| The complexâ… is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes.It couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The primary structures of the complexâ… subunits from several proteobacteria, Gram positive bacteria, members of the Thermus/Deinococcus group, and from the genus Aquifex are available now. The bacterial complex I, in general,is made up of 14 different subunits, representing a minimal structural form of a proton-pumping NADH:ubiquinone oxidoreductase. The 14 subunits sum up to a molecular mass of approx. 530 kDa. Little is known about their function, but they are most likely involved in quinone reduction and proton translocation.In addition to the homologues of the 14 bacterial complexâ… subunits, the mitochondrial complexâ… of eukaryotes contains up to 29 additional proteins. They add up to a molecular mass of approx.1 MDa. The homologues of the seven hydrophobic bacterial subunits are mitochondrially encoded in all eukaryotes. The function of the additional subunits in the mitochondrial complexâ… is not clear.They are all nuclear-encoded and show a weak but distinct sequence similarity between bovine and Neurospora crassa. They might build a scaffold around the 14 minimal subunits preventing the high energy electrons from escaping the complex and forming reactive oxygen species. Two of the additional subunits appear to have a biosynthetic function.One is an acyl carrier protein, the other is a 40 kDa subunit with a tightly bound NADPH belonging to a heterogeneous family of reductases/isomerases.Dunaliella salina is a unicellular green alga which is unique in its ability to adapt to hypersaline environment, from 0.1 mol/L to 5.5mol/L. The character of hypersaline make that respiratory chain offer more energy for themselves. The complexâ… is the first and key of the respiratory chain complexes,so it is involved in resisting the stresses. Based on the construction of cDNA library in Dunaliella salina, in this study we got these results:â… . Cloning full length 19-kD subunit of Complexâ… gene in Dunaliella salina. We also got the full length cDNA of this EST by 3' RACE and 5' RACE. During 3'RACE experiments of 19-kD subunit gene, two different full 3' ends were amplified and sequenced. Thus, Dunaliella salina has at least 2 full length of the 19-kD subunit, 682 bp and 727 bp, respectively.And encoding two distinct protein of the 19-kD subunit. Therefore we designed the reverse primer from the 3'-UTR of the 19-kD subunit cDNA. The RT-PCR screening for the 19-kD subunit used Dunaliella salina cells RNA and the specific primers. Surprisingly, the RT-PCR result gave rise to two bands after agarose gel electrophoresis. In addition to the smaller fragment (680 bp) a larger fragment (1128 bp) was present. The four genes are named variants A,B,C and D of 19-kD subunit, respectively.â…¡. Make sure that 19-kD subunit of Complexâ… gene in Dunaliella salina regulate by alternative splicing.The genomic DNA was amplified by PCR using a specific primer set corresponding to the 5' and 3' untranslated regions. The genomic DNA sequence of 19-kD subunit indicate that all the variant transcripts of 19-kD subunit are spliced by this genomic DNA sequence.â…¢. Bioinformatics analysis. The deduced proteins are analyed, and some features are found. For example, Analysis showed that these clones shared high identities with Complexâ… subunit from Chlamydomonas reinhardtii.â…£. Short-term changes in 19-kD subunit of Complexâ… gene mRNA levels corresponding to different stresses. This research was studyed by Real-Time Quantitative PCR. The results indicate that all the transcripts were down regulated under oxygen deficiency, salt treatment, and rotenone treatment.â…¤. Enzyme activity assay in different stresses. Found that 19-kD subunit may participate in the function of Complexâ… . Complexâ… from Dunaliella salina can strongly resist salinity treatment, and NaCl stress damaged Complexâ… via oxidative stress. Complexâ… from Dunaliella salina is rotenone-insensitive and specifically restrain by rotenone.We have cloned the gene of 19-kD subunit of Complexâ… from Dunaliella salina. It is the first time to find that the complexâ… subunit gene expresses four distinct mature transcripts by alternative splicing in algae. And used Real-Time Quantitative PCR analyse this phenomenon and found that the relation between 19-kD subunit and Complexâ… from Dunaliella salina, it will be important to understand the structure and function of Complexâ… .
|
PDF Full Text Request