In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.     

The heat-shock response in Drosophila KC 161 cells. mRNA competition is the main explanation for reduction of normal protein synthesis

The effect of heat shock on protein synthesis in the Drosophila melanogaster KC 161 tissue culture cell line was examined with a view to investigating the mechanism underlying the acute reduction in normal cellular protein synthesis typical of heat-shocked Drosophila cells. However, at 36-37 degrees C, the optimum temperature for induction of the 70-kDa heat-shock protein, this cell line did not show such a response. The synthesis of a very limited number of proteins was abruptly turned off following heat shock in the presence or absence of actinomycin, but the rate of synthesis of the majority of normal cellular proteins declined slowly over a three-hour period. Incubation of heat-shocked cells in hypertonic media increased the relative proportion of protein synthesis directed towards heat-shock proteins (as opposed to normal cellular proteins). Incubation with low concentrations of cycloheximide had the converse effect and resulted in a preferential increase in the size of polysomes translating normal cellular mRNAs, greater than the increase in size of polysomes synthesising heat-shock proteins. Heat shock also resulted in some mRNAs being almost completely displaced from polysomes into the postribosomal supernatant. These observations suggest that competition between normal cellular mRNAs and increasing amounts of heat-shock mRNAs with a higher affinity for the translation machinery was the main explanation for the gradual reduction in the synthesis of normal cellular proteins, although a slight reduction in overall translation initiation rates cannot be excluded as a subsidiary cause. The results demonstrate that the acute reduction in normal cellular protein synthesis seen in other Drosophila cell lines is not an integral and necessary feature of the heat-shock response in this organism, which makes it unlikely that the mechanism of this acute shut-off is intimately connected with the mechanism of induction of heat-shock mRNAs.     

A study by in situ hybridization of the stage of appearance and disappearance of the transition protein 2 and the mitochondrial capsule seleno-protein mRNAs during spermatogenesis in the mouse.

Transition protein 2 is a basic chromosomal protein which functions as an intermediate in the replacement of histones by protamines, and the mitochondrial capsule seleno-protein is a constituent of the outer membrane of mitochondria which functions in constructing the mitochondrial sheath surrounding the flagellum. To determine precisely the stages in spermatogenesis when these mRNAs are present, paraffin sections of sexually mature testes were hybridized to 35S- and 3H-labeled antisense RNAs and exposed to autoradiographic emulsion. The cell types hybridizing to probes in situ were determined by staining with hematoxylin and periodic acid Schiff. The in situ hybridizations reveal that the transition protein 2 mRNA is first detectable in step 7 round spermatids, persists at high levels through step 13, and is degraded before step 14. By contrast, the mitochondrial capsule seleno-protein mRNA is first detected in step 3 round spermatids and persists at high levels until step 16, the end of spermiogenesis. The mitochondrial capsule seleno-protein mRNA appears to be expressed only in haploid cells since low levels could not be detected in Northern blots of RNA from pachytene primary spermatocytes from 18 day prepubertal mice. These results demonstrate that the transition protein 2 and mitochondrial capsule seleno-protein mRNAs are transcribed and degraded at different times during the haploid phase of spermatogenesis.     

The rifampicin-inducible genes srn6 from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by kiiling of plasmid-free segregants

The gene systems srnB of plasmid F and pnd of plasmid R483 were discovered because of their induction by rifampicin. Induction caused membrane damage, RNase I influx, degradation of stable RNA and, consequently, cell killing. We show here that the srnB and pnd systems mediate efficient stabilization of a mini-R1 test-plasmid. We also show that the killer genes srnB' and pndA are regulated by antisense RNAs, and that the srnC- and pndB-encoded antisense RNAs, denoted SrnC- and PndB-RNAs, are unstable molecules of approximately 60 nucleotides. The srnB and pndA mRNAs were found to be very stable. The differential decay rates of the inhibitory antisense RNAs and the killer-gene-encoding mRNAs explain the induction of these gene systems by rifampicin. Furthermore, the observed plasmid-stabilization phenotype associated with the srnB and pnd systems is a consequence of this differential RNA decay: the newborn plasmid-free cells inherit the stable mRNAs, which, after decay of the unstable antisense RNAs, are translated into killer proteins, thus leading to selective killing of the plasmid-free segregants. Thus our observations lead us to conclude that the F srnB and R483 pnd systems are phenotypically indistinguishable from the R1 hok/sok system, despite a 50% dissimilarity at the level of DNA sequence.     

Kaposi's sarcoma-associated herpesvirus ORF57 protein enhances mRNA accumulation independently of effects on nuclear RNA export

The ORF57 protein expressed by Kaposi's sarcoma-associated herpesvirus (KSHV) during lytic replication is essential for KSHV virion production. ORF57 enhances gene expression by increasing accumulation of target gene mRNAs. ORF57 interacts with the cellular export factor REF and with RNA, suggesting that it may provide target mRNAs with access to REF, which mediates nuclear RNA export by binding to TAP/NXF1. A mutational analysis of ORF57 was performed to study the role of REF binding, RNA interaction, and multimerization in ORF57 function. ORF57 was shown to directly bind RNA. The ability to bind REF did not correlate with ORF57 function in enhancing mRNA accumulation. ORF57 enhanced the nuclear levels of mRNA and PAN, a nuclear KSHV RNA, and the activity of various ORF57 mutants on the levels of mRNA paralleled their ability to enhance nuclear PAN accumulation, suggesting that ORF57 may also act on messenger RNAs by export-independent effects on RNA stability. Finally, an ORF57 mutant lacking a region homologous to a nucleolar localization signal in herpesvirus saimiri was constructed. This mutant retained function, demonstrating that, unlike the ORF57 homolog in herpesvirus saimiri, nucleolar trafficking is not required for ORF57 function in enhancing mRNA accumulation.     

The p12I, p13II, and p30II proteins encoded by human T-cell leukemia/lymphotropic virus type I open reading frames I and II are localized in three different cellular compartments.

Three protein isoforms are encoded by the human T-cell leukemia/lymphotropic virus type I pX region open reading frames (ORF) I and II through alternative splicing. Both the singly and doubly spliced mRNAs from ORF I encode a single 12-kDa protein (p12I), whereas two distinct proteins of 13 kDa (p13II) and 30 kDa (p30II) are encoded from the ORF II alternatively spliced mRNA. Because the p12I protein is very hydrophobic and poorly immunogenic, we genetically engineered its cDNA by adding a short stretch of amino acids from the highly immunogenic epitope HA1 of influenza virus or the AU1 epitope of bovine papillomavirus. The HA1 epitope was also added to the p13II and p30II proteins, albeit rabbit immune sera raised against synthetic peptides were also available. To determine in which cellular compartments these proteins reside, we transfected the tagged and wild-type cDNAs in HeLa/Tat cells and studied their localization by indirect immunofluorescence. The p12I protein was identified in the cellular endomembranes and, particularly, in the perinuclear area. p13II and p30II were found in the nuclei and nucleoli of the transfected cells, respectively. The presence of the HA1 epitope at the carboxy terminus of p13II and p30II did not interfere with their cellular localization, since the rabbit immune sera demonstrated their presence in the same cellular compartments when the untagged proteins were expressed. The defined localization of these proteins in specific cellular compartments warrants further study of their function.     

NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila.

The conserved family of NXF proteins has been implicated in the export of messenger RNAs from the nucleus. In metazoans, NXFs heterodimerize with p15. The yeast genome encodes a single NXF protein (Mex67p), but there are multiple nxf genes in metazoans. Whether metazoan NXFs are functionally redundant, or their multiplication reflects an adaptation to a greater substrate complexity or to tissue-specific requirements has not been established. The Drosophila genome encodes one p15 homolog and four putative NXF proteins (NXF1 to NXF4). Here we show that depletion of the endogenous pools of NXF1 or p15 from Drosophila cells inhibits growth and results in a rapid and robust accumulation of polyadenylated RNAs within the nucleus. Fluorescence in situ hybridizations show that export of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs is inhibited. Depleting endogenous NXF2 or NXF3 has no apparent phenotype. Moreover, NXF4 is not expressed at detectable levels in cultured Drosophila cells. We conclude that Dm NXF1/p15 heterodimers only (but not NXF2-NXF4) mediate the export of the majority of mRNAs in Drosophila cells and that the other members of the NXF family play more specialized or different roles.     

Localization of the expression of types I, III, and IV collagen, TGF-beta 1 and c-fos genes in developing human calvarial bones

Total RNA extracted from developing calvarial bones of 15- to 18-week human fetuses was studied by Northern hybridization: in addition to high levels of type I collagen mRNAs, the presence of mRNAs for type III and type IV collagen, TGF-beta and c-fos was observed. In situ hybridization of sections containing calvarial bone, overlying connective tissues, and skin was employed to identify the cells containing these mRNAs. Considerable variation was observed in the distribution of pro alpha 1(I) collagen mRNA in osteoblasts: the amount of the mRNA in cells at or near the upper surface of calvarial bone was distinctly greater than that in cells at the lower surface, indicating the direction of bone growth. High levels of type I collagen mRNAs were also detected in fibroblasts of periosteum, dura mater, and skin. Type III collagen mRNA revealed a considerably different distribution: the highest levels were detected in upper dermis, lower levels were seen in fibroblasts of the periosteum and the fibrous mesenchyme between bone spiculas, and none was seen in osteoblasts. Type IV collagen mRNAs were only observed in the endothelial cells of blood capillaries. Immunohistochemical localization of type III and IV collagens agreed well with these observations. The distribution of TGF-beta mRNA resembled that of type I collagen mRNA. In addition, high levels of TGF-beta mRNA were observed in osteoclasts of the calvarial bone. These cells, responsible for bone resorption, were also found to contain high levels of c-fos mRNA. Production of TGF-beta by osteoclasts and its activation by the acidic environment could form a link between bone resorption and new matrix formation.