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

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

S.R. Syed Alwee^1*, L.C. Ching² and K. Harikrishna³

¹Malaysian Palm Oil Board, No. 6 Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor,
²PAMOL Plantation, P 0. Box 1, Kluang, 86007 Kluang, Johor
³Genome Centre, Institute of Bioscience, Universiti Putra Malaysia, 43300 UPM, Serdang, Selangor

Received 28 August 2000 / Accepted 12 November 2000

Abstract.
Flowering is an important part of oil palm crop production. The ability to control flowering will be economically beneficial to the oil palm industry. As an initial step towards understanding oil palm flowering, flower predominant genes were isolated. Differential screening and cold plaque screening techniques were employed to clone low to high abundance flower predominant genes from oil palm. Over 100 clones were isolated and some were characterized by Northern hybridization and shown to be expressed in a flower predominant manner. Genes isolated can be categorized based on their expression pattern. Sequence analysis was carried out on a subset of the isolated genes and this showed that a high percentage of these genes encoded novel proteins. This indicates that both techniques are reliable for isolation of tissue specific genes eventhough the cold plaque screening technique was more adaptable for the isolation of low abundance genes.

Keywords: Cold plaque; differential screening; flowering; oil palm; differential expressio

L. Eurwilaichitr^1,2*, P. Manitchotpisit³, C. Chutrakul¹ and S. Panyim¹

¹Institute of Molecular Biology and Genetics, Mahidol University, Salaya, Nakornpathorn 73170, Thailand; 2Training
Centre for Genetic Engineering and Biotechnology, National Center for Genetic Engineering and Biotechnology at
Institute of Molecular Biology and Genetics, Bangkok, Thailand; 3Microbiology Department,
Rangsit University, Bangkok, Thailand

Received I September 2000 / Accepted 30 November 2000

Abstract.
Giant catfish growth hormone (gcGH) cDNA was expressed under the control of two types of yeast promoters: TEFI, a constitutive promoter and GALI an inducible promoter. Yeast culturing was optimised by using rich buffered medium and high cell density culturing approach. The level of expression when the transformants were grown in BMGYrich medium was approximately 90 mg/l. The strength of the two promoters was not different in expressing gcGH. The gcGH secretion was directed by leader sequence of Saccharomyces cerevisiae a mating type factor. The level of gcGH secretion into medium was about 39-46% of total gcGH produced. The cleavage of a Kex2 recognition site at the N terminal region of the secreted gcGH was accurate, yielding the mature gcGH.

Keywords: Alpha mating factor, GALI promoter, giant catfish growth hormone, Saccharomyces cerevisiae, TEF1 promoter, yeast