The use of combined FISH/GISH in conjuction with DAPI counterstaining to identify chromosomes containing transgene inserts in amphidiploid tobacco

We have used combined fluorescent and genomic in situ hybridization (FISH/GISH) together with 4′,6-diamidino-2-phenylindole (DAPI) counterstaining to determine simultaneously the chromosomal integration site and subgenomic allocation of a transgene insert in amphidiploid tobacco (Nicotiana tabacum, 2n=4x=48). The procedure provides sufficient information on physical markers to identify at least 20 out of 24 chromosome pairs of two tobacco cultivars commonly used in studies on transgene expression and silencing (cv. Petit Havana SR1 and cv. Gatersleben). The chromosomes can be distinguished on the basis of diploid parental ancestry, size, morphology, the presence of rDNA loci and/or intergenomic exchanges, and the DAPI banding pattern, which is shown here for the first time forN. tabacum. From a single ISH experiment, it should now be possible in most cases to identify a tobacco chromosome carrying a transgene insert, thus permitting systematic studies of how the chromosomal location of transgenes influences expression levels. Edited by: D. Schweizer     

Rapid localization of transgenes in mouse chromosomes with a combined Spectral Karyotyping/FISH technique

We explored the feasibility of combined Spectral Karyotyping (SKY) and Fluorescence In Situ Hybridization (FISH) as means to rapidly map a chromosomally integrated renin/green fluorescent protein (GFP) fusion gene construct (Ren-GFP) in the transgenic mouse, Tg(Ren-GFP)1Kwg. A sequential hybridization with SKY probes followed by FISH gave consistently satisfactory results, demonstrating that multiple copies of the Ren-GFP transgene in this transgenic mouse line are integrated into a single chromosomal site of Chromosome (Chr) 4, most probably in the juxta-centromeric euchromatic region consisting of the A2-A3 domain. Chr 4 as a sole carrier of the transgene also was confirmed by co-hybridization to p1 BAC clone DNA containing telomeric sequences specific for mouse Chr 4 and the Ren-GFP construct in pGEM4Z vector. The hemizygosity of the Ren-GFP transgene is maintained not only in bone marrow cells, but also in lung cells proliferating in vitro, indicating that stable integration of the Ren-GFP transgene into chromosomal DNA was established at a very early embryonic stage. We conclude that the SKY/FISH technique is a reliable and facile method for establishing the integration site of a transgene. As such, this protocol has obvious advantages over traditional backcross methods in terms of time, cost and labor for determining the chromosomal location of transgenes.     

The genetic identity of alien chromosomes in potato breeding lines revealed by sequential GISH and FISH analyses using chromosome-specific cytogenetic DNA markers.

Genomic in situ hybridization (GISH) is one of the most popular and effective techniques for detecting alien chromatin introgressed into breeding lines; however, GISH analysis alone does not reveal the genetic identity of the alien chromosomes. We previously isolated a set of bacterial artificial chromosomes (BACs) specific to each of the 12 potato chromosomes. These BAC clones can be used as chromosome-specific cytogenetic DNA markers (CSCDMs) for potato chromosome identification. Here we demonstrate that GISH and fluorescence in situ hybridization (FISH), using CSCDMs, can be performed sequentially on the same chromosome preparations. Somatic metaphase chromosomes prepared using an enzymatic digestion and "flame-drying" procedure allows repeated probing up to five times without significant damage to chromosome morphology. The sequential GISH and FISH analyses reveal the genomic origin and genetic identity of the alien chromosomes in a single experiment and also determine whether an alien chromosome has been added to the genetic background of potato or is substituting for a homoeologous potato chromosome. The sequential GISH and FISH procedures should be widely applicable for germplasm characterization, especially in plant species with small-sized chromosomes.     

The impact of StuI digestion in situ on FISH to human chromosomes with satellite DNA probes

Human metaphase chromosomes were digested with StuI and subsequently hybridized in situ using chromosome 9 alphoid DNA and classical satellite III DNA as probes. The data obtained suggest that it is not possible to establish a general rule regarding the cytological effects induced by restriction enzymes in particular chromosome regions and that a number of factors, such as DNA sequences, DNA-protein interaction and enzyme structure, play a role in determining such effects.     

Identification of wheat-barley translocations by sequential GISH and two-colour FISH in combination with the use of genetically mapped barley SSR markers.

Five wheat-barley translocations in a wheat background were characterized through the combination of cytogenetic and molecular genetic approaches. The wheat chromosome segments involved in the translocations were identified using sequential GISH and two-colour FISH with the probes pSc119.2 and pAs1. The barley chromatin in these lines was identified using SSR markers. A total of 45 markers distributed over the total barley genome were selected from a recently published linkage map of barley and tested on the translocation lines. The following translocations were identified: 2DS.2DL-1HS, 3HS.3BL, 6BS.6BL-4HL, 4D-5HS, and 7DL.7DS-5HS. Wheat-barley disomic and ditelosomic addition lines for the chromosomes 3HS, 4H, 4HL, 5H, 5HL, and 6HS were used to determine the correct location of 21 markers and the position of the centromere. An intragenomic translocation breakpoint was detected on the short arm of the barley chromosome 5H with the help of SSR marker analysis. Physical mapping of the SSR markers on chromosomes 1H and 5H was carried out using the intragenomic and the interspecific translocation breakpoints, as well as the centromere, as physical landmarks.     

The presence of a chromatin boundary appears to shield a transgene in tobacco from RNA silencing

We present isogenic transgenic tobacco lines that carry at a given chromosomal position a beta-glucuronidase (GUS) reporter gene either with or without the presence of the matrix-associated region known as the chicken lysozyme A element. Plants were generated with the Cre-lox site-specific recombination system using heterospecific lox sites. Analysis of GUS gene expression in plant populations demonstrates that the presence of the A element can shield against RNA silencing of the GUS gene. Protection was observed in two of three independent tobacco transformants. Plants carrying an A element 5' of the GUS gene always had stable GUS activity, but upon removal of this A element, the GUS gene became silenced over time in two lines, notably when homozygous.     

The hamster transferrin receptor contains Ser/Thr-linked oligosaccharides: use of a lectin-resistant CHO cell line to identify glycoproteins containing these linkages.

We recently reported that the human transferrin receptor (TfR) contains O-linked GalNAc residues [1]. To investigate whether this modification is shared by transferrin receptors in other mammals, we investigated the glycosylation of TfR in hamster cells. To facilitate our analysis the lectin-resistant Chinese hamster ovary (CHO) cell line Lec8 was used. These cells are unable to galactosylate glycoproteins, resulting in truncation of the Ser/Thr-linked oligosaccharides to a single residue of terminal alpha-linked GalNAc. This structure is bound with high affinity by the lectin Helix pomatia agglutinin (HPA). The TfR was affinity purified from Lec8 cells metabolically radiolabeled with [3H]glucosamine and the receptor was found to bind tightly to HPA-Sepharose. Treatment of the purified TfR with mild alkaline/borohydride released [3H]GalNAcitol, demonstrating the presence of O-linked GalNAc. We also found that many other unidentified [3H]glucosamine-labeled glycoproteins from Lec8 cells were bound by HPA-Sepharose. The bound and unbound glycoproteins were separated by SDS/PAGE and individual species were selected for treatment with mild base/borohydride. Treatment of glycoproteins bound by HPA, but not those unbound, resulted in the release of [3H]GalNAcitol. These studies demonstrate both that the hamster TfR contains O-linked oligosaccharides and that this approach may have general utility for identifying the presence of these oligosaccharides in other glycoproteins.     

The use of dimenhydrinate in conjunction with dexamethasone for induction of parturition in beef cattle

Forty-eight crossbred Chianina cows (3 to 5 years of age), with an expected gestation length of 288 days, were randomly divided into four treatment groups to evaluate the use of dimenhydrinate (an antihistamine agent) in conjunction with dexamethasone (DEX) for inducing parturition in beef cattle. Group (A) received a 20 mg dose of DEX (im) on day 282 of gestation and a carrier vehicle (iv) 24 hours later (day 283); Group (B) was given a carrier vehicle (im) on day 282 and 500 mg of dimenhy-drinate (DMH) diluted in 200 ml of 2.5% dextrose-0.9% saline solution given (iv) on day 283 and Group (C) received 20 mg of DEX (im) on day 282 and 500 mg of DMH in solution (iv) on day 283 of gestation. The remaining 12 cows assigned to Group (D) were not handled and were allowed to calve under natural conditions. The number of cows calving and percent calving within 60 hours after the first injection were: 10(91%), none(0%) and 12(100%) for the DEX, DMH and DEX plus DMH groups, respectively. The mean gestation length of the control cows in Group (D) was 288.6 days. Frequency of dystocia was: 18.2, 8.3, 0 and 0% and retained placentae (>/=24 hours) was: 72.8, 16.6, 33.3 and 0% for DEX, DMH, DEX plus DMH and control groups, respectively. In this study, a 20 mg dose of DEX (im) followed 24 hours later with 500 mg of DMH (iv) was more successful for calving induction than when DEX or DMH was used alone. The combination DEX and DMH treatment induced calving in a shorter interval from treatment (P<.05) and decreased the incidence of retained placentae (P<.01) when compared with those induced following DEX treatment.     

The use of genomic in situ hybridization (GISH) to show transmission of recombinant chromosomes by a partially fertile bigeneric hybrid, Gasteria lutzii ×Aloe aristata (Aloaceae), to its progeny

Genomic in situ hybridization (GISH) was used to study somatic chromosomes of parental and progeny plants (all 2n=2x=14) of the bigeneric hybrid between Gasteria lutzii and Aloe aristata (Aloaceae), which is partially fertile, a rare occurrence in plants. GISH successfully distinguished between the two parental genomes in the F1 hybrid and revealed numerous genomic recombinations in chromosomes transmitted by the F1 to the back-cross progeny. The results indicate high levels of meiotic compatibility between the parental genomes, even though they differ in size by 20%. Recombination occurred at a frequency that was higher than that expected from the analysis of orcein-stained meiosis in the F1. The discrepancy suggests that terminalization may occur prior to or during metaphase I, reducing the apparent chiasma frequency, or possibly reveals an under-estimation caused by difficulties in resolving closely grouped chiasmata by eye. Received: 9 September 1996; in revised form: 21 October 1996 / Accepted: 21 October 1996     

The use of sequence-specific antibodies to identify a secondary binding site in thrombin

The peptide comprising residues 62-73 of the B-chain of human alpha-thrombin was synthesized and polyclonal antibodies raised against it. These antibodies were found to bind to the synthetic peptide, a CNBr fragment, and a proteolytic subfragment containing this sequence, as well as the entire thrombin molecule. The purified antibodies had no effect on the hydrolysis by thrombin of D-Phe-pipecolyl-Arg-p-nitroanilide and caused only a minimal decrease (20%) in the second-order rate constant for inactivation by antithrombin III. On the other hand, the antibodies competitively inhibited the binding of hirudin over the concentration range tested (0-43 nM), and a dissociation constant of 3.4 +/- 0.5 nM was found for the antibodies. The release of fibrinopeptide A from the A alpha-chain of fibrinogen by thrombin was competitively inhibited with an inhibition constant of 11.7 +/- 0.4 nM. The activation of protein C by thrombin in the presence of thrombomodulin was also inhibited by the antibodies, and an apparent inhibition constant of 10.7 +/- 1.5 nM was found. In contrast, the antibodies had no effect on the activation of protein C in the absence of thrombomodulin. These results are discussed in relation to data obtained recently on the interaction of well defined proteolytic derivatives of human alpha-thrombin with the ligands described above.     

The use of a laser microprobe to identify calcium oxalate in histological material

Summary Some birefringent renal deposits were found to give the usual histological reactions for calcium oxalate but were soluble in caustic alkalis. Comparison with known oxalate crystals using a laser microprobe mass analyser confirmed the presence of calcium oxalate. Similar crystals were found in liver tissue from a rat poisoned with ethylene glycol.     

The use of immunological methods to detect and identify bacteria in the environment

Immunological detection methods have become increasingly important in microbial ecology for the tracking of specific microorganisms and for community analysis. For a reliable application of these techniques, the monoclonal antibodies or polyclonal antisera used have to fulfill several quality criteria. Cross reactivity, cellular localization of the antigenic determinant, affinity characteristics and the expression of the antigenic determinant at environmental conditions have to be determined. Immunological methods can be used for the identification, quantification and enrichment of specific bacteria in extracts as well as for the visualization of cells in situ. The sensitivity of advanced immunological methods can be compared to PCR techniques. Using image processing of epifluorescence micrographs or confocal laser scanning microscopy, the immunofluorescence approach can now be applied to study complex environmental samples.     

The use of enzyme polymorphisms to identify genetic differences in the genus Trichinella

Seven isolates of the genus Trichinella were evaluated for their genetic identity and variability using starch gel electrophoresis. The various strains exhibited polymorphism in 8 of 11 enzyme systems tested and the strains could be segregated into at least 3 basic patterns for geographical isolates of Trichinella: pigs, wild carnivores and Trichinella spiralis var. pseudospiralis. There was also evidence for further differences between isolates from wild carnivores. The isozyme technique appears to have potential for zoogeographic studies in the genus Trichinella.     

The use of multi-frequency EPR techniques to identify the radicals produced in irradiated beta-blockers

The identification of radicals trapped in irradiated drugs can be very intricate. A multi-frequency electron paramagnetic resonance (EPR) study is proposed to resolve this problem. The Q-band (ca. 34 GHz) comparison with X-band (ca. 9 GHz) did not show significant differences for the four β-blockers studied (atenolol, esmolol, nadolol and propranolol). The use of a higher frequency (285 GHz) was required. It enabled us to determine the g-tensor values of the radicals present in atenolol and esmolol, respectively, g₁=2.0086, g₂=2.0059 and g₃=2.0021 and g₁=2.0066, g₂=2.0044 and g₃=2.0021. The latter was assigned as a phenoxyl radical, which can not be the case for the former. Therefore, radicals produced in esmolol may result from a more complex mechanism than the abstraction followed by the diffusion of an H atom inside the solid. In addition, two molecules as similar as atenolol and esmolol hydrochloride do not contain the same radicals after irradiation. These two conclusions drawn from the EPR results on β-blockers show clearly the importance of continuing the investigations on radiolytic mechanisms in solid-state drugs.     

Heterochromatin in the chromosomes of the gorilla: characterization with distamycin A/DAPI, D287/170, chromomycin A3, quinacrine, and 5-azacytidine

The chromosomes of the gorilla were extensively studied with various staining techniques labeling the different classes of heterochromatin. The chromosomal distribution of distamycin A/DAPI-, D287/170-, quinacrine-, and chromomycin A3-positive heterochromatic regions, as well as the nucleolus organizer regions, is described and compared with the karyotypes of other hominoid species. Lymphocyte cultures were treated with low doses of 5-azacytidine during the last hours of culture. This cytidine analog induces distinct undercondensation in 37 heterochromatic regions in the 24 gorilla chromosomes. The 5-azacytidine-induced undercondensations are localized not only in most of the distamycin A/DAPI-bright heterochromatic regions but also in many telomeric C-bands of the chromosomes. Furthermore, 5-azacytidine preserves the somatic pairing between heterochromatic regions from the interphase nuclei into the metaphase stage. The homeologies and differences in the chromosomal localization of the various classes of heterochromatin, 5-azacytidine-sensitive regions, 5-methylcytosine-rich DNA sequences, and satellite DNAs in the gorilla, chimpanzee, orangutan, and man are discussed.     

The use of reverse immunology to identify HLA-A2 binding epitopes in Tie-2

A potential target for a cancer vaccine would be receptors, such as Tie-2 which are over expressed on tumour endothelium. Using computer aided motif predictions for possible HLA class I epitopes, we have identified peptides from Tie-2 that should bind with a range of affinities to HLA-A*0201. No direct correlation between predicted values and actual binding affinities was observed. Although, the programs did produce a number of false positives, two epitopes were predicted that bound with relatively high affinity when compared with an influenza peptide. We have previously identified a Tie-2 epitope and shown that it was only immunogenic when we substituted preferred amino acids at key anchor residues to increase binding affinity. In this study we used a similar approach to generate modified epitopes. When HLA-A2 transgenic mice were immunised with peptides, CTL killing of the target cells was only achieved when the wild type epitope was presented at moderate levels. Moreover, the efficiency of immunisation was increased when we linked CD4 epitopes to CD8 epitopes. Caution should therefore be employed in the use of both reverse immunology and anchor modification of CTL epitopes in the identification of CTL epitopes for cancer vaccines.This article is a symposium paper from the “Robert Baldwin Symposium: 50 years of Cancer Immunotherapy”, held in Nottingham, Great Britain, on 30th June 2005.     

The transgene pyramiding tobacco with betaine synthesis and heterologous expression of AtNHX1 is more tolerant to salt stress than either of the tobacco lines with betaine synthesis or AtNHX1

Previous studies have shown that the overexpression of betA (encoding choline dehydrogenase from Escherichia coli ) or AtNHX1 (a vacuolar Na⁺/H⁺ antiport from Arabidopsis thaliana ) gene can improve the salt tolerance of transgenic plants. However, little is known about the effects of the transgene pyramiding of betA and AtNHX1 . Here, betA  +  AtNHX1 transgene pyramiding tobacco was produced by sexual crossing, and the salt tolerance was evaluated at the cellular and plant levels. In NaCl stress, the Na⁺ concentration in vacuoles and vacuolar membrane potential of transgene pyramiding cells were similar to those of AtNHX1 -transgenics, and much higher than those of betA -transgenics when detected using fluorescent dye staining; transgene pyramiding cells showed a higher protoplast viability and comparable mitochondrial activity as compared with single transgenics; and transgene pyramiding plants showed comparable Na⁺ content in leaves as compared with AtNHX1 -transgenics and remarkably higher than betA -transgenics; and transgene pyramiding lines exhibited higher percentage of seed germination, better seedling growth and higher fresh weight than lines that had betA or AtNHX1 alone. Based on the integrative analysis of salt tolerance, the consistency between the cellular level and the whole plant level was confirmed and the transgene pyramiding plants exhibited improved salt tolerance, but compared with the plants with betA or AtNHX1 alone, the differences were relatively small. Other mechanisms involved in salt tolerance should be considered to further enhance transgene pyramiding plants salt tolerance.     

The use of pH indicators to identify suitable environments for freezing samples in aqueous and mixed aqueous/nonaqueous solutions

The colours of frozen solutions containing pH indicators are shown to provide a test for changes in pH in the solvent environment which occur on freezing. Yeast alcohol dehydrogenase loses activity on freezing in phosphate buffer (a buffer in which pH indicator colour changes shows a marked decrease in pH on freezing) but when frozen in bis-tris, Hepes, or N-glycylglycine buffers (all of which show little change in the colour of universal pH indicator and hence of pH on freezing) is stable on freezing. The effects of freezing in different buffer systems on the rate of decomposition of NADPH, and on the rate hydrolysis of 4-nitrophenyl acetate, are rationalised in terms of the pH shifts in these buffers which were determined using universal pH indicator. It is proposed that a major reason for the instability of samples on freezing is the pH changes which occur when some systems are frozen. From the results a general scheme for selecting the best environment for safely freezing samples is proposed which is based on the use of pH indicators.     

The use of LC-MS to identify differentially expressed proteins in docetaxel-resistant prostate cancer cell lines

Docetaxel is a taxane-derived chemotherapy drug that has been approved for treatment of prostate cancer. While docetaxel is frequently used as a treatment for hormone-refractory prostate cancer, a subset of patients either do not respond to this treatment or those that do respond eventually become resistant to the drug over time. Resistance to docetaxel is complex and multi-factoral and further understanding of the cellular biochemistry underlying resistance is vital to improve treatment efficacy. To identify proteins altered in the resistant phenotype, three parental cell lines DU145, 22RV1 and PC-3, as well as their docetaxel resistant sub-lines, were subjected to quantitative label-free LC-MS proteomic profiling. A total of 189 significant (p < 0.05) protein abundance changes were identified in the DU145 resistant sub-lines, 254 in the 22RV1 sub-lines, and 51 and 72 in the 8 and 12 nM resistant PC-3 sub-lines, respectively. From these, 29 proteins demonstrated a significant (p < 0.05) fold change across two or more resistant variants. These included proteins indicative of an epithelial-to-mesenchemyl transition as well as altered heat shock response elements.