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Biomedical subjects

N Davidson

Publications and source records attributed to N Davidson.

At least 289 records · Page 16Linked to original sources
PubMed1978

Gene mapping and gene enrichment by the avidin-biotin interaction: use of cytochrome-c as a polyamine bridge.

A modification of previously described methods of electron microscopic gene mapping and of gene enrichment based on the avidin-biotin interaction is presented. The modification consists of coupling cytochrome-c instead of pentane diamine to the oxidized 2', 3' terminus of an RNA by Schiff base formation and BH-4 reduction. The RNA-cytochrome-c conjugate is purified by a simple chromatographic procedure; several biotins are attached to the cytochrome moiety by acylation. The extended arm between biotin and RNA gives efficient electron microscopic gene mapping of DNA:RNA-biotin hybrids with avidin-ferritin and avidin-polymethacylate sphere labels and efficient gene enrichment by buoyant banding of DNA:RNA-biotin:avidin-spheres in CsCl. A 1400 fold enrichment (thus, 25% pure) and a 90% yield of long Drosophila DNA strands with 5S RNA genes is achieved.

Avidin↗
PubMed1978

RNA:DNA hybrids are more stable than DNA:DNA duplexes in concentrated perchlorate and trichloroacetate solutions.

Rates of formation of RNA:DNA hybrids have been measured as a function of temperature and compared to DNA:RNA duplex denaturation temperatures in 4 M sodium perchlorate, 4 M NaClO4-6 M urea, and 3 M rubidium trichloracetate solvents. The usual bell shaped curves of reaction rate versus temperature were observed. The optimal temperatures for the RNA:DNA association reaction are 5 degrees to 12 degrees greater than the Tm's for DNA:DNA denaturation in these solvents, just as in formamide. R-loops of phi80d3ilv DNA with E. coli rRNA can be formed at high efficiency in these solvents.

DNA, Bacterial↗
PubMed1978

An electron microscopic method for the mapping of proteins attached to nucleic acids.

An electron microscopic method for demonstrating the presence of and mapping the positions of proteins specifically bound to nucleic acids is described. The nucleic acid-protein complex is treated with dinitrofluorobenzene under conditions such that dinitrophenyl (DNP) groups are attached to nucleophilic groups on the protein, with only a low level of random attachment to the nuclei acid. This product is treated with rabbit anti-DNP IgG. The position of the protein-(DNP)n(IgG)m complex on the nucleic acid strand can be observed by electron microscopy by protein free spreading methods and, in many cases, by cytochrome-c spreading. If necessary for visualization by the latter method, the size of the labeled region can be increased by treatment with goat anti-rabbit IgG. High efficiency of electron microscopic labeling is achieved. Examples studied are: the adenovirus-2 DNA terminal protein, a protein covalently bound to SV40 DNA, DNA polymerase I bound to DNA, E. coli RNA polymerase bound to T7 DNA, and proteins UV crosslinked to avian sarcoma virus RNA.

Adenoviruses, Human↗
PubMed1978

Structure of 50 to 70S RNA from Moloney sarcoma viruses.

The 50 to 70S RNAs of two clonal isolates of defective Moloney sarcoma-leukemia helper virus complex were analyzed by gel electrophoresis and electron microscopy. The RNAs extracted from both clone 3 and clone 124-5R of Moloney sarcoma-leukemia virus complex contained some large monomer subunits ca. 10,000 nucleotides in length (10 kilobases), which are believed to be the Moloney leukemia virus subunits. Both RNAs had an excess of a smaller, sarcoma-specific subunit, 5 kilobases (clone 3) or 6 kilobases (clone 124-5R) in length. Electron microscopy of intact 50 to 70S dimer RNA molecules showed for both clones many dimers of two small subunits, some dimers of two large subunits, but few if any heterodimers with one large and one small subunit. This result was unexpected because the sequences near the 5'end of the RNA subunits, which are believed to be involved in the dimer linkage, are probably homologous between the large and small subunits. We also observed that some small-small dimers migrated anomalously slowly on nondenaturing gels. The nature of this slow-migrating complex is unkown; it could be a higher aggregate of the small-small dimer with additional small or large subunits, or it could be an extended conformation of the small-small dimer.

Base Sequence↗
PubMed1978

High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures.

Several 50 to 70S tumor viral RNAs have previously been shown by electron microscopy to be dimers, with the two monomer subunits joined near their 5' ends. Five additional naturally occurring type C RNA tumor viruses have now been examined: AKR, and endogenous murine ecotropic virus; NZB, an endogenous murine xenotropic virus; and ecotropic and an amphotropic virus isolated from a wild mouse; and the avian reticuloendotheliosis virus (REV). All five 50 to 70S RNAs have similar 5'-to-5' dimer structures. Therefore, the observations support the hypothesis that the dimer linkage is a structural feature common to all type C mammalian viruses. REV is the first example of an avian virus with a clear 5'-to 5' dimer linkage. All of the mammalian viral RNAs, but not REV, showed symmetrically placed loops in each subunit of the dimer. Possible molecular structures and biological functions of the dimer linkages and loops are discussed.

AKR murine leukemia virus↗
PubMed1978

Heteroduplex analysis of the sequence relations between the RNAs of mink cell focus-inducing and murine leukemia viruses.

The sequence relationships betwen AKR ecotropic virus and an AKR-derived "mink cell focus-inducing" (MCF) isolate (AKR MCF 247), between Moloney murine leukemia virus (M-MLV) and an M-MLV MCF isolate (M-MLV83), and between AKR and M-MLV were studied by electron microscopic heteroduplex analysis. The MCF-specific sequences were found to map from 1.95 kilobases (kb) to 2.75 kb (+/- 0.15 kb) from the 3' end of the RNAs for both MCF isolates. The major sequence nonhomology regions between AKR and M-MLV lie between 0.9 and 3.5 kb from the 3' end. However, the AKR and M-MLV sequences immediately adjacent to the 1.95- and 2.75-kb junctions with MCF-specific sequences are relatively similar in AKR and M-MLV. Our results suggest that the env gene of MLVs maps from 1 kb to 3 kb from the 3' end of the genomic RNA and that the carboxyl end of the glycoprotein of each MCF strain is similar (or identical) to that of its ecotropic parent.

AKR murine leukemia virus↗
PubMed1977

Calcium-dependent increase in efflux of [1-3H] taurine from the superfused rat cerebellar cortex in vivo.

High (40 mM) potassium stimulation has been shown to increase the efflux in vivo of [1-3H] taurine from the superfused rat cerebellar cortex by 46%, P less than 0.001. During superfusion with calcium-free media this increase in efflux is abolished. The cellular location of [1-3H] taurine in the cerebellar cortex is not yet known, but this region of the brain contains exceptionally high levels of endogenous taurine. The calcium dependency of the release of labelled taurine raises the possibility that taurine has some neuro-humoral role in the cerebellar cortex.

Animals↗
PubMed1977

Comparison of bicyclic phosphorous esters with bicuculline and picrotoxin as antagonists of presynaptic inhibition in the rat cuneate nucleus.

The effects of 2 of a series of bicyclic phosphorous esters, the ethyl (EPTBO) and isopropyl (IPTBO) compounds, were compared with those of the GABA antagonists, picrotoxin and bicuculline, on presynaptic inhibition in the rat cunneate nucleus. Both EPTBO and IPTBO were found to be effective, reversible antagonists of presynaptic inhibition, with IPTBO approximately 10 times more potent than EPTBO and equipotent with bicuculline, EPTBO equipotent with picrotoxin.

Animals↗
PubMed1977

A method for gene enrichment based on the avidin-biotin interaction. Application to the Drosophila ribosomal RNA genes.

A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described. The purified RNA corresponding to the gene is covalently attached to biotin via a cytochrome c bridge. This modified RNA is hybridized to the total DNA. Those DNA strands which hybridize are separated from all the other DNA, using the avidin-biotin interaction, by one of two methods. Avidin is covalently attached to submicroscopic polymer spheres; the complexes of avidin spheres with the DNA: RNA-biotin hybrids band in CsCl at a much lower buoyant density than does free DNA. Alternatively, the DNA:RNA-biotin hybrids are isolated by affinity chromatography on an avidin-solid support column. These methods have been used to prepare long single strands of Drosophila ribosomal DNA (rDNA) in high yield and 42 to 80% pure.

Animals↗
PubMed1977

Positions of sea urchin (Strongylocentrotus purpuratus) histone genes relative to restriction endonuclease sites on the chimeric plasmids pSp2 and pSp17.

The positions of the several sea urchin histone genes on the eukaryotic fragments of the chimeric plasmids pSp2 and pSp17 have been mapped relative to the Eco RI and Hind III restriction endonuclease sites on the plasmids. Two principal mapping methods using the electron microscope have been used: (a) the R-loop procedure and a new modification thereof to map the genes on duplex DNA; (b) the gene 32-ethidium bromide technique to visualize RNA-DNA hybrids on single strands of DNA. It is known that there are two histone genes, H3 and H2A, on pSp17. There are two Eco RI sites at the two junctions of the procaryotic segment with the eucaryotic segment on the plasmid. We show, by an electron microscope method, that for H2A, with a length of 0.52 kilobases (kb), one end of the gene is situated 0.02 to 0.03 kb from one RI site, and that there is a Hind III site within this gene at about 0.13 kb from the end phe other RI site of this plasmid. The H4 gene lies between H2B and H1. The ms the incubation temperature is raised up to a temperature just below that at which strand dissociation of the duplex DNA occurs.

Animals↗
PubMed1977

A heteroduplex study of the sequence relationships between the RNAs of M-MSV and M-MLV.

The regions of sequence homology between the RNA genomes of a murine sarcoma virus (clone 124 Moloney-MSV) and its parental helper virus (clone 3 Moloney-murine leukemia virus (M-MLV)) have been mapped. Long complementary DNA transcripts of the M-MLV RNA were hybridized to M-MSV RNA, and the structures of the hybrids were observed in the electron microscope. Beginning at the 5' end, the two RNAs are homologous for a region of length 2.25 kb (kilobases). In the next region, of length approximately 4.2 kb on the MLV genome, there are several homology segments between MLV and MSV, but there are also several short sequences present on MLV and deleted in MSV. There is then a major substitution loop; with a sequence (beta L) of length 2.9 kb present on MLV and missing on MSV, and a sequence (beta S) of length 1.5 kb present on MSV and missing on MLV. At the 3' end, there is a homology sequence of length 0.8 kb. On the basis of these results, other data on genes expressed in M-MSV-transformed cells, and by analogy with the avian gene map, we suggest that the gag genes (internal structural proteins) lie in the 2.25 kb region of homology near the 5' ends of M-MSV and M-MLV RNAs, and that the beta S segment contains a sarcoma (src) gene. Some of the heteroduplexes and some of the MLV cDNA/MLV RNA homoduplexes are circular, thus showing that cDNA transcription is initiated at an internal position in the RNA, proceeds to the 5' end, and them "jumps" to the 3' end.

Base Sequence↗
PubMed1977

Sequence arrangement of the rDNA of Drosophila melanogaster.

The sequence arrangement of genes coding for stable rRNA species and of the interspersed spacers on long single strands of rDNA purified from total chromosomal DNA of Drosophila melanogaster has been determined by a study of the structure of rRNA:DNA hybrids which were mounted for electron microscope observation by the gene 32-ethidium bromide technique. One repeat unit contains the following sequences in the order given. First, an 18 S gene of length 2.13 +/- 0.17 kb. Second, an internal transcribed spacer (Spl) of length 1.58 +/- 0.15 kb. A short sequence coding for the 5.8S and perhaps the 2S rRNA species is located within this spacer. Third, the 28S gene with a length of 4.36 +/- 0.23 kb. About 55% of the 28S genes are unbroken or continuous (C genes). However, about 45% of the 28S genes contain an insertion of an additional segment of DNA that is not complementary to rRNA (l genes). The insertion occurs at a reproducible point 2.99 +/- 0.26 kb from the junction with Spl. The insertions are heterogeneous in length and occur in three broad size classes: 1.42 +/- 0.47, 3.97 +/- 0.55, and 6.59 +/- 0.62 kb. Fourth, an external spacer between the 28S gene and the next 18S gene which is presumably mainly nontranscribed and which has a heterogeneous length distribution with a mean length and standard deviation of 5.67 +/- 1.92 kb. Short inverted repeat stems (100-400 nucleotide pairs) occur at the base of the insertion. It is known from other studies that I genes occur only on the X chromosome. The present study shows that the I and C genes on the X chromosomes are approximately randomly assorted. The sequence arrangement on the plasmid pDm103 containing one repeat of rDNA (Glover et al., 1975) has been determined by similar methods. The I gene on this plasmid contains an inverted repeat stem. The occurrence of inverted repeat sequences flanking the insertion supports the speculation that these sequences are translocatable elements similar to procaryotic translocons.

DNA↗