Polyadenylated RNA complementary to repetitive DNA in mouse L-cells.

Complementary DNA, synthesized with L-cell polyadenylated RNA as template, renatured with total L-cell DNA to about 70%. About 30% complementary to unique sequence DNA and another 10 and 30% corresponded to sequences about 20- and 500-fold repetitive. Complementary DNA was fractionated after partial hybridization with total polyadenylated RNA to obtain preparations enriched or impoverished in complements of the most frequent polyadenylated RNA. Renaturation of these complementary DNA fractions with L-cell DNA revealed that most frequent RNAs are transcribed from repetitive DNA sequences, Complementary DNA, density labeled with bromodeoxyuridine, was fractionated by renaturation with L-cell DNA to yield fractions enriched in repetitive and unique sequence DNA. The denisty labeled complementary DNA was purified by equilibrium centrifiguation in an alkaline Cs2SO4 gradient. The complementary DNA representing mainly repetitive DNA sequences hybridized preferentially to frequent polyadenylated RNA.     

Structural organization of alpha-satellite DNA in a single monkey chromosome

The organization of α-satellite sequences in a single monkey chromosome has been studied by restriction endonuclease analysis and molecular cloning. A somatic cell hybrid containing the monkey chromosome was isolated by cloning after fusion of the mouse L-cell line B82 (thymidine kinase minus) with primary African green monkey kidney cells and selective growth in HAT medium. Unlike the mouse cells, the hybrid cells contain DNA that hybridizes with the α-satellite DNA of the monkey. The presence of a single α-satellite containing monkey chromosome was demonstrated by Giemsa-11 staining and by the absence of both this chromosome and monkey α-satellite DNA sequences in cells after back-selection in bromodeoxyuridine. Hybridization of restriction endonuclease-digested hybrid cell DNA with a cloned segment of African green monkey α-satellite DNA showed distinctly different patterns from those observed with monkey total DNA. In particular, EcoRI and HaeIII restriction endonuclease sites are much more abundant in the satellite sequences in the thymidine kinase-carrying chromosome than they are in total satellite. A library of hybrid DNA was constructed in a λ bacteriophage. Analyses of purified recombinant phage that hybridized with α-satellite also indicated an abundance of EcoRI and HaeIII sites. Of nine phage studied in detail, no two showed identical distributions of the two restriction sites in the α-satellite sequences, suggesting the independent evolution of different domains within the single chromosome. These results indicate that the thymidine kinase-carrying chromosome contains distinct subsets (domains) of the α-satellite DNA of the whole monkey genome and further, that while the satellite sequence on the single chromosome is distinctive, it is also complex.     

Nucleotide clusters in deoxyribonucleic acids. XII. The distribution of 5-methylcytosine in pyrimidine oligonucleotides of mouse L-cell satellite DNA and main band DNA.

Mouse L-cell DNA radioactively labeled in the 5-methylcytosine (5-MeC) residue was fractionated into satellite and main band DNA. Satellite DNA was found to contain about four times the molar concentration of 5-MeC than the main band DNA. Based on the known 5-MeC content of total L-cell DNA it was calculated that satellite DNA contains 3.5 – 4.6% 5-MeC. Both DNA fractions were depurinated and the pyrimidine oligonucleotides released separated by ionophoresis-homochromatography. In satellite DNA 5-MeC is distributed non-randomly. About 40% of the total 5-MeC is present in the sequence Pu - 5-MeC - Pu. The remainder occurs in the oligonucleotides CT, CT₃, C₂T₄, C₂T₅ and C₃T₅ only. The distribution of 5-MeC in main band DNA differs from that in satellite DNA indicating that two different fractions of the same nuclear DNA are methylated in different sequences.     

Availability of bases and nucleosides as precursors of nucleic acids in L cells and in the agent of meningopneumonitis

Tribby, Ilse I. E. (University of Chicago, Chicago, Ill.), and James W. Moulder. Availability of bases and nucleosides as precursors of nucleic acids in L cells and in the agent of meningopneumonitis. J. Bacteriol. 91:2362-2367. 1966.-Uninfected L cells and the meningopneumonitis agent propagated in L cells utilized exogenous adenine, guanine, and their ribonucleosides and deoxyribonucleosides for synthesis of both deoxyribonucleic acid (DNA) and ribonucleic acid. Cytosine, cytidine, and uridine were also incorporated into the nucleic acids of both host and parasite. L cells and the meningopneumonitis agent incorporated uracil, thymine, and deoxyuridine very poorly. L cells utilized thymidine and deoxycytidine almost exclusively for DNA synthesis, but the meningopneumonitis agent did not incorporate these nucleosides at all. Since the L cell had previously been shown to convert added thymidine to its nucleotides, mainly the triphosphate, it was concluded that the meningopneumonitis agent can utilize neither the thymidine nor the thymidine nucleotides of the L-cell pool, and that it can probably synthesize the thymidine triphosphate needed for DNA synthesis from the uridine of the L-cell pool.     

Scanning electron microscopy of the centromeric region of L-cell chromosomes after treatment with Hoechst 33258 combined with 5-bromodeoxyuridine

When mouse L-cells were treated with a combination of 5-bromodeoxyuridine (BrdUrd) and Hoechst 33258, the metaphase chromosomes revealed undercondensation of the chromatin fibers in the sister centromeres. The application of the osmium-thiocarbohydrazide technique to the air-dried chromosome preparations made it possible to elucidate the ultrastructure of the undercondensed centromeric region at the level of the 30 nm chromatin fiber. Scanning electron microscopy revealed that the undercondensed region consisted of a coiled fiber with a diameter of about 400 nm, and a gyre diameter of approximately 600 nm. The coiled fiber was composed of the 30 nm chromatin fiber loops. These findings indicate that a continuous coiled structure, which is the final higher order structure of the condensed chromatin fiber, exists throughout the entire length of the mouse L-cell metaphase chromosome.     

The use of restriction endonucleases to compare mitochondrial dna sequences in Mus musculus: A detailed restriction map of mitochondrial DNA from mouse L cells

The complete mitochondrial genome of a chloramphenicol-resistant, oligomycin-resistant mouse L cell has been cloned in E. coli. Using fragments of this DNA, purified by preparative agarose gel electrophoresis, we have mapped 139 restriction sites cleaved by 15 endonucleases. We have then compared the positions of these sites with those found in mtDNA purified from several other L-cell lines, from the tissue of several strains of laboratory mice, and from a plasmid containing mtDNA of a single, wild Mus musculus sample. The results indicate that all of the L-cell mtDNAs are identical except at a single EcoRI site, and that the L-cell sequence is identical to that found in mtDNA from normal tissue. They also suggest that mtDNA sequences found in North American Mus musculus populations may be much more homogeneous than expected from analysis of other rodents.     

Antibody to immunoselected L-cell antigens mimics stimulating activity of antibody to whole L cells

A heterogeneous IgG antibody raised in rabbits in response to injections of whole L cells was used to identify and select relevant antigens in a nonionic detergent extract of L cells prelabeled with [35S]methionine by means of immunoprecipitation and immunoaffinity chromatography. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the immunoprecipitate and immunoeluate contained far fewer protein bands than the whole cell extract but selectively retained a 42,000-MW protein species. In response to injections of the immunoprecipitate, rabbits produced a new antiserum which reacted predominantly with the 42,000-MW protein when reacted with L-cell proteins separated by sodium dodecyl sulfate-gel electrophoresis and transferred to nitrocellulose paper by the Western blot technique. The new antiserum (raised to the immunoprecipitate) and the original antiserum (raised to whole cells) were equipotent in stimulating calcium transport, phospholipid metabolism, and DNA synthesis in L cells. Binding of the IgG fractions of the two antisera displayed identical high affinity binding to L-cell surface antigens, with the same average association constant of 1.5 X 10(6) M-1. These studies have shown that an antiserum raised to whole L cells has a much narrower reactive spectrum with L-cell membrane antigens than might be imagined and has identified a 42,000-MW membrane protein as an important immunogen which itself elicits a potent immune response resulting in an antibody capable of mimicking the cell stimulatory properties of the original antiserum.     

L-Cell Virion Ribonucleic Acid

"L-cell virion," obtained from chronically infected 929 L-cell lines, was purified on 70% sucrose pads and its ribonucleic acid (RNA) was extracted and analyzed on sucrose gradients. Two RNA components were isolated from the virion, a rapidly sedimenting species (82S) and a more slowly sedimenting species (6S). When the fast-moving component was heated to 45 C, it yielded a homogeneous peak which now sedimented at approximately 72S. Upon heating to 70 C, the peak dissociates into a smaller homogeneous RNA species with a sedimentation coefficient of approximately 33S. Ribonuclease treatment and base composition analysis revealed that the fast-moving species is a single-stranded molecule. Base ratio analysis also revealed that the small (6S) RNA component is similar in its base composition to L-cell ribosomal RNA and distinctly different from the base composition of the large (72S) RNA component. Actinomycin D inhibits production of the L-cell virion by the L-cell line.     

Absence of ethidium bromide induced nicking and degradation of mitochondrial DNA in mouse L-cells.

The in vivo effects of ethidium bromide on the integrity of mitochondrial DNA have been studied in a mouse L-cell system in which this DNA may be nearly exclusively radiolabelled. This allows the detection of mitochondrial DNA in the presence of contaminating nuclear DNA and eliminates the need for extensive purification of mitochondria or the use of deoxyribonuclease. The mitochondrial DNA in treated cells rapidly attains a high negative superhelix density and is not substantially nickel or degraded over the course of several days.     

Specific interaction of murine colony-stimulating factor with mononuclear phagocytic cells

L-cell colony-stimulating factor (CSF) is identical to macrophage growth factor and stimulates macrophage proliferation (Stanley et al., 1976, J. Exp. Med. 143: 631-647). The nature of the interaction of iodinated L-cell CSF (125I-CSF) with murine peritoneal exudate macrophages was studied. On incubation with 10 pM 125I-CSF at 0 degrees C, cellular binding of 125I-CSF reaches a stable maximum within 15 h. This is in contrast to the association behavior at higher temperatures. At 37 degrees C, cell-associated 125I-CSF levels reach, within 45 min, an unstable maximum which is up to 10-fold less than that occurring under the same conditions at 0 degrees C. At 0 degrees C, binding is saturated (approximately 5 X 10(4) sites/cell) at CSF concentrations of 1 nM. A comparison of binding and competition experiments indicates that iodinated L-cell CSF binds as effectively as L-cell CSF and that human urinary CSF and L-cell CSF equipotently compete for 125I-CSF binding. Specificity of the CSF-binding site is demonstrated by the failure of other known growth factors and hormones to compete for 125I- CSF binding. These studies and other findings suggest that 125I-CSF binding is restricted to macrophages and their precursors and to macrophage cell lines and that the binding site(s) is the receptor mediating the biological action of this CSF.     

Structure of F-actin needles from extracts of sea urchin oocytes

The mouse L-cell line LD maintains its mitochondrial DNA genome in the form of a head-to-tail unicircular dimer of the monomeric 16,000 base-pair species. This situation permits a comparison of the mechanism of replication of this dimeric molecule with our previous studies of replication of monomeric mouse L-cell mitochondrial DNA. Whereas monomeric mitochondrial DNA requires about one hour for a round of replication, the dimeric molecule requires almost three hours. Denaturing agarose gel electrophoretic analyses of replicative intermediates reveals several discrete size classes of partially replicated daughter strands of dimeric mitochondrial DNA. This suggests that replication occurs with specific discontinuities in the rate of daughter strand synthesis. The strand specificity of these daughter strands was determined by hybridization with ³²P-labeled DNA representing either the heavy or light strand mitochondrial DNA sequence. The sizes and strand specificities of these discrete daughter strands indicate that the same set of control sequences is functional in both dimer and monomer mitochondrial DNA replication.Immediately following a round of replication, the majority of dimeric mitochondrial DNA molecules contain displacement loops, as assessed by their sensitivity to nicking within the displaced DNA strand by single-strand DNA specific S₁ nuclease under conditions which leave supercoiled DNA intact. This result is in contrast with the conformation of newly replicated monomeric mitochondrial DNA molecules, which lack both superhelical turns and displacement loops. This indicates that dimeric mitochondrial DNA proceeds through a different series of post-replicative processing steps than does monomeric mitochondrial DNA. We postulate that intermediates at late stages of dimeric mitochondrial DNA replication contain displacement loops which remain intact following closure of the full-length daughter strands.     

Localization of the human A1S9 gene complementing the ts A1S9 mouse L-cell defect in DNA replication and cell cycle progression to Xp11.2----p11.4

The temperature-sensitive ts A1S9 mouse L-cell mutant is defective in an X-linked gene essential for progression of cells through the S phase of the cell division cycle. A single copy fragment derived from the complementing human A1S9 gene was used as a probe to localize the gene on the X chromosome. Southern blot analysis of human x rodent hybrids and in situ hybridization to human metaphase chromosomes allowed the regional assignment of the human A1S9 gene to Xp11.2----p11.4.     

The regulation of haemopoiesis in long-term bone marrow cultures: I. Role of L-cell CSF

The effects of L-cell conditioned medium which contains granulocyte/macrophage colony stimulating factor (CSF); of highly purified L-cell CSF; and the antiserum directed against L-cell CSF, have been investigated in long-term murine bone marrow cultures. Treatment of cultures with CSF containing conditioned medium led to a rapid decline in haemopoiesis. However, this inhibition of in vitro haemopoiesis is probably caused by materials other than CSF, since the addition of highly purified L-cell CSF had no appreciable effect upon long-term haemopoietic cell proliferation or differentiation. Furthermore, the inhibitory activity of L-cell conditioned medium was not abrogated following neutralization of the CSF activity by CSF antiserum. The direct addition of CSF antiserum did not inhibit granulocyte or macrophage formation. These results suggest that long-term cultures of murine marrow cells may show extensive interactions with stromal cells which are not influenced by exogenous stimulatory or inhibitory factors.     

Dinitrophenol Inhibits the Rejoining of Radiation-Induced DNA Breaks by L-Cells

The production and rejoining of X-ray-induced single-stranded DNA breaks was studied using the alkaline sucrose density gradient technique and by measuring the disappearance of both 5' termini and 3'-OH termini using polynucleotide kinase and DNA polymerase, respectively. All studies were conducted using L-cell suspensions irradiated both in the presence and absence of 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation. Results show that the induction of single-stranded DNA breaks probably includes a nucleolytic component in addition to indirect free radical effects. A greater number of breaks were produced in the absence of DNP, suggesting that depressed adenosine triphosphate (ATP) levels reduce endogenous nucleolytic activity. The rejoining mechanism is enzymatic and requires an available ATP supply for operation. In the presence of DNP no DNA rejoining was observed following 30 min incubation after 10,000 rad. These results suggest that DNA breaks produced may be characterized by 5'-PO(4)-3'-OH termini and are rejoined by DNA ligase.     

Effect of Infection with the Meningopneumonitis Agent on Deoxyribonucleic Acid and Protein Synthesis by Its L-Cell Host

Cycloheximide, which had already been shown to inhibit protein synthesis in Earle's L cells (mouse fibroblasts) without having any effect on the multiplication or protein synthesis in Chlamydia psittaci (strain meningopneumonitis) infecting these host cells, also caused greater than 90% inhibition of deoxyribonucleic acid (DNA) synthesis in L cells after a 3-hr exposure to the drug. L cells infected with the meningopneumonitis agent and treated with cycloheximide were used to follow meningopneumonitis-specific DNA synthesis during intracellular growth of the parasite. The rate at which labeled precursors were incorporated into parasite DNA doubled every 2 hr. The effect of meningopneumonitis infection on L-cell DNA and protein synthesis was investigated in logarithmically growing and in stationary-phase (nondividing) populations of L cells. Host-specific DNA and protein synthesis appeared to be inhibited in infected L cells when compared with logarithmically growing control cells, whereas no inhibition was apparent when the comparison was made with stationary-phase control cells. The maximal amount of protein and DNA synthesis that occurred in meningopneumonitis-infected L cells was equal to the amount of DNA and protein synthesized in logarithmically growing, uninfected L cells. A possible explanation of these results is given.     

Mechanism of mitochondrial DNA replication in mouse L-cells: introduction of superhelical turns into newly replicated molecules

The first closed circular product of mouse L-cell mitochondrial DNA synthesis is a zero superhelix density molecule. Both of the asynchronously synthesized mitochondrial DNA daughter molecules pass through the zero superhelix density state. These molecules have a mean lifetime of approximately one hour before conversion to supercoiled molecules containing approximately 100 superhelical turns. A low frequency of intermediates in the conversion of these two closed circular forms is demonstrable by agarose gel electrophoresis. The degree of sensitivity to alkali has been used to demonstrate that newly replicated mitochondrial DNA has the same content of ribonucleotides as mass-labeled mitochondrial DNA.     

Related base sequences in the DNA of simple and complex organisms. IV. Evolutionary divergence of base sequence in mouse L-cell cytoplasmic and nucleus-restricted RNA

Nuclear and cytoplasmic RNAs of mouse L-cells were compared for base sequence relatedness between and within them. The relationship of each of these RNAs to the DNA of other rodents and the rabbit was determined. RNA restricted to the mouse L-cell nucleus is unrelated in base sequence to cytoplasmic RNA. There is less diversity of base sequence within the gene families transcribing nucleus-restricted RNA than within those transcribing cytoplasmic RNA, suggesting more recent evolutionary origin. The gene families which code for mouse L-cell unique nuclear RNA are far more divergent from those of other rodents than are cytoplasmic RNA gene families. They are unrelated in base sequence to any gene families in rabbit DNA, suggesting that these gene families evolved after the separation of the ancestors of the orders Rodentia and Lagomorpha. The known characteristics of the RNA restricted to the nucleus in eukaryotic cells are summarized.This investigation was supported by U.S. Public Health Service Training Grant GM 00182 and Research Grant GM 12449 from the Institute of General Medical Sciences.