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

在我们过去三年的研究中出现的一些关于玉米bz位点转座因子插入对功能等位基因表达的影响的有争议的线索将被调查。该研究将涉及Bz- m13（在Bz等位基因的第二外显子插入一个2.2 kb的缺陷Spm (dSpm)）及其状态衍生物的变化，以及Bz等位基因转录起始位点上游63个碱基对的Ds插入导致的Bz-wm，以及Bz-wm的稳定的Bz逆转录物。我们期望在以下领域获得有关基因表达控制的基本信息：1) 5'调控序列的扰动影响，该序列可调节UDPglucose: flavonoids glucosyl transferase （UFGT，由Bz等位基因编码的酶）的数量，并可能在组织特异性基因表达中发挥作用；由于Bz-wm中存在的Ds转座元件的切除而导致额外的核苷酸或小的缺失；2)反义dSpm信息在减少CS12等位基因前体mrna转录或加工中的作用，以及该反义dSpm信息是由插入CS12的dSpm转录还是从基因组其他位置的dSpm转录；3)玉米pre-mRNA加工系统在处理含有dSpm的转录本和系统响应最强烈的序列信号方面的灵活性；4)在CS10等位基因bz位点的第1和第2外显子上插入两个相距363bp的大dSpms对基因表达的影响和染色体断裂的可能性，以及当存在一个活跃的Spm时，这两个相同的插入可能在发育的不同时间切除的可能性。如果插入是相同的，则转座因子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因子的结构及其对基因表达的影响，通常是通过插入到结构基因中。然而，越来越明显的是，转座因子和它们所在的基因组之间可能发生更微妙的相互作用。像这样的研究将有助于澄清这种情况。

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.