Barbara R. Hough

General interspersion of repetitive with non-repetitive sequence elements in the DNA of Xenopus

The arrangement of repetitive and non-repetitive sequences was studied in the DNA of Xenopus. Labeled DNA sheared to various fragment lengths was reassociated to C_ot 50 (60 °C, 0.18 m-Na^+) with excess 450 nucleotide fragments of unlabeled DNA, and binding of the labeled DNA to hydroxyapatite was measured. Repetitive sequences monitored in this way are present on about 45% of the 450 nucleotide fragments. As DNA fragment length is increased, larger fractions of the DNA are found to contain repetitive elements. Up to 80% of the DNA binds at an average fragment length of 3700 nueleotides. Analysis of the data shows that a little more than 50% of the genome consists of closely interspersed repetitive and non-repetitive sequences. The average length of the repetitive sequence elements is 300±100 nueleotides, while the non-repetitive sequences separating adjacent repetitive sequence elements average 800±200 nueleotides. The remainder of the DNA is mainly non-repetitive, though most of it contains rare interspersed repetitive elements spaced at a minimum of 4000 nueleotides apart. It is concluded that a high degree of order exists in the arrangement of DNA sequences in the Xenopus genome.

Sequence complexity of heterogeneous nuclear RNA in sea urchin embryos

The sequence complexity of heterogeneous nuclear RNA is sea urchin gastrulas was measured by RNA-driven hybridization reactions with nonrepetitive sea urchin DNA. 28.5% of the sequence complexity of the genome is represented in the nuclear RNA. This amounts to 1.74 X 10(8) nucleotides of diverse sequence, more than 10 times the nucleotide complexity of the polysomal messenger RNA extracted from sea urchin embryos at the same stage. The complex set of nuclear RNA sequences driving this hybridization reaction was shown to be the same as the rapidly labeled hnRNA, using pulse-labeled nuclear RNA as driver.

Repetitive and non-repetitive sequence in sea urchin heterogeneous nuclear RNA.

Heterogeneous nuclear RNA was extracted from sea urchin gastrulae following a ten-minute pulse with [^3H]uridine. Hybridization reactions with excess sheared DNA were carried out using several methods. The fraction of the Hn[^3H]RNA in hybrids was determined by RNAase resistance and hydroxyapatite chromatography in a urea-phosphate medium. After incubation to a DNA C_0t value of 40, 20 to 25% of the Hn[^3H]RNA binds to hydroxyapatite. Approximately one third of these bound molecules are in repetitive DNA-RNA hybrids. The size of the bound RNA molecules measured after hybridization averages 1100 nucleotides. Up to 70% of the Hn[^3H]RNA forms hybrids at high DNA/RNA ratios (130,000/1) when reactions are carried out to high C0t values. Studies of the effect of increased DNA/RNA ratio on extent of hybridization at high DNA C_0t values show that at least two frequency classes of single copy transcript are present in the Hn[3H]RNA population. Both repetitive and single copy DNA transcripts are present within individual Hn[^3H]RNA molecules. Calculations indicate that at least 25% and possibly all of the Hn[^3H]RNA molecules contain repetitive sequence elements interspersed with non-repetitive sequences.

Structural genes adjacent to interspersed repetitive DNA sequences

The observation that repetitive and single copy sequences are interspersed in animal DNAs has suggested that repetitive sequences are adjacent to single copy structural gene sequences. To test this concept, single copy DNA sequences contiguous to interspersed repetitive sequences were prepared from sea urchin DNA by hydroxyapatite fractionation (repeat-contiguous DNA fraction). These single copy sequences included about one third of the total nonrepetitive sequence in the genome as determined by the amounts recovered during the hydroxyapatite fractionation and by reassociation kinetics. 3H-labeled mRNA from sea urchin gastrula was prepared by puromycin release from polysomes and used in DNA-driven hybridization reactions. The kinetics of mRNA hybridization reactions with excess whole DNA were carefully measured, and the rate of hybridization was found to be 3-5 times slower than the corresponding single copy DNA driver reassociation rate. The mRNA hybridized with excess repeat-contiguous DNA with similar kinetics relative to the driver DNA. At completion 80 percent of that mRNA hybridizable with whole DNA (approximately 65 percent) had reacted with the repeat-contiguous DNA fraction (50 percent). This result shows that 80-100 percent of the mRNA molecules present in sea urchin embryos are transcribed from single copy DNA sequences adjacent to interspersed repetitive sequences in the genome.

Studies on the repetitive sequence transcripts of Xenopus oocytes

RNA of the mature Xenopus oocyte was hybridized with an isolated repetitive DNA fraction of the Xenopus genome representing about 40% of the DNA and containing a major component with an average sequence-repetition frequency of about 1600. Ribonuelease-resistant hybrids which bind to hydroxyapatite represent the most stable fraction of the repetitive RNA-DNA duplexes. These hybrids were studied with CsCl equilibrium sedimentation, gel electrophoresis, and thermal chromatography from hydroxyapatite. It was concluded that after ribonuclease treatment the hybrids which bind to hydroxyapatite retain relatively long regions of duplex structure (over 10^2 nucleotides) but that, even so, they include significantly more nucleotide mismatch than do non-repetitive DNA-RNA hybrids formed with the same RNA. Hybrids were also detected by Cs_2SO_4 buoyant-density sedimentation in the material failing to bind to hydroxyapatite after ribonuclease treatment. These hybrids apparently retain only short duplex regions. RNA-driven hybridization reactions were carried out to obtain an estimate of the amount of repetitive DNA related in sequence to the RNA stored in the mature oocyte. In this procedure, the hybrids are not exposed to ribonuclease before binding to hydroxyapatite, and hybrids representing about 3.5% of the repetitive DNA were obtained. It was concluded that 2 to 5% of the total oocyte RNA is repetitive sequence transcript and that these transcripts are present in the oocyte at a much higher concentration than non-repetitive sequence transcripts.

Sequence repetition in the DNA of Nassaria (Ilyanassa) obsoleta

The reassociation kinetics of the DNA of Ilyanassa obsoleta have been studied in order to obtain a repetition frequency profile for the genome of this organism, a well known subject of embryological interest. At a criterion set about 25°C below the T_m, Ilyanassa DNA consists of about 38% nonrepetitive sequences; at least 12% of a repetitive sequence component averaging about 20 relatives per sequence; at least 15% of a repetitive sequence component averaging about 1000 relatives per sequence; and 18% of a component which reassociates extremely rapidly and either contains regions of internal homology or consists of a small number of extremely frequent sequences, or both.

The RNA of the Mature Xenopus Oocyte

The RNA of the mature oocyte of Xenopus laevis has been studied in many laboratories and more extensively characterized than that of any other oocyte. Some of the information which has been developed relative to this RNA is summarized briefly in the paragraphs below.