CHROMOSOMAL MAPPING, LINKAGE RELATIONSHIPS, AND EVOLUTIONARY STUDIES OF THE HUMAN ANONYMOUS DNA CLONE D1S1 (IN SITU HYBRIDIZATION, PRIMATES, GENE MAPPING, AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA, GENE DUPLICATION)

D1S1, an anonymous human DNA clone originally called (lamda)Ch4-H3 or (lamda)H3, was the first single copy mapped to a human chromosome (1p36) by in situ hybridization. The chromosomal assignment has been confirmed in other laboratories by repeating the in situ hybridization but not by another method. In the present study, hybridization to a panel of hamster-human somatic cell hybrids revealed copies of D1S1 on both chromosomes 1 and 3. Subcloning D1S1 showed that the D1S1 clone itself is from chromosome 3, and the sequence detected by in situ hybridization is at least two copies of part of the chromosome 3 copy. This finding demonstrates the importance of verifying gene mapping with two methods and questions the accuracy of in situ hybridization mapping.^ Non-human mammals have only one copy of D1S1, and the non-human primate D1S1 map closely resembles the human chromosome 3 copy. Thus, the human chromosome 1 copies appear to be part of a very recent duplication that occurred after the divergence between humans and the other great apes.^ A moderately informative HindIII D1S1 RFLP was mapped to chromosome 3. This marker and 12 protein markers were applied to a linkage study of autosomal dominant retinitis pigmentosa (ADRP). None of the markers proved linkage, but adding the three families examined to previously published data raises the ADRP:Rh lod score to 1.92 at (THETA) = 0.30. ^

Assignment of the porcine tumour necrosis factor alpha and beta genes to the chromosome region 7p11-q11 by in situ hybridization

The loci of the porcine tumour necrosis factor genes, alpha (TNFA) and beta (TNFB), have been chromosomally assigned by radioactive in situ hybridization. The genomic probes for TNFA and TNFB yielded signals above 7p11-q11, a region that has been shown earlier to carry the porcine major histocompatibility locus (SLA). These mapping data along with preliminary molecular studies suggest a genomic organization of the SLA that is similar to that of human and murine major histocompatibility complexes.

Induction of inhibitory factor κBα mRNA in the central nervous system after peripheral lipopolysaccharide administration: An in situ hybridization histochemistry study in the rat

In this study we investigate the mRNA expression of inhibitory factor κBα (IκBα) in cells of the rat brain induced by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). IκB controls the activity of nuclear factor κB, which regulates the transcription of many immune signal molecules. The detection of IκB induction, therefore, would reveal the extent and the cellular location of brain-derived immune molecules in response to peripheral immune challenges. Low levels of IκBα mRNA were found in the large blood vessels and in circumventricular organs (CVOs) of saline-injected control animals. After an i.p. LPS injection (2.5 mg/kg), dramatic induction of IκBα mRNA occurred in four spatio-temporal patterns. Induced signals were first detected at 0.5 hr in the lumen of large blood vessels and in blood vessels of the choroid plexus and CVOs. Second, at 1–2 hr, labeling dramatically increased in the CVOs and choroid plexus and spread to small vascular and glial cells throughout the entire brain; these responses peaked at 2 hr and declined thereafter. Third, cells of the meninges became activated at 2 hr and persisted until 12 hr after the LPS injection. Finally, only at 12 hr, induced signals were present in ventricular ependyma. Thus, IκBα mRNA is induced in brain after peripheral LPS injection, beginning in cells lining the blood side of the blood–brain barrier and progressing to cells inside brain. The spatiotemporal patterns suggest that cells of the blood–brain barrier synthesize immune signal molecules to activate cells inside the central nervous system in response to peripheral LPS. The cerebrospinal fluid appears to be a conduit for these signal molecules.

tRNA Nuclear Export in Saccharomyces cerevisiae: In Situ Hybridization Analysis

To understand the factors specifically affecting tRNA nuclear export, we adapted in situ hybridization procedures to locate endogenous levels of individual tRNA families in wild-type and mutant yeast cells. Our studies of tRNAs encoded by genes lacking introns show that nucleoporin Nup116p affects both poly(A) RNA and tRNA export, whereas Nup159p affects only poly(A) RNA export. Los1p is similar to exportin-t, which facilitates vertebrate tRNA export. A los1 deletion mutation affects tRNA but not poly(A) RNA export. The data support the notion that Los1p and exportin-t are functional homologues. Because LOS1 is nonessential, tRNA export in vertebrate and yeast cells likely involves factors in addition to exportin-t. Mutation of RNA1, which encodes RanGAP, causes nuclear accumulation of tRNAs and poly(A) RNA. Many yeast mutants, including those with the rna1-1 mutation, affect both pre-tRNA splicing and RNA export. Our studies of the location of intron-containing pre-tRNAs in the rna1-1 mutant rule out the possibility that this results from tRNA export occurring before splicing. Our results also argue against inappropriate subnuclear compartmentalization causing defects in pre-tRNA splicing. Rather, the data support “feedback” of nucleus/cytosol exchange to the pre-tRNA splicing machinery.

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [C-13]acetate was used in SIP to label the DNA of the denitrifiers. The [C-13]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the C-13 library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking Up [C-14] acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the waste-water industry to enhance denitrification.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Development of the Minimum Information Specification for in situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

We describe the creation process of the Minimum Information Specification for In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE). Modeled after the existing minimum information specification for microarray data, we created a new specification for gene expression localization experiments, initially to facilitate data sharing within a consortium. After successful use within the consortium, the specification was circulated to members of the wider biomedical research community for comment and refinement. After a period of acquiring many new suggested requirements, it was necessary to enter a final phase of excluding those requirements that were deemed inappropriate as a minimum requirement for all experiments. The full specification will soon be published as a version 1.0 proposal to the community, upon which a more full discussion must take place so that the final specification may be achieved with the involvement of the whole community. This paper is part of the special issue of OMICS on data standards.

Directed phase separation of PFO:PS blends during spin-coating using feedback controlled in situ stroboscopic fluorescence microscopy

Uniform thin-films of polymer blends can be produced through spin-coating, which is used on an industrial scale for the production of light emitting diodes, and more recently organic photovoltaic devices. Here, we present the results of the direct observation, and control, over the phase separation of polystyrene and poly(9,9′-dioctylfluorene) during spin-coating using high speed stroboscopic fluorescence microscopy. This new approach, imaging the fluorescence, from a blend of fluorescent + non-fluorescent polymers allows for intensity to be directly mapped to composition, providing a direct determination of composition fluctuations during the spin-coating process. We have studied the compositional development and corresponding structural development for a range of compositions, which produce a range of different phase separated morphologies. We initially observe domains formed by spinodal decomposition, coarsening via Ostwald Ripening until an interfacial instability causes break-up of the bicontinuous morphology. Ostwald ripening continues, and depending upon composition a bicontinuous morphology is re-established. By observing compositional and morphological development in real-time, we are able to direct and control morphological structure development through control of the spin coating parameters via in situ feedback. © 2013 The Royal Society of Chemistry.

Towards in situ Single Cell Systems Biology

Systems-level studies of biological systems rely on observations taken at a resolution lower than the essential unit of biology, the cell. Recent technical advances in DNA sequencing have enabled measurements of the transcriptomes in single cells excised from their environment, but it remains a daunting technical problem to reconstruct in situ gene expression patterns from sequencing data. In this thesis I develop methods for the routine, quantitative in situ measurement of gene expression using fluorescence microscopy.

The number of molecular species that can be measured simultaneously by fluorescence microscopy is limited by the pallet of spectrally distinct fluorophores. Thus, fluorescence microscopy is traditionally limited to the simultaneous measurement of only five labeled biomolecules at a time. The two methods described in this thesis, super-resolution barcoding and temporal barcoding, represent strategies for overcoming this limitation to monitor expression of many genes in a single cell. Super-resolution barcoding employs optical super-resolution microscopy (SRM) and combinatorial labeling via-smFISH (single molecule fluorescence in situ hybridization) to uniquely label individual mRNA species with distinct barcodes resolvable at nanometer resolution. This method dramatically increases the optical space in a cell, allowing a large numbers of barcodes to be visualized simultaneously. As a proof of principle this technology was used to study the S. cerevisiae calcium stress response. The second method, sequential barcoding, reads out a temporal barcode through multiple rounds of oligonucleotide hybridization to the same mRNA. The multiplexing capacity of sequential barcoding increases exponentially with the number of rounds of hybridization, allowing over a hundred genes to be profiled in only a few rounds of hybridization.

The utility of sequential barcoding was further demonstrated by adapting this method to study gene expression in mammalian tissues. Mammalian tissues suffer both from a large amount of auto-fluorescence and light scattering, making detection of smFISH probes on mRNA difficult. An amplified single molecule detection technology, smHCR (single molecule hairpin chain reaction), was developed to allow for the quantification of mRNA in tissue. This technology is demonstrated in combination with light sheet microscopy and background reducing tissue clearing technology, enabling whole-organ sequential barcoding to monitor in situ gene expression directly in intact mammalian tissue.

The methods presented in this thesis, specifically sequential barcoding and smHCR, enable multiplexed transcriptional observations in any tissue of interest. These technologies will serve as a general platform for future transcriptomic studies of complex tissues.

Lupin kernel fiber consumption modifies fecal microbiota in healthy men as determined by rRNA gene flourescent in situ by hybridization

Background Changes in the composition of gastrointestinal microbiota by dietary interventions using pro- and prebiotics provide opportunity for improving health and preventing disease. However, the capacity of lupin kernel fiber (LKFibre), a novel legume-derived food ingredient, to act as a prebiotic and modulate the colonic microbiota in humans needed investigation.

Aim of the study The present study aimed to determine the effect of LKFibre on human intestinal microbiota by quantitative fluorescent in situ hybridization (FISH) analysis.

Design A total of 18 free-living healthy males between the ages of 24 and 64 years consumed a control diet and a LKFibre diet (containing an additional 17–30 g/day fiber beyond that of the control—incorporated into daily food items) for 28 days with a 28-day washout period in a single-blind, randomized, crossover dietary intervention design.
Methods Fecal samples were collected for 3 days towards the end of each diet and microbial populations analyzed by FISH analysis using 16S rRNA gene-based oligonucleotide probes targeting total and predominant microbial populations.

Results Significantly higher levels of Bifidobacterium spp. (P = 0.001) and significantly lower levels of the clostridia group of C. ramosum, C. spiroforme and C. cocleatum (P = 0.039) were observed on the LKFibre diet compared with the control. No significant differences between the LKFibre and the control diet were observed for total bacteria, Lactobacillus spp., the Eubacterium spp., the C. histolyticum/C. lituseburense group and the Bacteroides–Prevotella group.
Conclusions Ingestion of LKFibre stimulated colonic bifidobacteria growth, which suggests that this dietary fiber may be considered as a prebiotic and may beneficially contribute to colon health.

Verrucomicrobia in situ abundance and water chemistry in a humic lake during the year 2000

Members of the highly diverse bacterial phylum Verrucomicrobia are globally distributed in various terrestrial and aquatic habitats. They are key players in soils, but little is known about their role in aquatic systems. Thus, we applied newly designed 16S rRNA-targeted probe set for the identification of Verrucomicrobia and of clades within this phylum to a study concerning the seasonal abundance of Verrucomicrobia in waters of the humic lake Große Fuchskuhle (Germany) by catalyzed reporter deposition fluorescence in situ hybridization. The Lake Große Fuchskuhle is located in the large Mecklenburg-Brandenburg lake district near Berlin (53°10'N, 13°02'E). The lake was artificially divided into four basins (northwest, northeast, southwest, and southeast). We chose the two most contrasting basins, the acidotrophic humic southwestern (SW) basin with a high influx of allochthonous dissolved organic carbon (DOC) and the more mesotrophic northeastern (NE) basin, to study abundance and seasonality of Verrucomicrobia. Lake water was collected from depths of 0.5 m (oxic) and 4.5 m (seasonally anoxic) approximately trimonthly in 2000 (March, June, September and December). The lake hosted diverse Verrucomicrobia clades in all seasons. Either Spartobacteria (up to 19%) or Opitutus spp. (up to 7%) dominated the communities, whereas Prosthecobacter spp. were omnipresent in low numbers (<1%). Verrucomicrobial abundance and community composition varied between the seasons, and between more and less humic basins, but were rather stable in oxic and seasonally anoxic waters.

Hibridación in situ fluorescente (FISH)

Background: At the end of 80s, cloning technologies with the increase of the antibodies’ sensibility made easier the development of technologies based on Fluorescence in situ Hibridation (FISH). Nowadays, It’s widely used in the field of basic investigation as much as clinic diagnostic. Method: FISH is a technique that combines molecular biology with histochemistry way to detect specific nucleotide sequences so that chromosome’s section or even whole chromosome can be marked on metaphases cells (cell in division) and on attached cellular nucleus. This detection is realized using DNA fluorescence probes (marked with fluorophores), that can be different according to the structures manage to detect: large single-locus probes, small unique-sequence probes, chromosome- or region-specific “paints” or repetitive sequence probes and genomic DNA probes. Some of the applications of this technique is that can be so useful in the detection of numerical and structural chromosomal alterations such as polyploidies or genomic rearrangement, to mapping metaphases cells and even to detect bacteria or another type of microorganism. In addition, FISH allows us to monitoring diseases (antitumor therapies, quantification of genomic altered cells…) and the precise location of chromosomic broken spots on tumor searching for new genes involved in cancer and detect and map interested known genes. Conclusion: FISH has many advantages ahead of conventional cytogenetic techniques (bands G karyotype) overall at the time of establish a clinic diagnostic to detect tumors and chromosomic aberration, presenting a higher sensibility and specificity as well as being a relative quick technique (24 hours).

Herceptest: HER2 expression and gene amplification in non-small cell lung cancer

HER2 is an erbB/HER type I tyrosine kinase receptor that is frequently over-expressed in malignant epithelial tumours. Herceptin, a humanised mouse monoclonal antibody to HER2, is proven therapeutically in the management of metastatic breast cancer, significantly prolonging survival when combined with cytotoxic chemotherapeutic agents. Immunohistochemical studies suggest that non-small-cell lung cancer (NSCLC) tumours may over-express HER2. Our aim was to evaluate HER2 gene amplification and semi-quantitative immuno-expression in NSCLC. A total of 344 NSCLC cases were immunostained for HER2 expression in 2 centres using the HercepTest. Fluorescence in situ hybridisation (FISH) analysis for HER2 gene amplification was performed on most positive cases and a subset of negative cases. Fifteen cases (4.3%) demonstrated 2+ or 3+ membranous HER2 immuno-expression. There was no correlation between immuno-expression and tumour histology or grade. Tumours from higher-stage disease were more often HercepTest-positive (p < 0.001). All 4 HercepTest 3 + cases demonstrated gene amplification. One of the 5 2+ cases tested for gene amplification showed areas of borderline amplification and areas of polyploidy. None of the 19 HercepTest-negative cases demonstrated gene amplification or polyploidy (p < 0.001). Gene amplification was demonstrated in all HercepTest 3+ scoring NSCLC cases. Unlike breast cancer, gene amplification and HER2 protein over-expression assessed by the HercepTest appeared to be uncommon in NSCLC. Herceptin may therefore target only a small proportion of NSCLC tumours and be of limited clinical value in this disease, particularly in the adjuvant setting. © 2001 Wiley-Liss, Inc.