Nooraini Abdulrashid* and Brian S. Hartley

*Faculty of Science, Universiti Teknologi Malaysia, Locked Bag 791, 80990 Johor Bahru, Malaysia, Centre for Biotechnology, Imperial College of Science, Technology and Medicine, London, SW7 2AZ, England

(Received 12 December 1997 / Accepted 16 February 1998)

Abstract.
The screening for the Aspergilus niger glucoamylase cDNA has led to the discovery that a full length cDNA in a pUC vector is lethal to Escherichia coli. Initial screening has indicated that full length glucoamylase cDNAs were initially present in the cDNA libraries but deletion mutants arose during the purification of the clones. The deletion mutants contained identical incomplete cDNAs lacking 35 nucleotides downstream of the initiation codon. The missing portion encodes part of an 18 amino acid signal peptide responsible for the translocation of the A. niger glucoamylase extracellularly. Therefore, to reconstruct a full length cDNA, synthetic oligonucleotides which represent the missing portion of the gene were ligated with the incomplete cDNA. However, several attempts to reconstruct a full length cDNA failed when the cDNA was in pUC18, a high copy number expression vector which utilizes the lac promoter. Fusion of a double stranded oligonucleotide representing the missing portion of the cDNA with a truncated cDNA led to the isolation of one deletion mutant which completely lost the entire glucoamylase cDNA except for the signal sequence. Incomplete cDNA lacking part of the signal sequence was then subcloned into a promoterless vector pSP72 and ligated with synthetic oligonucleotides representing the missing portion. This finally yielded full length 2.0 kb glucoamylase cDNA. In addition, it was found that subcloning of the full length glucoamylase cDNA into pT7T3 18U, another high copy number vector equipped with the lac promoter, also resulted in deletion of part of the cDNA to 1.5 kb.

Keywords: DNA Instability, Toxicity

Nakisah Mat Amin* and ¹Keith Vickerman

Unit of Biological Sciences, Faculty of Science and Professional Arts, Universiti Putra Malaysia Terengganu, Mengabang Telipot 21030 Kuala Terengganu, Malaysia, 1Division of Environmental Evolutionary Biology, Graham Kerr Building, University of Glasgow, G12 8QQ, Scotland, UK.

(Received 9 January 1998 / Accepted 23 February 1998)

Abstract.
A fluorescence technique has been employed to visualise nuclear division in Naegleria gruberi. The synchronously dividing Naegleria cells were stained with anti a-tubulin as a primay antibody and with a fluorescein isothiocyanate labelled goat anti mouse immunoglobin (FITC) as a secondary antibody. The cells were also stained with 4 1 6-diamidino-2-phenylindole DAP1) and propidium iodide (PI) to stain the nucleus. The results of this study indicated that by using anti (a tubulin, cytoplasmic microtubules; and mitotic spindles of Naegleria gruberi can be visualised under fluorescence microscopy. By this technique, a centrosome with associated microtubules was found to be present in the cytoplasm of Naegleria during nuclear division and this finding contradicts the previous idea that this organelle does not exist in Naegleria. The centrosome appeared to divide during prophase but it could not be detected later at metaphase telophase.

Keywords: Anti a tubulin, 4', 6 diamidino 2 phenylindole DAP1) Propidium Iodide (PI), Centrosome, Spindle Microtubules

Y,F. Ngeow*, L Rajan, V, Hema and CA Gaydos¹

Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
The Johns Hopkins University, Chlamydia Research Laboratory, Baltimore, USA.

(Received 29 December 1998 / Accepted 10 January 1998)

Abstract.
A major outer membrane protein (MOMP) based nested polymerase chain reaction (PCR) for the detection of Chlamydia trochomatios was evaluated for urine specimens collected from patients attending sexually trans. mitted disease clinic. A hundred and thirty nine samples were tooted by the nested VCR and a commercially available ligase chain reaction (LCR) ;assay called the LCx from Abbott Laboratories, USA, To resolve discrepant results, 87 of these specimen were also tested by a second LCR amplifying it di (OMPI) gene than the plasmi.d gene targetted in the LCx, The remainder 32 specimens were examined by the IDEIA Chlamydia, an enzyme immunoassay from DAKO Diagnostic@ Ltd,, UK, Using an expanded "gold standard" consisting of two positive test results, the sensitivity, specificity, positive find negative predictive values of the nested VCR were found to be 55.1%, 98,0%, 98,0% and 55.1% respectively. There was no significant difference in the test porformance for male and female urine

Keywords: Chlamydia Trachomatis, Urine, PCR

Bikash Chowdhury, A.B.Mandal* and A.K.Bandyopadhyay

Biotechnology Laboratory, Central Agricultural Research Institute, Post Box No.181, Port Blair 744101, India.

(Received 15 December 1997 / Accepted 19 February 1998)

Abstract.
Two indica rice varieties, a HYV viz. IR-18351-229 3 and a traditional cultivar C14-8, indigenous to Andamans were employed as experimental materials to recover aluminium (AI) tolerant genotype/s through in vitro screening with simulated excess Al stress in culture medium. Mature dehusked seeds were callused on MS medium with 250mg^-1 casein hydrolysate and 2mg^-12,4-D. Aluminium at 30, 60 and 90 ppm in the form of AICI₃. 6H₂O was supplemented in the callus induction medium (CIM) as a stressing agent. Seed germination, callus induction, callus health and morphogenetic responses in both varieties were found to be influenced by this abiotic stress. Induced calli were passaged three times over normal and two times on Al stressed callus maintenance medium (CMM). At the end of each selection, calli from 20 seeds in each treatment were separated into embryogenic and non embryogenic components under stereomicroscope to document percentage of embryogenic calli and note their health status. Putative Al tolerant genotypes were recovered from survivor calli of both varieties on Al free regeneration medium. However, only a few regenerants from C14 8 could qualify screening test under hydroponics with 30ppm Al stress. This clearly indicates differential tolerance norm at cellular and whole plant level in response to Al toxicity tolerance. These tolerant plants could flower and mature normally thus offering ample scope to developing Al tolerant lines. These lines may be used directly or through conventional recombination breeding.

Keywords: Aluminium Toxicity, Rice, in vitro Screening