Molecular cloning and expression of a stress-inducible gene highly homologous to human structural maintenance of chromosome 6

Guangping WANG; Weijun CAI

ABSTRACT

BACKGROUND:

Previous studies have demonstrated that the responses of myocardium to short periods of ischemia/reperfusion are associated with changes in the expressions of a variety of genes. However, the characteristics of these changes are unknown.

OBJECTIVE:

To clone and analyze a stress-inducible gene characteristics in order to study the molecular mechanism of myocardium to short periods of ischemia/reperfusion damage.

DESIGN:

A controlled animal study.

SETTING:

Department of Hematology, Xiangya Hospital of Central South University.MATERIALS Sixteen castrated male German Landrace-type domestic porcines, weighing between 21 and 39 kg, were provided by the laboratory of Department of Experimental of Cardiology at Bad Nauheim in Germany. The porcines were randomly divided into two groups ischemia/reperfusion group (n =14) and control group (n =2). The porcines in the ischemia/reperfusion group were observed at 0, 30 and 90 minutes of reperfusion after reocclusion, and ischemia and non-ischemia myocardial tissues were harvested from each pocrine.

METHODS:

The experiments were performed in the laboratory of Department of Experimental Cardiology of Max-Planck Institute for Physiological and Clinical Research at Bad Nauheim in Germany and the Department of Hematology, Xiangya Hospital of Central South University from July in 1998 to May in 2007. All the animals were anesthetized and thoracotomized. Following 30 minutes of stabilization, the left anterior descending coronary artery (LAD) was occluded for 10 minutes followed by 30-minute reperfusion and then another 10-minute reocclusion. The porcines were killed immediately at corresponding time points. The sham-operated animals were killed without occlusion. Experimental tissue was removed from the LAD area and control tissue from the region of the left circumflex coronary artery area. Firstly, a porcine heart cDNA library was screened, DNA and deduced amino acid sequences were then analyzed. Meanwhile, short periods of myocardium ischemia/reperfusion was performed by occluding porcine LAD followed by reperfusion as mentioned above, and total RNAs isolated from myocardium or a variety of other tissues were used for Northern blotting. Quantitation of mRNA levels was accomplished by using a PhosphorImager and Image Quant software, the ration of mRNA/18S rRNA was suggested as the level of gene expression.MAIN OUTCOME

MEASURES:

① DNA and amino acid sequences analysis of the cloned gene; ② Analysis of the cloned gene expression.

RESULTS:

All the 16 porcines were involved in the final analysis of results. After screening, a cDNA fragment with 3 461 base pairs was obtained. DNA sequencing and searching revealed that this cDNA shared 86% identity to human structural maintenance of chromosome 6 gene (hSMC6), and 84% identity to Mus musculus SMC6 (mSMC6). Furthermore, a largest polypeptide deduced from this cDNA contained 1007 amino acid residues, and protein homology searching indicated that the predicted polypeptide carried 92% and 89% identity to hSMC6 or mSMC6 protein respectively. Thus, the cloned cDNA was referred to as a porcine homology gene to hSMC6 (pSMC6). The results of Northern blotting showed that the expression of the pSMC6 mRNA in ischemia/reperfusion myocardium was increased, and its mRNA existed in all organs tested.

CONCLUSION:

A stress-inducible gene of pSMC6 from porcine myocardium has been cloned, which is high homology to hSMC6 gene, and the pSMC6 or along with other molecules is possible involved in the early repair of myocardium to DNA damage caused by ischemia/reperfusion in the porcine heart.