CONTEXT: Human DNA identification is a powerful tool for paternity cases as well as for criminal investigation, in which biological evidence is typed after collection from crime scenes and for the identification of human remains. OBJECTIVE: Identification of a criminal in a rape case with 4 suspects using STR and VNTR DNA analysis. TYPE OF STUDY: Forensic DNA analysis. SETTING: DNA Diagnostic Laboratory, Universidade Estadual do Rio de Janeiro, Brazil. PARTICIPANTS: Blood from 4 suspects and the victim, and skin from the fetus. PROCEDURES: Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). RESULTS: Three of the suspects were excluded and one of them was identified as the biological father of the fetus after typing with CTT and FFv Multiplexes. Complementary DNA typing at 3 VNTR loci was also carried out. CONCLUSIONS: After typing four suspects using 6 STR loci, one of them was identified as the biological father of the fetus. In order to significantly enhance the Combined Paternity Index (PI), complementary DNA typing in 3 VNTR loci was carried out. The included suspect was found to be the biological father with a PI of 412,860 (Probability of Paternity: 99.9997%).
The rate of renaturation for complementary DNA strands can be enhanced greater than 10(4)-fold by the addition of simple cationic detergents, and the reaction is qualitatively and quantitatively very similar to that found with purified heterogeneous nuclear ribonucleoprotein A1 protein. Under optimal conditions, renaturation rates are greater than 2000-fold faster than reactions run in 1 M NaCl at 68 degrees C. The reaction is second-order with respect to DNA concentration, and reaction rates approach or equal the rate with which complementary strands are expected to encounter each other in solution. Renaturation can even be observed well above the expected melting temperature of the duplex DNA, demonstrating that some cationic detergents have DNA double-helix-stabilizing properties. The reaction is also extremely rapid in the presence of up to a 10(6)-fold excess of noncomplementary sequences, establishing that renaturation is specific and relatively independent of heterologous DNA. This finding also implies that up to several thousand potential target sequences can be sampled per strand per second. Such reagents may be useful for procedures that require rapid nucleic acid renaturation, and these results suggest ways to identify and design other compounds that increase the kinetics of association reactions. Moreover...
Casino, A; Cipollaro, M; Guerrini, A M; Mastrocinque, G; Spena, A; Scarlato, V
Fonte: PubMedPublicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 25/03/1981Português
Relevância na Pesquisa
A Fortran computer algorithm has been used to analyze the nucleotide sequence of several structural genes. The analysis performed on both coding and complementary DNA strands shows that whereas open reading frames shorter than 100 codons are randomly distributed on both DNA strands, open reading frames longer than 100 codons ("virtual genes") are significantly more frequent on the complementary DNA strand than on the coding one. These "virtual genes" were further investigated by looking at intron sequences, splicing points, signal sequences and by analyzing gene mutations. On the basis of this analysis coding and complementary DNA strands of several eukaryotic structural genes cannot be distinguished. In particular we suggest that the complementary DNA strand of the human epsilon-globin gene might indeed code for a protein.
The method of Taylor etal., (11) has been used to transcribe complementary DNA probes from the four major RNA species of cucumber mosaic virus (RNAs 1 - 4 in order of decreasing molecular weight). Analysis of the kinetics of hybridization of these probes in homologous and heterologous complementary DNA-RNA hybridization reactions has shown that the sequence of the smallest RNA (RNA 4), which contains the coat protein gene, is present within RNA 3. RNAs 1 and 2 are unique RNA molecules while each has a region of approximately 300 nucleotides in common with RNA 4.
A cloned complementary DNA sequence has been isolated from a human placental cDNA library in the bacteriophage expression vector lambda gt11 after screening with polyclonal antibodies against human placental aromatase-system cytochrome P-450 (P-450Arom). A single recombinant clone, lambda hAROM1, was characterized by its ability to generate a beta-galactosidase fusion protein that reacted independently with polyclonal antibodies raised against beta-galactosidase and cytochrome P-450Arom and with monoclonal antibodies specific for cytochrome P-450Arom. The cDNA insert, which was found to be 1.8 kilobases in length, was radiolabeled and used to analyze poly(A)+ RNA isolated from human placenta and total RNA isolated from human adipose stromal cells cultured in the absence or presence of regulatory factors. The radiolabeled cDNA hybridized to several size species of mRNA in both placental and adipose stromal cell RNA fractions. Changes in the levels of adipose stromal cell RNA that hybridized to the cDNA insert were associated with comparable changes in the levels of translatable cytochrome P-450Arom mRNA and aromatase system activity. These findings are indicative that lambda hAROM1 contains DNA sequences complementary to human cytochrome P-450Arom mRNA and are suggestive that regulatory factors affect aromatase activity by altering the transcriptional activity of the cytochrome P-450Arom gene.
Bacteriophage G4 has physically separated origins of synthesis of its viral and complementary DNA strands. Chain termination and "plus and minus" DNA sequencing methods have been used to obtain the nucleotide sequence of these two origins. The unique origin at which the complementary DNA strand is initiated has located in the untranslated region between genes F and G. This sequence, which has considerable secondary structure, contains a stretch which is complementary to the RNA primer that is observed during synthesis in vitro of the G4 complementary DNA strand [Bouché, J.P., Rowen, L. & Kornberg, A. (1978) J. Biol. Chem., in press]. This G4 origin shows extensive sequence homology with the bacteriophage lambda origin of DNA replication [Denniston-Thompson, K., Moore, D. D., Kruger, D. E., Furth, M. E. & Blattner, F. R. (1977) Science 198, 1051-1056]. The sequence around the site in gene A at which G4 viral DNA strand synthesis is initiated by the nicking action of the cistron A protein is very similar to that of bacteriophage phiX174. An (A + T)-rich stretch flanked by (G + C)-rich sequences may be involved in the interaction between the DNA and protein.
Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize.
Free polyribosomal poly(A)-containing RNA isolated from normal rat liver was used to prepare a complementary DNA plasmid library in the Pst1 site of the plasmid pAT153 . A plasmid pGSTr155 complementary to mRNA coding for a glutathione transferase Ya subunit was selected by differential hybridization in situ and preliminary characterization was performed by hybrid-selected mRNA translation, immunoprecipitation and polyacrylamide-gel electrophoresis of the product synthesized in vitro. The nucleotide sequence of the complementary DNA contained within pGSTr155 was determined and shown to contain a single open reading frame corresponding to the first 129 amino acids of the N-terminus of the Ya subunit and a further 63 nucleotides upstream of the initiating methionine codon.
Considerable effort is currently being devoted to understand the functions of protein p53, a major regulator of cell proliferation. The protein p53 has been reported to catalyse the annealing of complementary DNA or RNA strands. We report that this activity is inhibited in the presence of the serine/threonine protein kinase CK2. It is shown that this inhibition can be explained by the occurrence of a high-affinity molecular association between p53 and CK2. The molecular complex involves an interaction between the C-terminal domain of p53 and the beta subunit of the oligomeric kinase. Accordingly, the isolated alpha subunit of the kinase was without effect. In addition, after phosphorylation by CK2, phosphorylated p53 lost its DNA annealing activity. Because the C-terminal domain of p53 is both involved in the association with CK2 and phosphorylated by it, our results suggest that either protein-protein interaction or phosphorylation of this domain might control the base pairing of complementary sequences promoted by p53 in processes related to DNA replication and repair.
A self-complementary nucleotide sequence can form both a unimolecular hairpin and a bimolecular duplex. In this study, the secondary structures of the self-complementary DNA and RNA oligonucleotides with different sequences and lengths were investigated under various solution conditions by gel electrophoresis, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy and a ultraviolet (UV) melting analysis. The DNA sequences tended to adopt a hairpin conformation at low cation concentrations, but a bimolecular duplex was preferentially formed at an elevated cationic strength. On the other hand, fully matched RNA sequences adopted a bimolecular duplex regardless of the cation concentration. The thermal melting experiments indicated a greater change in the melting temperature of the bimolecular duplexes (by ∼20°C) than that of the hairpin (by ∼10°C) by increasing the NaCl concentration from 10 mM to 1 M. Hairpin formations were also observed for the palindrome DNA sequences derived from Escherichia coli, but association of the complementary palindrome sequences was observed when spermine, one of the major cationic molecules in a cell, existed at the physiological concentration. The results indicate the role of cations for shifting the structural equilibrium toward a nucleotide assembly and implicate nucleotide structures in cells.
A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were characterized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.
Interleukin-12 (IL-12) has been shown to enhance cellular immunity in vitro and in vivo. The beneficial roles of IL-12 as a DNA vaccine adjuvant have been commonly observed. Here the impact of IL-12 complementary DNA (cDNA) as an adjuvant for a human papillomavirus (HPV) type 16 E7 DNA vaccine is investigated in a mouse tumour model. Coinjection of E7 DNA vaccine with IL-12 cDNA completely suppressed antigen-specific cytotoxic T-lymphocyte (CTL) responses, leading to a complete loss of antitumour protection from a tumour cell challenge. In addition, antigen-specific antibody and T helper cell proliferative responses were also suppressed by IL-12 cDNA coinjection. This inhibition was observed over different IL-12 cDNA doses. Furthermore, separate leg injections of IL-12 and E7 cDNAs suppressed antigen-specific CTL and tumour protective responses, but not antibody and T helper cell proliferative responses, suggesting different pathways for suppression of these two separate responses. Further knockout animal studies demonstrated that interferon-γ and nitric oxide are not directly associated with suppression of antigen-specific antibody responses by IL-12 cDNA coinjection. However, nitric oxide was found to be involved in suppression of antigen-specific CTL and tumour protective responses by IL-12 cDNA coinjection. These data suggest that coinjection of IL-12 cDNA results in suppression of E7-specific CTL responses through nitric oxide...
B-chromosomes are frequently argued to be genetically inert elements, but activity for some particular genes has been reported, especially for ribosomal RNA (rRNA) genes whose expression can easily be detected at the cytological level by the visualization of their phenotypic expression, i.e., the nucleolus. The B24 chromosome in the grasshopper Eyprepocnemis plorans frequently shows a nucleolus attached to it during meiotic prophase I. Here we show the presence of rRNA transcripts that unequivocally came from the B24 chromosome. To detect these transcripts, we designed primers specifically anchoring at the ITS-2 region, so that the reverse primer was complementary to the B chromosome DNA sequence including a differential adenine insertion being absent in the ITS2 of A chromosomes. PCR analysis carried out on genomic DNA showed amplification in B-carrying males but not in B-lacking ones. PCR analyses performed on complementary DNA showed amplification in about half of B-carrying males. Joint cytological and molecular analysis performed on 34 B-carrying males showed a close correspondence between the presence of B-specific transcripts and of nucleoli attached to the B chromosome. In addition, the molecular analysis revealed activity of the B chromosome rDNA in 10 out of the 13 B-carrying females analysed. Our results suggest that the nucleoli attached to B chromosomes are actively formed by expression of the rDNA carried by them...
A technique is described to identify the rare sequences within an RNA molecule that are available for efficient interaction with complementary DNA probes: the target RNA is digested by RNase H in the presence of a random pool of complementary DNA fragments generated from the same DNA preparation that was used for target RNA synthesis. The DNA region was amplified by PCR, partially digested with DNase and denatured prior to RNA binding. In the presence of single-stranded DNA fragments the RNA was digested with RNase H such that, on average, each molecule was cut once. Cleavage sites were detected by gel electrophoresis either directly with end-labeled RNA or by primer extension. The pattern of accessible sites on c- raf mRNA was determined and compared with the known profile of activity of oligonucleotides found in cells, showing the merit of the method for predicting oligonucleotides which are efficient for in vivo antisense targeting. New susceptible sites in the 3'-untranslated region of c- raf mRNA were identified. Also, four RNAs were probed to ascertain to what extent structure predicts accessibility: the P4-P6 domain of the Tetrahymena group I intron, yeast tRNAAsp, Escherichia coli tmRNA and a part of rat 18S rRNA.
Complementary DNA (cDNA) specific for gamma-globin nucleotide sequences has been prepared by hybridizing total cDNA made from cord blood messenger RNA (mRNA) as template to an excess of normal adult human globin mRNA and recovering the single-stranded cDNA from hydroxylapatite. The specificity of the gamma cDNA for gamma mRNA sequences is strongly supported by the hybridization of this cDNA at low Cot values (Co, concentration of RNA and t, time in seconds) to RNA samples containing large amounts of functional gamma globin mRNA and the lack of hybridization to RNA samples containing little, if any, gamma-globin mRNA. The absence of cross-hybridization of gamma cDNA with alpha, beta, and delta mRNAs is demonstrated by the complete hybridization of the gamma cDNA to mRNA samples completely lacking either alpha or beta and delta mRNA. An estimate of the number of gamma-globin genes in human cellular DNA was obtained by hybridization of purified gamma cDNA to DNA from spleen and white blood cells of normal and beta-thalassemia subjects and measurement of the percent of gamma cDNA hybridized at saturation. The results indicate that there are between one and two gamma-globin genes per total haploid gene DNA equivalent obtained from both normal and beta-thalassemia subjects. These values are consistent with genetic evidence for the presence of multiple gamma gene loci in human cells. The finding that the number of gamma-globin genes in beta-thalassemia DNA is similar to that in nonthalassemia DNA indicates that a deletion of gamma-globin genes cannot account for either the inadequate gamma-globin synthesis or indirectly for the decreased or absent beta-globin synthesis in beta-thalassemia cells.
The expression of heterologous mRNA in Xenopus oocytes was quantitatively inhibited by coinjection of single-stranded complementary DNA or synthetic complementary oligonucleotides. The lymphokines Interleukin-2 (IL-2) and Interleukin-3 (IL-3) were used as model systems to test the effectiveness of this procedure. Messenger RNA samples were hybridized to single stranded complementary DNA or oligonucleotides, injected into oocytes and the oocyte incubation medium assayed for the presence or absence of specific translation products 48 hours later. When IL-2 mRNA was hybridized to a large excess of long (490 bases) single stranded complementary DNA, the expression of IL-2 was effectively blocked (greater than 98%). Complementary oligonucleotides (18-23 bases) were almost as effective as the polynucleotide in inhibiting IL-2 activity (greater than 95%). Oligonucleotides derived from the 5' end, middle or 3' end of the coding sequence were all effective in arresting IL-2 mRNA translation. Oligonucleotide hybrid-arrest was effective even when no NaCl was present in the hybridization buffer, indicating that the annealing reaction could occur within the oocyte after injection. Definite proof that hybrid-arrest could occur in vivo was shown by the fact that oligonucleotides injected before or after mRNA injection...
Ovomucoid messenger RNA (mRNAom) comprises approximately 8% of the total mRNA in the estrogen-stimulated oviduct. The recombinant plasmid pOM100 contained DNA complementary to the 3' end of mRNAom. DNA complementary to the 5' end of mRNAom was obtained from a partially purified preparation of mRNAom by polymerization by reverse transcriptase in the presence of a restriction fragment primer from pOM100. The complementary DNA mixture was amplified by molecular cloning using poly dG/dC tailing to form recombinant bacterial plasmids. Recombinant plasmids containing ovomucoid DNA sequences were selected by in situ hybridization to 32P-labeled pOM100 fragments. The longest plasmid containing ovomucoid DNA sequences was designated pOM502. The complete DNA sequence of both pOM100 and pOM502 was determined. The two plasmids appear to contain sequences complementary to the entire length of mRNAom. The nucleic acid sequence agrees with the known amino acid sequences for both ovomucoid and its N-terminal signal peptide. Highly homologous sequences occur in two regions that coincide with structural domains of the protein. Comparison of the sequence of mRNAom with that for other eucaryotic mRNAs allowed identification of possible functional regions in the mRNA molecule.
A pneumopathic strain of bovine viral diarrhea virus was grown in cell culture and purified. Genomic ribonucleic acid was extracted, polyadenylated at the 3' end, and copied into complementary DNA after oligo-dT priming. Complementary DNA was male double stranded and cloned into the pUC9 plasmid. Approximately 200 complementary DNA clones varying in length from 0.5 to 2.5 kilobases were obtained. Hybridization assays indicated that the sequences isolated were specific for bovine viral diarrhea virus and that at least 5.5 kilobases of bovine viral diarrhea virus genome was represented in the library of complementary DNA clones, the majority of which may have originated from the 3' end of the virus genome. One cloned complementary DNA sequence was used as a 32P-labelled hybridization probe for bovine viral diarrhea virus detection. The probe hybridized with all cytopathic and noncytopathic strains of bovine viral diarrhea virus tested and was 100 times more sensitive than infectivity assays for the detection of bovine viral diarrhea virus. Hybridization did not occur with nucleic acids from bovine coronavirus, bluetongue virus, bovine adenovirus or uninfected cell cultures. Native plasmid DNA sequences, labelled with 32P, did not hybridize with bovine viral diarrhea virus ribonucleic acid.
Radioactive DNA complementary to nucleotide sequences in Moloney murine sarcoma virus (MSV) and Moloney leukemia virus (M-MuLV) complex was made by the endogenous reverse transcriptase reaction. These virus stocks contained a threefold excess of MSV over M-MuLV as measured by biological assay. The complementary DNA was an accurate copy of the viral RNA in that 86% of 35S viral RNA hybridized with complementary (cDNA) DNA at a 1.5 to 1 cDNA-RNA molar ratio. The complementary DNA, of a 4-6S size, was fractionated by sequential absorptions with MulV and the feline leukemia virus pseudotype of MSV, [MSV(FeLV)] RNA. In this manner three sets of nucleotide sequences whichrepresent different portions of the MSV viral complex were obtained: a sarcoma virus-specific fraction (cDNAsarc) with sequences that had no homology to M-MuLV RNA but which hybridized to MSV (FeLV) RNA, a sarcoma-leukemia fraction (cDNA common) with sequences common to MSV as well as M-MuLV viral RNA, and a cDNAleuk representing those nucleotide sequences found only in M-MuLV. Hybridization of MSV-MuLV viral 35S RNA with a threefold molar excess of cDNA's revealed that approximately 20% was hybridized with cDNAsarc, whereas approximately 75% was hybridized with cDNAcommon. M-MuLV 35S RNA alone did not hybridize with cDNAsarc but did hybridize 40 and 50% with cDNAleuk and cDNAcommon...
DNA double-strand breaks (DSBs) are a serious threat to the cell, for if not or miss-repaired, they can lead to chromosomal aberration, mutation and cancer. DSBs in human cells are repaired via non-homologous DNA end joining (NHEJ) and homologous recombination repair pathways. In the former process, the structure of DNA termini plays an important role, as does the genetic constitution of the cells, through being different in normal and pathological cells. In order to investigate the dependence of NHEJ on DSB structure in normal and cancer cells, we used linearized plasmids with various, complementary or non-complementary, single-stranded or blunt DNA termini, as well as whole-cell extract isolated from normal human lymphocytes, chronic myeloid leukemia K562 cells and lung cancer A549 cells. We observed a pronounced variability in the efficacy of NHEJ reaction depending on the type of ends. Plasmids with complementary and blunt termini were more efficiently repaired than the substrate with 3' protruding single-strand ends. The hierarchy of the effectiveness of NHEJ was on average, from the most effective to the least, A549/ normal lymphocytes/ K562. Our results suggest that the genetic constitution of the cells together with the substrate terminal structure may contribute to the efficacy of the NHEJ reaction. This should be taken into account on considering its applicability in cancer chemo-or radiotherapy by pharmacologically modulating NHEJ cellular responses.