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I L Cartwright

Publications and source records attributed to I L Cartwright.

27 records · Page 2Linked to original sources

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↗

Cleavage of chromatin with methidiumpropyl-EDTA . iron(II).

Methidiumpropyl-EDTA . iron(II) [MPE . Fe (II)] cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease. Moreover, digestions with MPE . Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases. Because of these properties MPE . Fe(II) would appear to be a superior tool for probing chromatin structure. We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE . Fe(II) and micrococcal nuclease cleavage. MPE . Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some double-stranded) cleavages in chromatin DNA. Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca. 190 base pairs. Under suitable conditions, relatively little other nicking is observed. Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE . Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion. The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats. In the case of the histone gene sequences a faint, but detectable, MPE . Fe(II) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern. The results indicate that MPE . Fe(II) will be very useful in the analysis of chromatin structure.

Animals↗

Analysis of chromatin structure and DNA sequence organization: use of the 1,10-phenanthroline-cuprous complex.

Limited treatment of Drosophila nuclei with the 1,10-phenanthroline-cuprous complex leads to rapid production of nucleosomal ladders indistinguishable from those obtained by micrococcal nuclease digestion. An investigation of the preferential sites of cleavage of protein-free DNA at locus 67B1 surprisingly indicated that both reagents recognized very similar features. Thus, a virtually identical pattern of preferential cleavages was generated over a 12 kb fragment encoding four transcripts at this locus. The distribution of cleavage sites was highly non-random, with major sites falling in the spacers between the genes. Both reagents cleaved certain chromatin-specific sites near the 5' ends of the genes. However, an analysis of preferential cleavages at the sequence level did not reveal the same close correspondence. We suggest that both reagents can recognize some localized secondary structural features of the DNA and that the particular distribution of sequences present at this locus results in a distinctive pattern of cleavage sites that delineates gene and spacer segments.

Animals↗

Azidopolynucleotides as photoaffinity reagents.

Polynucleotides containing adenosine and 8-azidoadenosine or inosine and 8-azidoinosine residues have been prepared from mixtures of nucleoside diphosphates using polynucleotide phosphorylase from Escherichia coli. These copolymers can form complexes with polyuridylic or polycytidylic acids respectively. Single stranded poly(adenylic, 8-azidoadenylic acid) [poly(A,z8A)] has been used as a photoaffinity reagent to explore the subunit topography of RNA polymerase from E. coli.

Adenosine↗

Inhibition of papain by N-acyl-aminoacetaldehydes and N-acyl-aminopropanones. Evidence for hemithioacetal formation by a cross-saturation technique in nuclear-magnetic resonance spectroscopy.

N-Acyl-aminoacetaldehydes are potent inhibitors of the proteolytic enzyme, papain. Although they exist predominantly in their hydrated form in aqueous solution only the aldehyde is an effective inhibitor. The binding constants for related amides and methyl ketones confirm that it is principally the lower steric requirement of the aldehyde rather than its increased electrophilicity which is responsible for its powerful inhibitor properties. Using nuclear magnetic resonance spectroscopy, evidence is provided for an N-acetyl-aminoacetaldehyde-papain complex. Using a cross-saturation technique evidence is also provided for a hemithioacetal, formed from the aldehyde and the active-site thiol group. Hemithioacetal formation has also been detected between N-benzoyl-aminoacetaldehyde and papain. This provides the first direct evidence for a tetrahedral adduct with papain and supports the proposed involvement of such intermediates in papain-catalysed hydrolyses.

Acetaldehyde↗

A simple, rapid preparation of alpha[32P]-labelled adenosine diphosphate.

Hexokinase (EC 2.7.1.1) will convert commercially available alpha-[(32)P]-labelled ATP into alpha-[(32)P]-labelled ADP. A simple, rapid isolation procedure for the alpha-[(32)P]-labelled ADP is described and this synthetic method can be used for the preparation of other alpha-[(32)P]-labelled nucleoside diphosphates.

Adenosine Diphosphate↗

The reaction between thiols and 8-azidoadenosine derivatives.

Thiols react at room temperature in dilute solution with 8-azidoadenosine and its nucleotides to give the corresponding 8-aminoadenosine derivatives. The reaction which takes place in the dark is base-catalysed and is particularly rapid when dithiols, e.g. dithiothreitol are used.

Adenine Nucleotides↗