基于Bioperl实现远程自动获取抗逆基因序列

BioPerl对于现今大量的生物数据来说,具有很好的获取和处理能力,并且NCBI提供了可以直接访问它下属的Entrez数据库（包括PubMed）的编程接口,即E-Utilities。为了从NCBI中自动获取大量抗逆基因序列,使得生物抗逆基因二次数据库得以搭建,该文基于BioPerl设计了远程自动获取大量抗逆基因序列的程序。此程序灵巧、精悍,Perl语言强大的文本处理功能使程序能实现不同类型基因序列的远程获取。     

Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.     

Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes

Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.     

Contact-based sequence alignment

This paper introduces the novel method of contact-based protein sequence alignment, where structural information in the form of contact mutation probabilities is incorporated into an alignment routine using contact-mutation matrices (CAO: Contact Accepted mutatiOn). The contact-based alignment routine optimizes the score of matched contacts, which involves four (two per contact) instead of two residues per match in pairwise alignments. The first contact refers to a real side-chain contact in a template sequence with known structure, and the second contact is the equivalent putative contact of a homologous query sequence with unknown structure. An algorithm has been devised to perform a pairwise sequence alignment based on contact information. The contact scores were combined with PAM-type (Point Accepted Mutation) substitution scores after parameterization of gap penalties and score weights by means of a genetic algorithm. We show that owing to the structural information contained in the CAO matrices, significantly improved alignments of distantly related sequences can be obtained. This has allowed us to annotate eight putative Drosophila IGF sequences. Contact-based sequence alignment should therefore prove useful in comparative modelling and fold recognition.     

Ion transit pathways and gating in ClC chloride channels

ClC chloride channels possess a homodimeric structure in which each monomer contains an independent chloride ion pathway. ClC channel gating is regulated by chloride ion concentration, pH and voltage. Based on structural and physiological evidence, it has been proposed that a glutamate residue on the extracellular end of the selectivity filter acts as a fast gate. We utilized a new search algorithm that incorporates electrostatic information to explore the ion transit pathways through wild-type and mutant bacterial ClC channels. Examination of the chloride ion permeation pathways supports the importance of the glutamate residue in gating. An external chloride binding site previously postulated in physiological experiments is located near a conserved basic residue adjacent to the gate. In addition, access pathways are found for proton migration to the gate, enabling pH control at hyperpolarized membrane potentials. A chloride ion in the selectivity filter is required for the pH-dependent gating mechanism.     

Immunocytochemical localization of Na+-HCO3- cotransporters and carbonic anhydrase dependence of fluid transport in corneal endothelial cells

In corneal endothelium, there is evidence for basolateral entry of HCO(3)(-) into corneal endothelial cells via Na(+)-HCO(3)(-) cotransporter (NBC) proteins and for net HCO(3)(-) flux from the basolateral to the apical side. However, how HCO(3)(-) exits the cells through the apical membrane is unclear. We determined that cultured corneal endothelial cells transport HCO(3)(-) similarly to fresh tissue. In addition, Cl(-) channel inhibitors decreased fluid transport by at most 16%, and inhibition of membrane-bound carbonic anhydrase IV by benzolamide or dextran-bound sulfonamide decreased fluid transport by at most 29%. Therefore, more than half of the fluid transport cannot be accounted for by anion transport through apical Cl(-) channels, CO(2) diffusion across the apical membrane, or a combination of these two mechanisms. However, immunocytochemistry using optical sectioning by confocal microscopy and cryosections revealed the presence of NBC transporters in both the basolateral and apical cell membranes of cultured bovine corneal endothelial cells and freshly isolated rabbit endothelia. This newly detected presence of an apical NBC transporter is consistent with its being the missing mechanism sought. We discuss discrepancies with other reports and provide a model that accounts for the experimental observations by assuming different stoichiometries of the NBC transport proteins at the basolateral and apical sides of the cells. Such functional differences might arise either from the expression of different isoforms or from regulatory factors affecting the stoichiometry of a single isoform.     

Multiple genetic processes result in heterogeneous rates of evolution within the major cluster disease resistance genes in lettuce

Resistance Gene Candidate2 (RGC2) genes belong to a large, highly duplicated family of nucleotide binding site-leucine rich repeat (NBS-LRR) encoding disease resistance genes located at a single locus in lettuce (Lactuca sativa). To investigate the genetic events occurring during the evolution of this locus, approximately 1.5- to 2-kb 3' fragments of 126 RGC2 genes from seven genotypes were sequenced from three species of Lactuca, and 107 additional RGC2 sequences were obtained from 40 wild accessions of Lactuca spp. The copy number of RGC2 genes varied from 12 to 32 per genome in the seven genotypes studied extensively. LRR number varied from 40 to 47; most of this variation had resulted from 13 events duplicating two to five LRRs because of unequal crossing-over within or between RGC2 genes at one of two recombination hot spots. Two types of RGC2 genes (Type I and Type II) were initially distinguished based on the pattern of sequence identities between their 3' regions. The existence of two types of RGC2 genes was further supported by intron similarities, the frequency of sequence exchange, and their prevalence in natural populations. Type I genes are extensive chimeras caused by frequent sequence exchanges. Frequent sequence exchanges between Type I genes homogenized intron sequences, but not coding sequences, and obscured allelic/orthologous relationships. Sequencing of Type I genes from additional wild accessions confirmed the high frequency of sequence exchange and the presence of numerous chimeric RGC2 genes in nature. Unlike Type I genes, Type II genes exhibited infrequent sequence exchange between paralogous sequences. Type II genes from different genotype/species within the genus Lactuca showed obvious allelic/orthologous relationships. Trans-specific polymorphism was observed for different groups of orthologs, suggesting balancing selection. Unequal crossover, insertion/deletion, and point mutation events were distributed unequally through the gene. Different evolutionary forces have impacted different parts of the LRR.