A simple and efficient method for the separation and detection of small DNA fragments by electrophoresis in formamide containing agarose gels and Southern blotting to DBM-paper

Sun, Yu; Xu, Yuan Zhong; Chambon, Pierre

doi:10.1093/nar/10.19.5753

Cited by 20 publications

(14 citation statements)

References 11 publications

(9 reference statements)

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“…The above observations suggest that in spite of its atypical nucleosomal ladder, the fl-globin region corresponding to the intronic probe d is organized in an array of nucleosome-like structures, which however are highly sensitive to further micrococcal nuclease digestion at 37°C when released in the monomer state. The existence of a nucleosome-like structure is also supported by results obtained when more extensive micrococcal nuclease digests were analysed using agarose-formamide gels (Sun et al, 1982) to achieve a better resolution in the 120-200 bp DNA fragment range. As shown in panels G and H of Figure 2, DNA fragments of 120 and 140 bp were revealed using the 3-globin and ovalbumin probes, suggesting that the ,B-globin gene sequences are organized into nucleosomal-and subnucleosomal- Erythrocyte nuclei were digested with micrococcal nuclease in the absence of spermine-spermidine (buffer A -Materials and methods) at 0°C or 20'C (300 U/mg DNA) or 37'C (100 U/mg DNA).…”

Section: Resultsmentioning

confidence: 69%

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Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

Sun¹,

Xu²,

Bellard³

et al. 1986

The EMBO Journal

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The structure of the chicken adult beta‐globin gene chromatin in immature and mature erythrocyte nuclei has been analysed using micrococcal nuclease digestion. The resulting DNA fragments were blotted onto DBM‐papers and probed with labelled DNA fragments spanning the adult beta‐globin gene and its 5′‐ and 3′‐flanking regions. The structure of the nucleosomes within and in the regions flanking the adult beta‐globin gene appears to be altered in at least two ways in erythrocyte chromatin, when compared with either bulk or inactive ovalbumin gene chromatin. First, oligomeric DNA fragments containing the beta‐globin gene are released faster than those of either bulk or ovalbumin gene chromatin. Second, although the difference in size of the liberated oligomeric DNA fragments is similar to the nucleosomal repeat length of bulk and ovalbumin gene chromatin, the individual oligomers are approximately 100 bp shorter than their bulk or ovalbumin gene counterparts, most noticeably when the nuclease digestion is performed at 37 degrees C. This results in an atypical ladder of approximately 300, 500, 700, 900 bp instead of the canonical chicken erythrocyte ladder which is an integral multiple of 207 bp. The same ladder was obtained from immature erythrocytes, in which the beta‐globin gene is actively transcribed, and from mature erythrocytes, in which it is considered to be inactive with RNA polymerase molecules clustered in the 5′ moiety of the gene. This indicates that the alteration of the nucleosomal structure is not due to transcription per se.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 69%

“…SV40 DNA HindH and HindIII restriction fragments were used as external or internal size markers (Bellard et al, 1982). Agarose-formamide gels (4% agarose-50% formamide) were run as previously described (Sun et al, 1982).…”

Section: Methodsmentioning

confidence: 99%

Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

Sun¹,

Xu²,

Bellard³

et al. 1986

The EMBO Journal

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“…A number of restriction enzymes cleave once in each repeat unit in the BR 2 a and 13 blocks, thus releasing the monomer units (13)(14)(15). To detect repeat-unit size variants, total C. tentans DNA was cleaved completely with Hinfl, separated in 5% agarose gels containing 40% formamide (19), and transferred to nitrocellulose filters. As seen from the end-labeled pBR322 marker fragments ( …”

Section: Resultsmentioning

confidence: 99%

Different evolutionary behavior of structurally related, repetitive sequences occurring in the same Balbiani ring gene in Chironomus tentans

Höög

Wieslander²

1984

Proc. Natl. Acad. Sci. U.S.A.

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The Balbiani ring 2 (BR 2) gene in Chironomus tentans is highly internally repeated. Two Many eukaryotic genes display structural features suggesting that they have evolved through the amplification of intragenic sequences (1-7). The genes encoding structural proteins have often maintained a conspicuous repetitive structure that is a valuable asset in the analysis of the evolutionary process. In one gene family of this type-the Balbiani ring (BR) genes in the dipteran Chironomus tentans-the repetitive feature of the genes is particularly prominent, with a hierarchic arrangement of the repetitive sequences. The BR genes contain the genetic information for large salivary secretory proteins (8, 9), which are used to spin tubes housing the aquatic larvae. The principal structures of the BR 1 (10-12) and BR 2 (13-15) genes in C. tentans, the BR 2 gene in Chironomus pallidivittattus (16), and the BR b (17) and BR c (18) genes in Chironomus thummi have been revealed, and, in all cases, the genes consist of a large number of 150-to 300-base-pair (bp)-long repeat units that are tandemly repeated. All repeat units have one constant region (C region) and one subrepeat region (SR region),t the latter being formed by a number of subrepeats. Furthermore, all BR genes show a high degree of sequence similarity, suggesting that they may share a common origin. The characterization of the various BR genes therefore allows the study of the evolution of a set of different but closely related genes within and between species, each gene consisting of several hundred repeat units specific to that gene but still being the result of amplifications starting from a common ancestor sequence (13). In BR 2 in C. tentans, there exist two different but related types of repeat units-the a and P types-which form separate sequence blocks and probably together cover most of the BR 2 gene (13,14). In this study we investigate the homogeneity of the blocks and record striking structural differences between them. These findings suggest that the blocks evolved at different times and that both sequence correction mechanisms and selection at the protein level operate to various extents on the different repeat structures along the gene. MATERIALS AND METHODSIsolation of DNA. Total C. tentans DNA was extracted from crude nuclei preparations as described (13).Cloning Procedures. The construction and cloning of cDNA transcripts from salivary gland 75S RNA has been described by Sumegi et al. (13).Southern Blot Analysis. Total C. tentans DNA was cleaved completely with Hinfl (New England Biolabs) and separated in a 5% agarose gel containing 40% formamide according to Sun et al. (19). The DNA fragments were transferred to nitrocellulose filters (20) and hybridized with the 32P-labeled cDNA inserts according to Thomas (21). The hybridization signal was quantified by scanning densitometry using a Joyce-Loebl densitometer.DNA Sequence Determination. Hinfl fragments from the recombinant plasmids were treated with DNA polymerase I (New England Biolabs) and...

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“…(14). Agarose gels were prepared according to (15,16). Polyacrylamide gels ( 1 : 20 ratio N,N'methylenbisacrylamide: acrylamide) were reinforced with 0.5% agarose.…”

Section: Introductionmentioning

confidence: 99%

Structure and function of an AT-rich, interspersed repetitive sequence fromChironomus thummi: solenoidal DNA, 142 bp palindrome-frame and homologies with the sequence for site-specific recombination of bacterial transposons

Israelewski

1983

Nucl Acids Res

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Chironomus thummi thummi contains a repetitive AT-rich 118 bp sequence mainly in the centromere regions and elsewhere in the genome (1). A large cluster of repeats is regularly present in the non-transcribed spacer of rDNA. Dimers and multimers of the repeat migrate slower in small pore gels than would be expected from their size. The results indicate a solenoidal structure with a coil girth of appr. 350 bp. This structure is most probably due to a highly periodic positioning of di-nucleotides of the type purinepurine or pyrimidine-pyrimidine with distances of appr. 10 bases. In a cluster~f 118 bp repeats, regions of dyad-symmetry are positioned such that a 142 -2 bp palindrome-frame is generated. Evidence is presented favouring the assumption that the repeat functions primarily in sister chromatid exchange.

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A simple and efficient method for the separation and detection of small DNA fragments by electrophoresis in formamide containing agarose gels and Southern blotting to DBM-paper

Cited by 20 publications

References 11 publications

Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

Different evolutionary behavior of structurally related, repetitive sequences occurring in the same Balbiani ring gene in Chironomus tentans

Structure and function of an AT-rich, interspersed repetitive sequence fromChironomus thummi: solenoidal DNA, 142 bp palindrome-frame and homologies with the sequence for site-specific recombination of bacterial transposons

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