Vol. 9 No. 3 September 2004

Volume 9 (2004) pp 409-422
Title PROTECTIVE EFFECTS OF NICOTINE AGAINST GLUTAMATEINDUCED NEUROTOXICITY IN PC12 CELLS
Authors Xiulan Sun1,2, Yue Liu1,3, Gang Hu2 and Hai Wang1*
Abstract This study aimed to assess whether nicotine prevented glutamate neurotoxicity in PC12 cells, and to identify the molecular mechanisms of any effects. The results showed that glutamate neurotoxicity in PC12 cells could be prevented by treatment with nicotine at concentrations of 10 nmol.l-1-1 mmol.l-1. This effect was in turn found to be inhibited by the application of the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine. Nicotine significantly decreased the basal level of intracellular free Ca2+ and enhanced the buffering action on Ca2+ overload induced by high concentrations of glutamate (5 mmol.l-1). In addition, nicotine treatment up-regulated the mRNA and protein expression of apoptosis-related factors including bcl-2 mRNA and protein, but down-regulated the expression of bax mRNA and protein. It is concluded that the protective effects of nicotine against the neurotoxicity induced by glutamate are mediated by nAChRs, due to the increased buffering action on Ca2+ and the modulation of apoptotic processes.
Address and Contact Information 1Beijing Institute of Pharmacology and Toxicology, Beijing 100850 ,P.R.China,
2Nanjing Medical University, Nanjing 210029 , P.R.China,
3Thadweik Academy of Medicine, Beijing 100850, P.R.China
* Corresponding author; tel: 86-10-66932651, fax: 86-10-6821-1656, e-mail: wh@nic.bmi.ac.cn
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Volume 9 (2004) pp 423-427
Title DNA REPLICATION REACTION IN XENOPUS CELL-FREE SYSTEM IS SUPPRESSED BY HIGH PRESSURE
Authors Hitoshi Takahashi1, Takeo Yamaguchi1, Masaaki Koga2, Hiroshi Kageura2 and Shigeyuki Terada1
Abstract Previously, we demonstrated that when mouse erythroleukemia cells are exposed to a pressure of 80 MPa, the cell-cycle progression of S-phase cells is retarded. To examine the effects of high pressure on DNA replication, we used a Xenopus cell-free system. From cell-cycle progression of sperm nuclei, it was found that sperm nuclei are stable to a pressure of 80 MPa, whereas egg extracts are susceptible to high pressure. Similarly, biotin-16-dUTP was incorporated into 80 MPa-treated sperm nuclei in pressure-untreated extracts, but not into naive sperm nuclei in 80 MPa-treated extracts. These results indicate that DNA replication in Xenopus cell-free system is suppressed by the susceptibility of the extracts to a pressure of 80 MPa.
Address and Contact Information 1Department of Chemistry and 2Department of Biology, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka, 814-80 Japan
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Volume 9 (2004) pp 429-437
Title SITE-SPECIFIC EXCISION OR PROTECTION OF AN ALPHA A GLOBIN GENE GENOMIC SITE IN APOPTOTIC TRANSFORMED CHICKEN ERYTHROBLASTS
Authors Nikolajs Sjakste
Abstract There is still no clarity on whether the endonuclease incisions in apoptotic cells are induced randomly in the genome or induced in some preferable sites. In order to evaluate the intensity of DNA fragmentation in the chicken alpha-globin domain, AEV-virus transformed chicken erythroblasts (HD3) were incubated in a serum free medium, and their DNA was Southern blotted and hybridised with probes representing different fragments of the domain. Probes corresponding to the upstream areas of the domain mostly hybridised with high molecular weight DNA. Unlike these, the probe corresponding to the 2 Kb BamHI-BamHI fragment, containing the alphaA globin gene (B18), revealed a 5 Kb band on the hybridisation autoradiographs. The probe to the neighbouring upstream fragment did not reveal this band, but it was clearly seen on hybridisations with a downstream 1 Kb BamHI-BamHI fragment. The intensity of the band increased with overall apoptotic DNA degradation, hence its appearance should be coupled to apoptosis. Hybridisation of BamHI-digested DNA with B18 probe revealed a shortening of the 2 Kb band in preparations of DNA from apoptotic cells. The presumable positions of the cuts correspond to the formerly described DNase hypersensitive sites in the domain. Slot-blot and Northern hybridisation of RNA extracted from apoptotic HD3 cells revealed that the excision of the area of the B18 gene is coupled to a decrease in the intensity of alphaA globin gene transcription. Transcription of the non-erythroid NIK gene, transcribed in the upstream part of the domain, did not depend on the level of apoptotic DNA fragmentation.
Address and Contact Information Faculty of Medicine of the University of Latvia, Sharlotes Street 1a, Riga LV1001, Latvia
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Volume 9 (2004) pp 439-449
Title SHOOT REGENERATION FROM GUS-TRANSFORMED TOMATO (Lycopersicon esculentum) HAIRY ROOT
Authors Reda E. A. Moghaieb1,2, Hirofumi Saneoka1 and Kounosuke Fujita1*
Abstract To study the influence of genetic background on the transformation and regeneration of cultivated tomato plants, hairy root lines of tomato (Lycopersicon esculentum) were obtained by inoculating the hypocotyl explants of three tomato cultivars with the Agrobacterium rhizogenes strain DCAR-2, which harbors the pBI-121 binary vector. The Ri-T-DNA transformation into the plant DNA was confirmed by both of mikimopine and GUS assay analyses. The regeneration efficiency from hairy root explants was assessed. The data indicated that white embryonic calli were formed within two weeks in the presence of 2 mgl-1 2, 4-D plus 0.25 mgl-1 kinetin. Adventitious shoots emerged from the embryonic callus in the presence of 1 mgl-1 GA3 along with 0.5 mgl-1 NAA. The regeneration frequency was higher in the cultivar UC-97, followed by Momotaro and then Edkawi. Molecular confirmation of the integration of the GUS gene into the hairy root-derived plants genomes was done via PCR using GUS-specific primers and also using Southern blotting analysis. Our data shows that regeneration is possible from hairy roots of the cultivated tomato and this system could be used to produce transgenic tomato plants expressing the genes present in Agrobacterium rhizogenes binary vectors.
Address and Contact Information 1Department of Environmental Dynamics and Management, Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima, 739-8528, Japan,
2Department of Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt
* Corresponding author: e-mail: fujiko@hiroshima-u.ac.jp
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Volume 9 (2004) pp 451-464
Title THE EFFECT OF ANTISENSE OLIGONUCLEOTIDE TREATMENT OF PLASMA MEMBRANE Ca2+- ATPase IN PC 12 CELLS
Authors Janusz Szemraj, Iwona Kawecka, Jacek Bartkowiak and Ludmiła Żyliłska*
Abstract Plasma membrane Ca2+-ATPase (PMCA), encoded by four separate genes, constitutes a high affinity system extruding Ca2+ outside the cell. The nerve growth factor-treated PC12 cell line possesses all four main PMCA isoforms. To evaluate the potential role of PMCA isoforms in the differentiation process, we transiently suppressed the expression of PMCA2 and 3 using the antisense oligonucleotides. In the transfected PC12 cells, we observed morphological changes, slowed neurite extension and diminished survival of the cells. The apparent transport activity and affinity of the calcium pump to Ca2+ were lower in the cells with suppressed PMCA2 and 3 isoforms than in the control cells. Moreover, in the transfected PC12 plasma membranes, the calcium pump was insensitive to stimulation by calmodulin. These findings suggest that PMCA2 and 3 isoforms may be involved in developmental and differentiation processes.
Address and Contact Information Neurochemical Laboratory, Department of Biochemistry, Medical University, ul. Mazowiecka 6/8, 92-215 Łódź, Poland
* Corresponding author: e-mail: luska@csk.am.lodz.pl
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Volume 9 (2004) pp 465-473
Title ISOLATION AND CHARACTERIZATION OF A SERINE/THREONINE PROTEIN KINASE SOS2 GENE FROM Brassica napus
Authors Jin Wang1,2,3, Weisheng Wu1, Kaijing Zuo2, Jiong Fei2, Xiaofen Sun1, Juan Lin1, Xufeng Li4 and Kexuan Tang1,2,*
Abstract A full-length cDNA of a new serine/threonine (Ser/Thr) protein kinase gene, designated as BnSOS2 (GenBank Acc. No.AY310413), was cloned from Brassica napus by rapid amplification of cDNA ends (RACE). The fulllength cDNA of BnSOS2 was 1779 bp and contained a 1539-bp open reading frame encoding a protein of 512 amino acids. Homology analysis shows that BnSOS2 strongly resembles other Ser/Thr protein kinase genes, and that its putative protein belongs to a typical Ser/Thr kinase family. Northern blot analysis reveals that BnSOS2 is salt-inducible. Our results indicate that BnSOS2 is a new member of the plant SOS2 gene family, which may play an important role in salt tolerance of plants.
Address and Contact Information 1State Key Laboratory of Genetic Engineering, School of Life Sciences, Morgan-Tan International Center for Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Fudan University, Shanghai 200433, China,
2Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200030, China,
3Mianyang Teacher’s College, Mianyang 621000, China,
4College of Life Science, Sichuan University, Chengdu 610064, China
*Corresponding author; tel: 021-65642772; fax: 021-65643552; e-mail: kxtang1@yahoo.com or kxtang1@sohu.com.
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Volume 9 (2004) pp 475-481
Title BIGLYCAN IS INTERNALIZED VIA A CHLORPROMAZINESENSITIVE ROUTE
Authors Martin Götte1,2,*, David Denis Sofeu Feugaing1 and Hans Kresse1†
Abstract The small leucine-rich proteoglycan biglycan (BGN) is abundantly expressed in mesenchymal tissues. Its expression level is related to the phenotypic differentiation of cells. A dysregulation in BGN expression occurs under several pathological conditions, including glomerulonephritis, mesothelioma, pancreatic cancer and a mouse model of osteoporosis. Since the extracellular concentration of BGN is regulated both by secretion and endocytosis, we performed mechanistic studies on BGN endocytosis in human skin fibroblasts in vitro, using inhibitors of different endocytic routes. Chlorpromazine, an inhibitor of the clathrin-coated pit-pathway reduced endocytosis of BGN in human skin fibroblasts by 40%, and decreased degradation of BGN by 66%. Filipin, an inhibitor of the caveolae pathway, and Tyrphostin AG 1478, a specific inhibitor of EGF-receptor phosphorylation that partially inhibits endocytosis of the structurally related proteoglycan decorin, had no influence on BGN internalization and degradation. Our data indicates that the classical clathrin-mediated endocytic pathway is a major route for the internalization of BGN. Based on the differential susceptibility to pharmacological inhibition, it appears that BGN endocytosis seems to be at least in part mechanistically different from decorin uptake.
Address and Contact Information 1Department of Physiological Chemistry and 2Department of Obstetrics and Gynecology, Münster University Hospital, Domagkstr. 11, D-48149 Münster, Germany
*Corresponding author; tel: (+49) 251 8356113, fax: (+49) 251 8356114, e-mail: mgotte@uni-muenster.de
Deceased
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Volume 9 (2004) pp 483-495
Title PROTECTIVE EFFECT OF VACCINATION WITH DNA OF THE H. pylori GENOMIC LIBRARY IN EXPERIMENTALLY INFECTED MICE
Authors Artur Dzwonek1, Michał Mikula1, Marek Woszczyłski2, Ewa Hennig1 and Jerzy Ostrowski1*
Abstract Immunologically mediated protection against H. pylori infection is an attractive alternative to antibiotic treatment. We compared the efficacy of conventional protein vaccination with that of genetic vaccination against experimental infection with H. pylori in mice. For oral immunization, we used the recombinant peptide of an antigenic fragment of UreB (rUreB) or H. pyloriwhole cell lysate antigens, and for genetic immunization, we used recombinant pcDNA and pSec plasmids inserted with the fragment of ureB or DNA of the H. pylori genomic library. Mice were challenged with the mouse stomachadapted H. pylori Sidney Strain. The detection of gastric bacterial colonization was performed by real-time PCR of a 26-kDa Helicobacter-specific gene, and the presence of serum H. pylori-specific antibodies was determined using direct ELISA assay. The most effective treatment appeared to be oral vaccination with rUreB and either intramuscular or intradermal vaccination with DNA of the H. pylori genomic library. Intradermal genetic vaccination with genomic library DNA significantly increased the IgG antibody response. Our study revealed acceptable efficacies of genetic vaccination with DNA of the H. pylori genomic library.
Address and Contact Information 1Department of Gastroenterology, Medical Center for Postgraduate Education, Maria Skłodowska-Curie Memorial Cancer Center, ul. Roentgena 5, 02-781 Warsaw, Poland,
2Department of Animal Genetics, Cancer Center and Institute of Oncology, Warsaw, Poland
*Corresponding author; tel: (48 22) 6440102, fax: (48 22) 6447601, e-mail: jostrow@warman.com.pl
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Volume 9 (2004) pp 497-509
Title THE DISTRIBUTION OF PERIPHERAL BLOOD DENDRITIC CELLS ASSAYED BY A NEW PANEL OF ANTI-BDCA MONOCLONAL ANTIBODIES IN HEALTHY REPRESENTATIVES OF THE POLISH POPULATION
Authors Joanna Narbutt1*, Aleksandra Lesiak1, Małgorzata Żakprelich1, Anna Woźniacka1, Anna Sysa-Jędrzejowska1, Marzena Tybura2, Tadeusz Robak2 and Piotr Smolewski2**
Abstract A growing number of studies are being performed on the role of dendritic cells (DCs) in the etiopathogenesis of various conditions. Therefore, it is extremely important to establish the best comparable methods for the determination of the absolute count of blood dendritic cells (BDCs) or their subsets, and the reference normal values for comparisons. The aim of our study was to assess a normal profile of BDCs in the non-cultured human blood of healthy Polish volunteers. BDCs were detected among peripheral blood mononuclear cells (PBMC) from 99 healthy people, aged 18- 56. Based on the panel of novel anti-BDCA1, BDCA2 and BDCA3 monoclonal antibodies (MoAbs), three main subpopulations of BDCs were distinguished: two myeloid types of BDCs, MDC1(BDCA-1+/ CD11c+ /HLA-DR+) or MDC2 (BDCA-3+/CD32-/CD64-/HLA-DR+), and a plasmacytoid subtype, PDC (BDCA-2+/CD123+/HLA-DR+). The number and percentage of BDCs were correlated with the age, gender, photosensitivity (phototype, minimal erythemal dose - MED) and morphological parameters of the healthy volunteers. BDCs represented 0.83% of the PBMC and the median total BDC number was 44.0 cell/ml. The total BDC number correlated with the WBC count (r=0.40; p<0.001) as well as with the lymphocyte and monocyte counts (r=0.20; p=0.045 and r=0.26; p=0.009, respectively). The median percentage of the MDC1 count (0.20%) was twice as high as the MDC2 count (0.10%). The median PDC count was 28.2 cell/ml, and these cells represented 0.50% of the PBMC. There was a positive correlation between PDC and skin photosensitivity (r=0.28; p=0.005). An inverse correlation between the PDC count and the age of the examined volunteers was also found (r=-0.22; p=0.029). Our study provides the first referential data on normal rates and counts of BDCs and their subpopulations, assessed by the new panel of anti-BDCA MoAbs, in healthy Polish subjects. The method used in the study allowed the determination of BDCs and their subset numbers in a relatively small blood volume.
Address and Contact Information 1Department of Dermatology, Medical University of Łódź, Poland,
2Department of Haematology, Medical University of Łódź, Poland
* tel/fax: (+4842) 6884565; e-mail: joanna.narbutt@onet.pl
** Corresondind author; tel: (+4842) 68951-91; fax: +4842689-51-93; e-mail: piotr_smolewski@wp.pl
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Volume 9 (2004) pp 511-518
Title THE EFFECT OF BRACHYTHERAPY ON ANTIOXIDANT STATUS AND LIPID PEROXIDATION IN PATIENTS WITH CANCER OF THE UTERINE CERVIX
Authors Celestyna Mila-Kierzenkowska1*, Kornelia Kędziorakornatowska2, Alina Woźniak1, Tomasz Drewa1,3, Bartosz Woźniak4, Sylwia Drewa5, Ewa Krzyżyłska-Malinowska1 And Roman Makarewicz6
Abstract The aim of this study was to investigate the effect of brachytherapy on lipid peroxidation and antioxidant status in patients with uterine cervix cancer. The study was conducted on 84 uterine cervix cancer patients from the Brachytherapy Department of the Regional Centre of Oncology in Bydgoszcz. Patients with uterine cervix cancer were found to have elevated levels of lipid peroxidation and antioxidant defence system impairment relative to healthy females. The results of the study indicate that brachytherapy has no direct effect on the antioxidant system of patients with uterine cervical carcinoma. However, the normalisation of catalase and glutathione peroxidase activity and erythrocyte TBARS level observed six months after the end of therapy may be due to the arrest of the progression of the disease.
Address and Contact Information 1Department of Medical Biology, 2Clinic of Geriatrics, 3Clinic of Urology, 4Clinic of Neurosurgery and Neurotraumatology, 5Department of Radiology, Ludwik Rydygier Medical University, Karłowicza 24, PL-85-092 Bydgoszcz, Poland,
6Regional Centre of Oncology, Romanowskiej 2, PL-85-796 Bydgoszcz, Poland
* Corresponding author; tel: 48 52 585 3737, fax: 48 52 585 3742, e-mail: celestyna@go2.pl
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Volume 9 (2004) pp 519-528
Title GENOTOXICITY OF LEAD IN LUPIN ROOT CELLS AS EVALUATED BY THE COMET ASSAY
Authors Renata Rucińska, Robert Sobkowiak and Edward A. Gwóźdź*
Abstract This paper presents the results of a study on the influence of lead (Pb2+) on DNA integrity on plant cells. The study was performed on the root tips of lupin (Lupinus luteus cv. Juno) seedlings treated with two selected concentrations of Pb(NO3)2: 150 and 350 mg l-1, which were found to inhibit root growth by 50% and 70%, respectively [Ruciłska et al. Plant Physiol. Biochem. 37 (1999) 37187-37194]. Roots exposed to those external lead concentrations took up about 50 and 70 mg l-1 Pb2+ g-1 fresh weight (FW) over 48 h of incubation. A dose-dependent increase in the degree of root injury was observed in the presence of both tested concentrations. The genotoxicity of lead in lupin root cells was analysed using a mild alkaline comet assay at pH 12.3, which allows the detection of single strand breaks. The quantity of the DNA fragments migrating away from the nuclear remnant (tail area) increased proportionally to the lead content inside the roots, and was positively correlated with the degree of root injury. At 150 mg l-1 Pb2+, a high frequency distribution of nuclei having large values of tail lengths and moments was observed. By contrast, the number of nuclei with minimum values of these parameters increased at 350 mg l-1 Pb2+. This data suggests that lead at low concentrations induces the formation of short, rapidly migrating DNA fragments, whereas at higher concentrations, lead probably causes other changes to DNA that result in slower DNA migration in the electric field.
Address and Contact Information Laboratory of Plant Ecophysiology, Faculty of Biology, Adam Mickiewicz University, Al. Niepodległości 14, 61-713 Poznań, Poland
*Corresponding author; e-mail: albus@amu.edu.pl; fax: (+4861)8294518.
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Volume 9 (2004) pp 529-541
Title EXTENSION OF THE MESSAPIA - dicoccoides LINKAGE MAP OF Triticum turgidum (L.) THELL.
Authors Antonio Blanco1*, Rosanna Simeone1, Alberto Cenci1, Agata Gadaleta1, Oronzo A. Tanzarella2, Enrico Porceddu2, Silvio Salvi3, Roberto Tuberosa3, Giovanni Figliuolo4, Pierluigi Spagnoletti4, Marion S. Röder5 and Victor Korzun5,6
Abstract A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the highdensity Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.
Address and Contact Information 1Department of Environmental and Agro-Forestry Biology and Chemistry, University of Bari, via Amendola, 165/A, 70126 Bari, Italy,
2Department of Agrobiology and Agrochemistry, University of Tuscia, 01100 Viterbo, Italy,
3Department of Agroenvironmental Science and Technology, University of Bologna, 40126 Bologna, Italy,
4Dipartimento di Biologia, Difesa e Biotecnologie Agro-Ambientali, University of Basilicata, Potenza, Italy,
5Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, D-06466, Gatersleben, Germany,
6present address: Lochow-Petkus GmbH, PF 1197, D-29296 Bergen, Germany
* Corresponding author; tel: 0039 080 5442992, fax: 0039 080 5442200, e-mail: blanco@agr.uniba.it
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Volume 9 (2004) pp 543-556
Title DNA METHYLATION POLYMORPHISM IN A SET OF ELITE RICE CULTIVARS AND ITS POSSIBLE CONTRIBUTION TO INTERCULTIVAR DIFFERENTIAL GENE EXPRESSION
Authors Yongming Wang1, Xiuyun Lin1, Bo Dong1, Yingdian Wang2 and Bao Liu1*
Abstract RAPD (randomly amplified polymorphic DNA) and ISSR (intersimple sequence repeat) fingerprinting on HpaII/MspI-digested genomic DNA of nine elite japonica rice cultivars implies inter-cultivar DNA methylation polymorphism. Using both DNA fragments isolated from RAPD or ISSR gels and selected low-copy sequences as probes, methylation-sensitive Southern blot analysis confirms the existence of extensive DNA methylation polymorphism in both genes and DNA repeats among the rice cultivars. The cultivar-specific methylation patterns are stably maintained, and can be used as reliable molecular markers. Transcriptional analysis of four selected sequences (RdRP, AC9, HSP90 and MMR) on leaves and roots from normal and 5-azacytidine-treated seedlings of three representative cultivars shows an association between the transcriptional activity of one of the genes, the mismatch repair (MMR) gene, and its CG methylation patterns.
Address and Contact Information 1Laboratory of Molecular Epigenetics, Institute of Genetics & Cytology, Northeast Normal University, Changchun 130024, China,
2Laboratory of Plant Developmental Biology, School of Life Sciences, Beijing Normal University, Beijing 100875, China
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Volume 9 (2004) pp 557-566
Title ISOLATION, CLONING AND CHARACTERISATION OF MOTIFS CONTAINING (GA/TC)n REPEATS ISOLATED FROM VETCH, VICIA BITHYNICA
Authors Tomasz Sakowicz1*, Richard Bowater2 and Paweł Parniewski3
Abstract Microsatellites are widely distributed in plant genomes and comprise unstable regions that undergo mutational changes at rates much greater than that observed for non-repetitive sequences. They demonstrate intrinsic genetic instability, manifested as frequent length changes due to insertions or deletions of repeat units. Detailed analysis of 1600 clones containing genomic sequences of Vicia bithynica revealed the presence of microsatellite repeats in its genome. Based on the screening of a partial DNA library of plasmids, 13 clones harbouring (GA/TC)n tracts of various lengths of repeated motif were identified for further analysis of their internal sequence organization. Sequence analyses revealed the precise length, number of repeats, interruptions within tracts, as well as sequence composition flanking the repeat motifs. Representative plasmids containing different lengths of (GA/TC)n embedded in their original flanking sequence were used to investigate the genetic stability of the repeats. In the study presented herein, we employed a well characterised and tractable bacterial genetic system. Recultivations of Escherichia coli harbouring plasmids containing (GA/TC)n inserts demonstrated that the genetic instability of (GA/TC)n microsatellites depends highly on their length (number of repeats). These observations are in agreement with similar studies performed on repetitive sequences from humans and other organisms.
Address and Contact Information 1University of Łódź, Department of Cytogenetics and Plant Molecular Biology, 90-237 Łódź, Banacha 12/16, Poland,
2University of East Anglia, School of Biological Sciences, Norwich NR4 7TJ, United Kingdom, 3Centre for Medical Biology, Polish Academy of Sciences, 93-232 Łódź, Lodowa 106, Poland
* Corresponding author; e-mail: tomeksakowicz@wp.pl
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