BIOCHEMICAL BASIS OF DEVELOPMENT IN DICTYOSTELIUM

盘基网柄菌的可重复转化技术 具有克隆序列的细胞使我们能够确定 5' 侧翼区域的必需顺式作用序列 发育控制基因（肌动蛋白 15） 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在 发展的起始和聚合过程中的积累 阶段。 我们想使用类似的方法来确定是否 一组细胞类型特异性基因，包括那些编码 孢子外壳蛋白，也共享共同的控制区域 确定它们所处的发育阶段和细胞类型 被转录。此外，我们还想分析一下 监测细胞外信号以确保正确 这些基因的转录。 我们分离出失败的细胞系 形成多细胞聚集体，因为它们缺乏肌球蛋白重 专链。 这些细胞系是在之后选择的 用携带部分肌球蛋白的载体进行转化 重链基因，使其反向转录 肌动蛋白 6 启动子（Knecht 和 Loomis，1987，见附录）。 令人惊奇的是，几乎完成了 肌球蛋白重链蛋白的缺乏并不致命，但会导致 阻止多细胞聚集体的形成以及所有后续的 分化，包括前孢子特异性的表达 基因。 我们将尝试绕过这个障碍到后期生化 通过改变发育条件来实现差异化 添加野生型发育细胞的提取物。 我们一直专注于主要孢子衣的基因 盘基网柄菌的蛋白质有几个原因。 那里 使用克隆序列有很大的优势， 产品是已知的并且可以被生化和生物化学识别 和免疫学技术。 孢子外壳蛋白 SP60， SP70和SP96在尖端坐标合成 发展的总体阶段（14小时）并累积 前孢子但不存在于前柄细胞中。 它们储存在前孢子中 在孢子形成过程中与质膜融合的囊泡 在每个孢子周围形成细胞外衣。 我们有 识别这些孢子外壳蛋白的抗体。 我们有 确定了 SP70 和 SP60 的 N 端氨基酸序列 并具有特征性的 cDNA 克隆，这些克隆似乎源自 来自每个孢子外壳蛋白的 mRNA。 我们计划 通过构建转化进一步表征这些克隆 整合时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 表明它们可以使预期的内源 mRNA 失活。 我们计划分离对应于每个的基因组克隆 用于观察 N 末端序列编码区的 cDNA。 由 直接确定必需的顺式作用序列 我们希望识别的转化体具有顺式作用序列 整合这组基因的表达。

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.