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PubMed1993

A short history of electrophoretic methods.

High resolution separation of proteins, based on charge differences, is possible with disc electrophoresis, displacement electrophoresis (isotachophoresis) and notably isoelectric focusing (IEF). Size separation is obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The combination of gel IEF, followed by SDS-PAGE in a second-dimensional slab gel, i.e. two-dimensional gel electrophoresis, affords the highest resolution with up to several thousand spots per gel. Staining of proteins gives high resolution patterns which can be scanned and stored in comprehensive databases. Over the last 10 years the electrophoretic separation in gels and subsequent visualization of nucleic acids (DNA, RNA) and even genes as well as nucleotides have been much improved, making possible efficient mapping of the genes in humans and all other organisms. This has led to the biggest concerted endeavor in the history of science, i.e. the mapping of the human genome, which will be of importance as long as mankind exists. In the last years electrophoresis in capillaries has attracted much interest because for numerous substances, such as proteins nucleic acids, pharmaceuticals, metabolites, and peptides, it offers high resolution on the analytical scale with over 1 million theoretical plates. Electrophoretic methods have unprecedented impact on life sciences, providing a basis for unique advances in biochemistry, molecular biology, genetics, gene technology and medicine.

Electrophoresis↗
PubMed1993

Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization.

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with T-cell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.

Aneuploidy↗
PubMed2004

Molecular mapping of resistance to Fusarium head blight in the spring wheat cultivar Frontana.

Fusarium head blight (FHB) is a destructive disease of wheat. The objective of this study was to characterise the FHB resistance of the Brazilian spring wheat cultivar Frontana through molecular mapping. A population of 210 doubled-haploid lines from a cross of Frontana (partially resistant) and Remus (susceptible) was evaluated for FHB resistance during three seasons. Spray and single-spikelet inoculations were applied. The severity, incidence and spread of the disease were assessed by visual scoring. The population was genotyped with 566 DNA markers. The major QTL effect associated with FHB resistance mapped to chromosome 3A near the centromere, explaining 16% of the phenotypic variation for disease severity over 3 years. The most likely position is in the Xgwm720-Xdupw227 interval. The genomic region on 3A was significantly associated with FHB severity and incidence in all years evaluated, but not with FHB spread, indicating the prominent contribution of this QTL to resistance against initial infection. The map interval Xgwm129-Xbarc197 on chromosome 5A also showed consistent association with FHB severity and accounted for 9% of the phenotypic variation. In addition, smaller effects for FHB severity were identified on chromosomes 1B, 2A, 2B, 4B, 5A and 6B in single years. Individual QTLs for resistance to FHB spread accounted for less than 10% of the variation in trait expression. The present study indicates that FHB resistance of Frontana primarily inhibits fungal penetration (type I resistance), but has a minor effect on fungal spread after infection (type II resistance).

Analysis of Variance↗
PubMed1999

Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization.

Detection of target RNA by in situ hybridization (ISH) in the classic and confocal fluorescence microscope was performed using strand-specific single-stranded RNA probes labeled directly with the fluorochromes fluorescein isothiocyanate or Texas red. The probes, produced by in vitro transcription from PCR-generated templates with T7 RNA polymerase and fluorochromized UTP, gave ISH signals directly visible by fluorescence microscopy without the use of any immunological detection step. In avoiding antibodies, it was possible to strongly increase the sensitivity of the ISH since antibodies may contain RNase which can reduce hybridization signals considerably, even beyond the detection limit. Fluorescent RNA probes thus allowed for the detection of low numbers of target molecules per cell, such as minus strand intermediates in picornavirus RNA replication. Using appropriate denaturing conditions, the targets could be visualized in a double-stranded configuration as well as in the presence of a 100-fold excess of complementary RNA. Furthermore, double ISH for the simultaneous detection of two different RNA species, such as plus and minus strand RNA of poliovirus, or of different regions of the viral genomic RNA was possible with appropriate fluorescent strand-specific probes labeled with different fluorochromes. Combination of ISH and immunofluorescence was found feasible if RNA was present in relatively large amounts. In addition to the investigation of virus replication, possible applications of fluorochromized RNA probes might include antisense RNA detection as well as plant virus resistance and gene silencing.

Fluorescein-5-isothiocyanate↗
PubMed2001

An evaluation of the use of two-dimensional gel electrophoresis in proteomics.

With whole genomes being sequenced almost routinely, the next logical step towards a better understanding of cellular mechanisms lies in studying the functional units of gene expression-proteins. One of the fundamental approaches in proteomics is the use of two-dimensional gel electrophoresis as a mode of separation and visualization of complex protein mixtures. Despite several limitations of the method, its ability to separate large numbers of proteins, including their post-translationally modified forms, ensures that it will continue to be popular in several well-defined areas of proteomics. In this article, we discuss the merits and drawbacks of two-dimensional gels and compare them with alternative systems such as one-dimensional gels and liquid chromatography-based separation methods. In the wake of recent advances in mass spectrometry and related areas, we outline areas where two-dimensional gels can best be utilized as the preferred separation method in proteomic strategies.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1987

Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.

The hepatoblastoma cell line Hep G2 was transfected with a plasmid carrying the gene that confers resistance to G418 and four 5'-3' tandem copies of the hepatitis B virus (HBV) genome positioned such that two dimers of the genomic DNA are 3'-3' with respect to one another. Cells of one clone that grew in the presence of G418 produce high levels of hepatitis B e antigen and of hepatitis B surface antigen. HBV DNA is carried by these cells as chromosomally integrated sequences and episomally as relaxed circular, covalently closed, and incomplete copies of the HBV genome. Viral DNA was detected also in conditioned growth medium at the buoyant densities characteristic for infectious Dane and immature core particles. Finally, HBV-specific components morphologically identical to the 22-nm spherical and filamentous hepatitis B surface antigen particles as well as 42-nm Dane particles were visualized by immunoelectron microscopic analysis. Therefore, we have demonstrated that the Hep G2 cell line can support the assembly and secretion not only of several of the replicative intermediates of HBV DNA but also of Dane-like particles. This in vitro system can now be used to study the life cycle of HBV and the reaction of immunocompetent cells with cells carrying HBV.

Carcinoma, Hepatocellular↗
PubMed2006

Production of anti-digoxigenin antibody HRP conjugate for PCR-ELISA DIG detection system.

There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates. Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2'-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti-digoxigenin antibody HRP conjugate for use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.

Animals↗
PubMed1988

DNA sequence (H) curves of the human immunodeficiency virus 1 and some related viral genomes.

The complete nucleotide sequences of several human immunodeficiency virus 1 (HIV-1) genomes were converted by computer to respective H curves. These three-dimensional space curves embody all the information contained in the sequence due to their abstract vectorial structure. For one sequence (HIV-1 isolate BRU) special efforts were made to maximize the available resolution (the number of nucleotides visually discernible within a unit length of the curve) when making a hard, master copy of the H curve. Using a computergraphic/photographic hybrid process the 9191 nucleotides of this HIV-1 sequence were condensed into an H curve of only 37.1 cm vertical length. Although each 1-mm segment of this curve represented 25 nucleotide residues, each of the individual nucleotides of the entire sequence was still distinguishable upon direct inspection using a simple magnifying lens. Several functionally important loci of the HIV-1 sequence could be recognized on the H curve owing to characteristic line forms at corresponding locations. Utilizing H curves of lower resolution, the total nucleotide sequences of several different HIV-1 isolates and related viral sequences [Visna, equine infectious anemia (EIAV), Moloney murine leukemia (Mo-MLV), bovine leukemia (BLV), and human T-cell leukemia, type I (HTLV-I)] were visually compared side by side. An interesting similarity was noted between the location of the S3 fragment of the EIAV sequence and that of a relatively G-C rich region on the env portion of the HIV-1 sequences.

Base Sequence↗
PubMed2001

Tissue microarray technology for high-throughput molecular profiling of cancer.

Tissue microarray (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time, either at the DNA, RNA or protein level. The technique facilitates rapid translation of molecular discoveries to clinical applications. By revealing the cellular localization, prevalence and clinical significance of candidate genes, TMAs are ideally suitable for genomics-based diagnostic and drug target discovery. TMAs have a number of advantages compared with conventional techniques. The speed of molecular analyses is increased by more than 100-fold, precious tissues are not destroyed and a very large number of molecular targets can be analyzed from consecutive TMA sections. The ability to study archival tissue specimens is an important advantage as such specimens are usually not applicable in other high-throughput genomic and proteomic surveys. Construction and analysis of TMAs can be automated, increasing the throughput even further. Most of the applications of the TMA technology have come from the field of cancer research. Examples include analysis of the frequency of molecular alterations in large tumor materials, exploration of tumor progression, identification of predictive or prognostic factors and validation of newly discovered genes as diagnostic and therapeutic targets.

Animals↗
PubMed2006

Families with the risk allele of DISC1 reveal a link between schizophrenia and another component of the same molecular pathway, NDE1.

We have previously reported a robust association between an allelic haplotype of 'Disrupted in Schizophrenia 1' (DISC1) and schizophrenia in a nationwide collection of Finnish schizophrenia families. This specific DISC1 allele was later identified to associate with visual working memory, selectively in males. DISC1 association to schizophrenia has since been replicated in multiple independent study samples from different populations. In this study, we conditioned our sample of Finnish families for the presence of the Finnish tentative risk allele for DISC1 and re-analyzed our genome-wide scan data of 443 markers on the basis of this stratification. Two additional loci displayed an evidence of linkage (LOD > 3) and included a locus on 16p13, proximal to the gene encoding NDE1, which has been shown to biologically interact with DISC1. Although none of the observed linkages remained significant after multiple test correction through simulation, further analysis of NDE1 revealed an association between a tag-haplotype and schizophrenia (P = 0.00046) specific to females, which proved to be significant (P = 0.011) after multiple test correction. Our finding would support the concept that initial gene findings in multifactorial diseases will assist in the identification of other components of complex genetic etiology. Notably, this and other converging lines of evidence underline the importance of DISC1-related functional pathways in the etiology of schizophrenia.

Alleles↗
PubMed1998

Rapid differentiation of closely related Candida species and strains by pyrolysis-mass spectrometry and Fourier transform-infrared spectroscopy.

Two rapid spectroscopic approaches for whole-organism fingerprinting of pyrolysis-mass spectrometry (PyMS) and Fourier transform-infrared spectroscopy (FT-IR) were used to analyze a group of 29 clinical and reference Candida isolates. These strains had been identified by conventional means as belonging to one of the three species Candida albicans, C. dubliniensis (previously reported as atypical C. albicans), and C. stellatoidea (which is also closely related to C. albicans). To observe the relationships of the 29 isolates as judged by PyMS and FT-IR, the spectral data were clustered by discriminant analysis. On visual inspection of the cluster analyses from both methods, three distinct clusters, which were discrete for each of the Candida species, could be seen. Moreover, these phenetic classifications were found to be very similar to those obtained by genotypic studies which examined the HinfI restriction enzyme digestion patterns of genomic DNA and by use of the 27A C. albicans-specific probe. Both spectroscopic techniques are rapid (typically, 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of successfully discriminating between closely related isolates of C. albicans, C. dubliniensis, and C. stellatoidea. We believe that these whole-organism fingerprinting methods could provide opportunities for automation in clinical microbial laboratories, improving turnaround times and the use of resources.

Candida↗
PubMed1999

Posterior column ataxia with retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32.

OBJECTIVE: To establish a genetic linkage between highly polymorphic microsatellite loci and the disease locus responsible for an autosomal recessive neurodegenerative syndrome that causes posterior column ataxia and retinitis pigmentosa. BACKGROUND: The authors reported previously a genetic syndrome that causes visual impairment, proprioceptive loss, sensory ataxia, and areflexia in affected individuals from a large, inbred family belonging to a sectarian population that has been genetically semi-isolated from mainstream society for several centuries. METHODS: To find the disease locus responsible for this condition, the authors performed a genome-wide search using genetic loci spaced at 10 to 20-cM intervals spanning human chromosomes (chr) 1-22. Pairwise linkage analysis, multipoint linkage analysis, and haplotype reconstruction were used to delineate the candidate region containing the disease gene. RESULTS: After testing 226 loci that covered the entire genome, the authors identified a maximum lod score of 8.94 at a recombination fraction of 0.00 for locus D1S2692. Additional analyses placed the disease gene, AXPC1, in an 8.3-cM interval flanked by markers D1S2692 and D1S414 on chr 1q31-q32. CONCLUSIONS: This study suggests that a single genetic mutation can cause selective degeneration of the posterior columns of the spinal cord and retina. Finding the gene responsible for this syndrome may increase our understanding of the molecular basis of diseases that affect sensory neurons.

Adolescent↗
PubMed2005

Male Pseudohermaphroditism in a raccoon dog (Nyctereutes procynoides).

A wild raccoon dog (Nyctereutes procynoides) which died due to a traffic accident on 18 October 2001, and was determined to be 4.5 years old, was examined. Visual appearance of the external genitalia in this animal showed to be female with a large penis-like clitoris protruding from the vulvar juncture. Visual examination of the internal genitalia revealed that the animal possessed both testes and uterus. Histological appearance of the removed gonads showed only Sertoli cells but no spermatogenesis. Using polymerase chain reaction with skin biopsy directed against the sex-determining region on the Y chromosome (SRY) gene, the genomic SRY gene was expressed as a single band and sequenced. Based on these findings, this raccoon dog was diagnosed as male pseudohermaphrodite.

Animals↗
PubMed1996

Relationships between angiotensin I converting enzyme gene polymorphism and renal complications in Korean IDDM patients.

OBJECTIVES: The prognosis of IDDM is mainly dependent on complicated diabetic nephropathy which is probably determined by both metabolic abnormalities and genetic predisposition. Angiotensin I converting enzyme (ACE) regulates systemic and renal circulations through angiotensin II formation and kinins metabolism. The insertion(i)/deletion(D) polymorphism in intron 16 of ACE gene is strongly associated with ACE levels, and subjects homozygote for deletion (genotype DD) have the highest plasma values. Recently, it was reported that I/D polymorphism of ACE gene is associated with diabetic nephropathy in Caucasian IDDM patients. We studied the relationship between the ACE gene polymorphism and diabetic nephropathy in Korean IDDM patients. METHODS: The study population consisted of 59 IDDM patients (duration > 5 yrs) and 107 control subjects. IDDM subjects were divided into 2 groups according to the presence or absence of diabetic nephropathy (with nephropathy: n = 31, without nephropathy: n = 28). After extraction of genomic DNA from peripheral blood leukocytes, PCR was performed using the sense primer (5' -GCC CTG CAG GTG TCT GCA GC-3') and anti-sense primer (3'-TGC CCA TAA CAG TGC TTC ATA -5'), respectively. The PCR products were electrophoresed in 2% agarose gels, and DNA was visualized directly with ethidium bromide staining. RESULTS: Frequencies for II, ID and DD genotypes were similar in IDDM subjects and controls (23: 19:17 vs 49:41:17, p = 0.142) and derived allele frequencies for I and D alleles were similar in both groups (0.551:0.449 vs 0.649:0.351, p = 0.098). The ACE genotype distributions were not different in diabetic subjects with or without nephropathy (12:9:10 vs 11:10:7, p = 0.78) and derived allele frequencies were also similar (0.532:0.468 vs 0.571:0.429, p = 0.81). CONCLUSION: The I and D allele frequency in our controls was different compared to ACE allele frequencies of Caucasian populations, but very similar compared to those of Chinese or Japanese subjects. We found that I/D polymorphism of ACE gene is not implicated in the diabetic nephropathy of Korean IDDM patients and may be explained by ethnic differences.

Adult↗
PubMed2004

Genome remodelling in three modern S. officinarumxS. spontaneum sugarcane cultivars.

This study provides evidence that nuclear and chromosome remodelling has taken place in sugarcane, a vegetative crop with a complex genome derived from interspecific hybridizations between Saccharum officinarum and S. spontaneum. Detailed knowledge on the chromosomal compositions of the three clones analysed was acquired. (1) All hybrid cultivars were found to be aneuploid, affecting both parental genomes (having chromosomes in addition to full genomes), with chromosome numbers from 2n=102-106 in My5514 and up to 2n=113-117 in C236-51. (2) Comparative in situ hybridization showed that about 16% of these chromosomes are inherited from S. spontaneum and less than 5% are recombinant or translocated chromosomes containing sequences of both S. officinarum and S. spontaneum. (3) Differences between the observed DNA contents (estimated by flow cytometry) and those expected from the number of chromosomes, allowed the introgression of additional S. spontaneum or S. officinarum DNA pieces into the B42231 and C236-51 cultivars to be estimated. (4) Size heterogeneity between S. officinarum homologous chromosomes carrying the 18S-5.8S-25S and 5S ribosomal genes (identified by FISH with pTa71 and pTa794, respectively) confirms remodelling occurred by chromosomal interchange events, at least in these homologous chromosomes. (5) Simultaneous visualization of nucleoli and NORs showed that all 18S-5.8S-25S loci were potentially functional in the three clones, independent of their origin and size.

Chromosome Mapping↗
PubMed2006

Unraveling transcriptional control and cis-regulatory codes using the software suite GeneACT.

Deciphering gene regulatory networks requires the systematic identification of functional cis-acting regulatory elements. We present a suite of web-based bioinformatics tools, called GeneACT http://promoter.colorado.edu, that can rapidly detect evolutionarily conserved transcription factor binding sites or microRNA target sites that are either unique or over-represented in differentially expressed genes from DNA microarray data. GeneACT provides graphic visualization and extraction of common regulatory sequence elements in the promoters and 3'-untranslated regions that are conserved across multiple mammalian species.

Animals↗
PubMed2006

Cis-motifs upstream of the transcription and translation initiation sites are effectively revealed by their positional disequilibrium in eukaryote genomes using frequency distribution curves.

BACKGROUND: The discovery of cis-regulatory motifs still remains a challenging task even though the number of sequenced genomes is constantly growing. Computational analyses using pattern search algorithms have been valuable in phylogenetic footprinting approaches as have expression profile experiments to predict co-occurring motifs. Surprisingly little is known about the nature of cis-regulatory element (CRE) distribution in promoters. RESULTS: In this paper we used the Motif Mapper open-source collection of visual basic scripts for the analysis of motifs in any aligned set of DNA sequences. We focused on promoter motif distribution curves to identify positional over-representation of DNA motifs. Using differentially aligned datasets from the model species Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster and Saccharomyces cerevisiae, we convincingly demonstrated the importance of the position and orientation for motif discovery. Analysis with known CREs and all possible hexanucleotides showed that some functional elements gather close to the transcription and translation initiation sites and that elements other than the TATA-box motif are conserved between eukaryote promoters. While a high background frequency usually decreases the effectiveness of such an enumerative investigation, we improved our analysis by conducting motif distribution maps using large datasets. CONCLUSION: This is the first study to reveal positional over-representation of CREs and promoter motifs in a cross-species approach. CREs and motifs shared between eukaryotic promoters support the observation that an eukaryotic promoter structure has been conserved throughout evolutionary time. Furthermore, with the information on positional enrichment of a motif or a known functional CRE, it is possible to get a more detailed insight into where an element appears to function. This in turn might accelerate the in depth examination of known and yet unknown cis-regulatory sequences in the laboratory.

Algorithms↗
PubMed1998

Characterization of viremia at different stages of varicella-zoster virus infection.

Varicella-zoster virus (VZV) viremia at different stages of infection was characterized. Different approaches were used, polymerase chain reaction (PCR), isothermal transcription based nucleic acid amplification (NASBA), and immunofluorescence to describe and quantitate viral infection of peripheral blood mononuclear cells (PBMC). In patients with acute varicella 200 to 5,000 copies of the viral genome in every 150,000 PBMC were found with quantitative competitive PCR (QCPCR). With NASBA, viral transcriptional activity was detected in these cells. RNA transcribed from the immediate early gene IE 63 as well as from the late gene 68 were found, indicating a productive infection. Glycoprotein gE specific immunofluorescence visualized by confocal laser scanning microscopy revealed that only 1 in 10,000 to 100,000 PBMC was infected. T and B lymphocytes as well as monocytes expressed viral protein on their surface. Similar results were obtained with PBMC from immunocompetent zoster patients. In some cases a transient viremia was found shortly after the onset of rash, although the viral load seemed to be lower than in patients with varicella. Examination of blood samples from 16 persons with postherpetic neuralgia (PHN) signs of viral replication in PBMC were not detected. In conclusion, the data suggest that VZV viremia is a frequent event in patients with varicella and zoster, but not in those with postherpetic neuralgia. Moreover, the results indicated that subclinical reactivations occur both in immunocompromised and immunocompetent individuals.

Aged↗