Nonrandom distribution of repeated DNA sequences with respect to supercoiled loops and the nuclear matrix.

The DNA in a eukaryotic nucleus is arranged into a series of supercoiled loops that are anchored at their bases to the nuclear matrix. We have analyzed the DNA sequences that are closest to the matrix attachment points for their relative content of specific repeated sequences. Sequences were enriched (mouse satellite, human Alu family) or depleted (mouse EcoRI repeat, monkey alpha component), depending on the specific sequence and species examined. These results can be understood in terms of a nonrandom arrangement of DNA sequences with respect to nuclear DNA loops.     

Sequence-specific interactions of nuclear factors with conserved sequences of human class II major histocompatibility complex genes

All class II major histocompatibility complex genes contain two highly conserved sequences, termed X and Y, within the promoter regions(s), which may have a role in regulation of expression. To study trans-acting factors that interact with these sequences, sequence-specific DNA binding activity has been examined by the gel electrophoresis retardation assay using the HLA-DQ2 beta gene 5' flanking DNA and nuclear extracts derived from various cell types. Several specific protein-binding activities were found using a 45-base-pair (bp) HinfI/Sau96I (-142 to -98 bp) and a 38-bp Sau96I/Sau96I (-97 to -60 bp) fragment, which include conserved sequence X (-113 to -100 bp) and conserved sequence Y (-80 to -71 bp), respectively. Competition experiments, methylation interference analysis, and DNase I foot-printing demonstrated that distinct proteins in a nuclear extract of Raji cells (a human B lymphoma line) bind to sequence X, to sequence Y, and to DNA 5' of the X sequence (termed sequence W). The factor binding site in the W sequence is also found to be conserved among beta-chain genes and is suggested to be a gamma-interferon control region.     

Analysis of the nucleotide sequence of an invertible controlling element.

The nucleotide sequence of the inversion region responsible for flagellar phase variation in Salmonella was determined. The inversion region is 995 base pairs (bp) in length and is bounded by a 14-bp inverted repeat sequence. A homologous recombination event between the 14-bp inverted repeat sequences would result in the inversion of the DNA segment between them. Sequence homologies with other systems suggest that the 14-bp inverted repeat sequences may have some general significance as sites for specific recombinational events. The gene which specifies H2 flagellin synthesis begins 16 bp outsie the inversion region. Within the inversion region, an open translational frame exists which could encode a low molecualr weight polypeptide (190 amino acids).     

Compact oligomers and nucleosome phasing.

Micrococcal nuclease (EC 3.1.4.7) digestion of histone H1- and H5-depleted chicken erythrocyte chromatin yields, in addition to 140-base-pair (bp) core particles, a series of nucleosome oligomers containing about 260 bp (compact dimer), 380 bp (compact trimer), etc. of DNA. These are postulated to represent members of a class of oligomers in which the DNA is tightly wound on stacked protein cores. The physical properties (melting, circular dichroism) as well as DNase I (EC 3.1.4.5) digestion patterns support this view. DNase I digestion of tight oligomers in which the 5' ends of the DNA have been labeled yields results consistent with this model and inconsistent with some other possible models. Several classes of such particles are postulated to exist, differing in DNA length by 10-bp increments. This may be an explanation of the 10-bp nucleosome "phasing" that has been observed in some nuclei.     

Structure of the chromosomal gene for murine interleukin 3.

We have isolated the mouse interleukin 3 (IL-3) gene from a mouse sperm DNA library based on homology with the mouse mast-cell growth-factor (MCGF) cDNA sequence. The nucleotide sequence determined for the gene and its flanking regions reveals that the IL-3 gene is composed of four introns and five exons. The nucleotide sequence of the exons agrees with that determined for the MCGF cDNA. A "TATA"-like sequence preceded by a G + C-rich region is found in the 5' flanking region. At the 3' region of the second intron are nine repeats of a closely related 14-base-pair (bp) sequence. These repeated sequences share extensive homology with a 20-bp repeated sequence found in the human genome, which was shown to have enhancer activity. Eight out of nine repeats form a 73-bp duplicated sequence and each 73-bp repeat contains sequences homologous to the core sequence suggested for enhancer elements. These sequences may play a role in the expression of the IL-3 gene in concanavalin A- or antigen-stimulated T lymphocytes. Stable transformants of L cells generated by co-transformation of the IL-3 gene with pSV2neo constitutively express MCGF activity indicating that the isolated gene is functional in vivo.     

Structural features of a phased nucleosome core particle.

Chicken erythrocyte inner histones associate with a cloned 260-base-pair (bp) segment of Lytechinus variegatus DNA in a unique location. The fragment contains a 120-bp segment encoding 5S rRNA, a 90-bp flanking sequence to the 5' side of the transcribed segment, and a 50-bp downstream flanking sequence. Association of DNA, uniquely labeled at one end or the other and at either the 3' or the 5' terminus of a given strand, with histones at 0.1 M ionic strength leads to formation of a compact complex which sediments at about 13 S. Analysis of cutting of the complex by DNase I shows that protection from the nuclease is confined to a region beginning 20 bp from the left end of the segment and extending to about 165 bp from the left end. Within the protected region, the two DNA strands differ in their susceptibilities to the nuclease, the precise location of nuclease cutting sites and the spacing between these sites, and the relative susceptibilities of specific cutting locations. It seems that information present in DNA and the histone octomer is sufficient to create a precisely phased nucleosome in which interactions of the two DNA strands with histones are not the same. The structure of this unique nucleosome is not predicted by the intellectual model based on studies of mixed populations of nucleosome core particles.     

The nuclear matrix prepared by amine modification

The nucleus is spatially ordered by attachments to a nonchromatin nuclear structure, the nuclear matrix. The nuclear matrix and chromatin are intimately connected and integrated structures, and so a major technical challenge in nuclear matrix research has been to remove chromatin while retaining a native nuclear matrix. Most methods for removing chromatin require first a nuclease digestion and then a salt extraction to remove cut chromatin. We have hypothesized that cut chromatin is held in place by charge interactions involving nucleosomal amino groups. We have tested this hypothesis by chemically modifying amino groups after nuclease digestion. By using this protocol, chromatin could be effectively removed at physiological ionic strength. We compared the ultrastructure and composition of this nuclear matrix preparation with the traditional high-salt nuclear matrix and with the third nuclear matrix preparation that we have developed from which chromatin is removed after extensive crosslinking. All three matrix preparations reveal internal nuclear matrix structures that are built on a network of branched filaments of about 10 nm diameter. That such different chromatin-removal protocols reveal similar principles of nuclear matrix construction increases our confidence that we are observing important architectural elements of the native structure in the living cell.     

Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP.

The interaction between the P1 recombinase protein Cre and the DNA site at which it acts, loxP, has been studied by using nuclease protection techniques. The region of DNA protected by Cre against nuclease attack by DNase I or neocarzinostatin is a 34-base-pair (bp) region containing two 13-bp inverted repeats separated by an 8-bp spacer region. These protected sequences have previously been shown to be required for efficient Cre-mediated recombination at loxP. The results of the above protection experiments suggest (i) that no more than 34 bp may be required for loxP recombination and (ii) that the asymmetry of loxP recombination is due to the 8-bp spacer sequence. With neocarzinostatin, a specific nucleotide within the 8-bp spacer region is not protected. This nucleotide is located in a 2-bp sequence shown to be involved in a loxP crossover event, suggesting that this region remains exposed after Cre binding. Protection experiments have also been done with loxP sites that have either the left or right inverted repeat deleted. The nuclease protection pattern of these sites reveals that each loxP site consists of two binding domains for Cre, each being composed of one 13-bp inverted repeat and the contiguous 4 bp of the 8-bp spacer region.     

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-β and ori-γ) being somewhat preferred. We show here that the chromatin in the ori-β and ori-γ regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G₁/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.     

The nuclear matrix revealed by eluting chromatin from a cross-linked nucleus

The nucleus is an intricately structured integration of many functional domains whose complex spatial organization is maintained by a nonchromatin scaffolding, the nuclear matrix. We report here a method for preparing the nuclear matrix with improved preservation of ultrastructure. After the removal of soluble proteins, the structures of the nucleus were extensively cross-linked with formaldehyde. Surprisingly, the chromatin could be efficiently removed by DNase I digestion leaving a well preserved nuclear matrix. The nuclear matrix uncovered by this procedure consisted of highly structured fibers, connected to the nuclear lamina and built on an underlying network of branched 10-nm core filaments. The relative ease with which chromatin and the nuclear matrix could be separated despite extensive prior cross-linking suggests that there are few attachment points between the two structures other than the connections at the bases of chromatin loops. This is an important clue for understanding chromatin organization in the nucleus.     

Artificial nucleosome positioning sequences.

We have used the emerging rules for the sequence dependence of DNA bendability to design and test a series of DNA molecules that incorporate strongly into nucleosomes. Competitive reconstitution experiments showed the superiority in histone octamer binding of DNA molecules in which segments consisting exclusively of A and T or G and C, separated by 2 base pairs (bp), are repeated with a 10-bp period. These repeated (A/T)3NN(G/C)3NN motifs are superior in nucleosome formation to natural positioning sequences and to other repeated motifs such as AANNNTTNNN and GGNNNCCNNN. Studies of different lengths of repetitive anisotropically flexible DNA showed that a segment of approximately 40 bp embedded in a 160-bp fragment is sufficient to generate nucleosome binding equivalent to that of natural nucleosome positioning sequences from 5S RNA genes. Bending requirements along the surface of the nucleosome seem to be quite constant, with no large jumps in binding free energy attributable to protein-induced kinks. The most favorable sequences incorporate into nucleosomes more strongly by 100-fold than bulk nucleosomal DNA, but differential bending free energies are small when normalized to the number of bends: a free energy difference of only about 100 cal/mol per bend (1 cal = 4.184 J) distinguishes the best bending sequences and bulk DNA. We infer that the distortion energy of DNA bending in the nucleosome is only weakly dependent on DNA sequence.     

Nuclease resistance and the enrichment of native nuclear acceptor sites for the avian oviduct progesterone receptor.

High-affinity nucleoprotein acceptor sites for the avian oviduct progesterone receptor (PR) have been enriched by a combination of nuclease digestion and centrifugation. These enriched binding elements exhibited markedly enhanced PR binding on a per mass DNA basis compared to chromatin (20- to 25-fold) or dehistonized chromatin (4- to 5-fold). Electrophoretic analysis of the nuclease-resistant DNA showed that there is a set of DNA fragments of 100-150 base pairs that are protected from digestion. Excessive digestion resulted in smaller DNA fragments and a loss of PR binding activity. The PR binding was saturable using a crude receptor preparation and displayed a competition with the same receptor preparation that was labeled with nonradioactive progesterone. The enhanced binding was also demonstrable using highly purified receptor preparations that exhibit two classes of binding sites both of which are of high affinity and saturable as assessed by Scatchard analyses. These two high-affinity classes of binding sites are shown to be competed by unlabeled purified PR. The nuclease resistance of these nucleoprotein acceptor sites from chromatin is a property similar to the nuclear matrix binding sites suggesting a relationship between these two classes of nuclear acceptor sites.     

恶性疟原虫FCC1/HN(CQS)株Pfmdr1、Cg1基因T-A克隆及序列分析

目的　将恶性疟原虫FCC1/HN株 (CQS)Pfmdr1和Cg1全基因编码区克隆入测序载体 ,测定其序列 ,为以后研究其与疟原虫耐药性的关系奠定基础。方法　利用PCR扩增技术 ,分 3个片段从恶性疟原虫FCC1/HN株基因组DNA中 ,特异扩增Pfmdr1全基因编码序列 ;分 2个片段特异扩增Cg1全基因编码序列。扩增产物经纯化回收后 ,T -A克隆入测序载体 pMD18-T ,转化大肠杆菌 (E coli)JM 10 9,筛选阳性克隆 ,并进行双酶切及PCR扩增鉴定 ,获得阳性重组质粒 ,用Sanger双脱氧链终止法进行DNA测定。结果　CQSFCC1/HN株Pfmdr1基因序列与CQSFc2 7株同源性高 ,全长 4 2 4 8bp ,编码 14 15个氨基酸 ;测得Cg1基因全长为 2 82 0bp ,编码 939个氨基酸 ,存在串联α重复序列。 结论　恶性疟原虫FCC1/HN(CQS)株Pfmdr1和Cg1全基因编码序列的测定 ,为以后研究耐药性虫株的上述基因以及它们与疟原虫耐药性的关系奠定基础。     

Cloning and expression of full-length cDNA encoding human vitamin D receptor.

Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3' noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of approximately equal to 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.     

利什曼原虫中国分离株SSU rDNA多变区序列分析

目的分析我国荒漠、山丘疫区利什曼原虫分离株的SSUrDNA多变区序列差异。方法nDNA进行PCR扩增，将扩增出的SSUrDNA基因的特异片段克隆于pGEMR-TEasyVector上，采用通用引物M13进行PCR扩增，全自动测序仪测序。结果序列分析显示本文报道的荒漠、山丘疫区的2株利什曼原虫（L.d.XJ771、L.d.SC6）的SSUrDNA序列大小均为392bp；序列差异发生在两个独特序列区（UQ-Ⅰ和UQ-Ⅱ），无移码突变；与GeneBank中的利什曼原虫比较分析，同源性在98%以上。结论我国荒漠、山丘疫区利什曼原虫分离株之间的SSUrDNA多变区的碱基序列有差异；荒漠疫区分离株L.d.XJ771与国际标准株L.d.DD8的SSUrDNA多变区的碱基序列完全相同。     

Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo.

We have examined the distribution of individual adducts in repetitive DNA sequences of rat liver in vivo after a single dose of the carcinogen N-hydroxy-2-acetyl-aminofluorene. Repetitive fragments [82, 125, 179, 225, and 370 base pairs (bp)] were isolated by digestion of hepatic DNA with HindIII restriction endonuclease (EC 3.1.23.21) and gel electrophoresis. As assayed by 32P postlabeling, no qualitative differences were observed between the DNA-bound metabolites in repetitive sequences and total DNA, but preferential binding to these sequences occurred. After 1 day of treatment, the amounts of N-hydroxy-2-acetylaminofluorene-induced adducts were found to be 13.8, 2.0, and 3.0 times higher in 179-, 225-, and 370-bp repeats, respectively, than in total DNA, while 82- and 125-bp repeats showed no differences. The relative distribution of individual adducts varied among the various sequences. After 9 days, all five sequences showed 1.3-1.7 times higher binding as compared to total DNA. In contrast, a random binding was observed when DNA reacted in vitro with an active metabolite, N-acetoxy-2-acetylaminofluorene. Taken together, these results suggest that the enrichment and differential excision of adducts in the repetitive DNA sequences may be a function of the nuclear organization of DNA. This application of the 32P assay constitutes a means to study the DNA damage and excision repair in vivo in chromatin structural components, including transcribed and nontranscribed multiple-copy genes, in a much more sensitive and precise way than has been hitherto possible.     

The homeo domain of a murine protein binds 5'' to its own homeo box.

Nuclear protein extracts from day 12.5 mouse embryos were used to study protein binding to DNA sequences 5' of the Hox 1.5 homeo box. Embryos of this developmental stage are known to express this gene. DNA binding protein blotting and retardation gel techniques show that murine embryonic nuclear proteins specifically bind a 753-base pair (bp) DNA fragment from the region upstream of the Hox 1.5 homeo box. A fusion protein containing the Hox 1.5 homeo domain constructed in lambda gt11 also binds the same 753-bp DNA fragment. Specific binding of the fusion protein to the upstream DNA fragment shows that the homeo box contains the sequences required for specific protein-DNA interactions, and the 753-bp fragment contains a homeo domain binding site. These results support the hypothesis that murine homeo boxes are DNA binding domains of proteins involved in the regulation of embryonic development.     

A random-walk/giant-loop model for interphase chromosomes.

Fluorescence in situ hybridization data on distances between defined genomic sequences are used to construct a quantitative model for the overall geometric structure of a human chromosome. We suggest that the large-scale geometry during the G0/G1 part of the cell cycle may consist of flexible chromatin loops, averaging approximately 3 million bp, with a random-walk backbone. A fully explicit, three-parametric polymer model of this random-walk/giant-loop structure can account well for the data. More general models consistent with the data are briefly discussed.