Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains.

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and phenotypic changes. Development of an alternative method for long-term preservation without such changes is therefore imperative. Despite previous successful attempts, cryopreservation of Drosophila embryos is still not of practical use because of low reproducibility. Here, we describe a protocol for primordial germ cell (PGC) cryopreservation and strain revival via transplantation of cryopreserved PGCs into agametic Drosophila melanogaster (D. melanogaster) host embryos. PGCs are highly permeable to cryoprotective agents (CPAs), and developmental and morphological variation among strains is less problematic than in embryo cryopreservation. In this method, PGCs are collected from approximately 30 donor embryos, loaded into a needle after CPA treatment, and then cryopreserved in liquid nitrogen. To produce donor-derived gametes, the cryopreserved PGCs in a needle are thawed and then deposited into approximately 15 agametic host embryos. A frequency of at least 15% fertile flies was achieved with this protocol, and the number of progeny per fertile couple was always more than enough to revive the original strain (the average progeny number being 77.2 ± 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 ± 0.2, and 9 out of 26 needles produced two or more fertile progeny. It was found that 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included. The agametic host makes it possible to revive the strain quickly by simply crossing newly emerged female and male flies. In addition, PGCs have the potential to be used in genetic engineering applications, such as genome editing.

Offspring production from cryopreserved primordial germ cells in Drosophila.

There is an urgent need to cryopreserve Drosophila stocks that have been maintained as living cultures for a long time. Long-term culture increases the risk of accidental loss and of unwanted genetic alteration. Here, we report that cryopreserved primordial germ cells (PGCs) can produce F1 progeny when transplanted into hosts. The cryopreserved donor PGCs could form germline stem cells in host gonads and contributed to continuous offspring production. Furthermore, the ability to produce offspring did not appear to vary with either differences between donor strains or cryopreservation duration. Therefore, we propose that our cryopreservation method is feasible for long-term storage of various Drosophila strains. These results underscore the potential usefulness of our cryopreservation method for backing up living stocks to avoid either accidental loss or genetic alteration.

Update of thermotolerant genes essential for survival at a critical high temperature in Escherichia coli.

Previous screening of a single-gene knockout library consisting of 3,908 disrupted-mutant strains allowed us to identify 51 thermotolerant genes that are essential for survival at a critical high temperature (CHT) in Escherichia coli [Murata M, Fujimoto H, Nishimura K, Charoensuk K, Nagamitsu H, Raina S, Kosaka T, Oshima T, Ogasawara N, Yamada M (2011) PLoS ONE 6: e20063]. In this study, we identified another 21 thermotolerant genes. E. coli thus has 72 thermotolerant genes in total. The genes are classified into 8 groups: genes for energy metabolism, outer membrane organization, DNA double-strand break repair, tRNA modification, protein quality control, translation control, cell division and transporters. This classification and physiological analysis indicate the existence of fundamental strategies for survival at a CHT, which seems to exclude most of the heat shock responses.

TMEM16A and TMEM16B channel proteins generate Ca2+-activated Cl- current and regulate melatonin secretion in rat pineal glands.

Pinealocytes regulate circadian rhythm by synthesizing and secreting melatonin. These cells generate action potentials; however, the contribution of specific ion channels to melatonin secretion from pinealocytes remains unclear. In this study, the involvement and molecular identity of Ca2+-activated Cl- (ClCa) channels in the regulation of melatonin secretion were examined in rat pineal glands. Treatment with the ClCa channel blockers, niflumic acid or T16Ainh-A01, significantly reduced melatonin secretion in pineal glands. After pineal K+ currents were totally blocked under whole-cell patch clamp conditions, depolarization and subsequent repolarization induced a slowly activating outward current and a substantial inward tail current, respectively. Both of these current changes were dependent on intracellular Ca2+ concentration and inhibited by niflumic acid and T16Ainh-A01. Quantitative real-time PCR, Western blotting, and immunocytochemical analyses revealed that TMEM16A and TMEM16B were highly expressed in pineal glands. siRNA knockdown of TMEM16A and/or TMEM16B showed that both channels contribute to ClCa currents in pinealocytes. Conversely, co-expression of TMEM16A and TMEM16B channels or the expression of this tandem channel in HEK293 cells mimicked the electrophysiological characteristics of ClCa currents in pinealocytes. Moreover, bimolecular fluorescence complementation, FRET, and co-immunoprecipitation experiments suggested that TMEM16A and TMEM16B can form heteromeric channels, as well as homomeric channels. In conclusion, pineal ClCa channels are composed of TMEM16A and TMEM16B subunits, and these fluxes regulate melatonin secretion in pineal glands.

Complete mitochondrial genome sequences of Brassica rapa (Chinese cabbage and mizuna), and intraspecific differentiation of cytoplasm in B. rapa and Brassica juncea.

The complete sequence of the mitochondrial genome was determined for two cultivars of Brassica rapa. After determining the sequence of a Chinese cabbage variety, 'Oushou hakusai', the sequence of a mizuna variety, 'Chusei shiroguki sensuji kyomizuna', was mapped against the sequence of Chinese cabbage. The precise sequences where the two varieties demonstrated variation were ascertained by direct sequencing. It was found that the mitochondrial genomes of the two varieties are identical over 219,775 bp, with a single nucleotide polymorphism (SNP) between the genomes. Because B. rapa is the maternal species of an amphidiploid crop species, Brassica juncea, the distribution of the SNP was observed both in B. rapa and B. juncea. While the mizuna type SNP was restricted mainly to cultivars of mizuna (japonica group) in B. rapa, the mizuna type was widely distributed in B. juncea. The finding that the two Brassica species have these SNP types in common suggests that the nucleotide substitution occurred in wild B. rapa before both mitotypes were domesticated. It was further inferred that the interspecific hybridization between B. rapa and B. nigra took place twice and resulted in the two mitotypes of cultivated B. juncea.

Spontaneous and nicotine-induced Ca2+ oscillations mediated by Ca2+ influx in rat pinealocytes.

The pineal gland regulates circadian rhythm through the synthesis and secretion of melatonin. The rise of intracellular Ca(2+) concentration ([Ca(2+)]i) following nicotinic acetylcholine receptor (nAChR) stimulation due to parasympathetic nerve activity downregulates melatonin production. Important characteristics and roles of Ca(2+) mobilization due to nAChR stimulation remain to be clarified. We report here that spontaneous Ca(2+) oscillations can be observed in ∼15% of the pinealocytes in slice preparations from rat pineal glands when this dissociation procedure is done within 6 h from a dark-to-light change. The frequency and half-life of [Ca(2+)]i rise were 0.86 min(-1) and 19 s, respectively. Similar spontaneous Ca(2+) oscillations were recorded in 17% of rat pinealocytes that were primary cultured for several days. Simultaneous measurement of [Ca(2+)]i and membrane potential revealed that spontaneous Ca(2+) oscillations were triggered by periodic membrane depolarizations. Spontaneous Ca(2+) oscillations in cultured pinealocytes were abolished by extracellular Ca(2+) removal or application of nifedipine, a blocker of voltage-dependent Ca(2+) channel (VDCC). In contrast, blockers of intracellular Ca(2+)-release channels, 2-aminoethoxydiphenylborate and ryanodine, have no effect. Our results also reveal that, in 23% quiescent pinealocytes, Ca(2+) oscillations were observed following the withdrawal of nicotine. Norepinephrine-induced melatonin secretion from whole pineal glands was significantly decreased by the coapplication of acetylcholine (ACh). This inhibitory effect of ACh was attenuated by nifedipine. In conclusion, both spontaneous and evoked Ca(2+) oscillations are due to membrane depolarization following activation of VDCCs. This consists of VDCC α1F subunit, and the associated Ca(2+) influx can strongly regulate melatonin secretion in pineal glands.

Molecular strategy for survival at a critical high temperature in Eschierichia coli.

The molecular mechanism supporting survival at a critical high temperature (CHT) in Escherichia coli was investigated. Genome-wide screening with a single-gene knockout library provided a list of genes indispensable for growth at 47°C, called thermotolerant genes. Genes for which expression was affected by exposure to CHT were identified by DNA chip analysis. Unexpectedly, the former contents did not overlap with the latter except for dnaJ and dnaK, indicating that a specific set of non-heat shock genes is required for the organism to survive under such a severe condition. More than half of the mutants of the thermotolerant genes were found to be sensitive to H(2)O(2) at 30°C, suggesting that the mechanism of thermotolerance partially overlaps with that of oxidative stress resistance. Their encoded enzymes or proteins are related to outer membrane organization, DNA double-strand break repair, tRNA modification, protein quality control, translation control or cell division. DNA chip analyses of essential genes suggest that many of the genes encoding ribosomal proteins are down-regulated at CHT. Bioinformatics analysis and comparison with the genomic information of other microbes suggest that E. coli possesses several systems for survival at CHT. This analysis allows us to speculate that a lipopolysaccharide biosynthesis system for outer membrane organization and a sulfur-relay system for tRNA modification have been acquired by horizontal gene transfer.