Eleven daughters of NANOG

Nanog is a recently discovered ANTP class homeobox gene. Mouse Nanog is expressed in the inner cell mass and in embryonic stem cells and has roles in self-renewal and maintenance of pluripotency. Here we describe the location, genomic organization, and relative ages of all human NANOG pseudogenes, comprising ten processed pseudogenes and one tandem duplicate. These are compared to the original, intact human NANOG gene. Eleven is an unusually high number of pseudogenes for a homeobox gene and must reflect expression in the human germ line. A pseudogene orthologous to NANOGP4 was found in chimpanzee and an expressed pseudogene in macaque. Examining pseudogenes of differing ages gives insight into pseudogene decay, which involves an excess of deletion mutations over insertions. The mouse genome has two processed pseudogenes, which are not clear orthologues of the primate pseudogenes.     

Comprehensive analysis of pseudogene HSPB1P1 and its potential roles in hepatocellular carcinoma

The incidence and mortality rate of hepatocellular carcinoma (HCC) nowadays is still at high levels. The regulatory roles of pseudogene in cancers have been gradually recognized in recent years. However, comprehensive investigation of abnormally expressed pseudogene and related mechanisms in HCC remains lacking. GSE124535 dataset was used to identify differentially expressed pseudogenes in HCC tissues compared with normal tissues. Prognostic value of these differentially expressed pseudogenes was analyzed at GEPIA. StarBase used to analyze microRNAs (miRNAs) can bind with pseudogene, while the targets for these miRNAs were analyzed at miRTarBase. Protein–protein interaction (PPI) network was then established for miRNA targets, after that hub genes were selected. Expression correlation of pseudogene and hub genes was analyzed at StarBase. In total, 16 upregulated and 17 downregulated pseudogenes were identified. Pseudogene HSPB1P1 was identified abnormally expressed in 20 types of human cancers and could be used as an indicator for poorer overall survival of patients with HCC. Functional analyses showed that HSPB1P1 was strongly correlated with signaling pathways related to cancer progression. Further studied revealed that HSPB1P1 could direct regulate the EZH2 expression in HCC. In summary, our study indicated that HSPB1P1 was a predictor for poorer overall survival of patients with HCC and may be potential therapeutic target against HCC.     

Structural basis for segmental gene conversion in generation of Anaplasma marginale outer membrane protein variants

Bacterial pathogens in the genus Anaplasma generate surface coat variants by gene conversion of chromosomal pseudogenes into single-expression sites. These pseudogenes encode unique surface-exposed hypervariable regions flanked by conserved domains, which are identical to the expression site flanking domains. In addition, Anaplasma marginale generates variants by recombination of oligonucleotide segments derived from the pseudogenes into the existing expression site copy, resulting in a combinatorial increase in variant diversity. Using the A. marginale genome sequence to track the origin of sequences recombined into the msp2 expression site, we demonstrated that the complexity of the expressed msp2 increases during infection, reflecting a shift from recombination of the complete hypervariable region of a given pseudogene to complex mosaics with segments derived from hypervariable regions of different pseudogenes. Examination of the complete set of 1183 variants with segmental changes revealed that 99% could be explained by one of the recombination sites occurring in the conserved flanking domains and the other within the hypervariable region. Consequently, we propose an 'anchoring' model for segmental gene conversion whereby the conserved flanking sequences tightly align and anchor the expression site sequence to the pseudogene. Associated with the recombination sites were deletions, insertions and substitutions; however, these are a relatively minor contribution to variant generation as these occurred in less than 2% of the variants. Importantly, the anchoring model, which can account for more variants than a strict segmental sequence identity mechanism, is consistent with the number of msp2 variants predicted and empirically identified during persistent infection.     

The ornithine aminotransferase-encoding gene family of rat: cloning, characterization, and evolutionary relationships between a single expressed gene and three pseudogenes

As a first step towards understanding the molecular mechanisms through which the expression of the gene (OAT) encoding ornithine aminotransferase (OAT) is regulated in a tissue-specific manner, we have used a near full length OAT cDNA to isolate related sequences from a rat genomic DNA library. Twenty-one unique clones representing five contigs and spanning approximately 140 kb of genomic DNA were isolated and characterized. From these clones we have identified a single expressed OAT gene and three processed pseudogenes. The comparison of the EcoRI, BamHI, and HindIII fragments contained within these genomic clones with those detected in total genomic DNA by the cDNA probe suggests that essentially all of the OAT-related sequences in the rat genome have been isolated. Thus, the tissue-specific regulation of OAT gene expression appears to be effected through a single expressed gene. Data are presented which suggest that the OAT-1, OAT-2, and OAT-3 pseudogenes arose approximately 28.5, 7.3, and 25.1 Myr ago, respectively. Mutation rates are presented for each codon position of the expressed rat and human OAT genes. The region of the rat genome flanking the boundary of the OAT-3 pseudogene is of additional interest as it shares considerable identity to sequences contained within expressed genes and flanking other processed pseudogenes.     

RT-PCR检测HUTP14A mRNA时排除假基因HUTP14C干扰的方法

人类U3蛋白14C基因(HUTP14C)是人类U3蛋白14A基因(HUTP14A)的假基因。两者转录本序列同源性高达95%。常规RT-qPCR技术在检测HUTP14A mRNA丰度时,HUTP14C的存在会影响检测结果。本研究旨在建立检测HUTP14A mRNA时排除HUTP14C干扰的RT PCR方法。本研究设计出能分别从多种肿瘤细胞DNA和RNA中特异性扩增HUTP14A和HUTP14C的引物,避免假基因HUTP14C对其同源基因HUTP14A检测的干扰。在检测细胞系HUTP14A mRNA时,通过DNaseⅠ消除RNA中污染的HUTP14C DNA,用靶向HUTP14C 3′-UTR的siRNA沉默HUTP14C mRNA后,再用RT PCR检测HUTP14A mRNA丰度,使结果更加准确。在18对肝癌及癌旁组织中,利用特异性引物进行RT PCR检测,HUTP14A和HUTP14C mRNA的表达略高于癌旁组织。本研究提示,针对有假基因存在的功能基因,对其mRNA丰度进行检测时,在提取细胞或组织总RNA后,用DNaseⅠ处理,再用RNA直接进行PCR扩增,排除DNA污染后,再进行RT-PCR或RT-qPCR扩增。大多假基因具有较长的3′-UTR区,在该区域设计siRNA特异性沉默假基因的mRNA后,用RT-qPCR检测功能基因的mRNA丰度,可以排除假基因mRNA的影响。在病理组织中检测功能基因的mRNA丰度时,可以根据假基因和其功能基因的序列差异设计出特异扩增功能基因的引物,从假基因的3′-UTR区设计特异扩增假基因的引物,通过RT-qPCR技术分别检测二者的mRNA。     

Evolution of antigen variation in the tick-borne pathogen Anaplasma phagocytophilum

Anaplasma phagocytophilum is an obligately intracellular tick-transmitted bacterial pathogen of humans and other animals. During the course of infection, A. phagocytophilum utilizes gene conversion to shuffle ～100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which codes for the major surface antigen and major surface protein 2 (MSP2). The role and extent of msp2(p44) recombination, particularly in hosts that only experience acute infections, is not clear. In the present study, we explored patterns of recombination and expression of the msp2(p44) gene of A. phagocytophilum in a serially infected mouse model. Even though the bacterium was passed rapidly among mice, minimizing the opportunities for the host to develop adaptive immunity, we detected the emergence of 34 unique msp2(p44) expression cassette variants. The expression of msp2(p44) pseudogenes did not follow a consistent pattern among different groups of mice, although some pseudogenes were expressed more frequently than others. In addition, among 263 expressed pseudogenes, 3 mosaic sequences each consisting of 2 different pseudogenes were identified. Population genetic analysis showed that genetic diversity and subpopulation differentiation tended to increase over time until stationarity was reached but that the variance that was observed in allele (expressed pseudogene) frequency could occur by drift alone only if a high variance in bacterial reproduction could be assumed. These findings suggest that evolutionary forces influencing antigen variation in A. phagocytophilum may comprise random genetic drift as well as some innate but apparently nonpurifying selection prior to the strong frequency-dependent selection that occurs cyclically after hosts develop strong adaptive immunity.