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PubMed2004

Suppressive subtractive hybridization as a tool for identifying genetic diversity in an environmental metagenome: the rumen as a model.

Molecular techniques previously used for genome comparisons of closely related bacterial species could prove extremely valuable for comparisons of complex microbial communities, or metagenomes. Our study aimed to determine the breadth and value of suppressive subtractive hybridization (SSH) in a pilot-scale analysis of metagenomic DNA from communities of microorganisms in the rumen. Suppressive subtractive hybridization was performed using total genomic DNA isolated from rumen fluid samples of two hay-fed steers, arbitrarily designated as tester or driver. Ninety-six subtraction DNA fragments from the tester metagenome were amplified, cloned and the DNA sequences were determined. Verification of the isolation of DNA fragments unique to the tester metagenome was accomplished through dot blot and Southern blot hybridizations. Tester-specific SSH fragments were found in 95 of 96 randomly selected clones. DNA sequences of subtraction fragments were analysed by computer assisted DNA and amino acid comparisons. Putative translations of 26 (32.1%) subtractive hybridization fragments exhibited significant similarity to Bacterial proteins, whereas 15 (18.5%) distinctive subtracted fragments had significant similarity to proteins from Archaea. The remainder of the subtractive hybridization fragments displayed no similarity to GenBank sequences. This metagenomic approach has exposed an unexpectedly large difference in Archaeal community structure between the rumen microbial populations of two steers fed identical diets and housed together. 16S rRNA dot blot hybridizations revealed similar proportions of Bacteria and Archaea in both rumen samples and suggest that the differences uncovered by SSH are the result of varying community structural composition. Our study demonstrates a novel approach to comparative analyses of environmental microbial communities through the use of SSH.

Animals↗
PubMed1980

Polyvalent strain-specific alloantisera as tools for routine genetic quality control of inbred and congenic strains of rats and mice.

Strain-specific polyvalent alloantisera may be obtained by injecting lymphocytes pooled from several different strains into an inbred recipient. 6 sera of this type were produced in rats and 23 in mice. A dye-exclusion microcytotoxic test was used to evaluate the strain specificity of such sera. A total of 663 out of 713 (93.0%) of the tests conformed with expectation, but there were 58 (6.7%) false negative results in which the test failed to detect non-authentic animals. There were also 2 (0.3%) false positive results, in which authentic animals were shown as non-authentic. These were attributed to technical errors. Most false negative results occurred when serum and test cell suspensions matched at the major histocompatibility complex. It was concluded that the use of strain-specific polyvalent immune sera, coupled with a simple immunological test such as the microcytotoxic test, offers a sensitive and quick new method for routine genetic quality control.

Animals↗
PubMed2005

Minos as a genetic and genomic tool in Drosophila melanogaster.

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 5'-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient "jump-starting" scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and approximately 55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila.

Animals↗
PubMed2005

PubMed, the New York Times and the Chicago Tribune as tools for teaching genetics.

An elementary course in human heredity for students not planning to major in the sciences can be based on current scientific literature and on the popular media. Examinations are constructed from questions on recent abstracts obtained from PubMed. The course is designed to promote writing skills in the sciences, and students write two papers in the course of a quarter. In the first paper, students trace the primary source of media reports on genetics and attempt to evaluate the reporter's translation. In a second paper, students write popular articles on the basis of current primary sources.

Curriculum↗
PubMed1999

Embryo transfer in the rat as a tool to determine genetic components of the gestational environment.

Embryo transfer, with the recipient dam nursing the transferred progeny, was used to study the impact of the gestational environment on adult blood pressure (BP) in three inbred rat strains, the hypertensive Dahl salt-sensitive SS/JrCtr, the normotensive Dahl salt-hypertension resistant SR/Jr, and the normotensive Dark Agouti rat. Rats that had been cross-fostered within 6 h of birth were included as a control for lactational and nurturing factors. Systolic BP was measured by tail-cuff plethysmography twice a week in rats after the age of 7 weeks. Embryo transfer success, measured as the percentage of embryos transferred resulting in pups weaned at 4 weeks, was 27% between the SS/JrCtr and SR/Jr and 53% for the SS/JrCtr and Dark Agouti. This assessment included all failures, some of which probably were not associated with the transfer. If only the number of embryos transferred to dams with successful pregnancies was included, the success rate was 48% between the SS/JrCtr and SR/Jr and 82% between the SS/JrCtr and Dark Agouti strains. Anomalies in pups were not evident. In contrast to the lactational environment, the gestational milieu had a profound effect on basal blood pressure of the hypertensive SS/JrCtr progeny, less of an effect on that of the Dark Agouti, and no effect on that of the SR/Jr. Although the SS/JrCtr strain is significantly larger than the SR/Jr and Dark Agouti strains, neither embryo transfer nor cross-fostering altered body weight of rats at the age of 6 weeks. These data indicate that embryo transfer can be an easy and efficient method of isolating genetically determined factors of the gestational environment.

Animals↗
PubMed1999

Using molecular genetics as a tool in understanding crawling cell locomotion in myoblasts.

We have used digitally recorded interference microscopy with automatic phase shifting (DRIMAPS) to investigate the crawling locomotion of normal and mutant mouse myoblasts. Contraction forces that give rise to cell body movement, tail retraction and cell adhesion to the substrate in myoblasts and other locomoting tissue cells arise from the interactions of actin and non-muscle myosin II. The activity of non-muscle myosin II is regulated differently from that of skeletal myosin. Using DRIMAPS, we found that crawling locomotion was altered in myoblasts that heterologously expressed human beta-cardiac myosin heavy chain (MHC); the cells moved more slowly and had reduced rates of protrusion and retraction. Immunolocalization demonstrated that MHC and non-muscle myosin II were not co-localized, suggesting that MHC does not compete directly with myosin II, but interferes with cell locomotion by binding inappropriately to actin filaments and possibly cross-linking them. Myosin I may be involved in protrusion of the lamellipodia. However, using DRIMAPS, we found that crawling locomotion was unaltered in myoblasts that heterologously expressed a truncated myosin I which lacked the membrane-binding tail domain. This suggests that, if endogenous myosin I is important for cell locomotion, this mutant was unable to interfere with its action. We conclude that the effects on locomotion of expressing foreign or mutant proteins of the cytoskeleton in vertebrate cells can be subtle and can be swamped by the intrinsic variability of the cells. Their characterization requires automated methods of acquiring data, such as DRIMAPS, and careful statistical analysis in order to take account of other sources of variation.

Actins↗
PubMed1998

Mitochondrial DNA: a tool for populational genetics studies.

Mitochondria are cellular organelles that have the function of the oxidative phosphorilation and the formation of ATP. In humans, the mtDNA is a double-stranded, circular, covalent closed molecule of 16.5 kb. The mtDNA is inherited as a haploid from the mother and heteroplasmy has been found rarely. From a populational perspective, it could be considered as a system of small, sexually isolated demes, or clonal lineages, with an evolutionary rate 5 to 10 times faster than the nuclear genome. All these characteristics make this molecule ideal for evolutionary studies. We present two applications of this molecule in genetical studies. One of these is referred to the Balearic Islands populations, Majorca, Minorca, Ibiza, and Chuetas. The other example is the populational dynamics of the different mitochondrial haplotypes in Drosophila subobscura. We also discuss the importance of nuclear markers to complete these studies as well as the study of the Y chromosome to compensate the bias produced by the study of only the mtDNA.

Animals↗
PubMed2001

Chromosome painting as a tool for rice genetics and breeding.

Chromosome painting and genomic in situ hybridization (GISH) are both effective methods for basic genetic research and practical breeding. These methods were applied even in the typically small chromosomes of rice. This manuscript describes in detail, highly reproducible, complete protocols for chromosome painting and GISH in rice chromosomes. Examples of useful applications of these methods are also presented.

Breeding↗
PubMedDate not supplied

New tools, new dilemmas: genetic frontiers.

The powerful new methods, expansive scope, and accelerated pace of human molecular genetics combine to catapult us into ethically unfamiliar territory. These features lend special urgency to questions of genetic ownership and privacy, disease and normalcy, identity and genetic determinism, and early diagnosis and therapy.

Biotechnology↗
PubMed1998

[The DNA-chip, a new tool for medical genetics].

The DNA (chip) technology, emerged in the last two years, provides an incredible technical development in rapid and automatised performance of genetic identification. The principle of procedure is, that by series of photolitographic and chemical steps on solid-phase of a small surface relatively dense oligonucleotide arrays can be generated. The oligonucleotides produced by the computer-directed in-situ microfabrication may bind complementary fluorescent-labeled DNA fragments from the unknown sample. After scanning, the registered position of positive signals provide information on the identity of DNA fragments. By this novel approach not only DNA sequencing can be performed, but genetic errors of known genes, presence of infectious organisms and the typing of genetic markers (such as HLA) used for transplantation and forensic medicine can be determined. The DNA-chip technology is a revolutionary new milestone in genetic diagnose.

DNA↗
PubMed2004

SARGE: a tool for creation of putative genetic networks.

SUMMARY: SARGE is a tool for creating, visualizing and manipulating a putative genetic network from time series microarray data. The tool assigns potential edges through time-lagged correlation, incorporates a clustering mechanism, an interactive visual graph representation and employs simulated annealing for network optimization. AVAILABILITY: The application is available as a .jar file from http://www.bioinformatics.cs.ncl.ac.uk/sarge/index.html.

Algorithms↗
PubMed1999

NetWork: an interactive interface to the tools for analysis of genetic network structure and dynamics.

We designed a Java applet called NetWork which enables a user to interactively construct and visualize a genetic network of interest, and to and to evaluate and explore its dynamics in the framework of a Boolean network model. NetWork displays the mechanism of gene interactions at the level of gene expression and enables the visualization of large genetic networks. NetWork can serve as an interactive interface to tools for the analysis of genetic network structure and behavior.

Algorithms↗
PubMed2004

Genetically modified animal models as tools for studying bone and mineral metabolism.

Genetic modification of mice is a powerful tool for the study of bone development and metabolism. This review discusses the advantages and disadvantages of various approaches used in bone-related research and the contributions these studies have made to bone biology. Genetic modification of mice is a powerful tool for the study of bone development and metabolism. This review discusses the advantages and disadvantages of various approaches used in bone-related research and the contributions these studies have made to bone biology. The approaches to genetic modification included in this review are (1) overexpression of genes, (2) global gene knockouts, (3) tissue-specific gene deletion, and (4) gene knock-in models. This review also highlights issues that should be considered when using genetically modified animal models, including the rigorous control of genetic background, use of appropriate control lines, and confirmation of tissue specificity of gene expression where appropriate. This technology provides a unique and powerful way to probe the function of genes and is already revolutionizing our approach to understanding the physiology of bone development and metabolism.

Animals↗
PubMed2006

ISSR-PCR: tool for discrimination and genetic structure analysis of Plutella xylostella populations native to different geographical areas.

The diamondback moth (DBM), Plutella xylostella (L.) is considered as the most destructive pest of Brassicaceae crops world-wide. Its migratory capacities and development of insecticide resistance in many populations leads to more difficulties for population management. To control movement of populations and apparitions of resistance carried by resistant migrant individuals, populations must be identified using genetic markers. Here, seven different ISSR markers have been tested as a tool for population discrimination and genetic variations among 19 DBM populations from Canada, USA, Brazil, Martinique Island, France, Romania, Austria, Uzbekistan, Egypt, Benin, South Africa, Réunion Island, Hong Kong, Laos, Japan and four localities in Australia were assessed. Two classification methods were tested and compared: a common method of genetic distance analyses and a novel method based on an advanced statistical method of the Artificial Neural Networks' family, the Self-Organizing Map (SOM). The 188 loci selected revealed a very high variability between populations with a total polymorphism of 100% and a global coefficient of gene differentiation estimated by the Nei's index (Gst) of 0.238. Nevertheless, the largest part of variability was expressed among individuals within populations (AMOVA: 73.71% and mean polymorphism of 94% within populations). Genetic differentiation among the DBM populations did not reflect geographical distances between them. The two classification methods have given excellent results with less than 1.3% of misclassified individuals. The origin of the high genetic differentiation and efficiency of the two classification methods are discussed.

Algorithms↗