bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine
Authors: Abeydeera Lalantha R. , Wang Wei-Hua , Cantley Thomas C. , Prather Randall S. , Day Billy N.
Published: July 2002

The present study examined the effect of different concentrations of cysteine in the presence of a thiol compound, β-mercaptoethanol (BME), during in vitro maturation (IVM) of pig oocytes on cumulus expansion, nuclear maturation, intracellular glutathione (GSH) level and subsequent embryonic development after in vitro fertilisation (IVF). In experiment 1, oocytes were matured in NCSU 23 medium containing 10% porcine follicular fluid, 25 μM BME, 0.5 μg/ml LH, 0.5 μg/ml FSH and 0, 0.1, 0.2 or 0.4 mg/ml cysteine for 20–22 h and then without hormonal supplements for an additional 20–22 h. After culture, cumulus cells were removed and a proportion of oocytes fixed to examine the rate of nuclear maturation. The remaining oocytes were co-incubated with spermatozoa for 5–6 h and putative zygotes were transferred to NCSU 23 medium containing 0.4% bovine serum albumin for 144 h. A proportion of putative zygotes were fixed 12 h after insemination to examine fertilisation parameters. In experiment 2, oocytes were matured as in experiment 1 and the GSH content was measured by a DTNB-GSSG reductase recycling assay. No mean differences among treatments were observed in nuclear maturation (78–89%). The mean differences in penetration rate (69–77%), polyspermy rate (31–40%), male pronuclear formation rate (93–96%) or mean number of sperm per oocyte (1.5-1.8) were not affected by the presence or absence of cysteine during oocyte maturation. Also no difference was observed in cleavage rates 48 h after insemination. However, compared with no addition (19%), the presence of 0.1-0.4 mg/ml cysteine during IVM increased (p < 0.001) the proportion of blastocysts (32–39%) at 144 h. In comparison with controls (5.6 pmol/oocyte), the GSH content of oocytes matured in the presence of cysteine was significantly (p < 0.001) higher (13–15 pmol/oocyte) with no mean differences among different cysteine concentrations. The results indicate that in the presence of a thiol compound, supplementation of IVM medium with cysteine can increase the GSH level and improve the developmental competence of pig oocytes following fertilisation. Further, no effect on either GSH level or embryo development was observed by increasing the levels of cysteine supplementation from 0.1 to 0.4 mg/ml.
The frontal aging hypothesis evaluated
Authors: GREENWOOD PAMELA M.
Published: July 2002

That the human frontal lobes are particularly vulnerable
to age-related deterioration has been frequently invoked
as an explanation of functional decline in aging. This
“frontal aging hypothesis” is evaluated in
this review by examining evidence of selectively reduced
frontal lobe function in aging. The frontal aging hypothesis
predicts that functions largely dependent on frontal regions
would decline in aging, while functions largely independent
of frontal lobes would remain relatively spared. The hypothesis
further predicts that age-related brain change would selectively
impact frontal regions. The literatures on working memory,
visuospatial attention, face recognition, and implicit
memory were reviewed as exemplars of functions dependent
on prefrontal, parietal, temporal and occipitotemporal
cortices, respectively, with a view to establishing mediating
structures and effects of aging. Age sensitivity was seen
both in functions dependent on frontal integrity as well
as in functions apparently independent of frontal integrity.
Further, although prefrontal areas exhibit age-related
decreases in regional volume, blood flow and metabolism,
nonfrontal cortical regions undergo similar declines. It
is concluded that while the frontal lobes are subject to
age-related changes reflected in both behavior and pathology,
there is only weak and conflicting evidence that frontal
regions are selectively and differentially affected by
aging. It is argued that a network-based theory of cognitive
aging has advantages over the localizationist approach
inherent in the frontal aging hypothesis. (JINS,
2000, 6, 705–726.)
Worth of watersheds: a producer surplus approach for valuing drought mitigation in Eastern Indonesia
Authors: PATTANAYAK SUBHRENDU K. , KRAMER RANDALL A.
Published: July 2002

This study combines hydrological modeling with applied micro-econometric techniques to value a complex ecosystem service: drought mitigation provided by tropical forested watersheds to agrarian communities. Spatial variation in current baseflow allows estimation of drought mitigation values as the marginal profit accruing to agricultural households. The paper shows that this uncommon focus on producer (not consumer) surplus measures is appropriate for valuation as long as markets for commodities related to the environmental services are complete. For the typical household, the estimated marginal profit is positive, validating the central hypothesis that baseflow makes positive contributions to agricultural profits. There is some evidence, however, that increased watershed protection will increase profits through greater baseflow only in watersheds with a unique mix of physio-graphic and climatic features. The paper evaluates and provides some support for the hypothesis, put forward by hydrological science and the Indonesian Government, that protected watersheds can supply latent and unrecognized ecosystem services to local people.
Controlling carbon emissions in China
Authors: GARBACCIO RICHARD F. , HO MUN S. , JORGENSON DALE W.
Published: July 2002

We examine the use of carbon taxes to reduce emissions of CO2 in China. To do so, we develop a dynamic computable general equilibrium (CGE) model of the Chinese economy. In addition to accounting for the effects of population growth, capital accumulation, technological change, and changing patterns of demand, we also incorporate into our model elements of the dual nature of China's economy where both plan and market institutions exist side by side. We conduct simulations in which carbon emissions are reduced by 5, 10, and 15 per cent from our baseline. After initial declines, in all of our simulations GDP and consumption rapidly exceed baseline levels as the revenue neutral carbon tax serves to transfer income from consumers to producers and then into increased investment. Although subject to a number of caveats, we find potential for what is in some sense a 'double dividend', a decrease in emissions of CO2 and a long run increase in GDP and consumption.
Algorithmic composition as a model of creativity
Authors: JACOB BRUCE L.
Published: July 2002

There are two distinct types of creativity: the flash out of the blue (inspiration? genius?), and the process of incremental revisions (hard work). Not only are we years away from modelling the former, we do not even begin to understand it. The latter is algorithmic in nature and has been modelled in many systems both musical and non-musical. Algorithmic composition is as old as music composition. It is often considered a cheat, a way out when the composer needs material and/or inspiration. It can also be thought of as a compositional tool that simply makes the composer’s work go faster. This article makes a case for algorithmic composition as such a tool. The ‘hard work’ type of creativity often involves trying many different combinations and choosing one over the others. It seems natural to express this iterative task as a computer algorithm. The implementation issues can be reduced to two components: how to understand one’s own creative process well enough to reproduce it as an algorithm, and how to program a computer to differentiate between ‘good’ and ‘bad’ music. The philosophical issues reduce to the question who or what is responsible for the music produced?
Seed storage behaviour of Fagus sylvatica and Fagus crenata
Authors: León-Lobos Pedro , Ellis Richard H.
Published: July 2002

The seed storage behaviour of Fagus sylvatica and F. crenata was investigated. A large fraction of seeds of both species survived desiccation to about 3% moisture content (MC) (in equilibrium with 10% relative humidity at 20°C). Nevertheless, viability was reduced significantly and progressively by desiccation from 14% to 3% MC. In addition, during subsequent hermetic storage at constant temperatures of 20 to –20°C in F. sylvaticaand 10 to –20°C in F. crenata seeds, viability was lost more rapidly with reduction in MC below about 7.6–11.5% (40–71%relative humidity at 20°C). Thus, Fagus sylvatica and F. crenataexhibited intermediate seed storage behaviour. Survival at –20°C with 7.8–11.5% (F. sylvatica) and 7.6% MC (F. crenata) was comparatively good, with 64–84% of seeds remaining able to germinate normally after 2 years of hermetic storage, although this was neither appreciably better nor worse than at 0–10°C. Optimum seed storage environments, within the range investigated, were provided by combining temperatures of –10 to –20°C with 7.8–11.5% (F. sylvatica) or 7.6–9.5%(F. crenata) MC.
Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus
Authors: Damiola Francesca , Le Minh Nguyet , Preitner Nicolas , Kornmann Benoı̂t , Fleury-Olela Fabienne , Schibler Ueli
Published: July 2002

In mammals, circadian oscillators exist not only in the suprachiasmatic nucleus, which harbors the central pacemaker, but also in most peripheral tissues. It is believed that the SCN clock entrains the phase of peripheral clocks via chemical cues, such as rhythmically secreted hormones. Here we show that temporal feeding restriction under light–dark or dark–dark conditions can change the phase of circadian gene expression in peripheral cell types by up to 12 h while leaving the phase of cyclic gene expression in the SCN unaffected. Hence, changes in metabolism can lead to an uncoupling of peripheral oscillators from the central pacemaker. Sudden large changes in feeding time, similar to abrupt changes in the photoperiod, reset the phase of rhythmic gene expression gradually and are thus likely to act through a clock-dependent mechanism. Food-induced phase resetting proceeds faster in liver than in kidney, heart, or pancreas, but after 1 wk of daytime feeding, the phases of circadian gene expression are similar in all examined peripheral tissues.
Delta signaling from the germ line controls the proliferation and differentiation of the somatic follicle cells during Drosophila oogenesis
Authors: St. Johnston Hernán López-Schier and Daniel
Published: July 2002

The body axes of Drosophila are established during oogenesis through reciprocal interactions between the germ line cells and the somatic follicle cells that surround them. The Notch pathway is required at two stages in this process: first, for the migration of the follicle cells around the germ line cyst and, later, for the polarization of the anterior-posterior (A-P) axis of the oocyte. Its function in these events, however, has remained controversial. Using clonal analysis, we show that Notch signaling controls cell proliferation and differentiation in the whole follicular epithelium.Notch mutant follicle cells remain in a precursor state and fail to switch from the mitotic cell cycle to the endocycle. Furthermore, removal of Delta from the germ line produces an identical phenotype, showing that Delta signals from the germ cells to control the timing of follicle cell differentiation. This explains the axis formation defects in Notch mutants, which arise because undifferentiated posterior follicle cells cannot signal to polarize the oocyte. Delta also signals from the germ line to Notch in the soma earlier in oogenesis to control the differentiation of the polar and stalk follicle cells. The germ line therefore regulates the development of the follicle cells through two complementary signaling pathways: Gurken signals twice to control spatial patterning, whereas Delta signals twice to exert temporal control.
The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis
Authors: Lee Horim , Suh Sung-Suk , Park Eunsook , Cho Euna , Ahn Ji Hoon , Kim Sang-Gu , Lee Jong Seob , Kwon Young Myung , Lee Ilha
Published: July 2002

The very late-flowering behavior of Arabidopsiswinter-annual ecotypes is conferred mainly by two genes,FRIGIDA (FRI) and FLOWERING LOCUS C(FLC). A MADS-domain gene, AGAMOUS-LIKE 20(AGL20), was identified as a dominant FRI suppressor in activation tagging mutagenesis. Overexpression of AGL20suppresses not only the late flowering of plants that have functionalFRI and FLC alleles but also the delayed phase transitions during the vegetative stages of plant development. Interestingly, AGL20 expression is positively regulated not only by the redundant vernalization and autonomous pathways of flowering but also by the photoperiod pathway. Our results indicate that AGL20 is an important integrator of three pathways controlling flowering in Arabidopsis.
Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRα and LXRβ
Authors: Repa Joyce J. , Liang Guosheng , Ou Jiafu , Bashmakov Yuriy , Lobaccaro Jean-Marc A. , Shimomura Iichiro , Shan Bei , Brown Michael S. , Goldstein Joseph L. , Mangelsdorf David J.
Published: July 2002

The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily that are bound and activated by oxysterols. These receptors serve as sterol sensors to regulate the transcription of gene products that control intracellular cholesterol homeostasis through catabolism and transport. In this report, we describe a novel LXR target, the sterol regulatory element-binding protein-1c gene (SREBP-1c), which encodes a membrane-bound transcription factor of the basic helix-loop-helix-leucine zipper family. SREBP-1c expression was markedly increased in mouse tissues in an LXR-dependent manner by dietary cholesterol and synthetic agonists for both LXR and its heterodimer partner, the retinoid X receptor (RXR). Expression of the related gene products, SREBP-1a and SREBP-2, were not increased. Analysis of the mouse SREBP-1c gene promoter revealed an RXR/LXR DNA-binding site that is essential for this regulation. The transcriptional increase in SREBP-1c mRNA by RXR/LXR was accompanied by a similar increase in the level of the nuclear, active form of the SREBP-1c protein and an increase in fatty acid synthesis. Because this active form of SREBP-1c controls the transcription of genes involved in fatty acid biosynthesis, our results reveal a unique regulatory interplay between cholesterol and fatty acid metabolism.
Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family
Authors: Pierce Sarah B. , Costa Michael , Wisotzkey Robert , Devadhar Sharmila , Homburger Sheila A. , Buchman Andrew R. , Ferguson Kimberly C. , Heller Jonathan , Platt Darren M. , Pasquinelli Amy A. , Liu Leo X. , Doberstein Stephen K. , Ruvkun Gary
Published: July 2002

The activity of the DAF-2 insulin-like receptor is required forCaenorhabditis elegans reproductive growth and normal adult life span. Informatic analysis identified 37 C. elegans genes predicted to encode insulin-like peptides. Many of these genes are divergent insulin superfamily members, and many are clustered, indicating recent diversification of the family. The ins genes are primarily expressed in neurons, including sensory neurons, a subset of which are required for reproductive development. Structural predictions and likely C-peptide cleavage sites typical of mammalian insulins suggest that ins-1 is most closely related to insulin. Overexpression of ins-1, or expression of human insulin under the control of ins-1 regulatory sequences, causes partially penetrant arrest at the dauer stage and enhances dauer arrest in weakdaf-2 mutants, suggesting that INS-1 and human insulin antagonize DAF-2 insulin-like signaling. A deletion of theins-1 coding region does not enhance or suppress dauer arrest, indicating a functional redundancy among the 37 ins genes. Of five other ins genes tested, the only other one bearing a predicted C peptide also antagonizes daf-2 signaling, whereas four ins genes without a C peptide do not, indicating functional diversity within the ins family.
Tn7 recognizes transposition target structures associated with DNA replication using the DNA-binding protein TnsE
Authors: Peters Joseph E. , Craig Nancy L.
Published: July 2002

We report that the bacterial transposon Tn7 selects targets by recognizing features associated with DNA replication using the transposon-encoded DNA-binding protein TnsE. We show that Tn7 transposition directed by TnsE occurs in one orientation with respect to chromosomal DNA replication, indicating that a structure or complex involved in DNA replication is likely to be a critical determinant of TnsE insertion. We find that mutant TnsE proteins that allow higher levels of transposition also bind DNA better than the wild-type protein. The increased binding affinity displayed by the TnsE high-activity mutants indicates that DNA binding is relevant to transposition activity and suggests that TnsE interacts directly with target DNAs. In vitro, TnsE interacts preferentially with certain DNA structures, indicating a mechanism for the TnsE-mediated orientation and insertion preference. The pattern of TnsE-mediated insertion events around the Escherichia coli chromosome provides insight into how DNA replication forks proceed in vivo.
Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors
Authors: Crispino John D. , Lodish Maya B. , Thurberg Beth L. , Litovsky Silvio H. , Collins Tucker , Molkentin Jeffery D. , Orkin Stuart H.
Published: July 2002

GATA-family transcription factors are critical to the development of diverse tissues. In particular, GATA-4 has been implicated in formation of the vertebrate heart. As the mouse Gata-4knock-out is early embryonic lethal because of a defect in ventral morphogenesis, the in vivo function of this factor in heart development remains unresolved. To search for a requirement for Gata4 in heart development, we created mice harboring a single amino acid replacement in GATA-4 that impairs its physical interaction with its presumptive cardiac cofactor FOG-2. Gata4ki/ki mice die just after embryonic day (E) 12.5 exhibiting features in common with Fog2−/− embryos as well as additional semilunar cardiac valve defects and a double-outlet right ventricle. These findings establish an intrinsic requirement for GATA-4 in heart development. We also infer that GATA-4 function is dependent on interaction with FOG-2 and, very likely, an additional FOG protein for distinct aspects of heart formation.
ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage
Authors: Maya Ruth , Balass Moshe , Kim Seong-Tae , Shkedy Dganit , Leal Juan-Fernando Martinez , Shifman Ohad , Moas Miri , Buschmann Thomas , Ronai Ze'ev , Shiloh Yosef , Kastan Michael B. , Katzir Ephraim , Oren Moshe
Published: July 2002

The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258–260 and 393–395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2.
Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development
Authors: Kamachi Yusuke , Uchikawa Masanori , Tanouchi Aki , Sekido Ryohei , Kondoh Hisato
Published: July 2002

Pax6 is a key transcription factor in eye development, particularly in lens development, but its molecular action has not been clarified. We demonstrate that Pax6 initiates lens development by forming a molecular complex with SOX2 on the lens-specific enhancer elements, e.g., the δ-crystallin minimal enhancer DC5. DC5 shows a limited similarity to the binding consensus sequence of Pax6 and is bound poorly by Pax6 alone. However, Pax6 binds cooperatively with SOX2 to the DC5 sequence, resulting in formation of a high-mobility form of ternary complex in vitro, which correlates with the enhancer activation in vivo. We observed Pax6 and SOX2-interdependent factor occupancy of DC5 in a chromatin environment in vivo, providing the molecular basis of synergistic activation by Pax6 and SOX2. Subtle alterations of the Pax6-binding-site sequence of DC5 or of the inter-binding-sites distance diminished the cooperative binding and caused formation of a non-functional low-mobility form complex, suggesting DNA sequence-guided and protein interaction-induced conformation change of the Pax6 protein. When ectopically expressed in embryo ectoderm, Pax6 and SOX2 in combination activate δ-crystallin gene and elicit lens placode development, indicating that the complex of Pax6 and SOX2 formed on specific DNA sequences is the genetic switch for initiation of lens differentiation.
Control of cell division by a retinoblastoma protein homolog in Chlamydomonas
Authors: Umen James G. , Goodenough Ursula W.
Published: July 2002

A key pathway that controls both cell division and differentiation in animal cells is mediated by the retinoblastoma (RB) family of tumor suppressors, which gate the passage of cells from G1 to S and through S phase. The role(s) of the RB pathway in plants are not yet clearly defined, nor has there been any evidence for its presence in unicellular organisms. Here we have identified an RB homolog encoded by the mat3 gene in Chlamydomonas reinhardtii, a unicellular green alga in the land plant lineage. Chlamydomonascells normally grow to many times their original size during a prolonged G1 and then undergo multiple alternating rounds of S phase and mitosis to produce daughter cells of uniform size.mat3 mutants produce small daughter cells and show defects in two size-dependent cell cycle controls: They initiate the cell cycle at a below-normal size, and they undergo extra rounds of S phase/mitosis. Unlike mammalian RB mutants, mat3 mutants do not have a shortened G1, do not enter S phase prematurely, and can exit the cell cycle and differentiate normally, indicating that the RB pathway in Chlamydomonas has a different role than in animals.
Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes
Authors: Fernández Ana M. , Kim Jason K. , Yakar Shoshana , Dupont Joëlle , Hernandez-Sanchez Catalina , Castle Arthur L. , Filmore Jonathan , Shulman Gerald I. , Le Roith Derek
Published: July 2002

Peripheral insulin resistance and impaired insulin action are the primary characteristics of type 2 diabetes. The first observable defect in this major disorder occurs in muscle, where glucose disposal in response to insulin is impaired. We have developed a transgenic mouse with a dominant-negative insulin-like growth factor-I receptor (KR–IGF-IR) specifically targeted to the skeletal muscle. Expression of KR–IGF-IR resulted in the formation of hybrid receptors between the mutant and the endogenous IGF-I and insulin receptors, thereby abrogating the normal function of these receptors and leading to insulin resistance. Pancreatic β-cell dysfunction developed at a relative early age, resulting in diabetes. These mice provide an excellent model to study the molecular mechanisms underlying the development of human type 2 diabetes.
Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genes
Authors: Hua Jian , Grisafi Paula , Cheng Shu-Hua , Fink Gerald R.
Published: July 2002

Wild-type Arabidopsis plants maintain a relatively constant size over a wide range of temperatures. Here we show that this homeostasis requires the BONZAI1 (BON1) gene becausebon1 null mutants make miniature fertile plants at 22°C but have wild-type appearance at 28°C. The expression of BON1 and a BON1-associated protein (BAP1) is modulated by temperature. Thus BON1 and BAP1 may have a direct role in regulating cell expansion and cell division at lower temperatures. BON1 contains a Ca2+-dependent phospholipid-binding domain and is associated with the plasma membrane. It belongs to the copine gene family, which is conserved from protozoa to humans. Our data suggest that this gene family may function in the pathway of membrane trafficking in response to external conditions.
Fine Mapping Suggests that the Goat Polled Intersex Syndrome and the Human Blepharophimosis Ptosis Epicanthus Syndrome Map to a 100-kb Homologous Region
Authors: Schibler Laurent , Cribiu Edmond P. , Oustry-Vaiman Anne , Furet Jean-Pierre , Vaiman Daniel
Published: July 2002

To clone the goat Polled Intersex Syndrome (PIS) gene(s), a chromosome walk was performed from six entry points at 1q43. This enabled 91 BACs to be recovered from a recently constructed goat BAC library. Six BAC contigs of goat chromosome 1q43 (ICC1–ICC6) were thus constructed covering altogether 4.5 Mb. A total of 37 microsatellite sequences were isolated from this 4.5-Mb region (16 in this study), of which 33 were genotyped and mapped. ICC3 (1500 kb) was shown by genetic analysis to encompass the PIS locus in a ∼400-kb interval without recombinants detected in the resource families (293 informative meioses). A strong linkage disequilibrium was detected among unrelated animals with the two central markers of the region, suggesting a probable location for PIS in ∼100 kb. High-resolution comparative mapping with human data shows that this DNA segment is the homolog of the human region associated withBlepharophimosis Ptosis Epicanthus inversus Syndrome(BPES) gene located in 3q23. This finding suggests that homologous gene(s) could be responsible for the pathologies observed in humans and goats.

[The sequence data, PCR primers and PCR conditions for STS and microsatellites described in this paper have been submitted to the GenBank data library under accession nos.AQ666547–AQ666579, AQ686084–AQ686129, AQ793920–793931,AQ810429–AQ810527, G41201–G41228, and G54270–G54286.]
Ab initio Gene Finding in Drosophila Genomic DNA
Authors: Salamov Asaf A. , Solovyev Victor V.
Published: July 2002

Ab initio gene identification in the genomic sequence ofDrosophila melanogaster was obtained using Fgenes(human gene predictor) and Fgenesh programs that have organism-specific parameters for human, Drosophila, plants, yeast, and nematode. We did not use information about cDNA/EST in most predictions to model a real situation for finding new genes because information about complete cDNA is often absent or based on very small partial fragments. We investigated the accuracy of gene prediction on different levels and designed several schemes to predict an unambiguous set of genes (annotation CGG1), a set of reliable exons (annotation CGG2), and the most complete set of exons (annotation CGG3). For 49 genes, protein products of which have clear homologs in protein databases, predictions were recomputed by Fgenesh+ program. The first annotation serves as the optimal computational description of new sequence to be presented in a database. Reliable exons from the second annotation serve as good candidates for selecting the PCR primers for experimental work for gene structure verification. Our results shows that we can identify ∼90% of coding nucleotides with 20% false positives. At the exon level we accurately predicted 65% of exons and 89% including overlapping exons with 49% false positives. Optimizing accuracy of prediction, we designed a gene identification scheme using Fgenesh, which provided sensitivity (Sn) = 98% and specificity (Sp) = 86% at the base level, Sn = 81% (97% including overlapping exons) and Sp = 58% at the exon level and Sn = 72% and Sp = 39% at the gene level (estimating sensitivity on std1 set and specificity on std3 set). In general, these results showed that computational gene prediction can be a reliable tool for annotating new genomic sequences, giving accurate information on 90% of coding sequences with 14% false positives. However, exact gene prediction (especially at the gene level) needs additional improvement using gene prediction algorithms. The Fgenesh program was also tested for predicting genes of human Chromosome 22 (the last variant of Fgenesh can analyze the whole chromosome sequence). This analysis has demonstrated that the 88% of manually annotated exons in Chromosome 22 were among the ab initio predicted exons. The suite of gene identification programs is available through the WWW server of Computational Genomics Group at http://genomic.sanger.ac.uk/gf.html.
Using GeneWise in the Drosophila Annotation Experiment
Authors: Birney Ewan , Durbin Richard
Published: July 2002

The GeneWise method for combining gene prediction and homology searches was applied to the 2.9-Mb region from Drosophila melanogaster. The results from the Genome Annotation Assessment Project (GASP) showed that GeneWise provided reasonably accurate gene predictions. Further investigation indicates that many of the incorrect gene predictions from GeneWise were due to transposons with valid protein-coding genes and the remaining cases are pseudogenes or possible annotation oversights.
Patterns of Variant Polyadenylation Signal Usage in Human Genes
Authors: Beaudoing Emmanuel , Freier Susan , Wyatt Jacqueline R. , Claverie Jean-Michel , Gautheret Daniel
Published: July 2002

The formation of mature mRNAs in vertebrates involves the cleavage and polyadenylation of the pre-mRNA, 10–30 nt downstream of an AAUAAA or AUUAAA signal sequence. The extensive cDNA data now available shows that these hexamers are not strictly conserved. In order to identify variant polyadenylation signals on a large scale, we compared over 8700 human 3′ untranslated sequences to 157,775 polyadenylated expressed sequence tags (ESTs), used as markers of actual mRNA 3′ ends. About 5600 EST-supported putative mRNA 3′ ends were collected and analyzed for significant hexameric sequences. Known polyadenylation signals were found in only 73% of the 3′ fragments. Ten single-base variants of the AAUAAA sequence were identified with a highly significant occurrence rate, potentially representing 14.9% of the actual polyadenylation signals. Of the mRNAs, 28.6% displayed two or more polyadenylation sites. In these mRNAs, the poly(A) sites proximal to the coding sequence tend to use variant signals more often, while the 3′-most site tends to use a canonical signal. The average number of ESTs associated with each signal type suggests that variant signals (including the common AUUAAA) are processed less efficiently than the canonical signal and could therefore be selected for regulatory purposes. However, the position of the site in the untranslated region may also play a role in polyadenylation rate.
Reverse Transcriptase-Polymerase Chain Reaction Validation of 25 “Orphan” Genes from Escherichia coli K-12 MG1655
Authors: Alimi Jean-Philippe , Poirot Olivier , Lopez Fabrice , Claverie Jean-Michel
Published: July 2002

Despite the accumulation of sequence information sampling from a broad spectrum of phyla, newly sequenced genomes continue to reveal a high proportion (50%–30%) of “uncharacterized” genes, including a significant number of strictly “orphan” genes, i.e., putative open reading frames (ORFs) without any resemblance to previously determined protein-coding sequences. Most genes found in databases have only been predicted by computer methods and have never been experimentally validated. Although theoretical evolutionary arguments support the reality of genes when homologs are found in a variety of distant species, this is not the case for orphan genes. Here, we report the direct reverse transcriptase-polymerase chain reaction assay of 25 strictly orphan ORFs of Escherichia coli. Two growth conditions, exponential and stationary phases, were tested. Transcripts were identified for a total of 19 orphan genes, with 2 genes found to be expressed in only one of the two growth conditions. Our results suggest that a vast majority of E. coli ORFs presently annotated as “hypothetical” correspond to bona fide genes. By extension, this implies that randomly occurring “junk” ORFs have been actively counter selected during the evolution of the denseE. coli genome.
A Systematic Analysis of Human Disease-Associated Gene Sequences In Drosophila melanogaster
Authors: Reiter Lawrence T. , Potocki Lorraine , Chien Sam , Gribskov Michael , Bier Ethan
Published: July 2002

We performed a systematic BLAST analysis of 929 human disease gene entries associated with at least one mutant allele in the Online Mendelian Inheritance in Man (OMIM) database against the recently completed genome sequence of Drosophila melanogaster. The results of this search have been formatted as an updateable and searchable on-line database called Homophila. Our analysis identified 714 distinct human disease genes (77% of disease genes searched) matching 548 unique Drosophila sequences, which we have summarized by disease category. This breakdown into disease classes creates a picture of disease genes that are amenable to study usingDrosophila as the model organism. Of the 548Drosophila genes related to human disease genes, 153 are associated with known mutant alleles and 56 more are tagged byP-element insertions in or near the gene. Examples of how to use the database to identify Drosophila genes related to human disease genes are presented. We anticipate that cross-genomic analysis of human disease genes using the power of Drosophilasecond-site modifier screens will promote interaction between human andDrosophila research groups, accelerating the understanding of the pathogenesis of human genetic disease. The Homophila database is available at http://homophila.sdsc.edu.
Impaired Memory Retention and Decreased Long-Term Potentiation in Integrin-Associated Protein-Deficient Mice
Authors: Chang Hui P. , Lindberg Frederik P. , Wang Hai L. , Huang A.M. , Lee Eminy H.Y.
Published: July 2002

Previously, we have demonstrated that integrin-associated protein (IAP) mRNA level is approximately fourfold higher in rats showing good retention performance (600 sec) than rats showing poor retention performance (<80 sec) in an inhibitory avoidance learning paradigm. In the present study, we have used the gene-targeted IAP-deficient mice to further investigate the role of IAP involved in memory formation and hippocampal long-term potentiation (LTP) in vivo. Results revealed that there was a significant impairment in memory retention and a significant reduction in the magnitude of LTP in IAP-deficient mice when compared with the wild-type and heterozygote mice, whereas the wild-type and heterozygote animals did not show marked differences on these measures. Furthermore, the impairment in retention performance of IAP-deficient mice was not due to different sensitivities of these animals to the electric shock. When we examined locomotor activity and rotarod treadmill performance, no differences were observed among these three groups of animals either. Western blot analysis confirmed the lack of IAP protein in IAP-deficient mice, whereas IAP expression was similar in both the wild-type and heterozygote controls. These results together demonstrate that IAP plays an important role in the process of memory formation and synaptic plasticity in mice.
God's Organism? The Chick as a Model System for Memory Studies
Authors: Rose Steven P.R.
Published: July 2002

The young chick is a powerful model system in which to study the biochemical and morphological processes underlying memory formation. Training chicks on a one trial passive avoidance task results in a molecular cascade in a specific brain region, the intermediate medial hyperstriatum ventrale. This cascade is initiated by glutamate release and engages a series of synaptic transients including increased calcium flux, up-regulation of NMDA-glutamate receptors, membrane protein phosphorylations, and the retrograde messenger NO. Expression of immediate early genes c-fosand c-junprecedes the synthesis, glycosylation, and redistribution, >4 hr downstream, of a number of synaptic membrane proteins, notably NCAM and L1. Other membrane proteins required in the early phase of memory formation include the amyloid precursor protein (APP) and apolipoprotein E. There are concomitant increases in dendritic spine number and changes in synaptic structure. Nonsynaptic factors, including corticosterone and BDNF, can modulate retention of the avoidance response, enhancing the salience of otherwise weakly retained memory. These results are discussed in relation to general concepts of memory formation and the spatio-temporal distribution of the putative memory trace.
Cellular and Molecular Mechanisms Underlying Learning and Memory Impairments Produced by Cannabinoids: Figure 1.
Authors: Sullivan Jane M.
Published: July 2002

Why does smoking marijuana impair learning and memory? Behavioral studies suggest that a disruption of normal hippocampal function contributes to these deficits. In vitro experiments find that cannabinoid receptor activation reduces neurotransmitter release below the levels required to trigger long-term changes in synaptic strength in the hippocampus. Cannabinoids reduce glutamate release through a G-protein-mediated inhibition of the calcium channels responsible for neurotransmitter release from hippocampal neurons. These mechanisms likely play a role in the learning and memory impairments produced by cannabinoids and by endogenous cannabinoid receptor ligands.
Purification and Characterization of Chaperonin 60 and Heat-Shock Protein 70 from Chromoplasts of Narcissus pseudonarcissus (Involvement of Heat-Shock Protein 70 in a Soluble Protein Complex Containing Phytoene Desaturase)
Authors: Bonk M. , Tadros M. , Vandekerckhove J. , Al-Babili S. , Beyer P.
Published: July 2002

In chromoplast differentiation during flower formation in Narcissus pseudonarcissus, the molecular chaperones chaperonin 60 (Cpn60; α- and [beta]) and heat-shock protein 70 (Hsp70) greatly increase in abundance. Both were purified and shown to be present in a functional form in chromoplasts, indicating their requirement in the extensive structural rearrangements during the chloroplast-to-chromoplast transition. The purified proteins, sequenced N terminally and from internal peptides, showed strong homology to plastid Cpn60 and Hsp70 representatives from other plant species. During chromoplast differentiation, the carotenoid biosynthetic pathway is strongly induced. The corresponding enzymes are all nuclear encoded and form a large, soluble, hetero-oligomeric protein complex after import but prior to their membrane attachment. By immunoprecipitations we have shown that the plastid Hsp70 is a structural constituent of a soluble entity also containing phytoene desaturase.
Sinapic Acid Ester Metabolism in Wild Type and a Sinapoylglucose-Accumulating Mutant of Arabidopsis
Authors: Lorenzen M. , Racicot V. , Strack D. , Chapple C.
Published: July 2002

Sinapoylmalate is one of the major phenylpropanoid metabolites that is accumulated in the vegetative tissue of Arabidopsis thaliana. A thin-layer chromatography-based mutant screen identified two allelic mutant lines that accumulated sinapoylglucose in their leaves in place of sinapoylmalate. Both mutations were found to be recessive and segregated as single Mendelian genes. These mutants define a new locus called SNG1 for sinapoylglucose accumulator. Plants that are homozygous for the sng1 mutation accumulate normal levels of malate in their leaves but lack detectable levels of the final enzyme in sinapate ester biosynthesis, sinapoylglucose:malate sinapoyltransferase. A study of wild-type and sng1 seedlings found that sinapic acid ester biosynthesis in Arabidopsis is developmentally regulated and that the accumulation of sinapate esters is delayed in sng1 mutant seedlings.
Targeting of the Arabidopsis Homomeric Acetyl-Coenzyme A Carboxylase to Plastids of Rapeseeds
Authors: Roesler K. , Shintani D. , Savage L. , Boddupalli S. , Ohlrogge J.
Published: July 2002

Acetyl-coenzyme A carboxylase (ACCase) occurs in at least two forms in rapeseed (Brassica napus): a homomeric (HO) and presumably cytosolic isozyme and a heteromeric, plastidial isozyme. We investigated whether the HO-ACCase of Arabidopsis can be targeted to plastids of B. napus seeds. A chloroplast transit peptide and the napin promoter were fused to the Arabidopsis ACC1 gene and transformed into B. napus, with the following results. (a) The small subunit transit peptide was sufficient to provide import of this very large protein into developing seed plastids. (b) HO-ACCase in isolated plastids was found to be biotinylated at a level comparable to extraplastidial HO-ACCase. (c) In vitro assays of HO-ACCase in isolated plastids from developing seeds indicate that it occurs as an enzymatically active form in the plastidial compartment. (d) ACCase activity in mature B. napus seeds is normally very low; however, plants expressing the SSU/ACC1 gene had 10- to 20-fold higher ACCase activity in mature seeds, suggesting that plastid localization prevents the turnover of HO-ACCase. (e) ACCase over-expression altered seed fatty acid composition, with the largest effect being an increase in oleic acid. (f) The total oil content of seeds was increased approximately 5% by the expression of HO-ACCase in plastids.
Three Different Polygalacturonases Are Expressed in Tomato Leaf and Flower Abscission, Each with a Different Temporal Expression Pattern
Authors: Kalaitzis P. , Solomos T. , Tucker M. L.
Published: July 2002

Abscission, or organ separation, is accompanied by a marked increase in hydrolases, which are responsible for the degradation of the middle lamella and the loosening of the primary cell wall surrounding cells in the separation layer. We recently reported on the cloning of a tomato (Lycopersicon esculentum) polygalacturonase (PG) cDNA, TAPG1, expressed during leaf and flower abscission. In addition to TAPG1, we have cloned two more PG cDNAs (TAPG2 and TAPG4) that are also expressed during leaf and flower abscission. The peptide sequences for the three abscission PGs are relatively similar (76–93% identity) yet different from the those of tomato fruit PG (38-–41% identity). None of the three abscission PG mRNAs are expressed in fruit, stems, petioles, or anthers of fully open flowers. An RNase protection assay revealed that all three PGs are expressed in leaf and flower abscission zones and in pistils of fully open flowers. TAPG4 mRNA is detected much earlier than TAPG1 and TAPG2 mRNA during both leaf and flower abscission.
The Arabidopsis 14-3-3 Multigene Family
Authors: Wu K. , Rooney M. F. , Ferl R. J.
Published: July 2002

The 14–3–3 proteins are ubiquitous eukaryotic proteins and are encoded by a gene family in many species. We examined the 14–3–3 gene family in Arabidopsis thaliana and found that it contains 10 members. Four new cDNAs, GF14[epsilon], GF14[kappa], GF14[mu], and GF14[nu], and two new genomic clones of GF14[phi] and GF14[upsilon] were isolated and characterized. Together with the six previously described 14–3–3 isoforms in Arabidopsis, they constitute a complete family of 10 distinct 14–3–3 proteins of 248 to 268 amino acids. Phylogenetic analysis revealed the presence of two ancient, distinct 14–3–3 gene classes in Arabidopsis and other plants. The [epsilon] forms diverged early from the other plant isoforms, and plant 14–3–3 genes displayed a different evolutionary course from that of mammals.
Influence of Salicylic Acid on H2O2 Production, Oxidative Stress, and H2O2-Metabolizing Enzymes (Salicylic Acid-Mediated Oxidative Damage Requires H2O2)
Authors: Rao M. V. , Paliyath G. , Ormrod D. P. , Murr D. P. , Watkins C. B.
Published: July 2002

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2, and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed.
Fusicoccin Counteracts then-Ethylmaleimide and Silver-Induced Stimulation of Oxygen Uptake inEgeria densaLeaves1
Authors: Teresa Marrè Maria , Albergoni Francesco
Published: July 2002

It was previously shown that a number of sulfhydryl [SH] group reagents (N-ethylmaleimide [NEM], iodoacetate, Ag+, HgCl2, etc.) can induce a marked, transitory stimulation of O2 uptake (QO2) in Egeria densa leaves, insensitive to CN− and salicylhydroxamic acid and inhibited by diphenylene iodonium and quinacrine. The phytotoxin fusicoccin (FC) also induces a marked increase in O2 consumption inE. densa leaves, apparently independent of the recognized stimulating action on the H+-ATPase. In this investigation we compared the FC-induced increase in O2consumption with those induced by NEM and Ag+, and we tested for a possible interaction between FC and the two SH blockers in the activation of QO2. The results show (a) the different nature of the FC- and NEM- or Ag+-induced increases of QO2; (b) that FC counteracts the NEM- (and Ag+)-induced respiratory burst; and (c) that FC strongly reduces the damaging effects on plasma membrane permeability observed in E. densa leaves treated with the two SH reagents. Two alternative models of interpretation of the action of FC, in activating a CN−-sensitive respiratory pathway and in suppressing the SH blocker-induced respiratory burst, are proposed.