Isolation of high quality DNA: a protocol combining “rennet” and glass milk

High quality DNA is essential for many molecular biology techniques. However, the reagents used for that purpose usually are expensive and/or cause a high environmental impact. Here, we describe two alternative protocols that use inexpensive reagents and are not hazardous to the environment. The first protocol utilizes the enzyme chymosin, normally used as “rennet” in cheese production and which is easily obtained on the commercial market. The second protocol uses “rennet DNA extraction protocol” combined with the DNA binding capacity of glass powder (glass milk), which can easily be “home made”. The first protocol is used when a high yield of DNA is needed, whereas the second protocol is used for production of a higher quality DNA, being able to work with sparse samples.

Genetic transformation of Drosophila willistoni using piggyBac transposon and GFP

Studies were carried out on the use of piggyBac transposable element as vector and the green fluorescent protein (EGFP) from the jellyfish, Aquorea victoria, as a genetic marker for the transformation of Drosophila willistoni. Preblastoderm embryos of D. willistoni white mutant were microinjected with a plasmid containing the EGFP marker and the piggyBac ITRs, together with a helper plasmid containing the piggyBac transposase placed under the control of the D. melanogaster hsp70 promoter. G0 adults transformants were recovered at a frequency of approximately 67 percent. Expression of EGFP in larvae, pupae and adults was observed up to the third generation, suggesting that this transposon was not stable in D. willistoni. Transformed individuals displayed high levels of EGFP expression during larvae and adult stages in the eye, abdomen, thorax and legs, suggesting a wide expression pattern in this species than reported to other species of Drosophilidae.

Descrevemos neste trabalho a transformação genética de Drosophila willistoni empregando o elemento transponível piggyBac como vetor e o gene EGFP (green fluorescent protein ) retirado da água-viva Aquorea victoria, como marcador de transformação. Embriões de D. willistoni em estágio pré-blastoderme, mutantes para o gene white, foram microinjetados com plasmídio contendo o marcador EGFP e as regiões ITRs do transposon piggyBac concomitantemente com um plasmídio auxiliar possuindo o gene da transposase de piggyBac sobre o controle do promotor do gene hsp70 de Drosophila melanogaster. Adultos transformantes Go foram gerados em uma taxa de 67 por cento. A expressão de GFP em larvas, pupas e adultos foi observada somente até a terceira geração, sugerindo que este transposon não é estável em D. willistoni. Os indivíduos transformados exigem um alto nível de expressão de EGFP durante os estágios de larva e, também em adultos o gene marcador é expresso nos olhos, abdome, tórax e patas, mostrando um padrão de expressão mais amplo nesta espécie do que o registrado para outros drosofilídeos.

Insertion sequences as variability generators in the Mycoplasma hyopneumoniae and M. synoviae genomes

We have analyzed the sequenced genomes of three strains of Mycoplasma hyopneumoniae and one strain of M. synoviae, and have found three and two different transposable element families, respectively in each species. In M. hyopneumoniae, the Insertion Sequences of the IS4 family is represented by ISMHp1, a putatively active element. The IS3 family is represented by several degenerated sequences. A third element called tMH was found, which shows some characteristics reminiscent of retrotransposons. In M. synoviae, three different possibly active IS4 elements are present (ISMHp1-like; ISMs1 and IS1634-like elements). The IS30 family is represented by the degenerated IS1630-like element. The IS1634-like element is shown to be involved in chromosomal rearrangements and horizontal gene transfer (HGT). The ISMHp1-like element is shown to relate to the HGT of a 25-kb region from M. gallisepticum to M. synoviae. The fractions of these genomes that correspond to mobile elements varied from 1.35 to 3.13 percent in M. hyopneumonia strains and was 2.08 percent in M. synoviae. Although these species possess reduced genomes, they maintain mobile elements, perhaps as a mechanism for genetic variability production.