Cytosine hypomethylation at CHG and CHH sites in the pleiotropic mutants of Mendelian inheritance in Catharanthus roseus

The 5S and 18S rDNA sequences of Catharanthus roseus cv ‘Nirmal’ (wild type) and its leafless inflorescence (lli), evergreen dwarf (egd) and irregular leaf lamina (ill) single mutants and lli egd, lli ill and egd ill double mutants were characterized. The lli, egd and ill mutants of Mendelian inheritance bore the names after their most conspicuous morphological feature(s). They had been chemically induced and isolated for their salt tolerance. The double mutants were isolated as morphological segregants from crosses between single mutants. The morphological features of the two parents accompanied salt tolerance in the double mutants. All the six mutants were hypomethylated at repeat sequences, upregulated and downregulated for many genes and carried pleiotropic alterations for several traits. Here the 5S and 18S rDNAs of C. roseus were found to be relatively low in cytosine content. Cytosines were preponderantly in CG context (53%) and almost all of them were methylated (97%). The cytosines in CHH and CHG (where H = A, T or C) contexts were largely demethylated (92%) in mutants. The demethylation was attributable to reduced expression of RDR2 and DRM2 led RNA dependant DNA methylation and CMT3 led maintenance methylation pathways. Mutants had gained some cytosines by substitution of C at T sites. These perhaps arose on account of errors in DNA replication, mediated by widespread cytosine demethylation at CHG and CHH sites. It was concluded that the regulation of cytosine methylation mechanisms was disturbed in the mutants. ILL, EGD and LLI genes were identified as the positive regulators of other genes mediating the RdDM and CMT3 pathways, for establishment and maintenance of cytosine methylation in C. roseus.     

Dose‐dependent fate of GFP‐expressing Escherichia coli in the alimentary canal of adult house flies

The adult house fly Musca domestica (L.) (Diptera: Muscidae) can disseminate bacteria from microbe‐rich substrates to areas in which humans and domesticated animals reside. Because bacterial abundance fluctuates widely across substrates, flies encounter and ingest varying amounts of bacteria. This study investigated the dose‐dependent survival of bacteria in house flies. Flies were fed four different ‘doses’ of green fluorescent protein (GFP)‐expressing Escherichia coli (GFP E. coli) (very low, low, medium, high) and survival was determined at 1, 4, 10 and 22 h post‐ingestion by culture and epifluorescent microscopy. Over 22 h, the decline in GFP E. coli was significant in all treatments (P < 0.04) except the very low dose treatment (P = 0.235). Change in survival (ΔS) did not differ between flies fed low and very low doses of bacteria across all time‐points, although ΔS in both treatments differed from that in flies fed high and medium doses of bacteria at several time‐points. At 4, 10 and 22 h, GFP E. coli ΔS significantly differed between medium and high dose‐fed flies. A threshold dose, above which bacteria are detected and destroyed by house flies, may exist and is likely to be immune‐mediated. Understanding dose‐dependent bacterial survival in flies can help in predicting bacteria transmission potential.     

Population Analysis of Licuala glabra Griff. var. glabra (Palmae) using RAPD Profiling

Random amplified polymorphic DNA (RAPD) profiling revealed geneticdiversity among three populations ofLicuala glabra var. glabra,an understorey palm growing in the dipterocarp forests of Terengganu,Malaysia. The Bukit Bauk population had individuals withoutaerial stems. The Bukit Besi individuals had stem heights to30 cm, while those of the Rasau Kerteh population had stemsup to 4 m in height. Southern blot analysis confirmed that anapparent monomorphic band generated by the primer OPU-17 amongthe three different populations corresponds to the same locus.Six primers were used to generate 87 RAPD bands that formed2-state character raw data for estimating genetic distancesusing the simple matching coefficient of similarity. The phenogramgenerated by UPGMA of the RAPD data grouped the individualsfrom the three populations under three clusters. This was inagreement with the morphological grouping. The infraspecificL. glabra var.selangorensis Becc. was grouped closer to theL. glabra var. glabra individuals than to the outgroupL. scortechiniiBecc. Principal component analysis supported the observationfrom the phenogram. The results show discrete genetic structuringin the populations studied and provide useful information forfuture conservation measures. Copyright 1999 Annals of BotanyCompany Licuala glabra Griff. var. glabra, RAPD, palm, AMOVA, population, genetic diversity, conservation.