Biochemical and Molecular Analysis of Mutations Caused by Transposable Element Insertions in the bz Locus of Maize

我们在过去三年的研究中发现了一些与玉米BZ基因座转座元件插入对功能(BZ)等位基因表达的影响有关的挑衅线索。这项研究将涉及BZ-M13(在BZ等位基因的第二外显子中插入2.2kb有缺陷的Spm(DSpm))及其状态衍生的变化，以及BZ-Wm，它是由BZ等位基因转录起始点上游插入63个碱基对所导致的BZ-Wm，以及来自BZ-Wm的稳定的BZ回复株。我们期望获得与基因表达控制有关的基本信息的领域是：1)5‘调控序列的扰动的影响，其调节UDP葡萄糖：类黄酮葡萄糖基转移酶(UFGT，其是由BZ等位基因编码的酶)的数量，并且可能在组织特异性基因表达中起作用，通过额外的核苷酸或由于切除BZ-Wm中存在的DS转座元件而导致的小片段缺失；2)反义dSpm信息在减少CS12等位基因中前-mRNA的转录或加工中的作用，以及该反义dSpm信息是从插入CS12的dSpm转录而来，还是从基因组中其他地方的dSpm转录而来；3)玉米前mRNA处理系统在处理含有dSpm的转录本和系统反应最强烈的序列信号时的灵活性；4)在CS10等位基因BZ基因座的外显子1和2的363个碱基内插入两个大的dSpm对基因表达和可能的染色体断裂的影响，以及当存在活跃的spm时，这两个可能相同的插入在发育的不同时间切除的可能性。如果插入片段被证明是相同的，那么转座元件两侧的序列可能是决定因素。分子遗传学使人们有可能了解转座元件的结构及其对基因表达的影响，通常是通过插入结构基因。然而，转座元件和它们所在的基因组之间可能会发生更微妙的相互作用，这一点变得越来越明显。像这样的研究将有助于澄清情况。

A number of provocative leads, which concern the effect of transposable element insertions in the bz locus of maize on the expression of functional (Bz) alleles and which have arisen during our research of the past three years will be investigated. The research will involve bz-m13 (an insertion of a 2.2 kb defective Spm (dSpm) in the second exon of a Bz allele) and the change of state derivatives therefrom as well as Bz-wm, which resulted from a Ds insertion 63 base pairs upstream from the transcription start site in a BZ allele, and the stable Bz revertant from Bz-wm. The areas in which we expect to derive basic information concerning control of gene expression are: 1) the effects of perturbation of a 5' regulatory sequence, which modulates the quantity of UDPglucose:flavonoid glucosyl transferase (UFGT, which is the enzyme encoded by Bz alleles) and which probably has a role in tissue-specific gene expression as well, by extra nucleotide or small deletions resulting from excision of the Ds transposable element present in Bz-wm; 2) the role of an antisense dSpm message in reducing the transcription or processing of the pre-mRNA in the CS12 allele and whether this antisense dSpm message is transcribed from the dSpm inserted in CS12 or from a dSpm elsewhere in the genome; 3) the flexibility of the maize pre-mRNA processing system in dealing with transcripts containing a dSpm and the sequence signals to which the system responds most strongly; and 4) the effect of having two large dSpms inserted within 363 bp of each other in exons 1 and 2 of the bz locus in the CS10 alleleon of gene expression and possible chromosome breakage as well as the possibility that the two insertions, which may be identical, excise at different times in development when an active Spm is present. If the insertions are shown to be identical, sequences flanking the transposable elements may be the determining factor. Molecular genetics has made it possible to learn a great deal about the structure of transposable elements and their effect upon gene expression, usually through insertion into a structural gene. However, it is becoming apparent that more subtle interactions maybe occuring between transposable elements and the genomes in which they reside. Studies such as this one will help to clarify the situation.