Molecular mechanisms of the origin of chromosome aberrations and the structural organisation of eukaryotic DNA

Summary A new hypothesis on the appearance of exchange chromosomal aberrations has been suggested. According to this hypothesis, temporal duplex polynucleotide structure should arise during G₁ and G₂ phases during the correction of DNA. The size of the duplex, as a rule, should be restricted to the size of complementary nucleotide sequences in the regions of repetitions. Any polynucleotide break in a duplex zone would result in chromosome breakage and if complementary broken ends interact with each other, then exchange chromosome aberrations may be formed. This hypothesis would explain such previously obscure phenomena as extremely high frequencies of exchanges after mutagen treatment, the nature of mitotic crossing-over, negative interference, change of aberration types before replication, the low frequency of damaged structural genes during aberration formation, etc.     

A negative image of the quiescent centre in regenerating root apices of Zea mays

Usually the presence of the quiescent centre in roots is demonstrated by the absence of labelled nuclei following treatment of the root with appropriate radioactive markers. By modification of the pulselabelling technique, a negative image of the quiescent center, showing more intense labelling from [³H]thymidine than the surrounding area, was obtained in regenerating root apices of Zea mays L.     

Cytophotometric investigation of DNA and RNA content in nuclei of active Strasburger cells in Pinus nigra var. austriaca (Hoess) Badoux

The nuclei of active, sieve cell-associated Strasburger cells in the secondary phloem of Pinus nigra var. austriaca (Hoess) Badoux have been studied for their structure and DNA and RNA content. No difference in size compared to those of ordinary ray cells was found. The nuclear surface is often increased by an ameboid or lobed shape. The amount of highly decondensed chromatin is greatly increased. Cytophotometric measurements of DNA content of both Feulgen and gallocyanine chromalum-stained nuclei showed normal DNA levels and proved absence of endomitotic polyploidization. RNA content, however, was significantly increased as compared to nuclei of young Strasburger cells and of ordinary ray parenchyma cells.Abbreviations StC₁ Strasburger cells in contact with young and immature sieve cells - StC₂ Srasburger cells in contact with mature and functionally active sieve cells - StC₃ dead Strasburger cells - eRPC pRPC erect and procumbent ray parenohyma cells, respectively - GCCA gallocyanine chromalum - T transmission - A absorbance Dedicated to Professor Dr. Wilhelm Halbsguth, Kiel, on the occation of his 65th birthday     

Precise localization of the origin of replication in a physical map of HeLa cell mitochondrial DNA and isolation of a small fragment that contains it

The precise positions of the origin of replication³ and of the D-loop within the HpaII restriction map of HeLa cell mitochondrial DNA have been investigated. For this purpose, 7 S DNA, which is the heavy-chain initiation sequence, was used as a template for fragment-primed DNA synthesis by Escherichia coli DNA polymerase I. The results indicate clearly that the origin of replication lies in HpaII fragment 8 at about 80 base-pairs from the border with fragment 17, and that the D-loop region extends from this site, through fragment 17, to a position in fragment 10 which is about 365 base-pairs from the border with fragment 17. Sequential digestion of fragment 8 with HaeIII enzyme has allowed the isolation of a subfragment, about 200 base-pairs long, that contains the origin of replication.     

Discontinuous DNA replication: accumulation of Simian virus 40 DNA at specific stages in its replication.

The number of proline residues in a protein should have very marked consequences for the rates of protein unfolding and refolding according to the model proposed by Brandts et al. (1975). Kinetic simulations of this model indicate that the half-time for refolding of a polypeptide chain with 20 proline residues should be greater than 10 minutes and should increase by about an order of magnitude for each additional 10 proline residues. Various means are considered by which the rate of protein folding in vivo and in vitro might be increased.     

Timing and Regulation of Nuclear and Cortical Events in the Cell Cycle of Tetrahymena pyriformis*

SYNOPSIS. Using continuous flow cultures based on the chemostat principle, we varied the cell generation times of the ciliate Tetrahymena pyriformis strain GL, from 4.9 to 22.2 hr and studied various parameters of the cell cycle at 28 C. These included: the duration of the periods required for oral morphogenesis, macronuclear division, cell division, G₁ S, and G₂. The size of individual cells was also measured. Independent of the growth rate, the period of oral morphogenesis occurred during the last 90 min of the cell cycle. In all cases macronuclear and cell divisions took place during the last part of these 90 min, and the final macronuclear separation occurred just before final cell separation. The S-period increased slightly, while the G₁ and G₂ both increased in roughly the same relative proportion to the increasing generation times. Slowly growing cells (generation time 20.5 hr) were shorter but broader and somewhat larger in volume than quickly growing cells (generation time 4.9 hr).     

DNA Throughout the Single Mitochondrion of a Kinetoplastid Flagellate: Observations on the Ultrastructure of Cryptobia vaginalis (Hesse, 1910)

SYNOPSIS. Cryptobia vaginalis (Hesse 1910) occurs as long thin and short broad forms in the vagina of the gnathobdelliform leeches Haemopis sanguisuga (Linnaeus) and Hirudo medicinalis Linnaeus. Cytochemical staining for DNA and transmission electron microscopy of sectioned material indicate that in the thin forms the kinetoplast DNA (kDNA) is dispersed irregularly through the mitochondrial network ( pankinetoplastic condition) rather than concentrated in the adbasal region of the mitochondrion ( eukinetoplastic condition) as in trypanosomatids and most other kinetoplastid flagellates. Light-microscopic studies on the rare broad forms, however, suggest that these have conventional adbasal location of the kinetoplast. Binary fission appears to occur in the thin forms, suggesting that the dispersed kinetoplast is either highly polyenergid or lacks a genetic function. In other features of its microanatomy, C. vaginalis is a conventional kinetoplastid. The flagellate has an incomplete corset of pellicular microtubules which may have a role in the cortical contractility characteristic of the genus Cryptobia . Feeding is by pinocytosis of vaginal colloids through a microtubule-lined cytopharynx, possibly after binding to a prominent filament-coated preoral ridge. A pulsatile (contractile) vacuole is present and appears to be responsible for defecation as well as osmoregulation. Some individuals have elongate bacterial epibionts attached to the body in parallel with the cortical microtubules. All individuals have 2–8 spheroplast-like endobiotic bacteria in the prenuclear cytoplasm.     

Use of lambda phasmids for deletion mapping of non-selectable markers cloned in plasmids

A nonselectable gene carried on a poorly selectable recombinant plasmid has been physically mapped by deletion analysis. Our method involved cloning the plasmid into a coliphage lambda vector and treating the recombinant phage with a chelator. Virtually all particles surviving this treatment carried large deletions within the plasmid insert. Further deletion analysis was done by inserting a selectable lambda sequence into one such deletion derivative and repeating the chelator selection. Chelator selection was also used to isolate deletions constructed in vitro. The deleted phage are readily characterized by restriction mapping, and the gene in question scored after infection of a mutant host strain. These techniques have enabled us to physically assign the cyclopropane fatty acid synthase gene of Escherichia coli to 0.8 kb of a 16-kb segment after characterizing only a small number of isolates. This approach should be generally useful in the mapping of plasmids for which no convenient method exists for selecting or scoring the gene in question.     

Immune response to human influenza virus hemagglutinin expressed in Escherichia coli

Cloned DNA fragments coding for parts of strain WSN (H1N1) influenza virus hemagglutinin (HA) were fused to a bacterial leader DNA derived from the Escherichia coli trp operon. Fusion proteins produced consisted of 190 amino acids of trpLE' protein at the amino terminus, and HA amino acids, either 1-308, 1-396, or 1-548 (complete HA), at the carboxyl terminus. These proteins were expressed at high levels (10-20% of total protein) in E. coli starved for tryptophan. A CNBr fragment (HA1-211) was derived from HA-308. Each of the proteins was purified and used for immunizing mice and rabbits. The antibody produced was shown to bind to (i) the HA fusion proteins, (ii) detergent-treated viral HA, (iii) HA, on intact virions, and (iv) the HA on the surface of cells infected with influenza virus. This shows that the HA fusion proteins expressed in bacteria can elicit antibodies that recognize at least some determinants of the native viral HA, and probably could lead to development of an anti-influenza vaccine.