Chromosome banding and 18S rDNA in situ hybridization analysis of seven species of the family Achiridae (Teleostei : Pleuronectiformes)

Achiridae is an important family of the order Pleuronectiformes widely distributed in North, Central, and South America with freshwater and marine species. In the present study cytogenetic analyses comprising conventional and molecular techniques were carried out in seven species of this family. The following diploid numbers (2n) and fundamental numbers (FN) were obtained: Achirus declivis 2n = 34, FN = 52; Achirus lineatus 2n = 40, FN = 66; Catathyridium jenynsi 2n = 40 and FN = 50; Gymnachirus nudus 2n = 36 and FN = 50; Hypoclinemus mentalis 2n = 38 and FN = 54; Trinectes paulistanus 2n = 42 and FN = 52; and Trinectes sp. 2n = 38 and FN = 54. All species presented a single nucleolar organizer region (NOR) bearing chromosome pair and C-band positive segments mainly distributed at the pericentromeric position. The wide variation observed in chromosome number and FN suggests the occurrence of larger chromosome rearrangements in the family Achiridae if compared with other families of the same order.

Physical mapping of the Nile tilapia (Oreochromis niloticus) genome by fluorescent in situ hybridization of repetitive DNAs to metaphase chromosomes - a review

The Nile tilapia (Oreochromis niloticus) has received increasing scientific interest over the past few decades for two reasons: first, tilapia is an enormously important species in aquaculture worldwide, especially in regions where there is a chronic shortage of animal protein; and second, this teleost fish belongs to the fascinating group of cichlid fishes that have undergone a rapid and extensive radiation of much interest to evolutionary biologists. Currently, studies based on physical and genetic mapping of the Nile tilapia genome offer the best opportunities for applying genomics to such diverse questions and issues as phylogeography, isolation of quantitative trait loci involved in behaviour, morphology, and disease, and overall improvement of aquacultural stocks. In this review, we have integrated molecular cytogenetic data for the Nile tilapia describing the chromosomal location of the repetitive DNA sequences, satellite DNAs, telomeres, 45S and 5S rDNAs, and the short and long interspersed nucleotide elements [short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)], and provide the beginnings of a physical genome map for this important teleost fish. (C) 2004 Elsevier B.V. All rights reserved.

Comparative cytogenetics of cichlid fishes through genomic in-situ hybridization (GISH) with emphasis on Oreochromis niloticus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An immunocytochemical and in situ hybridization analysis of annexin 1 expression in rat mast cells: Modulation by inflammation and dexamethasone

The presence and localization of the anti-inflammatory protein annexin 1 (also known as lipocortin 1) in perivenular rat mast cells was investigated here. Using the rat mesenteric microvascular bed and a combination of morphologic techniques ranging from immunofluorescence to electron microscopy analyses, we detected the presence of annexin 1 in discrete intracellular sites, both in the nucleus and in the cytoplasm. In resting mast cells, most of the protein pool (approximately 80% of the cytosolic portion) was localized to cytoplasmic granules. In agreement with other cell types, treatment of rats with dexamethasone (0.2 mg/kg, ip) increased annexin 1 expression in mast cells, inducing a remarkable appearance of dusters of protein immunoreactivity. This effect was most likely the result of de novo protein synthesis as determined by an increase in mRNA seen by in situ hybridization. Triggering an ongoing experimental inflammatory response (0.3 mg of carrageenin, ip) increased annexin 1 mRNA and protein levels. In conclusion, we report for the first time the localization of annexin 1 in connective tissue mast cells, and its susceptibility not only to glucocorticoid hormone treatment, but also to an experimental acute inflammatory response.

A rapid technique for analysis of formalin-fixed, paraffin-embedded tissues by fluorescent in situ hybridization with alpha-satellite probes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Epstein-Barr virus-associated gastric carcinoma in Brazil: comparison between in situ hybridization and polymerase chain reaction detection

Epstein-Barr virus (EBV) has been associated with 10% of gastric carcinomas. The aim of this study was to determine the frequency of EBV in gastric carcinomas in Brazil assessed by in situ hybridization (ISH) and PCR, which would contribute to the characterization of the clinical and pathological aspects of EBV-associated gastric carcinomas. One hundred and ninety-two gastric carcinoma cases were collected at hospitals in two Brazilian states. Seventy-three out of 151 cases were PCR(+), while 11/160 cases were ISH(+). Nine out of eleven ISH(+) cases displayed a diffuse staining pattern and 2 out of 11 a focal pattern. Both techniques showed that the EBV(+) cases were characterized by their association with males, older patients, lower gastric region, intestinal type, advanced stage and poorly to moderately differentiated tumors. The concordance between the two techniques was 55.8% (Cohen's kappa index = 0.034). Four cases were ISH(+)/PCR(-), while 49 cases were PCR(+)/ISH(-). Only two cases showed stained lymphocytes by ISH and one of them was PCR(-). The observed discrepancy between the two techniques could not be explained just by the elevated accuracy of PCR. ISH(+)/PCR(-) carcinomas may be encountered if EBV is not present in the whole tumor tissue or if there are polymorphisms in the sequences of the viral genome amplified. on the other hand, the high frequency of PCR(+) results associated with the absence of ISH staining in lymphocytes and/or tumors cells suggests that the virus may be present in tumor cells or other cell types without expressing EBER1, the target of the ISH technique.

Fluorescent in situ hybridization (FISH) as a diagnostic tool for Williams-Beuren syndrome

Fluorescent in situ hybridization (FISH) with commercial probes covering the elastin gene (ELN) was used to determine the frequency of the 7q11.23 deletion in 18 children clinically diagnosed with Williams-Beuren syndrome (WBS). A de novo deletion was detected in 15 of the children (83%). Diagnostic investigation for WBS started late in childhood (median = 5.8 years). All the children showed facial features typical of the syndrome, mental retardation and developmental delay. Over-friendliness was observed in the majority of cases. Clinodactyly of the 5th finger (n = 13), cardiovascular disease (n = 9), loquacity (n = 9), low birthweight (n = 8), and failure to thrive (n = 9) were observed only in those children with the deletion. Respiratory problems (n = 9), though not previously reported in the literature, was a common finding in the group studied. Our results confirmed that FISH is useful in identifying 7q11.23 deletions in cases of WBS. Clinical manifestations were more evident in the deletion-positive children.

Short interspersed repetitive elements (SINEs) from the cichlid fish, Oreochromis niloticus, and their chromosomal localization by fluorescent in situ hybridization

In the present study, we describe the cloning and characterization of a new SINE-like element from O. niloticus (ROn-2) and show the distribution of this SINE and a previously isolated SINE, ROn-1, in the chromosomes of O. niloticus. The ROn-2 element is 359 base pairs (bp) in length, contains short direct terminal repeats, a tRNA-related region similar to tRNA Val and tRNA Arg, a tRNA-unrelated region, and a poly-A tail. Analysis of the chromosomal distribution of ROn-1 and ROn-2 by fluorescent in situ hybridization showed that both SINE sequences are present in all chromosomes of tilapia, and organized in small clusters. The only exception was a large cluster of ROn-1 repeats found in the middle of the long arm of chromosome 1. In view of our data we discuss the hypothesis that the absence of large clusters of SINE sequences and the structural composition of these sequences may explain the absence of base-specific fluorochrome bands in the chromosomes of tilapia.

Fluorescent in situ hybridization mapping of the epidermal growth factor receptor gene in donkey

The physical localization of the epidermal growth factor receptor (EGFR) gene was performed on donkey chromosomes. Bacterial artificial chromosome DNA containing the equine EGFR gene was used to map this gene by fluorescent in situ hybridization on donkey metaphase chromosomes. The gene was mapped on donkey 1q21.1 region.

Fluorescent In Situ hybridization: a new tool for the direct identification and detection of F. psychrophilum

F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium ("Pan-Flavo") and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum.