Molecular characterization of the Spirometra mansonoides genome: renaturation kinetics, methylation, and hybridization to human cDNA probes

High molecular weight DNA from pleroceroid larvae of the tapeworm Spirometra mansonoides was purified from isolated nuclei by conventional techniques. The DNA so isolated has a melting temperature (Tm) of 87 degrees C and a guanine plus cytosine (G/C) content of 44%. 5-Methyl cytosine could not be detected in plerocercoid DNA by HPLC analysis of DNA hydrolysates, by radiolabeling 5'-termini of MspI digests with polynucleotide kinase, or by comparing restriction patterns generated by MspI and HpaII. Renaturation kinetics demonstrated that the genome of S. mansonoides contains repetitive as well as single copy sequences and has a genome size estimated at approx. 1.6 X 10(9) bp. Hybridization was carried out between plerocercoid DNA and cDNAs for human beta-actin, alpha-tubulin and growth hormone (hGH). Rationale for this analysis was based on known homologies among actin and tubulin genes in numerous species and on apparent similarities between hGH and a plerocercoid growth factor that may be reflected in similar DNA sequence. Scanning densitometry of dot blots demonstrated that the hGH probe annealed to the same extent at low stringency (1 M NaCl, 55 degrees C) to DNA from plerocercoids, rat liver and chicken erythrocytes; but this interaction was less than to DNA from human lymphocytes, calf thymus and mouse skin. Similar results were obtained when restriction endonuclease digests of these DNAs were analyzed by Southern transfer. Little or no hybridization of the growth hormone probe to plerocercoid DNA was evident at higher stringency (1 M NaCl, 65 degrees C). In contrast, human tubulin and actin probes showed extensive hybridization to pleroceroid restriction fragments under the high stringency conditions.     

The genes coding for 4 snRNAs of Drosophila melanogaster: localization and determination of gene numbers.

Four small nuclear RNAs (snRNAs) have been isolated from Drosophila melanogaster flies. They have been characterized by base analysis, fingerprinting, and injection into Axolotl oocytes. The size of the molecules and the modified base composition suggest that the following correlations can be made: snRNA1 approximately U2-snRNA; snRNA2 approximately U3-snRNA; snRNA3 approximately U4-snRNA; snRNA4 approximately U6-snRNA. The snRNAs injected into Axolotl oocytes move into the nuclei, where they are protected from degradation. The genes coding for these snRNAs have been localized by "in situ" hybridization of 125-I-snRNAs to salivary gland chromosomes. Most of the snRNAs hybridize to different regions of the genome: snRNA1 to the cytological regions 39B and 40AB; snRNA2 to 22A, 82E, and 95C; snRNA3 to 14B, 23D, 34A, 35EF, 39B, and 63A; snRNA4 to 96A. The estimated gene numbers (Southern-blot analysis) are: snRNA1:3; snRNA2:7; snRNA3:7; snRNA4:1-3. The gene numbers correspond to the number of sites labeled on the polytene salivary gland chromosomes.     

Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA

U1-snRNA is an integral part of the U1 ribonucleoprotein pivotal for pre-mRNA splicing. Toll-like receptor (TLR) signaling has recently been associated with immunoregulatory capacities of U1-snRNA. Using lung A549 epithelial/carcinoma cells, we report for the first time on interferon regulatory factor (IRF)-3 activation initiated by endosomally delivered U1-snRNA. This was associated with expression of the IRF3-inducible genes interferon-β (IFN-β), CXCL10/IP-10 and indoleamine 2,3-dioxygenase. Mutational analysis of the U1-snRNA-activated IFN-β promoter confirmed the crucial role of the PRDIII element, previously proven pivotal for promoter activation by IRF3. Notably, expression of these parameters was suppressed by bafilomycin A₁, an inhibitor of endosomal acidification, implicating endosomal TLR activation. Since resiquimod, an agonist of TLR7/8, failed to stimulate A549 cells, data suggest TLR3 to be of prime relevance for cellular activation. To assess the overall regulatory potential of U1-snRNA-activated epithelial cells on cytokine production, co-cultivation with peripheral blood mononuclear cells (PBMC) was performed. Interestingly, A549 cells activated by U1-snRNA reinforced phytohemagglutinin-induced interleukin-10 release by PBMC but suppressed that of tumor necrosis factor-α, indicating an anti-inflammatory potential of U1-snRNA. Since U1-snRNA is enriched in apoptotic bodies and epithelial cells are capable of performing efferocytosis, the present data in particular connect to immunobiological aspects of apoptosis at host/environment interfaces.     

拟南芥DNA探针的比较基因组原位杂交揭示的拟南芥与远缘植物基因组间的同源性

采用生物素标记的拟南芥基因组DNA探针在75%杂交严谨度下对双子叶植物番茄、蚕豆和单子叶植物水稻、玉米、大麦的染色体进行了比较基因组荧光原位杂交（comparative genomic in situ hybridization,cGISH）分析,以揭示拟南芥与远缘植物基因组间的同源性.cGISH信号代表了拟南芥基因组DNA中的重复DNA与靶物种染色体上同源序列的杂交.探针DNA在所有靶物种的全部染色体上都产生了杂交信号.杂交信号为散在分布,并呈现随基因组增大,杂交信号增多,且分布更加分散的趋势.所有靶物种的核仁组织区（NOR）都显示了明显强于其他区域的杂交信号,表明拟南芥基因组DNA探针可用于植物NOR的物理定位.在所有的靶物种中,信号主要分布在染色体的臂中间区和末端,着丝粒或近着丝粒区有少数信号分布.大麦染色体显示了与C-和N-带不同的独特的cGISH信号带型,表明此探针可用于不同植物染色体的识别.这些结果表明,拟南芥基因组与远缘植物基因组之间,除rDNA和端粒重复序列外,还存在其它同源的重复DNA;一些重复DNA序列在被子植物分歧进化为单子叶和双子叶植物之前就已存在,虽经历了长期的进化过程,至今在远缘物种之间仍保持了较高的同源性.结果还提示,大基因组中古老而保守的重复DNA在进化过程中发生了明显的扩增.     

Intragenomic DNA sequence homologies in the chicken and other members of the classAves: DNA re-association under reduced stringency conditions

Summary We have investigated the intragenomic DNA sequence homologies of twelve species of birds representing five orders, and emphasizing Galliformes. This study differs in two important ways from the classical approaches taken in constructing and evaluating phylogenies based on DNA sequence similarities. Comparisons are made on the basis of sequence homologieswithin genomes of related birds, rather than between genomes. DNA is reassociated at 50°C in 0.5M phosphate buffer; these conditions allow formation and detection of duplexes containing more mismatch than would normally be permitted using more stringent conditions, affording an opportunity to observe more ancient sequence homologies. Thermal stability profiles of DNA duplexes formed under these conditions are the basis of comparison; three general patterns were observed. This approach emphasizes differences in sequence composition between genomes while the more traditional method of intergenomic tracer DNA hybridization at higher stringency emphasizes sequence similarities.No correlation was found between taxonomic position and intragenomic sequence composition, either within or between lineages. The thermal stability profiles of DNA duplexes formed within avian genomes did not reflect the biological similarities inferred from morphology, karyotype, and studies of interspecific hybridization. While all of the differences observed could have occurred over geological time, it was surprising that the genomes of the domestic chicken and the Red Jungle Fowl (Gallus gallus) differ in their sequence compositions. It appears that amplification/reduction events and/or positional changes occur rather often during evolution of a lineage.Abbreviations SDS sodium dodecyl sulphate - PB equimolar sodium phosphate buffer pH 6.8 - Cot concentration of DNA in moles of nucleotide per liter times the incubation time in seconds - Equiv. or Equivalent Cot Cot corrected for the monovalent cation concentration effect on re-association rate - HAP hydroxylapatite - Te_1/2 temperature at which one-half the DNA has eluted from HAP - SSC 0.15M sodium chloride-0.015M sodium citrate     

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

Discrete regions of sequence homology between cloned rodent VL30 genetic elements and AKV-related MuLV provirus genomes

Southern blot analyses using reduced stringency hybridization conditions have been employed to search for sequence homologies between rodent VL30 genes and murine leukemia virus (MuLV) proviruses. These constitute two classes of transposon-like elements previously believed to be genetically unrelated. Our results demonstrate that cloned representatives of both ecotropic and xenotropic-like proviruses share discrete regions of sequence homology with VL30 genes of both rat and mouse origin. These regions of homology exist in both 3' and 5' halves of the MuLV genome but do not include extensive portions of the long terminal repeat (LTR) or a 0.4 Kbp segment of the env gene specific for recently acquired ecotropic-type MuLV proviruses. DNA sequencing, however, revealed that the short inverted terminal repeat sequence of MuLV proviral LTRs is almost perfectly conserved at the terminus of an integrated mouse VL30 gene. These results suggest that recombination events with rodent VL30-type sequences occurred during early MuLV evolution. The strong conservation of the inverted terminal repeat sequence may reflect a common integration mechanism for VL30 elements and MuLV proviruses.     

Characterization of Rous sarcoma virus-related sequences in the Japanese quail.

We detected sequences related to the avian retrovirus Rous sarcoma virus within the genome of the Japanese quail, a species previously considered to be free of endogenous avian leukosis virus elements. Using low-stringency conditions of hybridization, we screened a quail genomic library for clones containing retrovirus-related information. Of five clones so selected, one, lambda Q48, contained sequence information related to the gag, pol, and env genes of Rous sarcoma virus arranged in a contiguous fashion and spanning a distance of approximately 5.8 kilobases. This organization is consistent with the presence of an endogenous retroviral element within the Japanese quail genome. Use of this element as a high-stringency probe on Southern blots of genomic digests of several quail DNA demonstrated hybridization to a series of high-molecular-weight bands. By slot hybridization to quail DNA with a cloned probe, it was deduced that there were approximately 300 copies per diploid cell. In addition, the quail element also hybridized at low stringency to the DNA of the White Leghorn chicken and at high stringency to the DNAs of several species of jungle fowl and both true and ruffed pheasants. Limited nucleotide sequencing analysis of lambda Q48 revealed homologies of 65, 52, and 46% compared with the sequence of Rous sarcoma virus strain Prague C for the endonuclease domain of pol, the pol-env junction, and the 3'-terminal region of env, respectively. Comparisons at the amino acid level were also significant, thus confirming the retrovirus relatedness of the cloned quail element.     

Assignment of snRNA gene sequences to the large chromosomes of rat kangaroo and Chinese hamster isolated by flow cytometric sorting

Chromosomes from a rat kangaroo (Potorous tridactylus) cell line (PtK2) and from a Chinese hamster (Cricetulus griseus) cell line (CHV79) were isolated by means of fluorescence activated flow cytometric sorting. DAPI (4-6-diamino-2-phenylindole) was used as the DNA specific fluorescent dye. The karyotype of the PtK2 cells which exhibits 13 chromosomes was separated into 6, and the 22 chromosomes of the CHV79 cells were resolved into 11 fractions. DNA extracted from these chromosomal fractions was used for restriction enzyme digestion and blotting on nitrocellulose filters. The blots were challenged with gene probes corresponding to ribosomal RNA (18S and 28S) and small nuclear RNA (U1-snRNA) genes. The rRNA genes were exclusively assigned to chromosomes containing the nucleolus organizing region (in PtK2: X chromosome; in CHV79: chromosomes 4, 5, 6, and 11). — Solely the largest chromosomes in both cell lines hybridized with U1-snRNA indicating that these gene sequences are located on those chromosomes only. Further possible genetic and biochemical applications of this experimental system are discussed.     

An RFLP species-specific DNA sequence for the A genome of rice.

A DNA sequence, pOs6.20, was cloned from the nuclear genome of cultivated rice (Oryza sativa L.) by hybridization with a human minisatellite sequence. At high hybridization stringency, a subfragment of the rice sequence, pOs6.20.3, detected low-copy restriction fragment length polymorphisms (RFLPs), which behaved as Mendelian genetic markers. This subfragment detected multicopy RFLPs between an indica and a javanica cultivar at medium hybridization stringency. The sequences detected by pOs6.20.3 at high hybridization stringency did not occur in rice species possessing the B, C, D, E, and F genomes. These results suggest that the RFLPs detected by this sequence can be placed on a genetic map of rice and that this sequence can be used as a species-specific probe for the A genome of rice.     

Relationships betweenNor-loci from differentTriticeae species

TheNor-loci of polyploid wheats and their putative diploid progenitor species were assayed by probing isolated nuclear DNA with ribosomal DNA spacer sequences (spacer rDNA sequences, isolated by cloning), from theNor-loci of genomes B (Triticum aestivum), G (T. timopheevi), B (syn. S,T. speltoides), A (T. monococcum) and V (Dasypyrum villosum). DNA samples for analysis were digested with the restriction endonuclease Taq 1 and assayed by DNA-DNA hybridization under standard (37°C) and high stringency (64°C) conditions. The assay procedure emphasized differences between the divergent spacer sequences of the polyploid species and allowed relative homologies to the respective sequences in diploid species to be established. — The studies indicated thatT. timopheevi andT. speltoides contain different sets of spacer rDNA sequences which were readily distinguishable and, in the case ofT. timopheevi, assigned toNor-loci on different chromosomes. This contrast with the spacer rDNA sequences of the majorNor-loci on chromosomes 1 B and 6 B inT. aestivum, which were difficult to distinguish and were deduced to contain very similar sequences. Among the diploid progenitor species only the spacer rDNA fromT. speltoides shared close homology with polyploid wheat species. OneNor-locus inT. timopheevi (on chromosome 6 G) did not show close homology with any of the rDNA spacer probes available. — The data suggestsT. speltoides was the origin of someNor-loci for both theT. timopheevi andT. turgidum lines of tetraploid wheats. The possibility that the 6GNor-locus inT. timopheevi may have derived from an unknown diploid species by introgressive hybridization is discussed. The spacer rDNA sequence probe fromT. monococcum shared good homology with some accessions ofD. villosum and a line ofT. dicoccoides; the implications of this finding for evolution of present-day wheats are discussed.     

Heterology of mitochondrial DNA from mammals detected by electron microscopic heteroduplex analyses.

Heteroduplex analysis of mitochondrial DNA (mtDNA) from evolutionary closely related mammals (rat vs. mouse, man vs. monkey) are analyzed and compared to heteroduplex analysis of mt-DNA from more distantly related mammals (rat vs. man, rat vs. monkey, mouse vs. man, mouse vs. monkey and man vs. cow). Each analysis is transformed into a heteroduplex map and all maps are aligned to restriction enzyme maps and to genetic maps and where possible compared with the known sequence. We show that early evolutionary changes are seen mainly in URF2, URFA6L, URF6 and the D-loop region. The regions of rRNA, URF1, COI and COIII are generally very conserved regions but areas with some evolutionary activity can be localized. Heteroduplex analysis between distantly related species show much more heterology than do closely related species and the heteroduplex maps between all the distantly related species show a common pattern of heterology. Comparisons between the DNA sequence of mtDNA from man, cow and mouse and the equivalent heteroduplex maps show that base pair homologies higher than 73% are displayed as homologous regions. In the heteroduplex analysis of mtDNA's from more closely related species very few heterologies are displayed at 50% formamide but an increase in formamide concentration to 65-70% demonstrate also in these instances general heterologous regions.     

cDNA sequence of the rat U snRNP-associated protein N: description of a potential Sm epitope.

Anti-Sm antibodies from a patient with systemic lupus erythematosus (SLE) were used to isolate cDNA clones encoding the snRNP-associated protein N from a rat brain derived cDNA library. The predicted primary structure of the 240 amino acid protein has a proline rich carboxyl terminus and shares a region of sequence similarity with other snRNP polypeptides, A and B/B'. Anti-Sm sera recognize a beta-galactosidase fusion protein containing only the carboxyl-terminal 80 amino acids of N; antibodies eluted from this fusion protein also react with A, B/B' and N on immunoblots, suggesting that these proteins share an Sm epitope located within this segment. Polyclonal antibodies raised against a 23 amino acid synthetic peptide derived from this conserved region of N recognize A, N and B/B' on immunoblots and can immunoprecipitate the Sm class of U snRNAs. These results confirm that this sequence defines a potential Sm epitope. RNA blotting analyses demonstrate that a 1.6 kb mRNA expressed predominantly in brain encodes the N polypeptide in both rats and humans. At low stringency rat N cDNA also hybridizes to a 1.3 kb mRNA species which encodes B/B', suggesting that N is structurally related to, but distinct from B/B'. Although B/B' proteins are thought to be expressed in all human cells, only N and B, but not B', are observed on immunoblots of human brain proteins probed with anti-Sm sera. The apparent difference in the complement of proteins associated with snRNP particles in human brain versus elsewhere suggests a possible mechanism for the regulation of brain-specific mRNA splicing.     

Structural analysis of gene loci for rat U1 small nuclear RNA.

Four phage clones which hybridize with U1 small nuclear RNA were obtained from a rat gene library. Two clones contain a presumed pseudogene. A third clone includes two gene candidates that are co-linear with the rat U1-RNA, 3.6kb apart and in the opposite orientation. The two genes are surrounded by identical sequences of 491bp upstream and 178bp downstream. The upstream sequences do not contain a TATA box, but share many block homologies with those for the human U1-RNA gene(1-3). A 101bp "identifier (ID) sequence", which was reported to be specifically expressed in rat brain (4), is inserted immediately after the shared sequence downstream of one of the genes. In the fourth clone, there are two putative pseudogenes, which have one or three nucleotide changes, 3kb apart and in the same orientation. Southern blot analysis of total rat DNA reveals about 50 U1-RNA genes/pseudogenes in the genome.     

Nucleotide sequence at the site of junction between adenovirus type 12 DNA and repetitive hamster cell DNA in transformed cell line CLAC1.

The hamster cell line CLAC1 originated from a tumor induced by injecting human adenovirus type 12 (Ad12) into newborn hamsters. Each cell contained about 12 copies of viral DNA colinearly integrated at two or three different sites. We have cloned and sequenced a DNA fragment comprising the site of junction between the left terminus of Ad12 DNA and cellular DNA. The first 174 nucleotides of Ad12 DNA were deleted at the site of junction. Within 40 nucleotides, there were one tri-, two tetra-, one penta-, and one heptanucleotide which were identical in the 174 deleted viral nucleotides and the cellular sequence replacing them. In addition, there were patch-type homologies ranging from octa- to decanucleotides between viral and cellular sequences. There is no evidence for a model assuming adenovirus DNA to integrate at identical cellular sites. The cellular DNA sequence corresponding to the junction fragment was cloned also from BHK21 (B3) hamster cells and sequenced. Up to the site of linkage with viral DNA, this middle repetitive cellular DNA sequence was almost identical with the equivalent sequence from CLAC1 hamster cells. Taken together with the results of previously published analyses (11, 12), the data suggest a model of viral (foreign) DNA integration by multiple short sequence homologies. Multiple sets of short patch homologies might be recognized as patterns in independent integration events. The model also accounts for the loss of terminal viral DNA sequences.