Gomez-Escobar Natalia, Gregory William F, Britton Collette, Murray Linda, Corton Craig, Hall Neil, Daub Jen, Blaxter Mark L, Maizels Rick M
Institute of Cell, Animal and Population Biology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK.
Mol Biochem Parasitol. 2002 Nov-Dec;125(1-2):59-71. doi: 10.1016/s0166-6851(02)00219-0.
The genomic organisation of two abundant larval transcript (alt) genes from the filarial nematode Brugia malayi has been defined. The products of these genes are 78% identical in amino acid sequence, and are highly expressed in a stage-specific manner by mosquito-borne infective larvae. alt-1 is present as two near-identical copies organised in an inverted repeat of approximately 7.6 kb, occupying a total of 16 kb of the genome. alt-2 is a single-copy gene at a different locus to alt-1. The two alt-1 genes (alt-1.1 and -1.2) are 99.7% identical in coding sequence and 99.5% in intronic sequences. Both alt-1 and -2 contain 3 introns, and the third intron of alt-2 exhibits a size polymorphism evident in different individual parasites from the laboratory-maintained strain. Genomic sequence up- and down-stream from alt-1.1/1.2 (26 and 6 kb, respectively) and alt-2 (6 and 4 kb, respectively) show that neither gene is in a multiple array or an operon. Most notably, the neighbouring genes of alt-1 and -2 show no similarity to each other, or to the genes flanking the distant alt homologue in Caenorhabditis elegans. Despite this diversity in flanking genes, the 5' UTR tracts extending some 800 bp upstream of each B. malayi alt gene show a high degree of similarity (overall 59% identity with tracts of 77-86% identity). Surmising that this region may contain conserved promoter elements, constructs containing the B. malayi alt 5' UTR with or without coding sequence were made fused to beta-galactosidase reporter protein. These constructs were injected into the syncytical gonad of C. elegans and progeny stained for beta-gal expression. Our results show relatively strong expression in the gut cells of C. elegans for both alt-1 and -2 constructs, commencing in larval worms and continuing into adulthood. Moreover, expression was enhanced when constructs contained segments of alt-1 coding and intronic sequence in addition to the 5' UTR. We conclude that the high level of alt transcription in filarial L3s is not due to expression from a multi-copy gene family but to a set of strong promoter elements shared between the two alt genes.
马来丝虫两个丰富的幼虫转录本（alt）基因的基因组结构已被确定。这些基因的产物在氨基酸序列上有78%的同一性，并由蚊媒感染性幼虫以阶段特异性方式高度表达。alt-1以两个近乎相同的拷贝形式存在，以大约7.6 kb的反向重复排列，共占据基因组的16 kb。alt-2是位于与alt-1不同位点的单拷贝基因。两个alt-1基因（alt-1.1和-1.2）的编码序列同一性为99.7%，内含子序列同一性为99.5%。alt-1和-2都含有3个内含子，alt-2的第三个内含子在实验室保存菌株的不同个体寄生虫中表现出明显的大小多态性。alt-1.1/1.2上下游（分别为26 kb和6 kb）以及alt-2上下游（分别为6 kb和4 kb）的基因组序列表明，这两个基因都不在多基因阵列或操纵子中。最值得注意的是，alt-1和-2的相邻基因彼此之间没有相似性，也与秀丽隐杆线虫中遥远的alt同源物两侧的基因没有相似性。尽管侧翼基因存在这种多样性，但每个马来丝虫alt基因上游约800 bp的5' UTR区域显示出高度相似性（总体同一性为59%，片段同一性为77 - 86%）。推测该区域可能包含保守的启动子元件，构建了含有或不含有编码序列的马来丝虫alt 5' UTR与β-半乳糖苷酶报告蛋白融合的构建体。将这些构建体注射到秀丽隐杆线虫的合胞性腺中，并对后代进行β-半乳糖苷表达染色。我们的结果表明，alt-1和-2构建体在秀丽隐杆线虫的肠道细胞中均有相对较强的表达，从幼虫期开始并持续到成虫期。此外，当构建体除了5' UTR外还包含alt-1编码和内含子序列片段时，表达增强。我们得出结论，丝虫L3中alt的高水平转录不是由于多拷贝基因家族的表达，而是由于两个alt基因共享的一组强启动子元件。
