Towards Long Term Cultivation of Drosophila Wing Imaginal Discs In Vitro

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.     

The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.     

Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression

A series of vectors has been developed to provide improved positive and negative selection for allelic exchange. Based on homologous regions of DNA ranging in size from less than 200 bp to over 1 kb, we have successfully used these new plasmids to introduce or remove markers in chromosomal or plasmid DNA. Wild type fimbria genes were replaced both in Salmonella enteritidis (sefA, agfA and fimC) and Escherichia coli (fasA and fasH). Regulation of 987P fimbriation could be identified after replacement of fasA and fasH with allelic reporter fusions. The expression of fasA but not fasH is dependent upon the osmolarity of the growth medium in an HNS-dependent manner, but unlike some other fimbrial systems expression is not dependent on the exogenous iron concentration.     

Monitoring transgene expression levels in different genotypes of field grown maize (Zea mays L.)

Transgenes in commercially available genetically modified plants are generally controlled by strong constitutive promoters to ensure a high level of expression at all stages of cultivation. Constitutive promoters however are influenced by a wide range of factors, and expression profiles of the transgenes in multiple genetic backgrounds have not yet been extensively studied. In this study a powerful expression profiling methodology for transgenic maize (Zea mays L.) is demonstrated on a large scale, analysing thousands of data points from three genotypes of herbicide and insect pest tolerant transgenic maize. Martonvásár inbred lines were crossed with LH244 maize line containing the MON 88017 events, and leaf tissue from the sixth backcross generation was sampled at four relevant phenological phases. Relative expression levels were determined using 18S rRNA as a reference and detailed statistical analysis performed. Expression levels of both transgenes are varied throughout plant development, and the interaction between the genetic background and phenophase are significant (p < 0.05). Expression is present at a significant level throughout all the phenological stages. We found that the genetic background has a significant (p < 0.01) effect on transgene expression levels in the case of the CP4epsps transgene, but not in the case of cry3Bb1, implying that the sensitivity of different constitutive promoter constructs to the effects of the genetic background is different.     

Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR

The family B DNA polymerase gene from the euryarchaeon Thermococcus barophilus Ch5 (Tba5) contains an open reading frame of 6198 base pairs that encodes 2065 amino acid residues. The gene is split by three inteins that must be spliced out to form the mature DNA polymerase. A Tba5 DNA polymerase gene without inteins (genetically intein-spliced) was expressed under the control of the pET-28b(+)T7lac promoter in E. coli Rosetta 2(DE3)pLysS cells. The molecular mass of the purified Tba5 DNA polymerase was about 90 kDa consistent with the 90,470 Da molecular mass calculated based on the 776 amino acid sequence. The optimal pH for Tba5 DNA polymerase activity was 7.5 and the optimal temperature was 70–75 °C. The enzyme possessed 3′ → 5′ exonuclease activity and was activated by magnesium ions. PCR amplification using Tba5 DNA polymerase enables high-yield for 1- to 6-kb target DNA products, while 8- to 10-kb target DNA products were amplified at low or inefficient levels. To simultaneously improve product yield and amplification fidelity, Tba5 plus DNA polymerase mixtures were constituted with various amounts of Tba5 DNA polymerase mixed with Taq DNA polymerase. The Tba5 plus DNA polymerase mixtures robustly amplified up to 25-kb λ DNA fragments. In addition, the PCR error rate of Tba5 plus3 and Tba5 plus4 mixtures were much lower than those of wild-type Tba5 DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and Pfu plus DNA polymerase.     

Influence of homoeotic genes on the aldehyde oxidase pattern in imaginal discs of Drosophila melanogaster

In a study of the regulation of enzyme patterns in imaginal discs the aldehyde oxidase pattern was determined for some homoeotic mutations of D. melanogaster. Earlier indications that suggested that this pattern follows the determinitive state of compartments within imaginal discs were confirmed by the aldehyde oxidase (AO) pattern of both the wing and haltere discs from en¹; bx³, en¹; pbx, and en¹; bx³ pbx larvae and the antennal discs from Antp^73b and ss^a larvae. We additionally analyzed whether AO activity depended on the determinative state of an entire compartment or was expressed autonomously in clones. Homozygous engrailed clones were induced by mitotic recombination. From the AO clones found in normally negative areas of the posterior compartment it was concluded that enzyme activity depended upon the determinative state of the cells and was not a function of the compartment as a whole. The results are described with reference to a scheme in which compartmental and subcompartmental selector genes are thought to determine a binary code on which AO patterns depend.     

amlC,Another amylolytic gene maps close to the amlB locus in Streptomyces lividans TK24

The region located upstream of the α-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC. amlC Encodes a 993 amino acid (aa) protein with a calculated molecular weight of 107.054 kDa. On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1,4-α-d-glucan glucanohydrolase family. amlC is transcribed as a unique 3 kb leaderless monocistronic mRNA. Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences. amlC was successfully disrupted and was mapped at approx. 700 kb from a chromosomal end of S. lividans TK24, 100 kb on the right of the amplifiable unit AUD1 (Volff et al., 1996). Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI–II fragment of the chromosome.     

A physical map of the genome of ethanol fermentative bacterium Zymomonas mobilis ZM4 and localization of genes on the map

A physical map of the Zymomonas mobilis ZM4 genome has been constructed from the results of reciprocal Southern hybridization with PmeI, PacI, and NotI-digested genomic DNA fragments and linking cosmid clones. Restriction enzyme-digested Z. mobilis ZM4 genome was electrophoresed with phage lambda DNA concatemers as a size standard in a Bio-Rad CHEF-DRII pulsed-field gel electrophoresis (PFGE) system. The restriction enzyme PmeI generated 15 fragments (3–625 kb), and PacI produced 19 fragments (7–525 kb). Each size of restriction fragment was calculated by comparison to the size of phage lambda DNA concatemers, and the genome size of Z. mobilis ZM4 was estimated to be 2085.5 kb. The 19 known genes and three rrn operons were localized on the map.     

Antifeedant activity of numb and salty taste compounds against the larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

Helicoverpa armigera, an important polyphagous insect pest in agriculture, attacks more than 200 plant species of more than 30 families. Our previous study showed that the choice feeding percentages of H. armigera larvae to tobacco leaf discs treated with sweet, bitter, and hot taste substances were higher than the control leaf discs, while numb and salty substances could significantly inhibit their feeding. To quantitatively determine the synergistic effect of numb and salty substances, in this paper, the antifeeding activities of numb and salty substances and their mixtures blended in different doses or volume ratios were assayed on H. armigera larvae. The first bioassay was designed to elucidate the relative feeding preference of the larvae to the leaves from several common host species, each paired with tobacco leaf discs, and the result indicated that the most preferred host leaf by the larvae was tobacco leaf, followed by cotton and peanut leaves, suggesting that tobacco leaf was the most suitable matrix for the antifeeding bioassay, and the larval consumption of maize, pepper, or tomato leaves were significantly lower than that of tobacco leaf. The second bioassay was to test the choice feeding response of H. armigera larvae to tobacco leaf discs treated with Zanthoxylum bungeanum extracts obtained with different solvents, and the result showed that the antifeeding activity of the alcohol extracts was the strongest (93.38%), and the leaf consumption in the treatment and the control showed extremely significant difference (t = 4.23, t_0.01 = 3.25, P = 0.0022), followed by the dichloromethane extracts (47.64%), while the other three solvents (water, acetone, and n-hexane) could not extract the active antifeeding components from Z. bungeanum. The larval consumption of tobacco leaf discs treated with the alcohol extracts of Z. bungeanum and NaCl solution were significantly less than their corresponding controls. The mean larval consumption of the treated leaf discs decreased with ever-increasing dosage, and the consumption of tobacco leaf discs coated with different doses of alcohol extracts of Z. bungeanum or NaCl solution showed extremely significant difference (F_{alcohol extract of Z. bungeanum} = 3.88, F_0.01 = 3.58, P = 0.0064; F_{NaCl solution} = 54.29, F_0.01 = 3.58, P = 0.0000), with maximum antifeeding effects at a dosage of 30 μL per 1.5 cm ID leaf disc. We further tested the larval consumption of tobacco leaf discs treated with alcohol extracts of Z. bungeanum in saturated NaCl solution mixed in different volume ratios, and the result showed that the choice antifeeding percentages of the treatments with 15 μL or more Z. bungeanum alcohol extracts were higher than 90%, among which the mixture with 25:15 volume ratio of Z. bungeanum alcohol extracts and saturated NaCl solution exhibited the strongest antifeeding activity, and the mean consumed leaf area of tobacco leaf discs coated with this blend was only 0.10 mm². In the further test on feeding dose-response of the 25:15 mixture, the mean leaf consumption decreased linearly with ever-increasing dosage, with a regression equation y = ?3.9356x + 120.78(R² = 0.9998), and the 30 μL dose could completely inhibit H. armigera feeding.     

Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3 × 10⁻⁵) was roughly similar to that of Pfu DNA polymerase (4.8 × 10⁻⁵), but much lower than those of wild-type Neq DNA polymerase (57.2 × 10⁻⁵), Neq A523R DNA polymerase (13.1 × 10⁻⁵), and Neq N540R DNA polymerase (37.7 × 10⁻⁵). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.