BIOCHEMICAL BASIS OF DEVELOPMENT IN DICTYOSTELIUM

网柄藻的可重复转化技术 带有克隆序列的细胞让我们能够确定 必需的顺式作用序列在5'侧翼区的一个 发育控制基因（actin 15）， 另一个共表达的基因（肌动蛋白6）（Cohen et al 1986）。 这两个基因的转录本在基因组的转录后立即出现。 在聚集过程中开始发育和积累 阶段 我们想使用类似的方法来确定 一组细胞类型特异性基因，包括那些编码 孢子外壳蛋白也具有共同控制区， 决定发育阶段和细胞类型， 都被转录了此外，我们还想分析 细胞外信号被监测，以确保适当的 这些基因的转录。 我们已经分离出细胞系， 形成多细胞聚集体，因为它们缺乏重肌球蛋白 具体的链。 这些细胞系是在 用携带部分肌球蛋白的载体转化 重链基因，使其在逆转录 在肌动蛋白6启动子的控制下定向（Knecht和 卢米斯，1987年，见附录）。 令人惊讶的是，几乎完全 缺乏肌球蛋白重链蛋白并不致命，但会导致 阻止多细胞聚集体的形成和所有随后的 分化，包括前孢子特异性的表达 基因. 我们将尝试绕过这个街区， 通过改变发展条件， 再加入野生型发育细胞的提取物。 我们一直专注于主要孢子外壳的基因 Dictyosteelium discoideum的蛋白质的几个原因。 那里 是克隆序列的强大优势， 产品是已知的，可以通过生物化学和生物化学识别， 和免疫学技术。 孢子外壳蛋白，SP 60， SP 70和SP 96在端部配位合成， 聚集发育阶段（14小时），并在 前孢子，但不存在于前柄细胞。 它们储存在前孢子中 在孢子形成过程中与质膜融合的小泡 在每个孢子周围形成细胞外膜。 我们有 识别这些孢子外壳蛋白的抗体。 我们有 测定了SP 70和SP 60的N端氨基酸序列 并对cDNA克隆进行了鉴定， 每种孢子外壳蛋白的mRNA。 我们计划 通过构建转化子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， 表明它们可以抑制预期的内源性mRNA。 我们计划分离出对应于每一个基因的基因组克隆， cDNA以观察N-末端序列编码区。 通过 直接确定了重要的顺式作用序列， 我们希望识别的转化体共享顺式作用序列 整合了这组基因的表达。

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.