| Homologous genes of different eukaryotic species share similar gene structures after the long-term evolution,especially those with important functions,they share not only highly similar protein-coding sequences,but also highly conserved intron-exon structure.Single-copy genes are often most important genes and highly conserved during evolution.Therefore,the structure conservation of intron-exon of single-copy genes would provide a very good subject for studies on the most basic event of intron evolution,namely,loss and gain of introns.In this study,we selected the highly-conserved regions of 105 homologous genes,and compared their intron-exon structure in great detail.3574 introns and 1001 conserved sites from human,mouse,rat,drosophila,anopheles and nematodes were comparatively analyzed to infer intron-evolution process in different species.By comparative analysis of loss and gain of introns and careful verification of each site,it was found that vertebrates have more introns compared to insects and nematodes.It was speculated that in the early stages of evolution,a large number of introns exist in the common ancestor of vertebrates,insects and nematodes,which were lost later in large numbers in insects and nematodes,especially the former.Meanwhile,new introns were also introduced,which far exceeds the number of those lost in vertebrates and slightly exceeds the number of intron loss in nematodes.On the contrary,gain of introns was far less than intron loss.After statistical analysis of the 105 homologous single-copy genes,we proposed a hypothesis to explain the difference in intron number and distribution in these eukaryotes.It was speculated that the large number of introns in vertebrates may not be gradually gained in the later stage of evolution,but explosively obtained by the common ancestor of vertebrates.Such hypothesis also explains the difference in the number of introns,namely,introns exist in much larger number in vertebrates and less in insects.Meanwhile,insertion phase shift distribution ratio of introns in insects was significantly different from that in vertebrates and nematodes.Previous studies demonstrated a 5:3:2 ratio of insertion phase shift distribution(0:1:2),but it was found in our study a 4:3:3 ratio in insects,and a 5:2.5:2.5 ratio in both vertebrates and nematodes,which may be due to a larger number of 0-phase intron losses in insects.In addition,since loss and gain of introns is frequent during evolution,it should also be observed between evolutionarily close species.To prove this inference,we select dozens of single-copy genes and compared their sequence among 33 species of Drosophila melanogaster,and the most significant difference in gene sequence was found in the Hp27(Heat shock protein 27)gene.Extra 72-92 base pairs were found in the species subgroup of D.ananassae,D.bipectinata,D.malerkotliana and D.parabipectinata(according to sequence of D.melanogaster from http://flybase.org/reports/FBgn0001226.html).According to RT-PCR results,this extra sequence was spliced from the mature mRNA,which makes it an intron.We performed phylogenetic analysis of the sequences of these Drosophila species subgroups.Specifically,we chose evolutionarily closer D.persimilis and D.pseudoobscura as outgroup,the tree constructed by neighbor-joining method is similar to the trees constructed by maximum-likelihood and Bayesian method based on Hsp27 and COI,COII,ND1,andND4L gene sequences.The results suggested that the melanogaster species subgroups significantly clustered into pedigrees:ananassae species subgroup differentiated first;followed by the montium species subgroup;and a third branch consisting of the melanogaster species subgroup and the "suzukii,elegans,eugracilis,ficusphila and takahashii" species subgroup.Analysis of evolution relationship of the 4 species of ananasae species subgroup using the three methods yield exactly the same results,with high degree of confidence.Namely,D.ananassae differentiated first,followed by evolutionarily close D.bipectinata,D.parabipectinata and D.malerkotliana.Since only the 4 species of the D.ananassae subgroup(D.ananassae,D.bipectinata,D.malerkotliana and D.parabipectinata)had this intron,while D.persimilis and D.pseudoobscura don't,it was speculated that the Hsp27 gene of the D.ananassae species subgroup gained this intron during evolution.In summary,taken together the earlier findings by our group that the Dfak gene of D.melanogaster species subgroup lost an intron,there must be continuous loss and gain of introns in genome of certain insect species,though we can't be sure whether there was explosive loss and/or gain of introns in the early differentiation of insects.Idiopathic basal gaglia calcification(IBGC),also known as Fahr disease(FD)or familial idiopathic basal ganglia calcification(FIBGC),is a rare neurological disease that can be familial or disseminated.This disease is characterized by abnormal calcification of hypothalamus,dentate nucleus and other parts of the brain that control motion of the body,and is often accompanied by language disability,Parkinson's disease,chorea,epilepsy,dementia and other neuropsychiatric symptoms.SLC20A2 is considered the most common disease-causing gene of IBGC,but the pathogenic mechanism has not been clarified.The homologous gene of human SLC20A2 in Drosophila is CG42575(dSLC20A2),with a 42%consistency.Therefore,to establish a Drosophila model of IBGC would greatly facilitate studies on the function of this molecule in the cell and during tissue development,providing valuable tools for studies on its functions in the nervous system.The following results have been obtained.1)Drosophila models with mutations in C42575 gene have been obtained,including a normal-UAS-CG42575 transgenic drosophila strain that spatial-and temporal-specifically expresses high level of CG42575;4 mutated-UAS-CG42575 transgenic strains(UAS-CG42575-S123C,UAS-CG42575-E562K,UAS-CG42575-T582M and UAS-CG42575-S588W)that resemble mutations in human disease;2 transgenic strains highly expressing CG42575 with a GFP tag;2 strains of CG42575-deficient strains by Cas9 method;and prepared CG-42575-specific rabbit polyclonal antibody which has been proved effective.2)Studies on these Drosophila models have yield some preliminary results:A,using CG42575 and CG42575-GFP transgenic flies,it was found that CG42575 is located on the membrane of brain cells and neuro muscular junction of third-instar larvae.B,nonfunctional mutation in CG42575 causes embryonic death,which can be rescued by nervous system-specific expression of wild-type CG42575:flies can live to adult.C,high expression of wild-type CG42575 in the nervous system causes no obvious phenotypes,while high expression of mutated UAS-CG42575-S588W cause significant abnormality in the neuromuscular synapse:synaptic bouton junction significantly shortened and smeared,with many satellite synaptic boutons,which may be caused by the dominant negative effect of non-functional CG42575 inserted in the membrane.Previous studies of our group proved that abnormalities in synaptic bouton are often accompanied by synaptic dysfunction.Therefore,the above results suggest an important role of CG42575 in development and function of the neural system. |
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