DNA microarrays with unmodified oligonucleotides are a cost-effective alternative to cDNA microarrays. This study examined how purity, length, homology and GC content of the oligonucleotide probes influence the sensitivity and specificity of the method using cyanobacterial genes. Oligonucleotide purification by high pressure liquid chromatography was omitted without significant reduction in hybridization sensitivity. For two of three genes tested, a reduction in oligonucleotide length did not reduce hybridization sensitivity, and maximum sensitivity was achieved with probes that were 45 nt long. Oligonucleotide probes with ≤71% contiguous sequence identity to non-target DNA cross-hybridized with the sequences at a rate of < 8%. Cross-hybridization decreased as the GC content decreased from 65% to 55% or 35%. These results support the following criteria for selecting unmodified oligonucleotide probes that generate sensitive and specific microarrays: length of 45 nt, ≤71% identity to non-target sequences, and ≤55% GC content. In most of the bacterial species tested, oligonucleotide probes meeting these criteria were successfully designed for more than 95% of genes.
Septins, which are involved in cytokinesis, have been identified in a variety of fungi and animal cells. For analysis of the function of septin, drugs targeting septin would be useful; however, no such drugs have been available hitherto. By serendipity, we found that forchlorfenuron (FCF, N-(2-chloro-4-pyridyl)-N-phenylurea, 4PU300), a synthetic plant cytokinin, disturbed cytokinesis in Saccharomyces cerevisiae. Upon administration of FCF, septin structures at the bud neck became deformed and filament-like septin appeared outside of the neck. Under these conditions, the localization of actin was normal and Gin4, which is localized at the bud neck in a septin-dependent manner, was found to remain at the location of apparently normal septin at the bud neck, whereas it was not co-localized to the deformed septin at the bud neck or to septin seen outside the bud neck. FCF administration immediately induced production of sporadic septin structures outside the bud neck, and these structures disappeared promptly upon removal of the drug. Taken together, these findings indicate that FCF maybe a promising drug for investigating the structure and function of septin.
Plastid protein import 2 (ppi2), a mutant of Arabidopsis thaliana, lacks a homologue of a component of the translocon at the outer envelope membrane of chloroplasts (Toc), designated Toc159 of the pea. Toc159 is thought to be essential for the import of photosynthetic proteins into chloroplasts. In order to investigate the effect of protein import on the plant development, we examined the morphologies of the developing leaves and the shoot apical meristems (SAM) in the ppi2 plants. Our histological analysis revealed that the development of leaves is severely affected in ppi2, while the structure of SAM is normal. Abnormalities in leaves became obvious in the later stages of leaf development, resulting in the gen-eration of mature leaves with fewer mesophyll cells and more intercellular spaces as compared with the wild type. Palisade and spongy tissues of the mature leaves were indistinguishable in ppi2. Replication of chloroplast DNA was also suggested to be impaired in ppi2. Our results suggest that protein import into chloroplasts is important for the normal development of leaves.
The complicated genetic pathway regulates the developmental programs of male reproductive organ, anther tissues. To understand these molecular mechanisms, we performed cDNA microarray analyses and in situ hybridization to monitor gene expression patterns during anther development in rice. Microarray analysis of 4,304 cDNA clones revealed that the hybridization signal of 396 cDNA clones (271 non-redundant groups) increased more than six-fold in every stage of the anthers compared with that of leaves. Cluster analysis with the expression data showed that 259 cDNA clones (156 non redundant groups) were specifically or predominantly expressed in anther tissues and were regulated by developmental stage-specific manners in the anther tissues. These co-regulated genes would be impor-tant for development of functional anther tissues. Furthermore, we selected several clones for RNA in situ hybridization analysis. From these analyses, we found several novel genes that show temporal and spatial expression patterns during anther development in addition to anther-specific genes reported so far. These results indicate that the genes identified in this experiment are controlled by different programs and are specialized in their developmental and cell types.
The polyploid nature of wheat is a key characteristic of the plant. Full-length complementary DNAs (cDNAs) provide essential information that can be used to annotate the genes and provide a functional analysis of these genes and their products. We constructed a full-length cDNA library derived from young spikelets of common wheat, and obtained 24056 expressed sequence tags (ESTs) from both ends of the cDNA clones. These ESTs were grouped into 3605 contigs using the phrap method, representing expressed loci from each of the three genomes. Using BLAST, 3605 contigs were grouped into 1902 gene clusters, showing that loci of the three genomes are not always expressed. A homology search of these gene clusters against a wheat EST database (15964 gene clusters) and a rice full-length cDNA database (21447 gene clusters) revealed that a quarter of the wheat full-length cDNAs were novel. A protein database of Arabidopsis was used to examine the functional classification of these gene clusters. The GC-content in the 5´-UTR region of wheat cDNAs was compared to that of rice. Forty-three genes (3.5% of wheat cDNAs homologous to those of rice) possessed distinct GC-content in the 5´-UTR region, suggesting different breeding behaviors of wheat and rice.
The colonization of America by Drosophila subobscura has been a unique exper iment in nature that has allowed us to explore the effects of evolution on a continental scale. To analyze this evolutionary event, nucleotide sequences of the Odh (Octanol dehydrogenase) gene were obtained for 43 lethal chromosomal lines from colonizing populations of North America and 5 from South America, in addition to 5 chromosomal lines from Europe with different viabilities and 2 from laboratory marker stocks. Since 10 different Odh haplotypes were found in America, the minimum number of colonizers would be 5 (or 3 mated females). Only one Odh haplotype was found in American O5 inversions confirming that only one copy of this inversion was included among the sample of colonizers. The same Odh haplotypes were detected in association with the same chromosomal arrangements and with identical lethal genes in both North and South America indicating that exactly the same chromosome types reached both hemispheres. These observations indicate that the two continental colonizations are not independent. They are derived from the same colonization event. The population from which the colonization started should contain the O5 inversion, a non-negligible frequency of the O3+4+7 arrangement and all other arrangements found in America. So far the only populations that fulfill all these requirements are those from Greece, indicating that these populations can be considered good candidates as a starting point for an in depth analysis of the origin of the American colonization by D. subobscura.
We attempted to develop a PCR-based marker that detects various segments of rye chromosome incorporated into wheat. We designed three sets of PCR primers based on the nucleotide sequence data of a rye repetitive sequence previously reported. One of the primer sets amplified a clear ca. 1.4 kb fragment in a rye cultivar but not in any form of wheat, diploid, tetraploid or hexaploid. We used this critical primer set for PCR of various wild species and cultivars of rye, an array of wheat plants carrying different rye chromosomes or small segments from different regions of rye chromosome 1R, and plants carrying parts of the rye B chromosome. The PCR amplified the 1.4 kb fragment in all the plant materials examined. We believe this PCR primer set will be useful as a universal PCR-based marker for the introgression of rye chromosome segments in the wheat genome.
The jawless fish occupy an important phylogenetic position for understanding the evolution of body plans, the origin of adaptive immunity and genome evolution in chordates. We describe here the construction of a large-insert bacterial artificial chromosome (BAC) library from the inshore hagfish, Eptatretus burgeri. The BAC library contains 93,978 clones with an average insert size of 100 kb and is estimated to represent threefold genome-equivalent coverage. The library was organized in three-dimensional pools to facilitate screening by PCR. We have screened this library by PCR and isolated several BAC clones; the average number of positive clones was compatible with the estimated genome coverage of the library. This BAC library, constructed for the first time from the jawless fish, should serve as a useful resource for the scientific community.