Multistranded Structures Research Articles

The splitting of human chromosomes into chromatids in the absence of either DNA or protein synthesis

If cells are irradiated during most of the G 1 portion of the cell cycle, chromosome aberrations are produced. If, however, they are irradiated while in S or G 2, chromatid aberrations are formed. The best estimate of when the transition occurs is late G 1. That is, in both plant and animal cells, the chromosome splits into chromatids before DNA synthesis. This doubling of the chromosome conceivably could be caused by either a synthesis of chromosomal protein prior to S, or by a loosening of a multistranded struture. Experiments were carried out with phytohemagglutinin-stimulated human lymphocytes to test which of the two possible explanations cited above would be the more likely. Phytohemagglutinin-stimulated lymphocytes were cultured in the presence of 2·10 −3 M hydroxyurea and triated thymidine for 40 h. The hydroxyurea was used to inhibit DNA synthesis and to enrich the population of cells at the end of G 1. Cells were then irradiated with 200 R of X-rays and recultured in the absence of tritium. When the cells reached metaphase, unlabeled cells were scored for the types of aberrations induced by the prior irradiation. Chromatid aberrations were found, indicating that in human cells, too, the split occurs in G 1 in the absence of DNA synthesis. Similar experiments in which the protein synthesis inhibitor cycloheximide was added after the cells were in culture for 16 h (before any DNA synthesis had occurred) and kept in until the fortieth hour showed that the transition occurred when protein synthesis was inhibited, too. The experiments indicate that the human somatic chromosome is a multistranded structure that loosens in late G 1 to form functional chromatids.

Requirement of a stable secondary structure for the antiviral activity of polynucleotides.

Several polyribonucleotides, both as single species and when annealed in pairs, are able to induce interferon production in mammalian cells and cellular resistance to virus infection. These studies suggest that a stable secondary, presumably multistranded structure is required for this activity.

Chromosome structure--a model based on DNA replication.

A model of chromosome structure is proposed which assumes: (i) that DNA replication is accomplished via right-hand (RH) rotation; and (ii), that where replicating DNA segments arc very long RH-rotation will not proceed with absolute freedom. It is expected that inhibition of rotation in daughter molecules will lead to the formation of left-hand-individual (LH-I) coiling systems in the two daughter molecules. It is also expected that inhibition of rotation in the parental molecule will cause the LH-I coiled daughters to be held together in a right-hand-relational (RH-R) association. The interaction between LH-I and RH-R coiling is expected to cause separation of the daughter molecules. Multistranded DNA-containing structures are expected to show, in addition to the LH-I coiling heirarchies formed within individual strands, an RH-R coiling heirarchy formed by the complex as a whole. An LH-I coiling heirarchy was looked for, and found, in Cleveland's drawings of flagellate chromosomes. Evidence for the existence of RH-R coiling was also found. Results of electron-microscope studies on chromosome structure were briefly examined, as were the structures of lampbrush chromosomes, salivary chromosomes and "normal" chromosomes. These studies provided additional, though less direct, evidence in favor of the replication hypothesis and the predictions developed from it.

The replicative organization of DNA in polytene chromosomes of Drosophila hydei.

Salivary-gland nuclei ofDrosophila hydei were pulse-labeledin vitro with3H-thymidine and studied autoradiographically in squash preparations. The distribution of radioactive label over the length of the polytene chromosomes was discontinuous in most of the labeled nuclei; in some nuclei the pattern of incorporation was continuous. Comparison of the various labeling patterns of homologous chromosome regions in different nuclei showed that specific replicating units are replicated in a specific order. By combining autoradiography with cytophotometry of Feulgen-stained chromosomes, it was possible to correlate thymidine labeling of specific bands with their DNA content. The resulting data indicate that during the S-period many or perhaps all of the replicating units in a salivary-gland nucleus start DNA synthesis simultaneously but complete it at different times. Furthermore, the data support the hypothesis that the chromomere is a unit of replication or replicon. The DNA content of haploid chromomeres was found to be about 5×10-4 pg for the largest bands inDrosophila hydei. From the results of H3-thymidine autoradiography and Feulgen-cytophotometry on neuroblast and anlage nuclei it was concluded that during growth of the polytenic nucleus heterochromatin is for the most part excluded from duplication. The results of DNA measurements in interbands of polytene chromosomes do not agree with a multistrand structure for the haploid chromatid. A chromosome model is proposed which is in accordance with the reported results and with current views concerning the replicative organization of chromosomes.

The Preparation and Properties of a Helix-rich Fraction Obtained by Partial Proteolysis of Low Sulfur S-Carboxymethylkerateine from Wool

Abstract 1. The low sulfur fraction of S-carboxymethyl wool keratin, which has an α-helix content of about 50% in aqueous solution, has been subjected to partial proteolysis with the enzyme Pronase P to yield an acid-precipitable fraction with a helix content of about 85%. 2. The optimum conditions for the preparation of this fraction have been studied with respect to time, temperature, pH, enzyme concentration, and Ca++ concentration. 3. In dilute solution the material appears to consist chiefly of particles with a molecular weight of about 41,000 and an axial ratio of 8:1 to 10:1. The dimensions are consistent with a multistranded, rod-like particle made up of probably three α-helical chains. 4. In 8 m urea, the particles break down irreversibly to a heterogeneous mixture, with molecular weights in the range 2000 to 5000, which suggests that peptide bonds are broken in the helical segment during proteolysis, but the integrity of the particle is maintained by its multistranded structure. Both gel chromatography and gel electrophoresis indicate that the helix-rich particle is derived from the major helical components of the low sulfur fraction of S-carboxymethyl wool keratin. 5. Amino acid analysis of the helix-rich fraction shows that, compared to the low sulfur fraction of S-carboxymethyl wool keratin, it is enriched in the helix-favoring residues lysine, leucine, alanine, and glutamic and aspartic acids and depleted in the helix-inhibiting residues S-carboxymethylcysteine, proline, serine, threonine, and glycine.

Enzymic synthesis of polynucleotides. III. Phosphorolysis of natural and synthetic ribopolynucleotides

1. 1. Ribopolynucleotides can be classified into three groups according to their susceptibility to cleavage by polynucleotide phosphorylase of Azotobacter vinelandii: ( a) rapidly phosphorolyzed; this group includes synthetic polynucleotides, such as polyadenylic or polyuridylic acid, containing only one kind of nucleotide; ( b) slowly phosphorolyzed; this group includes synthetic (poly AGUC), yeast, and bacterial RNA, as well as the aggregate (poly A + U) formed by mixing solutions of poly-adenylic and polyuridylic acid; ( c) phosphorolyzed at an intermediaterate; this is the case with tobacco mosaic virus RNA. The slow phosphorolysis of polynucleotides of group ( b) can be related to the fact that, contrary to those of group ( a), they consist largely of multistranded rather than single-stranded chains. In view of this, the intermediate rate of phosphorolysis of tobacco mosaic virus RNA might reflect a slight tendency of this compound to assume a multistranded structure. 2. 2. Adenylic acid tri- and tetranucleotides with 5′-phosphomonoester end groups, obtained by hydrolysis of polyadenylic acid with an enzyme from liver nuclei, are slowly phosphorolyzed by Azotobacter polynucleotide phosphorylase. 3. 3. The nucleoside 5′-diphosphates produced by phosphorolysis of tobacco mosaic virus RNA have been identified chromatographically.