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Question

Fig.2.A nonsense mutation is found in the two splicing variants of the ninaD gene.(A)We performed PCR analyses with genomic DNA from homozygous
ninaDP245 and wt flies by using several sets of oligonucleotide primers overlapping the entire genomic region of the predicted gene CG5750.Sequencing of the
PCR products revealed a base pair exchange(G to A)in homozygous ninaDP245 flies,resulting in a stop codon at position 71 of the predicted amino acid sequence
of CG5750.(B)To judge whether CG5750 consists of two genes,we performed PCR and RT-PCR analyses on genomic DNA and mRNA preparations from wt flies
by using three different primer pairs.Primer pair 1 spanned exons 3 to 6,primer pair 2 was specific for exon 3,and primer pair 3 was specific for exon 6 of the
predicted gene CG5750(see D).Agarose gel electrophoresis revealed PCR products with genomic DNA for all three primer pairs;an RT-PCR product could only
be obtained with primer pair 2.(C)3-RLM-RACE-PCR with a 5 oligonucleotide primer specific for exon 3 of the predicted gene CG5750 resulted in two different
products,showing the existence of two splicing variants.(D)The predicted exon intron structure of CG5750(red)and the determined exon intron structures
of the two splicing variants of the putative ninaD gene(blue).The longer splicing variant consists of five exons,the fifth not primarily predicted in GC5750,with
164,303,812,91,and 366 bases,respectively.The shorter variant includes only the first three exons.Coding regions are colored,and the position of the point
mutation found in the putative ninaDP245 allele is indicated by asterisks.(E)Northern blot analyses with mRNA preparations obtained from wt,heterozygous
(het),and homozygous(hom)ninaDP245 flies confirmed the existence of the two splicing variants of the putative ninaD gene.In heterozygous ninaDP245 flies,
the levels of the putative ninaD mRNAs were strongly reduced compared with wt flies;in homozygous ninaDP245 flies(ninaD)they were hardly detectable.(F)
Primary structure of the putative NinaD protein.The positions of the N-terminal and C-terminal transmembrane domains are indicated in blue and red;the
positions of the putative N-glycosylation sites are indicated with green-filled circles,and clustered cysteine residues are indicated by a‘‘C.’’

Answer 1

Fig.2.A无义突变是在两个基因剪接变异体ninaD发现。（一）我们完成了与基因组DNA的PCR分析合子
ninaDP245和WT苍蝇通过使用多套重叠寡核苷酸引物的整个预测的基因组区域的CG5750.Sequencing
PCR产物碱基对揭示了交换（G到A）在纯ninaDP245苍蝇，造成71位置在终止密码子的氨基酸序列
对CG5750。（二）判断是否CG5750的两个基因组成，我们进行基因组DNA和mRNA的筹备工作从野生PCR和RT - PCR分析苍蝇
通过对三种不同的引物pairs.Primer跨越外显子1 3至6日，引物2为3外显子特异性，引物对三是为特定的6外显子
。预测基因CG5750（见四）琼脂糖凝胶电泳显示PCR扩增引物对所有三种基因组DNA的产品;的RT - PCR产物只能
得到引物对2。（C）的寡核苷酸引物5外显子的基因CG5750 3的具体预测3 RLM - RACE技术- PCR技术产生了两个不同的
产品，显示了两个剪接变异体的存在。（四）预测外显子CG5750（红色）内含子的结构和外显子内含子结构的确定
这两个假定的ninaD基因（蓝色）。剪接变异的时间越长由五个外显子剪接变异体，第五主要不是预测GC5750，同
164,303,812,91和366基地，分别只包含短变体的前三个地区是exons.Coding着色，该点的位置
突变发现，在假定ninaDP245是由星号表示等位基因。（E）的基因从野生得到的筹备工作，杂Northern杂交分析
（过度紧张）和纯合子（磡）ninaDP245苍蝇确认了两个假定ninaD gene.In杂ninaDP245苍蝇剪接变异体的存在，
假定的ninaD mRNA的水平与野生型相比，大大减少苍蝇，在纯ninaDP245苍蝇（ninaD）他们很难探测（女）。
假定的NinaD的N端和C端跨膜结构域protein.The职位主要是在蓝色和红色表示;的
在假定的N -糖基化位位置以表示绿满圈，聚集半胱氨酸残基是由a''C表示。