Rab8b and its interacting partner TRIP8b are involved in regulated secretion in AtT20 cells.

Rab proteins are a family of small GTPases that regulate intracellular vesicle traffic. Rab8b, because of its homology with Rab8, has been suggested to function in vesicle transport to the plasma membrane. Using the yeast two-hybrid system, we identified a Rab8b interacting clone, termed TRIP8b, from a rat brain cDNA library. The gene encodes a 66-kDa protein with homology to the peroxisomal targeting signal 1 receptor. The interaction between Rab8b and TRIP8b was further verified by in vitro binding assays and co-immunoprecipitation studies. Additional experiments with Rab8b mutants demonstrated that Rab8b requires a guanine nucleotide but not prenylation for its interaction with TRIP8b. Western immunoblot analysis showed that TRIP8b was primarily expressed in brain. Subcellular fractionation of AtT20 cells revealed that TRIP8b was present in both cytosolic and membrane fractions. To investigate the function of Rab8b and TRIP8b in secretion, we examined the release of ACTH from AtT20 cells. Results from stable cell lines expressing Rab8b or TRIP8b indicated that both proteins had a stimulatory effect on cAMP-induced secretion of ACTH. In summary, these data suggest that Rab8b and TRIP8b interact with each other and are involved in the regulated secretory pathway in AtT20 cells.

Characterisation of independent DNA and multiple Zn-binding domains at the N terminus of human DNA-(cytosine-5) methyltransferase: modulating the property of a DNA-binding domain by contiguous Zn-binding motifs.

We report here a detailed mapping and characterisation of a DNA-binding domain at the N terminus of human DNA-(cytosine-5) methyltransferase. A small region, B1 (codon 202 to 369), was first identified by its Zn- and gross DNA-binding properties. Further fine-mapping using deletion and point mutation analysis shows that the DNA- and Zn-binding domains involve separate peptide motifs, KRRKTTPKEPTEKK (codons 202 to 215) for a bipartite DNA-binding oligopeptide (DB1) and CX2CX13HX2D(X)23EX2EX13CX3H (codons 232 to 297) for possibly two contiguous Zn-binding domains (AZn), which can function independently. However, B1 (containing DB1 and AZn) differs from DB1 because it does not bind to a 30 base-pair duplex. Interestingly, H3 codons 202 to 974, which encloses B1 and B2 (containing the Zn-binding CX2CX2CX4CX2CX2C motif from codon 533 to 550) binds preferentially to 0.8 kb duplexes, as compared with 0.4 to 0.6 kb duplexes. As the homologous murine B1, which targets the murine methylase to replication foci, also binds to DNA and Zn, it is possible that the N terminus of mammalian methylase may be involved in sensing the appropriate length of newly synthesized DNA before methylation by its C terminus. This may enable a time delay for the transient existence of hemi-methylation sites for their unknown biological functions in mammals.

The amplification refractory mutation system (ARMS): a rapid and direct prenatal diagnostic technique for beta-thalassaemia in Singapore.

beta-Thalassaemia major patients have chronic anaemia and since 3-4 per cent of Singaporeans carry the beta-gene, prenatal diagnosis is essential. We evaluated the amplification refractory mutation system (ARMS) technique as a routine test for prenatal diagnosis of beta-major. Six mutations along the beta-gene were studied--41-42 (-TCTT), IVSII #654 (C-T), 17 beta (A-T), -28 TATA (A-G), IVSI #5 (G-C), and IVSI #1 (G-T). Our results indicate that prenatal diagnosis using these mutations can be offered to 90 per cent (35/39) of our Chinese couples and 54.6 per cent (12/22) of our Malay couples at risk. Confirmation of ARMS results was carried out using allele-specific oligonucleotide hybridization. Prenatal diagnosis using ARMS was successfully carried out in nine cases which included a set of triplets and twins. The triplets were diagnosed with the beta-trait carrying the 41-42 mutation. The couple with twins possessed the #654 mutation and one twin was diagnosed with the beta-trait and the other with #654 homozygosity. Genomic sequencing of the undefined mutations in the Chinese couples revealed rarer mutations at -29 and an ATG-AGG base substitution at the initiation codon for translation. In the Malay couples, genomic sequencing detected mutations at codon 15 (TGG-TAG) and codon 26 (GAG-AAG). We conclude that ARMS with its direct detection of amplified products by gel electrophoresis provides an accurate, rapid, and simpler method for our beta-thalassaemia prenatal diagnosis programme in Singapore.

Amplification of cDNA via RT-PCR using RNA extracted from postmortem tissues.

Analysis of cDNA derived from messenger RNA is of advantage over using genomic DNA in genetic analysis of large genes, especially those with lengthy intron sequences. However, because of its instability and rapid degradation, RNA extraction from postmortem tissues has not been attempted. Here, we report the successful extraction of intact mRNA from various postmortem tissues from accidental and sudden death cases. Subsequently with reverse transcriptase-polymerase chain reaction (RT-PCR), we were able to amplify cDNA fragments of different lengths up to 0.9 kb. The described method therefore provides a useful tool in genetic analysis of postmortem tissues.

The diagnosis of tuberculous meningitis using the polymerase chain reaction.

AIM: DNA amplification by the polymerase chain reaction (PCR) was evaluated as a means for rapid diagnosis of tuberculous meningitis (TBM). METHODS: A 240 bp region (nts 460-700) from the MPB 64 protein coding gene specific for Mycobacterium tuberculosis (TB) was selected for amplification. Nineteen clinical samples were studied. Six were obtained from patients with TBM diagnosed by culture (4/6) or by response to therapy (2/6). The remaining 13 samples were obtained from patients with febrile seizu es (8/13), aseptic meningitis (3/13) and septic meningitis (2/13), and these served as negative controls. RESULTS: We detected TB DNA in all the 6 CSF specimens obtained from patients with TBM. PCR alone was sufficient to detect TB DNA in 5 of these 6 samples. However, one sample was positive only when PCR was followed by oligonucleotide hybridisation. In the 2 patients whose CSF were obtained only after commencement of TB therapy, TB cultures were negative but positive on PCR nd oligoprobe labelling. The diagnosis of TBM was confirmed based on their remarkable response to therapy. Twelve of the thirteen negative controls were TB DNA negative. There was one false positive sample, which was thought to be due to TB DNA contamination. CONCLUSION: Taken together, our results indicate that DNA amplification using PCR, followed by oligonucleotide hybridisation offers a rapid (5 working days) means of diagnosis of TBM, provided care is taken to ensure that cross contamination of DNA samples is avoided.

DNA amplification by the polymerase chain reaction for the rapid diagnosis of tuberculous meningitis. Comparison of protocols involving three mycobacterial DNA sequences, IS6110, 65 kDa antigen, and MPB64.

DNA amplification of three Mycobacterium tuberculosis-specific DNA sequences by the polymerase chain reaction (PCR) were evaluated as a means for rapid diagnosis of tuberculous meningitis (TBM). The DNA sequences amplified were a 123 bp region of the IS6110 insertion elements which occur in multiple copies in the mycobacterial genome, a 240 bp region (nts 460-700) from the MPB 64 protein coding gene, and the 383 bp region of the 65 kDa heat shock protein (HSP) antigen. Twenty-seven cerebrospinal fluid (CSF) specimens were studied. Six were obtained from patients with TBM diagnosed by culture (4/6) or by the patients' response to anti-tuberculous therapy (2/6). The remaining 21 specimens were obtained from patients with febrile seizures (3/21), aseptic meningitis (3/21), septic meningitis (14/21), and cryptococcal meningitis (1/21), and these served as negative controls. Our results indicate that although the protocols involving the 3 DNA sequences were able to detect TB DNA in the 6 TBM specimens, the main drawback was their extreme sensitivity, thus giving rise to false positive results. In particular, the repeat copy sequence, IS6110, and the 65 kDa HSP gave unacceptably large numbers of false positive results (62% and 33%, respectively).