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Structural and functional analysis of rice genome.

Rice is an excellent system for plant genomics as it represents a modest size genome of 430 Mb. It feeds more than half the population of the world. Draft sequences of the rice genome, derived by whole-genome shotgun approach at relatively low coverage (4-6 X), were published and the International Rice Genome Sequencing Project (IRGSP) declared high quality (>10 X), genetically anchored, phase 2 level sequence in 2002. In addition, phase 3 level finished sequence of chromosomes 1, 4 and 10 (out of 12 chromosomes of rice) has already been reported by scientists from IRGSP consortium. Various estimates of genes in rice place the number at >50,000. Already, over 28,000 full-length cDNAs have been sequenced, most of which map to genetically anchored genome sequence. Such information is very useful in revealing novel features of macro- and micro-level synteny of rice genome with other cereals. Microarray analysis is unraveling the identity of rice genes expressing in temporal and spatial manner and should help target candidate genes useful for improving traits of agronomic importance. Simultaneously, functional analysis of rice genome has been initiated by marker-based characterization of useful genes and employing functional knock-outs created by mutation or gene tagging. Integration of this enormous information is expected to catalyze tremendous activity on basic and applied aspects of rice genomics.

Chromosomes, Plant↗

In vivo structure-function analysis of Drosophila Hairless.

Hairless plays an important role as the major antagonist in the Notch signalling pathway in Drosophila. It appears to be a direct inhibitor of the signal transducer Su(H). Hairless encodes a pioneer protein which was dissected in a structure-function analysis; a series of deletion constructs was tested for wild type and gain of function activity in the fly as well as for Su(H) binding. Thereby, the Hairless protein was subdivided into the absolutely essential Su(H)-binding domain, similarly important N- and C-terminal domains and a central antimorphic domain. Therefore, Hairless protein might have additional functions apart from Su(H) binding and may antagonize Notch mediated cell-cell communication in a more complex way than currently anticipated.

Animals↗

Discriminant function analysis of the central portion of the innominate.

Terry Collection innominates of 260 American whites and blacks (65 males and 65 females of each race) were analyzed by discriminant function analysis for sex assessment, race known, and for simultaneous race and sex assessment. Measurements from the preservationally favored central portion of the innominate were chosen for their potential usefulness in forensic casework and to objectify long-recognized "formal" differences between the sexes in the greater sciatic notch. These included acetabular diameter, greater sciatic notch height, and position of greatest notch depth. Accuracy of sex prediction when race is known is roughly 90% in both the base sample of 260 and a test sample of 200 (50 additional individuals for each group). For simultaneous race and sex assessment, accuracy of prediction is roughly 60% for each group in both samples. This represents about a 45% reduction in error over random assignment by race and sex.

Basal Ganglia↗

Functional analysis of secreted and transmembrane proteins critical to mouse development.

We describe the successful application of a modified gene-trap approach, the secretory trap, to systematically analyze the functions in vivo of large numbers of genes encoding secreted and membrane proteins. Secretory-trap insertions in embryonic stem cells can be transmitted to the germ line of mice with high efficiency and effectively mutate the target gene. Of 60 insertions analyzed in mice, one-third cause recessive lethal phenotypes affecting various stages of embryonic and postnatal development. Thus, secretory-trap mutagenesis can be used for a genome-wide functional analysis of cell signaling pathways that are critical for normal mammalian development and physiology.

Animals↗

[Results of functional analysis on an obstructive model of the lung using an Ergojet CVT high-frequency jet respirator. Comparison with the normal pattern].

This paper expands a previous study on functional analysis of high frequency jet (HFJV) "Ergojet CVT" ventilation module inserted in Ergotronic 3 (Temel SA) respirator. The analysis was made on a pulmonary monoalveolar model with a compliance of 50 ml/cmH2 = and a resistance of 20 cmH2O/l/se simulating an obstructive pattern. Results were also compared with those obtained in a previous study on normal lung. HFJV was applied using a jet with or without additional gas input (VE) at respiratory rates of 90, 120, 150, 180, 210, 240, 270, and 300 per minute varying in each frequency the releasing pressure of the jet (PG 1, 2, and 3 kg/cm2) and the inspiratory time (Ti 30 to 50%) giving rise to a total of 96 different situations. In each of these conditions we measured the air way pressure (Paw) and the alveolar pressure (PA) and we calculated the tidal volume (VT) and the trapped volume (VAT). These measurements were correlated with those monitored by the respirator and those obtained in the normal lung. Results show that the VT is composed almost exclusively by the jet volume (VTjet), being only of consideration the VE with a Ti of about 33% and a PG of 2 to 3 kg/cm2. When the normal pattern was compared with the obstructive one, a small reduction in VT (average of 15%) and a marked increase in peak Paw (average of 75%) were observed. The use of a Ti of 50% has no clinical interest due to the large VAT that it produces.(ABSTRACT TRUNCATED AT 250 WORDS)

High-Frequency Jet Ventilation↗

FASTSNP: an always up-to-date and extendable service for SNP function analysis and prioritization.

Single nucleotide polymorphism (SNP) prioritization based on the phenotypic risk is essential for association studies. Assessment of the risk requires access to a variety of heterogeneous biological databases and analytical tools. FASTSNP (function analysis and selection tool for single nucleotide polymorphisms) is a web server that allows users to efficiently identify and prioritize high-risk SNPs according to their phenotypic risks and putative functional effects. A unique feature of FASTSNP is that the functional effect information used for SNP prioritization is always up-to-date, because FASTSNP extracts the information from 11 external web servers at query time using a team of web wrapper agents. Moreover, FASTSNP is extendable by simply deploying more Web wrapper agents. To validate the results of our prioritization, we analyzed 1569 SNPs from the SNP500Cancer database. The results show that SNPs with a high predicted risk exhibit low allele frequencies for the minor alleles, consistent with a well-known finding that a strong selective pressure exists for functional polymorphisms. We have been using FASTSNP for 2 years and FASTSNP enables us to discover a novel promoter polymorphism. FASTSNP is available at http://fastsnp.ibms.sinica.edu.tw.

Gene Frequency↗

Functional analysis of RRD1 (YIL153w) and RRD2 (YPL152w), which encode two putative activators of the phosphotyrosyl phosphatase activity of PP2A in Saccharomyces cerevisiae.

In the context of the cooperative project for functional analysis of novel genes uncovered during the systematic sequencing of the Saccharomyces cerevisiae genome, we deleted two paralogous ORFs: YIL153w and YPL152w. Based on the resulting phenotypes, the corresponding genes were named RRD1 and RRD2, respectively. Rrd proteins show significant similarity to the human phosphotyrosyl phosphatase activator (PTPA). Both single mutants, rrd1delta and rrd2delta, were viable. Deletion of RRD1 caused pleiotropic phenotypes under a wide range of conditions, including sensitivity to Ca2+, vanadate, ketoconazole, cycloheximide and Calcofluor white, and resistance to caffeine and rapamycin. The only phenotypes found for rrd2delta - resistance to caffeine and rapamycin - were weaker than the corresponding phenotypes of rrd1delta. The double mutant rrd1,2delta was inviable on rich glucose medium, but could grow in the presence of an osmotic stabilizer. The rrd1,2delta mutant was partially rescued by inactivation of HOG1 or PBS2, suggesting an interaction between the RRD genes and the Hog1p signal transduction pathway. Introduction of slt2delta into the rrd1,2delta background improved the growth of rrd1,2delta on sorbitol-containing medium, indicating that the Rrd proteins also interact with the Slt2p/Mpk1p signaling pathway. Suppression of the lethal phenotype of the rrd1,2delta mutant by overexpression of PPH22 suggested that the products of the RRD genes function positively with catalytic subunits of PP2A. The synthetic lethality was also suppressed by the "viable" allele (SSD1-v1) of the SSD1 gene.

Alleles↗

CDNA-AFLP combined with functional analysis reveals novel genes involved in the hypersensitive response.

To identify genes required for the hypersensitive response (HR), we performed expression profiling of tomato plants mounting a synchronized HR, followed by functional analysis of differentially expressed genes. By cDNA-AFLP analysis, the expression profile of tomato plants containing both the Cf-4 resistance gene against Cladosporium fulvum and the matching Avr4 avirulence gene of this fungus was compared with that of control plants. About 1% of the transcript-derived fragments (442 out of 50,000) were derived from a differentially expressed gene. Based on their sequence and expression, 192 fragments, referred to as Avr4-responsive tomato (ART) fragments, were selected for VIGS (virus-induced gene silencing) in Cf-4-transgenic Nicotiana benthamiana. Inoculated plants were analyzed for compromised HR by agroinfiltration of either the C. fulvum Avr4 gene or the Inf1 gene of Phytophthora infestans, which invokes a HR in wild-type N. benthamiana. VIGS using 15 of the ART fragments resulted in a compromised HR, whereas VIGS with fragments of ART genes encoding HSP90, a nuclear GTPase, an L19 ribosomal protein, and most interestingly, a nucleotide binding-leucine rich repeat (NB-LRR)-type protein severely suppressed the HR induced both by Avr4 and Inf1. Requirement of an NB-LRR protein (designated NRC1, for NB-LRR protein required for HR-associated cell death 1) for Cf resistance protein function as well as Inf1-mediated HR suggests a convergence of signaling pathways and supports the recent observation that NB-LRR proteins play a role in signal transduction cascades downstream of resistance proteins.

Algal Proteins↗

Identification and functional analysis of human Tom22 for protein import into mitochondria.

Mitochondria have a receptor complex in the outer membrane which recognizes and translocates mitochondrial proteins synthesized in the cytosol. We report here the identification and functional analysis of human Tom22 (hTom22). hTom22 has an N-terminal negatively charged region exposed to the cytosol, a putative transmembrane region, and a C-terminal intermembrane space region with little negative charge. Tom22 forms a complex with Tom20, and its cytosolic domain functions as an import receptor as in fungi. An import inhibition assay, using pre-ornithine transcarbamylase (pOTC) derivatives and a series of hTom22 deletion mutants, showed that the C-terminal segment of the cytosolic domain is important for presequence binding, whereas the N-terminal domain is important for binding to the mature portion of pOTC. No evidence for pOTC interaction with the Tom22 intermembrane space domain was obtained. Binding studies revealed that the presequence is critical for pOTC binding to Tom20, whereas both the presequence and mature portion are important for binding to Tom22. A cell-free immunoprecipitation assay indicated that an internal segment of the Tom22 cytosolic domain is important for interaction with Tom20.

Amino Acid Sequence↗

Functional analysis of six genes from chromosomes XIV and XV of Saccharomyces cerevisiae reveals YOR145c as an essential gene and YNL059c/ARP5 as a strain-dependent essential gene encoding nuclear proteins.

We report here basic functional analysis of strains deleted for six open reading frames (ORFs), YNL059c and YNL148c from chromosome XIV and YOR145c, YOR152c, YOR161c and YOR162c from chromosome XV of Saccharomyces cerevisiae. ORFs were replaced with the KanMX4 resistance marker using a long flanking homology PCR strategy in FY1679 and W303 diploid strains. Replacement cassettes were constructed in plasmid pUG7 and the cognate wild-type genes were recovered by gap repair. Sporulation and tetrad analysis revealed that deletion of YNL059c/ARP5 was lethal for vegetative growth in strain W303 and caused severe growth defects in strain FY1679 while YOR145c was essential for growth in both strains. Fusion of the green fluorescent protein (GFP) gene to the 3' ends of the YNL059c/ARP5 and YOR145c coding sequences created functional chimeric genes at the respective chromosomal loci. Both Arp5-GFP and Yor145-GFP localized to the nucleus, Yor145-GFP concentrating in the nucleolus. The vectors containing the deletion cassettes and the cognate wild-type genes, the oligonucleotides, and the deletant strains are available from the EUROFAN resource centre EUROSCARF (Frankfurt).

Actins↗

Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae patovar savastanoi.

The sequence of a 1823 base-pair region containing the replication functions of pPS10, a narrow host-range plasmid isolated from a strain of Pseudomonas savastanoi, is reported. The origin of replication, oriV, or pPS10 is contained in a 535 base-pair fragment of this sequence that can replicate in the presence of trans-acting function(s) of the plasmid. oriV contains four iterons of 22 base-pairs that are preceded by G+C-rich and A+T-rich regions. A dnaA box located adjacent to the repeats of the origin is dispensable but required for efficient replication of pPS10; A and T are equivalent bases at the 5' end of the box. repA, the gene of a trans-acting replication protein of 26,700 Mr has been identified by genetic and functional analysis. repA is adjacent to the origin of replication and is preceded by the consensus sequences of a typical sigma 70 promoter of Escherichia coli. The RepA protein has been identified, using the minicell system of E. coli, as a polypeptide with an apparent molecular mass of 26,000. A minimal pPS10 replicon has been defined to a continuous 1267 base-pair region of pPS10 that includes the oriV and repA sequences.

Amino Acid Sequence↗

Discriminant function analysis of clinical and psychological variables in temporomandibular joint pain dysfunction.

Detailed clinical, psychological and illness behaviour information was obtained from 43 patients with temporomandibular joint pain dysfunction. A discriminant function analysis was performed on the multiple variables to test their effectiveness as predictors of a successful response to simple conservative therapy. The questionnaire and statistical methodology employed are described in detail. It was found that clinical, psychological, and illness behaviour factors when used alone were poor predictors (less than 60 per cent) of treatment outcome. However, grouping clinical and psychological factors resulted in over 80 per cent successful prediction.

Adult↗

Computer assistance in clinical functional analysis.

The use of computers in the dental practice has been primarily restricted to the acquisition of billing data. Additional possibilities for use of PCs exist in diagnostic data acquisition and evaluation; clinical functional analysis seems a particularly suitable application. Such software is now available: CMDfact. Dentally, it is based on a previously developed and published examination and documentation system, the graphic user interface of which is used in the newly developed software. After the examination data have been acquired by mouse click or numerical entry, these are available for evaluation. A special function, the "Diagnosis pilot" is integrated to support the user. This helps in the assignment of the appropriate "Initial diagnoses", since it brings together the individually existing principal symptoms and suitable diagnoses for the initial diagnosis in question and also states which diagnoses "would be appropriate" for this, but are not available. With 3D animation, the software also helps the dentist to explain aspects of CMD to patients. The software also assists the dentist with a detailed multimedia help system, which provides context-sensitive help for every examination step. These help functions explain the sense of the relevant examinations, their performance and evaluation in the form of short texts and explanatory photographs and videos.

Computer Graphics↗

Cloning and functional analysis of a cDNA encoding Ginkgo biloba farnesyl diphosphate synthase.

Farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2. 5.1.10) catalyzes the synthesis of farnesyl diphosphate, and provides precursor for biosynthesis of sesquiterpene and isoprenoids containing more than 15 isoprene units in Ginkgo biloba. Here we report the cloning, characterization and functional analysis of a new cDNA encoding FPS from G. biloba. The full-length cDNA (designated GbFPS) had 1731 bp with an open reading frame of 1170 bp encoding a polypeptide of 390 amino acids. The deduced GbFPS was similar to other known FPSs and contained all the conserved regions of trans-prenyl chain-elongating enzymes. Structural modeling showed that GbFPS had the typical structure of FPS, the most prominent feature of which is the arrangement of 13 core helices around a large central cavity. Southern blot analysis revealed a small FPS gene family in G. biloba. Expression analysis indicated that GbFPS expression was high in roots and leaves, and low in stems. Functional complementation of GbFPS in an FPS-deficient strain confirmed that GbFPS mediates farnesyl diphosphate biosynthesis.

Alkyl and Aryl Transferases↗

Functional analysis of problem behavior in children with mental retardation.

Pediatric nurses are frequently asked to give advice on behavior management. This can become considerably more difficult when the child in question has limited cognitive abilities and communication skills. It is especially complex when the behavior in question involves self-injury or aggression. Frequently used behavior modification strategies such as time-out may actually reinforce the inappropriate behavior in these children by allowing them to escape from an aversive task. Likewise, other common behavior management strategies such as discussion, saying "no," or yelling at the child, may reinforce the inappropriate behavior by providing attention for it. It is sometimes difficult to identify the antecedents of self-injurious or aggressive behavior, making appropriate behavior management plans elusive. However, functional analysis can be used to identify the functions that serve to maintain a child's inappropriate behavior, and may provide a basis for developing and implementing an individualized behavior management plan. The possibility that discomfort from an untreated or undertreated physiologic problem is exacerbating the behavior should not be overlooked.

Behavior Therapy↗

Functional analysis of a novel GATA3 mutation in a family with the hypoparathyroidism, deafness, and renal dysplasia syndrome.

The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of a member of the GATA-binding family of transcription factors, GATA3. This dual zinc finger transcription factor binds DNA with its C-terminal zinc finger (ZnF2) and stabilizes this binding with its N-terminal zinc finger (ZnF1). ZnF1 also interacts with other zinc finger proteins, notably Friend of GATA (FOG). The HDR syndrome has been described in patients with mutations affecting both ZnF1 and ZnF2 domains; the former result in inefficient interaction with FOG, and the latter result in disruption of DNA binding. We report a patient with renal failure, hypoparathyroidism, and bilateral hearing loss. Assessment of family members indicated that the disease arose as a de novo mutation in her mother. Analysis of GATA3 in the family revealed a heterozygous missense mutation resulting in a nonconservative change of a single amino acid (R276P) in the ZnF1 domain. Functional analysis using dissociation electrophoretic mobility shift and yeast two-hybrid assays showed reduced binding affinity to the GATA motifs but normal interaction with FOG in vitro. These results are consistent with the predicted functions of human GATA3-ZnF1 from three-dimensional molecular modeling and with HDR being a result of GATA3 haploinsufficiency.

Adolescent↗

Differences among species in compact bone tissue microstructure of mammalian skeleton: use of a discriminant function analysis for species identification.

In order to develop an identification key for distinguishing between several mammalian species, bone structure of their compact bone tissue was analyzed using qualitative and quantitative characteristics. Ninety femora of adult male humans, pigs, cows, sheep, rabbits, and rats were studied. The average area, perimeter, minimum, and maximum diameter of 1863 Haversian canals and 1863 secondary osteons were measured using a digital image device. The observed data were first used to evaluate inter- and intraspecies diversity. After that, we applied a discriminant function analysis for differentiation of the species by these variables. Classification functions for investigated species give cross-validated correct classification rates for 76.17% of cases. This percentage value can be increased by integrating conclusions from the qualitative analysis.

Adult↗

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA. III. Functional analysis of the 690 loop.

An instant-evolution experiment was performed on the eight nucleotides comprising the loop region of the 690 hairpin in Escherichia coli 16 S ribosomal RNA. Positions 690 to 697 were randomly mutated and 101 unique functional mutants were isolated, sequenced and analyzed for function in vivo. Non-random nucleotide distributions were observed at each of the mutated positions except 693 and 694. Nucleotide identity significantly affected ribosome function at positions 690, 695, 696 and 697. Pyrimidines were absent at position 696 in the instant-evolution pool as were C at position 691 and G at position 697. A highly significant covariation was observed between nucleotides 690 and 697. No functional double mutants at positions 691 and 696 were obtained from the instant-evolution pool. In our NMR structure of the 690 loop, both the G690.U697 and G691.A696 form sheared hydrogen-bonded mismatches. To further examine the functional constraints between these paired nucleotides, one set of site-directed mutations was constructed at positions 690:697 and another set was constructed at positions 691:696. Functional analysis of the site-directed mutants is consistent with our instant-evolution findings and revealed constraints on the placement of specific functional groups observed in the NMR structure. Ten instant-evolution mutants were isolated that are more functional than the wild-type. Hyperactivity in these mutants correlates with a single mutation at position 693.

Base Pair Mismatch↗