We have developed a new T-DNA vector, pGA2715, which can be used for promoter trapping and activation tagging of rice (Oryza sativa) genes. The binary vector contains the promoterless ␤-glucuronidase (GUS) reporter gene next to the right border. In addition, the multimerized transcriptional enhancers from the cauliflower mosaic virus 35S promoter are located next to the left border. A total of 13,450 T-DNA insertional lines have been generated using pGA2715. Histochemical GUS assays have revealed that the GUS-staining frequency from those lines is about twice as high as that from lines transformed with the binary vector pGA2707, which lacks the enhancer element. This result suggests that the enhancer sequence present in the T-DNA improves the GUS-tagging efficiency. Reverse transcriptase-PCR analysis of a subset of randomly selected pGA2715 lines shows that expression of the genes immediately adjacent to the inserted enhancer is increased significantly. Therefore, the large population of T-DNA-tagged lines transformed with pGA2715 could be used to screen for promoter activity using the gus reporter, as well as for creating gain-of-function mutants.Recent completion of the draft sequence for the rice (Oryza sativa) genome has resulted in an explosion of information on rice genes (Goff et al., 2002; Yu et al., 2002). The challenge for the post-sequencing era is to identify the biological functions for these genes. Of all the approaches used to discover gene function, the most direct is to disrupt the genes and analyze the consequences. Various methods have been developed in plants for this purpose. These include using ethyl methanesulfonate, fast-neutron treatment, or insertion of an element, such as a transposable element or T-DNA (Koornneef et al., 1982;Sundaresan, 1996;Krysan et al., 1999). Insertional mutagenesis has the advantage that the inserted element acts as a tag for gene identification. However, all gene disruption approaches also have some limitations. For example, it is difficult to identify the function of redundant genes, or of genes required in early embryogenesis or gametophyte development.To overcome those limitations, modified insertional elements have been developed. One of these modified designs is the gene trap system that involves creating fusions between the tagged genes and a reporter gene, such as ␤-glucuronidase (gus) or green fluorescent protein (gfp; Sundaresan et al., 1995;Springer, 2000). This system provides a way of identifying novel genes based on their expression patterns. Insertion of the promoterless reporter not only destroys normal gene function but also activates expression of the reporter gene. Because expression levels can be monitored in heterozygote plants, the gene trap system is useful for studying the patterns of most plant genes, including essential genes that cause lethal mutations. This system is convenient for observing mutant phenotypes because reporter activation indicates the location, condition, and time of expression for the disrupted gene. In Arabidopsis, ...

bWe developed one-step sequence-and ligation-independent cloning (SLIC) as a simple, cost-effective, time-saving, and versatile cloning method. Highly efficient and directional cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature.T he need for high-throughput recombinant DNA technology is rising because of the rapid increase in interest in functional genomic studies following the surge of data generated by nextgeneration sequencing. Construction of a library or conversion of an existing library into a different context requires a highthroughput gene cloning method, but conventional methods suffer from high cost, prolonged manipulation, or sequence restriction.Ligation-independent cloning (LIC) is based on the 3=-to-5= exonuclease activity of T4 DNA polymerase and has been used for 2 decades as a high-throughput method due to its uniformity and cost-effectiveness but requires a specifically designed vector containing a long stretch of sequence that lacks a particular deoxynucleoside triphosphate (1,(3)(4)(5)(6)9). Sequence-and ligation-independent cloning (SLIC) overcomes the sequence restraint of LIC and allows the assembly of multiple overlapping fragments simultaneously, but the cloning efficiency of SLIC in the absence of RecA is rather low (10). Various recombinase-based cloning methods, including Gateway cloning (Invitrogen), Cre-lox recombination, Red/ET recombination (Gene Bridges), In-Fusion (Clontech), Cold Fusion (System Biosciences), and CloneEZ (GenScript), have been developed, but the general use of these methods has been hampered by high cost and restrictions in the sequence or hosts (2, 12-15).We have optimized SLIC to make it comparable to commercial methods in terms of simplicity, time saving, and cloning efficiency. One-step SLIC utilizes only T4 DNA polymerase but shows cloning efficiencies similar to those of the original SLIC method in the presence of RecA and of commercial methods. An overview of one-step SLIC is illustrated in Fig. 1A. First, the vector needs to be linearized by either restriction enzyme digestion or inverse PCR. An insert(s) is prepared by PCR with primers with a 15-bp or longer extension homologous to each end of the linearized vector. Second, the vector and insert(s) are mixed and incubated at room temperature for 2.5 min with T4 DNA polymerase to generate 5= overhangs. For optimal results, a 1:2 to 1:4 molar vector-to-insert ratio is desirable. Third, the reaction mixture is placed on ice for 10 min for single-strand annealing and then competent Escherichia coli cells are transformed with the annealed DNA complex directly. The annealed complex turns into seamless recombinant DNA through homologous recombination in vivo with high efficiency. For a detailed description of the method, see the supplemental material.pUC118-HMG (11) was cleaved with BamHI, and a 1-kb insert containing Ton_0709 from the genomic DNA of Thermococcus onnurineus NA1 ...

The shift from terrestrial to aquatic life by whales was a substantial evolutionary event. Here we report the whole-genome sequencing and de novo assembly of the minke whale genome, as well as the whole-genome sequences of three minke whales, a fin whale, a bottlenose dolphin and a finless porpoise. Our comparative genomic analysis identified an expansion in the whale lineage of gene families associated with stress-responsive proteins and anaerobic metabolism, whereas gene families related to body hair and sensory receptors were contracted. Our analysis also identified whale-specific mutations in genes encoding antioxidants and enzymes controlling blood pressure and salt concentration. Overall the whale-genome sequences exhibited distinct features that are associated with the physiological and morphological changes needed for life in an aquatic environment, marked by resistance to physiological stresses caused by a lack of oxygen, increased amounts of reactive oxygen species and high salt levels.

Flowering plants have evolved various stratagems to prevent inbreeding and promote outcrosses. One such mechanism, gametophytic self-incompatibility, provides a genetic barrier to self-fertilization, and in the simplest cases is controlled by the highly polymorphic S locus. Growth of a pollen tube in the style is arrested when the S allele carried by the pollen matches one of the two S alleles carried by the pistil. Putative S allele proteins of the pistil have been identified in several solanaceous species based on their co-segregation with S alleles, and they have been shown to be ribonucleases. So far, there has been only correlative or indirect evidence for the claim that these S allele-associated proteins (S proteins) are involved in recognition and rejection of self pollen. Here we show that inhibition of synthesis of S3 and S2 proteins in Petunia inflata plants of S2S3 genotype by the antisense S3 gene resulted in failure of the transgenic plants to reject S3 and S2 pollen. We further show that expression of the transgene encoding S3 protein in P. inflata plants of S1S2 genotype confers on the transgenic plants the ability to reject S3 pollen. The self-incompatibility behaviour of the pollen was not affected by the transgene in either set of experiments. Taken together, these findings provide direct in vivo evidence that S proteins control the self-incompatibility behaviour of the pistil.