1. Ismail^*1, L.D. Bellis^1,2 and S.M. Smith¹

¹Institute of Cell and Molecular Biology, University of Edinburgh, the King's Buildings,
Mayfield Road, Edinburgh, EH9 3JH, United Kindom;
²Dipartimento di Biologia Piante Agrarie, sez. Fisiologia Vegetale, Universita di Pisa,
via Mariscoglia 34, 56124 Pisa Italy

Received 16 June 2000 / Accepted 14 September 2000

Abstract.
A series of cucumber isocitrate lyase (ICL) gene promoter linked to GUS were introduced into Nicotiana plumbaginifolia by leaf disc transformation using Agrobacterium tumefaciens. The plants were self fertilised and seeds germinated for GUS assay. With 2900 bp ICL promoter, the GUS activity rose from stage 0 to its peak at stage 4 and then fell at stage 7. Deletion to 1568 bp greatly reduced the GUS activity suggesting for a cis acting sequences between the two regions required for seeds germination. Further deletions of ICL promoter retained a low level of GUS activity. Nevertheless, GUS histochemical assay showed that constructs with ICL promoter of 1568 lip, 1367 bp, 1193 and 572 bp directed GUS expression suggesting a qualitative germination response. All of the ICL promoter constructs caused organ specific pattern of GUS expression in cotyledons and expression was also observed in the root hairs of the seedlings.

Keywords: Agrobactetium tumefaciens, isocitrate lyase promoter, GUS, germinating seeds, tobacco

M. Maizan, A.R. Mohd. Ali and S.H. Sharifah*

Veterinary Research Institute, 59, Jalan Sultan Golan Shah 31400, Ipoh Perak

Received 28 August 2000 / Accepted 12 December 2000

Abstract.
A cytopathogenic infectious agent isolated from the lungs of dead and sick pigs with severe pneumonia during the 1998/1999 Nipah virus outbreak was used for the development of a RT-PCR, for the rapid diagnosis of Nipah virus infection. Oligonucleotide primers were constructed and reverse transcriptase polymerase chain reaction (RT-PCR) and nested PCR were established for the detection of Nipah and Hendra viruses. The primary and nested PCR based on the primers of specific regions within the N genes of both viruses amplified a 397 bp and a 300 bp product respectively. The nucleotide sequence of the amplified 397 bp fragments of the 1998 pig isolate and the naturally occurring Nipah virus from the buffy coat of blood of pigs from a recently identified farm was compared with the published N gene sequences of the Nipah and Hendra viruses. The sequences of the 397 bp PCR product derived from tissue culture infected cells and buffy coats showed 99% and 70.8% homology with Nipah and Hendra virus respectively. The amplified products were further identified as Hendra or Nipah virus by restriction enzyme analysis using EcoRV.

Keywords: Nipah virus, RT PCR, sequencing, restriction enzyme analysis

L.L. Choo ¹, T. Tamura², R.A. Rahim ¹, A.M. Ali¹, H. M. Ghazali¹, K. Inagaki and H. Tanaka^2*

¹Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
²Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, 1 1 1, Tsushima naka, Okayama, 700 8530, Japan

Abstract.
This paper reports a convenient screening method for detecting methionine g-lyase activity in microorganisms which can grow on DL methionine as the sole nitrogen source. L Methionine g-lyase has been known to have the most versatile catalytic properties among other pyridoxal 5' phosphate dependent enzymes, but only known from a limited number of biological sources. For an efficient screening of the enzyme, we employed 5, 5' dithiobis (2 nitrobenzoic acid) (DTNB) to detect the enzyme reaction product methanethiol, which spontaneously reduces DTNB and develops a yellow coloration on and around colonies. The yellow color has emerged and faded away within 72 h, suggesting that the reduced DTNB may be oxidized by spontaneous and/or enzymatic formation of disulfide. This method, despite reduction in specificity compared to the standard assay, allowed us to select candidate microorganisms out of hundreds of other similar colonies merely by observing the yellow coloration.

Keywords: L Methionine g-lyase, 5, 5' Dithiobis (2 nitrobenzoic acid)

A. Janna Ong¹, M.Marziah ^1*, G.K.A. Ahmad Parveez²

¹Department of Biochemistry and Microbiology, Faculty of Science and Environmental Studies,
University Putra Malaysia, 43400 UPM, Serdang, Selangor Darn 1 Ehsan, Malaysia
²Palm Oil Research Institute of Malaysia, Biology Division, 50720 Kuala Lumpur, Malaysia

Received 5 February 2000 / Accepted 17 July 2000

Abstract.
Protocorm like bodies (PLBs) of Dendrobium Sonia 17 were bombarded with 1.0 mm gold microparticles at 1100 psi using the PDS 1000/helium powered biolistic gun. At two weeks post bombardment the PLBs were subjected to different concentrations of antibiotic selections. The five selective agents tested were hygromycin, geneticin, Basta, paromomycin, and kanamycin each at 0, 25, 50, 100, 150, 200, and 300 mg/L. For hygromycin, the experiment was further carried out at lower concentrations of 0, 5, 10, 15, 20, and 25 mg/L. After one month into selection, fresh weights of the PLBs were taken and the number of surviving PLBs counted. It was observed that hygromycin was the best selective agent as it gave a total kill off (all PLBs died) at 25 mg/L. Geneticin was second with 100 % dead at 150 mg/L. For Basta, 10 % of the PLBs still survived at the highest concentration, 300 mg/L, tested. As for paromomycin and kanamycin, all the PLBs survived at 300 mg/L.

Keywords: Dendrobium orchid, transformation, selective agent