Drosophila melanogaster eggshell development: Localization of the s19 chorion gene

Summary Further IF screening ofDrosophila melanogaster geographic strains has revealed a variant of the s19 major chorion protein. Developmental analysis of F1 hybrids indicates that the source of the variation is found in the structural gene for this protein. The linkage group of the variant gene was determined to be the third, and the gene was localized by several methods of recombination analysis. The s19 gene was found to be tightly linked to thesepia locus, as had been previously found for the s18 gene (Yannoni and Petri 1980). Lack of recombination between the s19 and s18 genes in double heterozygotes suggested that these two genes are within 0.3 map units of each other. Although more precise localization of the s19 gene failed, the s18 gene could be more specifically located to the right ofsepia, betweensepia andhairy. Contrary to our prediction (ibid.), the s19 and s18 genes have been found to be tightly linked in spite of the fact that they display somewhat different developmental stage specificity.     

In vitro development of theDrosophila chorion in a chemically defined organ culture medium

Summary TheDrosophila chorion is produced normally in isolated follicles in Robb's chemically defined culture medium. The complex architecture of the shell developed in vitro from follicles as young as early stage 10 is completely normal morphologically. In addition, the time required for in vitro development closely approximates that observed for in vivo development. Comparisons of insect culture media developed by Robb, Grace, Schneider, and Echalier show large variations in their ability to supportDrosophila chorion development.     

Crosslinking of theDrosophila chorion involves a peroxidase

Summary TheDrosophila chorion contains an endogenous peroxidase activity which remains inactive until late stage 14 when it catalyzes the crosslinking of the chorionic proteins. Using explanted follicles developing in vitro, premature, but otherwise normal crosslinking can be induced with hydrogen peroxide and normal crosslinking can be prevented with peroxidase inhibitors. Inhibition or premature activation of the shell peroxidase allows characterization of chorionic filament specific proteins and establishes new criteria for the identification of eggshell components.     

Invasion of thehobo transposable element studied byin situ hybridization on polytene chromosomes ofDrosophila melanogaster

The invasion kinetics ofhobo transposable element in theDrosophila melanogaster genome was studied byin situ hybridization on the polytene chromosomes. Six independent lines ofDrosophila melanogaster flies that had been previously transformed by microinjection of the pHFL1 plasmid containing a completehobo element were followed over 50 generations. We observed thathobo elements were scattered on each of the chromosome arms, with more insertion sites on the 3R arm. The total number of insertion sites remains quite small, between four and six, at generation 52. On the 2R arm, a short inversion appeared once at generation 52. Most of the integration sites reported here were already described for several transposons but some of them appear to be hotspots forhobo elements.     

Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

In situ localization of PCR-amplified DNA and cDNA

Combining the high sensitivity of PCR with the cell localizing ability ofin situ hybridization allows for the reproducible detection of low copy targets in intact cells. This article describes several key variables that include fixation, protease digestion, the hot start maneuver, stringency, and, for RNA analysis, DNase digestion that are important to successfulin situ PCR. Also stressed is the importance of performing and interpreting controls with each experiment. Important controls include omission of key components, use of samples known either to contain or lack the target of interest and, most importantly, the in-built controls invariably present in the heterogeneous component of any given tissue type.     

人单拷贝及重复DNA序列的原位PCR

在培养的人小肠癌转移腹水细胞系细胞中进行了Y染色体特异的重复序列及单拷贝序列的原位扩增与检测.结果显示原位PCR法的灵敏度比直接的原位杂交法明显提高.     

Characterization by isoelectric focusing of chorion protein variants inDrosophila melanogaster and their use in developmental and linkage analysis

Summary Drosophila melanogaster chorion proteins are characterized on one-dimensional isoelectric focusing (IF) gels. The six major chorion components previously identified on SDS gels are shown to resolve into at least 11 components in our IF system. IF screening of 102 geographic strains ofDrosophila melanogaster revealed seven cases of variation in major chorion components. Two strains, Crimea and Falsterbo, which were monomorphic for a variant B1 protein and two strains, Skafto and Lausanne, which were monomorphic for a variant C1 protein, were chosen for further study. After IF developmental analysis of F1 hybrids had indicated that the sources of the variation resided in the structural genes for these proteins, each variant was crossed to a multiply marked and inverted strain (BLT) to determine the linkage group of the variant gene. To localize genes to more specific sites multiply marked 3rd (SKERO) or X-chromosomal (CB1) (X-PLE) mapping strains were used. In both Crimea and Falsterbo the gene for the B1 protein is located near map location 26 on the 3rd chromosome. In both Lausanne and Skafto the C1 gene is located on the X chromosome. Hence, for the first time, we have demonstrated genetically the non-linkage of two chorion genes, B1 and C1.     

The use ofin situ hybridization histochemistry for the study of neuropeptide gene expression in the human brain

1. The application of in situ hybridization histochemistry to the study of neuropeptide gene expression in human brain postmortem tissues is reviewed. We focus on neuropeptides preferentially expressed in hypothalamus and basal ganglia. 32P-labeled oligonucleotides were used as hybridization probes. 2. Autoradiography combined with computerized image analysis was used to visualize and quantify the hybridization signal. 3. Several criteria were considered in order to ascertain the specificity of the signal, including Northern analysis, use of heterologous probes, competition assays, and thermal stability of the hybrids. 4. In control human striatum high levels of hybridization signal were observed for somatostatin, neuropeptide Y, and preproenkephalin A mRNAs. In contrast, no detectable signal was observed with the cholecystokinin, arginine-vasopressin, and oxytocin probes in this area. In the hypothalamus high levels of oxytocin and arginine-vasopressin mRNAs were visualized in several nuclei. Preproenkephalin A and somatostatin mRNAs were also observed in this region, while cholecystokinin mRNA was not detected. 5. No significant correlations were found between the density of the hybridization signal and parameters such as postmortem delay, age, and gender in the population studied. 6. Finally, alterations of mRNA levels for some of these peptides were found in Parkinson's disease and Huntington's chorea striatal tissues. 7. These results show that in situ hybridization histochemistry can be used to examine at the microscopic level neuropeptide gene expression in postmortem materials.     

Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus:In situ hybridization histochemistry and immunocytochemistry

1. In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem. 2. Alternate serial sections were hybridized with a 48-base, 35S-labeled synthetic oligonucleotide probe encoding SP using in situ hybridization histochemistry and processed either histochemically for AChE or immunocytochemically for choline acetyltransferase (ChAT). 3. In addition, serial section analysis was used to demonstrate the correlation between SP and SP mRNA in the same cells of the LDT. 4. These studies reveal that the cholinergic neurons of the LDT synthesize SP.     

Genetic and biochemical analysis of brown eye mutation in Drosophila nasuta nasuta and Drosophila nasuta albomicans

By analyzing the progeny of crosses involving brown eye mutants and the wild types in two members of Drosophila nasuta subgroup namely D. n. nasuta and D. n. albomicans we could show that the mutant gene is recessive, located in the chromosome 2 and the alleles of this gene are present at different loci. A study of fitness in the eye color mutants in comparison with the wild types revealed that D. n. nasuta mutant has higher viability at both 25 ± 1°C and ambient temperatures; while D. n. albomicans mutant has faster rate of development only at 25 ± 1°C. Quantitative analysis of eye pigments in the mutants revealed that there is biosynthesis of both pteridines and xanthommatins unlike in bw/bw of D. melanogaster, where only xanthommatins are synthesized. In both the species, the pteridine quantities in mutants are similar; whereas xanthommatin quantity in is 10 times higher than that of . Further, the F₁ progeny of intraspecific crosses (wild type X mutant) are found to have high amounts of pteridine, even when compared with parental wild type. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quantitative aspects of anin situ hybridization procedure for detecting mRNAs in cells using 96-well microplates

The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitativein situ hybridization (ISH) using either image analysis or fluorescencein situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by thep-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.     

Specific detection of RNA molecules by fluorescent in situ hybridization in living cells

The antisense therapeutic strategy makes the assumption that sequence-specific hybridization of an oligonucleotide to its target can take place in living cells. The present work provides a new method for the detection of intracellular RNA molecules using in situ hybridization on living cells. The first step consisted in designing nonperturbant conditions for cell permeabilization using streptolysin O. In a second step, intracellular hybridization specificity was evaluated by incorporating various types of fluorescently labeled nucleic acid probes (plasmids, oligonucleotides). Due to its high expression level, the 28S ribosomal RNA was retained as a model. Results showed that: (1) no significant cell death was observed after permeabilization; (2) on living cells, 28S RNA specific probes provided bright nucleoli and low cytoplasmic signal; (3) control probes did not lead to significant fluorescent staining; and (4) comparison of signals obtained on living and fixed cells showed a colocalization of observed fluorescence. These results indicate the feasibility of specific hybridization of labeled nucleic acid probes under living conditions, after a simple and efficient permeabilization step. This new detection method is of interest for investigating the dynamics of distribution of various gene products in living cells, under normal or pathological conditions.Abbreviations PI propidium iodide - SLO streptolysin O     

Patterns of replication in the neo-sex chromosomes ofDrosophila nasuta albomicans

Drosophila nasuta albomicans (with 2n = 6), contains a pair of metacentric neo-sex chromosomes. Phylogenetically these are products of centric fusion between ancestral sex (X, Y) chromosomes and an autosome (chromosome 3). The polytene chromosome complement of males with a neo-X- and neo-Y-chromosomes has revealed asynchrony in replication between the two arms of the neo-sex chromosomes. The arm which represents the ancestral X-chromosome is faster replicating than the arm which represents ancestral autosome. The latter arm of the neo-sex chromosome is synchronous with other autosomes of the complement. We conclude that one arm of the neo-X/Y is still mimicking the features of an autosome while the other arm has the features of a classical X/Y-chromosome. This X-autosome translocation differs from the other evolutionary X-autosome translocations known in certain species ofDrosophila.     

Chromosomal mapping of T-DNA inserts in transgenicPetunia byin situ hybridization

The chromosomal location of T-DNa inserts in ten independently derived and confirmed transgenic plants ofP. hybrida was detected byin situ hybridization. Nine transgenic plants had the T-DNA inerts at single sites distributed among each of the seven chromosomes; in one plant the T-DNA inserts were detected on two different chromosomes. Although the T-DNA inserts were integrated randomly among the chromosomes, seven of the 11 total inserts were located at or near the telomere. Thus, T-DNA inserts appear to have potential for tagging chromosomes and chromosome fragments.     

Sequential gene activation by ecdysteroids in polytene chromosomes ofDrosophila melanogaster

Summary Changes in polytene chromosome 3 L puffing patterns in the fat body ofDrosophila melanogaster larvae and prepupae are compared to those in the salivary gland. While some general features are common to the two tissues, there are differences which reflect their different developmental roles. In vitro experiments with fat body chromosomes show that they have a distinct response to ecdysteroids which is different from that of salivary gland chromosomes, and which does not,in this culture system, reproduce the changes observed in normal development. In short term culture experiments, the fat body chromosomes appear more sensitive to ecdysteroids than the salivary gland chromosomes and, although 20-OH ecdysone is more active than ecdysone in these assays, the possibility is not excluded that ecdysone has a role in normal development as it appears to alter gene activity at physiological levels in these cells.     

Strategies for signal amplification in nucleic acid detection

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.     

In situ nutrient assays of periphyton growth in a lowland Costa Rican stream

Nutrient limitation of primary production was experimentally assessed using an in situ bioassay technique in the Quebrada Salto, a third-order tropical stream draining the northern foothills of the Cordillera Central in Costa Rica. Bioassays employed artificial substrata enriched with nutrients that slowly diffuse through an agar-sand matrix (Pringle & Bowers, 1984). Multiple comparisons of regression coefficients, describing chlorophyll-a accrual through time for different nutrient treatments, revealed positive micronutrient effect(s). Micronutrient treatment combinations (Fe, B, Mn, Zn, Co, Mo, EDTA), supplemented with and without nitrate and phosphate, exhibited significantly greater chlorophyll-a accrual over all other treatments (P < 0.05), supporting over three times that of the control after 14-d of substratum colonization. Neither of the major nutrients (N or P) produced a significant stimulation, although the N treatment displayed 50% more chlorophyll-a than the control after 14-d. Similarly, Si, EDTA, and Si + N + P treatments did not exhibit chlorophyll-a response curves that were significantly different from the control. During the experiment, mean NH₄-N and (NO₂ + NO₃)-N concentrations in the Salto were 2.0 µM (28.6 µg · l^–1) and 7.2 µM (100.2 µg · l ^–1), respectively. High concentrations of PO₄-P ( = 2.0 µM; 60.9 µg · l^–1) and TP ( = 3.0 µM; 94.0 µg · l^–1) were also found, and consequently low molar N:P ratios = 4.7). Despite the potential for N limitation in the system, both N and P appear to be at growth saturating levels. This may be due to micronutrient limitation and/or light limitation of periphyton growth in densely shaded upstream portions of the stream.