Roy J. Britten

Analysis of repeating DNA sequences by reassociation.

Repetitive DNA occurs widely, if not universally, among higher organisms. A variety of procedures has been developed or adapted to examine its characteristics and a body of concepts and language has grown up to deal with its complexities. This chapter attempts to summarize this body of knowledge and technique. Owing to human frailty and the real problems of the subject, it tends to emphasize the approaches and conceptual position. The chapter describes techniques for the analysis of repeating DNA sequences by reassociation and a method for the evaluation of rate constants. The purity of the DNAs used in reassociation is critical, as the presence of contaminating proteins and metal ions can markedly alter reassociation results and reproducibility. Metal ions can be removed from DNA by passing it over Chelex 100 (Bio-Rad Labs) that has been neutralized and equilibrated with buffer.

High-Resolution Density Gradient Sedimentation Analysis

The principle of stability for a sample layered in a density-gradient liquid column is discussed, and a method for separating ribonucleoprotein particles by means of sedimentation in the ultracentrifuge is described.

Organization, Transcription, and Regulation in the Animal Genome

This review concerns recent experimental information in areas of animal cell molecular biology which are relevant to the mechanism of gene regulation. New data regarding interspersion and clustering of repetitive sequence elements in DNA are considered. Molecular characteristics of animal structural genes and mRNAs are discussed, with particular reference to the frequency of structural gene sequences, mRNA turnover, and the interpretation of dipteran complementation groups. The molecular characteristics of nuclear RNAs, the primary transcription products, are reviewed. Evidence for transcription level regulation is summarized and the relation of nuclear and mRNA examined. The protein activator branch of the Britten-Davidson model for gene regulations is further developed and considered in ligh of current knowledge.

Reduction in the rate of DNA reassociation by sequence divergence.

An estimate is made of the effect of imperfectly complementary sequences on the rate of reassociation of DNA. Rate measurements are reported for the reassociation of deaminated DNA and for the pairing of DNAs from related bacteria. A method is presented for separating the effect of the incubation temperature on the rate from the effect of sequence divergence. After correction to the optimum incubation temperature, the rate of DNA reassociation appears to be reduced by a factor of two for each 10 deg. C reduction in melting temperature due to sequence divergence. For most typical cases this effect is modest. However it can be quite important for measurements of the relation between the DNAs of different species.

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.

Structural gene sets active in embryos and adult tissues of the sea urchin

Structural gene sequences active in a variety of sea urchin adult and embryo tissues are compared. A single-copy 3H-DNA fraction, termed mDNA, was isolated, which contains sequences complementary to the messenger RNA present on gastrula stage polysomes. Gastrula message sequences are 50 fold concentrated in the mDNA compared to total single-copy DNA. mDNA reactions were carried out with excess mRNA from blastula, pluteus, exogastrula, adult ovary, tubefoot, intestine, and coelomocytes, and with excess total mature oocyte RNA. A single-copy 3H-DNA fraction totally devoid of gastrula message sequences, termed null mDNA, was also reacted with these RNAs. Large differences in the extent of both mDNA and null mDNA reaction with the various RNAs were observed, indicating that in each state of differention a distinct set of structural genes is active, generally characterized by several thousand specific sequences. The complexity of gastrula mRNA was shown in previous work to be about 17 X 10(6) nucleotides. In units of 10(6) nucleotides, the complexities of the RNA sequence reacting with mDNA and with null mDNA in each tissue are, respectively, as follows: intestine mRNA: 2.1 and 3.7; coelomocyte mRNA: 3.5 and less than or equal to 1.4; tubefoot mRNA: 2.7 and less than or equal to 0.4; ovary mRNA: 13 and 6.7; oocyte total RNA: 17 and 20; blastula mRNA: 12 and 15; pluteus mRNA: 14 and less than or equal to 0.6; exogastrula mRNA: 14 and less than or equal to 0.6. The total complexity of each mRNA polulation is the sum of these values, as verified for several cases by reactions with total single-copy DNA. A relatively small set of mRNAs, the complexity of which is about 2.1 X 10(6) nucleotides, appears to be shared by several of the tissues studied.

Nucleotide Sequence Repetition: A Rapidly Reassociating Fraction of Mouse DNA

The separated complemnentary strands of a minor component in mouse DNA reassociate with each other much more rapidly than do the complementary strands of other DNAs including those of the principal part of mouse DNA. This difference in capacity of the strands to reassociate can be used to effect a preparative separation of the minor component from the principal fraction. The rate constant for reassociation of the minor component, compared with those of viral and bacterial DNAs, indicates that the minor component consists of a short nucleotide sequence present in about one million copies.

Divergence between samples of chimpanzee and human DNA sequences is 5%, counting indels

Five chimpanzee bacterial artificial chromosome (BAC) sequences (described in GenBank) have been compared with the best matching regions of the human genome sequence to assay the amount and kind of DNA divergence. The conclusion is the old saw that we share 98.5% of our DNA sequence with chimpanzee is probably in error. For this sample, a better estimate would be that 95% of the base pairs are exactly shared between chimpanzee and human DNA. In this sample of 779 kb, the divergence due to base substitution is 1.4%, and there is an additional 3.4% difference due to the presence of indels. The gaps in alignment are present in about equal amounts in the chimp and human sequences. They occur equally in repeated and nonrepeated sequences, as detected by repeatmasker (http://ftp.genome.washington.edu/RM/RepeatMasker.html).

A Measurement of the Sequence Complexity of Polysomal Messenger RNA in Sea Urchin Embryos

The first measurement has been made of the number of diverse mRNA sequences (mRNA sequence complexity) in the total polysomes of a eucaryotic system, the sea urchin gastrula. mRNA was purified of nuclear RNA and any other heterogeneous RNA contaminants by release from polysomes with puromycin. Trace quantities of labeled nonrepetitive DNA fragments were hybridized with an excess of mRNA. The hybridization reaction followed ideal first order kinetics in mRNA concentration. At completion of the hybridization reaction, 1.35% of the nonrepetitive DNA was present as mRNA-DNA hybrid. The hybridized DNA was extracted and was at least 70% hybridizable with mRNA, demonstrating a 50-fold purification of the expressed sequences. This purified DNA fraction reassociated with excess unfractionated sea urchin DNA at a rate identical to that of the total nonrepetitive DNA tracer. The mRNA had therefore been hybridized to nonrepetitive DNA sequence, and the amount of hybrid could be used as a direct measure of the mRNA sequence complexity. The complexity of the gastrula mRNA can be calculated as about 17 million nucleotides, sufficient to comprise some 14,000 distinct structural genes. This result also provides an estimate of the number of diverse proteins being translated in the gastrula. From the rate of mRNA-DNA hybrid formation, we estimate that about 8% of the mRNA belongs to this complex class, and that less than 500 copies of each species of message in this class exist per embryo. Most of the mRNA population consists of a relatively small number of diverse species represented a much larger number of times.

Interspersion of repetitive and non-repetitive DNA sequences in the sea urchin genome

Abstract Measurements are reported which lead to the conclusion that repetitive and nonrepetitive sequences are intimately interspersed in the majority of the DNA of the sea urchin, Strongylocentrotus purpuratus. Labeled DNA was sheared to various lengths, reassociated with a great excess of 450 nucleotide-long fragments to cot 20, and the binding of the labeled DNA to hydroxyapatite was measured. Repetitive sequences measured in this way are present on about 42% of the 450 nucleotide-long fragments. As the DNA fragment length is increased, larger and larger fractions of the fragments contain repetitive sequences. Analysis of the measurements leads to the following estimate of the quantitative features of the pattern of interspersion of repetitive and nonrepetitive sequences. About 50% of the genome consists of a short-period pattern with 300–400 nucleotide average length repetitive segments interspersed with about 1000 nucleotide average length nonrepetitive segments. Another 20% or more consists of a longer period interspersed pattern. About 6% of the genome is made up of relatively long regions of repetitive sequences. The remaining 22% of the genome may be uninterrupted single copy DNA, or may have more widely spaced repeats interspersed. The similarity of these results to previous measurements with the DNA of an amphibian suggests that this interspersion pattern is of general occurrence and selective importance.