Regulation of glyoxysomal enzymes during germination of cucumber. Temporal changes in translatable mRNAs for isocitrate lyase and malate synthase

The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)-rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X-ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light.     

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans.

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA, and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting the expression of nonregulated genes. The three genes code for poly(A)+ RNAs that begin to accumulate at different times during conidiation. The brlA- and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.     

Cloning and Expression Studies of Novel Small RNAs in Tetraploid Cotton

Small RNAs are a group of non-coding RNAs that downregulate gene expression in a sequence-specific manner to control plant growth and development. The objective of the present study was to clone and characterize several small RNAs in cotton. To identify small RNAs that are involved in the development of cotton bolls and fibers, we generated cDNA libraries from cotton bolls at 13?days post-anthesis from two cotton cultivars, Pima Phy 76 (Gossypium bardadense) and Acala 1517?C99 (Gossypium hirsutum). Screening of these libraries identified eight small RNAs, seven of which have not been reported in other plant species and appear to be absent in the known sequences of other plant species. Their predicted target genes are known to be involved in cotton fiber development. The cloned small RNAs displayed lower and differential expression in the examined boll developmental stages using RT-PCR and quantitative RT-PCR. The genetic polymorphism of the small RNAs at the DNA level was evaluated by miRNA-amplified fragment length polymorphism (AFLP) analysis using primers designed from the small miRNA genes in combination with AFLP primers. Homologous small RNA gene sequences were further isolated using this homology-based genotyping approach, and potential hairpin structures were identified. The results represent a novel method to isolate small including miRNA genes at the RNA and DNA levels in many plant species where genome sequences are not available or expressed sequence tags are limited.     

Coordinate accumulation of five transcripts in the primary mesenchyme during skeletogenesis in the sea urchin embryo

The sea urchin larval skeleton is produced by the primary mesenchyme (PM), a group of 32 cells descended from the four micromeres of the 16-cell embryo. The development of this lineage proceeds normally in isolated cultures of micromeres. A complementary DNA (cDNA) library was generated from cytoplasmic polyadenylated RNA isolated from differentiated micromere cultures of Strongylocentrotus purpuratus. Five clones were selected on the basis of their enrichment in differentiated PM cell RNA as compared to the polyribosomal RNAs of other embryonic cell types and other developmental stages. Each cloned cDNA hybridized to a distinct RNA that was abundant in the polyribosomes of differentiated PM cells, but absent from larval ectoderm and from 16-cell embryos. These RNAs were encoded by single or low copy genes. In situ hybridization analysis of the most abundant of these RNAs (SpLM 18) demonstrated that it was specifically limited to the skeletogenic PM of intact embryos. During the development of the PM, all five RNAs exhibited the same schedule of accumulation, appearing de novo, or increasing abruptly just before PM ingression, and remaining at relatively high levels thereafter. This pattern of RNA accumulation closely paralleled the pattern of synthesis of PM-specific proteins in general (Harkey and Whiteley, 1983) and of the SpLM 18-encoded protein specifically (Leaf et al., 1987). These results indicate that at least five distinct genes in the sea urchin, each of which encodes a PM-enriched or PM-specific mRNA, are expressed with tight coordination during development of the larval skeleton. They also demonstrate that expression of these genes in the PM is regulated primarily at the level of RNA abundance rather than RNA utilization.     

Thermodynamic studies of the interaction of alpha-chymotrypsin with water. I. Determination of the isosteric enthalpies and entropies of water binding to the native enzyme

RNA polymerase enzymes isolated from soybean hypocotyl tissue during successive developmental stages (2-8 days old) have been fractionated by Sephadex column isoelectric focusing. Both the enzymes bound to chromatin and those enzymes free in the soluble phase were investigated during development with respect to their distribution within these two pools. All observed activites were classified according to their alpha-amanitin sensitivity and isoelectric points. Two Class I subspecies (Ia, Ib) and two Class III subspecies (IIIa, IIIb) were continually present bound to chromatin throughout the developmental sequences except the IIb form which was absent at the latest stage. However, a great multiplicity (9 total) of Class II activities (totally inhibited by alpha-amanitin) were observed to be bound to chromatin at the 2nd day stage. These forms were first released from the chromatin complex and recovered in a soluble pool (4th day stage). Subsequent hypocotyl development was accompanied by the gradual disappearance of these Class II subspecies from this pool (6th day) until only two soluble species and one chromatin-bound Class II activity remained (8th day). These observations indicate that the early development of this tissue is accompanied by a dramatic alteration in the conplexity of chromatin-bound RNA polymerase subspecies. Such events may in part determine the domain of RNA secies synthesized at successive developmental stages.     

A 5-fold reduction in sister-chromatid exchange following implantation of mouse embryos is not directly related to the expression of embryonic genes responsible for oxygen radical metabolism

We studied the spontaneous sister chromatid exchange (SCE) frequencies in mice at different stages of development; early preimplantation: 2 days post conception (p.c.); late preimplantation: 4 days p.c.; post implantation: day 10 and 13 p.c. The SCE level in preimplantation embryos is 5 times higher than any of the stages following implantation. The explanation for such observations may include a direct impact of maternal circulatory system as a result of implantation or onset of expression of a set of embryonic genes. Here, we studied the expression and developmental profile of the three enzymes of oxygen radical metabolism (superoxide dismutase, glutathione peroxidase, and catalase) during development. Our results suggest that the onset and increase in the activity of these enzymes with in-utero differentiation, development and growth is not directly associated with the drop in SCEs/cell following implantation of the embryos. This unique developmental phenomenon may be mediated by the maternal circulatory system or expression of some other embryonic genes, possibly the genes involved in DNA repair.     

Identification of Most Stable Endogenous Control Genes for MicroRNA Quantification in the Developing Mouse Lung

MicroRNAs (miRNAs) are endogenous small non coding RNAs acting as negative regulators. miRNA are involved in lung development and pulmonary diseases. Measurement of their levels by qPCR is directly influenced by the stability of normalization gene(s), which can be affected by the experimental conditions. The developing lung is a changing tissue and one normalization gene showing stability on one developmental day may be modulated over time. Moreover, some developmental events are affected by sex, which also has to be considered. In this study, we compared stability of five putative control genes in the lung between sexes from the pseudoglandular to the alveolar stages and in adult lungs. Expression of sno135, sno142, sno202, sno234, and sno251 was studied by qPCR in male and female lung samples collected at seven time points from GD 15.5 to PN 30. Cq values of sno251 showed the highest variation across the different developmental stages, while sno234 was the most stable gene. Gene expression stability was studied by geNorm, NormFinder and BestKeeper. Our data showed that ranking of genes based on expression stability changed according to developmental time and sex. sno135/sno234 and sno142/sno234 were proposed as best combinations of normalization genes when both sexes and all the studied developmental stages are considered. Normalization of let7-a RNA levels with different pairs of control genes proposed by geNorm and NormFinder gave similar data, while the use of less stable genes introduced a statistically significant difference on PN 0. In conclusion, variations in stability of normalization gene expression are observed over time and according to sex during lung development. Best pairs of normalization genes are presented for specific developmental stages, and for the period extending from the pseudoglandular to the alveolar stages. The use of normalization genes selected for their expression stability is essential in lung development studies.     

Control of developmental timing in animals

The molecular mechanisms that time development are now being deciphered in various organisms, particularly in Caenorhabditis elegans. Key recent findings indicate that certain C. elegans timekeeping genes are conserved across phyla, and their developmental expression patterns indicate that a timing function might also be conserved. Small regulatory RNAs have crucial roles in the timing mechanism, and the cellular machinery required for production of these RNAs intersects with that used to process double-stranded RNAs during RNA interference.     

Expression of imprinted Igf2 and Peg1/Mest genes in postimplantation parthenogenetic mouse embryos treated with transforming growth factor alpha in vitro

The effect of transforming growth factor alpha (TGFt) on the expression of imprinted Igf2 and Peg1/Mest genes was studied in diploid parthenogenetic embryos (PEs) of (CBA x C57BL/6)F1 mice during the postimplantation period of embryogenesis. The PEs were treated with TGFalpha in vitro at the morula stage and, after they developed to the blastocyst stage, were implanted into the uterus of false-pregnant females. On the tenth day of pregnancy, the PEs were explanted for subsequent in vitro culturing for 24 or 48 h. The expression of the imprinted Igf2 and Peg1/Mest genes was studied by means of whole mount in situ hybridization using digoxigenin-labeled antisense RNAs. The expression of the imprinted Igf2 and Peg1/Mest genes was studied in embryos on the tenth day of in utero development before culturing and after 24 and 48 h of culturing in vitro. The expression of Igf2 before culturing was detected only in the brain of 60% of PEs on the tents day of pregnancy (the 21-to 25-somite stages); while the Peg1/Mest expression was not detected at all. In control (not treated with TGFalpha) PEs, neither gene was expressed at the same 21- to 25-somite stages. After 24 h of culturing, the Igf2 expression was detected in the brain of 71% of PEs at the 30- to 35-somite stages, while the Peg1/Mest expression was not detected. In control (untreated) PEs, neither imprinted gene was expressed at the 30- to 35-somite stage. After 48 h of culturing, Igf2 was expressed in the regions of the brain, developing jaws, heart, liver, and somites of all TGFalpha-treated PEs at the 40- to 45-somite stages; and Peg1/Mest was expressed in the brain, heart, and liver of these embryos. In control (untreated) PEs, neither Igf2 nor Peg1/Mest was expressed at these stages The expression patterns of the imprinted Igf2 and Peg1/Mest genes in PEs at the most advanced developmental stages (40-45 somites) and in normal (fertilized) embryos at the same stages were similar; however, their expression rate in PEs was substantially lower than in normal embryos. These data indicate that exogenous TGFalpha can reactivate the expression of the two imprinted genes, modulating the effects of genomic imprinting in such a way that the PE development is improved and substantially prolonged.     

Dipeptidase-C inDrosophila melanogaster: Genetic,ontogenetic, and tissue-specific variation

Dip-A, Dip-B, and Dip-C constitute structural genes for three peptidic enzymes in Drosophila melanogaster distinct from the leucine aminopeptidases. Their ontogenetic and tissue distributions of activities suggest the involvement of these enzymes in a general metabolic role, such as the regulation of amino acid and oligopeptide pools to make amino acids available for protein synthesis. Screening of chromosome substitution isogenic lines for DIP-C activity indicated that, like DIP-A and DIP-B, unlinked activity modifiers exist for Dip-C. The developmental profiles of dipeptidase activities are very similar, except in the pupal stage, during which DIP-C activity is markedly low compared to the other two enzymes. Intercorrelations of dipeptidase activities vary ontogenetically, which is consistent with the need for coordinate expression of these enzymes during certain developmental stages. Tissue-specific expression of dipeptidases in larvae and adults are also similar, although the relative levels of DIP-A activity differ from those of DIP-B and DIP-C in certain organs and body parts. Some of the differences among chromosome substitution lines for dipeptidase activities appear to be systemic, while others are developmental stage-specific and tissue-specific. Second- and third-chromosome variants for DIP-C activity differed in their tissue distribution. This is consistent with the presence of temporal and spatial variants in natural populations for other Drosophila enzymes.     

Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes

MicroRNAs (miRNAs) and small interfering RNAs are important regulators of plant development and seed formation, yet their population and abundance in the oil crop Brassica napus are still not well understood, especially at different developmental stages and among cultivars with varied seed oil contents. Here, we systematically analyzed the small RNA expression profiles of Brassica napus seeds at early embryonic developmental stages in high-oil-content and low-oil-content B. napus cultivars, both cultured in two environments. A total of 50 conserved miRNAs and 9 new miRNAs were identified, together with some new miRNA targets. Expression analysis revealed some miRNAs with varied expression levels in different seed oil content cultivars or at different embryonic developmental stages. A large number of 23-nucleotide small RNAs with specific nucleotide composition preferences were also identified, which may present new classes of functional small RNAs.