EXPRESSION OF H-2 ANTIGENS IN SJL/J TUMORS

SJL/J 肿瘤中 H-2 抗原的表达

• 批准号: 3187315
• 负责人: Kirk W. Beisel
• 金额: $ 11.69万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3187315
• 关键词: T lymphocyte; chemical carcinogen; gel electrophoresis; gene expression; histocompatibility antigens; immunochemistry; lymphoma; methylcholanthrene; molecular cloning; molecular oncology; monoclonal antibody; neoplasm /cancer genetics; neoplasm /cancer immunology; phase contrast microscopy; sarcoma; serology /serodiagnosis; tissue /cell culture

SJL/J lymphomas can be used as a model of tumor progression from a slow-growing malignancy to an aggressive tumor. We have previously shown that one of the class I MHC antigens, Ds, is not expressed in some SJL/J lymphomas. The Ds-negative tumors are not rejected and grow rapidly. The goal of this research is to determine the structural defect(s) that prevents expression of the Ds antigens. Loss of expression could be due to gross deletion or rearrangement of the gene or small alterations such as point mutations or gene conversion. We will identify and sequence genomic Ds genes from normal tissue and from several malignant SJL/J lymphoma lines. A comparison of the sequences will locate the gene(s). Since the amino acid sequence of the Ds glycoprotein is not known, the Ds gene(s) must be identified from the other 33-35 cross-hybridizing class I genes. A combination of two approaches will be used to identify and determine the number of Ds genes: (1) several cDNA probes will be made from mRNA isolated by immunopurification of polysomes. Size-selected, double-stranded cDNA will be used for insertion into pBR322. These cDNA clones will be tested for their ability to cross-hybridize with a class I probe and then will be sequenced; and (2) the genomic Ds gene(s) will be identified from the other 33-35 cross-hybridizing class I genes on a Southern blot by comparing the DNA from various H-2 congenic and intra-MHC recombinant mouse strains as well as from many SJL/J lymphoma lines. The Ds gene(s) will be identified by its "unique" restriction enzyme site polymorphism. This gene(s) will be cloned into a cosmid vector for sequence analysis. The identify of the genomic Ds gene(s) will be confirmed by cloning the appropriate restriction fragment(s) containing the Ds gene(s) and comparing the sequence of the genomic gene(s) with the Ds cDNA sequence(s). The Ds genes will be subcloned into the pSV2-neo vector for both sequence and immunochemical analyses. Immunochemical analyses of the glycoproteins produced in transformed mouse L cells will confirm the production of clones containing the Ds gene(s). Defective Ds genes will then be cloned from various SJL/J lymphoma lines and sequenced. Such alterations in gene structure may be detected by restriction site analyses on Southern blots followed by sequencing of the appropriate segment of the tumor Ds gene. These studies will, therefore, identify the genetic mechanisms that prevent the expression of cell surface antigens in malignancies. (AG)

SJL/J淋巴瘤可以用来作为肿瘤从 由缓慢生长的恶性肿瘤转变为侵袭性肿瘤。我们之前已经展示了 在某些SJL/J中，I类MHC抗原DS不表达 淋巴瘤。DS阴性的肿瘤不会被排斥，而且生长迅速。这个 本研究的目的是确定结构缺陷(S) 阻止DS抗原的表达。表达的丧失可能是由于 基因的严重缺失或重排或小的改变，如 点突变或基因转换。我们将对基因组进行鉴定和测序 正常组织和几种恶性SJL/J淋巴瘤的DS基因 台词。序列的比较将确定该基因的位置(S)。自.以来 DS糖蛋白的氨基酸序列未知，即DS基因(S) 必须从其他33-35个杂交I类基因中鉴定出来。一个 将使用两种方法的组合来识别和确定 DS基因的数量：(1)将从mRNA中制备几个cdna探针 通过免疫提纯多聚体分离得到。大小选定， 将双链基因插入到pBR322中。这些cdna 克隆人将接受测试，以确定它们与I类生物杂交的能力 然后进行测序；以及(2)基因组DS基因(S)将被 从其他33-35个杂交I类基因中鉴定出 比较不同H-2同源基因和MHC内DNA的Southern杂交 重组小鼠品系以及许多SJL/J淋巴瘤株。这个 DS基因(S)将通过其“唯一”的限制性内切酶位点进行鉴定 多态。该基因(S)将被克隆到粘粒载体中，用于 序列分析。基因组DS基因(S)的鉴定将是 通过克隆包含该基因的适当限制片段(S)确认 DS基因(S)与基因组基因序列(S)的比较 双链c DNA序列(S)。将DS基因亚克隆到pSV2-neo中 用于序列分析和免疫化学分析的载体。免疫化学 转化的小鼠L细胞产生的糖蛋白分析 确认含有ds基因的克隆的生产(S)。有缺陷的 然后将从各种SJL/J淋巴瘤株中克隆DS基因，并 已排序。这种基因结构的变化可以通过以下方式检测到 Southern杂交的限制性内切酶切点分析及序列测定 肿瘤DS基因的适当片段。因此，这些研究将： 识别阻止细胞表面表达的遗传机制 恶性肿瘤中的抗原。(AG)