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Results for “Sequence Homology, Amino Acid”

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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1998

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

NASA Discipline Plant Biology↗
NASA NTRS2002

Conservation of Pax gene expression in ectodermal placodes of the lamprey

Ectodermal placodes contribute to the cranial ganglia and sense organs of the head and, together with neural crest cells, represent defining features of the vertebrate embryo. The identity of different placodes appears to be specified in part by the expression of different Pax genes, with Pax-3/7 class genes being expressed in the trigeminal placode of mice, chick, frogs and fish, and Pax-2/5/8 class genes expressed in the otic placode. Here, we present the cloning and expression pattern of lamprey Pax-7 and Pax-2, which mark the trigeminal and otic placodes, respectively, as well as other structures characteristic of vertebrate Pax genes. These results suggest conservation of Pax genes and placodal structures in basal and derived vertebrates.

NASA Discipline Evolutionary Biology↗
NASA NTRS1993

Kinetic Induction of Oat Shoot Pulvinus Invertase mRNA by Gravistimulation and Partial cDNA Cloning by the Polymerase Chain Reaction

An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)(+) RNA, isolated from oat pulvini, and two oli-gonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 hour after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

Wu, Liu-Lai↗
NASA NTRS1998

Assessment of changes in microbial community structure during operation of an ammonia biofilter with molecular tools

Biofiltration has been used for two decades to remove odors and various volatile organic and inorganic compounds in contaminated off-gas streams. Although biofiltration is widely practiced, there have been few studies of the bacteria responsible for the removal of air contaminants in biofilters. In this study, molecular techniques were used to identify bacteria in a laboratory-scale ammonia biofilter. Both 16S rRNA and ammonia monooxygenase (amoA) genes were used to characterize the heterotrophic and ammonia-oxidizing bacteria collected from the biofilter during a 102-day experiment. The overall diversity of the heterotrophic microbial population appeared to decrease by 38% at the end of the experiment. The community structure of the heterotrophic population also shifted from predominantly members of two subdivisions of the Proteobacteria (the beta and gamma subdivisions) to members of one subdivision (the gamma subdivision). An overall decrease in the diversity of ammonia monooxygenase genes was not observed. However, a shift from groups dominated by organisms containing Nitrosomonas-like and Nitrosospira-like amoA genes to groups dominated by organisms containing only Nitrosospira-like amoA genes was observed. In addition, a new amoA gene was discovered. This new gene is the first freshwater amoA gene that is closely affiliated with Nitrosococcus oceanus and the particulate methane monooxygenase gene from the methane oxidizers belonging to the gamma subdivision of the Proteobacteria.

NASA Discipline Life Support Systems↗
NASA NTRS1994

The sequence, and its evolutionary implications, of a Thermococcus celer protein associated with transcription

Through random search, a gene from Thermococcus celer has been identified and sequenced that appears to encode a transcription-associated protein (110 amino acid residues). The sequence has clear homology to approximately the last half of an open reading frame reported previously for Sulfolobus acidocaldarius [Langer, D. & Zillig, W. (1993) Nucleic Acids Res. 21, 2251]. The protein translations of these two archaeal genes in turn are homologs of a small subunit found in eukaryotic RNA polymerase I (A12.2) and the counterpart of this from RNA polymerase II (B12.6). Homology is also seen with the eukaryotic transcription factor TFIIS, but it involves only the terminal 45 amino acids of the archaeal proteins. Evolutionary implications of these homologies are discussed.

Non-NASA Center↗
NASA NTRS2000

A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

Non-NASA Center↗
NASA NTRS1995

Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

NASA Discipline Neuroscience↗
NASA NTRS1995

Isolation, characterization, and primary structure of rubredoxin from the photosynthetic bacterium, Heliobacillus mobilis

Rubredoxin is a small nonheme iron protein that serves as an electron carrier in bacterial systems. Rubredoxin has now been isolated and characterized from the strictly anaerobic phototroph, Heliobacillus mobilis. THe molecular mass (5671.3 Da from the amino acid sequence) was confirmed and partial formylation of the N-terminal methionyl residue was established by matrix-assisted laser desorption mass spectroscopy. The complete 52-amino-acid sequence was determined by a combination of N-terminal sequencing by Edman degradation and C-terminal sequencing by a novel method using carboxypeptidase treatment in conjunction with amino acid analysis and laser desorption time of flight mass spectrometry. The molar absorption coefficient of Hc. mobilis rubredoxin at 490 nm is 6.9 mM-1 cm-1 and the midpoint redox potential at pH 8.0 is -46 mV. The EPR spectrum of the oxidized form shows resonances at g = 9.66 and 4.30 due to a high-spin ferric iron. The amino acid sequence is homologous to those of rubredoxins from other species, in particular, the gram-positive bacteria, and the phototrophic green sulfur bacteria, and the evolutionary implications of this are discussed.

NASA Discipline Number 52-30↗
NASA NTRS1993

Molecular Basis of the Increase in Invertase Activity Elicited by Gravistimulation of Oat-Shoot Pulvini

An asymmetric (top vs. bottom) increase in invertase activity is elicited by gravistimulation in oatshoot pulvini starting within 3h after treatment. In order to analyze the regulation of invertase gene expression in this system, we examined the effect of gravistimulation on invertase mRNA induction. Total RNA and poly(A)(+)RNA, isolated from oat pulvini, and two oligonucleotide primers, corresponding to two conserved amino-acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the Polymerase Chain Reaction (PCR). A partial-length cDNA (550 base pairs) was obtained and characterized. There was a 52 % deduced amino-acid sequence homology to that of carrot beta-fructosi- dase and a 48 % homology to that of tomato invertase. Northern blot analysis showed that there was an obvious transient accumulation of invertase mRNA elicited by gravistimulation of oat pulvini. The mRNA was rapidly induced to a maximum level at 1h following gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher (five-fold) than that in the top half of the pulvinus tissue. The induction of invertase mRNA was consistent with the transient enhancement of invertase activity during the graviresponse of the pulvinus. These data indicate that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional and/or translational levels. Southern blot analysis showed that there were four genomic DNA fragments hybridized to the invertase cDNA. This suggests that an invertase gene family may exist in oat plants.

Wu, Liu-Lai↗
NASA NTRS2003

Senseless acts as a binary switch during sensory organ precursor selection

During sensory organ precursor (SOP) specification, a single cell is selected from a proneural cluster of cells. Here, we present evidence that Senseless (Sens), a zinc-finger transcription factor, plays an important role in this process. We show that Sens is directly activated by proneural proteins in the presumptive SOPs and a few cells surrounding the SOP in most tissues. In the cells that express low levels of Sens, it acts in a DNA-binding-dependent manner to repress transcription of proneural genes. In the presumptive SOPs that express high levels of Sens, it acts as a transcriptional activator and synergizes with proneural proteins. We therefore propose that Sens acts as a binary switch that is fundamental to SOP selection.

NASA Program Fundamental Space Biology↗
NASA NTRS2000

New genes and new biological roles for expansins

Expansins are extracellular proteins that loosen plant cell walls in novel ways. They are thought to function in cell enlargement, pollen tube invasion of the stigma (in grasses), wall disassembly during fruit ripening, abscission and other cell separation events. Expansins are encoded by two multigene families and each gene is often expressed in highly specific locations and cell types. Structural analysis indicates that one expansin region resembles the catalytic domain of family-45 endoglucanases but glucanase activity has not been detected. The genome projects have revealed numerous expansin-related sequences but their putative wall-loosening functions remain to be assessed.

NASA Program Fundamental Space Biology↗
NASA NTRS1999

Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

NASA Discipline Plant Biology↗
NASA NTRS1994

The beetle Tribolium castaneum has a fushi tarazu homolog expressed in stripes during segmentation

The genetic control of embryonic organization is far better understood for the fruit fly Drosophila melanogaster than for any other metazoan. A gene hierarchy acts during oogenesis and embryogenesis to regulate the establishment of segmentation along the anterior-posterior axis, and homeotic selector genes define developmental commitments within each parasegmental unit delineated. One of the most intensively studied Drosophila segmentation genes is fushi tarazu (ftz), a pair-rule gene expressed in stripes that is important for the establishment of the parasegmental boundaries. Although ftz is flanked by homeotic selector genes conserved throughout the metazoa, there is no evidence that it was part of the ancestral homeotic complex, and it has been unclear when the gene arose and acquired a role in segmentation. We show here that the beetle Tribolium castaneum has a ftz homolog located in its Homeotic complex and expressed in a pair-rule fashion, albeit in a register differing from that of the fly gene. These and other observations demonstrate that a ftz gene preexisted the radiation of holometabolous insects and suggest that it has a role in beetle embryogenesis which differs somewhat from that described in flies.

NASA Discipline Developmental Biology↗
NASA NTRS1994

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

Schistosoma japonicum/enzymology/genetics/immunolo↗
NASA NTRS1998

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

Non-NASA Center↗
NASA NTRS1998

Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.

Non-NASA Center↗
NASA NTRS2003

The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

NASA Discipline Plant Biology↗