Light Sheet Microscopy for Single Molecule Tracking in Living Tissue

Single molecule observation in cells and tissue allows the analysis of physiological processes with molecular detail, but it still represents a major methodological challenge. Here we introduce a microscopic technique that combines light sheet optical sectioning microscopy and ultra sensitive high-speed imaging. By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample. Thereby we could directly observe and track small and large tracer molecules in aqueous solution. Furthermore, we demonstrated the feasibility to visualize the dynamics of single tracer molecules and native messenger ribonucleoprotein particles (mRNPs) in salivary gland cell nuclei of Chironomus tentans larvae up to 200 µm within the specimen with an excellent signal quality. Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems

Cotranscriptional recruitment of the nuclear poly(A)-binding protein Pab2 to nascent transcripts and association with translating mRNPs

Synthesis of the pre-mRNA poly(A) tail in the nucleus has important consequences on the translational activity of the mature mRNA in the cytoplasm. In most eukaryotes, nuclear polyadenylation of pre-mRNAs is thought to require the nuclear poly(A)-binding protein (PABP2/PABPN1) for poly(A) tail synthesis and ultimate length control. As yet, however, the extent of the association between PABP2 and the exported mRNA remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) assays to show that the fission yeast ortholog of mammalian PABP2 (Pab2) is cotranscriptionally recruited to active genes. Notably, the association of Pab2 to genes precedes that of a typical 3′-processing/polyadenylation factor, suggesting that Pab2 recruitment during the transcription cycle precedes polyadenylation. The inclusion of an RNase step in our ChIP and immunoprecipitation assays suggests that Pab2 is cotranscriptionally recruited via nascent mRNA ribonucleoprotein (mRNPs). Tandem affinity purification coupled with mass spectrometry also revealed that Pab2 associates with several ribosomal proteins as well as general translation factors. Importantly, whereas previous results suggest that the nuclear poly(A)-binding protein is not present on cytoplasmic mRNAs, we show that fission yeast Pab2 is associated with polysomes. Our findings suggest that Pab2 is recruited to nascent mRNPs during transcription and remains associated with translated mRNPs after nuclear export

Gene and genon concept: coding versus regulation: A conceptual and information-theoretic analysis of genetic storage and expression in the light of modern molecular biology

We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term “genon”. In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various pieces, as steered by the genon. It emerges finally as an uninterrupted nucleic acid sequence at mRNA level just prior to translation, in faithful correspondence with the amino acid sequence to be produced as a polypeptide. After translation, the genon has fulfilled its role and expires. The distinction between the protein coding information as materialised in the final polypeptide and the processing information represented by the genon allows us to set up a new information theoretic scheme. The standard sequence information determined by the genetic code expresses the relation between coding sequence and product. Backward analysis asks from which coding region in the DNA a given polypeptide originates. The (more interesting) forward analysis asks in how many polypeptides of how many different types a given DNA segment is expressed. This concerns the control of the expression process for which we have introduced the genon concept. Thus, the information theoretic analysis can capture the complementary aspects of coding and regulation, of gene and genon

The complexity of 75S premessenger RNA in balbiani ring granules studied by a new RNA band retardation assay.

Under normal growth conditions, Balbiani ring granules constitute premessenger ribonucleoprotein (RNP) particles synthesized in two chromosomal puffs, Balbiani ring (BR) 1 and 2, in the larval salivary glands of Chironomus tentans. At least three genes encoding 75S RNA are present in these two BRs: one in BR1 and two in BR2 (BR2.1 and BR2.2). The complexity of BR granule 75S RNA was studied by agarose gel electrophoresis under non-denaturing conditions. We recorded three main bands, designated I, II and III. Experiments with denaturing gels demonstrated that the differences in migration reflected mainly, but not exclusively, conformational differences. Northern blotting experiments showed that band I contained BR1 sequences, band II contained BR2.1 sequences, and band III contained BR2.2 sequences. To study whether additional genes contributed to the BR granule 75S RNA, an RNA band shift assay was developed. When an oligodeoxyribonucleotide complementary to repetitive BR1 and BR2.2 sequences was hybridized to 75S RNA prior to electrophoresis, bands I and III were retarded but not band II. An oligonucleotide complementary to a repetitive BR2.1 sequence only shifted band II. Since no detectable 75S RNA remained unchanged in these experiments, and all bands were identified by Northern blotting, all the BR granules are likely to originate from the BR1, BR2.1 and BR2.2 genes; no additional genes have to be invoked. Possible applications of the new RNA band shift assay are discussed