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Biomedical subjects

N Fedoroff

Publications and source records attributed to N Fedoroff.

33 records · Page 2Linked to original sources
PubMed1987

Studies on the introduction and mobility of the maize Activator element in Arabidopsis thaliana and Daucus carota.

We have co-transformed carrot (Daucus carota) and Arabidopsis thaliana with an Agrobacterium tumefaciens non-tumorigenic T-DNA carrying the maize transposable element Activator (Ac) and an Agrobacterium rhizogenes Ri T-DNA. We present evidence that the Ac element transposes in transformed root or root-derived callus cultures of both species. We show that fertile plants can be regenerated from transformed, root-derived callus cultures of Arabidopsis, demonstrating the utility of the Ri plasmid for introducing the maize Ac element into plants. We also present evidence that Ac elements that excise from the transforming T-DNA early after transformation continue to be mobile in carrot root cultures.

Cells, Cultured↗
PubMed1987

Phenotypic assay for excision of the maize controlling element Ac in tobacco.

We describe a phenotypic assay designed to detect excision of the maize controlling element Ac from a selectable marker gene, neomycin phosphotransferase II (NPT II). An NPT II gene which expresses kanamycin resistance in tobacco cells, and contains a unique restriction enzyme site in the untranslated leader region, was constructed. Ac, or a defective Ac element (Ac big up tri, open), was inserted into the leader region of this gene. The transposon insertions inactivated the NPT II gene as determined by transient NPT II expression assays. The three plasmids were inserted into the T DNA of Agrobacterium tumefaciens Ti plasmid vectors, and transferred to tobacco protoplasts. The transformed protoplasts were selected with 100 or 200 microg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed 25% as many calli resistant to 100 or 200 microg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac big up tri, open did not form any calli resistant to 200 microg/ml of kanamycin when transformed under similar conditions. Southern blot hybridization analyses of seven kanamycin-resistant calli or plants obtained after transformation by the NPT II gene interrupted by Ac revealed that in all cases Ac had excised, restoring the structure of the NPT II gene. This assay is therefore useful to monitor the activity of a transposable element such as Ac and to define the regions of this element involved in transposition activity.

Journal Article↗
PubMed1986

Transposition of the maize controlling element "Activator" in tobacco.

Transposition of the maize autonomous controlling element Activator (Ac) and a nonautonomous derivative, Dissociation (Ds), was investigated in tobacco cells. Tobacco protoplasts were transformed with Ti-plasmid vectors that contained Ac or Ds flanked by short maize wx gene sequences. The structures of the elements and surrounding wx and T-DNA sequences were investigated in nine Ac and five Ds tobacco transformants by digestion with restriction enzymes, Southern blotting, and hybridization using specific probes. In four of the nine Ac transformed lines, Ac had excised from its original position in the T-DNA and inserted at new sites in the tobacco genome. Ds did not excise from its original T-DNA position in any of the transformants examined. Two Ac fragments and cellular flanking sequences were cloned from a line of tobacco in which Ac had transposed. Fragments, comprised of sequences flanking the newly integrated Ac elements, were used as hybridization probes to normal tobacco DNA and to the tobacco DNA from which they were isolated. The Ac copies were integrated into repetitive tobacco DNA sequences. Two tobacco fragments containing empty Wx donor sites were cloned from the DNA of the same Ac transformant and sequenced. Both sequences are among the types of excision products observed to result from Ac-catalyzed excision events in maize. Our results indicate that the maize controlling element Ac is capable of self-catalyzed transposition in tobacco.

Journal Article↗
PubMed1984

Analysis of sh-m6233, a mutation induced by the transposable element Ds in the sucrose synthase gene of Zea mays.

The unstable allele sh-m6233 caused by insertion of the transposable element Ds into the sucrose synthase gene of maize, was cloned. The mutation is caused by the insertion of an 4 kb DNA segment, consisting of two identical Ds elements of 2000 bp length, of which one is inserted into the center of the other in inverted orientation. This structure is, at the level of restriction mapping and partial DNA sequencing, identical to the double Ds element found in a larger insert in the mutant allele sh-m5933. 8 bp of host DNA are duplicated upon insertion. In a revertant, a 6-bp duplication is retained.

Journal Article↗
PubMed1983

Molecular identification and isolation of the Waxy locus in maize.

The Waxy (Wx) locus in maize determines the amylose content of pollen and endosperm tissue. There are several mutant alleles of the locus caused by insertion of transposable controlling elements. In the present study, we have used the properties of controlling element alleles to identify the Wx locus and its gene product, with the subsequent objective of isolating the elements causing the mutations. We present evidence that the Wx locus encodes a starch granule-bound 58 kd polypeptide that is synthesized in vitro as a 65 kd precursor. We describe the isolation of recombinant plasmids containing cDNA inserts homologous to Wx mRNA and a recombinant lambda phage containing a genomic Eco RI fragment encompassing most or all of the Wx transcription unit. We show that a mutation caused by the controlling element Dissociation (Ds) is attributable to an insertion of approximately 2.4 kb at the Wx locus.

Alleles↗
PubMed1983

Isolation of the transposable maize controlling elements Ac and Ds.

Restriction endonuclease fragments containing part of the Waxy (Wx) locus have been cloned from strains with insertion mutations at the locus caused by the controlling elements Activator (Ac) and Dissociation (Ds). Evidence is presented that the genetically defined Ac element corresponds to a 4.3 kb insertion, while the two Ds elements correspond to 4.1 kb and 2.0 kb insertions, all near the 3' end of the Wx transcription unit. The 4.1 kb Ds is almost completely homologous to the Ac element, differing by a central deletion of less than 0.2 kb. The 2.0 kb Ds element is homologous to the ends of the Ac element. Sequences homologous to the ends of the Ac element are present in many copies in the genomes examined, while there are ten or fewer copies of a sequence with homology to the center of the cloned Ac element. The Ac element at the Wx locus can be distinguished structurally from the other Ac-like sequences in the genome.

Alleles↗
PubMed1983

The controlling element Ds at the Shrunken locus in Zea mays: structure of the unstable sh-m5933 allele and several revertants.

We have analyzed the structure of the Shrunken (Sh) locus in a strain containing an unstable recessive mutation, sh-m5933, caused by the transposable controlling element Dissociation (Ds). We have also analyzed nine spontaneous Sh revertant alleles. The sh-m5933 allele contains a 30 kb insertion at the Sh locus, as well as a duplication that includes part of the insertion and the Sh locus sequence on the 5' side of the insertion site. The revertants continue to show Ds-mediated chromosome breakage at the Sh locus, have an intact Sh locus from which the insertion has been excised, and retain the duplication. One of the nine revertant alleles has a 2 kb deletion at the junction between the Sh locus and the insertion sequence in the duplicated segment of the locus. The revertant also shows a temporal change in the pattern of somatic chromosome breakage, implicating the junction sequence as the site of Ds-mediated chromosome breakage.

Alleles↗
PubMed1983

Molecular studies on mutations at the Shrunken locus in maize caused by the controlling element Ds.

The structure and expression of the Shrunken (Sh) locus have been examined in several maize strains with mutations at the locus caused by the controlling element Dissociation (Ds). Three of the strains (sh-m6233, sh-m5933, and sh-m6258) are of independent origin, and the fourth (sh-m6795) represents a spontaneous recessive sh allele derived from an Sh revertant of strain sh-m6258. The sh-m6233 and sh-m5933 strains produce undetectable levels of the Sh-encoded sucrose synthetase and very small amounts (less than 1%) of an apparently normal sucrose synthetase mRNA in immature endosperm tissue. Both strains have rearrangements affecting the structure of the locus near the 5' end of the transcription unit. The Sh locus in the related strains sh-m6258 and sh-m6795 is interrupted by an insertion or a rearrangement having one breakpoint in an intervening sequence near the 3' end of the mRNA coding sequence. The 5' end of the gene is transcribed in immature kernels of both mutants, giving aberrant poly (A)+ mRNAs that are homologous to the normal transcript up to the insertion or rearrangement breakpoint and lack homology to the 3' end of the gene. The aberrant transcripts from both strains are translated in vivo and in vitro, yielding 82- and 85-kD proteins that are immunologically related to the 92-kD Sh-encoded sucrose synthetase monomer.

Gene Expression Regulation↗
PubMed1977

Intermolecular duplexes in heterogeneous nuclear RNA from HeLa cells.

Rapidly sedimenting hnRNA complexes contain regions of stable intermolecular duplex. Disruption of such complexes, as judged by a reduction in sedimentation rate, requires conditions sufficient to denature the duplex regions. Rapidly sedimenting molecules reappear only when the complementary sequences reanneal-that is, the formation of such complexes is dependent upon time and the concentration of homologous RNA. These experiments lead us to the conclusion that rapidly sedimenting hnRNA complexes consist of two or more largely single-stranded RNA molecules held together by short duplex regions. Precisely such structures have been visualized in the electron microscope. Rapidly sedimenting fractions of native nuclear RNA from preparative sucrose gradients consist primarily of large, multi-molecular complexes interconnected by duplex regions averaging 300 base pairs in length. Exposure of the RNA to severely denaturing conditions eliminates such complexes. Reannealing of the RNA reconstitutes complexes which are indistinguishable from those observed in preparations before denaturation.

Base Sequence↗
PubMed1976

Investigation of the organization of mammalian chromosomes at the DNA sequence level.

New developments in DNA sequencing techniques permit rapid progress in the determination of both repetitious and single-copy mammalian sequences. Three distinct families of highly repetitious satellite DNA's from the kangaroo rat Dipodomys ordii have been sequenced. With the MS satellite it was possible to show that the basic repeat sequence and its variants were arranged in a nonrandom order suggesting a hierarchy of repeats. The HS-alpha satellite from D. ordii was shown to resemble the guinea pig alpha satellite, a long term evolutionary persistence inconsistent with previous models. Sequences from hemoglobin mRNA were determined using hemoglobin complementary DNA as template for transcription in vitro. Seven of the largest fragments have been assigned to untranslated regions of the mRNA whereas 15 others have been tentatively located within the structural genes. From correlations with sequences from corresponding regions in the human hemoglobin mRNA's we have been able to make the first direct measurements of the rate of fixation of mutations that do not change the amino acid sequence. The minimum estimate for this rate is greater than the highest previously estimated rates of fixation of neutral mutations (calculated for fibrinopeptide A. A new technique, deoxysubstitution sequencing, which should speed determination of the complete mRNA sequences, is described.

Animals↗
PubMedDate not supplied

Direct Agrobacterium tumefaciens-mediated transformation of Hyoscyamus muticus hairy roots using green fluorescent protein.

Hyoscyamus muticus hairy root segments were infected with Agrobacterium tumefaciens ASE containing the binary vector pCGN1548 with a green fluorescent protein (GFP) reporter gene under the control of the CaMV 35S promoter. The roots were incubated on callus-inducing medium to generate transformed cells. Transformants were selected on medium containing 50 and 100 mg/L kanamycin and screened by visual inspection for GFP expression. Highly fluorescent cells were incubated on phytohormone-free medium for regeneration of the hairy root phenotype. This infection technique can be applied directly to existing hairy root cultures which have been previously characterized and selected for desirable physiological traits. These studies also indicate that GFP is not toxic to H. muticus plant tissue and that H. muticus hairy roots have minimal autofluorescence which allows for clear observation of GFP.

Cell Culture Techniques↗