Oviedo Norma-Angélica, Salgado Heladia, Collado-Vides Julio, Guarneros Gabriel
Departmento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, A.P. 14-740, México DF 07000, Mexico.
Gene. 2004 Mar 31;329:115-24. doi: 10.1016/j.gene.2003.12.031.
The bar loci in the chromosome of bacteriophage lambda inhibit phage vegetative growth in bacteria defective for peptidyl-tRNA hydrolase (Pth). Expression of the bar regions results in accumulation of peptidyl-tRNA, inhibition of protein synthesis, and arrest of mutant cell growth. These effects have been ascribed to the expression of two-codon ORFs present in translatable sequences named 'minigenes' in the lambda bar regions. To investigate the nature, frequency, and distribution of minigenes in the phage genome, we conducted a survey of their location in lambda DNA. A short-fragment random genomic DNA library was constructed for the identification of clones inhibitory of Pth-defective cells (bar-like phenotype). Three new bar-like minigenes were identified in the library but only one was on the sense strand and it had a rare initiation codon. This result contrasted with the in silico identification of over a hundred putative minigenes using an ad hoc computer program on both strands of lambda DNA. Unlike bar constructs, most of the toxic constructed clones were also toxic to wild-type bacteria, thus suggesting a different inhibition mechanism. Sequence analysis of these cloned inserts showed that they harbored minigenes, mini-ORFs, gene starts, gene ends, or combinations thereof. Our data suggest that minigene-like sequences may, at least partly, account for toxicity in wild-type cells. We propose that clustering of minigenes at gene ends may play a role in gene expression. Other minigenes identified in silico were non-toxic. It is still an open question what the in vivo function of these and toxic minigenes might be.
噬菌体λ染色体上的bar基因座会抑制肽基-tRNA水解酶（Pth）缺陷型细菌中的噬菌体营养生长。bar区域的表达会导致肽基-tRNA积累、蛋白质合成受到抑制以及突变细胞生长停滞。这些效应归因于λ bar区域中名为“小基因”的可翻译序列中存在的双密码子开放阅读框（ORF）的表达。为了研究噬菌体基因组中小基因的性质、频率和分布，我们对它们在λ DNA中的位置进行了调查。构建了一个短片段随机基因组DNA文库，用于鉴定对Pth缺陷型细胞具有抑制作用的克隆（类bar表型）。在该文库中鉴定出了三个新的类bar小基因，但只有一个位于有义链上，并且它具有罕见的起始密码子。这一结果与使用专门的计算机程序在λ DNA的两条链上通过计算机模拟鉴定出的一百多个推定小基因形成了对比。与bar构建体不同，大多数有毒的构建克隆对野生型细菌也有毒性，因此表明存在不同的抑制机制。对这些克隆插入片段的序列分析表明，它们含有小基因、小ORF、基因起始位点、基因终止位点或它们的组合。我们的数据表明，类小基因序列可能至少部分地解释了野生型细胞中的毒性。我们提出，小基因在基因末端的聚集可能在基因表达中起作用。在计算机模拟中鉴定出的其他小基因是无毒的。这些小基因以及有毒小基因在体内的功能仍然是一个悬而未决的问题。
