Nuclease activity of Plasmodium falciparum Alba family protein PfAlba3

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A variant in the restriction endonuclease cleavage pattern of mitochondrial DNA in the domestic fowl, Gallus gallus domesticus

Summary. The cleavage patterns of mitochondrial DNAs (mtDNAs) were investigated from 15 lines of domestic fowls, Gallus gallus domesticus . using 11 restriction endonucleases. The cleavage patterns with 10 restriction endonucleases were identical in all the lines. A variant was found in a line of White Leghorn in the pattern with Mspl digestions. Cleavage patterns of the red jungle fowl, Gallus gallus gallus , were identical to the common patterns shown by the 14 lines of domestic fowls.     

Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant ofNeisseria gonorrhoeae

A technique that allows for easy identification of transformants ofNeisseria gonorrhoeae in the absence of selective pressure has been developed. A suicide vector that contains a gonococcal DNA uptake sequence was constructed to aid in DNA uptake. In this transformation procedure, a limiting number of cells is incubated with an excess amount of DNA, and the mixture is plated onto a non-selective medium. At least 20% of the resulting colonies contained cells that had been transformed. This strategy was utilized to construct specific deletions of the S.NgoI, II, IV, V and VII restriction-modification (R/M) genes. All five deletions were successfully incorporated into the chromosome of FA19, producing strain JUG029. Strain JUG029 could be transformed with non-methylated plasmid DNA while strain FA19 could not be transformed with such DNA. The development of a simple, non-selective transformation technique, coupled with the construction of a strain that is more permissive for DNA-mediated transformation, will aid in genetic manipulations of the gonococcus.     

Detection and differentiation of whitefly-transmitted geminiviruses in plants and vector insects by the polymerase chain reaction with degenerate primers

Degenerate oligonucleotide primers, designed for amplification of an approx. 500 bp fragment of DNA-A of five well characterised whitefly-transmitted geminiviruses, were used in the polymerase chain reaction (PCR) to detect known or putative geminiviruses infecting seven plant species and originally obtained from Africa, India, America or Europe. Although nucleotide sequences are published for only four of the viruses, all 13 were detected. Six of the viruses were also detected in single viruliferous whiteflies (Bemisia tabaci). Virus was detected both in fresh B. tabaci and in specimens that were frozen and dried before being dispatched from their country of origin. Individual viruses could be distinguished by the patterns of DNA fragments obtained by the action of restriction endonucleases on the PCR products. This approach also allowed six virus isolates from leaf curl-affected tomato to be assigned to four country-specific forms.     

Homing in juvenile salmon in response to imposed and spontaneous displacement: experiments in an artificial stream

Displacement of juvenile Atlantic salmon Salmo salar within an artificial stream was either spontaneous (fish left areas of shallow water in response to experimental reduction in water level) or imposed (fish were removed by the experimenter from areas of shallow water and placed at a distance from their home site). Prior to displacement, the fish showed a high degree of site fidelity in terms of preferential use of specific areas within the stream, but the extent to which this persisted once they had left/been removed from their preferred sites was variable. Direction of displacement was not a critical factor, but homing was significantly less likely to occur following spontaneous as opposed to imposed displacement. In the case of imposed displacement, fish that were more strongly site attached prior to displacement were more likely to return to their home site after this manipulation.     

Improved Large-Scale Preparation of Phage T4 Endonuclease VII Overexpressed in E. coli

Using PCR, we cloned T4 gene 49, which encodes the endonucleaseVII, and the inactive mutant gene 49 amE727 into vector pET-11a.In combination with Escherichia coli host strain BL21(DE3),this system provided excellent repression of the expressionof the highly toxic protein before induction with IPTG. Afterinduction, the proteins were made in high quantities while remainingsoluble. Dilution of the crude lysate at 1 : 10,000 continuedto show a highly specific activity in the case of the wild-typeenzyme. The protein was purified to homogeneity with a recoveryof 33% using two chromatography steps. The yield was 20 timeshigher and the specific activity 500 times higher than thatobtained by using the previously published protocol.