BIOCHEMISTRY OF INVERTEBRATE GLOBINS AND HEMOCYANINS

血红蛋白在大多数无脊椎动物中广泛但零星分布。 门。大多数这些血红蛋白都有15-17kD的链，是 胞内(小于或等于65Kd的分子)或胞外 分子量达到几百万。一些节肢动物和软体动物有 具有由2到20个肌红蛋白样单位组成的链的血红蛋白 由多肽键连接。许多血红蛋白表现出很高的 协同作用的O2结合，并受pH强烈影响。其他人则是 不合作，完全不受pH的影响。这非同寻常 形式和功能的多样性提供了一个有吸引力的系统，其中 研究基因结构和进化。我们计划确定cDNAs 和4种无脊椎动物珠蛋白的基因结构：Urechis (胞内，约14KD链)，地龙(胞外，15-18Kd) 链)，Barbatia(细胞内，约33Kd链，含2个血红素 结构域和17Kd链)和Cardita(细胞外，各290Kd链 约有20个血红素域)。我们将了解到，如果，似乎最不可能的是， 结构域由独特的RNA加工事件引起。如果这个领域的基因 珠蛋白显示了预期的重复结构，然后显示了 它们应该表明一种重复的模式。现有数据表明， Cardita珠蛋白中的大约20个结构域可能都非常相似。基因 结构应允许评估不平等可能起的作用 互换和基因转化。豆科植物的豆类血红蛋白基因 植物有3个内含子和4个外显子，与脊椎动物的珠蛋白基因形成对比 它有2个内含子。目前，唯一已知的结构是 无脊椎动物的珠蛋白基因是摇蚊幼虫的基因，但这 基因没有内含子，因此没有揭示祖先珠蛋白的性质 基因与内含子的数量有关。 测定了8种链中的一种的氨基酸序列 双核铜蛋白，血蓝蛋白，来自马蹄蟹， 西门子。我们建议确定其余7个链的结构 通过重组DNA技术，并将其作为探针用于检测 血蓝蛋白基因。血蓝蛋白已被证明与 脉孢子菌酪氨酸酶。因此我们计划分离酪氨酸酶，如果 有可能是从河蟹和/或其他已知的节肢动物和软体动物中 同时具有酪氨酸酶和血蓝蛋白。我们将分离Poly(A)+RNA， 并制备cDNAs，进行克隆和筛选。 寡核苷酸。然后我们将使用该cDNA来筛选基因组文库。 酪氨酸酶和血蓝蛋白基因。

Hemoglobins are widely but sporadically distributed in most invertebrate phyla. Most of these hemoglobins have 15-17 Kd chains and are either intracellular (molecules less than or equal to 65Kd) or extracellular with molecular weights to several million. Some arthropods and molluscs have hemoglobins which have chains composed of 2 to 20 myoglobin-like units joined by peptide bonds. Many of the hemoglobins exhibit highly cooperative O2 binding and are strongly affected by pH. Others are non-cooperative and completely independent of pH. This extraordinary diversity of both form and function provides an attractive system in which to investigate gene structure and evolution. We plan to determine the cDNA and gene structure of globins from 4 invertebrates: Urechis (intracellular, about 14Kd chains), Lumbricus (extracellular, 15-18Kd chains), Barbatia (intracellular, about 33Kd chains with 2 heme-containing domains and 17Kd chains), and Cardita (extracellular, 290Kd Chains each with about 20 heme-domains). We will learn if, as seems most unlikely, the domains arose by unique RNA-processing events. If the genes of the domain globins show the expected repeating structure, then the differences between them should indicate a pattern of duplication. Existing data suggest that the about 20 domains in Cardita globin may all be very similar. The gene structure should allow assessment of the possible roles of unequal crossing-over and gene conversion. The leghemoglobin gene of leguminous plants has 3 introns and 4 exons in contrast to the vertebrate globin gene which has 2 introns. At this time the only known structure of an invertebrate globin gene is that of the larval insect, Chironomus, but this gene has no introns so sheds no light on the nature of the ancestral globin gene with regard to the numbers of introns. We have determined the amino acid sequence of one of the 8 kinds of chain of the binuclear copper protein, hemocyanin, from the horseshoe crab, Limulus. We propose to determine the structures of the remaining 7 chains by recombinant DNA techniques and to use the cDNA as a probe for the hemocyanin genes. Hemocyanins have been shown to be homologous with Neurospora tyrosinase. We plan therefore to isolate tyrosinase, if possible, from Limulus and/or other arthropods and molluscs known to possess both tyrosinase and hemocyanin. We will isolate the poly(A)+RNA, and prepare cDNA which will be cloned and screened with appropriate oligonucleotides. We will then use the cDNA to screen genomic libraries for the tyrosinase and hemocyanin genes.