BIOCHEMISTRY OF INVERTEBRATE GLOBINS AND HEMOCYANINS

无脊椎动物球蛋白和血蓝蛋白的生物化学

• 批准号: 3289145
• 负责人: AUSTEN F RIGGS
• 金额: $ 13.61万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1990-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289145
• 关键词: Invertebrata; complementary DNA; gene conversion; genetic crossing over; genetic library; genetic manipulation; genetic regulation; globulins; hemocyanin; hemoglobin; molecular cloning; monophenol monooxygenase; nucleic acid sequence; saltwater environment

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.

血红蛋白在大多数无脊椎动物中广泛但零星分布， 门 这些血红蛋白中的大多数具有15-17 Kd链，并且是 细胞内（分子小于或等于65 Kd）或细胞外， 分子量达到几百万。 一些节肢动物和软体动物 具有由2至20个肌红蛋白样单位组成的链的血红蛋白 通过肽键连接。 许多血红蛋白表现出高度的 合作O2结合，并强烈受pH值的影响。其他是 非合作和完全独立的pH值。这一非凡的 形式和功能的多样性提供了一个有吸引力的系统， 来研究基因结构和进化 我们计划确定 四种无脊椎动物球蛋白的基因结构 蚯蚓（Lubricus）（胞内，约14 Kd链）、蚯蚓（胞外，15- 18 Kd Barbatia（胞内，约33 Kd链，含2个血红素 结构域和17 Kd链）和Cardita（细胞外，每个290 Kd链 具有约20个血红素结构域）。 我们将了解，如果，似乎最不可能， 结构域由独特的RNA加工事件产生。 如果该域的基因 球蛋白显示出预期的重复结构， 它们应表明一种重复模式。 现有数据表明， Cardita珠蛋白中的约20个结构域可能都非常相似。 基因 结构应允许评估不平等 交换和基因转换。 豆科植物的豆血红蛋白基因 与脊椎动物珠蛋白基因相比，植物珠蛋白基因具有3个内含子和4个外显子 它有两个内含子 目前已知的唯一一种 无脊椎动物的珠蛋白基因是幼虫昆虫摇蚊的基因，但这 一个基因没有内含子，因此无法解释祖先球蛋白的性质 基因的内含子数。 我们测定了8种链中的一种的氨基酸序列 双核铜蛋白，血蓝蛋白，来自鲎， 鲎 我们建议确定其余7个链的结构 通过重组DNA技术，并使用cDNA作为探针， 血蓝蛋白基因 血蓝蛋白已被证明与 脉孢菌酪氨酸酶。 因此，我们计划分离酪氨酸酶，如果 可能来自鲎和/或其他节肢动物和软体动物， 同时具有酪氨酸酶和血蓝蛋白。 我们将分离poly（A）+RNA， 并制备cDNA，其将被克隆并用适当的引物筛选， 寡核苷酸 然后我们将使用cDNA筛选基因组文库 酪氨酸酶和血蓝蛋白基因