Chief Editor: Muhammad Ahkam Subroto. Associate Editors: Bambang Sunarko, Adi Santoso, Syahruddin Said, Satya Nugroho. Editorial Assistant: Siti Elly Faisholyah. Consulting Editors: Asrul Muhammad Fuad, Yantyati Widyastuti, Witjaksono, Tri Muji Ermayanti, Nita Rosalinda, Wien Kusharyanto, Arief Budiono, Novik Nurhidayat, Ira Nurhayati Djajanegara. Cover: Wien Kusharyoto.
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Content

Sensitivity Improvement of a Direct Competitive Elisa for Atrazine by Exploiting Low Cross-Reactivity of an Atrazine-Specific Recombinant Antibody Fab-Fragment
Wien Kusharyoto and Rolf D. Schmid

Abstract
The hapten-binding site of the antibody Fab-fragment K411B specific towards the herbicide atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine) was modified by means of structural modeling and site-directed mutagenesis. A triple mutant (GlnL89Glu/ValH37Ile/GluL3Val) of the Fab-fragment showed an increased affinity towards the hapten H/Cl/C6 (4-amino-6-chloro-1,3,5-triazine-2-(6-aminohexanecarboxylic acid)) compared to the affinity of the wild-type Fab-fragment towards the same hapten. However, the mutant exhibited substantially lower affinity towards the hapten H/Cl/C6 than towards atrazine and the hapten iPr/Cl/C6 (4-chloro-6-(isopropylamino)-1,3,5-triazine-2-(6-aminohexanecarboxylic acid)), which is usually 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/Cl/C6 to improve the sensitivity of a direct-competitive ELISA for atrazine. H/Cl/C6 was covalently conjugated with horseradish peroxidase (HRP), and the conjugate H/Cl/C6-HRP was used as enzyme tracer in the ELISA for atrazine. An eight-fold improvement in sensitivity of a direct-competitive ELISA for atrazine could be achieved using the tracer H/Cl/C6-HRP compared to the sensitivity of ELISA using the tracer iPr/Cl/C6-HRP. The detection limit for atrazine was as low as 0.01μg/l.

Keywords: atrazine, direct-competitive ELISA, cross-reactivity, recombinant Fab-fragment, structural modeling


An Application of Reverse Transcriptase Polymerase Chain Reaction in a Relative Quantification of Gene Expression
Adi Santoso

Abstract
The ability to quantify steady state levels of individual messenger-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 (NPA) have been widely used for detecting mRNA, these techniques have critical limitations. The most obvious limitation of these two techniques is the required number of target mRNAs to be detected. Reverse transcription-polymerase chain reaction (RT-PCR), 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 sensitivity and reliability of RT-PCR is dependent upon both the RT and PCR steps. The PCR step has been problematic because of the exponential nature of this reaction where small variation can lead to dramatic changes in final result. Therefore, the use of RT-PCR for quantification of gene expression requires pre-experimental planning and design. In this experiment, the procedure for pre experimental planning, linear range determination, and subsequent relative quantification of gene expression are described in detail. A study of ornithine decarboxylase gene, a gene involved in the polyamine biosynthesis and temporally expressed, during embryogenesis of Musca domestica (housefly) was used as the model. The results show that during early embryogenesis (t-1 to t-4) the expression level 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-10 to t-12.

Keywords: RT-PCR; Northern blot; NPA; gene expression, quantification.


Maternal Contribution in Revealing the Effects of Methoxyacetic Acid (MAA) Administered Before Implantation on the Embryonic Development of Swiss Webster Mice (Mus Musculus)
Ekayanti M. Kaiin, Sony H. Sumarsono, Tien W. Surjono, Sri Sudarwati.

Abstract
Maternal contribution to and direct action of methoxyacetic acid (MAA) on the embryonic development had been examined by conducting embryo transfer. To reveal the maternal contribution, 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 mmol/kg 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 blastocysts, similarly 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 solvent. Observations on fetuses resulting from embryo transfer were carried out on day 16 of gestation. Administration of MAA to the donors tended to decrease the implantation rate and the survival rate of the implanted embryos. When 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) if compared to those of MAA treated donors. The intrauterine death tended to increase either in the treated donors or recipients. There was no effect 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 more important for their survival.

Keywords: methoxyacetic acid (MAA), preimplantation embryo, donor, recipient, embryo transfer, mice.


The Use of 16S RNA and NODC Gene Sequence in Resolving the Phylogenetic Relationship of Rhizobia Associated with Paraserianthes Falcataria (L.) Nielsen Plant
Titik K. Prana, Teruo Sone, Hiroko Kawasaki-Nakagawa, Atsushi Yokota ,Tatsuji Seki, and Fusao Tomita

Abstract
Studies on genetic position of several isolates living symbiotically on Paraserianthes falcataria have been carried out using amplification and sequencing techniques of 16S rDNA and specific gene for nodulation (nodC). The “phylogenetic tree” constructed based on 165S rDNA showed that rhizobia growing symbiotically on Paraserianthes falcataria consisted of 3 groups. The first group, was fast-growing rhizobia which have close relationship to Rhizobium tropicii, the second and the third groups were slow-growing rhizobia which have close relationship to Bradyrhizobium elkanii and B.japonicum. However, the “phylogenetic tree” constructed on the basis of partial nodC gene indicated the existence of an independent group, since they did not show any significant degree of relationship with the existing groups. This was also supported by differences in physiological characteristics i.e Indole Acetic Acid production and salt tolerance of the isolates. These differences shows us that “direct sequencing” method of certain specific genes could give a more specific result that would display more clearly the degree of relationship at species level.

Keywords: NodC gene, 16S rRNA, rhizobia, phylogenetic tree