Uncoupling of leading- and lagging-strand DNA replication during lesion bypass in vivo

Numerous agents attack DNA, forming lesions that impair normal replication. Specialized DNA polymerases transiently replace the replicative polymerase and copy past lesions, thus generating mutations, the major initiating cause of cancer. We monitored, in Escherichia coli, the kinetics of replication of both strands of DNA molecules containing a single replication block in either the leading or lagging strand. Despite a block in the leading strand, lagging-strand synthesis proceeded further, implying transient uncoupling of concurrent strand synthesis. Replication through the lesion requires specialized DNA polymerases and is achieved with similar kinetics and efficiencies in both strands.     

A previously undetected MHC gene with an unusual periodic structure

The major histocompatibility complex is a chromosomal segment embodying several gene clusters among which those with immune functions are the best characterized. This region is suspected to host other as yet undetected genes whose characterization may shed light on the population genetics and evolution of the whole gene complex and thus on its unexplained character of marker locus for a number of diseases of nonimmune or unknown pathogenesis. A novel gene was identified that is transcribed in all tissues tested and is located in mouse and man between the CA and Bf genes of the H-2 and HLA complexes, respectively. From the nucleotide sequence, derived from liver complementary DNA clones, it is predicted that this novel single-copy gene encodes a 42-kilodalton polypeptide that bears no recognizable relation to the protein families known so far, but it displays striking hallmarks of natural selection.     

Global identification of human transcribed sequences with genome tiling arrays

Elucidating the transcribed regions of the genome constitutes a fundamental aspect of human biology, yet this remains an outstanding problem. To comprehensively identify coding sequences, we constructed a series of high-density oligonucleotide tiling arrays representing sense and antisense strands of the entire nonrepetitive sequence of the human genome. Transcribed sequences were located across the genome via hybridization to complementary DNA samples, reverse-transcribed from polyadenylated RNA obtained from human liver tissue. In addition to identifying many known and predicted genes, we found 10,595 transcribed sequences not detected by other methods. A large fraction of these are located in intergenic regions distal from previously annotated genes and exhibit significant homology to other mammalian proteins.     

Human immunodeficiency virus may encode a novel protein on the genomic DNA plus strand

The genome of the human immunodeficiency virus (HIV) is known to contain eight open reading frames (ORFs) on the minus strand of the double-stranded DNA replicative intermediate. Data presented here indicate that the DNA plus strand of HIV contains a previously unidentified ORF in a region complementary to the envelope gene sequence. This ORF could encode a protein of approximately 190 amino acid residues with a relative molecular mass of 20 kilodaltons if translation began from the first initiation codon. The predicted protein is highly hydrophobic and thus could be membrane associated. It is possible, therefore, that the HIV genome encodes a protein on antisense messenger RNA.     

Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation

DNMT1, the major maintenance DNA methyltransferase in animals, helps to regulate gene expression, genome imprinting, and X-chromosome inactivation. We report on the crystal structure of a productive covalent mouse DNMT1(731-1602)-DNA complex containing a central hemimethylated CpG site. The methyl group of methylcytosine is positioned within a shallow hydrophobic concave surface, whereas the cytosine on the target strand is looped out and covalently anchored within the catalytic pocket. The DNA is distorted at the hemimethylated CpG step, with side chains from catalytic and recognition loops inserting through both grooves to fill an intercalation-type cavity associated with a dual base flip-out on partner strands. Structural and biochemical data establish how a combination of active and autoinhibitory mechanisms ensures the high fidelity of DNMT1-mediated maintenance DNA methylation.     

Replicating form of a single-stranded DNA virus: isolation and properties

The replicating form of single-stranded DNA virus has been isolated in pure form by chromatography on columns of methylated albumin. Its buoyant density in CsCl and "melting temperature" are characteristic of a double stranded DNA structure containing 43 percent guanine-cytosine. The nearest neighbors to uridylate were compared in the RNA synthesized when replicating-form DNA and mature single-stranded DNA were employed as templates in an in vitro system. The mature DNA component of the replicating duplex does not serve as the sole source of complementary RNA. The results agree best with the assumption that both strands of the replicating form function as templates. It is important to note that this is contrary to the situation found in the intact cell where only one of the two strands appears to be transcribed into message.     

GnRH受体基因的结构和行为特征

在分析了人ＧｎＲＨ受体基因结构特征的基础上，对小鼠、大鼠和绵羊的ＧｎＲＨ受体基因进行了描述。不同动物ＧｎＲＨ受体基因的结构特征基本相同。ＰＣＲ分析表明编码ＧｎＲＨ受体的基因位于染色体的特定位置。在ＧｎＲＨ受体基因上存在多个启动子和转录起始位点与多重调节序列，表明ＧｎＲＨ受体基因的活动是复杂的和高度调节的。     

DNA-functionalized nanotube membranes with single-base mismatch selectivity

We describe synthetic membranes in which the molecular recognition chemistry used to accomplish selective permeation is DNA hybridization. These membranes contain template-synthesized gold nanotubes with inside diameters of 12 nanometers, and a "transporter" DNA-hairpin molecule is attached to the inside walls of these nanotubes. These DNA-functionalized nanotube membranes selectively recognize and transport the DNA strand that is complementary to the transporter strand, relative to DNA strands that are not complementary to the transporter. Under optimal conditions, single-base mismatch transport selectivity can be obtained.     

Plus and minus single-stranded DNA separately encapsidated in adeno-associated satellite virions

Based on physical and chemical determinations, the mnolecular weight of the type 4 adeno-satellite virus is 5.4 X 10(6) daltons, and the virion contains 1.4 X 10(6) daltons of DNA. Denaturation and renaturation studies indicate that the viral genome is a single-stranded DNA molecule and that each virion contains either a minus or a plus strand. Upon extraction, the minus and plus strands unite to form double-stranded DNA molecules with no obvious excess of unpaired strands.     

Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1 beta

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), a widespread environmental contaminant, may elicit its effects by altering gene expression in susceptible cells. Five TCDD-responsive complementary DNA clones were isolated from a human keratinocyte cell line. One of these clones encodes plasminogen activator inhibitor-2, a factor that influences growth and differentiation by regulating proteolysis of the extracellular matrix. Another encodes the cytokine interleukin-1 beta. Thus, TCDD alters the expression of growth regulatory genes and has effects similar to those of other tumor-promoting agents that affect both inflammation and differentiation.     

RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination

The vast repertoire of immunoglobulins and T cell receptors is generated, in part, by V(D)J recombination, a series of genomic rearrangements that occur specifically in developing lymphocytes. The recombination activating gene, RAG-1, which is a gene expressed exclusively in maturing lymphoid cells, was previously isolated. RAG-1 inefficiently induced V(D)J recombinase activity when transfected into fibroblasts, but cotransfection with an adjacent gene, RAG-2, has resulted in at least a 1000-fold increase in the frequency of recombination. The 2.1-kilobase RAG-2 complementary DNA encodes a putative protein of 527 amino acids whose sequence is unrelated to that of RAG-1. Like RAG-1, RAG-2 is conserved between species that carry out V(D)J recombination, and its expression pattern correlates precisely with that of V(D)J recombinase activity. In addition to being located just 8 kilobases apart, these convergently transcribed genes are unusual in that most, if not all, of their coding and 3' untranslated sequences are contained in single exons. RAG-1 and RAG-2 might activate the expression of the V(D)J recombinase but, more likely, they directly participate in the recombination reaction.     

Transcription of nonrepeated DNA in mouse brain

Under normal conditions of DNA renaturation, about 60 percent of mouse DNA fragments renature at a rate consistent with their being present only once per sperm. These nonrepeated sequences (also called single-copy or unique) may be used in RNA-DNA hybridization experiments to provide quantitative estimates of RNA diversity. About 10 percent of the mouse single-copy sequences are transcribed in mouse brain tissue. Estimates of about 3 percent were obtained for mouse liver and kidney RNA's. If only one of the complementary DNA strands is transcribed, this hybridization value implies that the equivalent of at least 300,000 different sequences of 1000 nucleotides are expressed in mouse brain tissue. It is suggested that the large amount of DNA in mammals is functionally important, and that a substantial proportion of the genome is expressed in the brain.     

Visualization of nucleolar genes

The presence of extrachromosomal nucleoli in amphibian oocytes has permitted isolation and electron microscopic observation of the genes coding for ribosomal RNA precursor molecules. Visualization of these genes is possible because many precursor molecules are simultaneously synthesized on each gene. Individual genes are separated by stretches of DNA that apparently are not transcribed at the time of synthesis of precursor rRNA in the extrachromosomal nucleoli.     

Biological damage from intranuclear tritium: DNA strand breaks and their repair

Isotopic decay in tritiated thymidine in the DNA of frozen (-196 degrees C) Chinese hamster cells causes breaks in DNA strands to accumulate at a rate of 2.1 breaks per decay. After DNA is thawed the tritium-induced breaks repair rapidly with a half-time of 15 minutes at 37 degrees C. In comparison to breakage by x-rays, the efficiency of DNA strand breakage by tritium is equivalent to 0.48 rad per decay. This dose per decay is close to that predicted by simple dosimetric considerations (0.38 rad per decay) for irradiation by the beta particles from tritium.     

Three-dimensional structure of the Tn5 synaptic complex transposition intermediate

Genomic evolution has been profoundly influenced by DNA transposition, a process whereby defined DNA segments move freely about the genome. Transposition is mediated by transposases, and similar events are catalyzed by retroviral integrases such as human immunodeficiency virus-1 (HIV-1) integrase. Understanding how these proteins interact with DNA is central to understanding the molecular basis of transposition. We report the three-dimensional structure of prokaryotic Tn5 transposase complexed with Tn5 transposon end DNA determined to 2.3 angstrom resolution. The molecular assembly is dimeric, where each double-stranded DNA molecule is bound by both protein subunits, orienting the transposon ends into the active sites. This structure provides a molecular framework for understanding many aspects of transposition, including the binding of transposon end DNA by one subunit and cleavage by a second, cleavage of two strands of DNA by a single active site via a hairpin intermediate, and strand transfer into target DNA.     

TRANSCRIPTION IN VIVO OF DNA FROM BACTERIOPHAGE SP8

The DNA of bacteriophage SP8, when denatured, yields two components differing in buoyant density in cesium chloride gradients and separable by chromatography on a column of methylated bovine serum albumin and kieselguhr. The denser of the two strands (H) contains more pyrimidines and fewer purines than the lighter (L) strand. Only the H strand forms hybrids with the RNA synthesized by the infected host. The L strand is capable of annealing with complementary RNA synthesized in vitro with it as primer in reactions catalyzed by RNA polymerase. During the vegetative development of phage, host-specific messenger RNA is also synthesized.