Multiple fluorescence in situ hybridization

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.     

Chromosome mapping of the human ras-related rab3A gene to 19p13.2

The rab genes belong to one of the three main branches of the ras super family. The encoded rab proteins share 38 to 75% amino acid identity with the yeast YPT1 and SEC4 proteins. We used the human rab3A cDNA to map the corresponding gene on human chromosomes by chromosome sorting and in situ hybridization. Both techniques allowed the assignment of the rab3A gene to chromosome 19 with a regional localization on 19p13.2 obtained by in situ hybridization.     

Quantification of fluorescence in situ hybridization signals by image cytometry.

In this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluated. Quantification of the fluorescence ISH signals was performed using an epi-fluorescence microscope with a multi-wavelength illuminator, equipped with a cooled charge couple device (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimen. Specific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non-uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min-max filter) to estimate local background intensities. The choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of field. The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double-labeled probes.     

Regional chromosomal assignment of the human mineralocorticoid receptor gene to 4q31.1

Summary The gene for human mineralocorticoid receptor (hMR), previously mapped to chromosome 4, has been further localized to 4q31.1 by in situ hybridization using a biotinylated 3.75kb human cDNA clone encoding the primary amino acid sequence of hMR as a probe. Preliminary comparative mapping studies in orangutan (Pongo pygmaeus) suggest localization of the probe to the long arm of chromosome 3.     

Combined microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ

We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial "black box" should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the alpha subdivision of the class Proteobacteria (alpha-Proteobacteria) and of the Cytophaga-Flavobacterium group obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined ( approximately 60% of the universal probe-labeled cells and approximately 50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the alpha-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the "dissection" of microbial communities by type and function.     

Amino acid sequence of rat apolipoprotein A-II deduced from the nucleotide sequence of cloned cDNA

A rat apolipoprotein A-II cDNA clone was isolated from a rat liver cDNA library by in situ hybridization of bacteriophage plaques using a 32P-labeled human apoA-II cDNA as a probe. The cDNA insert from this clone was characterized by DNA sequencing. The amino acid composition derived from the DNA sequence data matched well with that of rat apoA-II reported earlier (Herbert et al. 1974. J. Biol Chem. 249: 5718-5724), indicating that the cDNA insert coded for rat apoA-II. Further evidence was provided by a comparison of the amino acid sequence of rat apoA-II obtained here with that of human apoA-II (Brewer et al. 1972. Proc. Natl. Acad. Sci. USA. 69: 1304-1308). While the rat apoA-II cDNA insert did not code for the entire presegment, it had the same COOH-terminal residues of the presegment as well as the same prosegment (Ala-Leu-Val-Arg-Arg) as in human preproapoA-II, suggesting that rat apoA-II was also synthesized initially as preproapoA-II. Mature rat apoA-II contains 79 amino acids. Residue 6 of mature rat apoA-II is Asp, while it is Cys in human apoA-II, and this would account for the absence of dimeric forms of rat apoA-II in plasma. While the overall amino acid sequence homology between rat and human apoA-II is about 50%, the amphipathic alpha-helical structures, which are responsible for lipid-binding, seem to be conserved in the two proteins. The size of rat apoA-II mRNA was estimated to be about 600 nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of cDNAs for human phosphoribosylpyrophosphate synthetases 1 and 2 and X chromosome localization of PRPS1 and PRPS2 genes

Cloned cDNAs representing the entire, homologous (80%) translated sequences of human phosphoribosylpyrophosphate synthetase (PRS) 1 and PRS 2 cDNAs were utilized as probes to localize the corresponding human PRPS1 and PRPS2 genes, previously reported to be X chromosome linked. PRPS1 and PRPS2 loci mapped to the intervals Xq22-q24 and Xp22.2-p22.3, respectively, using a combination of in situ chromosomal hybridization and human x rodent somatic cell panel genomic DNA hybridization analyses. A PRPS1-related gene or pseudogene (PRPS1L2) was also identified using in situ chromosomal hybridization at 9q33-q34. Human HPRT and PRPS1 loci are not closely linked. Despite marked cDNA and deduced amino acid sequence homology, human PRS 1 and PRS 2 isoforms are encoded by genes widely separated on the X chromosome.     

Suprabasal marker proteins distinguishing keratinizing squamous epithelia: Cytokeratin 2 polypeptides of oral masticatory epithelium and epidermis are different

Terminal differentiation of squamous epithelia is usually characterized by the synthesis of a subset of cytokeratins (CKs) in suprabasal cell layers which become major components of the intermediate filament (IF) bundle cytoskeleton of the maturing cells. We have examined the significance, molecular nature and pattern of synthesis of the elusive human CK 2 by analyzing mRNAs from certain stratified epithelia, using in vitro translation, cDNA cloning. Northern blotting and in situ hybridization. We show that genuine polypeptides with the typical gel electrophoretic mobility of CK 2 exist but that the CK 2 present in the masticatory epithelia of hard palate and gingiva (CK 2p) differs from that found in epidermis (CK 2e) by its amino acid sequence and is encoded by a different gene. The two CKs 2 show only limited sequence homology (71% identical amino acid positions in the rod domain), and the oral CK 2p is more closely related to the corneal CK 3 (86%), as is also indicated by the cross-reaction of monoclonal antibody AE5. By in situ hybridization and immunocytochemistry, we further show that both CK 2e and CK 2p are expressed only in suprabasal cell layers of the specific epithelia where they can accumulate to represent major cytoskeletal proteins. We discuss this tissue-type specificity of CK 2 synthesis in otherwise morphologically and biochemically similar epithelia in relation to differences of IF appearance and packing in upper strata between epidermal and masticatory epithelia as well as to tissue formation and differentiation during development.     

Isolation and chromosomal assignment of a novel human gene, CORO1C, homologous to coronin-like actin-binding proteins

We have isolated a gene, termed CORO1C (human coronin-like actin-binding protein 1C), that encodes a new member of the coronin-like family of proteins. The cDNA consists of 3,857 nucleotides, with an open reading frame of 1,422 bp encoding a 474 amino acid protein. The deduced amino acid sequence shared 65% identity with p57 (human coronin-like actin-binding protein), as well as 46% identity with coronin, a protein first isolated from the slime mold Dictyostelium discoideum. Computer analysis predicted that the product of the CORO1C gene would contain five WD repeats in its N-terminal region and a coiled-coil motif in its C-terminal region, both of which are conserved among coronin-like proteins. CORO1C was ubiquitously expressed in all human tissues examined, in contrast to other known coronin-like molecules, each of which is expressed in a tissue-specific manner. Immunocytochemical staining demonstrated that CORO1C was co-localized with F-actin; therefore, the gene product is likely to be important in cytokinesis, motility, and signal transduction, as are the other members of this molecular family. We assigned this novel gene to chromosome 12q24.1 by fluorescence in situ hybridization.     

A novel rat carboxypeptidase, CPA2: characterization, molecular cloning, and evolutionary implications on substrate specificity in the carboxypeptidase gene family

A new member of the carboxypeptidase gene family, carboxypeptidase A2 (CPA2), has been identified from the predicted amino acid sequence of a rat pancreatic cDNA clone. In vivo recombination and in situ hybridization techniques employing the CPA2 cDNA resulted in the isolation of two genomic clones spanning the 25-kilobase pair rat CPA2 gene. Evolutionary trees built from the amino acid sequences of the known pancreatic carboxypeptidases show that CPA2 and carboxypeptidase A1 (CPA1) are the products of genes which duplicated before the mammalian radiation, and that bovine CPA is of the A1 type. The substrate specificities of CPA1 and CPA2 isolated from rat pancreas are similar to bovine CPA in that carboxyl-terminal amino acids with aromatic or branched aliphatic side chains are preferred. However, the substrate preference of rat CPA1 is skewed toward smaller amino acids, while that of rat CPA2 is skewed toward bulkier amino acids as compared to bovine CPA. The differences in the substrate specificities of these three carboxypeptidases are compatible with the nature of the amino acid replacements in their binding pockets for the carboxylterminal amino acid of the substrate.     

Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification

The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.     

Chromosomal assignment of retinoic acid receptor (RAR) genes in the human, mouse, and rat genomes.

The human genes encoding the alpha and beta forms of the retinoic acid receptor are known to be located on chromosomes 17 (band q21.1:RARA) and 3 (band p24:RARB). By in situ hybridization, we have now localized the gene for retinoic acid receptor gamma, RARG, on chromosome 12, band q13. We also mapped the three retinoic acid receptor genes in the mouse, by in situ hybridization, on chromosomes 11, band D (Rar-a); 14, band A (Rar-b); and 15, band F (Rar-g), respectively, and in the rat, using a panel of somatic cell hybrids that segregate rat chromosomes, on chromosomes 10 (RARA), 15 (RARB), and 7 (RARG), respectively. These assignments reveal a retention of tight linkage between RAR and HOX gene clusters. They also establish or confirm and extend the following homologies: (i) between human chromosome 17, mouse chromosome 11, and rat chromosome 10 (RARA); (ii) between human chromosome 3, mouse chromosome 14, and rat chromosome 15 (RARB); and (iii) between human chromosome 12, mouse chromosome 15, and rat chromosome 7 (RARG).     

Differential influence of the 4F2 heavy chain and the protein related to b(0,+) amino acid transport on substrate affinity of the heteromeric b(0,+) amino acid transporter

We provide evidence here that b(0,+) amino acid transporter (b(0, +)AT) interacts with 4F2 heavy chain (4F2hc) as well as with the protein related to b(0,+) amino acid transporter (rBAT) to constitute functionally competent b(0,+)-like amino acid transport systems. This evidence has been obtained by co-expression of b(0, +)AT and 4F2hc or b(0,+)AT and rBAT in human retinal pigment epithelial cells and in COS-1 cells. The ability to interact with 4F2hc and rBAT is demonstrable with mouse b(0,+)AT as well as with human b(0,+)AT. Even though both the 4F2hc x b(0,+)AT complex and the rBAT x b(0,+)AT complex exhibit substrate specificity that is characteristic of system b(0,+), these two complexes differ significantly in substrate affinity. The 4F2hc x b(0,+)AT complex has higher substrate affinity than the rBAT x b(0,+)AT complex. In situ hybridization studies demonstrate that the regional distribution pattern of mRNA in the kidney is identical for b(0,+)AT and 4F2hc. The pattern of rBAT mRNA expression is different from that of b(0,+)AT mRNA and 4F2hc mRNA, but there are regions in the kidney where b(0,+)AT mRNA expression overlaps with rBAT mRNA expression as well as with 4F2hc mRNA expression.     

Localization of epidermal growth factor precursor in tooth and lung during embryonic mouse development

The murine epidermal growth factor (EGF) precursor is a 1217 amino acid protein which contains mature EGF (amino acid residues 977-1029) as well as eight EGF-like repeats. Although the highest levels of EGF are found in the adult male mouse submandibular gland, the results of in situ hybridization studies and mRNA analyses suggest that EGF precursor mRNA is synthesized in several adult mouse tissues including the lung and the incisor. To determine if EGF precursor gene expression is intrinsic to the developmental program for either embryonic tooth or lung organogenesis, sense and antisense oligodeoxyribonucleotide probes corresponding to amino acids 1070-1081 of the precursor were used to localize cellular sites of synthesis of EGF precursor mRNA by in situ hybridization. Antibodies directed against amino acid residues 348-691 of the precursor were used in immunodetection techniques to identify either EGF precursor protein or processed derivatives. In contrast to earlier reports indicating that embryonic mouse tissues do not synthesize EGF precursor mRNA, we found that EGF precursor mRNA is present in clusters of ectoderm-, mesoderm-, and ectomesenchyme-derived cells associated with embryonic teeth and lung organs. Moreover, epitopes common to the EGF precursor were immunolocalized in both the epithelial and mesenchymal tissues of embryonic mouse tooth and lung organs. These results suggest that the EGF precursor and/or motifs contained within the precursor molecule, including mature EGF, may play an instructive or permissive role in epithelial-mesenchymal interactions pursuant to organogenesis.     

Diagnostic nucleic acid hybridizations for infectious diseases

The use of nucleic acid hybridization techniques has expanded into many areas, including studies of gene structure and function, routine diagnosis of human, animal and plant diseases, and also forensic science. In situ hybridization is one of the techniques currently available for nucleic acid hybridization and has some distinct advantages compared with standard techniques such as dot-blot, Southern or Northern hybridization, in which the histological structure is lost during extraction of nucleic acids. On the other hand, immunohistochemical staining is one branch of histochemistry that has received considerable attention in recent years as a very sensitive method for localization of specific proteins and other antigenic macromolecules within tissues and cells. This technique has also been widely used for clinical diagnosis and in various fields of research in medical science and biology. Automation of colorimetric in situ hybridization and immunohistochemistry would greatly contribute to the ease of introducing these techniques for routine pathological diagnosis and would improve the reproducibility of the assay. In this review, author will describe the development of an automated method for in situ hybridization and immunohistochemical staining using an automatic machine for both procedures.     

Cloning of human lysyl hydroxylase: complete cDNA-derived amino acid sequence and assignment of the gene (PLOD) to chromosome 1p36.3----p36.2.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. A deficiency in this enzyme activity is known to exist in patients with the type VI variant of the Ehlers-Danlos syndrome, but no amino acid sequence data have been available for the wildtype or mutated human enzyme from any source. We report the isolation and characterization of cDNA clones for lysyl hydroxylase from a human placenta lambda gt11 cDNA library. The cDNA clones cover almost all of the 3.2-kb mRNA, including all the coding sequences. These clones encode a polypeptide of 709 amino acid residues and a signal peptide of 18 amino acids. The human coding sequences are 72% identical to the recently reported chick sequences at the nucleotide level and 76% identical at the amino acid level. The C-terminal region is especially well conserved, a 139-amino-acid region, residues 588-727 (C-terminus), being 94% identical between the two species and a 76-amino-acid region, residues 639-715, 99% identical. These comparisons, together with other recent data, suggest that lysyl hydroxylase may contain functionally significant sequences especially in its C-terminal region. The human lysyl hydroxylase gene (PLOD) was mapped to chromosome 1 by Southern blot analysis of human-mouse somatic cell hybrids, to the 1p34----1pter region by using cell hybrids that contain various translocations of human chromosome 1, and by in situ hybridization to 1p36.2----1p36.3. This gene is thus not physically linked to those for the alpha and beta subunits of prolyl 4-hydroxylase, which are located on chromosomes 10 and 17, respectively.     

Physiology and in situ identification of putative aminergic neurotransmitters in the nervous system of Gyrocotyle fimbriata, a parasitic flatworm

The longitudinal nerve cords (LNC) in Gyrocotyle fimbriata do not change their activity in response to catecholamines but are excited by the amino acids glutamate and aspartate at concentrations less than 10(-5)M. This activity can be blocked by application of 2-amino-4-phosphonobutyrate (APB), an aminergic blocking agent. In situ measurements of fluorescent spectra from LNC neurites have a lambdamax at 513.6 nm. Treatment with HCI shifts the peak to 510 nm. Both glutamate and aspartate fluoresce and behave similarly with peak emissions of 512.5 nm before HCI treatment and 511 nm and 509 nm, respectively, after treatment. Dopamine and norepinephrine both fluoresce at 512.2 nm but the peaks shifts to 516.5 and 528, respectively, following acid treatment. These experiments indicate that the fluorescing substance in the neurones is not a known catecholamine and could be an amino acid.