ISOLATION AND CHARACTERIZATION OF THE GENE ENCODING THE SURFACE-MEMBRANE 3'-NUCLEOTIDASE NUCLEASE OF LEISHMANIA-DONOVANI
ISOLATION AND CHARACTERIZATION OF THE GENE ENCODING THE SURFACE-MEMBRANE 3'-NUCLEOTIDASE NUCLEASE OF LEISHMANIA-DONOVANI
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DOI：
10.1016/0166-6851(95)00035-y
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发表时间：
1995-04-01
影响因子：
1.5
通讯作者：
DWYER, DM
中科院分区：
文献类型：
--
作者：
DEBRABANT, A;GOTTLIEB, M;DWYER, DM
杜氏利什曼原虫和相关的锥虫原生动物具有能够水解3 '-核苷酸和核酸的外向表面膜酶。凭借这些活性，这种3 '-核苷酸酶/核酸酶（3'-NT/Nu），以前被证明类似于真菌和植物I类单链特异性核酸酶，被认为在嘌呤的挽救中起关键作用，嘌呤对这些生物体的生存至关重要。从L. Donovani前鞭毛体和胰蛋白酶处理。四个释放的胰蛋白酶肽片段产生氨基酸序列信息（Pept-1至Pept-4），其为制备用于PCR扩增约100个核苷酸的寡核苷酸引物提供了基础。300 bp的DNA片段。该片段被克隆、测序并用于检测L. donovani粘粒文库。对从粘粒克隆中分离的4.5 kb SmaI片段进行核苷酸序列分析，发现一个1434 nt的开放阅读框架（ORF），编码477个氨基酸的蛋白质。将Pept-1至Pept-4定位到ORF推导的蛋白质序列上。合成对应于Pept-1至Pept-4的肽并用于免疫家兔。产生的抗肽抗体在Western印迹上识别43-kDa蛋白质，并免疫沉淀来自L. donovani膜提取物。此外，ORF推导的蛋白质共享显着的序列同一性，S1和P1真菌核酸酶的曲霉和桔青霉，分别。累积起来，这些结果证明ORF对应于杜氏乳杆菌3 '-核苷酸酶/核酸酶的基因。在北方印迹中，特异于3 '-NT/Nu基因的核苷酸探针与单个2.5-kb信使RNA杂交。Southern印迹分析结果与3 '-NT/Nu由单拷贝基因编码一致。这些数据构成了这种独特的锥虫表面膜酶的基因的第一份报告。
Leishmania donovani and related trypanosomatid protozoa possess an externally oriented surface membrane enzyme capable of hydrolyzing both 3'-nucleotides and nucleic acids. By virtue of these activities, this 3'-nucleotidase/nuclease (3'-NT/Nu), previously shown to be analogous to fungal and plant class-I single-strand-specific nucleases, is thought to play a critical role in the salvage of purines, essential for the survival of these organisms. The 43-kDa 3'-NT/Nu was purified from L. donovani promastigotes and trypsin treated. Four of the released tryptic peptide fragments yielded amino-acid sequence information (Pept-1 to Pept-4) which provided the basis for the preparation of oligonucleotide primers used for PCR amplification of an approx. 300-bp DNA fragment. This fragment was cloned, sequenced and used to probe a genomic L. donovani cosmid library. Nucleotide sequence analysis of a 4.5-kb SmaI fragment, isolated from a cosmid clone, revealed an open reading frame (ORF) of 1434 nt encoding a 477-amino-acid protein. Pept-1 to Pept-4 were mapped onto the ORF-deduced protein sequence. Peptides corresponding to Pept-1 to Pept-4 were synthesized and used to immunize rabbits. The resulting anti-peptide antibodies recognized the 43-kDa protein on Western blots and immunoprecipitated the native 3'-nucleotidase activity from L. donovani membrane extracts. Further, the ORF-deduced protein shared significant sequence identity with the S1 and P1 fungal nucleases of Aspergillus oryzae and Penicillium citrinum, respectively. Cumulatively, these results demonstrated that the ORF corresponded to a gene for the L, donovani 3'-nucleotidase/nuclease. In Northern blots a nucleotide probe specific for the 3'-NT/Nu gene hybridized to a single 2.5-kb messenger RNA. Results of Southern blot analyses were consistent with the 3'-NT/Nu being encoded by a single copy gene. These data constitute the first report of the gene for this unique trypanosomatid surface membrane enzyme.