Aucsia gene silencing causes parthenocarpic fruit development in tomato

In angiosperms, auxin phytohormones play a crucial regulatory role in fruit initiation. The expression of auxin biosynthesis genes in ovules and placenta results in uncoupling of tomato (Solanum lycopersicum) fruit development from fertilization with production of parthenocarpic fruits. We have identified two newly described genes, named Aucsia genes, which are differentially expressed in auxin-synthesis (DefH9-iaaM) parthenocarpic tomato flower buds. The two tomato Aucsia genes encode 53-amino-acid-long peptides. We show, by RNA interference-mediated gene suppression, that Aucsia genes are involved in both reproductive and vegetative plant development. Aucsia-silenced tomato plants exhibited auxin-related phenotypes such as parthenocarpic fruit development, leaf fusions, and reflexed leaves. Auxin-induced rhizogenesis in cotyledon explants and polar auxin transport in roots were reduced in Aucsia-silenced plants compared with wild-type plants. In addition, Aucsia-silenced plants showed an increased sensitivity to 1-naphthylphthalamic acid, an inhibitor of polar auxin transport. We further prove that total indole-3-acetic acid content was increased in preanthesis Aucsia-silenced flower buds. Thus, the data presented demonstrate that Aucsia genes encode a novel family of plant peptides that control fruit initiation and affect other auxin-related biological processes in tomato. Aucsia homologous genes are present in both chlorophytes and streptophytes, and the encoded peptides are distinguished by a 16-amino-acid-long (PYSGXSTLALVARXSA) AUCSIA motif, a lysine-rich carboxyl-terminal region, and a conserved tyrosine-based endocytic sorting motif.     

A peptidomimetic siRNA transfection reagent for highly effective gene silencing

RNA interference (RNAi) techniques hold forth great promise for therapeutic silencing of deleterious genes. However, clinical applications of RNAi require the development of safe and efficient methods for intracellular delivery of small interfering RNA (siRNA) oligonucleotides specific to targeted genes. We describe the use of a lipitoid, a cationic oligopeptoid-phospholipid conjugate, for non-viral transfection of synthetic siRNA oligos in cell culture. This peptidomimetic delivery vehicle allows for efficient siRNA transfection in a variety of human cell lines with negligible toxicity and promotes extensive downregulation of the targeted genes at both the protein and the mRNA level. We compare the lipitoid reagent to a standard commercial transfection reagent. The lipitoid is highly efficient even in primary IMR-90 human lung fibroblasts in which other commercial reagents are typically ineffective.     

The maize b1 paramutation control region causes epigenetic silencing in Drosophila melanogaster

Paramutation is an epigenetic process in which a combination of alleles in a heterozygous organism results in a meiotically stable change in expression of one of the alleles. The mechanisms underlying paramutation are being actively investigated, and examples have been described in both plants and mammals, suggesting that it may utilize epigenetic mechanisms that are widespread and evolutionarily conserved. Paramutation at the well-studied maize b1 locus requires a control region consisting of seven 853 bp tandem repeats. To study the conservation of the epigenetic mechanisms underlying seemingly unique epigenetic processes such as paramutation, we created transgenic Drosophila melanogaster carrying the maize b1 control region adjacent to the Drosophila white reporter gene. We show that the b1 tandem repeats cause silencing of the white reporter in Drosophila. A single copy of the tandem repeat sequence is sufficient to cause silencing, and silencing strength increases as the number of tandem repeats increases. Additionally, transgenic lines with the full seven tandem repeats demonstrate evidence of either pairing-sensitive silencing and silencing in trans, or epigenetic activation in trans. These trans-interactions are dependent on repeat number, similar to maize b1 paramutation. Also, as in maize, the tandem repeats are bidirectionally transcribed in Drosophila. These results indicate that the maize b1 tandem repeats function as an epigenetic silencer and mediate trans-interactions in Drosophila, and support the hypothesis that the mechanisms underlying such epigenetic processes are conserved.     

Designing of highly effective complementary and mismatch siRNAs for silencing a gene

In past, numerous methods have been developed for predicting efficacy of short interfering RNA (siRNA). However these methods have been developed for predicting efficacy of fully complementary siRNA against a gene. Best of author's knowledge no method has been developed for predicting efficacy of mismatch siRNA against a gene. In this study, a systematic attempt has been made to identify highly effective complementary as well as mismatch siRNAs for silencing a gene.Support vector machine (SVM) based models have been developed for predicting efficacy of siRNAs using composition, binary and hybrid pattern siRNAs. We achieved maximum correlation 0.67 between predicted and actual efficacy of siRNAs using hybrid model. All models were trained and tested on a dataset of 2182 siRNAs and performance was evaluated using five-fold cross validation techniques. The performance of our method desiRm is comparable to other well-known methods. In this study, first time attempt has been made to design mutant siRNAs (mismatch siRNAs). In this approach we mutated a given siRNA on all possible sites/positions with all possible nucleotides. Efficacy of each mutated siRNA is predicted using our method desiRm. It is well known from literature that mismatches between siRNA and target affects the silencing efficacy. Thus we have incorporated the rules derived from base mismatches experimental data to find out over all efficacy of mutated or mismatch siRNAs. Finally we developed a webserver, desiRm (http://www.imtech.res.in/raghava/desirm/) for designing highly effective siRNA for silencing a gene. This tool will be helpful to design siRNA to degrade disease isoform of heterozygous single nucleotide polymorphism gene without depleting the wild type protein.     

DNA sequence of the coding region of the HLA-B44 gene

An HLA-B44 cDNA clone was identified in a cDNA library constructed from an HLA-B44 homozygous cell line. The DNA sequence was determined and was found to contain the complete coding sequence but for (probably) the three N-terminal codons. Comparisons of the derived amino acid sequence with other HLA-A and -B locus amino acid sequences revealed four HLA-B44-specific substitutions including a new polymorphic site. Regions of strong sequence conservation for HLA-B-locus products were found at the nucleotide and amino acid levels.     

35S驱动Pib基因启动区和编码区的转基因初步分析

将包含Pib基因启动区及下游完整编码区的9.9 kb DNA片段克隆到双元载体pPZP2Ha3(+)中, 构建了35S驱动的正义表达载体pNAR701(20.3 kb); 同时将Pib基因编码区6 986~9 392 bp之间的DNA片段, 克隆到双元载体pPZP2Ha3(-)中, 构建了35S驱动的反义表达载体pNAR703(12.8 kb); 用农杆菌介导法转入中感稻瘟病水稻品种R109中。PCR、Southern blot鉴定以及转基因T0代种子的潮霉素抗性鉴定证明, 目的基因已经整合到R109基因组中, 并能在后代稳定遗传。Northern blot分析表明含有启动区及下游完整编码的Pib基因片段在35S驱动下能够在转基因后代中表达。对T1代苗期转基因植株和分蘖期离体叶片进行抗稻瘟病初步分析, 结果显示pNAR701转基因植株对稻瘟病生理小种ZD1和ZG1的抗性较对照增强, 而转反义片段的pNAR703转基因植株对稻瘟病的抗性较对照减弱。     

Nucleotide sequence of the coding region of the mouse N-myc gene.

A genomic clone for the mouse N-myc gene was isolated and the total nucleotide sequence (4807 bp) of the two coding exons and an intron located between them was determined. The amino acid sequence of the N-myc protein was deduced from the DNA sequence. This protein is composed of 462 amino acids, slightly larger than human and mouse c-myc proteins, and is rich in proline like the c-myc protein. Comparison of the amino acid sequences of the mouse N-myc and c-myc proteins showed that conserved sequences are located in eight regions: four regions are in the N-terminal half of the N-myc protein and are separated from each other by regions poorly homologous to those of the c-myc protein, and the four others are located in the C-terminal half, throughout which certain homology exists. A remarkable sequence containing 13 successive acidic amino acids is present in one of the conserved regions located in the middle of the N-myc protein.     

Identification of template strand in heteroduplex DNA directing cell-free enzyme synthesis

Summary Cell-free synthesis of galactose enzymes was directed by heteroduplex DNA carrying an amber mutation in thegal T gene on one DNA strand, the wildtype configuration on the complementary strand. Comparison with the corresponding homoduplex DNA preparations shows that the presence of the wildtype base sequence in the l-strand is necessary and sufficient for the synthesis of active enzyme. Single stranded DNA of either configuration is inactive in this system.