DNA SEQUENCES INVOLVED IN THE HEAVY CHAIN SWITCH

在免疫球蛋白的抗原驱动分化过程中- 产生细胞，从mu到gamma的转换， 可以发生链合成。 这种蛋白质开关是一种 将V区从供体C μ移动到C μ的DNA缺失 γ、C β或C α基因。 此删除开始和结束 在开关片段中，DNA区域由串联重复的 序列的 我们建议用分子克隆和测序技术， 在6至10个重排γ 3中转换重组位点， 杂交瘤DNA的γ 1基因。 伽玛3和伽玛1 每个开关区域包括特征49个碱基对 串联重复序列。 我们以前的数据表明， 伽马3开关均匀分布在其49聚体中， 共有序列，但S γ 1开关是 定位于其49聚体共有序列的一部分。 此外，伽马开关位点附近的序列表明， 一种酶可以介导所有四种酶的切割/再连接 小鼠的伽马基因。 我们将使用新的序列数据 从伽马转换点测试这些初步结论。 我们 将确定开关重组是否发生在串联 仅重复序列，或者如果它可以发生在非49聚体中， 序列在S γ 1区。 我们还将使用来自 从克隆的开关区域的核苷酸序列来理解 两者之间的转换点是否存在相关关系 来自单个免疫球蛋白表达细胞的染色体。 在同一组实验中，我们将研究 从具有简单的开关的单元重新排列开关区域的结构 开关模式（典型开关）和复杂开关 模式（非典型开关）。 我们还将检查开关 使用α-寡核苷酸产生的鼠杂交瘤的区域含量 表达骨髓瘤配偶体。 我们会问两个脾细胞- 衍生的Igh基因座倾向于重排到相同的开关区域，并且 重链转换中同种型特异性的指示 重组事件 这样我们就可以了解 开关的序列识别、特异性和遗传学 我们建议选择组成型重组酶的细胞系， 产生这些酶。

During antigen-driven differentiation of immunoglobulin- producing cells, a switch from mu to gamma, epsilon, or a heavy chain synthesis can occur. This protein switch is the result of a DNA deletion that moves a V region from a donor C mu to a C gamma, C epsilon, or C alpha gene. This deletion begins and ends in switch segments, DNA regions composed of tandemly repeated sequences. We propose to molecularly clone and sequence the switch recombination sites in six to ten rearranged gamma 3 and gamma 1 genes from hybridoma DNA. The gamma 3 and gamma 1 switch regions each include a characterisitic 49 base pair tandemly repeated sequence. Our previous data indicate that the gamma 3 switches are uniformly distributed throughout its 49mer consensus sequence, but that the S gamma 1 switches are localized to a part of its 49mers consensus sequence. Furthermore, sequences near gamma switch sites suggest that a single enzyme may mediate the cutting/religation for all four gamma genes in the mouse. We will use our new sequence data from gamma switch sites to test these tentative conclusions. We will determine if switch recombination occurs in the tandemly repeated sequences only, or if it can occur in non-49mer sequences in the S gamma 1 region. We will also use data derived from nucleotide sequences of cloned switch regions to understand any correlative relationships in switch sites on the two chromosomes from a single immunoglobulin expressing cell. Under the same group of experiments, we will examine the structure of rearranged switch regions from cells with simple switching patterns (typical switches) and complex switching patterns (atypical switches). We will also examine the switch region content of murine hybridomas generated using an alpha- expressing myeloma partner. We will ask if the two spleen cell- derived Igh loci tend to rearrange to the same switch region, and indication of isotype specificity in the heavy chain switch recombinational event. So that we might understand the DNA sequence recognition, specificity, and genetics of switch recombinases we propose to select a cell line that constitutively produces these enzymes.