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High efficiency method to obtain supercoiled DNA with a commercial plasmid purification kit

100%

Carbone A., Fioretti F. M., Fucci L., Ausió J., Piscopo M.

Acta Biochimica Polonica

2012

vol. 59

issue 2

275-278

Supercoiled state corresponds to the active form for plasmid applications. The relaxed circular form of plasmids is often inactive or poorly active. To obtain significant amounts of almost fully supercoiled DNA, we modified the standard protocol of a commercially available Qiagen plasmid purification kit. Our changes led to isolation of almost 100% of the plasmids in the supercoiled state. The modified protocol was used to purify different plasmids with consistent results. The purified plasmids maintain supercoiled state for about two months. The modified protocol is very advantageous because it allows easy DNA production with high degree of supercoiled form at low cost.

Protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on male mollusk Mytilus galloprovincialis (Lamarck 1819)

59%

Piscopo M., Trifuoggi M., Notariale R., Labar S., Troisi J., Giarra A., Rabbito D., Puoti R., de Benedictis D., Brundo M., Basile A., Good K., Ausió J., Guerriero G.

Acta Biochimica Polonica

2018

vol. 65

issue 2

259-267

Here we report the industrial pollution effects due to cadmium on the reproductive health of Mytilus galloprovincialis. Mussels were removed from the biofouling of a Conatex panel after one year exposition at a polluted site near a disposal metallurgical factory. A high cadmium bioaccumulation was observed in the testis of mussels housed at the polluted site, with respect to a control site, as determined by inductively coupled plasma-mass spectrometry, along with a 10 fold increase in metallothionein 20 kDa gene (mt20) expression levels determined by qPCR. Furthermore, mussels transferred into laboratory tanks from the reference site, and exposed to 1.5, 5 and 10 µM CdCl2, revealed a 1.7, 3.2 and 4.5 fold expression increase in the testis mt20, respectively, and a positive correlation with cadmium bioaccumulation was found. To evaluate a potential detrimental risk of such alterations on spermatozoa, we carried out electrophoretic analyses on their protamine-like proteins. As determined by AU-PAGE, after 1.5 µM CdCl2 exposure, protamine-like proteins also display major alterations with respect to those obtained after 5 and 10 µM CdCl2 exposure. All protamine-like proteins isolated from the polluted biofouling were in an aggregated form and displayed the same reduced DNA binding affinity of the protamine-like proteins obtained after 1.5 µM CdCl2 as demonstrated EMSA with sperm genomic DNA. Our results contribute to the studies concerning cadmium induced testis alterations and highlight protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on Mytilus galloprovincialis, for the sensitivity of the in vivo and in vitro changes of protamine-like proteins' state and their DNA binding affinity.

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2 Acta Biochimica Polonica

1 2018

1 2012

2 Ausió J.

2 Piscopo M.

1 Basile A.

1 Brundo M.

1 Carbone A.

1 Fioretti F. M.

1 Fucci L.

1 Giarra A.

1 Good K.

1 Guerriero G.

1 Labar S.

1 Notariale R.

1 Puoti R.

1 Rabbito D.

1 Trifuoggi M.

1 Troisi J.

1 de Benedictis D.

Search results

High efficiency method to obtain supercoiled DNA with a commercial plasmid purification kit

Protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on male mollusk Mytilus galloprovincialis (Lamarck 1819)