Nucleotide sequence of a highly repetitive component of rat DNA

Pech, Michael; Igo‐Kemenes, Tibor; Zachau, Hans G.

doi:10.1093/nar/7.2.417

Cited by 93 publications

(45 citation statements)

References 37 publications

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“…This reflects that all EcoKI sites are not equally accessible and hence repeats of various lengths are generated. It is also reported that EcoRl sites present adjacent to methylated residues show slow accessibility to EcoRI [20]. Furthermore, a major portion of satellite DNA in high molecular weight range reflects higher order organization of the chromatin.…”

Section: Resultsmentioning

confidence: 96%

Age‐related analysis of EcoRI generated satellite DNA‐containing chromatin of rat liver

1996

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SummaryEcoRI digestion of nuclei and their subsequent lysis with EDTA solubilizes 45% and 36% of chromatin DNA from the liver of young (18+2 weeks) and old (100+5 weeks) rats, respectively. After hybridization with 185 bp rat satellite I DNA, these soluble fractions are found to be enriched in specific DNA sequences such as satellite DNA. Besides regular repeat pattern, a major portion of the satellite chromatin forms higher order organization Digestion kinetics confirms condensation of satellite DNAcontaining chromatin similar to that of bulk chromatin in old age. Furthermore, densitometric scanning of the slot-blot of soluble chromatin fractions reveals loss of satellite DNA in the okL However, an increase in the linker histone HI and its subfraction HI ° in the satellite DNA-emiched fraction of chromatin from old rats suggests greater compaction. These results provide the first evidence that the satellite DNAcontaining chromatin differs in the liver of young and old rats.

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Section: Resultsmentioning

confidence: 96%

Age‐related analysis of EcoRI generated satellite DNA‐containing chromatin of rat liver

1996

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“…Panel d shows the photograph and densitometric trace of a stained gel of total rat DNA digested with HaeIII. The sizes in kilobases of the indicated fragments were determined by comparison with the migration of appropriately sized marker DNAs and, for the blots shown in panels b and c, by rehybridizing these blots (after removing the first probe with alkali [65]) with a clone of rat satellite I DNA to detect the "ladder" of 370-bp monomer and its higher multiples that are generated by HaeIII digestion of satellite I (13,41). At least 70% of the stained 0.35-kb band in panel d is due to the HaeIII fragments derived from the ca.…”

Section: Methodsmentioning

confidence: 99%

“…Perhaps as striking is the fact that satellite DNA sequences account for only 1 to 2% of the total rat genome but 10 to 20% of the total DNA in mice or primates (38,41,52). Understanding the biological significance of these rather large-scale differences among the DNA composition of these animals should help determine the role or effect of repeated DNA.…”

Section: Methodsmentioning

confidence: 99%

Structure of the highly repeated, long interspersed DNA family (LINE or L1Rn) of the rat.

D’Ambrosio¹,

Waitzkin²,

Witney³

et al. 1986

Mol. Cell. Biol.

108

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We present the DNA sequence of a 6.7-kilobase member of the rat long interspersed repeated DNA family (LINE or LlRn). This member (LINE 3) is flanked by a perfect 14-base-pair (bp) direct repeat and is a full-length, or close-to-full-length, member of this family. LINE 3 contains an approximately 100-bp A-rich right end, a number of long (>400-bp) open reading frames, and a ca. 200-bp G+C-rich (ca. 60%) cluster near each terminus. Comparison of the LINE 3 sequence with the sequence of about one-half of another member, which we also present, as well as restriction enzyme analysis of the genomic copies of this family, indicates that in length and overall structure LINE 3 is quite typical of the 40,000 or so other genomic members of this family which would account for as much as 10% of the rat genome. Therefore, the rat LINE family is relatively homogeneous, which contrasts with the heterogeneous LINE families in primates and mice. Transcripts corresponding to the entire LINE sequence are abundant in the nuclear RNA of rat liver. The characteristics of the rat LINE family are discussed with respect to the possible function and evolution of this family of DNA sequences.Repeated DNA sequences are present in the genomes of all metazoans (6). A class of highly repeated DNA that has been studied in primate and mouse genomes (1,10,14,18,19,31,32,37,49) has been called long interspersed repeated DNA (referred to as LINES [52] or, more recently, the Li family [59]). These families contain long members (several kilobases) that are repeated >20,000 times per genome and are responsible for the prominently stained electrophoretic bands that are seen when total genomic DNA is digested with the appropriate restriction endonuclease; the names of the endonucleases were originally used to denote these families.Extensive studies on mouse and primate LINEs (see references 43 and 53 for recent reviews) revealed several major features that these families share. First, many cloned members have, at what has been called the right or 3' end, a putative polyadenylation site, AATAAA, followed by an A-rich sequence (14,54,60). Second, although full-length members are 6 to 7 kilobases (kb) long (1, 14, 19), many cloned members are truncated and most often are missing a variable portion from their left end. Furthermore, there appear to be many more genomic copies of the right end than of the left end of certain cloned members of these families (14,19,59 by RNA polymerase 11 (20,50,56), although there are conflicting results about the extent to which transcripts are poly(A) + (26, 50) and to what extent LINE transcription may be asymmetric (50,56). It has been suggested that the truncated LINE members that end in A-rich 3' ends are incomplete DNA copies (retrotranscripts) of poly(A)+ LINE transcripts (14,43,55,59,60).The rat also contains a highly repeated family of transcribed LINE sequences (65). Members of this family have been (or still are) undergoing transposition in the rat genome, since at least three single-copy loci are polymorphic ow...

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“…Sequence homologies between C6 and 9L cells were recognised in the 167 and 203 bp fragments; these were identical to the consensus sequence for the 370 bp satellite DNA I obtained from Hind III-digested rat DNA. 8 Reaction of the end-labelled DNA fragments with ACNU or MCNU generated prominent bands, corresponding to positions of guanine in the original sequence through chemical cleavage (fig 2). The amount of radioactivity in bands corresponding to breakage at guanine positions was considerably higher than that observed for any other base.…”

Section: Resultsmentioning

confidence: 99%

DNA lability induced by nimustine and ramustine in rat glioma cells.

Mineura

Fushimi

Itoh

et al. 1988

Journal of Neurology, Neurosurgery & Psychiatry

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suMNARY The DNA labile sites induced by two nitrosoureas, nimustine (ACNU) and ramustine (MCNU) synthesised in Japan, have been examined in highly reiterated DNA sequences ofrat glioma cells. Reiterated fragments of 167 and 203 base pairs (bp), obtained after Hind III and Hae III restriction endonuclease digestion of rat glioma cells DNA, were used as target DNA sequences to determine the labile sites. In vitro reaction with ACNU and MCNU resulted in scission products corresponding to the locations ofguanine. Subsequent piperidine hydrolysis produced more frequent breaks of the phosphodiester bonds at guanine positions, thus forming alkali-labile sites.Nitrosourea compounds have been used frequently as effective chemotherapeutic agents against malignant gliomas.' The synthesised antitumour drugs 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (nimustine, ACNU) and methyl-6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-a-D-glucopyranoside (ramustine, MCNU) have also been reported as enhancing therapeutic effectiveness in the chemotherapy of malignant brain tumours.23 However, the mechanisms involved in their tumour cell-killing effects and DNA interactions are still unclear.This paper presents a DNA sequencing analysis of in vitro interactions ofACNU or MCNU with cellular DNA, and characterises the DNA sites labile to these nitrosoureas. Materials and methodsC6 and 9L cell lines were established from gliomas in rats,4 5 and were maintained in Eagle's MEM Earle medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 in air. Cells at the exponential growing stage were trypsinised, and were lysed in 1% sodium dodecyl sulphate in 0-15 M sodium chloride, 0015 M sodium citrate (pH 74) buffer. The lysate was digested by RNase (100 pg/ml) for 60 min and then Pronase (500 pg/ml) for at least 3 hours at 37°C. After protein removal using chloroform-isoamyl alcohol (19:1 vol/vol) and distilled phenol, the extracted DNA was dialysed extensively against Tris-EDTA buffer (10 mM Tris-HCI, 1 mM EDTA pH 8 0).The DNA was digested with Hind III restriction endonuclease (Boehringer, I unit/ug DNA) for 3 hours in medium salt buffer (20 mM Tris-HCl pH 7-6, 50 mM NaCl, 6 mM MgCI2, 0-1 mg/ml gelatin, 6 mM 2-mercaptoethanol). The reacted DNA was located on a 4% polyacrylamide gel and stained with 1Oyg/ml ethidium bromide for 15 minutes. A 370 base pair (bp) fragment was recovered from the gel by electrophoresis.' The 370 bp fragment was labelled at the 3'-end with 3000 Ci/mmol) for 30 min at 150C by the Klenow fragment of Ecoli DNA polymerase I (Boehringer). The reaction mixture contained 20 mM Tris-HCI (pH 7 6), 6 mM MgCl,, 6 mM NaCl, 6 mM 2-mercaptoethanol, 0-1 mg/ml gelatin, 100 uCi of[a-`2P]dATP, 100 pM dGTP, 100 pM 1TP, 100 pM dCTT, 1-5 pg DNA, and I unit of enzyme in 50 p1. After ethanol precipitation, labelled DNA was once again digested with Hae III in medium salt buffer at 37°C for 2 hours. 167 and 203 bp fragments were isolated on a 4% polyacrylamide gel. ...

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Nucleotide sequence of a highly repetitive component of rat DNA

Cited by 93 publications

References 37 publications

Age‐related analysis of EcoRI generated satellite DNA‐containing chromatin of rat liver

Age‐related analysis of EcoRI generated satellite DNA‐containing chromatin of rat liver

Structure of the highly repeated, long interspersed DNA family (LINE or L1Rn) of the rat.

DNA lability induced by nimustine and ramustine in rat glioma cells.

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