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PubMed1974

Nucleotide sequences of human globin messenger RNA.

Globin messenger RNA, isolated from human peripheral blood reticulocytes, was transcribed into complementary DNA by use of the RNA-dependent DNA polymerase of avian myeloblastosis virus. The complementary DNA was then transcribed into (32)P-labeled complementary RNA by E. coli RNA polymerase in the presence of alpha-(32)P-labeled ribonucleoside triphosphates. The fingerprint pattern obtained from ribonuclease T1 digests of human globin complementary RNA was specific and reproducible. Different patterns were obtained from digests of duck, mouse, and rabbit globin complementary RNA. The fingerprint patterns obtained from digests of purified natural human 10S globin messenger RNA, labeled in vitro with (125)I or with [gamma-(32)P]ATP and polynucleotide kinase, were similar to that of the complementary RNA but contained some additional oligonucleotides. Sufficient nucleotide sequence information has been obtained from about 50% of the intermediate sized oligonucleotides (8-14 base residues long), to make possible examination of correspondence between these nucleotide sequences and globin amino-acid sequences. Approximately 70% of these oligonucleotide sequences can be matched to unique amino-acid sequences in the alpha- or beta-globin chains. The other 30% do not match known amino-acid sequences and presumably correspond to untranslated portions of the mRNA; some of these sequences, however, can be matched to amino-acid sequence in the abnormally long segment of the alpha chain of hemoglobin Constant Spring, which is thought to result from a chain-termination mutation.

Amino Acid Sequence↗
PubMedDate not supplied

Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates.

Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.

Binding Sites↗
PubMed1988

Complementary oligodeoxynucleotide probes of RNA conformation within the Escherichia coli small ribosomal subunit.

The large RNA molecule within each ribosomal subunit is folded in a specific and compact form. The availability of specific 16S RNA sequences on the surface of the small ribosomal subunit has been probed by using complementary oligodeoxynucleotides. The hybridization of 8-15-nucleotide-long oligomers to their RNA complements within the subunit was quantitated by using a nitrocellulose membrane filter binding assay. The probes have been grouped into classes on the basis of sequence-specific binding ability under different conditions of ionic environment, incubation temperature, and subunit activation state [as defined by the ability to bind phenylalanyl-tRNA in response to a poly(uridylic acid) message]. Oligodeoxynucleotides complementary to nucleotides flanking 7-methylguanosine residue 527 and to the 3'-terminal sequence bound 30S subunits regardless of the activation state. Oligodeoxynucleotides that complement 16S ribosomal RNA residues 1-16, 60-70, 685-696, and 1330-1339 and the sequence adjacent to the colicin E3 cleavage site at residue 1502 all bound efficiently only to subunits in an inactivated conformation. Probes complementary to residues 1-11 and 446-455 bound only inactivated subunits, and then with low efficiency. Sequences complementary to nucleotides 6-16, 99-109, 1273-1281, and 1373-1383 bound 30S subunits poorly regardless of the activation state. With one exception, each probe was bound by native or heat-denatured 16S ribosomal RNA (as determined by size-exclusion chromatography). We conclude that complementary oligodeoxynucleotide binding efficiency is a sensitive measure of the availability of specific RNA sequences under easily definable conditions.

Base Sequence↗
PubMed2002

Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction.

OBJECTIVE: There is increasing evidence that T cell-independent pathways, such as the up-regulation of protooncogenes and the production of growth factors and matrix-degrading enzymes, lead to progressive destruction of affected joints. Therefore, identification of differentially regulated genes restricted to rheumatoid arthritis (RA) synovial fibroblasts is essential. A combination of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and complementary DNA (cDNA) array with defined genes was used for a highly sensitive differential screening using small amounts of RNA. METHODS: RNA was extracted from cultured synovial fibroblasts obtained from 6 patients with RA and 6 patients with osteoarthritis (OA). RAP-PCR was performed using different arbitrary primers for first- and second-strand synthesis. PCRs were hybridized to cDNA array membranes. RA samples were compared with OA samples for differentially expressed genes. RESULTS: In contrast to standard cDNA array, the identification of 12 differentially expressed genes in RA compared with OA (approximately 6%) was possible. Differentially expressed genes of interest were confirmed using semiquantitative RT-PCR and in situ hybridization. CONCLUSION: Numerous variants of the differential display method and continuous improvements, including RAP-PCR, have proven to be both efficient and reliable for examining differentially regulated genes. Our results show that RAP-PCR combined with cDNA arrays is a suitable method for identifying differentially expressed genes in rheumatoid synovial fibroblasts, using very small amounts of RNA.

Arthritis, Rheumatoid↗
PubMed1979

Isolation and characterization of cDNA complementary to transient maternal poly(A)+ RNA from the Drosophila oocyte.

cDNA complementary to total oocyte poly(A)+ RNA from Drosophila melanogaster was enriched for sequences complementary to transient maternal sequences; that is, those sequences which disappear from the oocyte during subsequent. A seven- to ten-fold enrichment factor was obtained, from 5.3% to about 50% of the total cDNA. Kinetic analysis of this enriched fraction indicates that the transient maternal sequences include 44 +/- 14 different sequences.

Animals↗
PubMed1975

Mapping of adenovirus 2 RNA sequences in lytically infected cells and transformed cell lines.

The strands of the six EcoRI fragments and the HpaI fragments E and C of Ad2 DNA were separated by electrophoresis in agarose gels. Using 32P-labeled fragment strands in solution hybridization experiments, the fraction of each strand complementary to RNA extracted from infected or transformed cells was assayed by chromatography on hydroxylapatite. In this manner, a tentative map of the cytoplasmic RNA sequences has been constructed for viral RNA extracted from cells both early and late during infection (see Fig. 16; in the map shown, the two strands of Ad2 are named the r and l strands following the bacteriophage convention). Since early cytoplasmic RNA anneals to four distinct regions of the genome, Ad2 probably codes for at least four early gene functions. Summation experiments have shown that all RNA sequences found in the cytoplasm of cells early during infection are also present in the cells' cytoplasm at late times. Viral RNA sequences in five independently isolated and cloned transformed rat cell lines were also mapped on the Ad2 genome. One class of Ad2-transformed rat cells contains RNA sequences complementary to only the segment of Ad2 DNA from 0.03-0.10 on the physical map, and this corresponds to one of the four regions of the genome expressed early during infection. If a viral gene product is necessary to maintain the transformed phenotype of the cell or codes for the virus-specific tumor (T) antigen, this genetic information must be at the left end of the genome (see Fig. 16). The two other classes of Ad2-transformed rat cells contain viral RNA sequences complementary to two or three of the regions of the genome transcribed into early cytoplasmic RNA. At both early and late times during the lytic cycle, the nucleus of the infected cell contains viral RNA sequences that are not transported to the cell's cytoplasm, suggesting that RNA processing and selection may play a role in the regulation of viral mRNA production.

Adenoviridae↗
PubMed1989

Mouse RNAase MRP RNA is encoded by a nuclear gene and contains a decamer sequence complementary to a conserved region of mitochondrial RNA substrate.

Using complementary oligonucleotide probes, we have isolated the nuclear gene for the RNA moiety of RNAase MRP; it is present as a single copy and encodes an uncapped primary transcript of 275 nucleotides. Direct sequence analysis revealed that the 136 nucleotide RNA that copurifies with RNAase MRP represents the 3' half of the 275 nucleotide primary transcript. The 5'-flanking region of the gene has putative transcriptional control elements homologous to the promoters of RNA polymerase II-transcribed U-series snRNA genes; however, the coding region possesses a box A sequence and terminates at four T residues, both features characteristic of polymerase III-transcribed genes. A decamer sequence, 5'-CGA-CCCCUCC-3', complementary to a conserved sequence adjacent to the enzymatic cleavage site on the mitochondrial RNA substrate, is present in the RNAase MRP RNA. Isolation of a nuclear gene for the RNA component of a mitochondrial enzyme implies that nucleic acids can be transported across mitochondrial membranes.

Animals↗
PubMed2003

Inhibition of HIV tat-TAR interactions by an antisense oligo-2'-O-methylribonucleoside methylphosphonate.

An antisense oligo-2'-O-methylribonucleotide having alternating methylphosphonate/phosphodiester linkages, 1676, whose sequence is complementary to the apical stem-loop of HIV-1 TAR RNA, was prepared to determine its effects on Tat protein-TAR interaction and Tat-mediated gene transactivation in cell culture. This oligomer and its all-phosphodiester analogue, 1707, were shown to: (1) bind to TAR at 37 degrees C with K(d)'s in the low nM concentration range; (2) inhibit Tat-TAR complex formation; and (3) inhibit expression of a chloramphenicol reporter gene under control of the HIV LTR in HeLa HL3T1 cells in culture.

Base Sequence↗
PubMed2003

A new route to 2'-O-alkyl-2-thiouridine derivatives via 4-O-protection of the uracil base and hybridization properties of oligonucleotides incorporating these modified nucleoside derivatives.

Oligonucleotides containing 2-thiouridine (s2U) in place of uridine form stable RNA duplexes with complementary RNAs. Particularly, this modified nucleoside has proved to recognize highly selectively adenosine, the genuine partner, without formation of a mismatched base pair with the guanosine counterpart. In this paper, we describe new methods for the synthesis of 2-thiouridine and various 2'-O-alkyl-2-thiouridine derivatives. Oligoribonucleotides having these modified nucleoside derivatives were synthesized, and their hybridization and structural properties were studied in detail by the 1H NMR analysis of these modified nucleosides and Tm experiments of RNA duplexes with their complementary RNA strands.

Magnetic Resonance Spectroscopy↗
PubMed1974

Interspecific recombination of mitochondrial DNA molecules in hybrid somatic cells.

The mitochondrial DNA (mtDNA) in rodent-human hybrid somatic cells was studied in strains that contain nucleotide sequences from both parental mtDNAs. A test for linkage of rodent to human mtDNA was devised on the basis of the density and sequence differences between these DNAs. When a mixture of rodent and human mtDNAs was banded in a CsCl gradient and each fraction hybridized with a mixture of complementary [(3)H]RNA transcribed from human mtDNA and complementary [(32)P]RNA transcribed from rodent mtDNA, each DNA was detected as a distinct and separate band at its expected density by specific hybridization with its complementary RNA. In some of the hybrid cell strains, the mtDNA sequences derived from the two species did not separate in the CsCl gradients. This result is interpreted as evidence for linkage between sequences from the two parental mtDNAs. While the exact nature of the linkage and the structure of the molecules containing both types of sequences are not known, the evidence supports the conclusion that the linkage is realized by a covalent bond. An event leading to the covalent bonding of these different sequences may be described as recombination. Among 18 hybrid cell strains examined, 13 contained large proportions of recombinant molecules. These molecules were found in both mouse-human and rat-human strains, and in strains containing more human or more rodent mtDNA sequences.

Animals↗
PubMed1992

Yeast site-specific ribonucleoprotein endoribonuclease MRP contains an RNA component homologous to mammalian RNase MRP RNA and essential for cell viability.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA sequence complementary to mammalian mitochondrial origins of replication in a manner consistent with a role in primer RNA metabolism. The same activity in the yeast Saccharomyces cerevisiae has recently been identified; it cleaves an RNA substrate complementary to a yeast mitochondrial origin of replication at an exact site of linkage of RNA to DNA. We have purified this yeast enzyme further and detect a single, novel RNA of 340 nucleotides associated with the enzymatic activity. The single-copy nuclear gene for this RNA was sequenced and mapped to the right arm of chromosome XIV. The identity of the clone, as encoding the RNA copurifying with enzymatic activity, was confirmed by a match to the directly determined sequence of the RNA. The gene sequence also identified a 340-nucleotide RNA in total yeast RNA and in purified RNase MRP enzyme preparations. Inspection of the sequence of the yeast RNA revealed homologies to the RNA component of mouse RNase MRP, 49% overall with specific regions of much greater similarity. The flanking regions of the gene showed characteristics of an RNA polymerase II transcription unit, including a TATAAA box and a 7/8 match to the yeast cell cycle box UAS. The RNase MRP RNA gene was deleted by insertional replacement and found to be essential for cellular viability, indicating a critical nuclear role for RNase MRP.

Animals↗
PubMed1976

Genome expression and mRNA maturation at late stages of productive adenovirus type 2 infection.

RNA from adenovirus 2-infected KB cells was annealed in liquid with RNA in vast excess to viral heavy (l) and light (r) 32P-labeled DNA strands. Hybridization kinetics were analyzed by computer to estimate the number of viral RNA abundance classes, their relative concentrations, and the fraction of each DNA strand from which they originated. Early whole cell RNA extracted 5 h postinfection annealed rapidly to 10 to 15% of l and r strands and then slowly to final values of 60 and 40% of l and r strands. By 9 h postinfection the expression of late genes was apparent and whole cell RNA annealed to 20 and 75% of l and r strands. Whole cell RNA extracted between 12 and 36 h postinfection annealed to 7 to 15% and 75 to 90% of l and r strands. Late nuclear RNA hybridized to 10 and 90% of l and r strands, and late polyribosomal RNA hybridized to 20 and 75% of l and r strands. Based upon kinetic analyses, we estimate that mRNA synthesized exclusively during late stages arises from about 6 to 8% and 45 to 49% of l and r strands. This assumes that the early class I mRNA (in low concentration late) originates from 8 to 10% and 6 to 10% of l and r strands and that early class II mRNA (in high concentration late) is derived from 2% and 8 to 13% of l and r strands. Mixing experiments indicated that early mRNA is a subset of RNA extracted from polyribosomes late after infection and that late nuclear RNA contains sequences complementary to early l strand class I nRNA. RNA-RNA hybrids were isolated from late mRNA containing sequences from 60% of l and r strands, but it is not known when these were synthesized, and therefore whether complementary RNA transcripts are synthesized late after infection, as they are known to be synthesized early. These results demonstrate that portions of the genome are transcribed into RNA sequences that remain confined to the nucleus and are not exported to polyribosomes as mRNA.

Adenoviruses, Human↗
PubMed1997

Cloning and functional expression of the luteinizing hormone receptor complementary deoxyribonucleic acid from the marmoset monkey testis: absence of sequences encoding exon 10 in other species.

Based on sequence homologies among the human, porcine, rat, and mouse genes for the LH receptor (LHR), overlapping partial fragments of LHR complementary DNAs (cDNAs) were multiplied from marmoset monkey testicular RNA using reverse transcription-PCR. Ligations of the individual cDNA fragments generated a full-length monkey LHR cDNA (2031 bp) containing the complete amino acid-coding sequence (676 amino acids). Northern hybridization analysis of monkey testicular RNA, using a complementary RNA probe corresponding to the full-length cDNA, demonstrated major transcripts of 5.5 and 1.4 kilobases and minor ones of 4.0, 2.7, and 1.9 kilobases. Sequence analysis of the monkey LHR cDNA revealed a striking feature, i.e. the absence of an 81-bp nucleotide sequence corresponding to exon 10, present in the LHR cDNAs of all other species studied to date. The monkey LHR cDNA displayed 83-94% overall sequence homology with the other mammalian LHR cDNAs. Reverse transcription-PCR with human exon 10-specific primers demonstrated the total absence of this sequence from the monkey LHR messenger RNA. Southern hybridization of monkey genomic DNA using a human exon 10 probe demonstrated its presence in the monkey gene and that it is totally spliced out from the primary transcript. COS cells transfected with the monkey LHR cDNA showed similar high affinity (Kd = 0.25 nmol/liter) of [125I]iodo-hCG binding as those transfected with human LHR cDNA (Kd = 0.20 nmol/liter). The cells expressing the recombinant monkey and human LHR displayed similar responses of extracellular cAMP and inositol trisphosphate to hCG. In conclusion, marmoset monkey LHR seems to lack the sequence corresponding to exon 10 of the LHR gene in other mammalian species. The truncation does not alter LHR function, as the monkey receptor protein bound hCG and evoked cAMP and inositol trisphosphate responses comparable to those of the human LHR containing the exon 10-encoded structure. As the sequence homologous to exon 10 is missing in the other two glycoprotein receptors, i.e. those of FSH and TSH, this extra exon is apparently inserted into the LHR messenger RNA of some species during evolution from intronic sequences by a change in alternative splicing.

Alternative Splicing↗
PubMed1980

Studies in AMV DNA. I. Physical properties and sequence composition of DNA present in AMV virions (AMV DNA).

The basic physical properties of AMV DNA (labeled with 3H-thymidine) as well as sequence homology between 3H-AMV DNA and viral or host cell genomes were investigated. It was found that native AMV DNA is in part single-stranded (20% of the total DNA). Using CsCl density gradient centrifugation AMV DNA was separated into two bands with buoyant densities of 1.702 g/cm3 (L-AMV DNA) and 1.722 g/cm3 (H-AMV DNA) in the peak fractions. Corresponding melting temperatures were estimated to be 82.0 degrees C and 81.3 degrees C for L- and H-AMV DNA, respectively. Hybridization experiments revealed that AMV DNA is not complementary to the AMV genomic RNA but complementary prevalently to the sequences with low frequency of repetition and to the unique sequences of the myeloblast genome. These unique sequences, comprising about 40% of the total 3H-AMV DNA, were not detected in the nuclear DNA of leukosis-free chicken embryo fibroblasts. After infection of embryo cells with AMV the extent of hybridization in the region of unique sequences rose roughly by 20%. Renaturation of thermally denatured 3H-AMV DNA using the phenol emulsion reassociation technique was observed. The results suggest that AMV DNA is composed of a number of different special sequences of the host genome.

Animals↗
PubMed1987

Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain.

Neurotransmitter-related messenger RNAs were detected by in situ hybridization in sections of rat and mouse brains by using 35S-radiolabelled RNA probes transcribed from cDNAs cloned in SP6 promoter-containing vectors. The distribution of messenger RNAs for glutamic acid decarboxylase, tachykinins (substance P and K), and tyrosine hydroxylase was examined in the striatum, pallidum, and substantia nigra. Dense clusters of silver grains were observed with the RNA probe complementary of the cellular messenger RNA for glutamic acid decarboxylase (antisense RNA) over most large neurons in the substantia nigra pars reticulata and medium-sized to large neurons in all pallidal subdivisions. A few very densely and numerous lightly labelled medium-sized neurons were present in the striatum. Among the areas examined, only the striatum contained neurons labelled with the antisense tachykinin RNA. Most of these neurons were of medium size, and a few were large. With the antisense tyrosine hydroxylase RNA, silver grains were found over neurons of the substantia nigra pars compacta and adjacent A10 and A8 dopaminergic cell groups. No signal was observed with RNAs identical to the cellular messenger RNA for glutamic acid decarboxylase or tachykinin (sense RNA). These results show a good correlation with immunohistochemical studies, suggesting that documented differences in the distribution and the level of glutamic acid decarboxylase, tyrosine hydroxylase, and substance P immunoreactivities in neurons of the basal ganglia are related to differences in the level of expression of the corresponding genes rather than to translation accessibility, stability, or transport of the gene products.

Animals↗
PubMed1994

Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization.

In situ hybridization experiments were carried out to detect avocado sunblotch viroid (ASBVd) in foliar tissue of avocado, using a digoxigenin-labelled RNA probe complementary to the ASBVd-RNA in sections of aldehyde-fixed, LRGold-embedded leaf samples. Detection of the probe was made through anti-digoxigenin antibody and protein-A colloidal gold (20 nm). Seventy to 80% of the signals came from chloroplast while the cytoplasm and vacuole were labelled with ca. 10% of the gold particles. This is in contrast with the subcellular localization of potato spindle tuber viroid and some other related viroids, which are mainly found in the nucleus.

Chloroplasts↗
PubMed1997

Responses of bone to titania-hydroxyapatite composite and nacreous implants: a preliminary comparison by in situ hybridization.

The effect of two biomaterials on bone formation in vivo by in situ hybridization, was compared by using RNA probes complementary to collagen alpha1(I) RNA, osteonectin RNA and osteocalcin RNA. Holes were drilled into the midshafts of rat femurs. Titania-hydroxyapatite composite (THA) or nacre cylinders were implanted and the bone-implant regions collected 14 days after operation. Cuboidal osteoblasts, intensely labelled with the three probes, were seen to be lining the newly formed bone surrounding the THA implant. Between the implant and the new bone, a layer of un-labelled, apparently non-osteogenic cells was observed. By contrast, the nacre implant was bonded to the newly formed bone without any soft tissue interference. Osteoblasts lining the distal surface of the newly formed bone were stained with all three RNA probes, although weaker than in the THA sample. Some of the osteoblasts were flattened. We concluded from the appearance of the osteoblasts that the bone formation in the nacre samples had progressed beyond the phase of maximal synthetic activity. Around the THA implant, the labelling indicated that bone-forming activity was still high. It was concluded that the bioactivity of nacre was higher than that of THA.

Journal Article↗
PubMed1974

Nucleotide sequence analysis of RNA synthesized from rabbit globin complementary DNA.

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as template for in vitro synthesis of (32)P-labeled RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T(1) and alkaline phosphatase digestion have been determined. Several fragments were long enough to fit uniquely with the alpha or beta globin amino-acid sequences. These data demonstrate that the cDNA was copied from globin mRNA and contained no detectable contaminants.

Alkaline Phosphatase↗