DNA sequence organization in the pea genome

Abstract

The reassociation kinetics of pea (Pisum sativum L.) DNA fragments (300 nucleotides) were measured with hydroxylapatite. The most slowly reassociating fragments do so with a rate constant of 2 X 10(-4) L mol-1s-1, as determined from experiments with total DNA as well as with a tracer enriched for slowly renaturing sequences. This rate is about 1000 times slower than that observed for Escherichia coli DNA included as an internal kinetic standard, indicating a kinetic complexity of 4.5 X 10(9) nucleotide pairs or 4.6 pg of DNA per haploid nucleus. This estimate is in good agreement with previous chemical and cytophotometric measurements. The majority (85%) of the 300 nucleotide fragments contain repetitive sequences. While the reassociation of repetitive DNA could be modeled with two theoretical second-order components, the data did not specify a unique solution. The reassociation kinetics of isolated high- and low-frequency fractions indicate that repetitive sequence families in pea DNA probably cover a broad range of frequencies ranging from 100 to 10 000 or more copies per haploid genome. Single-copy sequences account for about 30% of the DNA, but because of extensive interpersion of repetitive sequences only about 15% of 300 nucleotide fragments reassociate with single-copy kinetics. From studies of hydroxylapatite binding as a function of fragment length, we conclude that the major class of single-copy sequences has a modal length of about 300 nucleotides. Long tracer reassociation kinetics indicate that sequences with an apparent repetition frequency of about 10 000 copies are interspersed at intervals of less than 1300 nucleotides throughout 75% of the genome. At a detection limit of about 3%, we find no single-copy sequences longer than 1000 nucleotides.

Single copy DNA homologies in Atriplex. I. Cross reactivity estimates and the role of deletions in genome evolution

“…On the other hand, all the Atriplex genomes and spinach are small by comparison with many plant and animal genomes for which sizes have been measured previously (Bachmann et a!., 1972;Bennett and Smith, 1976). Taking the average Atriplex haploid genome size as roughly 54>< 108 NTP, it corresponds to about 12 per cent of the pea genome (Bennett and Smith, 1976;Murray et al, 1978), or 20 per cent of an average mammalian genome (Bachmann et a!., 1972).…”

DNase I sensitivity of transcriptionally active genes in intact nuclei and isolated chromatin of plants.

“…DNA was purified by a modification of the Marmur procedure (20). The resulting EtOH precipitates were dissolved, treated sequentially with RNase A, proteinase K, and CHCl3/octanol (24:1), and again precipitated with EtOH (21). Total wheat reference DNA was isolated directly from unimbibed wheat embryos as described (21) or from chromatin isolated as described above.…”

“…The resulting EtOH precipitates were dissolved, treated sequentially with RNase A, proteinase K, and CHCl3/octanol (24:1), and again precipitated with EtOH (21). Total wheat reference DNA was isolated directly from unimbibed wheat embryos as described (21) or from chromatin isolated as described above. Unless otherwise noted, DNA used for reassociation studies was sheared to a singlestrand mass average length of 250-270 nucleotides (NT) as determined by alkaline agarose electrophoresis (21,22).…”

“…Polysomal RNA was prepared from 3-hr-imbibed wheat embryos. Poly(A)+RNA was purified by using poly(U)-Sepharose and [3H]cDNA was prepared as described (24 (21). Reassociation was monitored by using hydroxylapatite (20,21).…”

“…Poly(A)+RNA was purified by using poly(U)-Sepharose and [3H]cDNA was prepared as described (24 (21). Reassociation was monitored by using hydroxylapatite (20,21). Data are presented in the form of Cot curves and all Cot values are corrected to the equivalent Cot for 0.12 M sodium phosphate buffer (pH 6.8) at 60'C (20,25).…”