RNA synthesis and chromatin structure in mammalian cells. In situ detection of template changes in living and formalin fixed L cell nuclei

An autoradiographic study was carried out to compare the mean rates of RNA synthesis in exponential and stationary L cell monolayer cultures, with the ability of the cell nuclei to rapidly bind ³H-actinomycin D (AMD) in vivo and to serve as templates for polyadenylic acid, poly(A), synthesis in vitro.     

In situ hybridization of iodinated ribosomal RNA to human chromosomes

Iodinated ribosomal RNA was hybridized to human metaphase chromosomes in a test of the effectiveness of iodinated products for in situ hybridization studies in a diploid system. The results indicate that ¹²⁵I is a feasible alternative as a source of radioactivity for autoradiographic mapping studies.     

In vivo BUdR labeling of mammalian chromosomes

A technique is described for labeling mammalian chromosomes in vivo with BUdR. Rats and mice are given BUdR by tail vein infusion over a 24-h period at a concentration of 25 μg/g wt/h. Metaphase cells that have gone through two or three cycles of DNA synthesis reveal characteristic differential chromatid fluorescence after staining with Hoechst dye. Sister chromatid exchanges can then be easily detected in these cells.     

In situ methylation of human chromosomes with methylase-AluI

A method for in situ DNA methylation with the prokaryotic methylase AluI has been developed for use on fixed human chromosomes. Incorporation of methyl groups into the chromosomal DNA has been shown by autoradiography using a labeled substrate. The methylation prevents the digestion of chromosomal DNA by AluI, allowing direct visualization of clusters of nonmethylated AGCT targets along the human complement.     

In situ random primer extension of metaphase chromosomes

We have developed a technique of random primer extension of fixed chromosomes that is applicable to both mouse and man. Human chromosomes are not homogeneously labeled with this technique; those regions corresponding to R-bands appear to be more sensitive than those identified as G-bands, whereas centromeric regions are not labeled. These results not only corroborate specific structural differences between distinct regions of mammalian genomes but also open up the possibility of assays with specific primers to test whether primer extension is useful for the identification of genes and families of sequences on chromosomes.     

Chromosomal RNA synthesis in polytene chromosomes of Chironomus tentans

The presence of heterogeneous RNA of high molecular weight has been demonstrated on the giant chromosomes, in the nuclear sap and in the cytoplasm of the salivary glands in Chironomus tentans. The kinetic properties of this heterogeneous RNA have also been outlined in some detail. — Salivary glands were incubated for different time intervals (20, 45 and 180 min) in haemolymph, supplemented with tritiated cytidine and uridine. The different cellular components were isolated by micromanipulation and RNA extracted with an SDS-pronase solution and analysed with electrophoresis in agarose. — Heterogeneous, high molecular weight RNA with a peak around 35 S was saturated with label on chromosome I, II and III in 45 min, although the synthetic capacity was unchanged during at least 180 min incubation. This indicated a complete turnover of heterogeneous RNA on the chromosomes in less than 45 min. The turnover time in the giant puffs (the so called Balbiani rings) on the fourth chromosome, was even shorter and estimated to less than 30 min. No shift in the electrophoretic pattern of this heterogeneous RNA was found to occur on the chromosomes during long incubation times or during actinomycin D experiments. These labelling characteristics of heterogeneous RNA on the chromosomes indicate that all the different molecules in the heterogeneous RNA have a similar and rapid turnover. A conversion to smaller, stable molecules was excluded. — Heterogeneous RNA of a distribution corresponding to that on the chromosomes was found in the nuclear sap and also in the cytoplasm. The activity in both these cellular compartments increased between 45 and 180 min incubation. The distribution pattern for high molecular weight RNA was in all experiments similar on the chromosomes, in the nuclear sap and in the cytoplasm. It appears that at least a considerable part of the high molecular weight RNA leaves the chromosomes to enter the nuclear sap and lateron to some extent the cytoplasm in this high molecular form. Stable molecules of smaller size (6–15 S) did not appear during 180 min incubation. The data indicate, however, also a substantial breakdown of heterogeneous RNA to acid soluble products during this time.     

Activity of the endogenous RNA polymerase on fixed polytene chromosomes.

Fixed polytene chromosomes can serve as templates for RNA synthesis in situ, using the endogenous chromosomal DNA-dependent RNA polymerase. Labelling is mainly localized in band regions. However, radioactivity can also be found in interbands and puffs similar to that which occurs in vivo. It is also found by this technique that the nucleolar RNA polymerase appears to be active in these preparations and requires Mg²⁺ for activity. Since the pattern of the RNA transcribed in situ with the DNA-dependent RNA polymerase from E. coli of native chromosomes differs from that with endogenous RNA polymerase and resembles the one obtained with heat-treated chromosomes, it is suggested that the polymerase from E. coli does not act specifically on eukaryotic chromosomes.     

In situ fluorescent nick translation procedure for plant chromosomes

Pectinase and cellulase, which are used to macerate plant material, always show traces of DNase activities that result in DNA nicking. Moreover, the DNA polymerase I usually applied in the in situ nick translation techniques shows both 5' to 3' and 3' to 5' exonuclease activities. As a result, significant nonspecific labeling appears in control preparations that are not digested by a restriction endonuclease. Our procedure includes blocking nonspecific nick labeling before incubation with restriction enzymes (HpaII and HaeIII). This is achieved by incorporation of ddGTP into DNA by the Taq polymerase which lacks 3' to 5' exonuclease activity. This method gives satisfactory results because it eliminates nonspecific nick translation signals that are present after applying the methods described for animal material.     

In situ hybridization and chromosome banding in mammalian species

Chromosome banding is often required in conjunction with fluorescent in situ hybridization of labelled probes for chromosome painting, satellite DNA and low-copy sequences to allow identification of chromosomes and simultaneous probe localization. Here, we present a method that reveals both patterns with only one observation step. The band pattern is produced by restriction-enzyme digestion of chromosomes, followed by fixation with paraformaldehyde in PBS, a short chromosome denaturation step in hybridization solution, and then standard in situ hybridization, washing and detection protocols. Using a range of different mammalian species, chromosome-banding patterns were immediately recognizable, although synchronisation procedures normally required for high- resolution G-banding were not needed. Unlike other methods available, only one round of observation is required using a conventional fluorescence microscope, the method works without modification in many species, and in situ hybridization is not used for chromosome identification (allowing multiple targets and minimizing background). The banding pattern is probably generated by a combination of DNA dissolution and heterochromatin reorganisation after enzyme digestion, followed by paraformaldehyde fixation of the new chromatin structure and incomplete denaturation. The method is of widespread utility in comparative genomics and genome organization programmes.     

In situ hybridization to somatic metaphase chromosomes of potato

Summary An in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a ladderpattern in genomic Southern's of Solanum tuberosum and Solanum phureja, but not in those of Solanum brevidens and two Nicotiana species. This sequence hybridized predominantly on telomeric and centromeric regions of all chromosomes, although chromosomes 7, 8, 10 and 11 were not always labeled clearly.     

In situ methylation of insect chromosomes with methylase HpaII

A method of in situ DNA methylation with the prokaryotic methylase HpaII has been developed on fixed mitotic and meiotic chromosomes of the insect species Baetica ustulata. Incorporation of methyl groups into the chromosomal DNA is revealed by autoradiography using a labelled substrate and by its ability to prevent endonuclease digestion. The method allows direct visualization of clusters of methylatable CCGG sites. The distribution of these clusters in the chromosome complement of Baetica shows two separate domains of heterochromatic DNA which differ in their methylation patterns. Each is distributed at equivalent locations in both homologous and nonhomologous chromosomes. The existence of two compartments, one methylated and the other unmethylated, in the heterochromatic DNA could be interpreted as a remnant of the ancestral echinoderm-like pattern of methylation.     

Effect of cycloheximide on RNA synthesis in Chironomus polytene chromosomes

Modifications in the synthesis of salivary gland RNA were induced by treatments with 10 /ml cycloheximide (CHM) on 4th instar larvae of Chironomus pallidivitattus. After 3, 6 and 24 h CHM treatment, RNA was labeled in vitro, by incubating the salivary glands in a medium containing H³-uridine. The electrophoretical analyses corresponding to the 3 and 6 h treatment showed a stimulation of the non-ribosomal components of the newly synthesized RNA, while preribosomal RNA synthesis appeared depressed. This fact was also confirmed at cytological level, since autoradiograms made after 3 h of CHM treatment showed a reduced H³-uridine label over the nucleolus and an increase of diffuse labeling over the chromosomes. Longer treatments (24 h) causes a considerable inhibition of the synthesis of all RNA species. The role played by protein synthesis inhibition in the aforementioned effects is discussed. — Some of the morphological implications of CHM treatment, such as modifications of the nucleolar structure (nucleolar segregation) are also reported. The use of a squash technique based on glutaraldehyde fixation of the salivary glands, considerably facilitates such studies.     

In situ synthesis of protein arrays

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.     

In situ synthesis of artificial lipids

Lipids constitute one of the most enigmatic family of biological molecules. Although the importance of lipids as basic units of compartmental structure and energy storage is well-acknowledged, deciphering the biosynthesis and precise roles of specific lipid species has been challenging. To better understand the structure and function of these biomolecules, there is a burgeoning interest in developing strategies to produce noncanonical lipids in a controlled manner. This review covers recent advances in the area of in situ generation of synthetic lipids. Specifically, we report several approaches that constitute a powerful toolbox for achieving noncanonical lipid synthesis. We describe how these methodologies enable the direct construction of synthetic lipids, helping to address fundamental questions related to the cell biology of lipid biosynthesis, trafficking, and signaling. We envision that highlighting the current advances in artificial lipid synthesis will pave the way for broader interest into this emerging class of biomimetic molecules.