Sequence Organization and Genomic Distribution of the Major Family of Interspersed Repeats of Mouse DNA (original) (raw)
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A family of moderately repetitive sequences in mouse DNA
Nucleic Acids Research, 1980
When mouse DNA is digested to completion with restriction endonuclease Eco Rl, a distinct band of 1.3 kb segments comprising about 0.5-3% of thegenomeis observed upon agarose gel electrophoresis. This DNA is not tandemlyrepeated in the genome and is not derived from mouse satellite DNA. Restriction endonuclease analysis suggested that the 1.3 kb segments are heterogeneous. Specific sequences were selected from the 1.3 kb segments and amplified by cloning in plasmid pBR322. Southern transfer experiments indicated that three separately cloned mouse DNA inserts hybridized predominantly to the Eco R1 1.3 kbband and to the conspicuous subsegments generated by secondary restriction endonuclease cleavage of the sucrose gradient purified 1.3 kb segments. Segments were also excised by Hha I (Hha I segments) from the chimeric plasmids containing mouse DNA inserts and subjected to restriction endonuclease and cross-hybridization analysis. It was found that the three Hha I segments were different, although two of them exhibited partial sequence homology. Cot analysis indicated that each of the Hha I segments are repeated about 104 times in the mouse genome. These findings indicate that a family of related but non-identical, moderately repetitive DNA sequences, rather than a single homogeneous repeat, is present in the 1.3 kb Eco RI band.
Characterization of a highly repetitive sequence DNA family in rat
Journal of Molecular Biology, 1981
A 92 to 93 base-pair highly reiterated DNA from rat liver has been isolated by digestion with EcoRI and cloned in pBR322. Three recombinant plasmids have been studied in detail; these are p93-2 and p93-10, both of which contain two 93 base-pair inserts, and p93-15, which contains one 92 base-pair and two 93 base-pair inserts. Analysis of these seven cloned DNA fragments reveals that 93 base-pair highly repetitive DNA exhibits as much aa 50% overall sequence heterogeneity although several families can be identified on the basis of a high degree of sequence homology among particular cloned inserts. Individual families have also been identified on the basis of specific restriction endonucleaae sites. This property, coupled with a limited degree of cross-hybridization among the groups of inserts in filter hybridizations, has been utilized to demonstrate that the three 93(2) base-pair families identified here are repeated in a regularly interspersed, tandem manner with each family repeated no more frequently than once every fourth sequence. Similar conclusions have been reached by analysis of a specific fraction of 370 basepair rat repeated DNA .
Studies of a novel repetitive sequence family in the genome of mice
European Journal of Biochemistry, 1988
A new middle repetitive sequence is described in the mouse genome. It has been revealed with a recombinant clone isolated from a Mus musculus BamHI gene library constructed in pBR322 and containing an insertion of 1.73 kb. When digests of genomic DNA were subjected to Southern blot hybridization, using the 1.73-kb insert as probe, we obtained a light smear and discrete bands, indicating a dispersion in the mouse genome of this sequence. This 1.73-kb sequence seems to be a part of a greater repetitive sequence at least 6 kb in length.
Nucleic Acids Research, 1983
A new molecular hybridization approach to the analysis of complex genomes has been developed. Tracer and driver DNAs were d ested with the same restriction enzyme(s), and tracer DNA was labeled with P using T4 DNA polymerase. Tracer DNA was mixed with an excess amount of driver, and the mixture was electrophoresed in an agarose gel. Following electrophoresis, DNA was alkali-denatured in situ and allowed to reanneal in the gel, so that tracer DNA fragments could hybridize to the driver only when homologous driver DNA sequences were present at the same place in the gel, i.e. within a restriction fragment of the same size. After reannealing, unhybridized single-stranded DNA was digested in situ with Si nuclease. The hybridized tracer DNA was detected by autoradiography. The general applicability of this technique was demonstrated in the following experiments. The common EcoRI restriction fragments were identified in the genomes of E. coli and four other species of bacteria. Two of these fragments are conserved in all Enterobacteriaceae. In other experiments, repeated EcoRI fragments of eukaryotic DNA were visualized as bands of various intensity after reassociation of a total genomic restriction digest in the gel. The situation of gene amplification was modeled by the addition of varying amounts of x phage DNA to eukaryotic DNA prior to restriction enzyme digestion. Restriction fragments of X DNA were detectable at a ratio of 15 copies per chicken genome and 30 copies per human genome. This approach was used to detect amplified DNA fragments in methotrexate (MTX)-resistant mouse cells and to identify commonly amplified fragments in two independently derived MTX-resistant lines.
1991
A strategy for the rapid isolation of DNA probes from radiation-fusion Chinese hamster cell hybrids containing overlapping portions of the murine X chromosome based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) previously used with human somatic cell hybrids has been developed. This specific amplification of mouse DNA on a hamster background depends on the use of primers directed to the B2 short interspersed repeat element family and the R repeat, from the long interspersed repeat element family, Ll. Two sets of amplification conditions, which gave specific amplification of mouse DNA from either a mouse X-monochromosomal hybrid or irradiation-fusion hybrids having reduced X content, were defined. The mouse X-only chromosome hybrid yielded approximately 20 discrete reproducible bands, while the irradiation-fusion hybrids yielded between 1 and 10 discrete products. Comparison of different irradiation-fusion hybrids has allowed the definition of both specific and shared products corresponding to different regions within the overlapping X-chromosome fragments present within these hybrids. Use of such hybrids and the IRS-PCR technique has allowed the isolation of probes corresponding to the central region of the mouse X chromosome that contains the X-inactivation center. The method should be widely applicable to the isolation of mouse DNA sequences from mouse hybrid cell lines on either human or Chinese hamster backgrounds.
The Major Components of the Mouse and Human Genomes. 2. Reassociation Kinetics
European Journal of Biochemistry, 1981
The reassociation kinetics of DNA fragments obtained from the major components of the mouse and human genomes (recently isolated in our laboratory) have been investigated. I t has been found that the relative amounts of interspersed repeated and unique sequences strikingly differ in the different major components of each genome and in the corresponding major components of the two genomes. Furthermore, within each major component, the interspersed repeated and unique sequences do not differ in dG + dC contents. These findings lead to the general conclusion that the sequence organization of mammalian genomes is not uniform in different chromosomal regions and that it exhibits remarkable variations in different mammals.
An alternative view of mammalian DNA sequence organization
Journal of Molecular Biology, 1981
The biochemical and biophysical techniques originally introduced by Davidson et al. (1973) and for the determination of the general organization and length of repetitive and non-repetitive sequences in eukaryotic DNA have been extended and modified. Improvements in the experimental methods employed in these pioneering works have led to novel interpretations and conclusions about mammalian DNA sequence organization. In what is commonly referred to as an interspersion experiment, the average spacing of repetitive DNA regions is inferred from the length dependence of hydroxyapatite binding of radiolabeled tracer DNAs reassociated with an excess of short 200 nucleotide repetitive sequence driver DNA. Studies on Syrian hamster DNA, using an improved procedure for conducting interspersion experiments, suggest that either a frequent cluster in the distribution of non-repetitive DNA sequence lengths occurs at 7200 ( +2000) nucleotides or that repetitive sequences are randomly spaced on a number average basis. In contrast, measurements obtained using the traditional methods suggest that a frequent cluster in the distribution of non-repetitive DNA sequence lengths occurs at approximately 1000 nucleotides. When reassociations were conducted at elevated temperatures, to allow only well-matched repetitive sequences to hybridize, the amount of DNA operationally observed as "repetitive" was reduced. Interspersion experiments conducted with Syrian hamster DNA at a reassociation temperature of 75°C yielded data similar to those obtained by Manning et al. (1975) for Lbosophila melalzogaster DNA reassociated at 60°C.