A one-step efficient and specific non-radioactive non-fluorescent method for in situ hybridization of banded chromosomes

A non-radioactive method for in situ hybridization of cosmid probes to metaphase chromosomes is described. Two procedures are involved: (i) hybridization with a cosmid probe labelled by nick translation in the presence of digoxigenin dUTP. The signal is visualized by an alkaline phosphatase conjugated antibody. (ii) FPG banding of the chromosomes. The steps involved in these two procedures are combined in an order which allows simultaneous observation of the banding pattern and the hybridization signal. The metaphases can thus be analysed after a single photographic step. This technique is considerably simpler than the method used previously.     

Microevolutionary divergence pattern of the segmentation gene hunchback in Drosophila

To study the microevolutionary processes shaping the evolution of the segmentation gene hunchback (hb) from Drosophila melanogaster, we cloned and sequenced the gene from 12 isofemale lines representing wild-type populations of D. melanogaster, as well as from the closely related species Drosophila sechellia, Drosophila orena, and Drosophila yakuba. We find a relatively low degree of sequence variation in D. melanogaster (theta = 0.0017), which is, however, consistent with its chromosomal location in a region of low recombination. Tests of neutrality do not reject a neutral-evolution model for the whole region. However, pairwise tests with different subregions indicate that there is a relative excess of polymorphic sites in the leader and the intron. Codon usage pattern analysis shows a particularly biased codon usage in the highly conserved regions, which is in line with the hypothesis that selection on translational accuracy is the driving force behind such a bias. A comparison of the expression pattern of hb in different sibling species of D. melanogaster reveals some regulatory changes in D. yakuba, which could be interpreted as changes in the timing of secondary expression domains.     

Comparison of the gap segmentation gene hunchback between Drosophila melanogaster and Drosophila virilis reveals novel modes of evolutionary change.

We have cloned and sequenced a large portion of the hunchback (hb) locus from Drosophila virilis. Comparison with the Drosophila melanogaster hb sequence shows multiple strong homologies in the upstream and downstream regions of the gene, including most of the known functional parts. The coding sequence is highly conserved within the presumptive DNA-binding finger regions, but more diverged outside of them. The regions of high divergence are correlated with regions which are rich in short direct repeats (regions of high 'cryptic simplicity'), suggesting a significant influence of slippage-like mechanisms in the evolutionary divergence of the two genes. Staining of early D.virilis embryos with an hb antibody reveals conserved and divergent features of the spatial expression pattern at blastoderm stage. It appears that the basic expression pattern, which serves as the gap gene function of hb, is conserved, while certain secondary expression patterns, which have separate functions for the segmentation process, are partly diverged. Thus, both slippage driven mutations in the coding region, which are likely to occur at higher rates than point mutations and the evolutionary divergence of secondary expression patterns may contribute to the evolution of regulatory genes.     

Cellular localization of induced human interferon-beta mRNA by non-radioactive in situ hybridization

Induced interferon-beta (IFN-beta) mRNA was localized in human FS-4 fibroblasts by in situ hybridization using biotinylated probes. The hybridization sites were detected by incubation with a nick-translated genomic DNA probe (1.8 kb) via streptavidin-colloidal gold followed by silver contrast enhancement. The positive signals were observed by reflection-contrast light microscopy. IFN-beta mRNA was transiently induced by poly r(I): r(C) in fibroblasts 2-4 h after induction. Induction in the presence of cycloheximide and actinomycin D (superinduction conditions) exhibited an enhanced level of IFN-beta mRNA with a maximum at 4-8 h. The kinetics of the IFN-beta mRNA expression in the cytoplasm as revealed by in situ hybridization proved to be compatible with the results of Northern blotting experiments of total cellular RNA.     

Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization

Summary We report a general procedure which allows the application of whole cosmid cloned genomic sequences for non-radioactive in situ hybridization. The presence of highly repetitive sequences, like Alu and Kpn fragments, is eliminated through competition hybridization with Cot-1 DNA. The method has been tested and optimized with several randomly chosen cosmids of the human thyroglobulin (Tg) gene (8q24). At present, the procedure can be performed with three of the four tested individual cosmids. In cases where a single clone does not result in a specific signal, a larger fragment may be required, which can be accomplished by using two (partially overlapping) cosmids of the same region. The advantages and further potentialities of such a hybridization approach are discussed.     

Developmental expression of crystallin genes: in situ hybridization reveals a differential localization of specific mRNAs

The time and place of the accumulation of alpha A-, beta B1- and gamma-crystallin RNA in the developing rat lens have been studied by in situ hybridization. alpha A- and gamma-crystallin RNA were first detected in the lens vesicle, while beta B1-crystallin RNA could be seen only after elongation of the primary fiber cells. Both beta B1- and gamma-crystallin RNA were confined to the fiber cells of fetal lenses, while alpha A-crystallin mRNA could also be detected in the epithelial cells. A quantification of the hybridization pattern obtained in the differentiation zone of the newborn rat lens showed that alpha A-crystallin RNA is concentrated in the cortical zone. alpha B-crystallin mRNA has the same distribution pattern. beta B1-crystallin RNA was relatively poorly detectable by in situ hybridization in both fetal and newborn rat lenses. The grain densities obtained with this probe increased from the periphery of the lens toward the interior, indicating that beta B1-crystallin RNA accumulated during differentiation of the secondary fiber cells. A similar accumulation pattern was obtained for gamma-crystallin mRNA, but, unexpectedly, this RNA could also be detected in the elongating epithelial cells. Our results show that gamma-crystallin RNA starts to accumulate as soon as visible elongation of epithelial cells occurs, during differentiation of the primary as well as the secondary fiber cells.     

A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome-labelled RNA

A new method has been developed for the detection of in situ hybridization by fluorescence microscopy. It is based on the covalent binding of commercially available fluorochromes to the 3′-terminus of RNA.     

A simplified and efficient method for in situ hybridization to whole Drosophila embryos, using electrophoresis for removing non-hybridized probes

We report a simplified and reliable method for non-radioactive in situ hybridization to whole Drosophila embryos. In the previous method (Tautz and Pfeifle, 1989) the post-hybridization wash, or the procedure for washing non-hybridized probe away from embryos depends simply on diffusion. We modified the method with application of electrophoresis to the wash. After hybridized with RNA probe, embryos were transferred to a small well where an electric charge was given to drive non-hybridized probe away from the embryos. This procedure enables us to acquire a much higher signal-to-noise ratio than that obtained from a conventional method. Furthermore, this is a time-saving method. We propose a term "electro-wash" for this procedure.     

Tissue- and stage-specific control of homeotic and segmentation gene expression in Drosophila embryos by the polyhomeotic gene

The distributions of the products of the homeotic genes Sex combs reduced (Scr) and Ultrabithorax (Ubx) and of the segmentation genes, fushi tarazu (ftz), even skipped (eve) and engrailed (en) have been monitored in polyhomeotic (ph) mutant embryos. None of the genes monitored show abnormal expression at the blastoderm stage in the absence of zygotic ph expression. Both Scr and Ubx are ectopically expressed in the epidermis of ph embryos, confirming the earlier proposal, based on genetic analysis, that ph+ acts as a negative regulator of Antennapedia (ANT-C) and bithorax (BX-C) complex genes. At the shortened germ band stage, en is also ectopically expressed, mainly in the anterior region of each segment. In contrast to these effects in the epidermis, the expression of en, Ubx, Scr and ftz is largely or completely suppressed in the central nervous system, whereas eve becomes ectopically expressed in most neurones.     

hunchback, a gene required for segmentation of an anterior and posterior region of the Drosophila embryo

The locus hunchback (hb) is a member of the gap class of segmentation genes of Drosophila. A number of X-ray-induced deletions locate the hb locus at the chromosomal site 85A3-B1, to the right of the pink locus, which maps in the same interval. A total of 14 EMS and 3 X-ray-induced hb alleles have been studied. Homozygous mutant embryos show deletions of segments in two separate regions. In the six strong alleles, the labium and all three thoracic segments are deleted anteriorly while posteriorly the 8th abdominal segment and adjacent parts of the 7th abdominal segment are lacking. The eight weak alleles show smaller deletions both in the thoracic and posterior abdominal region. In the weakest allele only part of the mesothorax is deleted. Three hb alleles produce a homoeotic transformation: superimposed on a strong or weak deletion phenotype, head or thoracic segments are transformed into abdominal segments, respectively. This suggests that hb might also be involved in the regulation of genes in the Bithorax complex (BX-C). Fate mapping of the normal-appearing segments in strong mutant embryos using the UV-laser beam ablation technique (Lohs-Schardin et al., 1979) shows that these segments arise from the normal blastoderm regions. The mutant phenotype can be recognized soon after the onset of gastrulation in a failure to fully extend the germ band. In 6-hr-old mutant embryos, two clusters of dead cells are observed in the thoracic and posterior abdominal region. These observations indicate region specific requirement of hb gene function. The analysis of germ line chimeras by transplantation of homozygous mutant pole cells shows that hb is already expressed during oogenesis. Homozygous mutant embryos derived from a homozygous mutant germ line have a novel phenotype. The anterior affected region is enlarged, including all three gnathal segments and the anterior three abdominal segments. In addition three abdominal segments with reversed polarity are formed between the remaining head structures and the posterior abdomen. Heterozygous mutant embryos derived from a homozygous mutant germ line develop normally, indicating that maternal gene expression is not required for normal development.     

Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues

For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to decalcify the tissues. We evaluated the usefulness of various acid-based decalcifying reagents with reference to 28 S rRNA staining by in situ hybridization using a thymine-thymine dimerized oligonucleotide probe. The reagents evaluated were 10% nitric acid, 10% HCl, 5% formic acid, 5% trichloroacetic acid, Morse’s solution, Plank-Rychlo’s solution, and K-CX solution, all of which are commonly used to decalcify tissues, and their effects on retention of morphology and RNA were compared with EDTA-based solutions. When normal mouse mandible was used as a model tissue, well-preserved morphology of ameloblasts was obtained from sections decalcified with Morse’s solution, 10% HCl, Plank-Rychlo’s solution, and K-CX solution, and best retention of 28 S rRNA was obtained with 5% formic acid and Morse’s solution. We recommend Morse’s solution to decalcify tissues to be processed for the rapid analysis of specific RNA expression. Indeed, we detected specific mRNAs strongly in sections treated with Morse’s solution, and quantitative analysis showed that the ratio of signal intensities of 28 S rRNA and the specific mRNAs correlated with each other depending on decalcifying solutions. Accepted: 3 January 2000     

Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos

In Drosophila, polar plasm contains polar granules, which deposit the factors required for the formation of pole cells, germ line progenitors. Polar granules are tightly associated with mitochondria in early embryos, suggesting that mitochondria could contribute to pole cell formation. We have previously reported that mitochondrial large and small rRNAs (mtrRNAs) are transported from mitochondria to polar granules prior to pole cell formation and the large rRNA is essential for pole cell formation. Here we show that the localization of mtrRNAs is diminished in embryos laid by tudor mutant females, although the polar granules are maintained. We also found that Tud protein was colocalized with mtrRNAs at the boundaries between mitochondria and polar granules when the transport of mtrRNAs takes place. These observations suggest that Tud mediates the transport of mtrRNAs from mitochondria to polar granules.