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K Lowenhaupt

Publications and source records attributed to K Lowenhaupt.

35 records · Page 2Linked to original sources
PubMed1995

Double-stranded RNA adenosine deaminase binds Z-DNA in vitro.

A Z-DNA binding protein of 140,000 M(r) has been purified from chicken lungs by sedimentation through 40%(w/w) sucrose and Z-DNA affinity chromatography. Specificity of the protein for Z-DNA was confirmed by competition with polyd(CG) that had been stabilized in the Z-DNA conformer by chemical bromination and also with a supercoiled plasmid that contains a Z-DNA-forming insert. In addition to a Z-DNA binding site, the protein also has a separate binding site for double-stranded RNA. Peptide sequence of the protein shows that it has high similarity to the RNA editing enzyme double-stranded RNA adenosine deaminase (dsRAD), which deaminates adenosine in dsRNA to form inosine. The Z-DNA binding protein has this enzymatic activity, confirming its identity to dsRAD. Recombinant human dsRAD also binds to Z-DNA. Z-DNA is stabilized in a sequence-dependent manner by negative supercoiling, which occurs in actively transcribed genes upstream to RNA polymerase. It is proposed that Z-DNA links editing to transcription by localizing dsRAD to a particular region of a gene and thus determines the efficiency with which an RNA is edited. The presence of Z-DNA forming elements in many genes raises the possibility that RNA editing by dsRAD is far more prevalent than is currently thought.

Adenosine Deaminase↗
PubMed1993

Z-DNA binding protein from chicken blood nuclei.

A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.

Animals↗
PubMed1991

Cloning and characterization of Rrp1, the gene encoding Drosophila strand transferase: carboxy-terminal homology to DNA repair endo/exonucleases.

We previously reported the purification of a protein from Drosophila embryo extracts that carries out the strand transfer step in homologous recombination (Lowenhaupt, K., Sander, M., Hauser, C. and A. Rich, 1989, J. Biol. Chem. 264, 20568). We report here the isolation of the gene encoding this protein. Partial amino acid sequence from a tryptic digest of gel purified strand transfer protein was used to design a pair of degenerate oligonucleotide primers which amplified a 635 bp region of Drosophila genomic DNA. Recombinant bacteriophage were isolated from genomic and embryo cDNA libraries by screening with the amplified DNA fragment. These bacteriophage clones identify a single copy gene that expresses a single mRNA transcript in early embryos and in embryo-derived tissue culture cells. The cDNA nucleotide sequence contains an open reading frame of 679 amino acids within which are found 5 tryptic peptides from the strand transfer protein. Expression of this cDNA in E. coli produces a polypeptide with the same electrophoretic mobility as the purified protein. The deduced protein sequence has two distinct regions. The first 427 residues are basic, rich in glutamic acid and lysine residues and unrelated to known proteins. The carboxy-terminal 252 residues are average in amino acid composition and are homologous to the DNA repair proteins, Escherichia coli exonuclease III and Streptococcus pneumoniae exonuclease A. This protein, which we name Rrp1 (Recombination Repair Protein 1), may facilitate recombinational repair of DNA damage.

Amino Acid Sequence↗
PubMed1991

Drosophila Rrp1 protein: an apurinic endonuclease with homologous recombination activities.

A protein previously purified from Drosophila embryo extracts by a DNA strand transfer assay, Rrp1 (recombination repair protein 1), has an N-terminal 427-amino acid region unrelated to known proteins, and a 252-amino acid C-terminal region with sequence homology to two DNA repair nucleases, Escherichia coli exonuclease III and Streptococcus pneumoniae exonuclease A, which are known to be active as apurinic endonucleases and as double-stranded DNA 3' exonucleases. We demonstrate here that purified Rrp1 has apurinic endonuclease and double-stranded DNA 3' exonuclease, activities and carries out single-stranded DNA renaturation in a Mg(2+)-dependent manner. Strand transfer, 3' exonuclease, and single-stranded DNA renaturation activities comigrate during column chromatography. The properties of Rrp1 suggest that it could promote homologous recombination at sites of DNA damage.

Amino Acid Sequence↗
PubMed1989

Drosophila melanogaster strand transferase. A protein that forms heteroduplex DNA in the absence of both ATP and single-strand DNA binding protein.

The purification of a Drosophila strand transfer protein is described, which involves Bio-Rex 70, Superose 6, Mono S, and single-stranded DNA-agarose chromatography. A 105,000-dalton polypeptide copurifies with the strand transfer activity on the last two column steps. The strand transferase carries out strand transfer at an unusually low protein:single-stranded DNA ratio and requires neither a nucleotide cofactor nor exogenous single-strand DNA binding protein to form heteroduplex DNA. Biochemical analysis of the reaction products has established that one strand of the DNA duplex is displaced during the reaction. Several properties, including the kinetics and stoichiometry of strand transfer, differentiate this activity from previously characterized strand transferases.

Adenosine Triphosphate↗
PubMed1989

Nonrandom distribution of long mono- and dinucleotide repeats in Drosophila chromosomes: correlations with dosage compensation, heterochromatin, and recombination.

Long stretches of (dC-dA)n.(dT-dG)n, abbreviated CA/TG, have a distinctive distribution on Drosophila chromosomes (M.L. Pardue, K. Lowenhaupt, A. Rich, and A. Nordheim, EMBO J. 6:1781-1789, 1987). The distribution of CA/TG suggests a correlation with the overall transcriptional activity of chromosomal regions and with the ability to undergo meiotic recombination. These correlations are conserved among Drosophila species and may indicate one or more chromosomal functions. To test the generality of these findings, we analyzed the distribution of the rest of the six possible mono- and dinucleotide repeats (A/T, C/G, AT/AT, CA/TG, CT/AG, and CG/CG). All but CG/CG were present at significant levels in the genomes of the six Drosophila species studied; however, A/T levels were an order of magnitude lower than those of the other sequences. Data base analyses suggested that the same sequences are present in other eucaryotes. Like CA/TG, both CT/AG and C/G showed increased levels on dosage-compensating chromosomes; however, the individual sites clearly differed for each sequence. In contrast, A/T and AT/AT, although present in Drosophila DNA, could not be detected in situ in polytene chromosomes. We also used in situ hybridization to analyze the neo-Y chromosome of Drosophila miranda, an ancestral autosome that has become attached to the Y chromosome and is now partially heterochromatic. The neo-Y has acquired repeated DNA sequences; we found that the added sequences are as devoid of mono- and dinucleotide repeats as other heterochromatin. The distribution and function of these sequences are likely to result from both their repetitious nature and base contents.

Animals↗
PubMed1988

Sensitive homologous recombination strand-transfer assay: partial purification of a Drosophila melanogaster enzyme and detection of sequence effects on the strand-transfer activity of RecA protein.

A sensitive homologous recombination strand-transfer assay is described that employs short radiolabeled double-stranded DNA fragments from the lac/polylinker region of plasmid pUC18 and (+)viral M13mp18 single-stranded DNA as substrates. Substitution of a short radiolabeled double-stranded fragment for full-length linear M13 double-stranded DNA results in an assay whose sensitivity is improved greater than 8-fold. In addition, it is less sensitive to interference from nucleases or ligases than previous assays. The assay was used to partially purify an ATP-independent strand-transfer activity from a crude nuclear extract of Drosophila melanogaster embryos. We have also tested the efficiency with which various short double-stranded DNA segments are assembled into plectonemic joints by RecA protein with this assay and found 5- to 10-fold differences. These results are interpreted as evidence for DNA sequence-specific effects in RecA-mediated homologous pairing in vitro.

Animals↗
PubMed1987

(dC-dA)n.(dG-dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function.

In situ hybridization of (dC-dA)n.(dG-dT)n to the polytene chromosomes of Drosophila melanogaster reveals a clearly non-random distribution of chromosomal sites for this sequence. Sites are distributed over most euchromatic regions but the density of sites along the X chromosome is significantly higher than the density over the autosomes. All autosomes show approximately equal levels of hybridization except chromosome 4 which has no detectable stretches of (dC-dA)n.(dG-dT)n. Another striking feature is the lack of hybridization of the beta-heterochromatin of the chromocenter. The specific sites are conserved between different strains of D. melanogaster. The same overall chromosomal pattern of hybridization is seen for the other Drosophila species studied, including D. simulans, a sibling species with a much lower content of middle repetitive DNA, and D. virilis, a distantly related species. The evolutionary conservation of the distribution of (dC-dA)n.(dG-dT)n suggests that these sequences are of functional importance. The distribution patterns seen for D. pseudoobscura and D. miranda raise interesting speculations about function. In these species a chromosome equivalent to an autosomal arm of D. melanogaster has been translocated onto the X chromosome and acquired dosage compensation. In each species the new arm of the X also has a higher density of (dC-dA)n.(dG-dT)n similar to that seen on other X chromosomes. In addition to correlations with dosage compensation, the depletion of (dC-dA)n.(dG-dT)n in beta-heterochromatin and chromosome 4 may also be related to the fact that these regions do not normally undergo meiotic recombination.

Animals↗
PubMed1986

Analysis of Z-DNA in fixed polytene chromosomes with monoclonal antibodies that show base sequence-dependent selectivity in reactions with supercoiled plasmids and polynucleotides.

Five monoclonal anti-Z-DNA antibodies were characterized with respect to their binding of synthetic nucleic acid polymers and of supercoiled circular plasmid DNA. All of the antibodies reacted only with DNA in the Z-conformation; however, they fell into two classes on the basis of sequence specificity. One class, with broad specificity, reacted well with all sequences in the Z-form, including poly(dG-dC), poly(dG-dm5C), and poly (dG-dBr5C) in linear polymers and poly(dG-dC)n and poly[(dC-dA)n.(dT-dG)n] sequences in supercoiled plasmids. The other class bound only Z-DNA formed by poly(dG-dC). Binding of the monoclonal antibodies specifically to inserts of Z-DNA-forming sequences in plasmids was mapped directly by cross-linking of antibody to the DNA, digestion with restriction nuclease, and electrophoretic analysis of both the unbound fragments and the bound fragments recovered from immune complexes. The monoclonal antibodies were used for indirect immunofluorescence staining of Drosophila polytene chromosomes fixed by two procedures. One procedure yielded chromosomes with Z-specific antibody binding in many interbands, a few specific bands, and parts of some puffs. On chromosomes fixed by the second procedure, antibody staining appeared to follow the DNA concentration, staining all bands brightly. For each fixation procedure, chromosomes showed the same staining pattern with each of the broad specificity monoclonal antibodies that had been seen with polyclonal antibodies. The antibodies that reacted only with poly(dG-dC) and poly (dG-dC)n plasmid inserts did not stain chromosomes fixed by either protocol. We conclude that stretches of poly(dG-dC)n sequences do not contribute significantly to the presence of Z-DNA in fixed polytene chromosomes of Drosophila melanogaster.

Animals↗
PubMed1983

Chromatin structure in pre- and postblastula embryos of Drosophila.

Early in embryogenesis of Drosophila melanogaster, DNA synthesis is extremely rapid while RNA synthesis is virtually undetectable. We have examined the chromatin structure of nuclei from preblastula embryos to determine whether these unusual rates of replication and transcription correlate with any alteration in the chromatin. DNase I-hypersensitive sites at the 5' end of genes have been postulated to be necessary but not sufficient for activity of the associated gene and have been shown to be established prior to the onset of transcription. In order to ascertain whether the apparent transcriptional incompetence of the early embryos is the result of the absence of such chromatin structure, we have examined nuclei from cleavage-stage embryos to determine whether the DNase I-hypersensitive sites have been established. A variety of genes, including inducible heat-shock genes, a constitutively expressed ribosomal protein gene, and two developmentally regulated genes, have been examined. In every case the pattern of DNase I-hypersensitive sites in preblastula embryos duplicates that in the later (6-18 hr after oviposition) embryos. In addition, two extra sites are observed in the early embryos, one at the 5' end of the hsp 70 gene and one in a gene at chromosomal locus 67B1. These sites do not correlate with any known function; however, neither can functional significance be ruled out. In a further investigation of the chromatin structure of early embryos, a nucleosomal array was generated. The pattern produced from nuclei of early embryos is extremely similar to that from 6- to 18-hr embryos, but somewhat less distinct. Both nucleosomal arrays and DNase I-hypersensitive sites must therefore be established very rapidly following DNA replication. The chromatin structure of cleavage-stage embryos detected by these tests appears to be essentially the same as that of older embryos, both in general, and at specific loci.

Animals↗
PubMed1982

Nucleosome repeat structure is present in native salivary chromosomes of Drosophila melanogaster.

The regularly repeating periodic nucleosome organization is clearly resolved in the chromatin of the isolated salivary chromosomes of Drosophila melanogaster. A new microsurgical procedure of isolation in buffer A of Hewish and Burgoyne (1973, Biochem. Biophys. Res. Commun., 52:504-510) yielded native Drosophila salivary chromosomes. These chromosomes were then swollen and spread by a modified Miller procedure, stained or shadowed, and examined in the electron microscope. Individual nucleoprotein fibers were resolved with regularly repeated nucleosomes of approximately 10 nm diameter. Micrococcal nuclease digestion of isolated salivary nuclei gave a family of DNA fragments characteristic of nucleosomes for total chromatin, 5S gene, and simple satellite (rho = 1.688 g/cm3) sequences.

Animals↗
PubMed1981

DNase I hypersensitive sites in Drosophila chromatin occur at the 5' ends of regions of transcription.

By using a map of the unique region of DNA encoding the fur small heat-shock proteins of Drosophila melanogaster (hsp 22, hsp 23, hsp 26, and hsp 28), and a simple mapping technique, the positions of the DNase I hypersensitive sites of chromatin in the vicinity of these genes have now been determined. The major chromatin-specific sites occur at the 5' ends of each of the four heat-shock protein genes in embryo nuclei. These genes are not active in the nuclei analyzed but can be quickly induced in these cells by the heat-shock stimulus. The chromatin structure indicated by DNase I hypersensitivity may be a necessary factor in the general mechanism of gene activation.

Animals↗
PubMed1979

Synthesis and turnover of globin mRNA in murine erythroleukemia cells induced with hemin.

When murine erythroleukemia (MEL) cells are induced with hemin, they carry out several early functions of the erythroid program. However, they do not become committed to terminal differentiation nor do they become benzidine positive. This is in contrast to MEL cells induced with dimethyl sulfoxide (Me(2)SO) which undergo a more complete program of erythroid differentiation. In order to determine the relationship between commitment and various events in the erythroid program, we compared the induction of MEL cells with hemin and with Me(2)SO. The amount of globin mRNA accumulated in inducing MEL cells and the rate of its synthesis and turnover were quantitated. Although MEL cells induced with hemin accumulated significantly less globin mRNA than did cells induced with Me(2)SO, the rate of synthesis of globin mRNA was the same in fully induced cells, irrespective of inducer. Therefore, there is no evidence that induction with hemin produces an early program that is different or altered from that which is part of Me(2)SO induction. MEL cells induced with Me(2)SO specifically destabilize their globin mRNA after 4 days of induction. This raises the question of whether this destabilization of globin mRNA is an independently programmed late event, as suggested by the time of its occurrence, or, alternatively, whether it might be the inevitable consequence of an early event(s). For instance, destabilization might be linked to the synthesis or translation of globin mRNA. Because MEL cells induced with hemin do not destabilize their globin mRNA, we have concluded that this turnover of globin mRNA is a late event, occurring only in a committed cell population.

Animals↗
PubMed1978

A change in the stability of globin mRNA during the induction of murine erythroleukemia cells.

The stability of globin mRNA in murine erythroleukemia cells (Friend cells) before and during DMSO-induced differentiation was investigated. Cells were exposed to 3H-uridine for 2 hr and then transferred to medium without the radioactive precursor. The loss of radioactivity in total RNA, poly(A)-containing RNA and globin mRNA was followed. The globin mRNA was isolated using a highly specific globin cDNA column. In uninduced cells and cells early in differentiation, the globin mRNA decays with a half-life of less than 50 hr. After 4 days of induction, the globin mRNA decays with a half-life of 17 hr, demonstrating a change in stability during the induction process. Although the stability of globin mRNA changes during induction, this is not true for total poly(A)-containing RNA. At all times of induction, the poly(A)-containing RNA decays as two populations, one with a half-life of 6 hr and the other with a half-life of 36 hr. The half-life of the rRNA also remains unchanged during differentiation.

Cell Differentiation↗