亮氨酸拉链结构蛋白在大肠杆菌重组子中的表达

酵母转录因子GCN4是通过亮氨酸拉链(bZIP)结构结合DNA的蛋白质之一，当GCN4二聚体与DNA结合时，亮氨酸拉链区的2个单体结合为平行的卷曲螺旋结构，而其基区由无规线团结构变为α螺旋．为探讨亮氨酸拉链蛋白与DNA的结合机理，设计了含有GCN4亮氨酸拉链蛋白基区结合DNA的必需氨基酸的折叠片段，并将其克隆到Escherichia coli BL21，讨论了此亮氨酸拉链蛋白的表达条件．在蛋白质的小量表达试验中，重组子Escherichia coli BL21于5mL含有50μg／mL氨节青霉素和34μg／mL氯霉素的LB液体培养基中培养至对数期，加入不同浓度的IPTG，继续培养以诱导蛋白质的表达，在不同的时间(如：诱导前，诱导2，4，6，8h)取样100μL到1．5mL离心管中、离心收集沉淀，将沉淀悬浮于样品缓冲液中，用10％SDS-PAGE检测；在10L含氨苄青霉素和氯霉素的LB液体培养基中进行了大量表达，根据小量表达的试验结果确定了IPTG的浓度和诱导时间．结果表明：含有这种拉链蛋白质的重组子Escherichia coli BL21在37℃下小量培养时，0．1-0．8mmol的IPTG均可在2-10h内诱导该蛋白质表达；而大量培养时，0．2mmol和0．4mmol的IPTG在37℃均不可能诱导表达，只在28℃时才表达；小量培养和大量培养的最佳诱导时间为4-6h，诱导剂IPTG的浓度为0．2mmol，大量表达的温度为28℃而不是37℃．     

Insertional mutagenesis of the myc locus by a LINE-1 sequence in a human breast carcinoma

The proto-oncogene c-myc is the cellular homologue of the transforming sequence carried by the avian myelocytomastosis virus MC29. A growing body of evidence implicates structural and functional alterations in and around proto-oncogenes such as c-myc in tumorogenesis. Here we report that comparison of the structure of myc from a ductal adenocarcinoma of the breast and from normal breast tissue of the same patient (Sc) revealed a tumour-specific rearrangement of one myc locus and amplification of the other myc locus. (For myc reviews see refs 1-4; for myc involvement in breast neoplasia see refs 5-7.) Within the second intron of the rearranged locus was a non-myc sequence with nearly complete homology to a long interspersed repetitive element (a LINE-1 sequence or L1). In this case, the L1 sequence has functioned as a mobile genetic element to produce a somatic mutation.