Nucleosome packing in chromatin as revealed by nuclease digestion.

Chromatin DNA of rat thymus nuclei was cleaved by Serratia marcescens endonculease. The fragments have been examined by polyacrylamide gel electrophoresis under denaturing conditions. The results obtained are interpreted to mean that the internucleosomal DNA is cleaved by the endonuclease into fragments which are multiples of 10 nucleotides. The 10 nucleotide periodicity in fragmentation of internucleosomal DNA is independent of the presence of histone H1 and is likely to be determined by the interaction of this DNA stretch with the histone core of nucleosomes. Such interaction implies a close association between the nucleosomes in the chromatin thread. Quasi-limit chromatin digest (50--55% of DNA hydrolysis) contains undegraded DNA fragments with length of up to 1000 nucleotides or more. A part of this resistant DNA consists of single-stranded fragments or contains single stranded regions. These data may be accounted for by a very compact nucleosome packing in the resistant chromatin in which one of the DNA stands is more accessible to the endonuclease action.     

On the degree of unwinding of the DNA helix by ethidium. II. Studies by electron microscopy.

When a negatively twisted covalently closed DNA is annealed with single-stranded fragments of the same DNA, under proper conditions a loop (or loops) may form by the disruption of a segment (or segments) of base pairs between the complementary strands of the covalently closed DNA, and the formation of base pairs between the strands of the covalently closed DNA and the single-stranded fragments. Since such a process involves essentially no net gain or loss of the number of base pairs, it is driven by the free energy favoring the reduction of the number of superhelical turns. If the fragments are sufficiently long or are present at a sufficiently hig concentration during annealing, the most stable product between a covalently closed DNA and the DNA fragments (under conditions favoring the formation of double-stranded DNA) is a looped molecule devoid of superhelical turns. The size of the looped region or regions, which can be measured by electron microscopy, provides a way to determine the degree of superhelicity of the covalently closed DNA in the absence of the fragments. When this is compared with the degree of superhelicity of the covalently closed DNA determined by titration with the intercalative dye ethidium, the unwinding angle of the DNA double helix due to the intercalation of an ethidium can be calculated. Such measurements were done on two samples of phage PM2 DNA with different extents of supercoiling. The results are in agreement with the value 26 degree obtained recently by alkaline titration of covalently closed PM2 DNA samples in CsC1 density gradients (Wange, J.C., (1974) J. Mol. Biol. 89, 783-801).     

Functional analogues of bleomycin: DNA cleavage by bleomycin and hemin-intercalators

New hemin-intercalators (Hem-G's) that cleave DNA were synthesized, on the basis of 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole (Glu-P-1) as an intercalator moiety. Hem-G's, which possess an intramolecular ligand of the ferrous ion (a histidine or imidazole moiety), cleave DNA very efficiently and act at guanine-pyrimidine sequences preferentially. Bleomycin (BLM) also cleaved DNA with the same base-sequence selectivity shown by Hem-G's. The 5'-terminus of the DNA fragments cleaved by Hem-G's or by BLM is a phosphoryl group, while the 3'-terminus of the cleaved DNA fragments does not possess a 3'-phosphoryl group. There are more than three kinds of 5'-end 32P-labeled DNA fragments, which can be substrates of terminal deoxynucleotidyl transferase (TdT). One of the 3'-termini of the cleaved DNA fragments is a 3'-hydroxy group. The mobility of the 3'-end 32P-labeled DNA fragment cleaved by Hem-G's or by BLM corresponds to the removal of pyrimidine bases having guanine at the 5'-side. The mobility of one kind of the cleaved 5'-end 32P-labeled DNA fragments corresponds to the removal of guanine having pyrimidine at the 3'-side, followed by 3'-dephosphorylation. We propose that there exist plural mechanisms for DNA cleavage by Hem-G's or by BLM. The deduced structures of the cleaved DNA fragments suggest that one of the mechanisms involves deletion of two nucleotide units from DNA.     

Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.     

Effects of mutation position on frequency of marker rescue by homologous recombination.

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

Characterization of nascent DNA fragments produced by excision of uracil residues in DNA.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d(pRib[32P]poly(T)) and d(Rib[32P]poly(T)). Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escherichia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.     

Thermal denaturation of DNA in the chromatin fragments released by mild micrococcal nuclease digestion of HTC cell nuclei

In chromatin a minor fraction melts at a temperature lower than deproteinized DNA, which may be assigned to DNA destabilizing proteins. We attempted to localize the destabilized DNA in the various chromatin fragments separated by electrophoresis after a mild micrococcal nuclease digestion. The small released fragments are enriched in coding sequences. About 20% of the DNA extracted from the released nucleosomes are single-stranded, 60% of the DNA in these fragments are digested by nuclease S₁ after incubation at low temperature, which suggests that the DNA destabilizing proteins are present in the released nucleosomes. Hybridization studies have shown that 25% of the DNA in nucleosomes are specific of this class of fragments. DNA destabilizing proteins could be associated with the specific sequences.     

General method for cloning amplified DNA by differential screening with genomic probes.

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.     

平末端DNA随机连接构建重组质粒

目的:拼接DNA片段并克隆。方法:用T4DNA连接酶将DNA片段以平末端随机连接,随后用限制性内切酶切割,琼脂糖电泳分离酶切产物,挑选特定片段纯化回收,与线性化的载体质粒连接,转化大肠杆菌感受态细胞。结果:通过以上步骤,成功拼接了不同DNA片段,构建了含有目的拼接片段的重组质粒。结论:该方法简便、易行、可靠,可作为拼接、克隆DNA的备选方案,在分子生物学研究和基因工程中应用。     

Cathepsin D is specifically inhibited by deoxyribonucleic acids

A cathepsin D (CD) inhibitor was searched using mouse embryonic fibroblasts deficient for CD. Synthetic DNA fragments specifically inhibited CD activity in a dose-dependent manner, but not the activities of other serine or cysteine proteinases. Cathepsin E activity was also inhibited by DNA fragments when hemoglobin was used as a substrate. CD inhibition by DNA fragments appeared to be electrostatic in nature and dependent on Tm values. Moreover, CD activity was partly inhibited by exogenously ingested DNA fragments, suggesting that DNA fragments with high Tm values are potent inhibitors of CD in vitro and partly in vivo.     

Characterization of mouse ribosomal gene fragments purified by molecular cloning.

Four mouse ribosomal gene fragments cloned in lambda gtWES were studied by restriction enzyme mapping and Southern transfer experiments. These fragments were found to contain 18S DNA and transcribed as well as non-transcribed spacer DNA. Variation in the structure of these mouse DNA inserts was limited to one region of spacer DNA. This variation may reflect real structural differences found in mouse ribosomal genes or possibly deletion events which occurred during cloning. The transcribed regions of the inserts appear identical to one antoher and restriction enzyme fragments from this region correspond to fragments observed in digests of total mouse DNA. These clones will be useful in studying the structure of transcribed spacer DNA including the ribosomal gene promoter.     

Sequence-Dependent DNA Separation by Anion-Exchange High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) system with a new nonporous anion-exchange resin, DNA–NPR, made it possible to rapidly separate DNA fragments up to 20 kbp with high resolution. In order to further characterize this chromatographic DNA separation system, we prepared a mixture of double-stranded DNAs of constant length carrying a fully degenerated 50-bp region and analyzed their chromatographic behavior on the DNA–NPR column. The results indicated that the separation of DNA fragments on the anion-exchange HPLC was governed not only by size, but also by nucleotide sequence: even DNA fragments with the same size and the same base content could be separated on this column. Taking advantage of this characteristic feature of the anion-exchange HPLC, we could readily fractionate human cDNAs with practically acceptable recovery and high resolution. Furthermore, the combination of HPLC and gel electrophoresis realized separation of a mixture of DNA fragments in a two-dimensional pattern.     

Formalin removal from archival tissue by critical point drying

The extraction of high-quality nucleic acid may be problematic in formalin-fixed tissues because of cross-linking between proteins and DNA. Old fixed tissue specimens do produce fragmented DNA (<1.2 kb), which is only used for PCR amplification. Here we show that high molecular weight DNA (>194 kb) can be successfully extracted from fixed tissue samples (16-70 years old) by gradual dehydration and critical point drying. The reliability of extracted DNA was measured by its ability to serve as a template for the amplification of mtDNA fragments (403 and 1198 bp) and an nDNA fragment (1844 bp). In addition, fingerprinting analysis was performed using DNA from fixed human tissue to ensure the ability of extracted DNA to hybridize with the DNA probe. DNA derived by this method can be subject to amplification, complete digestion by restriction endonuclease, and hybridization.     

Transformation by subgenomic fragments of Rous sarcoma virus DNA

Subgenomic fragments of Rous sarcoma virus (RSV) DNA, generated by Eco RI digestion of DNA of RSV-infected chicken cells, induced transformation of NIH/3T3 mouse cells with efficiencies that were 100–1000 fold lower than the efficiency of transformation by intact RSV DNA. Analysis of the DNAs of NIH cells transformed by Eco RI-digested RSV DNA indicated that these cells contained no more than 2 × 10⁶ daltons of RSV DNA, and did not contain sequences from the 5′ terminus of RSV RNA which are included in the leader sequence of subgenomic src mRNA of RSV-infected cells. The product of the RSV src gene (pp60^src), however, was produced in apparently similar quantities by NIH cells transformed by Eco RI fragments of RSV DNA and by intact RSV DNA. Thus expression of the src gene of RSV in NIH cells transformed by subgenomic fragments of RSV DNA did not require the terminal sequences of the RSV genome, which appear to be involved in synthesis and processing of src mRNA in RSV-infected cells. DNAs of NIH cells transformed by Eco RI-digested RSV DNA were found to induce transformation in secondary transfection assays with efficiencies that were similar to the efficiency of transformation by intact RSV DNA. These results suggest that transformation by subgenomic fragments of RSV DNA may be a consequence of integration of src gene-containing DNA fragments in the vicinity of a promoter site in the recipient cell genome, leading to efficient expression of the RSV src gene.     

DNA breaks induction by mimosine

Mouse erythroleukemic F4 N cells were treated with mimosine, etoposide, Fe(II)-EDTA, and Cu(II) in the presence of ascorbate. DNA was isolated and subjected to agarose gel electrophoresis and the size and distribution of the DNA fragments produced by the agents were compared. With increasing concentration of Cu(II) the production of DNA fragments was increased without decrease of the average length of the fragments, and their sizes were similar to those produced by etoposide as expected for cleavage of DNA at the nuclear matrix attachments sites. In contrast, mimosine and Fe(II) produced fragments of random size and with the progression of the reaction the average length of the fragments decreased. These results indicate that mimosine cuts DNA in a random fashion, regardless of its higher order chromatin organization. A conclusion is drawn that the DNA fragments obtained after mimosine treatment are a result of mimosine-assisted, Fe(II) dependent Fenton-like reactions randomly cutting chromosomal DNA.     

以gfp为报告基因的肺炎链球菌启动子诱捕文库的构建与分析

首先构建一个以gfp(green fluorescence protein)为报告基因的自杀质粒pEVP3-SDGFP,将肺炎链球菌基因组DNA的随机酶切片段(200bp～800 bp)克隆到该质粒gfp基因上游的多克隆位点,得到约58000个含有肺炎链球茵基因组DNA随机酶切片段的重组子,提取质粒即为质粒库,该库大约覆盖肺炎链球菌基因组全长的5倍,插入率达到90%以上,且有较强的随机性,质量较高.将该质粒库转化入肺炎链球菌TIGR4菌株,带有随机片段的报告质粒通过同源重组的方式将gfp基因融合于细菌染色体上该随机片段之后,利用质粒的抗生素抗性基因筛选出重组菌株,从而构建出相应的菌株库,共获得包含约500000个肺炎链球菌转化子的菌株库,经体内、外实验表明,其包含插入了S.pn体内、外表达基因片段的细菌,可以报告特定条件下的基因表达,并可通过流式细胞仪识别、分选.该文库的构建为进一步利用差异荧光诱导技术筛选肺炎链球菌体内诱导基因奠定了基础.     

Characterization of DNA fragmentation events caused by genotoxic and non-genotoxic agents

Leukemic cells have been shown to generate several classes of DNA fragments after treatment with cytotoxic cancer chemotherapy agents. However, it is unclear which of these fragmentation events are a direct effect of DNA-damaging chemotherapy agents, and which fragmentation events are caused by downstream processes, such as apoptosis. We have performed a detailed analysis of DNA fragmentation events which occur following cytotoxic chemotherapy in four representative leukemic cell lines (HL-60, Jurkat, K562, and Molt-4). We used a DNA topoisomerase II inhibitor (etoposide), an alkylating agent (melphalan), a nucleoside analog (cytosine arabinoside), and a non-genotoxic agent (N-methylformamide) to induce cell death. We studied high molecular weight and low molecular weight DNA fragmentation events, as well as the specific cleavage of the MLL breakpoint cluster region (bcr). The DNA fragments produced at late time points were largely independent of the agents used, while those generated at earlier time points showed clear differences in terms of fragment size and relative abundance, depending on the agent used. In addition, there were clear differences between cell lines in terms of size, relative abundance, and rate at which DNA fragments were produced by treatment with the same agents. We think that this survey documents the importance of studying several different cell lines, time points, and assays before reaching conclusions about the types of DNA fragments produced during treatment with cytotoxic agents, and provides a useful framework for studying a wide range of DNA fragments produced by cytotoxic agents.     

The Euglena gracilis chloroplast genome: analysis by restriction enzymes.

1. Chloroplast DNA was isolated from autotrophically and mixotrophically grown Euglena gracilis cells. 2. Aliquots of chloroplast DNA were mechanically degraded to an average molecular weight of 4-7 X 10(6) and G+C-rich DNA fragments (density 1.701 g/cm3) were separated from the bulk DNA (density 1.685 g/cm3) using preparative CsCl density gradients. 3. Total chloroplast DNA and its DNA subfractions, which first were characterized with respect to average G+C content and hybridization capacity for chloroplast rRNA, were hydrolysed with restriction endonucleases (endo R-EcoRI, end R-HindII, endoR-HindIII, endo R-HindII+III, endoR-Hpal, endo R-HpaII and endoR-HaeIII). The fragments were separated on gels under a variety of electrophoretic conditions. 4. With each enzyme tested, a rather large number of bands was obtained. In all cases, different banding patterns were obtained for total DNA, and the DNA subfractions. 5. Chloroplast DNA from autotrophically and mixotrophically grown cells gave identical banding patterns. 6. Digestion of total DNA with the endoR-HaeIII yielded 51-52 fragments separated in the gels in a total of 36 bands of which 11-12 bands were composed of 2-3 fragments as estimated by densitometry. The molecular weights of all fragments combined was 87 X 10(6) or 95% of the genome (92 X 10(6)). 7. Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome (Mr92 X 10(6)). These cistrons are located on seven different types of endo R-HaeIII fragments. The hybridising fragments are preferentially found in the G+C-rich subfraction and in bands which are composed of 2-3 fragments.