1-methyl Adenine Research Articles

Echinoderm eggs as a model for discoveries in cell biology.

I happen to have been trained in cell and developmental biology in the early 1970s, which was near the beginning of the explosive growth of the field of cell biology. The American Society for Cell Biology had been founded in 1960 and so the field was in its early days. Cell biology research was dominated by the use of the electron microscope and by protein biochemistry. Molecular biology and the use of genetics were in their infancy. When we track the path of discoveries in cell biology contributed by research using echinoderm eggs, we follow the development of new technologies in genetics, molecular biology, biochemistry and biophysics, bioengineering, and imaging. The changes in approaches and methods have led to many key discoveries in cell biology through the use of sea urchin, sand dollar and sea star eggs. These include the discovery of cyclin, cytoplasmic dynein, rho activation for cytokinesis, new membrane addition as a late event in cytokinesis, multiple kinesins playing multiple roles, how flagella beat, the dynamics of microtubules in the mitotic apparatus, control over centrosomes and cell cycle checkpoints, the process of nuclear envelope breakdown for cell division, the discovery of 1-methyl adenine (hormones) as the trigger for meiotic maturation, Ca++ transients controlling cell activation and exocytosis among others. What I hope to provide in this perspective is to highlight some of those wonderful discoveries as my own career evolved to contribute to the field.

A nonradioactive restriction enzyme-mediated assay to detect DNA repair by Fe(II)/2-oxoglutarate-dependent dioxygenase

The Escherichia coli DNA repair enzyme AlkB belongs to the Fe(II)/2-oxoglutarate-dependent dioxygenase family. It removes methyl groups from 1-methyl adenine (1-meA) and 3-methyl cytosine (3-meC) lesions present in single-stranded DNA by oxidative decarboxylation. In the current article, we describe an in vitro assay that permits rapid detection of AlkB activity. To achieve this, we generated methylated oligonucleotide using methyl methanesulfonate and then monitored DNA repair using a methylation-sensitive restriction enzyme and novel agarose gel electrophoresis system capable of resolving small oligonucleotides. Our approach overcomes several drawbacks of NAD+-dependent formaldehyde dehydrogenase-coupled assay and radioisotope-based assay for determining AlkB DNA repair activity.

(-)-Sclerotiorin from an Unidentified Marine Fungus as an Anti-meiotic and Antifungal Agent

(-)-Sclerotiorin, isolated and purified from the fermented broth of an unidentified marine fungus (98F134), inhibited the maturation of starfish oocytes induced by 1-methyl adenine with an IC50 value of 0.50 microM. (-)-Sclerotiorin also showed antifungal activity against Saccharomyces cerevisiae, Aspergilus fumigatus, Alternaria longipes, Piricularia oryzae, Verticillium albo-atrum, Fusarium culmorum, and F. nivale with IC50 values less than 20 microg/mL.

Studies on 1-methyl adenine transfer RNA methyltransferase of Mycobacterium smegmatis.

The presence of 1-methyl adenine in transfer RNA is a feature that Mycobacterium smegmatis shares with only a few other prokaryotes. The enzyme 1-methyl adenine tRNA methyl transferase from this source has been purified and the preliminary results show the presence of two activity peaks with different substrate specificity.

Calcium transients during early development in single starfish (Asterias forbesi) oocytes.

Maturation and fertilization of the starfish oocyte are putative calcium-dependent events. We have investigated the spatial distribution and temporal dynamics of this calcium dependence in single oocytes of Asterias forbesi. We used the calcium photoprotein, aequorin, in conjunction with a microscope-photomultiplier and microscope-image intensifier. Surprisingly, in contrast to earlier work with Marasthenias glacialis, there is no detectable increase in intracellular-free calcium in the oocyte of A. forbesi in response to the maturation hormone 1-methyl adenine. During fertilization of the same, matured, A. forbesi oocyte there is a large increase in intracellular-free calcium. The calcium concentration increases to approximately 1 microM at the point of insemination and the region of elevated free calcium expands across the oocyte in approximately 20 s (17-19 degrees C). After the entire oocyte reaches an elevated concentration of free calcium, the concentration decreases uniformly throughout the oocyte over the next several minutes.

Studies on the effects ofin vitro methyiation on aminoacylation of transfer RNA

In vitro methyiation ofEscherichia coli transfer ribonucleic acid by cell free extracts ofMycobacterium smegmatis leads exclusively to the formation of 1-methyl adenine [Vani, B. R., Ramakrishnan, T., Taya, Y., Noguchi, S., Yamaiuzumi, Z. and Nishimura, S. (1978)J. Bact., 137, 1085]. We have studied the effect of this modification on aminoacylationof Escherichia coli tRNA by mycobacterial enzymes. Aminoacylation with total algal protein hydrolysate as well as several individual aminoacids like methionine, valine, tyrosine, aspartic acid and lysine were monitored. In all the cases methyiation had a positive effect on the extent of aminoacylation by mycobacterial enzymes. Decreased aminoacylationin vitro was observed when hypomethylated transfer RNA from ethionine treated cells was used as the substrate for aminoacylation

Methionine sulfoximine in vivo modifies the tRNALys pool of developing rat brain.

tRNA was extracted from brains of 3-, 8-, and 18-day-old rats that were injected intracerebrally, 45 min before death, with [3H]methyl methionine or [8-3H]guanosine, and intraperitoneally, 3 h before death, with L-methionine-dl-sulfoximine (MSO), a methylation-activating convulsant agent. Although there was no effect of age or of MSO on the per gram yield of tRNA, its specific radioactivity (dpm/A260) was highest at 3 days in both the control and the MSO groups. Age- and MSO-related changes in tRNALys content of the brain tRNA pool were investigated by means of benzoylated DEAE-cellulose (BDC) and reverse-phase chromatography (RPC). BDC chromatography revealed tRNALys species in the brains of the MSO-treated animals that were absent in control brains. Of particular interest was the finding that differences in RPC-5 chromatographic mobility between control and MSO-tRNALys species were abolished by conversion of lysyl-tRNA, suggesting that the MSO-elicited change(s) in tRNALys structure involved the binding site(s) for lysine. Two additional findings were made: (a) lysine acceptance by the [3H]methyl-labeled tRNALys purified from brains of the MSO-treated animals was higher than that of controls at 18 days; and (b) omission of the BDC chromatographic step accentuated the differences in mobility on RPC-5 columns between tRNALys species of control and MSO-treated animals. Lastly, we found that some tRNALys species of control and MSO-treated brains contained significantly different proportions of N2-methyl guanine and 1-methyl adenine, relative to controls. These MSO-elicited changes in the methyl base content of tRNALys of immature rat brain are the first evidence of an alteration of brain tRNA structure by a centrally acting excitatory agent.

Spermatogenic columns, somatic cells, and the microenvironment of germinal cells in the testes of asteroids.

Sixty-seven specimens of the common North Atlantic asteroid, Asterias vulgaris, were collected at seasonal intervals over a 2-year period and their testes observed with both light and electron microscopy. In the germinal epithelium, a predictable series of interactions between versatile somatic cells and germinal cells is repeated annually in relation to sequential events in spermatogenesis. For example, massive proliferation and differentiation of spermatogenic cells depend on the elaboration of thousands of spermatogenic columns, which are distinct cellular subdivisions of the germinal epithelium. Each fully developed column is composed of at least one somatic cell surrounded by ≈︁ 400 germinal cells. Such columns form only after intensive spermatogonial mitosis begins in the germinal epithelium. Single annual periods of spermatogenic proliferation and differentiation are initiated from 1 to 3 months out of phase in different individuals and overlap incompletely. Therefore, it is possible to observe testes that are entirely in the proliferative phase, entirely in the differentiative phase, or in both phases simultaneously. Detailed ultrastructural observations and preliminary autoradiographic data demonstrate that columns maintain their height for a variable period of time as germinal cells are generated near their bases, pass along their lengths, and differentiate near their tips; therefore, simultaneous proliferation and differentiation of more than one generation of germinal cells occur in the same column. Finally, formation of primary spermatocytes ceases basally, (terminating proliferation), and remaining columns degrade completely as germinal cells composing them differentiate or are phagocytized (terminating differentiation and spermatogenesis); resulting spermatozoa ultimately accumulate in the expandable lumen. It is proposed that spermatogenic columns provide the structural basis for organization of the microenvironment of small groups of spermatogenic cells (≈︁ 400 at a time) during proliferation and differentiation. Preliminary evidence from A. vulgaris and other species also suggests that somatic cells are temporally pluripotent and are variously involved in the formation, structure, and activities of columns, in extensive phagocytosis, and probably in contributing intrinsic (e.g., 1-methyl adenine and steroids) and mediating extrinsic (e.g., gamete shedding substance and nutrients) microenvironmental factors influential during spermatogenesis in asteroids. The prodigious spermatogenic capabilities of asteroids apparently depend on the generation of spermatogenic columns, on the progressive interaction of germinal and somatic cells before, during, and after columns form, and on the predictable effects of microenvironmental factors received and interpreted at the structural level of the spermatogenic column.

Nucleotide sequence of Bombyx mori L. tRNA1Gly

THE functional adaptation of the tRNA population in the posterior silk gland of the silkworm Bombyx mori L during growth and secretion of fibroin1 is the result of changes in the predominant isoaccepting tRNA species to accord with the frequency of various codons in fibroin mRNA (refs 2–4). tRNA adaptation to fibroin biosynthesis can be demonstrated by polyacrylamide gel electrophoresis maps. The main glycine codons GGU and GGA of fibroin mRNA (ref. 3) are translated by means of two prevalent iso-tRNAGly species: iso-tRNA1Gly decodes GGU and GGC whereas iso-tRNA2Gly decodes GGA (refs 4–6). The glycine tRNA1, the first sequenced tRNA from an insect6, has 74 nucleotides and relatively few modified residues7: one thymidine, one pseudouridine, one dihydrouridine, one 1-methyl adenine, 1-methyl guanosine, four 5-methyl cytidine and one unusal 2′-O-methyl uridine in the acceptor stem. It carries the expected GCC anticodon4. Comparison with three other homodecoding tRNAGCCGly species reveals a high degree of homology.

The developmental pattern of homologous and heterologous tRNA methylation in rat brain differential effect of spermidine

UsingS-adenosyl-L-[Me-(14)C] methionine, rat cerebral cortex methyltransferase activity was determined during the early postnatal period in the absence of addedEscherichia coli tRNA and in its presence. [Me-(14)C] tRNA was purified from both systems and its [Me-(14)C] base composition determined. The endogenous formation of [Me-(14)C] tRNA (homologous tRNA methylation) was totally abolished in the presence of 2.5 mM spermidine, whereasE. coli B tRNA methylation (heterologous methylation) was markedly stimulated. Only [Me-(14)C] 1-methyl guanine and [Me-(14)C]N (2)-methyl guanine were formed by homologous methylation, there being an inverse shift in their relative proportions with age. Heterologous tRNA methylation led, additionally, to the formation of [Me-(14)C]N 2 (2) -dimethyl guanine, 5-methyl cytosine, 1-methyl adenine, 5-methyl uracil, 2-methyl adenine, and 1-methyl hypoxanthine. A comparison of heterologous tRNA methylation between the whole brain cortex (containing nerve and glial cells) and bulk-isolated nerve cell bodies revealed markedly lower proportions of [Me-(14)C]N 2-methyl andN 2 (2) -dimethyl guanine and significantly higher proportions of [Me-(14)C] 1-methyl adenine in the neurons. The present findings suggest (1) that homologous tRNA methylation may provide developing brain cells with continuously changing populations of tRNA and (2) that neurons are enriched in adenine residue-specific tRNA methyltransferases that are highly sensitive to spermidine.

The methylated constituents of L cell messenger RNA: Evidence for an unusual cluster at the 5′ terminus

An analysis of the methylated constituents of L cell mRNA by a combination of chromatographic methods and enzymatic treatments indicates that they comprise both 2′-0-methyl nucleosides and N 6-methyl adenine, and/or 1-methyl adenine, and suggests that the 2′-0-methyl nucleotides, Ym, are part of an unusual class of sequences forming the 5′ terminus of mRNA. These sequences seem to contain two 2′-0-methyl residues and a terminal residue that is not phosphorylated but, nevertheless, is blocked with respect to polynucleotide kinase reactivity. A strong candidate is a sequence of the type XppY 1mpY 2mpZp …, where X represents a blocking group which is itself occasionally methylated. The sequences isolated from total poly(A) + mRNA contain all four species of 2′-0-methylated nucleoside, indicating some variability among different mRNA species. The methylated sequences do not appear to be enriched in the mRNA which hybridizes with repetitive DNA. The average L cell mRNA molecule also contains three residues of N 6-methyl adenine. These residues are not part of the poly(A) segment, but appear to be located internal to the poly(A) near the 3′ end of the mRNA molecules.

Active contraction of Starfish oocyte follicle cells after treatment with 1-methyl adenine.

Active contraction of Starfish oocyte follicle cells after treatment with 1-methyl adenine.

Methylation in vivo of guanine in the nucleic acids of rat testes by methyl methane sulphonate.

TREATMENT of male rats with a single intraperitoneal dose of 50 or 100 mg/kg body weight of methyl methane sulphonate results in infertility during the second and third week after the injection1. This infertility is caused not by aspermia but by a dominant lethal mutation in the sperm which are selectively affected at the late spermatid or early spermatozoa stage. The mutation does not interfere with the fertilization of the ova but the resulting embryos die in utero2. It has been assumed that this biological action of methyl methane sulphonate is connected with its properties as an alkylating agent. When a bacteriophage is inactivated by treatment with methyl methane sulphonate, guanine and adenine of the bacteriophage DNA are methylated to 7-methyl guanine, 3-methyl adenine, and 1-methyl adenine in proportion 78 : 20 : 2 (ref. 3). We have now found that an intravenous injection of methyl methane sulphonate methylates the 7 position of the guanine of DNA and RNA in rat testes.