ANALYSIS OF TRYPANOSOME MRNA SYNTHESIS BY GENE TRANSFER

人和动物的锥虫病有主要的医学和兽医用 后果。锥虫的两个显著和不同寻常的特征是 与他们的基因表达有关。锥虫成熟的mRNA是 由可能涉及大型多顺反子解剖的过程形成的 通过39个核苷酸微小外显子的反应获得初级RNA转录 以反式剪接形成成熟mRNA的5‘端。39位核苷酸 迷你外显子最初由短的、非多聚腺苷化的RNA的5‘端组成 这是从一组重复的基因转录而来的。尽管 编码基因中特定DNA序列的微小功能的基因 由于缺乏遗传系统，基因的表达受到了阻碍。这个 布氏锥虫、西莫氏细单胞菌和利什曼原虫 Enrietti最近被证明对DNA介导的 后两者可以被稳定地转化为 含有抗药性标记。这些技术的可用性意味着 这是第一次顺式和反式作用因素涉及 锥体转录、剪接和翻译的调节是 可以进行遗传分析。 两个结构性表达基因，迷你外显子供体RNA(MedRNA)和 磷酸甘油酸激酶(PGK B和C)基因可能具有重要作用 在锥虫体内。迷你外显子是所有mRNA上的5‘外显子，可能是 对信使核糖核酸的稳定性和翻译很重要。在布氏锥虫中，PGK基因 编码两种略有不同的蛋白质，PGK B基因产物驻留在 细胞质和PGK C基因产物位于糖体，即 锥虫体内糖酵解的主要部位。尽管新生的RNA PGK B和C基因水平相当，它们的转录 可能是源于一种常见的上游启动子，PGK B的mRNA水平是 比来自PGK C基因的基因要大得多。这一比率，可在 布鲁氏原环(昆虫形)毛滴虫的血液形态发生逆转。 有机体。调控这些基因表达的序列将是 使用标记的PGK基因进行解剖，其中所有正常侧翼序列 都存在于转染性构建体上。序列在中的作用 对微小外显子加成效率的调节将通过分析 野生型和体外合成突变序列的能力 作为标记的PGK基因结构上的剪接受体信号。 MedRNA内的剪接信号将通过分析 突变MedRNAs作为特定基因微小外显子供体的能力 MRNA.锥虫启动子从未被定义过。MedRNA基因 启动子将通过对标记的MedRNA基因的突变分析来确定 呈现在一种转化的DNA结构上。归根结底，结合了 锥体中这些序列的定向突变和测试将 揭示锥体如何调节MedRNA的表达和 通过微小外显子添加和聚腺苷酸化，Pre-mRNA成熟为 特定的mRNAs。

The human and animal trypanosomiases have major medical and veterinary consequences. Two salient and unusual characteristics of trypanosomes are related to expression of their genes. Mature mRNAs of trypanosomatids are formed by a process that may involve dissection of large polycistronic primary RNA transcripts by a reaction in which a 39 nucleotide mini-exon is spliced in trans to form the 5' end of the mature mRNA. The 39-nucleotide mini-exon initially comprises the 5' end of a short, non-polyadenylated RNA that is transcribed from a set of tandemly reiterated genes. Although the genes that encode the mini-function of specific DNA sequences in gene expression have been stymied by the absence of a genetic system. The trypanosomatids Trypanosoma brucei, Leptomonas seymouri, and Leishmania enrietti recently have been shown to be amenable to DNA-mediated transfection; the latter two can be stably transformed by plasmids that contain drug-resistant markers. The availability of these techniques means that for the first time the cis and trans-acting factors involved in the regulation of trypanosome transcription, splicing and translation are accessible to genetic analyses. Two constitutively expressed genes, the mini-exon donor RNA (medRNA) and phosphoglycerate kinase (PGK B and C) genes probably have essential roles in trypanosomes. The mini-exon is the 5' exon on all mRNAs and may be important for mRNA stability and translation. In T. brucei, the PGK genes encode two slightly different proteins, the PGK B gene product resides in the cytoplasm and the PGK C gene product locates to the glycosome, which is the main site of glycolysis in trypanosomes. Although the nascent RNA levels of the PGK B and C genes are equivalent and their transcription probably is derived from a common, upstream promoter, PGK B mRNA levels are much greater than are those from the PGK C gene. This ratio, found in procyclic (insect form) T. brucei is reversed in bloodstream form organisms. Sequences that regulate the expression of these genes will be dissected using marked PGK genes, in which all normally flanking sequences are present on the transfecting constructs. The function of sequences in modulating the efficiency of mini-exon addition will be studied by assaying wild-type and in vitro synthesized mutant sequences for their ability to serve as splice acceptor signals on the marked PGK gene constructs. Splicing signals within the medRNA will be determined by assaying the ability of mutant medRNAs to function as mini-exon donors to a specific mRNA. Trypanosome promoters have never been defined. The medRNA gene promoter will be identified by mutation analysis of a marked medRNA gene present on a transforming DNA construct. Ultimately, a combination of directed mutagenesis and testing of these sequences in trypanosomes will reveal how trypanosomes regulate the expression of medRNA and the maturation, via mini-exon addition and polyadenylation, of pre-mRNA to specific mRNAs.