Annales Bogorienses

Number 2, 2004

39-49
MODIFICATIONS OF PLASMIDS AND CRY GENES OF Bacillus thuringiensis subsp. kurstaki lID-I AFTER TREATMENT WITH ETHYLMETHANOSULFONATE (EMS) AND UV LIGHT

Authors: Eddy Jusuf

Abstract

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Bacillus thllringiensis subsp. kllrstaki HD-J is a potential insecticidal bacterium producing five type of a -endotoxin crystal proteins. This bacterium is widely commercialized due to its wide spectrum toxicity against both Lepidopteran and Dipteran larvae . The objective of this work was to create autolysin deficient mutant causing cell fails to lyse. This mutant yield intact cell s within spore and a -endotoxin crystal protein protected inside, from which an undamaged active bio-insecticide wou ld be obtained . Two methods of mutagenesis, 2ry( of ethylmethanosulfonate and 10, 25, and 50 second of UV light expo ure, resu lted in mor loss of motility) mutation. Observation showed that 14 of 20 survived mutants have lost some of its plasmids (varied from one to five). wh ile the other six maintained their plasmids. By employing the polymerase chain reaction (PCR) the change on the cry genes was studied.

50-58
CYTOLOGICAL ANALYSIS OF ROOT CULTURES OF Artemisia cina

Authors: Tri Muji Ermayanti, Oktavia Yanti, Erwin Al Hafiizh

Abstract

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Artemisia cina is a medicinal plant species producing bioactive compounds which are potential as antitumor, antifungal and antibacterial. The aim of this study was to analyze the stabililY of chromosome number in root cultures of A. cina. Transformed root culture was established by infection of leaves of A. cina with Agrobacterium rhizogenes strains 07-20001 . ATCC-15834. A4 and A. tumefaciens strain RlOOO . Roots isolated from glasshouse plants, plantlets grown in solid and liquid MS medium were utilized for investigation of chromosome examination of untransformed roots. Chromosome examination was conducted by squashing method and chromosome numbers were calculated under microscope. The .results showed that both untransformed and transforme root had instability in the chromosome number, but had the modal number of chromosome x=8 with the diploid number of 2n = 4x = 32. Roots isolated from glasshouse plants of A. Cina had 53.7% of cell with the diploid numbers of 2n = 32 and 46.3% of cells had chromosome numbers ranged from 2n = 12 to 2n = 64. Untransformed roots isolated from plantlets cultured in solid medw had only 36.1 % or cells with chromo orne number of 2n = 32, and unlran fomled ro t5 grown in liquid medium had 49.4% of cells with 2n = 32. The chromosome numbers of A. Cina transformed roots was affected by trains of Agrobacterium. Root transformed with the bacterium Strain 07 -20001 showed lhe highest in normal chromosome number of 2n = 32 (62.4%) followed by roots lransformed wiLll strains ATCC-15834 (61.9 %). R1000 (43.6%) and A4 (43.0%). The range of the chromosome number untransformed roots was from 2n= 17 to 2n=64 whilst that of transformed roots was from 2n= 1 l to 20=66

72-79
DEVELOPMENT OF SOMATIC EMBRYO IN Lithospermum erythrorhizon Siebb. et Zucc AND THE STUDY ON THE EFFECT OF METHYL JASMONATE ON ITS MATURATION

Authors: Totik Sri Mariani, Octavia Ramayanti, Kazufumi Yazaki, Hiroshi Miyake

Abstract

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A research on the somatic embryogenesis in Lithospermum erythrorhizol1 has been conducted. Embryogenic callus was inoculated in EIM9 liquid medium (modification ofLS medium), i.e. L2PVP (initiation medium) and the development of somatic embryo was observed. Ten J.1M and 100).lM methyl jasmonate was added into L3PVP medium (differentiation medium) for somatic emb yo maLUration. The purposes of this research were to observe tlle development of somatic embryo and to observe the effect of methyl jasmonate on maturation of the somatic embryo in L erylhrorhizol/ . The results showed that somatic embryogenesis in L erythrorhizon derived from single cells differentiated further forming proembryo, globular. heart, torpedo and cotyledon stage. Treatment with 10 f.1M and 100).lM methyl jasmonate induced maturation of somatic embryos (cotyledon stage) after transferring them to embryo development medium (L4PVP).

80-86
PCR AMPLIFICATION OF ORNITHINE DECARBOXYLASE (ODC) GENE FRAGMENT FROM TOBACCO (Nicotiana tabacum L.) cv. TEMANGGUNG

Authors: Ira Djajanegara, Sabar Pambudi, Retno Lestari, Nina Artanti

Abstract

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In order Lo create an antisense construct of the gene encoding Ornithine Decarboxylase (ODC) from tobacco (Nicotiana tabacum L.) cv. Temanggung, the target gene must be isolated. In this paper. we present the PCR amplification of a fragment from putative gene encoding ODC from tobacco cv. Temanggung. Leaf genomic DNA was i olated and used as the template for PCR. PCR optimization was done by adjusting the annealing temperature and the cycle number. Verification of the fragment obtained was also done using the second primer pairs .

Number 1, 2004

1-8
SENSITIVITY IMPROVEMENT OF A DIRECT COMPETITIVE ELISA FOR ATRAZINE BY EXPLOITING LOW CROSS-REACTIVITY OF AN ATRAZINE-SPECIFIC RECOMBINANT ANTmODY FAB-FRAGMENT

Authors: Wien Kusharyoto, Rolf D. Schmid

Abstract

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The hapten-binding site of the antibody Fab-fragment K411 B specific towards the herbicide atrazine (2-cllloro-4-(ethylamino)-6-(isopropyJamjno)- 1,3,5-triazine) was modified by means of structural modeling and site-directed mutagenesis. A triple mutant (GlnL89GlulValH3711e/GluL3Val) of the Fab-fragment showed an increased affinity towards the hapten HlCVC6 (4-arnino-6-chloro-l ,3,5-triazine-2-(6-aminohexanecarboxylic acid» compared to the affinity ofthe wild-type Fab-fragment lowards the same hapten. However, the mutant exhibited substantially lower affinity towards the hapten H/CVC6 Ihan towards atrazine and the hapten iPr/ CVC6 (4-chloro-6-(isopropylamino)-1,3,5-triazioe-2-(6-aminohexanecarboxylic acid», which is usual ly used in the synthesis of enzyme tracers in ELISA for atrazine. Advantage was taken of the low cross-reactivity and increased affinity of the mutant Fab fragment towards H/CVC6 to improve the sensitivity of a direct-competitive ELISA tor atrazine. HlCIlC6 was covalently conjugated with horseradish peroxidase (HRP), and the conjugate H/CI/C6-HRPwas used as enzyme tracer in the ELISA for atrazine. An eight-fold improvement in sensitivity ofa direct-competitive ELISA for atrazine could be achieved using the tracer H/CVC6-HRP compared to the sensitivity of ELISA using the tracer iPr/CVC6-HRP. The detection limit for atrazine was as low as 0.01 i g/l.

9-19
AN APPLICATION OF REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION IN A RELATIVE QUANTIFICATION OF GENE EXPRESSION

Authors: Adi Santoso

Abstract

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The ability to quantify steady state levels of individual mes enger-RNA (mRNA) transcripts has been the key issue for study on the control of gene expression. Although two available techniques, Northern blot and nuclease protection assays (NI)A) have been widely used tor detecting mRNA, these techniques have critical limitations. The most obvious limitation of these two techn iques is the required number of target mRNAs to be detected. Reverse transcription-polymerase chain reaction (RT-P R), which has been accepted as a highly sensitive and specific method, provides a means for detecting and quantifying gene expression using, theoretically only a single molecule of mRNA. The ensitivity and reliability ofRT-PCR i dependent upon both the RT and PCR steps. The PCR step ha been problematic because of the exponential nature of this reaction where small variation can lead Lo dramatic changes in final result. here fore, the use of RT-P R for quanti fication of gene expres ion requires pre-experimental planntng and de ign. In thiS experiment, the procedure fOT pre-experimental planning, linear range determination and subsequent relative quantification of gene expression are described in detail. study of ornithine decarboxyJase gene, a gene involved in the polyamine biosynthesis and temporally expressed, dunng embryogenesis of Musca domestica (housefly) was used as the model. The re ults show that during early embryogenesis (t-l to t-4) the expression lev I was very low. The increase In expression profile was observed started at t-5, peaked at t-9, and followed by substantial decrease from t-l 0 to t- 12.

20-26
MATERNAL CONTRIBUTION IN REVEALING THE EFFECTS OF METHOXYACETIC ACID (MAA) ADMINISTERED BEFORE IMPLANTATION ON THE EMBRYONIC DEVELOPMENT OF SWISS WEBSTER MICE (Mus musculus)

Authors: Ekayanti Mulyawati Kaiin, Sony H. Sumarsono, Tien W. Surjono, Sri Sudarwati

Abstract

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Maternal contribution to and direct action of methoxyacetic acid (MAA) on the embryonic development bad been examined by conducting embryo tTansfer. To reveal the maternal contribut ion, compacted morulae and early blastocysts, which were collected from untreated Swiss Webster donor mice on day 3 of gestation, were transferred to day 2 pseudopregnant recipients, after having been treated with 2 .0 mmollkg body weight (b.w.) MAA by gavage on day 1 of pseudopregnancy. Direct effect of MAA on the embryonic development were observed by transferring compacted morulae and early bla tocysts, simi larly recovered from day 3 pregnant donor mice, after MAA treatment on day 2 of gestation with the same method and dosing, to untreated day 2 pseudopregnant recipients. Control donor mice and recipient were given distilled water only as the MAA olvent. Observations on fetuses resulting fTom embryo transfer wert: carried out on day 16 of gestatlOn . Administration of MAA to the donors tended to decrease the unplantatlOn rate and the survival rate of the implanted embryos. W11en MAA was given to the recipients the implantation rate and survival rate of embryos transferred decreased significantly (p<0 .05) but the survival rate of implanted embryos were significantly higher (p<0.05) Lf compared to those of MAA treated donors. The intrauterine death tended to inc rease either in the treated donors or recipients. Th re was no et1ect of MAA on the fetal body weight and in producing fetal malformations . It is concluded that at the beginning of implantation, maternal contribution in revealing the effects of MAA on the embryonic development of Swiss Webster mice is predominant, whereas after Implantation took place, the quality of the embryos become ';lore important for their survival.

27-36
THE USE OF 16s RNA AND NODe GENE SEQUENCE IN RESOLVING THE PHYLOGENET IC RELATIONSHIP OF Rhizobia A SOCIATED WITH Paraseriaflthes fa/cataria (L.) Nielsen PLANT

Authors: Titik K. Prana, Teruo Sone, Hiroko Kawasaki Nakagawa, Atsushi Yokota, Tatsuji Seki, Fusao Tomita

Abstract

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Studies on genet ic position of several iso lates liv ing symbiot i a lly on ParaseriCllltli esfalcalaria have been carried out using amplification and se uenc ing tec hni ques of 16S rDNA and specific gene fo r nodulation (/lode) . The "p hylogenetic LTee" cons tructed based On 165S rONA showed that rh izobia growing symbiotically on Paraserianthes fa/ca taria consisted f 3 group . TIl . fi r t group, was fasl- growing rhIzobia wh ich have close r lationshlp to Rhizobium tropicii, the second and the third groups were slow-growing r!lI zobia wh ic h have c lose relationsh ip to Bradyrhizobillnl elkanJi and B j aponiclIfll. However, the "phy logenetic tTee" constructed on the ba is of part ln l ll odC gene md icatcd the existence of an JIldependen t group, si nce they did not show any siglU tica nt degree of relationsh ip w ith the ex isting groups. T hi s was also supported by diffi rences in pby iological characteri tiC i.e Indole Ace ti c Acid productIOn and salt tolerance of the iso lates . These differe nces shows uS that "direct _equenc ing" method of certain specdic genes coul d give a more specific re ult tha n ou ld di p lay more clearly the degree of relat ionship at species leve l.