CLONING AND CHARACTERIZATION OF A CERVICAL MUCIN

宫颈粘蛋白的克隆和表征

基本信息

批准号：
2674064
负责人：
Beverly S. Chilton
金额：
$ 6.32万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-08-01 至 2000-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2674064
关键词：
animal genetic material tag cervix complementary DNA gene induction /repression gene mutation glycosylation hormone regulation /control mechanism human genetic material tag laboratory rabbit molecular cloning mucins nucleic acid probes polymerase chain reaction protein sequence protein structure function site directed mutagenesis steroid hormone

项目摘要

DESCRIPTION: (Adapted from applicant's description) Endocervical mucins are central to fertility/infertility regulation as hormone dependent-changes in their viscoelastic properties control sperm and bacterial access to the upper reproductive tract. The ultimate objective of this research is to understand how steroids regulate mucin biosynthesis in the mammalian endocervix. The aim of this proposal is to clone and characterize the cDNA for the high molecular weight rabbit cervical mucin which has been isolated, purified and partially deglycosylated. This goal will be achieved by 1) deglycosylating the high molecular weight cervical mucin and obtaining microsequence data; 2) using the microsequence data to prepare gene specific probes/PCR primers as tools to clone and characterize the cDNA for the mucin. The high molecular weight cervical mucin will be maximally deglycosylated with minimal or no protein degradation by a combination of enzymatic and chemical steps as follows: cervical mucin will be incubated with neuraminidase (sialidase) to produce asialo-mucin. The asialo-mucin will be treated sequentially with trifluoromethanesulfonic acid (TFMSA)/anisole to remove peripheral sugars, followed by oxidation and beta-elimination. In conjunction with TFMSA/anisole treatment, the asialo-mucin may be treated with exo- and endo-alpha-N-acetylgalactosaminidases to remove GalNAc and Gal---GalNAc residues. Reaction products will be sent to Harvard Microchemistry where in situ digestion, tryptic peptide separation and microsequence analysis will be performed. Microsequence data will be used in a computer search of GenEMBL and SWISS-PROT data bases to verify that the cervical mucin is new. Microsequence will be used to design gene-specific probes for use with conventional cloning techniques, and primers for adaptor mediated PCR, i.e., Marathon TM 5' and 3' RACE (rapid amplification of cDNA ends), to obtain full-length cDNA sequence. Northern blot analyses and RNase protection assays will be used to determine whether the size and number of mRNAs are altered during cervical differentiation and/or in response to steroid hormones. Future direction will include the use of site-directed mutagenesis to provide templates for the synthesis of new proteins whose structural and functional characteristics can be tested in vitro, and the use of the rabbit cDNAs to clone the human cervical mucin.

描述：（改编自申请人的描述）宫颈内粘蛋白是生育/不孕调节的核心，因为激素依赖性变化它们的粘弹性特性控制精子和细菌进入上生殖道。这项研究的最终目标是了解类固醇如何调节哺乳动物的粘蛋白生物合成子宫颈内膜。本提案的目的是克隆和表征 cDNA 对于已分离的高分子量兔宫颈粘蛋白，纯化并部分去糖基化。这一目标将通过以下方式实现：1) 将高分子量宫颈粘蛋白去糖基化并获得微序列数据； 2）利用微序列数据制备基因特异性探针/PCR 引物作为克隆和表征 cDNA 的工具粘蛋白。高分子量宫颈粘蛋白将最大限度地通过以下组合进行去糖基化，蛋白质降解极少或没有：酶促和化学步骤如下：宫颈粘蛋白将被孵育与神经氨酸酶（唾液酸酶）一起产生脱唾液酸粘蛋白。脱唾液酸粘蛋白依次用三氟甲磺酸处理 (TFMSA)/苯甲醚去除外围糖，然后氧化和 β-消除。与 TFMSA/苯甲醚处理相结合，脱唾液酸粘蛋白可以用外切和内切-α-N-乙酰氨基半乳糖苷酶去除 GalNAc 和 Gal---GalNAc 残留物。反应产物将被送往哈佛微化学研究所原位消化、胰蛋白酶肽分离和微序列分析将被执行。微序列数据将用于计算机搜索 GenEMBL和SWISS-PROT数据库验证宫颈粘蛋白是新的。微序列将用于设计基因特异性探针，用于常规克隆技术和接头介导的 PCR 的引物，即 Marathon TM 5'和3'RACE（cDNA末端快速扩增），获得全长 cDNA 序列。 Northern 印迹分析和 RNase 保护检测将用于确定 mRNA 的大小和数量是否符合要求。在宫颈分化期间和/或响应类固醇而改变荷尔蒙。未来的方向将包括使用站点定向诱变为合成新蛋白质提供模板结构和功能特性可以在体外进行测试，并且使用兔 cDNA 克隆人宫颈粘蛋白。