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BIOCHEMICAL BASIS OF DEVELOPMENT IN DICTYOSTELIUM

盘基网柄菌发育的生物化学基础

基本信息

批准号：
3271875
负责人：
WILLIAM F LOOMIS
金额：
$ 17.62万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
1979
资助国家：
美国
起止时间：
1979-04-01 至 1994-07-31
项目状态：
已结题

项目摘要

Techniques for the reproducible transformation of Dictyostelium cells with cloned sequences have allowed us to determine essential cis-acting sequences in the 5' flanking region of a developmentally controlled gene (actin 15) that are shared by another gene that is co-expressed (actin 6) (Cohen et al 1986). Transcripts of both of these genes appear immediately after the initiation of development and accumulate during the aggregation stage. We want to use a similar approach to determine whether a set of cell-type specific genes, including those that code for the spore coat proteins, also share common control regions that determine the stage in development and cell type in which they are transcribed. Moreover, we would like to analyze the extracellular signals that are monitored to ensure proper transcription of these genes. We have isolated cell-lines that fail to form multicellular aggregates because they lack myosin heavy chain specifically. These cell lines were selected after transformation with a vector that carries a portion of the myosin heavy chain gene such that it is transcribed in the reverse orientation under the control of the actin 6 promotor (Knecht and Loomis, 1987, see Appendix). Surprisingly, the almost complete lack of myosin heavy chain protein is not lethal but results in a block to formation of multicellular aggregates and all subsequent differentiations including the expression of pre-spore specific genes. We will attempt to by-pass this block to late biochemical differentiations by altering the conditions of development and adding back extracts of wild-type developing cells. We have been concentrating on the genes for the major spore coat proteins of Dictyostelium discoideum for several reasons. There are strong advantages to working with cloned sequences whose products are known and can be recognized by both biochemical and immunological techniques. The spore coat proteins, SP60, SP70, and SP96 are coordinately synthesized at the tipped aggregate stage of development (14 hr) and accumulate in prespore but not in prestalk cells. They are stored in prespore vesicles that fuse with the plasma membranes during sporulation to form the extracellular coats around each spore. We have antibodies that recognize these spore coat proteins. We have determined the N-terminal amino acid sequence of SP70 nd SP60 and have characterized cDNA clones that appear to be derived from mRNAs for each of the spore coat proteins. We plan to further characterize these clones by constructing transformation vectors that will disrupt their endogenous genes when integrated by homologous recombination (De Lozanne and Spudich, 1987). We also plan to construct anti-sense transformation vectors and show that they can inactivate the expected endogenous mRNAs. We plan to isolate genome clones corresponding to each of the cDNAs to observe the N-terminal sequence coding regions. By directly determining the essential cis-acting sequences in transformants we hope to recognize share cis-acting sequences that integrate expression of this set of genes.

网柄网柄菌可重复转化技术研究具有克隆序列的细胞使我们能够确定 A基因5‘侧翼区的基本顺式作用序列发育控制基因(肌动蛋白15)，由另一个共表达的基因(肌动蛋白6)(Cohen等人，1986)。这两个基因的转录本都出现在在聚集过程中启动发展和积累舞台。我们希望使用类似的方法来确定一个一组细胞类型的特定基因，包括那些编码孢子壳蛋白，也共享共同的控制区，确定它们所处的发育阶段和细胞类型都被转录了下来。此外，我们还想分析一下被监测的细胞外信号，以确保适当这些基因的转录。我们已经分离出了失败的细胞系形成多细胞聚集体，因为它们缺乏肌球蛋白重具体地说是链条。这些细胞系是在利用携带部分肌球蛋白的载体进行转化重链基因，这样它就被反向转录肌动蛋白6启动子控制下的定向(KNECHTT和 Loomis，1987，见附录)。令人惊讶的是，几乎完成了缺乏肌球蛋白重链蛋白不是致命的，但会导致阻止形成多细胞聚集体和所有随后的分化包括前孢子特异性的表达基因。我们将试图绕过这一障碍，转到Late Biotics 通过改变发展和发展的条件来区分加入野生型发育细胞的提取液。我们一直在关注主孢子皮的基因。盘基网眼菌的蛋白质有几个原因。那里是处理克隆序列的强大优势，这些克隆序列产品是已知的，并且可以被两个生物化学公司识别和免疫学技术。孢子壳蛋白SP60， SP70和SP96是在末端配位合成的综合发展阶段(14小时)，并在前孢子，但不在前柄细胞中。它们储存在预孢子中。在孢子形成过程中与质膜融合的小泡以形成每个孢子周围的胞外衣。我们有识别这些孢子壳蛋白的抗体。我们有 SP70和SP60的N-端氨基酸序列测定并鉴定了看似源自来自每种孢子外壳蛋白的mRNAs。我们计划通过构建转换来进一步表征这些克隆整合后会破坏其内源基因的载体通过同源重组(de Lozanne和Spudich，1987)。我们还计划构建反义转化载体和表明它们可以灭活预期的内源性mRNAs。我们计划分离出与每一个观察N-末端序列编码区。通过直接测定基本的顺式作用序列我们希望识别的转化子具有相同的顺式作用序列整合了这一组基因的表达。