Chromatin organization in the oomycete Achlya ambisexualis.

Nuclei from the O?mycete Achlya ambisexualis and rabbit kidney nuclei were digested with micrococcal nuclease and the resultant DNA fragments analyzed on slab gels. The average DNA repeat size was found to be 159 +/- 1.2 base pairs for Achlya and 199.8 +/- 3.7 base pairs for rabbit kidney. The presence of a DNA repeat size of 159 base pairs for Achlya extends the characterization of eukaryotic chromatins to this most primitive and perhaps unique microbe.     

Deoxyribonucleic acid sequence organization of a yeast plasmid.

Two-micrometer deoxyribonucleic acid (DNA), a circular plasmid of Saccharomyces cerevisiae, contains two nontandem repeated sequences which are inverted with respect to one another. These repeated sequences together account for 21% of the molecule length. Restriction endonuclease analysis and electron microscopy demonstrated the existence of two forms of 2-mum DNA differing in the orientation of the interstitial segments bounded by the inverted repeated sequences. The two forms of 2-mum DNA could result from an intramolecular reciprocal recombination between inverted repeat elements. A map containing the restriction endonuclease sites and the units of the inverted repeat has been constructed.     

Analysis of deoxyribonucleic acid replication in human X chromosomes by fluorescence microscopy.

The genetically inactive, late-replicating human female X chromosome can be effectively distinguished from its more active, earlier-replicating homologue, when cells are grown according to the appropriate BrdU-33258 Hoechst protocol. Results obtained from a fluorescence analysis of DNA replication in X chromosomes are consistent with those from previous autoradiographic studies, but reflect additional sensitivity and resolution offered by the BrdU-Hoechst methodology. Both qualitative and quantitative differences in 33258 Hoechst fluorescence intensity, reflecting alterations in replication kinetics, can be detected between the two X chromosomes in female cells. The pattern of replication in the single X chromosome in male cells is indistinguishable from that of the early female X. Intercellular fluctuations in the distribution of regions replicating early or late in S phase, particularly with reference to the late female X, can be localized to structural bands, suggesting multifocal control of DNA synthesis in X chromosomes.     

Homology between the invertible deoxyribonucleic acid sequence that controls flagellar-phase variation in Salmonella sp. and deoxyribonucleic acid sequences in other organisms.

The invertible deoxyribonucleic acid (DNA) segment cloned from Salmonella sp. was radioactively labeled and used as a probe to search for homologous sequences by Southern hybridization. Only one copy of the invertible segment could be found on the Salmonella sp. genome. Partial sequence homology with the invertible region was detected in bacteriophage Mu and P1 DNA by low-stringency hybridization. Under these conditions, no homology was detected with Escherichia coli DNA. A strain of Salmonella sp. defective in phase variation carrying the vH2- allele was also analyzed by DNA-DNA hybridization. The results show that there is sequence divergence between diphasic and vH2- strains within the invertible sequence.     

DNA sequence organization in soybean investigated by electron microscopy

An electron microscopic analysis of the DNA sequence organization in the soybean genome is reported. This analysis employed the gene 32 proteinethidium bromide spreading technique, a procedure which produces striking contrast between double and single-stranded DNA regions. To investigate the arrangement of repetitive sequences differing in genomic frequency, three kinetic fractions of 5 kb DNA fragments were isolated by reassociation and hydroxyapatite chromatography. Renatured structures in each fraction were then visualized in the electron microscope. The majority of repeated sequences, irrespective of frequency, were shown to be relatively non-divergent, to exceed 1.5 kbp in length (number-average), and to be organized primarily into long regularly repeating tandem or clustered arrays. Duplex regions >5 kbp were commonly visualized. A small fraction of low frequency repeats (<100 copies per genome), however, was observed to have a distinctly different form of arrangement. These repeats averaged 0.2 kbp in length, contained divergent sequences, and were contiguous to single copy DNA sequences having an average length of 1.15 kbp. Repeats which flanked a given single copy sequence did not appear to be homologous. Neither short clustered permuted repeats nor interspersion of repeats which differed significantly in reiteration frequency were found to be major features of soybean genome organization.     

Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis.

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.     

Sequence organization in nuclear deoxyribonucleic acid from Physarum polycephalum. Physical properties of foldback sequences.

An investigation was performed with the use of physical techniques, to determine the nature and organization of inverted repeat sequences in nuclear DNA fragments from Physarum polycephalum. From the average size of foldback duplexes (550 nucleotide pairs), and the foldback duplex yield as determined by treatment of DNA with S1 deoxyribonuclease followed by hydroxyapatite chromatography, it is estimated that there are at least 25000 foldback sequences in the Physarum genome. Foldback DNA molecules exhibit properties intermediate between single-stranded DNA and native duplexes on elution from hydroxyapatite with a salt gradient. In addition, thermal-elution chromatography of foldback DNA from hydroxyapatite crystals shows that foldback duplexes are less stable than native DNA. These properties can be explained on the basis that inverted repeat sequences are mismatched when in the foldback configuration. The results of experiments in which the binding of foldback DNA molecules to hydroxyapatite was determined, by using fragments of different single-chain size, agree with previous studies indicating that inverted repeat sequences are located, on average, every 7000 residues throughout the Physarum genome. The inverted repeats are derived from both the repetitive and single-copy components in Physarum nuclear DNA.     

Determination of the size of rat ribosomal deoxyribonucleic acid repeating units by electron microscopy.

The empoyment of a novel method of affinity chromatography, which makes use of antibodies that specifically bind DNA/RNA hybrids, has made it possible to enrich for rat rDNA molecules which contain R loops formed with the 18S and 28S rRNAs. An approximately 150-fold enrichment of the ratrRNA coding sequences was obtained by this affinity chromatography procedure. This degree of enrichment made it possible to visualize these R loop containing molecules in the electron microscope and, thus, to obtain a map of the transcribed and spacer regions of rat rDNA. Eleven of the molecules that were observed contained either 3 or 4 R loops, or else 2 R loops separated by a long spacer. Thus, these molecules provided direct information in regard to the length of rat rDNA repeating units. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. Within the errors of the measurements, these could all represent repeating units of exactly the same length, although a certain degree of length heterogeneity, possibly up to 4 or 5 kbp, cannot be ruled out by the data. If significantly longer or shorter rDNA repeating units exist in the rat genome, they are probably much less common than the 37.2 kbp unit. These electron microscopic measurements provide the most definitive data yet available on the size of the repeating units of mammalian rRNA genes.