乙酰胆碱受体在神经突触传导过程中具有重要作用，也是氯化胆碱类杀虫剂的作用靶标。采用RACE技术，成功地从棉蚜中克隆了3个nAChR亚基，其中2个为α亚型，1个为β亚型，分别命名为Agα1 、Agα2和A gβ1。通过锚定mRNA 的5'mG结构，5'RACE 结果表明Agβ1有三个不同的剪接变体，具有不同长度的5′UTR区，表明Agβ1亚基具有多重的转录起始位点。其中，最短的剪接变体A gβ1C 在蛋白编码区域也存在选择性剪接，位于D环区域的186 bp碱基缺失。3'RACE 实验结果表明，Agα1亚基虽然具有ploy (A) 和加尾信号AATAAA等完整的mRNA基因结构，但缺失了终止子和乙酰胆碱受体α亚基保守的第4个跨膜区，文中对此做了进一步分析。分子进化树的分析表明，昆虫乙酰胆碱受体亚基应当被划分为三个不同的亚类群αⅠ,αⅡandβ。本文的研究揭示了昆虫乙酰胆碱受体亚基复杂的基因结构。

A simple and effective method was set up to purify acetylcholinesterase (AChE, EC3.1.1.7) from the cotton aphid, Aphis gossypii Glover. The procedure involved filtration on a sephadex G-25 column, separation with sephadex G-200 and procainamide affinity column. AChE from both susceptible and resistant strains were purified to a single band as resolved on denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity increased by 35,100- and 33, 680-fold with a yield of 30.3 and 29.8%, respectively. The molecular mass of the purified AChE was about 63,500 Dalton as determined by SDSPAGE. However, three bands resolved on PAGE gel electrophoresis, leading to the inference that native AChE exists in three forms. The optimum conditions for measuring the activity of purified AChE with kinetic method were 0.02M phosphate buffer, pH7.2, 0.02mM 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB), and 25℃. Investigation also revealed that crude extract and purified AChE had different kinetic characteristics and inhibitory properties. They responded differently to varied DTNB, ATChI, and phosphate buffer ion concentrations, as well as pH, temperature, and inhibitors. The purified AChE was more sensitive to eserine, methamidophos, and pirimicarb. Especially for resistant aphids, the sensitivity of purified AChE to methamidophos and pirimicarb was enhanced 6.43 and 11.73 times, respectively. We infer that one or more factors in the crude extract from the resistance strain have more influence on AChE sensitivity. Further study is needed to investigate the basis of these observations. Arch.

Two acetylcholinesterase (AChE) genes, Ace1 and Ace2, have been cloned from cotton aphid, Aphis gossypii Glover, using the rapid amplification of cDNA ends (RACE) technique. To the best of our knowledge, this should be the first direct molecular evidence that multiple AChE genes exist in insects. The Ace1 gene was successfully amplified along its full length of 2371 bp. The open reading frame is 2031 bp long and encodes 676 amino acids (GenBank accession No. AF502082). The Ace2 gene was amplified as a mega-fragment of 2130 bp lacking part of 5′-end untranslated region (UTR). The open reading frame is 1992 bp long and ecodes a protein of 664 amino acids (GenBank accession No. AF502081). Both genes have the conserved amino acids and features shared by the AChE family, but share only 35% identity in amino acid sequence. The Ace1 gene is highly homologous to the AChE gene of Schizaphis graminum (AF321574) with 95% identity, and Ace2 to that of Myzus persicae (AF287291) with 92% identity. Phylogenetic analysis showed that the two cloned AChEs of A. gossypii are different in evolution. The phylogenetic tree generated by the PHYLIP program package inferred that AChE2 of A. gossypii is a more ancestral form of AChE. Homology modeling of structures using Torpedo californica (2ACE_) and Drosophila melanogaster (1Q09: A) native acetylcholinesterase structure as main template indicated that the two AChEs of Aphis gossypii might have different three-dimensional structures. Alternative splicing of Ace1 near the 5′-end resulting in two proteins differing by the presence or absence of a fragment of four amino acids is also reported.

The systematic analysis of very short alternative splicing (VSAS) in the human genome showed that VSAS might contribute more to protein-function diversity than expected. More than 65% of VSAS fragments have different secondary structures from flanking regions. They tend to have a non-loop structure and have an important influence on protein functions, as shown by the predicted 3D structure of human IL-4d2. The observed VSAS events can be classified into two groups depending on whether they insert new structure domains in the proteins, and they might be of different evolutionary status.

Two acetylcholinesterase genes, Ace1 and Ace2, have been fully cloned and sequenced from both organophosphate-resistant and susceptible clones of cotton aphid. Comparison of both nucleic acid and deduced amino acid sequences revealed considerable nucleotide polymorphisms. Further study found that two mutations occurred consistently in all resistant aphids. The mutation F139L in Ace2 corresponding to F115S in Drosophila acetylcholinesterase might reduce the enzyme sensitivity and result in insecticide resistance. The other mutation A302S in Ace1 abutting the conserved catalytic triad might affect the activity and insecticide sensitivity of the enzyme. Phylogenetic analysis showed that insect acetylcholinesterases fall into two subgroups, of which Ace1 is the paralogous gene whereas Ace2 is the orthologous gene of Drosophila AChE. Both subgroups contain resistance-associated AChE genes. To avoid confusion in the future work, a nomenclature of insect AChE is also suggested in the paper.

Understanding recombination rate variation is very important for studying genome diversity and evolution, and for investigation of phenotypic association and genetic diseases. Recombination hotspots have been observed in many species and are well studied in yeast. Recent study demonstrated that recombination hotspots are also a ubiquitous feature of the human genome. But the nature of human hotspots remains largely unknown. We have developed and validated a novel computational method for testing the existence of hotspots as well as for localizing them with either unphased or phased genotyping data. To study the characteristics of hotspots within or close to genes, we scanned for unusually high levels of recombination using the European population samples in the SeattleSNPs database, and found evidence for the existence of human a hotspots similar to those of yeast. This type of hotspots, found at promoter regions, accounts for about half of the total detected and appears to depend on some specific transcription factor binding sites (such as CGCCCCCGC). These characteristics can explain the observed weak correlation between hotspots and GC-content, and their variation may contribute to the diversity of hotspot distribution among different individuals and species. These long-sought putative human a recombination hotspots should deserve further experimental investigations.

The importance of U1 snRNA binding free energy in the regulation of alternative splicing has been studied in some genes with site-directed mutagenesis. Here we report a large-scale analysis of its impact on 50 splice site (50ss) selection in human genome. The results show that free energy exerts different effects on alternative 50ss choice in different situations and 8.1 kcal/mol is a hreshold. When both free energies of two competing 50ss are larger than 8.1 kcal/mol, the 50ss with lower free energy is more frequently used. However, in other pairs of 50ss, lower-free-energy 50ss does not seem to be favored and even the other 50ss is used more frequently, which suggests that very low binding free energy would impair splicing. Some observations hold true only for those alternative 50 splicing with short alternative exons (<50nt), which implies a complex mechanism of 50ss selection involving both U1 snRNA binding free energy and regulatory factors.

Motivation: MicroRNAs (miRNA) are 22 nt long non-coding RNAs that are derived from larger hairpin RNA precursors and play important regulatory roles in both animals and plants. The short length of the miRNA sequences and relatively low conservation of pre-miRNA sequences restrict the conventional sequence-alignment-based methods to finding only relatively close homologs. On the other hand, it has been reported that miRNA genes are more conserved in the secondary structure rather than in primary sequences. Therefore, secondary structural features should be more fully exploited in the homologue search for new miRNA genes. Results: In this paper, we present a novel genome-wide computational approach to detect miRNAs in animals based on both sequence and structure alignment. Experiments show this approach has higher sensitivity and comparable specificity than other reported homologue searching methods. We applied this method on Anopheles gambiae and detected 59 new miRNA genes. Availability: This programis available at http://bioinfo.au.tsinghua.edu.

Background: MicroRNAs (miRNAs) are a group of short (～22 nt) non-coding RNAs that play important regulatory roles. MiRNA precursors (pre-miRNAs) are characterized by their hairpin structures. However, a large amount of similar hairpins can be folded in many genomes. Almost all current methods for computational prediction of miRNAs use comparative genomic approaches to identify putative pre-miRNAs from candidate hairpins. Ab initio method for distinguishing premiRNAs from sequence segments with pre-miRNA-like hairpin structures is lacking. Being able to classify real vs. pseudo pre-miRNAs is important both for understanding of the nature of miRNAs and for developing ab initio prediction methods that can discovery new miRNAs without known homology. Results: A set of novel features of local contiguous structure-sequence information is proposed for distinguishing the hairpins of real pre-miRNAs and pseudo pre-miRNAs. Support vector machine (SVM) is applied on these features to classify real vs. pseudo pre-miRNAs, achieving about 90% accuracy on human data. Remarkably, the SVM classifier built on human data can correctly identify up to 90% of the pre-miRNAs from other species, including plants and virus, without utilizing any comparative genomics information. Conclusion: The local structure-sequence features reflect discriminative and conserved characteristics of miRNAs, and the successful ab initio classification of real and pseudo pre-miRNAs opens a new approach for discovering new miRNAs.

Alternative translation is an important cellular mechanism contributing to the generation of proteins and the diversity of protein functions. Instead of studying individual cases, we systematically analyzed the alteration of protein subcellular location and domain formation by alternative translational initiation in eukaryotes. The results revealed that 85.7% of alternative translation events generated biological diversity, attributed to different subcellular localizations and distinct domain contents in alternative isoforms. Analysis of isoelectric point values revealed that most N-terminal truncated isoforms significantly lowered their isoelectric point values targeted at different subcellular localizations, whereas they had conserved domain contents the same as the fulllength isoforms. Furthermore, Fisher's exact test indicated that the two ways-targeting at different cellular compartments and changing domain contents-were negatively associated. The N-term truncated isoforms should have only one way to diversify their functions distinct from the full-length ones. The peculiar consequence of subcellular relocation as well as change of domain contents re-flected the very high level of biological complexity as alternative usage of initiation codons. Proteins.