bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization and functional analysis of three wheat genes with homology to the CONSTANS flowering time gene in transgenic rice
Authors: Nemoto Yasue , Kisaka Mayumi , Fuse Takuichi , Yano Masahiro , Ogihara Yasunari 
Published: September 2003

SummaryThe CONSTANS (CO) gene of Arabidopsis plays a key role in the photoperiodic flowering pathway. To investigate photoperiod responses in cereals in more detail, we isolated three kinds of CO/Hd1 (rice ortholog of CO) homolog from hexaploid wheat, derived from the A, B, and D genomes and designated as wheat ortholog of CO from A genome (TaHd1‐1), TaHd1‐2, and TaHd1‐3, respectively. They were highly similar to each other and to Hd1, and in addition harbored two conserved regions: two zinc finger motifs and CONSTANS, CONSTANS‐LIKE and TIMING OF CAB EXPRESSION 1 (CCT) domain like CO/Hd1. They were located on the long arm of the homoeologous chromosome 6. TaHd1‐2 harbored a 63‐bp deletion at the promoter region containing the GATA‐1 box, and consequently, we detected no subsequent transcript. The TaHd1‐1 genomic clone was introduced to a rice line deficient in Hd1 function. Transgenic plants complemented the functions of rice Hd1: they promoted heading under short‐day (SD) conditions and delayed it under long‐day (LD)/natural conditions, indicating that Hd1 proteins from SD and LD plants share common structures and functions.
Distribution and characterization of over 1000 T‐DNA tags in rice genome
Authors: Chen Shuangyan , Jin Weizheng , Wang Mingyi , Zhang Fan , Zhou Jie , Jia Qiaojun , Wu Yunrong , Liu Feiyan , Wu Ping 
Published: September 2003

SummaryWe generated T‐DNA insertions throughout the rice genome for saturation mutagenesis. More than 1000 flanking sequences were mapped on 12 rice chromosomes. Our results showed that T‐DNA tags were not randomly spread on rice chromosomes and were preferentially inserted in gene‐rich regions. Few insertions (2.4%) were found in repetitive regions. T‐DNA insertions in genic (58.1%) and intergenic regions (41.9%) showed a good correlation with the predicted size distribution of these sequences in the rice genome. Whereas, obvious biases were found for the insertions in the 5′‐ and 3′‐regulatory regions outside the coding regions both at 500‐bp size and in introns rather than in exons. Such distribution patterns and biases for T‐DNA integration in rice are similar to that of the previous report in Arabidopsis, which may result from T‐DNA integration mechanism itself. Rice will require approximately the same number of T‐DNA insertions for saturation mutagenesis as will Arabidopsis. A database of the T‐DNA insertion sites in rice is publicly available at our web site (http://www.genomics.zju.edu.cn/ricetdna).
Establishment of a high‐efficiency SNP‐based framework marker set for Arabidopsis
Authors: Törjék O. , Berger D. , Meyer R. C. , Müssig C. , Schmid K. J. , Rosleff Sörensen T. , Weisshaar B. , Mitchell‐Olds T. , Altmann T. 
Published: September 2003

SummaryThe major goal of this project was the establishment of a tool for rapid mapping of new mutations and genotyping in Arabidopsis consisting of at least 100 evenly spaced framework markers. We assembled a single nucleotide polymorphism (SNP)‐based marker set consisting of 112 polymorphic sites with average spacing of 1.15 Mbp derived from an SNP database that we recently developed. This information was used to set up efficient SNP detection reactions based on multiplexed primer extension assays. The 112 Columbia (Col‐0)/C24 framework markers were used to assemble 18 multiplexed SNaPshot assays with which up to eight separate loci can be genotyped in a single‐tube/single‐capillary format. In addition, for 110 framework markers matrix‐assisted laser desorption/ionization time of flight (MALDI‐ToF) assays have been established for high throughput analyses. We demonstrated the usefulness and the robustness of both procedures of this tool by genotyping 48 BC3F1 individuals created between the accessions Col‐0 and C24. Subsets of 10–62 of the established markers discriminate between various combinations of the accessions Col‐0, C24, Landsberg erecta (Ler), Cape Verdi Islands (Cvi) and Niederzenz (Nd). Using a subset of 17 evenly distributed and established SNP markers that are also polymorphic between Ler and Col‐0, we were able to rapidly map a mutant gene (tbr1) to an interval of 2.3 Mbp in an Ler (tbr1) × Col‐0 cross.
The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis
Authors: Kim Jeong Hoe , Choi Dongsu , Kende Hans 
Published: September 2003

SummaryPreviously, we identified a novel rice gene, GROWTH‐REGULATING FACTOR1 (OsGRF1), which encodes a putative transcription factor that appears to play a regulatory role in stem elongation. We now describe the GRF gene family of Arabidopsis thaliana (AtGRF), which comprises nine members. The deduced AtGRF proteins contain the same characteristic regions—the QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) domains—as do OsGRF1 and related proteins in rice, as well as features indicating a function in transcriptional regulation. Most of the AtGRF genes are strongly expressed in actively growing and developing tissues, such as shoot tips, flower buds, and roots, but weakly in mature stem and leaf tissues. Overexpression of AtGRF1 and AtGRF2 resulted in larger leaves and cotyledons, as well as in delayed bolting of the inflorescence stem when compared to wild‐type plants. In contrast, triple insertional null mutants of AtGRF1–AtGRF3 had smaller leaves and cotyledons, whereas single mutants displayed no changes in phenotype and double mutants displayed only minor ones. The alteration of leaf growth in overexpressors and triple mutants was based on an increase or decrease in cell size, respectively. These results indicate that AtGRF proteins play a role in the regulation of cell expansion in leaf and cotyledon tissues.
Characterization of a proximal element in the rat preadipocyte factor‐1 (Pref‐1) gene promoter
Authors: Takemori Hiroshi , Doi Junko , Katoh Yoshiko , Halder Sunil K , Lin Xing‐zi , Horike Nanao , Hatano Osamu , Okamoto Mitsuhiro 
Published: September 2003

Preadipocyte factor‐1 (Pref‐1) was shown to negatively regulate adipocyte differentiation. We recently reported that ZOG, a rat homolog of Pref‐1, was specifically expressed in the adrenal zona glomerulosa. Results of the investigation of Pref‐1 expression in preadipocyte and in undifferentiated adrenal cortex suggested that down‐regulation of Pref‐1 gene was closely correlated with the differentiation process. In this study we demonstrate that an upstream region (from −76 to −47) of the rat Pref‐1 gene was essential for its expression in adrenocortical carcinoma‐derived H295R cells. A nucleotide sequence found in this region, GCGTGGGCGTGGGCGGGGG (Egr/GC‐box), seemed to contain three elements, two early growth response (Egr) elements and one GC‐box, overlapping each other. Mutations of four or five nucleotides in a 7‐nucleotides‐stretch in the midst of the Egr/GC‐box eliminated the binding of Sp1/3, abolished the activation by Egr‐factor(s) and diminished the Pref‐1 promoter activity. When mutations were introduced into the outside of the middle portion, the binding of Sp1/3 to the Egr/GC‐box was abolished similarly. However, the decrease in the promoter activity was less than that found with the construct mutated at the middle. These results indicated that an element present at the 7‐nucleotides‐stretch in the midst of the Egr/GC‐box might be important for the Pref‐1 promoter activity, and this proximal element was possibly activated by a still‐unidentified nuclear factor(s). This element would function as the promoter of the Pref‐1 gene in H295R cells, but not in HeLa cells.
Identification and characterization of novel chromogranin B‐derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS
Authors: Wang Zesheng , Vandenberghe Isabel , Depreitere Jan , Devreese Bart , Clerens Stefan , Nouwen Etienne J , Van Beeumen Jozef , De Potter Werner 
Published: September 2003

Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocrine cells. It can be processed proteolytically to small peptide fragments. In the present study three proteolytic products of porcine CgB were obtained after size‐exclusion, immunoaffinity, and reversed‐phase chromatography, and then identified by electrospray tandem MS. One novel peptide was identified as S586–R602 (SR‐17) and is phosphorylated at one or two serine residues. Another novel peptide H603–Q636 (HQ‐34), with molecular mass 3815.56 Da, was found to be oxidized at the methionine residue. In addition, a secretolytin‐like peptide fragment (KR‐11), which is two amino acids shorter than the bovine secretolytin, was found. This is the first report that the C‐terminal region of CgB, the homologue of human CCB, is proteolytically processed further into three small peptide fragments.
Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam
Authors: Yoshioka Shiro , Takahashi Satoshi , Hori Hiroshi , Ishimori Koichiro , Morishima Isao 
Published: September 2003

To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450cam, we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450cam from the inclusion bodies. The C357H mutant in the ferrous‐CO form exhibited the Soret peak at 420 nm and the Fe‐CO stretching line at 498 cm−1, indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate‐binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O–O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450cam is important for the construction of the heme pocket and the heterolysis of the O–O bond.
Evaluating low level sequence identities
Authors: Nicholas Hugh B. , Arst Herbert N. , Caddick Mark X. 
Published: September 2003

A review published several years ago [Hawkins, A.R. & Lamb, H.K. (1995) Eur. J. Biochem. 232, 7–18] proposed that genetic, biochemical and physiological data can override sequence comparison in the determination of homology in instances where structural information is unavailable. Their lead example was the hypothesis that the transcriptional activator protein for quinate catabolism in Aspergillus nidulans, QUTA, is derived from the pentafunctional AROM protein by a gene duplication followed by cleavage [Hawkins, A.R., Lamb, H.K., Moore, J.D. & Roberts, C.F. (1993) Gene136, 49–54]. We tested this hypothesis by a sensitive combination of position‐specific log‐odds scoring matrix methods. The position‐specific log‐odds scoring matrices were derived from a large number of 3‐dehydroquinate synthase and 5‐enolpyruvylshikimate‐3‐phosphate synthase domains that were proposed to be the domains from the AROM protein that gave rise to the transcriptional activator protein for quinate metabolism. We show that the degree and pattern of similarity between these position‐specific log‐odds scoring matrices and the transcriptional activator protein for quinate catabolism in A. nidulans is that expected for random sequences of the same composition. This level of similarity provides no support for the suggested gene duplication and cleavage. The lack of any trace of evidence for homology following a comprehensive sequence analysis indicates that the homology hypothesis is without foundation, underlining the necessity to accept only similarity of sequence and/or structure as evidence of evolutionary relatedness. Further, QUTA is homologous throughout its entire length to an extended family of fungal transcriptional regulatory proteins, rendering the hypothesized QUTA–AROM homology even more problematic.
The C‐terminal cysteine‐rich region dictates specific catalytic properties in chimeras of the ectonucleotidases NTPDase1 and NTPDase2
Authors: Heine Petra , Braun Norbert , Sévigny Jean , Robson Simon C. , Servos Jörg , Zimmermann Herbert 
Published: September 2003

Ecto‐nucleoside triphosphate diphosphohydrolases (E‐NTPDases) comprise a novel family of ectonucleotidases that are important in the hydrolysis of extracellular nucleotides. The related NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) share a common membrane topography with a transmembrane domain at both the N‐ and C‐terminus, an extensive extracellular loop with five ‘apyrase conserved regions’ (ACR1 to ACR5), and a cysteine‐rich C‐terminal region. Whereas NTPDase1 expressed in CHO cells hydrolyzes ATP and ADP equivalently, NTPDase2 has a high preference for the hydrolysis of ATP over ADP. In addition recombinant NTPDase1 hydrolyzes ATP to AMP with the formation of only minor amounts of free ADP. In contrast, ADP appears as the major free product when ATP is hydrolyzed by NTPDase2. In order to determine molecular domains responsible for these differences in catalytic properties, chimeric cDNAs were constructed in which N‐terminal sequences of increasing length of NTPDase1 were substituted by the corresponding sequences of NTPDase2 and vice versa. The turnover points were contained within ACR1 to ACR5. Chimeric cDNAs were expressed in CHO cells and surface expression was verified by immunocytochemistry. ATP and ADP hydrolysis rates and ADP and AMP product formation were determined using HPLC. Amino‐acid residues between ACR3 and ACR5 and in particular the cysteine‐rich region between ACR4 and ACR5 conferred a phenotype to the chimeric enzymes that corresponded to the respective wild‐type enzyme. Protein structure rather than the conserved ACRs may be of major relevance for determining differences in the catalytic properties between the related wild‐type enzymes.
Identification of tyrosine‐phosphorylated proteins associated with the nuclear envelope
Authors: Otto Henning , Dreger Mathias , Bengtsson Luiza , Hucho Ferdinand 
Published: September 2003

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation‐dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine‐phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine‐phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine‐specific antibodies, tryptic in‐gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2β, the heat shock protein hsc70, and the DNA/RNA‐binding proteins PSF, hypothetical 16‐kDa protein, and NonO, which copurify with the nuclear envelope.
Phosphatidyl inositol‐4,5‐bisphosphate bound to bovine cardiac Na+/Ca2+ exchanger displays a MgATP regulation similar to that of the exchange fluxes
Authors: Asteggiano Carla , Berberián Graciela , Beaugé Luis 
Published: September 2003

This work shows the existence of a phosphatidylinositol 4,5‐bisphosphate (PtdIns‐4,5‐P2) bound form of the cardiac sarcolemmal Na+/Ca2+ exchanger. That was demonstrated in Western blots and cross‐immunoprecipitation by using specific antibodies against the NCX1 exchanger (NCX1) and against PtdIns‐4,5‐P2. In addition, PtdIns‐4,5‐P2 bound to the Na+/Ca2+ exchanger and the Na+/Ca2+ exchange fluxes displayed a similar MgATP regulation: (a) both increase by 100–130% when membrane vesicles are incubated (15–20 s at 37 °C) with 1 mm MgATP and 1 µm Ca2+ (b) in the presence of 100 µm Ca2+, MgATP fails to stimulate the exchange fluxes and does not modify the levels of PtdIns‐4,5‐P2 bound to the exchanger. In addition, in the absence of Ca2+, the net synthesis of total membrane PtdIns‐4,5‐P2 is greatly reduced compared with that in the presence of 1 µm Ca2+. Furthermore, in the absence of Ca2+ there is no effect of MgATP on the levels of PtdIns‐4,5‐P2 bound to the exchanger. These results indicate that, in bovine heart, MgATP‐stimulation of Na+/Ca2+ exchange is associated with intracellular Ca2+‐dependent levels of PtdIns‐4,5‐P2 bound to the exchanger molecule.
Involvement of Rel factors in the expression of antimicrobial peptide genes in amphibia
Authors: Miele Rossella , Björklund Gunnel , Barra Donatella , Simmaco Maurizio , Engström Ylva 
Published: September 2003

Genes coding for antimicrobial peptides in amphibia reveal a remarkably high number of structural motifs for response elements, previously identified in the genes of insect antimicrobial peptides and in those of the mammalian acute phase response. This study focuses on the functional analysis of the bombinin gene promoter in a Drosophila blood cell line, and the identification of κB‐binding factors in skin secretions of the frog Bombina orientalis. Transfection experiments demonstrated that the bombinin gene promoter was activated in a lipopolysaccharide‐dependent manner, and that insect Rel factors target specific sequences in the amphibian gene promoter. After bathing frogs in bacteria, their skin secretions contained κB‐specific binding complexes, indicating that Rel factors are crucial components in the response against gram‐negative bacteria in this species. These results suggest that a common ancestral control mechanism governs the expression of the first line host‐defence from insects to vertebrates.
The low‐molecular‐mass subunit of the cell wall channel of the Gram‐positive Corynebacterium glutamicum
Authors: Lichtinger Thomas , Rieß Franziska G. , Burkovski Andreas , Engelbrecht Fredi , Hesse Dörte , Kratzin Hartmut D. , Krämer Reinhard , Benz Roland 
Published: September 2003

The 5‐kDa protein PorA of the Gram‐positive bacterium Corynebacterium glutamicum is the subunit of the cell wall channel. Antibodies raised against PorA specifically detected the protein on the cell surface. PorA was sequenced using Edman degradation and a gas phase sequencer. The primary sequence was used to create degenerate oligonucleotide primers. The gene of the channel‐forming protein and its flanking regions were obtained by PCR followed by inverse PCR. The gene porA comprises 138 bp and encodes a 45‐amino‐acid‐long acidic polypeptide with an excess of four negatively charged amino acids in agreement with the high cation selectivity of the PorA cell wall channel. PorA does not contain an N‐terminal extension. A ribosomal‐binding site was recognized 6 bp before the start codon ATG of porA. It codes for the smallest subunit of a membrane channel known so far and for the first cell wall channel protein of a corynebacterium. Southern blots demonstrated that only the chromosomes of corynebacteria contain homologous sequences to porA; no hybridization could be detected with DNA from other mycolata.
Crystal structures of bovine β‐lactoglobulin in the orthorhombic space group C2221
Authors: Oliveira Kesley M. G. , Valente‐Mesquita Vera L. , Botelho Michelle M. , Sawyer Lindsay , Ferreira Sérgio T. , Polikarpov Igor 
Published: September 2003

The crystal structures of β‐lactoglobulin genetic variants A and B have been determined in the orthorhombic space group C2221 (lattice Y) by X‐ray diffraction at 2.0 Å and 1.95 Å resolution, respectively. The structural comparison shows that both variants exhibit the open conformation of the EF loop at the pH of crystallization (pH 7.9), in contrast to what has been reported for the same genetic variants at pH 7.1 in the trigonal space group P3221 (lattice Z) [Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N. & Jameson, G.B. (1999) Protein Sci.8, 75–83]. Furthermore, it was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue.
Effect of Non‐Steroidal Aromatase Inhibitor on Blood Plasma Ovarian Steroid and Thyroid Hormones in Laying Hen (Gallus domesticus)
Authors: Sechman A. , Rząsa J. , Paczoska‐Eliasiewicz H. 
Published: September 2003

SummaryHyLine Brown laying hens at 30 weeks of age were treated twice daily with Fadrozole, a non‐steroidal aromatase inhibitor (AI; 1 mg/kg body weight; i.m.) for six consecutive days; control hens received saline. Blood was collected every day 0.5 h after oviposition, i.e. just before AI treatment. Ovarian steroids: progesterone (P4), testosterone (T) and estradiol (E2), and iodothyronines: thyroxine (T4), triiodothyronine (T3) and reverse‐triiodothyronine (rT3) were measured in blood plasma by radioimmunoassay methods. In AI‐treated hens a gradual delay in oviposition time was observed. AI significantly decreased P4 and E2 levels, maximally by 43% on day 4 and by 74% on day 5, respectively, and elevated T level, maximally by 248% on day 4. Simultaneously, the increases in T4 and T3 levels with no changes in rT3 levels were observed. The maximal effect of AI on T4 and T3 levels was found on day 4 (60% increase) and day 5 (312% increase), respectively. Moreover, statistically significant, negative coefficient of correlation between E2 and T3 (r = −0.51), and positive coefficient of correlation between T and T3 (r = 0.42) in AI‐treated hens were found. The results obtained indicate that in mature laying hens there is a strong relationship between ovarian steroids and thyroid hormones, and suppression of E2 synthesis not only disrupts ovarian function but also affects the activity of the thyroid gland and peripheral metabolism of thyroid hormones.