Loss of CpG dinucleotides from DNA. VI. Methylation of mitochondrial and chloroplast genes

From nucleotide sequences of mitochondrial and chloroplast genes the probable frequency of the CpG----TpG + CpA substitutions was determined. These substitutions may indicate the level of prior DNA methylation. It was found that the level of this methylation is significantly lower in mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) than in nuclear DNA (nDNA) of the same species. The species (taxon) specificity of mtDNA and chDNA methylation was revealed. A correlation was found between the level of CpG methylation in nDNA, and mtDNA and chDNA in different organisms. It is shown that cytosine residues in CpG were not subjected to significant methylation in the fungi and invertebrate mtDNA and also in the algae chDNA. In contrast, the vertebrate mtDNA bears the impress of CpG-supression, which is confirmed by direct data on methylation of these DNA. Here the first data on the possible enzymatic methylation of the plant mtDNA and chDNA were obtained. It was shown that the degree of CpG-suppression in the 5S rRNA nuclear genes of lower and higher plants is significantly higher in the chloroplast genes of 4,5S and 5S rRNA. From data on pea chDNA hydrolysis with MspI and HpaII it was established that in CCGG sequences this DNA is not methylated. The role of DNA methylation in increasing the mutation rate and in accelerating the evolutionary rates of vertebrate mtDNA is discussed.     

Hormone-dependent insertional Arabidopsis thaliana mutants with decreased viability and fertility

We present data on the phenotype identification and genetic analysis of offspring in three lines of dominant morphological mutants of Arabidopsis thaliana having drastically reduced fertility (a sterile calluslike mutant, a flower mutant, and a dwarf mutant) and in five lines of recessive morphological mutants (four mutants with lethal seedlings and one pigmentation mutant). The mutants were selected from a collection of transgenic plants that had genomes carrying a T-DNA insertion of plasmid vectors pLD3 and pPCVRN4; the collection was created earlier via agrobacterial transformation of germinating seeds. The results presented here were obtained using compensation of hormonal imbalance in the insertional morphological mutants of A. thaliana by exogenous hormones.     

Saving insertional recessive lethal mutants of Arabidopsis thaliana

A group of 13 recessive lethal mutants was selected on the basis of the collection of Arabidopsis thaliana transgenic plants with insertions of T-DNA vector plasmid pLD3 or pPCVRN4, which was produced by agrobacterial transformation of germinating seeds. The use of media containing exogenous hormones made it possible to compensate the lethal effect, identify phenotypes, and characterize six lines of recessive lethal germlings using genetic and molecular-genetic methods.     

Identification of the Gene Whose Mutation Leads to the Appearance of Recessive Lethal Germlings in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The data are presented on genetic and molecular-genetic analysis of a mutant from the collection of morphological insertion mutants of Arabidopsis thaliana we obtained earlier, which belongs to the phenotypic class of recessive lethal germlings. A nucleotide DNA sequence, 147 bp in size, was identified, which adheres to the left border area of T-DNA insertion. The site of localization of the insertion was determined using computer analysis.__________Translated from Ontogenez, Vol. 36, No. 3, 2005, pp. 222–224.Original Russian Text Copyright © 2005 by Ogarkova, Tomilov, Tomilova, Tarasov.     

Identification of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> genes whose expression determines changes in the shoot structure

A system was created to obtain and select Arabidopsis thaliana genes whose superexpression causes development of a mutant phenotype. Three morphological mutants (two with a markedly retarded growth and one with a fasciated stem) associated with the superexpression of genes At5g10080, At1g33390, and At5g13760 were generated with the use of this system. Localization, structure, and a possible functional organization of these genes were determined.     

Identification of the gene whose mutation leads to hypocotyl tropism in Arabidopsis thaliana

Genetic and molecular analysis of a mutant of Arabidopsis thaliana with bended hypocotyl from a previously obtained collection of insertion mutants is presented. The examined mutation was shown to be recessive and based on a single insertion of pLD3 vector T-region into the A. thaliana genome. Computer-aided analysis of a DNA region adjacent to the left border of the insertion revealed a putative site of T-DNA insertion, the At1g15760 gene from 609-bp chromosome 1 represented by a single exon.     

Identification of a gene, included in control of root system development in Arabidopsis thaliana

Genetic and molecular genetic analysis of a lethal root mutant of Arabidopsis thaliana was carried out. The mutant was obtained from a collection created earlier by means of insertion mutagenesis. The mutation was found to be recessive. It was caused by an insertion of the T region of vector pLD3 used for transformation of germinating seeds when creating the collection of insertion mutants. A 118-bp DNA fragment flanking the left border of the insertion was isolated using the TAIL PCR technique, and its nucleotide sequence was determined. Computer analysis of this DNA region demonstrated that it was located in exon 32 of the YUP8HI2R.44 gene in chromosome 1.     

Identification of mutant gene responsible for cotyledons necrosis in developing seedlings of Arabidopsis thaliana]

Genetic and molecular analyses of an Arabidopsis thaliana mutant with necrotic cotyledons from the collection of insertion mutants obtained earlier were conducted. The mutation under study showed incomplete dominance and represented a single insertion of the T region of pLD3 vector used for transformation of germinating seeds to the plant genome during the creation of the collection. Using TAIL-PCR, a fragment of the mutant DNA adjacent to the left border of the T-DNA insertion was isolated and sequenced. Computer r-aided analysis showed that the insertion was located on the left arm of chromosome 1. The open reading frame containing the insertion has one exon and encodes a protein of 446 amino acids, whose functions are unknown.     

Creation of a collection of morphological insertional mutants of Arabidopsis thaliana

A collection of transgenic Arabidopsis thaliana plants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12-18% of the total number of A. thaliana genes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.     

Identification of the gene whose mutation leads to the morphological changes in the hypocotyl of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The results of genetic and molecular genetic analysis of line 176 of Arabidopsis thaliana with reduced hypocotyls obtained from a previously developed collection of insertion mutants, are presented. The examined mutation proved to be recessive and based on a single insertion of the T-DNA vector pLD3 into the A. thaliana genome. Computer-aided analysis of the amplified in TAIL-RCR DNA region adjacent to the left border of the insertion revealed a putative site of T-DNA insertion, the 2.5-kb At2g09920 gene located in the long arm of chromosome 2, near the centromere.Translated from Genetika, Vol. 41, No. 2, 2005, pp. 166–170.Original Russian Text Copyright © 2005 by Ogarkova, Tomilov, Tomilova, Pogorelko, Tarasov.