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Dendritic molecules are highly-branched arborescent structures and have found applications as chemical reagents, lubricants, contrast media for magnetic resonance, and others. Dendritic nucleic acids could be extremely useful for the development of nucleic acid diagnostics as signal amplification tools and potentially as drug (antisense) delivery vehicles. Further, due to the relatively large size of nucleic acid molecules, nucleic acid dendrimers could be readily labeled with numerous fluorescent compounds and/or protein moieties with limited steric hindrance and/or quenching. Herein, we present a physical-mathematical model of a new class of dendrimers, constructed entirely from unique nucleic acid monomers that are designed such that sequential hybridization adds successive layers of monomer in a geometric expansion of both mass and free single-stranded sequences, called arms, at the surface. The specially designed monomer is a heterodimer of two single-stranded nucleic acid oligomers possessing a central double-stranded waist and four single-stranded arms for binding. Assembly of a dendrimer is initiated from a single monomer and proceeds in layers, the first comprising four monomers, which provides 12 single-stranded arms. Thus, the second layer adds 12 monomers resulting in 36 single-stranded arms. After addition of the 6th layer, the dendrimer is comprised of 1457 monomers, of which 972 reside in the 6th layer, which possesses 2916 single-stranded arms. The accompanying mathematical description of a dendrimer's growth is generic. A natural consequence and limiting condition of the growth process we describe is a saturated solution of nucleic acid, which is, in effect, a "nucleic acid membrane".

HeLa cell 5 S ribosomal RNA, labeled in vitro at 40°, 60°, or 70°C with (125)I, has been digested with RNase T1 and fingerprinted by conventional methods. Regardless of the temperature of iodination, (125)I was bound (exclusively) to virtually all CMP residues. Characterization of labeled oligonucleotides yielded sequence information consistent with that derived from in vivo labeled RNA.

The feline oncornavirus-associated cell membrane antigen (FOCMA) was defined as a tumor antigen common to cat lymphomas and fibrosarcomas induced by feline leukemia virus (FeLV) and feline sarcoma virus (FeSV), respectively. The antigen was recognized by sera from cats thought to be resistant to leukemogenesis. We report here that a common denominator in the activity of naturally occurring viremic cat antisera to FOCMA is, in fact, their reactivity to FeLV C antigenic determinants. The cat antisera, monoclonal antibodies to FOCMA, and monoclonal antibodies to FeLV C, all reacted in immunofluorescence assays with FeLV C-infected cells and immunoprecipitated a molecule electrophoretically indistinguishable from envelope glycoprotein of FeLV. Viremic cat antisera to FOCMA bound to budding virus particles of FeLV C-infected cells, even though some of them could not be absorbed by mature virion proteins. Thus, the unusual feature of cat antibodies to FOCMA is their binding to nascent but not to mature virus particles. FOCMA-positive cat lymphomas expressed antigenic determinants of FeLV-C gp70, with or without productive infection. FeLV-negative tumors not expressing FeLV C gp70 were also FOCMA negative. Furthermore, most of the viremic cat sera and the monoclonal antibodies to FOCMA did not react with FeSV-transformed nonproducer cells. The absence of FOCMA from these cells and from FeLV-negative lymphoid tumors and its presence in FeLV-C infected fibroblasts indicated that this antigen is virus encoded and not a cellular tumor-specific antigen.

Globin mRNA isolated from a number of beta0 thalassemia patients of different ethnic origins was analyzed by RNA-cDNA hybridization and, in two cases, by fingerprint analysis of 125I-labeled mRNA. Quantitation of the relative amounts of alpha- and beta-mRNA by hybridization to purified alpha-and beta-cDNA revealed that in approximately half the cases, there was less than 1% as much beta-mRNA as alpha-mRNA. In the rest of the cases, low levels of beta-like mRNA were detected in amounts 4-12% as abundant as alpha-mRNA. There was variability in the yield of beta-like mRNA in patients of the same racial group, in the same patient at different times and in similarly affected siblings: beta-mRNA was virtually absent in some samples, whereas low but significant levels were found in other samples. In one patient, beta-like mRNA was not detected in peripheral blood RNA, but was present in the RNA of bone marrow cells. In one case, the thermal stability of the beta0 thalassemia mRNA-beta-cDNA hybrid was measured and found to be slightly lower than that of the authentic beta-mRNA-beta-cDNA hybrid. In none of the cases tested was there synthesis of beta-globin chains directed by beta0 thalassemia mRNA in a cell-free protein-synthesizing system, even when beta-like mRNA was detected in the sample by hybridization assays. mRNA from two patients was labeled in vitro with 125I, digested with T1 RNAase and fractionated in two dimensions. Analysis of the resulting fingerprints revealed the presence of prominent alpha chain-specific oligonucleotides without detectable beta chain-specific oligonucleotides, and thereby confirmed the results of hybridization assays showing absent or very low levels of beta-mRNA in the same RNA samples. Our results support the concept that beta0 thalassemia is heterogeneous in its molecular basis even within the same racial group: in some patients, it is associated with absent beta globin mRNA, whereas in other patients, it is associated with low but significant levels of nonfunctional beta or beta-like globin mRNA. The variable amounts of beta-like mRNA detected in different samples from the same patient, and in patients with the same genotype, indicate that as yet undefined factors can influence the yield of beta-like mRNA observed in beta0 thalassemia.

A long-acting thymidine pellet consisting of 190 mg of cholesterol and 60 mg of thymidine has been developed for the study of thymidine metabolism and reutilization in vivo. Implantation of such a pellet s.c. in adult mice will maintain the blood plasma concentration of thymidine at levels between 40 and 8 X 10(-6) M, which are from 36 to 7 times those of normal mice, for periods up to 48 hr. During this period, in vivo uptake and reutilization of [125I]iododeoxyuridine, a thymidine analog, into intestinal and tumor DNA were almost completely suppressed. While iododeoxyuridine reutilization is not large in normal proliferative tissue even in the absence of pellet implants, reutilization of over 30% was measured in large, rapidly growing ascites tumors. The inhibition of iododeoxyuridine incorporation by elevated thymidine blood levels is directly proportional to serum concentration. This appears to be due to a thymidine pool in rapid equilibrium with blood thymidine. This pool is at least 10 times larger than the 4-nmole pool of extracellular thymidine.

An H4-IIE-C3 hepatoma cell line derived from an ACI rat has been shown to have differentially stained regions attached to the short arms of chromosomes 3, 11 and 13 and the long arm of an unidentified small chromosome. There is cell to cell variability in the number and size of the differentially stained regions, which contain, on the average, about 5% of the total DNA. A series of secondary constrictions occur at intervals along the length of each differentially stained region. These stain with silver by the Ag-AS method, indicating that the differentially stained regions contain sites of active 45S ribosomal precursor RNA transcription. In situ hybridization to metaphase chromosomes shows that the hepatoma cells have a 10 fold increase in DNA coding for 18S and 28S ribosomal RNA, 90% of it located in the differentially stained regions, and no change in the number of genes coding for 5S RNA. These results have been confirmed by filter disc hybridization.

Metaphase chromosomal aberrations were produced by 125I-labeled iododeoxyuridine (125I-UdR) incorporated into Chinese hamster Don cells at the end of the S-period of the cell cycle. Chromosome damage and the number of autoradiographic silver grains were recorded for whole cells, for chromosome pairs No. 4 and No. 5, and for the X and the Y chromosomes. The X and the Y chromosomes, which label late in S, were at least twice as heavily labeled as chromosome pairs No. 4 and No. 5--two readily recognizable autosomes of similar size. The incidence of chromosome damage was at least six times that which would have been expected from equivalent doses of X-rays and the incidence of damage was directly related to the number of silver grains over each chromosome. We estimate that it takes four to ten disintegrations to produce a visible chromosome aberration. The finding that chromosome damage is localized at the site of the 125I decay is most readily explained by the high flux of low energy Auger electrons occurring at the site of the decay of the incorporated 125I atom.

Cessation of mitosis was brought about in Vicia faba roots incubated for 24 hours in the thymine analogue, 5-aminouracil. Recovery of mitotic activity began 8 hours after removal from 5-aminouracil and reached a peak at 15 hours. If colchicine was added 4 hours before the peak of mitoses, up to 80 per cent of all cells accumulated in mitotic division stages. By use of single and double labeling techniques, it was shown that synchrony of cell divisions resulted from depression in the rate of DNA synthesis by 5-aminouracil, which brought about an accumulation of cells in the S phase of the cell cycle. Treatment with 5-aminouracil may have also caused a delay in the rate of exit of cells from the G(2) period. It appeared to have no effect on the duration of the G(1) period. When roots were removed from 5-aminouracil, DNA synthesis resumed in all cells in the S phase. Although thymidine antagonized the effects of 5-aminouracil, an exogenous supply of it was not necessary for the resumption of DNA synthesis, as shown by incorporation studies with tritiated deoxycytidine.

Carrier-free iodination of 5S RNA with (125)I yields a probe suitable for use in cytological RNA-DNA hybridization studies. The genes coding for 5S ribosomal RNA in Drosophila could be localized by autoradiography after a 2-day exposure, whereas a 2-month exposure is needed when the best available [(3)H]RNA is used. The procedure introduces one covalently bound (125)I atom per 100 nucleotides, resulting in a specific activity of over 10(8) dpm/mug of RNA. The method may be readily applied to other problems involving molecular hybridization techniques.

Several techniques of RNA and DNA finger-printing and determination of sequence have been applied to nucleic acids labeled with (125)I. Fingerprints of human 5S RNA and bacteriophage f2 RNA resemble those of their noniodinated counterparts both in complexity and in specific pattern. Iodination as used here is thus a general labeling procedure, and appears principally to label cytidine residues. This iodination method shows little sensitivity to potential structure in single-stranded RNA molecules, yields stable oligonucleotide products in a reproducible manner, and does not change the specificity of several ribonucleases and deoxyribonucleases.