host
disease, and can also be applied to defining the molecular phenotype of a
variety of other normal and pathological processes.
Genome Analysis of F. nucleatum sub spp vincentii and Its Comparison With the Genome of F. nucleatum ATCC 25586
Authors: Kapatral Vinayak , Ivanova Natalia , Anderson Iain , Reznik Gary , Bhattacharyya Anamitra , Gardner Warren L. , Mikhailova Natalia , Lapidus Alla , Larsen Niels , D'Souza Mark , Walunas Theresa , Haselkorn Robert , Overbeek Ross , Kyrpides Nikos
Published: July 2003

We present the draft genome sequence and its analysis for Fusobacterium nucleatum sub spp. vincentii (FNV), and compare that genome with F. nucleatum ATCC 25586 (FN). A total of 441 FNV open reading frames (ORFs) with no orthologs in FN have been identified. Of these, 118 ORFs have no known function and are unique to FNV, whereas 323 ORFs have functional orthologs in other organisms. In addition to the excretion of butyrate, H2S and ammonia-like FN, FNV has the additional capability to excrete lactate and aminobutyrate. Unlike FN, FNV is likely to incorporate galactopyranose, galacturonate, and sialic acid into its O-antigen. It appears to transport ferrous iron by an anaerobic ferrous transporter. Genes for eukaryotic type serine/threonine kinase and phosphatase, transpeptidase E-transglycosylase Pbp1A are found in FNV but not in FN. Unique ABC transporters, cryptic phages, and three types of restriction-modification systems have been identified in FNV. ORFs for ethanolamine utilization, thermostable carboxypeptidase, γ glutamyl-transpeptidase, and deblocking aminopeptidases are absent from FNV. FNV, like FN, lacks the classical catalase-peroxidase system, but thioredoxin/glutaredoxin enzymes might alleviate oxidative stress. Genes for resistance to antibiotics such as acriflavin, bacitracin, bleomycin, daunorubicin, florfenicol, and other general multidrug resistance are present. These capabilities allow Fusobacteria to survive in a mixed culture in the mouth.
Computational Discovery of Internal Micro-Exons
Authors: Volfovsky Natalia , Haas Brian J. , Salzberg Steven L.
Published: July 2003

Very short exons, also known as micro-exons, occur in large numbers in some eukaryotic genomes. Existing annotation tools have a limited ability to recognize these short sequences, which range in length up to 25 bp. Here, we describe a computational method for the identification of micro-exons using near-perfect alignments between cDNA and genomic DNA sequences. Using this method, we detected 319 micro-exons in 4 complete genomes, of which 224 were previously unknown, human (170), the nematode Caenorhabditis elegans
(4), the fruit fly Drosophila melanogaster (14), and the mustard plant Arabidopsis thaliana (36). Comparison of our computational method with popular cDNA alignment programs shows that the new algorithm is both efficient and accurate. The algorithm also aids in the discovery of micro-exon-skipping events and cross-species micro-exon conservation.
Mitochondrial Genome Variation and Evolutionary History of Australian and New Guinean Aborigines
Authors: Ingman Max , Gyllensten Ulf
Published: July 2003

To study the evolutionary history of the Australian and New Guinean
indigenous peoples, we analyzed 101 complete mitochondrial genomes including
populations from Australia and New Guinea as well as from Africa, India,
Europe, Asia, Melanesia, and Polynesia. The genetic diversity of the
Australian mitochondrial sequences is remarkably high and is similar to that
found across Asia. This is in contrast to the pattern seen in previously
described Y-chromosome data where an Australia-specific haplotype was found at
high frequency. The mitochondrial genome data indicate that Australia was
colonized between 40 and 70 thousand years ago, either by a single migration
from a heterogeneous source population or by multiple movements of smaller
groups occurring over a period of time. Some Australian and New Guinea
sequences form clades, suggesting the possibility of a joint colonization
and/or admixture between the two regions.
Evolutionary Conservation of Regulatory Elements in Vertebrate Hox Gene Clusters
Authors: Santini Simona , Boore Jeffrey L. , Meyer Axel
Published: July 2003

Comparisons of DNA sequences among evolutionarily distantly related genomes permit identification of conserved functional regions in noncoding DNA. Hox genes are highly conserved in vertebrates, occur in clusters, and are uninterrupted by other genes. We aligned (PipMaker) the nucleotide sequences of the HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human, and mouse, which are separated by approximately 500 million years of evolution. In support of our approach, several identified putative regulatory elements known to regulate the expression of Hox
genes were recovered. The majority of the newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac database). The regulatory intergenic regions located between the genes that are expressed most anteriorly in the embryo are longer and apparently more evolutionarily conserved than those at the other end of Hox clusters. Different presumed regulatory sequences are retained in either the Aα or Aβ duplicated Hox clusters in the fish lineages. This suggests that the conserved elements are involved in different gene regulatory networks and supports the duplication-deletion-complementation model of functional divergence of duplicated genes.
Large-Scale Identification and Analysis of Genome-Wide Single-Nucleotide Polymorphisms for Mapping in Arabidopsis thaliana
Authors: Schmid Karl J. , Sörensen Thomas Rosleff , Stracke Ralf , Törjék Ottó , Altmann Thomas , Mitchell-Olds Tom , Weisshaar Bernd
Published: July 2003

Genetic markers such as single nucleotide polymorphisms (SNPs) are essential tools for positional cloning, association, or quantitative trait locus mapping and the determination of genetic relationships between individuals. We identified and characterized a genome-wide set of SNP markers by generating 10,706 expressed sequence tags (ESTs) from cDNA libraries derived from 6 different accessions, and by analysis of 606 sequence tagged sites (STS) from up to 12 accessions of the model flowering plant Arabidopsis thaliana. The cDNA libraries for EST sequencing were made from individuals that were stressed by various means to enrich for transcripts from genes expressed under such conditions. SNPs discovered in these sequences may be useful markers for mapping genes involved in interactions with the biotic and abiotic environment. The STS loci are distributed randomly over the genome. By comparison with the Col-0 genome sequence, we identified a total of 8051 SNPs and 637 insertion/deletion polymorphisms (InDel). Analysis of STS-derived SNPs shows that most SNPs are rare, but that it is possible to identify intermediate frequency framework markers that can be used for genetic mapping in many different combinations of accessions. A substantial proportion of SNPs located in ORFs caused a change of the encoded amino acid. A comparison of the density of our SNP markers among accessions in both the EST and STS datasets, revealed that Cvi-0 is the most divergent accession from Col-0 among the 12 accessions studied. All of these markers are freely available via the internet.
Internet Contig Explorer (iCE)—A Tool for Visualizing Clone Fingerprint Maps
Authors: Fjell Christopher D. , Bosdet Ian , Schein Jacqueline E. , Jones Steven J.M. , Marra Marco A.
Published: July 2003

Fingerprinted clone physical maps have proven useful in various applications, supporting both whole-genome and region-specific DNA sequencing as well as gene cloning studies. Fingerprint maps have been generated for several genomes, including those of human, mouse, rat, the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, Arabidopsis thaliana and rice. Fingerprint maps of other genomes, including those of fungi, bacteria, poplar, and the cow, are being generated. The increasing use of fingerprint maps in genomic research has spawned a need in the research community for intuitive computer tools that facilitate viewing of the maps and the underlying fingerprint data. In this report we describe a new Java-based application called iCE (Internet Contig Explorer) that has been designed to provide views of fingerprint maps and associated data. Users can search for and display individual clones, contigs, clone fingerprints, clone insert sizes and markers. Users can also load into the software lists of particular clones of interest and view their fingerprints. iCE is being used at our Genome Centre to offer up to the research community views of the mouse, rat, bovine, C. briggsae, and several fungal genome bacterial artificial chromosome (BAC) fingerprint maps we have either completed or are currently constructing. We are also using iCE as part of the Rat Genome Sequencing Project to manage our provision of rat BAC clones for sequencing at the Human Genome Sequencing Center at the Baylor College of Medicine.
Kinase Pathway Database: An Integrated Protein-Kinase and NLP-Based Protein-Interaction Resource
Authors: Koike Asako , Kobayashi Yoshiyuki , Takagi Toshihisa
Published: July 2003

Protein kinases play a crucial role in the regulation of cellular functions. Various kinds of information about these molecules are important for understanding signaling pathways and organism characteristics. We have developed the Kinase Pathway Database, an integrated database involving major completely sequenced eukaryotes. It contains the classification of protein kinases and their functional conservation, ortholog tables among species, protein–protein, protein–gene, and protein–compound interaction data, domain information, and structural information. It also provides an automatic pathway graphic image interface. The protein, gene, and compound interactions are automatically extracted from abstracts for all genes and proteins by natural-language processing (NLP).The method of automatic extraction uses phrase patterns and the GENA protein, gene, and compound name dictionary, which was developed by our group. With this database, pathways are easily compared among species using data with more than 47,000 protein interactions and protein kinase ortholog tables. The database is available for querying and browsing athttp://kinasedb.ontology.ims.u-tokyo.ac.jp/.