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At least 1,063 records · Page 59Linked to original sources
PubMed2006

Pulsed-field gel electrophoresis: a versatile tool for analysis of fungal genomes. A review.

The separation of chromosome-size DNA molecules by pulsed-field gel electrophoresis (PFGE) has become a well-established technique in recent years. Although it has very wide-ranging applications, it made a real breakthrough for fungal genome analysis. Because of the small size of fungal chromosomes, their investigation was not possible earlier. Different PFGE approaches allowed the separation of DNA molecules larger than 10 megabase pairs in size, and electrophoretic karyotypes for numerous previously genetically uncharacterized fungal species could be established. This review discusses the applicability of these electrophoretic karyotypes for the investigation of genome structure, for strain identification and for species delimitation.

Chromosomes, Fungal↗
PubMed2006

Dynamic analysis of a genomic island in Magnetospirillum sp. strain AMB-1 reveals how magnetosome synthesis developed.

The entire structure of a 98 kb genomic region that abounds in genes related to magnetosome synthesis was first described in the Magnetospirillum sp. strain AMB-1. The deletion of this 98 kb genomic region and the circular form after excision from the chromosome was detected by PCR amplification. This strongly suggests that the region has undergone a lateral gene transfer. The region has the characteristics of a genomic island: low GC content, location between two repetitive sequences, and the presence of an integrase in the flanking region of the first repetitive sequence. This 98 kb genomic region has the potential for transfer by the integrase activity. Comparative genome analysis revealed other regions with a high concentration of orthologs in magnetic bacteria besides the 98 kb region, and magnetosome synthesis seemed to need not only the exogenous 98 kb region, but also other orthologs and individually originating genes.

Gene Transfer Techniques↗
PubMed2005

Analysis of genomic DNA alterations and mRNA expression patterns in a panel of human pancreatic cancer cell lines.

Genomic alterations influencing the expression and/or activity of tumor suppressors or oncogenes such as KRAS2, CDKN2A, TP53, and DPC4 have been directly implicated in the initiation and progression of human pancreatic adenocarcinoma. In an effort further to systematically characterize the genomic alterations that occur in this disease, we conducted a genome wide analysis of alterations in gene copy number using array-based comparative genomic hybridization (CGH). For this analysis, we utilized a panel of 25 human pancreatic cancer cell lines derived from either primary or metastatic tumors. This panel also included a metastatic progression series of cell lines derived from COLO 357 cells. Array CGH permitted the identification of alterations in the copy number of genes that might participate in the aberrant behavior of pancreatic cancer cells. In addition, the acquisition of invasive and metastatic potential by derivatives of COLO 357 cells was accompanied by additional focal genomic alterations including point mutations and amplification of KRAS2. To complement the array CGH analysis, we also conducted an analysis of mRNA expression patterns in a subset of these cells using cDNA microarrays. By this means, we identified a set of candidate genes, including those regulated by RAS signaling, that may contribute to the process of cancer cell invasion and metastasis. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http://www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html.

Cell Line, Tumor↗
PubMed1999

Genome type analysis of Brazilian adenovirus strains of serotypes 1,2,3,5, and 7 collected between 1976 and 1995.

A collection of 92 epidemiologically unrelated isolates of Ad1 (n = 14), Ad2 (n = 29), Ad3 (n = 19), Ad5 (n = 16), and Ad7 (n = 14) collected in the cities of Belem do Pará (1 degree S 48 degrees W) and Rio de Janeiro (23 degrees S 43 degrees W) between 1976 and 1995 from patients with respiratory disease and conjunctivitis were characterized by restriction enzyme analysis of genomic DNA. Among the strains of subgenus B, two different genome types of serotype 7, 7b and 7e, were identified. The analysis of their temporal distribution throughout the study period suggested an alternating appearance of these two DNA variants. Only one genome type of Ad3, 3p, was detected during the sampling period. Further analysis with Xba I, Bcl I, and Hpa I indicated that it is a p1-like genome type. Both previously described and new genomic variants were identified among subgenus C strains. Genome types D1, D7, D10, and one not previously described were identified among the 14 Ad1 strains analyzed. Genome types D2, D5, D25, and 13 new DNA variants were identified among the 29 Ad2 isolates. Genome type D38 and 5 new variants were found among the 16 strains of Ad5. In spite of the relatively small size of the sample analyzed, the results of this study confirm the important genetic variability previously observed for members of subgenus C by other authors.

Adenoviridae↗
PubMed2001

Low frequency of genetic lesions in Wilms tumors by representational difference analysis.

Genomic representational difference analysis (RDA) was carried out on a total of nine Wilms tumors and one cystic partially differentiated nephroblastoma (CPDN; a sub-type of Wilms) to look for novel genetic deletions involving tumor suppressor genes. Genomic DNA from either short-term cultured Wilms tumor cells or a WT xenograft was used to create driver representations, and genomic DNA from matched normal kidney or normal kidney cells grown in short-term culture was used to create the tester. Genuine difference products were obtained from only one of the tumors. However, none of these fragments were found to be deleted in the original tumor biopsy, microdissected tumor or in the lung metastasis from this patient. It is, therefore, likely that the deletions were due to random losses associated with the genetic instability of the cultured cells from this particular tumor. We did not isolate difference products from any of the other tumors, showing that they did not have chromosomal losses, homozygous deletions or regions of LOH that were detectable by RDA.

Animals↗
PubMed2005

Identification of estrogen-responsive genes using a genome-wide analysis of promoter elements for transcription factor binding sites.

We developed a pipeline to identify novel genes regulated by the steroid hormone-dependent transcription factor, estrogen receptor, through a systematic analysis of upstream regions of all human and mouse genes. We built a data base of putative promoter regions for 23,077 human and 19,984 mouse transcripts from National Center for Biotechnology Information annotation and 8793 human and 6785 mouse promoters from the Data Base of Transcriptional Start Sites. We used this data base of putative promoters to identify potential targets of estrogen receptor by identifying estrogen response elements (EREs) in their promoters. Our program correctly identified EREs in genes known to be regulated by estrogen in addition to several new genes whose putative promoters contained EREs. We validated six genes (KIAA1243, NRIP1, MADH9, NME3, TPD52L, and ABCG2) to be estrogen-responsive in MCF7 cells using reverse transcription PCR. To allow for extensibility of our program in identifying targets of other transcription factors, we have built a Web interface to access our data base and programs. Our Web-based program for Promoter Analysis of Genome, PAGen@UIC, allows a user to identify putative target genes for vertebrate transcription factors through the analysis of their upstream sequences. The interface allows the user to search the human and mouse promoter data bases for potential target genes containing one or more listed transcription factor binding sites (TFBSs) in their upstream elements, using either regular expression-based consensus or position weight matrices. The data base can also be searched for promoters harboring user-defined TFBSs given as a consensus or a position weight matrix. Furthermore, the user can retrieve putative promoter sequences for any given gene together with identified TFBSs located on its promoter. Orthologous promoters are also analyzed to determine conserved elements.

Algorithms↗
PubMed2001

Genomics: leprosy - a degenerative disease of the genome.

Analysis of the genome of the leprosy bacillus uncovers evidence of extensive deletion and inactivation of genes. Secluded in a specialised niche, it has discarded much of its genetic heritage, though retaining just enough to be a major human pathogen.

Genome, Bacterial↗
PubMed2003

Genome-scale analysis of the uses of the Escherichia coli genome: model-driven analysis of heterogeneous data sets.

The recent availability of heterogeneous high-throughput data types has increased the need for scalable in silico methods with which to integrate data related to the processes of regulation, protein synthesis, and metabolism. A sequence-based framework for modeling transcription and translation in prokaryotes has been established and has been extended to study the expression state of the entire Escherichia coli genome. The resulting in silico analysis of the expression state highlighted three facets of gene expression in E. coli: (i) the metabolic resources required for genome expression and protein synthesis were found to be relatively invariant under the conditions tested; (ii) effective promoter strengths were estimated at the genome scale by using global mRNA abundance and half-life data, revealing genes subject to regulation under the experimental conditions tested; and (iii) large-scale genome location-dependent expression patterns with approximately 600-kb periodicity were detected in the E. coli genome based on the 49 expression data sets analyzed. These results support the notion that a structured model-driven analysis of expression data yields additional information that can be subjected to commonly used statistical analyses. The integration of heterogeneous genome-scale data (i.e., sequence, expression data, and mRNA half-life data) is readily achieved in the context of an in silico model.

Bacterial Proteins↗
PubMed1995

PCR restriction analysis of genome composition and stability of cold-adapted reassortant live influenza vaccines.

Using cold-adapted master donor strains of influenza virus as a model, an approach was developed that exploits unique nucleotide differences between the donor strains and wild-type influenza viruses for rapidly and simply determining the genome composition and genetic stability of live attenuated vaccine reassortants. The approach is based on PCR amplification of approximately 150-300-nucleotide-long regions of individual RNA segments that include the unique nucleotide positions, followed by restriction nuclease treatment of the DNAs obtained with specific restriction endonucleases. Restriction sites recognized by chosen nucleases either existed or were created during PCR in the genome of one (but not the other) parent strain. The technique requires a minimal amount of infectious virus (approx. 100 microliters of allantoic or tissue culture fluid with a haemagglutination titre 1:4-1:8 or less) and allows rapid (within about 10 h) determination of the origin of the RNA segment or the presence of a mutation. The method is beneficial for genome composition analysis of reassortant vaccine strains as well as for investigation of the genetic stability of live attenuated vaccines during replication in vaccinees.

Acclimatization↗
PubMed1988

Southern analysis of genomic DNA with unique and degenerate oligonucleotide probes: a method for reducing probe degeneracy.

We have optimized conditions for genomic Southern analysis of eukaryotic DNA with both unique and degenerate oligonucleotide probes. Using the human Factor IX gene, optimal probe concentrations and hybridization times were determined, and washing conditions with sodium chloride and tetramethylammonium chloride (TMA-Cl) were compared. With TMA-Cl, the washing temperature was independent of GC content. With these conditions, the Factor IX gene was detected in genomic DNA using a 128-fold degenerate 20-mer and a 32-fold degenerate 17-mer. This permits the detection of a gene prior to cloning and the reduction of probe degeneracy, and facilitates the isolation of that gene. We apply this method of probe degeneracy reduction using probes for the human Factor VIII gene.

Base Sequence↗
PubMed1999

Analysis of genomic integrity and p53-dependent G1 checkpoint in telomerase-induced extended-life-span human fibroblasts.

Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279-282, 1998; A. G. Bodnar et al., Science 279:349-352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21(Waf1/Cip1/Sdi1). TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.

Cell Survival↗
PubMed1993

Genome type analysis of South American adenoviruses of subgenus C collected over a 7-year period.

A collection of 148 adenovirus strains of subgenus C collected in Argentina, Chile, and Uruguay were studied by restriction enzyme analysis of genomic DNA with endonucleases BamHI, BglII, BstEII, EcoRI, HindIII, KpnI, and SmaI. Only strains corresponding to serotypes 1, 2, and 5 were identified representing both already described and new genome types. The most frequently detected type was Ad 2 followed by Ad 1 and Ad 5. Three different genomic variants of both Ad 1 and Ad 5 were discriminated. Ad 2 with 9 genome types exhibited the greatest variability. Novel profiles emerged only in strains corresponding to this serotype by restriction with BglII and BstEII. The circulation of prototype-like strains of Ad 1 throughout the study period is note-worthy. Thirty-six of 43 strains corresponded to genome type D 1. Clearly, one genome type of each serotype predominated over the 7-year period (Ad 1 D 1, n = 36; Ad 2 D 5, n = 32, Ad 5 D 36, n = 17). Some genomic variants were detected sporadically and only in Argentina, Chile, or Uruguay whereas others were widely distributed and circulated for years. A high proportion of the analysed strains was isolated from children under one year and males were more frequently infected than females.

Adenoviruses, Human↗
PubMed1996

Genomic DNA analysis from mouse toe lysates.

The protocol described in this report provides a simple, accurate and efficient assay for detection of transgenes and mutations in large colonies of rodents, using crude lysates prepared from the digit cut from the animals for identification purposes. This can be done as early as 6 days of age, minimizing trauma to the mice and allowing assays to be completed long before weaning.

Animals↗
PubMed2005

[Genetic analysis of osteoporosis susceptibility: future perspectives on genome wide analysis].

Genetic susceptibility of osteoporosis has been analyzed mainly via sib-pair analysis and association study on candidate genes, however these approaches might have substantial limitation. Recently proposed genome wide analysis of association using high density polymorphic markers has not been established on this field. Preferable phenotypes and suitable subjects have to be defined as well as for tag-SNP selection and the method of multi-step screening.

Genetic Predisposition to Disease↗
PubMed1998

Genome size determination in peronosporales (Oomycota) by Feulgen image analysis.

Genome size was determined, by nuclear Feulgen staining and image analysis, in 46 accessions of 31 species of Peronosporales (Oomycota), including important plant pathogens such as Bremia lactucae, Plasmopara viticola, Pseudoperonospora cubensis, and Pseudoperonospora humuli. The 1C DNA contents ranged from 0.046 (45. 6 Mb) to 0.163 pg (159.9 Mb). This is 0.041- to 0.144-fold that of Glycine max (soybean, 1C = 1.134 pg), which was used as an internal standard for genome size determination. The linearity of Feulgen absorbance photometry method over this range was demonstrated by calibration of Aspergillus species (1C = 31-38 Mb) against Glycine, which revealed differences of less than 6% compared to the published CHEF data. The low coefficients of variation (usually between 5 and 10%), repeatability of the results, and compatibility with CHEF data prove the resolution power of Feulgen image analysis. The applicability and limitations of Feulgen photometry are discussed in relation to other methods of genome size determination (CHEF gel electrophoresis, reassociation kinetics, genomic reconstruction) that have been previously applied to Oomycota.

Aspergillus↗
PubMed1996

Large-scale Homologous Analysis of Genome Sequence.

We described a new method for the large-scale homologous analysis of genome sequences, which used hashing technique combined with sparse dynamic programming to get a sequence alignment. Three examples, the plant chloroplast genomes, the mammalian T-cell receptors C(alpha)/C(delta) gene loci and the mammalian gamma-crystallin gene clusters were analysed. The results showed that the method was more rapid to obtain accurate enough data and might be useful in genome analysis.

Journal Article↗
PubMed2002

Comparative genomic hybridization analysis of primary cutaneous B-cell lymphomas: identification of common genomic alterations in disease pathogenesis.

To investigate genetic alterations in primary cutaneous B-cell lymphomas (PCBCLs), we have analyzed 29 cases of PCBCL. Comparative genomic hybridization showed chromosome imbalances (CIs) in 12 cases (41%). The mean number of CIs per sample was 2.05 +/- 2.97, with gains (1.48 +/- 2.38) more frequent than losses (0.56 +/- 1.40). The common regions of gains were 18/18q (50%), 7/7p (42%), 3/3q (33%), 20 (33%), 1p (25%), 12/12q (25%), and 13/13q (25%), whereas loss of 6q was frequent (42%). Among the different subsets of PCBCLs, CI was seen in 50% of diffuse large-cell lymphomas (DLCLs), 33% of marginal zone lymphomas, and 8% of follicle center cell lymphomas and unclassified lymphomas. A similar pattern of CI was observed in these lymphomas, but loss of 6q and gains of 3/3q were present only in DLCLs. Microarray-based genomic analysis of four DLCL cases identified oncogene gains of SAS/CDK4 (12q13.3) in three cases and MYCL1 (1p34.3), MYC (8q24), FGFR2 (10q26), BCL2 (18q21.3), CSE1L (20q13), and PDGFB (22q12-13) in two cases, whereas losses of AKT1 (14q32.3), IGFR1 (15q25-26), and JUNB (19p13.2) were identified in three cases, and losses of FGR (1p36), ESR (6q25.1), ABL1 (9q34.1), TOP2A (17q21-22), ERBB2 (17q21.2), CCNE1 (19q13.1), and BCR (22q11) were each identified in two cases. In addition, real-time-polymerase chain reaction detected amplification of BCL2 in 5 of 29 cases. These findings suggest that there are complex but consistent genetic alterations associated with the pathogenesis of PCBCLs.

Chromosome Aberrations↗
PubMed1998

Detection of genomic instability in lung cancer tissues by random amplified polymorphic DNA analysis.

Genomic instability resulting in multiple mutations is believed to be a driving force in the carcinogenic process. In this study, the random amplified polymorphic DNA (RAPD) technique, a simple PCR-based DNA polymorphism assay system, was used for detecting genomic instability in lung cancer tissues. DNAs from 20 lung cancer (18 non-small cell lung cancers and two small cell lung cancers) and their corresponding normal tissues were amplified individually by RAPD with seven different 10-base arbitrary primers. PCR products from RAPD were electrophoretically separated in agarose gels and banding profiles were visualized by ethidium bromide staining. The ability to detect genomic instability in 20 cancer tissues by each single primer ranged from 15 to 75%. DNA changes were detected by at least one primer in 19 (95%) cancer tissues. These results seem to indicate that genomic rearrangement is associated with lung carcinogenesis and that RAPD analysis is useful for the detection of genomic instability in lung cancer tissues.

Biopsy↗