Functional promoter analysis using an approach based on an in vitro evolution strategy

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the Goldfinger isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter Junction. The selective-shotgun approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional linker-scanning deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the, prospect of identifying interacting promoter regions.

Emerging viral diseases of South-East Asia and the Western Pacific: A brief review

Over the past 6 years, a number of zoonotic and vectorborne viral diseases have emerged in Southeast Asia and the Western Pacific. Vectorborne disease agents discussed in this article include Japanese encephalitis, Barmah Forest, Ross River, and Chikungunya viruses. However, most emerging viruses have been zoonotic, with fruit bats, including flying fox species as the probable wildlife hosts, and these will be discussed as well. The first of these disease agents to emerge was Hendra virus, formerly called equine morbillivirus. This was followed by outbreaks caused by a rabies-related virus, Australian bat lyssavirus, and a virus associated with porcine stillbirths and malformations, Menangle virus. Nipah virus caused an outbreak of fatal pneumonia in pigs and encephalitis in humans in the Malay Peninsula. Most recently, Tioman virus has been isolated from flying foxes, but it has not yet been associated with animal or human disease. Of nonzoonotic viruses, the most important regionally have been enterovirus 71 and HIV.