A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligonucleotide primed in situ labelling (PRINS)

The centromeric alpha satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence and cannot be easily distinguished by mean of probes for Southern blot or in situ hybridisation. We have used the oligonucleotide-primed in situ (PRINS) labelling technique with primers defined from the alpha satellite sequence of chromosome 13. One primer was found to label specifically the centromeric region of chromosomes 13 and allowed the detection of a polymorphism between two chromosome 13 homologues in one individual.     

Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney

The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.     

The distribution of sequences complementary to human satellite DNAs I,II and IV in the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus)

Human satellite DNAs I, II and IV were transcribed to yield radioactive complementary RNAs (cRNAs). These cRNAs were hybridised to metaphase chromosomes of man, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus). The results of this in situ hybridisation were analysed quantitatively and compared with accepted chromosome homologies based on Giemsa banding patterns. The cRNA to satellite II (cRNAII) did not hybridise to chimpanzee chromosomes, although its hybridisation to chromosomes of gorilla and orang utan yielded more autoradiograph grains than hybridisation to human chromosomes, and cRNAIV hybridised to many chromosomes of gorilla and chimpanzee but was almost entirely restricted to the Y chromosome in orang utan. Most sites of hybridisation were located on homologous chromosomes in all four species, but there were a number of sites which showed no correspondence between satellite DNA location and chromosome banding patterns, and others where a given chromosomal location hybridised with different cRNAs in each species. These results are in contrast to those found for many transcribed DNA sequences, where the same sequence is usually located at homologous chromosome sites in different species, and appear to cast doubt on many proposed models of satellite DNA function.     

Local enrichment with homopolymeric (dA/dT) DNA in genomes of some lower dipterans and Drosophila melanogaster

An investigation into the chromosomal localization of homopolymeric dA/dT was carried out with species of the genera Rhynchosciara, Chironomus, Drosophila and several other taxa. In situ hybridisation probing mitotic and polytene chromosomes with RNA homopolymers was performed, followed by immunological detection of the DNA/RNA hybrid. Use of this method allowed us to assess specific regions of some dipteran genomes, where the signal was generally, but not always, located in heterochromatic regions. Human and Drosophila chromosome regions known to contain dA/dT runs of up to 153 bp were devoid of consistent labelling. The stability of the rA/dT hybrid formed in situ was in agreement with the T(m) for long rA/dT hybrid complexes, suggesting that the method used in this work is able to identify unusually long homopolymeric dA/dT tracts.     

Comparison of different methods of introducing a label into the DNA of microorganisms

Different methods of isotope introduction into plasmid and chromosomal DNA have been compared. The efficiency of label introduction into DNA was estimated by the results of DNA--DNA hybridisation and by the thermostability of hybrid duplexes. Using the enzymatic methods of label introduction levels of DNA specific radioactivity and label binding in homologous and heterologous reactions were close. DNA labeled by the method of enzymatic methylation forms more thermostable hybrid duplexes than "nick-translation" DNA. The advantages of enzymatic methods of label introduction for creating a microorganism reference DNA bank are discussed.     

Satellite DNA and evolution of sex chromosomes

The satellite DNA (satellite III) which is mainly represented in the female of Elaphe radiata (Ophidia, Colubridae) has been isolated and its buoyant density has been determined (=1.700 g cm^–3). In situ hybridisation of radioactive complementary RNA of this satellite DNA with the chromosomes of different species has revealed that it is mainly concentrated on the W sex chromosome and its sequences are conserved throughout the sub-order Ophidia. From hybridisation studies these sequences are absent from the primitive family Boidae which represents a primitive state of differentiation of sex chromosomes. Chromosome analysis and C-banding have also revealed the absence of heteromorphism and of an entirely heterochromatic chromosome in the species belonging to the primitive family and their presence in the species of highly evolved families. It is suggested that the origin of satellite DNA (satellite III) in the W chromosome is the first step in differentiation of W from the Z in snakes by generating asynchrony in the DNA replication pattern of Z and W chromosomes and thus conceivably reducing the frequency of crossing-over between them which is the prerequisite of differentiation of sex chromosomes. Presence of similar sex chromosome associated satellite DNA in domestic chicken suggests its existence in a wider range of vertebrates than just the snakes.     

Ribonucleic acid from the higher plant Matthiola incana. Molecular weight measurements and DNA-RNA hybridisation studies.

The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo. In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured. 1. The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography. The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6). The rRNA precursor has a molecular weight of approx. 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5). 2. The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA. In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found. A rapidly-hybridising component is attributed to small amounts of contaminating rRNA. 3. M. incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1. The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1. 4. S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component. None of the RNAs tested hybridised to the satellite DNA. The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA. 4. 5 and 4 S RNA preparations contain fragments which hybridise to sequences complementary to high-molecular-weight rRNA. This spurious hybridisation can be eliminated by competition with unlabelled high-molecular-weight RNA.     

Multiplicity and diversity of cloned zein cDNA sequences and their chromosomal localisation

We have constructed and screened cDNA libraries from total maize endosperm poly(A) RNA or from a mRNA fraction enriched in zein sequences. From these libraries we have isolated clones representative of the major classes of zein cDNA sequences and have characterised them by crosshybridisation, by hybrid-selected translation, by in situ hybridisation to maize chromosomes, and hybridisation to genomic Southern blots. We conclude that at least four types of non cross-hybridising zein sequences are present, two coding for light chains and two for heavy chains. At least in the case of the light zeins, there is considerable sequence diversity among the clones which hybridise to each type. Similar results are obtained by translation of the mRNAs selected by each clone. In situ hybridisation shows that the light chain zein genes are located on chromosomes 4, 7, and 10, whilst genes coding for some of the heavy chain zeins are confined to the distal part of the long arm of chromosome 4.     

Satellite DNA of Anopheles stephensi liston (Diptera: Culicidae)

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.     

The location of four human satellite DNAs on human chromosomes.

In situ hybridisation was carried out with ³H-cRNAs transcribed from four human satellite DNAs. The human metaphase chromosomes used were stained with quinacrine and photographed prior to hybridisation. This allowed accurate karyotyping of the autoradiographs. A method of quantitative analysis of grain distribution permitted identification of minor sites of hybridisation which could be distinguished from purely random grains. The hybridisation patterns for each of the transcribed satellite cRNAs were similar, with the C-band of chromosome 9 and the Y chromosome being the most heavily labelled sites. Other detectable sites of hybridisation were the centromeric regions of chromosomes 1, 5, 7, 10, 12, 13, 14, 15, 17, 20, 21 and 22. The cRNA transcribed from DNA satellite II, however, was the only one to hybridise to the centromere region of chromosome 16. The evolution of the human satellite DNAs and the validity of current models of satellite DNA function are discussed in the light of the present results.     

Ultrastructural localisation of intramuscular expression of BDNF mRNA by silver-gold intensified non-radioactive in situ hybridisation

A non-radioactive in situ hybridisation method is described for the detection of low intramuscular levels of brain-derived neurotrophic factor (BDNF) mRNA at the electron microscope level. Application of high-grade silver-gold intensification of the diaminobenzidine end product of in situ hybridisation revealed that in adult rat muscle the constitutive expression of muscular BDNF is confined to the myofibres; satellite cells, Schwann cells, endothelial cells, fibroblasts or axons do not appear to contribute to BDNF production in normal muscle. Although muscular BDNF is a neurotrophic factor for innervating motoneurons and supposedly released only at the motor endplates, the production of BDNF mRNA appears to occur along the entire length of the myofibres and is not confined to nuclei in the postsynaptic regions.     

A comparative study of in situ hybridization procedures using cRNA applied to Drosophila hydei salivary gland chromosomes

The effect of different denaturation and hybridization procedures on the efficiency of in situ ³H-cRNA hybridization with DNA in the polytene chromosomes of Drosophila hydei was investigated.Denaturation of the DNA in the squash preparations with 90% formamide in 0.1 × SSC at 65 °C for 2.5 h gave a significantly higher retention of radioactivity following in situ hybridization than did denaturation by 30 sec incubation in boiling 0.1 × SSC.A comparison of the effect of various SSC concentrations in the hybridization mixture revealed that among the SSC concentrations tested, 3 × SSC or 4 × SSC gave the highest efficiency of hybrid formation.Hybridization in 50% formamide at 20 °C resulted in continuing hybrid formation over a period of 3.5 h, the majority of the cRNA/DNA hybrids being formed within the first 10 min of the incubation period. The thermal dissociation profile of in situ cRNA/DNA hybrids formed in 50% formamide, 4 × SSC at 20 °C, as determined in 0.1 × SSC indicated a T_m of 66 °C. The shape of the profile and the results of competition experiments suggested a high fidelity of base-matching in the in situ ³H-cRNA/DNA hybrids.Non-chromosomal background labeling in autoradiographs of polytene chromosomes hybridized with ³H-cRNA was effectively reduced by adding a 200–1000 fold excess of cold 28S + 18S RNA.     

The chromosomal localisation of satellite DNA in Ptyas mucosus (Ophidia,Colubridae)

Ptyas mucosus male DNA has a repetitious DNA satellite (p= 1.700 g cm^?3) constituting 5% of the haploid genome. In situ hybridisation of radioactive complementary RNA (cRNA) has revealed that satellite sequences are located in the centromeric region of one pair of macrochromosomes and in the terminal region of 8 pairs of microchromosomes. These regions are constitutively heterochromatic as revealed by C-banding. The possibility of involvement of satellite rich microchromosomes in nucleolus organisation is discussed.     

Vitamin U and RNA metabolism in prokaryotes

The paper is concerned with a study of the vitamin U effect on the rate of 14C-uridine incorporation into various categories of RNA in E. coli MRE-600 cells. It is found that cells grown with vitamin U (0.06 mg/ml) and incubated with 14C-uridine for 5 min are able to produce a 10-12-fold increase of the label incorporation into 4 S and 5 S RNA and a 14-fold increase into high polymeric RNA in comparison with the control cells. Under longer intervals of incubation (20 min) the intensity of high-polymeric RNA formation was half as high as for 4 S and 5 S RNA formation. MAK column chromatography of high-polymeric RNA in salt and temperature gradients showed the presence of the RNA temperature fraction in bacteria cells. Vitamin U stimulates the formation of various categories of RNA and causes a quantitative increase in the RNA temperature fraction.     

The effects of mercury-substitution on the hybridisation characteristics of nucleic acids.

The effect of different levels of mercury substitution on the rate and extent of hybridisation of globin mRNA with a complementary DNA (cDNA) copy has been investigated. It was found that mercuration significantly reduces both the rate of hybridisation and the extent of the reaction, but that these effects are abolished when at least stoichiometric amounts of 2-mercaptoethanol are included in the hybridisation medium. As a preliminary to using this technique to isolate specific groups of sequences after long-term hybridisations, we have investigated both the rate of demercuration of RNA and its retention on thiol-sepharose columns after extended incubation under commonly employed hybridisation conditions at 43 degrees or 60 degrees. Retention was essentially quantitative even after incubation times of 300 hours at 43 degrees, but decreased significantly after 48 hours at 60 degrees. It is concluded that thiol-sepharose chromatography offers considerable advantages over hydroxyapatite chromatography for the recovery of hybridised sequences, particularly with regard to the lower levels of non-specific binding obtained and its ability to distinguish directly between DNA-DNA and DNA-Hg RNA hybrids.