bluuebunny/crossref_metadata_2025 huggingface dataset:
The Major Histocompatibility Complex–related Fc Receptor for IgG (FcRn) Binds Albumin and Prolongs Its Lifespan
Authors: Chaudhury Chaity , Mehnaz Samina , Robinson John M. , Hayton William L. , Pearl Dennis K. , Roopenian Derry C. , Anderson Clark L.
Published: February 2003

The inverse relationship between serum albumin concentration and its half-life suggested to early workers that albumin would be protected from a catabolic fate by a receptor-mediated mechanism much like that proposed for IgG. We show here that albumin binds FcRn in a pH dependent fashion, that the lifespan of albumin is shortened in FcRn-deficient mice, and that the plasma albumin concentration of FcRn-deficient mice is less than half that of wild-type mice. These results affirm the hypothesis that the major histocompatibility complex–related Fc receptor protects albumin from degradation just as it does IgG, prolonging the half-lives of both.
Second Class Minors
Authors: Sahara Hiroeki , Shastri Nilabh
Published: February 2003

CD4 T cells regulate immune responses that cause chronic graft rejection and graft versus host disease but their target antigens remain virtually unknown. We developed a new method to identify CD4 T cell–stimulating antigens. LacZ-inducible CD4 T cells were used as a probe to detect their cognate peptide/MHC II ligand generated in dendritic cells fed with Escherichia coli expressing a library of target cell genes. The murine H46 locus on chromosome 7 was thus found to encode the interleukin 4–induced IL4i1 gene. The IL4i1 precursor contains the HAFVEAIPELQGHV peptide which is presented by Ab major histocompatibility complex class II molecule via an endogenous pathway in professional antigen presenting cells. Both allelic peptides bind Ab and a single alanine to methionine substitution at p2 defines nonself. These results reveal novel features of H loci that regulate CD4 T cell responses as well as provide a general strategy for identifying elusive antigens that elicit CD4 T cell responses to tumors or self-tissues in autoimmunity.
Molecular Action of Lidocaine on the Voltage Sensors of Sodium Channels
Authors: Sheets Michael F. , Hanck Dorothy A.
Published: February 2003

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Qmax) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel–gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Qmax of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaine's most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near −100 mV. These findings suggest that lidocaine's actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.
Titin Determines the Frank-Starling Relation in Early Diastole
Authors: Helmes Michiel , Lim Chee Chew , Liao Ronglih , Bharti Ajit , Cui Lei , Sawyer Douglas B.
Published: February 2003

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF–SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.
Inactivation of BK Channels by the NH2 Terminus of the β2 Auxiliary Subunit: An Essential Role of a Terminal Peptide Segment of Three Hydrophobic Residues
Authors: Xia Xiao-Ming , Ding J.P. , Lingle Christopher J.
Published: February 2003

An auxiliary β2 subunit, when coexpressed with Slo α subunits, produces inactivation of the resulting large-conductance, Ca2+ and voltage-dependent K+ (BK-type) channels. Inactivation is mediated by the cytosolic NH2 terminus of the β2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH2 terminus in the inactivation process. The β2 NH2 terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2–4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH2-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process.
Involvement of Helices at the Dimer Interface in ClC-1 Common Gating
Authors: Duffield Michael , Rychkov Grigori , Bretag Allan , Roberts Michael
Published: February 2003

ClC-1 is a dimeric, double-pored chloride channel that is present in skeletal muscle. Mutations of this channel can result in the condition myotonia, a muscle disorder involving increased muscle stiffness. It has been shown that the dominant form of myotonia often results from mutations that affect the so-called slow, or common, gating process of the ClC-1 channel. Mutations causing dominant myotonia are seen to cluster at the interface of the ClC-1 channel monomers. This study has investigated the role of the H, I, P, and Q helices, which lie on this interface, as well as the G helix, which is situated immediately behind the H and I helices, on ClC-1 gating. 11 mutant ClC-1 channels (T268M, C277S, C278S, S289A, T310M, S312A, V321S, T539A, S541A, M559T, and S572V) were produced using site-directed mutagenesis, and gating properties of these channels were investigated using electrophysiological techniques. Six of the seven mutations in G, H, and I, and two of the four mutations in P and Q, caused shifts of the ClC-1 open probability. In the majority of cases this was due to alterations in the common gating process, with only three of the mutants displaying any change in fast gating. Many of the mutant channels also showed alterations in the kinetics of the common gating process, particularly at positive potentials. The changes observed in common gating were caused by changes in the opening rate (e.g. T310M), the closing rate (e.g. C277S), or both rates. These results indicate that mutations in the helices forming the dimer interface are able to alter the ClC-1 common gating process by changing the energy of the open and/or closed channel states, and hence altering transition rates between these states.
Influence of Pore Residues on Permeation Properties in the Kv2.1 Potassium Channel. Evidence for a Selective Functional Interaction of K+ with the Outer Vestibule
Authors: Consiglio Joseph F. , Andalib Payam , Korn Stephen J.
Published: February 2003

The Kv2.1 potassium channel contains a lysine in the outer vestibule (position 356) that markedly reduces open channel sensitivity to changes in external [K+]. To investigate the mechanism underlying this effect, we examined the influence of this outer vestibule lysine on three measures of K+ and Na+ permeation. Permeability ratio measurements, measurements of the lowest [K+] required for interaction with the selectivity filter, and measurements of macroscopic K+ and Na+ conductance, were all consistent with the same conclusion: that the outer vestibule lysine in Kv2.1 interferes with the ability of K+ to enter or exit the extracellular side of the selectivity filter. In contrast to its influence on K+ permeation properties, Lys 356 appeared to be without effect on Na+ permeation. This suggests that Lys 356 limited K+ flux by interfering with a selective K+ binding site. Combined with permeation studies, results from additional mutagenesis near the external entrance to the selectivity filter indicated that this site was located external to, and independent from, the selectivity filter. Protonation of a naturally occurring histidine in the same outer vestibule location in the Kv1.5 potassium channel produced similar effects on K+ permeation properties. Together, these results indicate that a selective, functional K+ binding site (e.g., local energy minimum) exists in the outer vestibule of voltage-gated K+ channels. We suggest that this site is the location of K+ hydration/dehydration postulated to exist based on the structural studies of KcsA. Finally, neutralization of position 356 enhanced outward K+ current magnitude, but did not influence the ability of internal K+ to enter the pore. These data indicate that in Kv2.1, exit of K+ from the selectivity filter, rather than entry of internal K+ into the channel, limits outward current magnitude. We discuss the implications of these findings in relation to the structural basis of channel conductance in different K+ channels.
Potential theory on Lipschitz domains in Riemannian manifolds: The case of Dini metric tensors
Authors: Mitrea Marius , Taylor Michael
Published: February 2003

We study the applicability of the method of layer potentials in the treatment of boundary value problems for the Laplace-Beltrami operator on Lipschitz sub-domains of Riemannian manifolds, in the case when the metric tensor

g

j
k

d

x
j

⊗
d

x
k

g_{jk} dx_j\otimes dx_k

has low regularity. Under the assumption that
\[
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="StartAbsoluteValue g Subscript j k Baseline left-parenthesis x right-parenthesis minus g Subscript j k Baseline left-parenthesis y right-parenthesis EndAbsoluteValue less-than-or-equal-to upper C omega left-parenthesis StartAbsoluteValue x minus y EndAbsoluteValue right-parenthesis comma">

|

g

j
k

(
x
)
−

g

j
k

(
y
)

|

≤
C

ω
(

|

x
−
y

|

)
,

|g_{jk}(x)-g_{jk}(y)|\leq C\,\omega (|x-y|),

\]
where the modulus of continuity

ω
\omega

satisfies a Dini-type condition, we prove the well-posedness of the classical Dirichlet and Neumann problems with

L
p

L^p

boundary data, for sharp ranges of

p
p

’s and with optimal nontangential maximal function estimates.
Oscillation and variation for singular integrals in higher dimensions
Authors: Campbell James , Jones Roger , Reinhold Karin , Wierdl Máté
Published: February 2003

In this paper we continue our investigations of square function inequalities in harmonic analysis. Here we investigate oscillation and variation inequalities for singular integral operators in dimensions

d
≥
1

d \geq 1

. Our estimates give quantitative information on the speed of convergence of truncations of a singular integral operator, including upcrossing and

λ
\lambda

jump inequalities.
A black-box group algorithm for recognizing finite symmetric and alternating groups, I
Authors: Beals Robert , Leedham-Green Charles , Niemeyer Alice , Praeger Cheryl , Seress Ákos
Published: February 2003

We present a Las Vegas algorithm which, for a given black-box group known to be isomorphic to a symmetric or alternating group, produces an explicit isomorphism with the standard permutation representation of the group. This algorithm has applications in computations with matrix groups and permutation groups. In this paper, we handle the case when the degreennof the standard permutation representation is part of the input. In a sequel, we shall treat the case when the value ofnnis not known in advance. As an important ingredient in the theoretical basis for the algorithm, we prove the following result about the orders of elements ofSnS_n: the conditional probability that a random elementσ∈Sn\sigma \in S_nis annn-cycle, given thatσn=1\sigma ^n=1, is at least1/101/10.
Levi’s parametrix for some sub-elliptic non-divergence form operators
Authors: Bonfiglioli Andrea , Lanconelli Ermanno , Uguzzoni Francesco
Published: January 2003

We construct the fundamental solutions for the sub-elliptic operators in non-divergence form∑i,jai,j(x,t)XiXj−∂t{\textstyle \sum _{i,j}} a_{i,j}(x,t)\,X_iX_j-\partial _tand∑i,jai,j(x)XiXj{\textstyle \sum _{i,j}}a_{i,j}(x)\,X_iX_j, where theXiX_i’s form a stratified system of Hörmander vector fields andai,ja_{i,j}are Hölder continuous functions belonging to a suitable class of ellipticity.
A 𝐶⁰-theory for the blow-up of second order elliptic equations of critical Sobolev growth
Authors: Druet Olivier , Hebey Emmanuel , Robert Frédéric
Published: February 2003

Let(M,g)(M,g)be a smooth compact Riemannian manifold of dimensionn≥3n \ge 3, andΔg=−divg∇\Delta _g = -div_g\nablathe Laplace-Beltrami operator. Also let2⋆2^\starbe the critical Sobolev exponent for the embedding of the Sobolev spaceH12(M)H_1^2(M)into Lebesgue spaces, andhha smooth function onMM. Elliptic equations of critical Sobolev growth like\[Δgu+hu=u2⋆−1\Delta _gu + hu = u^{2^\star -1}\]have been the target of investigation for decades. A very niceH12H_1^2-theory for the asymptotic behaviour of solutions of such an equation is available since the 1980’s. In this announcement we present theC0C^0-theory we have recently developed. Such a theory provides sharp pointwise estimates for the asymptotic behaviour of solutions of the above equation.
Feedback control of mammalian Hedgehog signaling by the Hedgehog-binding protein, Hip1, modulates Fgf signaling during branching morphogenesis of the lung
Authors: Chuang Pao-Tien , Kawcak T'Nay , McMahon Andrew P.
Published: February 2003

Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue is how negative regulation of Hh signaling might contribute to generating differential responses over tens of cell diameters. In cells that respond to Hh, two proteins that are up-regulated are Patched1 (Ptch1), the Hh receptor, a general target in both invertebrate and vertebrate organisms, and Hip1, a Hh-binding protein that is vertebrate specific. To address the developmental role of Hip1 in the context of Hh signaling, we generatedHip1mutants in the mouse. Loss ofHip1function results in specific defects in two Hh target issues, the lung, a target of Sonic hedgehog (Shh) signaling, and the endochondral skeleton, a target of Indian hedgehog (Ihh) signaling. Hh signaling was up-regulated inHip1mutants, substantiating Hip1's general role in negatively regulating Hh signaling. Our studies focused on Hip1 in the lung. Here, a dynamic interaction between Hh and fibroblast growth factor (Fgf) signaling, modulated at least in part by Hip1, controls early lung branching.
EctodermalWnt3/β-cateninsignaling is required for the establishment and maintenance of the apical ectodermal ridge
Authors: Barrow Jeffery R. , Thomas Kirk R. , Boussadia-Zahui Oreda , Moore Robert , Kemler Rolf , Capecchi Mario R. , McMahon Andrew P.
Published: February 2003

The formation of the apical ectodermal ridge (AER) is critical for the distal outgrowth and patterning of the vertebrate limb. Recent work in the chick has demonstrated that interplay between theWntandFgfsignaling pathways is essential in the limb mesenchyme and ectoderm in the establishment and perhaps the maintenance of the AER. In the mouse, whereas a role forFgfsfor AER establishment and function has been clearly demonstrated, the role ofWnt/β-cateninsignaling, although known to be important, is obscure. In this study, we demonstrate thatWnt3, which is expressed ubiquitously throughout the limb ectoderm, is essential for normal limb development and plays a critical role in the establishment of the AER. We also show that the conditional removal ofβ-cateninin the ventral ectodermal cells is sufficient to elicit the mutant limb phenotype. In addition, removingβ-cateninafter the induction of the ridge results in the disappearance of the AER, demonstrating the requirement for continuedβ-cateninsignaling for the maintenance of this structure. Finally, we demonstrate thatWnt/β-cateninsignaling lies upstream of theBmpsignaling pathway in establishment of the AER and regulation of the dorsoventral polarity of the limb.
Systematic Discovery of New Genes in the Saccharomyces cerevisiae Genome
Authors: Kessler Marco M. , Zeng Qiandong , Hogan Sarah , Cook Robin , Morales Arturo J. , Cottarel Guillaume
Published: February 2003

We used genome-wide comparative analysis of predicted protein sequences to identify many novel small genes, named smORFs forsmall open readingframes, within the budding yeast genome. Further analysis of 117 of these new genes showed that 84 are transcribed. We extended our analysis of one smORF conserved from yeast to human. This investigation provides an updated and comprehensive annotation of the yeast genome, validates additional concepts in the study of genomes in silico, and increases the expected numbers of coding sequences in a genome with the corresponding impact on future functional genomics and proteomics studies.
Genome-Scale Reconstruction of the Saccharomyces cerevisiae Metabolic Network
Authors: Förster Jochen , Famili Iman , Fu Patrick , Palsson Bernhard Ø. , Nielsen Jens
Published: February 2003

The metabolic network in the yeast Saccharomyces cerevisiaewas reconstructed using currently available genomic, biochemical, and physiological information. The metabolic reactions were compartmentalized between the cytosol and the mitochondria, and transport steps between the compartments and the environment were included. A total of 708 structural open reading frames (ORFs) were accounted for in the reconstructed network, corresponding to 1035 metabolic reactions. Further, 140 reactions were included on the basis of biochemical evidence resulting in a genome-scale reconstructed metabolic network containing 1175 metabolic reactions and 584 metabolites. The number of gene functions included in the reconstructed network corresponds to ∼16% of all characterized ORFs in S. cerevisiae. Using the reconstructed network, the metabolic capabilities of S. cerevisiae were calculated and compared with Escherichia coli. The reconstructed metabolic network is the first comprehensive network for a eukaryotic organism, and it may be used as the basis for in silico analysis of phenotypic functions.

[Supplemental material is available online at www.genome.org. The detailed genome-scale reconstructed model ofSaccharomyces cerevisiae can be found athttp://www.cpb.dtu.dk/models/yeastmodel.html orhttp://geneticcircuits.ucsd.edu/organisms/yeast.html.]
Linkage Disequilibrium and Haplotype Diversity in the Genes of the Renin–Angiotensin System: Findings From the Family Blood Pressure Program
Authors: Zhu Xiaofeng , Yan Denise , Cooper Richard S. , Luke Amy , Ikeda Morna A. , Chang Yen-Pei C. , Weder Alan , Chakravarti Aravinda
Published: February 2003

Association studies of candidate genes with complex traits have generally used one or a few single nucleotide polymorphisms (SNPs), although variation in the extent of linkage disequilibrium (LD) within genes markedly influences the sensitivity and precision of association studies. The extent of LD and the underlying haplotype structure for most candidate genes are still unavailable. We sampled 193 blacks (African-Americans) and 160 whites (European-Americans) and estimated the intragenic LD and the haplotype structure in four genes of the renin–angiotensin system. We genotyped 25 SNPs, with all but one of the pairs spaced between 1 and 20 kb, thus providing resolution at small scale. The pattern of LD within a gene was very heterogeneous. Using a robust method to define haplotype blocks, blocks of limited haplotype diversity were identified at each locus; between these blocks, LD was lost owing to the history of recombination events. As anticipated, there was less LD among blacks, the number of haplotypes was substantially larger, and shorter haplotype segments were found, compared with whites. These findings have implications for candidate-gene association studies and indicate that variation between populations of European and African origin in haplotype diversity is characteristic of most genes.

[The sequence data described in this paper are available in GenBank under the following accession nos: AGT, MIM 106150; Renin, MIM 179820;ACE, MIM 106180; Angiotensin receptor I, MIM 106165. Supplementary material is available online athttp://www.genome.org.]
A Complexity Reduction Algorithm for Analysis and Annotation of Large Genomic Sequences
Authors: Chuang Trees-Juen , Lin Wen-Chang , Lee Hurng-Chun , Wang Chi-Wei , Hsiao Keh-Lin , Wang Zi-Hao , Shieh Danny , Lin Simon C. , Ch'ang Lan-Yang
Published: February 2003

DNA is a universal language encrypted with biological instruction for life. In higher organisms, the genetic information is preserved predominantly in an organized exon/intron structure. When a gene is expressed, the exons are spliced together to form the transcript for protein synthesis. We have developed a complexity reduction algorithm for sequence analysis (CRASA) that enables direct alignment of cDNA sequences to the genome. This method features a progressive data structure in hierarchical orders to facilitate a fast and efficient search mechanism. CRASA implementation was tested with already annotated genomic sequences in two benchmark data sets and compared with 15 annotation programs (10 ab initio and 5 homology-based approaches) against the EST database. By the use of layered noise filters, the complexity of CRASA-matched data was reduced exponentially. The results from the benchmark tests showed that CRASA annotation excelled in both the sensitivity and specificity categories. When CRASA was applied to the analysis of human Chromosomes 21 and 22, an additional 83 potential genes were identified. With its large-scale processing capability, CRASA can be used as a robust tool for genome annotation with high accuracy by matching the EST sequences precisely to the genomic sequences.

[Supplementary material is available online at http://www.genome.organdhttp://crasa.sinica.edu.tw/bioinformatics/Supplementary.htm.]
Evolutionary Implications of Microbial Genome Tetranucleotide Frequency Biases
Authors: Pride David T. , Meinersmann Richard J. , Wassenaar Trudy M. , Blaser Martin J.
Published: February 2003

We compared nucleotide usage pattern conservation for related prokaryotes by examining the representation of DNA tetranucleotide combinations in 27 representative microbial genomes. For each of the organisms studied, tetranucleotide usage departures from expectations (TUD) were shared between related organisms using both Markov chain analysis and a zero-order Markov method. Individual strains, multiple chromosomes, plasmids, and bacteriophages share TUDs within a species. TUDs varied between coding and noncoding DNA. Grouping prokaryotes based on TUD profiles resulted in relationships with important differences from those based on 16S rRNA phylogenies, which may reflect unequal rates of evolution of nucleotide usage patterns following divergence of particular organisms from a common ancestor. By both symmetrical tree distance and likelihood analysis, phylogenetic trees based on TUD profiles demonstrate a level of congruence with 16S rRNA trees similar to that of both RpoA and RecA trees. Congruence of these trees indicates that there exists phylogenetic signal in TUD patterns, most prominent in coding region DNA. Because relationships demonstrated in TUD-based analyses utilize whole genomes, they should be considered complementary to phylogenies based on single genetic elements, such as 16S rRNA.
Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array–CGH
Authors: Lage José M. , Leamon John H. , Pejovic Tanja , Hamann Stefan , Lacey Michelle , Dillon Deborah , Segraves Richard , Vossbrinck Bettina , González Antonio , Pinkel Daniel , Albertson Donna G. , Costa Jose , Lizardi Paul M.
Published: February 2003

Structural genetic alterations in cancer often involve gene loss or gene amplification. With the advent of microarray approaches for the analysis of the genome, as exemplified by array–CGH (Comparative GenomicHybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density. However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array–CGH analysis. We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples. We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias. The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours. Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines. Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples. Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material.
Complex Events in the Evolution of the Human Pseudoautosomal Region 2 (PAR2)
Authors: Charchar Fadi J. , Svartman Marta , El-Mogharbel Nisrine , Ventura Mario , Kirby Patrick , Matarazzo Maria R. , Ciccodicola Alfredo , Rocchi Mariano , D'Esposito Maurizio , Graves Jennifer A. Marshall
Published: February 2003

The 320-kb human pseudoautosomal region 2 (PAR2) at the tips of the long arms of the X and Y chromosomes is thought to have been duplicated onto the Y chromosome recently in primate evolution. The four genes within PAR2 have been proposed to constitute two zones with different base ratios and transcription, one of which was added recently to the X chromosome. To test this hypothesis, we cloned and mapped PAR2 genes in other species, the lemur, the cat, and a marsupial, the tammar wallaby. None of the human PAR2 genes colocalized with human PAR1 genes in the marsupial genome, confirming that the human PAR1 and PAR2 evolved independently. Of the four PAR2 genes, only SYBL1 was located on the X chromosome in all species, including marsupials, so it was part of the ancient X. HSPRY3 localized to the X in all the eutherians, but not marsupial, so it must have been added to the X 80–130 million years ago. CXYorf1 was present on the X in primates and also in mouse, but autosomal in wallaby, suggesting a later addition 70–130 million years ago, and IL9R was on the X only in primate, suggesting addition 60–70 million years ago. The results therefore demonstrate that at least two independent additions were necessary for PAR2 evolution. The present gene order on the human X also requires two inversions. The complicated evolutionary pathway supports the hypothesis that terminal interchromosomal rearrangements are common in regions unpaired at meiosis.

[The sequence data from this study have been submitted to GenBank under accession nos. AF544202, AF544203, AF544204, and AF544205.]
Polymorphism Ratio Sequencing: A New Approach for Single Nucleotide Polymorphism Discovery and Genotyping
Authors: Blazej Robert G. , Paegel Brian M. , Mathies Richard A.
Published: February 2003

Polymorphism ratio sequencing (PRS) combines the advantages of high-throughput DNA sequencing with new labeling and pooling schemes to produce a powerful assay for sensitive single nucleotide polymorphism (SNP) discovery, rapid genotyping, and accurate, multiplexed allele frequency determination. In the PRS method, dideoxy-terminator extension ladders generated from a sample and reference template are labeled with different energy-transfer fluorescent dyes and coinjected into a separation capillary for comparison of relative signal intensities. We demonstrate the PRS method by screening two human mitochondrial genomes for sequence variations using a microfabricated capillary array electrophoresis device. A titration of multiplexed DNA samples places the limit of minor allele frequency detection at 5%. PRS is a sensitive and robust polymorphism detection method for the analysis of individual or multiplexed samples that is compatible with any four-color fluorescence DNA sequencer.
A Microarray-Based Antibiotic Screen Identifies a Regulatory Role for Supercoiling in the Osmotic Stress Response of Escherichia coli
Authors: Cheung Kevin J. , Badarinarayana Vasudeo , Selinger Douglas W. , Janse Daniel , Church George M.
Published: February 2003

Changes in DNA supercoiling are induced by a wide range of environmental stresses in Escherichia coli, but the physiological significance of these responses remains unclear. We now demonstrate that an increase in negative supercoiling is necessary for transcriptional activation of a large subset of osmotic stress-response genes. Using a microarray-based approach, we have characterized supercoiling-dependent gene transcription by expression profiling under conditions of high salt, in conjunction with the microbial antibiotics novobiocin, pefloxacin, and chloramphenicol. Algorithmic clustering and statistical measures for gauging cellular function show that this subset is enriched for genes critical in osmoprotectant transport/synthesis and rpoS-driven stationary phase adaptation. Transcription factor binding site analysis also supports regulation by the global stress ς factor rpoS. In addition, these studies implicate 60 uncharacterized genes in the osmotic stress regulon, and offer evidence for a broader role for supercoiling in the control of stress-induced transcription.
Coexpression of Neighboring Genes in Caenorhabditis Elegans Is Mostly Due to Operons and Duplicate Genes
Authors: Lercher Martin J. , Blumenthal Thomas , Hurst Laurence D.
Published: February 2003

In many eukaryotic species, gene order is not random. In humans, flies, and yeast, there is clustering of coexpressed genes that cannot be explained as a trivial consequence of tandem duplication. In the worm genome this is taken a step further with many genes being organized into operons. Here we analyze the relationship between gene location and expression in Caenorhabditis elegans and find evidence for at least three different processes resulting in local expression similarity. Not surprisingly, the strongest effect comes from genes organized in operons. However, coexpression within operons is not perfect, and is influenced by some distance-dependent regulation. Beyond operons, there is a relationship between physical distance, expression similarity, and sequence similarity, acting over several megabases. This is consistent with a model of tandem duplicate genes diverging over time in sequence and expression pattern, while moving apart owing to chromosomal rearrangements. However, at a very local level, nonduplicate genes on opposite strands (hence not in operons) show similar expression patterns. This suggests that such genes may share regulatory elements or be regulated at the level of chromatin structure. The central importance of tandem duplicate genes in these patterns renders the worm genome different from both yeast and human.

[Supplemental material is available online at http://www.genome.org.]
Comparative Genome Analysis of the Primary Sex-Determining Locus in Salmonid Fishes
Authors: Woram Rachael A. , Gharbi Karim , Sakamoto Takashi , Hoyheim Bjorn , Holm Lars-Erik , Naish Kerry , McGowan Colin , Ferguson Moira M. , Phillips Ruth B. , Stein Jake , Guyomard René , Cairney Margaret , Taggart John B. , Powell Richard , Davidson William , Danzmann Roy G.
Published: February 2003

We compared the Y-chromosome linkage maps for four salmonid species (Arctic charr, Salvelinus alpinus; Atlantic salmon, Salmo salar; brown trout, Salmo trutta; and rainbow trout,Oncorhynchus mykiss) and a putative Y-linked marker from lake trout (Salvelinus namaycush). These species represent the three major genera within the subfamily Salmoninae of the Salmonidae. The data clearly demonstrate that different Y-chromosomes have evolved in each of the species. Arrangements of markers proximal to the sex-determining locus are preserved on homologous, but different, autosomal linkage groups across the four species studied in detail. This indicates that a small region of DNA has been involved in the rearrangement of the sex-determining region. Placement of the sex-determining region appears telomeric in brown trout, Atlantic salmon, and Arctic charr, whereas an intercalary location forSEX may exist in rainbow trout. Three hypotheses are proposed to account for the relocation: translocation of a small chromosome arm; transposition of the sex-determining gene; or differential activation of a primary sex-determining gene region among the species.
Centromere Satellites From Arabidopsis Populations: Maintenance of Conserved and Variable Domains
Authors: Hall Sarah E. , Kettler Gregory , Preuss Daphne
Published: February 2003

The rapid evolution of centromere sequences between species has led to a debate over whether centromere activity is sequence-dependent. TheArabidopsis thaliana centromere regions contain ∼20,000 copies of a 178-bp satellite repeat. Here, we analyzed satellites from 41 Arabidopsis ecotypes, providing the first broad population survey of satellite variation within a species. We found highly conserved segments and consistent sequence lengths in theArabidopsis satellites and in the published collection of human α-satellites, supporting models for a functional role. Despite this conservation, polymorphisms are significantly enriched at some sites, yielding variation that could restrict binding proteins to a subset of repeat monomers. Some satellite regions vary considerably; at certain bases, consensus sequences derived from each ecotype diverge significantly from the Arabidopsis consensus, indicating substitutions sweep through a genome in less than 5 million years. Such rapid changes generate more variation within the set ofArabidopsis satellites than in genes from the chromosome arms or from the recombinationally suppressed centromere regions. These studies highlight a balance between the mechanisms that maintain particular satellite domains and the forces that disperse sequence changes throughout the satellite repeats in the genome.

[Supplemental material is available online atwww.genome.org.]
Transcriptional Interactions Between Yeast tRNA Genes, Flanking Genes and Ty Elements: A Genomic Point of View
Authors: Bolton Eric C. , Boeke Jef D.
Published: February 2003

Retroelement insertion can alter the expression of nearby genes. TheSaccharomyces cerevisiae retrotransposons Ty1–Ty4 are transcribed by RNA polymerase II (pol II) and target their integration upstream of genes transcribed by RNA polymerase III (pol III), mainly tRNA genes. Because tRNA genes can repress nearby pol II-transcribed genes, we hypothesized that transcriptional interference may exist between Ty1 insertions and pol III-transcribed genes, the preferred targets for Ty1 integration. Ty1s upstream of two pol III-transcribed genes (SNR6 and SUP2) were recovered and analyzed by RNA blot analysis. Ty1 insertions were found to exert a neutral or modest stimulatory effect on the expression of these genes. Further RNA analysis indicated a modest tRNA position effect on Ty1 transcription. To investigate the possible genomic relevance of these expression effects, we compiled a comprehensive tRNA gene database. This database allowed us to analyze a genome's worth of tRNA genes and Ty elements. It also enabled the prediction and experimental confirmation of tRNA gene position effects at native chromosomal loci. We provide evidence supporting the hypothesis that tRNA genes exert a modest inhibitory effect on adjacent pol II promoters. Direct analysis of PTR3transcription, promoted by sequences very close to a tRNA gene, shows that this tRNA position effect can operate on a native chromosomal gene.

[The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: S. Sandmeyer and J. McCusker.]
Genomic Sequence and Transcriptional Profile of the Boundary Between Pericentromeric Satellites and Genes on Human Chromosome Arm 10p
Authors: Guy Jane , Hearn Tom , Crosier Moira , Mudge Jonathan , Viggiano Luigi , Koczan Dirk , Thiesen Hans-Jurgen , Bailey Jeffrey A. , Horvath Julie E. , Eichler Evan E. , Earthrowl Mark E. , Deloukas Panos , French Lisa , Rogers Jane , Bentley David , Jackson Michael S.
Published: February 2003

Contiguous finished sequence from highly duplicated pericentromeric regions of human chromosomes is needed if we are to understand the role of pericentromeric instability in disease, and in gene and karyotype evolution. Here, we have constructed a BAC contig spanning the transition from pericentromeric satellites to genes on the short arm of human chromosome 10, and used this to generate 1.4 Mb of finished genomic sequence. Combining RT-PCR, in silico gene prediction, and paralogy analysis, we can identify two domains within the sequence. The proximal 600 kb consists of satellite-rich pericentromerically duplicated DNA which is transcript poor, containing only three unspliced transcripts. In contrast, the distal 850 kb contains four known genes (ZNF248, ZNF25, ZNF33A, andZNF37A) and up to 32 additional transcripts of unknown function. This distal region also contains seven out of the eight intrachromosomal duplications within the sequence, including the p arm copy of the ∼250-kb duplication which gave rise to ZNF33Aand ZNF33B. By sequencing orthologs of the duplicatedZNF33 genes we have established that ZNF33A has diverged significantly at residues critical for DNA binding butZNF33B has not, indicating that ZNF33B has remained constrained by selection for ancestral gene function. These results provide further evidence of gene formation within intrachromosomal duplications, but indicate that recent interchromosomal duplications at this centromere have involved transcriptionally inert, satellite rich DNA, which is likely to be heterochromatic. This suggests that any novel gene structures formed by these interchromosomal events would require relocation to a more open chromatin environment to be expressed.

[Supplemental material is available online atwww.genome.org and also at http://www.ncl.ac.uk/ihg/10p11.htm. The sequence data from this study have been submitted to EMBL under accession nos. AL391686, AL161931, AL133350, AL121927, AL132657,AL135791, AL132659, AL117337, AL117339, AL132658, AL133217,AL133216, AJ245587, AJ245588, AJ251655, AJ275023–AJ275036,AJ250940–AJ250950, AJ275024–AJ275036, AJ492195, AJ492196,AJ491691–AJ491697. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: W. Amos.]
Sequence Analysis of a Functional Drosophila Centromere
Authors: Sun Xiaoping , Le Hiep D. , Wahlstrom Janice M. , Karpen Gary H.
Published: February 2003

Centromeres are the site for kinetochore formation and spindle attachment and are embedded in heterochromatin in most eukaryotes. The repeat-rich nature of heterochromatin has hindered obtaining a detailed understanding of the composition and organization of heterochromatic and centromeric DNA sequences. Here, we report the results of extensive sequence analysis of a fully functional centromere present in theDrosophila Dp1187 minichromosome. Approximately 8.4% (31 kb) of the highly repeated satellite DNA (AATAT and TTCTC) was sequenced, representing the largest data set of Drosophilasatellite DNA sequence to date. Sequence analysis revealed that the orientation of the arrays is uniform and that individual repeats within the arrays mostly differ by rare, single-base polymorphisms. The entire complex DNA component of this centromere (69.7 kb) was sequenced and assembled. The 39-kb “complex island” Maupiti contains long stretches of a complex A+T rich repeat interspersed with transposon fragments, and most of these elements are organized as direct repeats. Surprisingly, five single, intact transposons are directly inserted at different locations in the AATAT satellite arrays. We find no evidence for centromere-specific sequences within this centromere, providing further evidence for sequence-independent, epigenetic determination of centromere identity and function in higher eukaryotes. Our results also demonstrate that the sequence composition and organization of large regions of centric heterochromatin can be determined, despite the presence of repeated DNA.

[Supplemental material is available online atwww.genome.org. The sequence data from this study have been submitted to GenBank under accession nos.:Beagle = AY183918, F = AY183919,412 = AY183920, Bel = AY183921, You = AY183922, Maupiti = AY183926, AATAT = AY183925, AY183931–AY184007, and TTCTC = AY183923–AY183924, AY183927–AY183930,AY184008–AY184069].
A Recent Polyploidy Superimposed on Older Large-Scale Duplications in the Arabidopsis Genome
Authors: Blanc Guillaume , Hokamp Karsten , Wolfe Kenneth H.
Published: February 2003

The Arabidopsis genome contains numerous large duplicated chromosomal segments, but the different approaches used in previous analyses led to different interpretations regarding the number and timing of ancestral large-scale duplication events. Here, using more appropriate methodology and a more recent version of the genome sequence annotation, we investigate the scale and timing of segmental duplications in Arabidopsis. We used protein sequence similarity searches to detect duplicated blocks in the genome, used the level of synonymous substitution between duplicated genes to estimate the relative ages of the blocks containing them, and analyzed the degree of overlap between adjacent duplicated blocks. We conclude that the Arabidopsis lineage underwent at least two distinct episodes of duplication. One was a polyploidy that occurred much more recently than estimated previously, before theArabidopsis/Brassica rapa split and probably during the early emergence of the crucifer family (24–40 Mya). An older set of duplicated blocks was formed after the monocot/dicot divergence, and the relatively low level of overlap among these blocks indicates that at least some of them are remnants of a larger duplication such as a polyploidy or aneuploidy.
Identification and Functional Analysis of Human Transcriptional Promoters
Authors: Trinklein Nathan D. , Aldred Shelley J. Force , Saldanha Alok J. , Myers Richard M.
Published: February 2003

Genomic and full-length cDNA sequences provide opportunities for understanding human gene structure and transcriptional regulatory elements. The simplest regulatory elements to identify are promoters, as their positions are dictated by the location of transcription start sites. We aligned full-length cDNA clones from the Mammalian Gene Collection to the human genome rough draft sequence to estimate the start sites of more than 10,000 human transcripts. We selected genomic sequence just upstream from the 5′ end of these cDNA sequences and designated these as putative promoters. We assayed the functions of 152 of these DNA fragments, chosen at random from the entire set, in a luciferase-based transfection assay in four human cultured cell types. Ninety-one percent of these DNA fragments showed significant transcriptional activity in at least one of the cell lines, whereas 89% showed activity in at least two of the lines. We analyzed the distributions of strengths of these promoter fragments in the different cell types and identified likely alternative promoters in a large fraction of the genes. These data indicate that this approach is an effective method for predicting human promoters and provide the first set of functional data collected in parallel for a large set of human promoters.

[Supplemental material is available online atwww.genome.org and http://www-shgc.stanford.edu/myerslab/.]