Transformation of Chlamydomonas reinhardtii CW-15 with the Hygromycin Phosphotransferase Gene as a Selectable Marker

To transform Chlamydomonas reinhardtiiDang. cells, plasmid pCTVHyg was constructed with the use of theEscherichia colihygromycin phosphotransferase gene (hpt) controlled by the SV40 early promoter. Cells of the CW-15 mutant strain were transformed by electroporation, with the yield reaching 10³ hygromycin-resistant (Hyg^R) clones per 10⁶ recipient cells. The exogenous DNA integrated in the Ch. reinhardtii nuclear genome showed stable transmission for approximately 350 cell generations, while hygromycin resistance was expressed as an unstable character. Codon usage was compared for the hptgene and Ch. reinhardtiinuclear genes. The results testified that codon usage bias, which is characteristic of Ch. reinhardtii, is not the major factor affecting foreign gene expression. The advantages of the selective system for studying Ch. reinhardtii transformation with heterologous genes are discussed.     

The pattern of chromosome folding in interphase is outlined by the linear gene density profile

Spatial organization of chromatin in the interphase nucleus plays a role in gene expression and inheritance. Although it appears not to be random, the principles of this organization are largely unknown. In this work, we show an explicit relationship between the intranuclear localization of various chromosome segments and the pattern of gene distribution along the genome sequence. Using a 7-megabase-long region of the Drosophila melanogaster chromosome 2 as a model, we observed that the six gene-poor chromosome segments identified in the region interact with components of the nuclear matrix to form a compact stable cluster. The six gene-rich segments form a spatially segregated unstable cluster dependent on nonmatrix nuclear proteins. The resulting composite structure formed by clusters of gene-rich and gene-poor regions is reproducible between the nuclei. We suggest that certain aspects of chromosome folding in interphase are predetermined and can be inferred through in silico analysis of chromosome sequence, using gene density profile as a manifestation of "folding code."     

Phylogenetic shadowing of a histamine-gated chloride channel involved in insect vision

A recently identified gene, hclA (synonym: ort), codes for an ionotrophic histamine receptor subunit in Drosophila melanogaster, and known hclA mutations lead to defects in the visual system, neurologic disorders and changed responsiveness to neurotoxins. To investigate whether this novel class of receptors is common across the Insecta, we analysed the genomes of 15 other insect species (Diptera, Hymenoptera, Coleoptera, Lepidoptera) and revealed orthologs of hclA in all of them. The predicted receptor domain of HCLA is extensively conserved (86-100% of identity) among the 16 proteins. Minor changes in the amino acid sequence that includes the putative transmembrane domains (TMs) 1-3 were found in non-drosophilid species only. Substantial amino acid variability was observed in the signal polypeptides, the intracellular loop domains and in TM4, in good accordance with known data on sequence variations in ligand-gated ion channels. Pairwise comparisons revealed three consensus sequences for N-glycosylation, conserved in HCLAs of all species studied, as well as a drosophilid-specific putative phosphorylation site. Real-time PCR analysis demonstrated that hclA-mRNA is abundant in heads of adult Drosophila. However, species- and sex-specific variations of the hclA expression levels were also observed.     

Clustering of Pathogen‐Response Genes in the Genome of Arabidopsis thaliana

Previously, we used heterologous expressed sequence tag (EST) mapping to generate a profile of 4 935 pathogen‐response genes of Arabidopsis thaliana. In this work, we performed a computer analysis of this profile, revealing 1 594 non‐homologous clustered genes distributed among all A. thaliana chromosomes, whose co‐regulation may be related to host responses to pathogens. To supplement computer data, we arbitrarily selected two clusters and analyzed their expression levels in A. thaliana ecotypes Col‐0 and C24 during infection with the yellow strain of Cucumber mosaic virus CMV(Y). Ecotype Col‐0 is susceptible to CMV(Y), whereas C24 contains the dominant resistance gene RCY1. Upon infection with CMV(Y), all clustered genes were significantly activated in the resistant ecotype C24. In addition, we demonstrated that posttranslational histone modifications associated with trimethylation of histone H3 lysine 27 are most likely involved in regulation of several cluster genes described in this study. Overall, our experiments indicated that pathogen‐response genes in the genome of A. thaliana may be clustered and co‐regulated.