A novel rice genomic sequence encoding coding segments homologous to other metallothionein-like genes was isolated from Oryza sativa genomic library. This sequence, hereby designated as rgMT (rice genomic metallothionein-like gene), consists of two exons and one intron. From the coding sequence, it is predicted that rgMT encodes one protein of 74 amino acids. Differential expression of rgMT in rice plants was observed as mature transcripts were more abundant in roots than in leaves and sheaths. Under different stress conditions, such as excess heavy metals and heat shock, expression of rgMT was significantly elevated. This was especially noticeable with 250 microM CuCl2 for 16 h, 40 degrees C heat for 2 h and 0.06% DMSO for 1 h. Under sucrose starvation, rgMT transcripts also increased with time up to 72 h. During recovery from sucrose starvation, the transcripts declined slightly within 12 h of recovery. rgMT transcripts were also seen to have increased expression in senescent leaves. These results support the notion that rgMT is a stress-inducible gene in rice heretofore unreported.

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A type 2 metallothionein-like gene from rice, OsMT-2 (Oryza sativa metallothionein-like gene-2), was isolated in its cDNA form and sequenced. By northern analyses OsMT-2 expression was shown to be induced under stress by sucrose starvation, heat shock and, to a lesser extent, abscisic acid, but not excess metals, including copper. Its response to sucrose starvation was transient and different from OsMT-1, a type 1 metallothionein-like gene of rice inducible by copper. These results suggest that while OsMT-2 is also involved in cellular response to stress, its function may be complementary to that of OsMT-1.

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We report on a novel application of real-time reverse transcription-loop-mediated isothermal amplification (real-time RT-LAMP) to identify the presence of a specific body fluid using blood as a proof-of-concept model. By comparison with recently developed methods of body fluid identification, the RT-LAMP assay is rapid and requires only one simple heating-block maintained at a single temperature, circumventing the need for dedicated equipment. RNA was extracted from different body fluids (blood, semen, saliva, menstrual blood, sweat, and urine) for use in real-time RT-LAMP reaction. The 18S rRNA locus was used as the internal control and hemoglobin beta (HBB) as the blood-specific marker. Reverse transcription and LAMP reaction were performed in the same tube using a turbidimeter for real-time monitoring the reaction products within a threshold of 60 min. HBB LAMP products were only detected in blood and not in any of the other body fluid, but products from the 18S rRNA gene were detected in all the tested body fluids as expected. The limit of detection was a minimum of 10(-5) ng total RNA for detection of both 18S rRNA and HBB. Augmenting the detection of RT-LAMP products was performed by separation of the products using gel electrophoresis and collecting the fluorescence of calcein. The data collected indicated complete concordance with the body fluid tested regardless of the method of detection used. This is the first application of real-time RT-LAMP to detect body fluid specific RNA and indicates the use of this method in forensic biology.

A novel stress-inducible metallothionein-like gene from rice, designated as rgMT-1 (rice genomic metallothionein-like gene-1), was isolated and sequenced. From the sequence analysis of its 5'-flanking region, two putative TATA boxes, one CAAT box, and several short sequences homologous to regulatory cis-elements previously reported were identified. Two direct repeats, one 10 bp in length (CAAAATCAAA) and the other 11 bp (GTGAAAATACT), respectively, were also found. By transient GUS (beta-glucuronidase) assay, the expression of GUS, in vitro, was enhanced by the presence of the rgMT-1 intron. The critical region which controls the basal transcription was shown to lie between -73 and -36 upstream of rgMT-1, in which one of the two putative TATA boxes was located. The promoter activity was lost completely when both putative TATA boxes were deleted. This is the first report describing the genomic structure and regulation of a monocotyledonous metallothionein-like gene critical to the response of stress.