Molecular cloning and restriction enzyme analysis of a long repetitive DNA sequence in rice.

Rice long repetitive DNA (9-20 kbp) reassociating at Cot 50 M.s was cloned in pBR325. Out of several recombinants (Camr Ampr Tets), only a few were selected randomly for further characterization. The insert size in all these clones was 3-4 kbp. Restriction enzyme analysis showed the absence of EcoRI and BclI sites, presence of a single PstI and PvuII site and multiple sites for AluI in 3 clones namely pRLl, pRL7 and pRL10. The BamHI-PstI fragment of about 0·4 kbp in the pRL7 insert DNA (pRL7-0·4 kbp) was subcloned in M13mpl8 and partially sequenced using Sanger’s dideoxynucleotide chain termination method. Dot matrix comparison of this sequence with rice rDNA sequences revealed low homology with the 25S rDNA sequence of rice, however, hybridisation did not indicate any homology.

Use of high resolution thermal denaturation approach in preliminary identification of plant DNAs.

High resolution thermal denaturation (HRTD) profiles of the DNAs of six related millets, oat and rice showed several maxima and shoulders skewed towards the GC-rich side. Linear regression and correlation analyses exhibited no correlation between the repetitive DNA content/nuclear DNA content and the number of peaks, while there existed a very good correlation between delta T and repetitive DNA content/nuclear DNA content indicating that increase in repetitive DNA content has resulted in a greater sequence heterogeneity in these DNAs. In addition, specific melting characteristics of each of the eight plant DNAs were identified, which showed species specificity.

5-Methylcytosine content and methylation status in six millet DNAs.

High performance liquid chromatographic analysis of the total nuclear DNAs of 6 millets plant species indicates that the 5-methylcytosine content ranges from 3% in barn yard millet to 9·6% in great millet while the fraction of cytosines methylated varies between 14% in little millet to 31 % in pearl millet. Digestion of millet DNAs with MspI/HpaII suggests that CpG methylation is more in great millet DNA while CpC methylation is more in the other 5 millet DNAs. Digestion of millet DNAs with MboI, Sau3AI and DpnI indicates that some of the 5’ GATC3’ sequences are methylated at adenine and/or cytosine residues except in little millet where adenine methylation of the 5’GATC3’ sequences is insignificant and there is a predominance of cytosine methylation in these sequences.

Molecular organization of great millet (Sorghum vulgare) DNA.

Approximately 52% of the nuclear genome of great millet (Sorghum vulgare) consists of repetitive DNA which can be grouped into very fast, fast and slow components. The reiteration frequencies of the fast and slow reassociating components are 7000 and 92 respectively. Approximately 90% of the genome consists of repeated sequences interspersed amongst themselves and with single copy sequences. The interspersed repeat sequences are of three sizes viz. > 1·5 kilobase pairs, 0·5–1·0 kilobase pairs and 0·15–0·30 kilobase pairs while the size of the single copy sequences is 3·0 kilobase pairs. Hence the genome organization of great millet is essentially of a mixed type.

Analysis of bovidae genomes: Arrangement of repeated and single copy DNA sequences in bovine, goat and sheep.

Approximately 43-60% of the total genome in bovine, goat and sheep consisted of interspersed repeated and single copy DNA sequences. Most of the interspersed repeated DNA sequences were 1500-2400 nucleotide pair long while a minor portion was more than 4000 nucleotide pair long in goat and sheep and 3200 nucleotide pair long in bovine. About 1/3rd of single copy sequence were interspersed and their length was in the range of 1000-1500 nucleotide pairs.

DNA Sequence organization In finger millet (Eleusine coracana).

Approximately 39 to 49% of the genome of finger millet consists of repetitive DNA sequences which intersperse with 18% of single copy DNA sequences of 1900 nucleotide pairs. Agarose gel filtration and electrophoresis experiments have yielded the sizes of interspersed repeated sequences as 4000-4200 nucleotide pairs and 150-200 nucleotide pairs. Approximately 20% of the repeated DNA sequences (4000-4200 nucleotide pairs) are involved in long range interspersion pattern, while 60% of the repeated DNA sequences (150-200 nucleotide pairs) are involved in short period interspersion pattern. Based on the data available in literature and the results described here on DNA sequence organization in plants, it is proposed that plants with haploid DNA content of more than 2.5 pg exhibit mostly the short period interspersion pattern, while those with haploid DNA content of less than 2.5 pg show diverse patterns of genome organization.