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At least 19 recordsLinked to original sources
PubMed1990

Sex determination and the Y chromosome: the application of molecular genetic technique to behavioral genetics.

In mammals, the Y chromosome mediates both gonadogenesis and spermatogenesis. It is also known to influence such traits as histocompatibility, sperm head morphology, pubertal (but not adult) testosterone level, sexual behavior, and aggressive behavior. An immediate goal in my laboratory is the isolation and characterization of the Y chromosomal gene responsible for initiating differentiation of the primitive bipotential gonads to become testes: the Y chromosomal gonadogenesis gene. Function of this gene initiates a cascade of events involving large numbers of other genes scattered throughout the genome, but it is not responsible for initiating development of all of the male phenotype; where : is XXSxr karyotype males, bearing the Sxr region of the Y chromosome which includes this gene, are sterile. It is not known if this gene influences those behaviors known to be influenced by the Y chromosome. If animals with an XXSxr karyotype, transgenic for specific Y chromosomal genes, could be produced, questions such as this could be answered. The developmental biology of the testis, molecular genetics of the Sxr region of the Y chromosome, and isolation of the testis determination gene from DNA of XXSxr males are discussed. Also discussed are the production of transgenic mice and the prospects for using such animals as coisogenic strains, differing by precisely known DNA sequences, in behavior genetic analysis. Such animals could be used both to test for behavioral phenotype and to dissect out biochemical and neurological mechanisms responsible for the behavior.

Animals↗
PubMed1994

Genetic diversity of cultured Penaeus vannamei shrimp using three molecular genetic techniques.

Three molecular genetic techniques, restriction fragment length polymorphisms (RFLPs), random amplification of polymorphic DNA (RAPD), and allozyme variability, were used to evaluate the genetic diversity of two specific-pathogen-free (SPF) populations (numbers 1 and 2) and one candidate SPF population (number 4) of Penaeus vannamei developed and maintained by the U.S. Marine Shrimp Farming Program. A total of 114 individuals were tested, which included 30 each from families 1.5 and 1.6 of population 1 and from population 2, and 24 from population 4. Two HhaI mitochondrial DNA polymorphisms (A and B) were found in all the animals examined, with family 1.5 and population 2 showing type A and family 1.6 showing type B. After scoring 73 bands obtained with six different RAPD primers, the percentage of polymorphic bands was: 55% for families 1.5 and 1.6 of population 1, 48% for population 2, and 77% for population 4, suggesting that population 4 is the most polymorphic of all three populations. The allozymic variation at 30 loci showed no fixed differences in isozyme genotypes between families 1.5 and 1.6. The percentage of polymorphic loci, under the criterion that the frequency of the most common allele was less than 0.95 in each population, was 6.67%, 3.33% and 16.67% for family 1.5 of population 1, family 1.6 of population 1, and population 2, respectively. Mean heterozygosities (+/- SE) were 0.023 +/- 0.017, 0.018 +/- 0.016, and 0.064 +/- 0.026, respectively. The low levels of allozyme polymorphisms indicate that mitochondrial DNA and nuclear DNA techniques are more useful for examining genetic diversity in order to follow individual stocks within a breeding program and to correlate genotypes with desirable growth and reproductive performance of SPF P. vannamei stocks.

Animals↗
PubMed2002

Characterization of Trichodesmium spp. by genetic techniques.

The genetic diversity of Trichodesmium spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a hetR fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of Trichodesmium spp. from the two hemispheres. Culture isolates of Trichodesmium thiebautii, Trichodesmium hildebrandtii, Trichodesmium tenue, and Katagnymene spiralis displayed remarkable similarity when these techniques were used, suggesting that K. spiralis is very closely related to the genus TRICHODESMIUM: The largest genetic variation was found between Trichodesmium erythraeum and all other species of Trichodesmium, including a species of KATAGNYMENE: Our data obtained with all three techniques suggest that there are two major clades of Trichodesmium spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.

Bacterial Proteins↗
PubMed2006

TILLING is an effective reverse genetics technique for Caenorhabditis elegans.

BACKGROUND: TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetic technique based on the use of a mismatch-specific enzyme that identifies mutations in a target gene through heteroduplex analysis. We tested this technique in Caenorhabditis elegans, a model organism in which genomics tools have been well developed, but limitations in reverse genetics have restricted the number of heritable mutations that have been identified. RESULTS: To determine whether TILLING represents an effective reverse genetic strategy for C. elegans we generated an EMS-mutagenised population of approximately 1500 individuals and screened for mutations in 10 genes. A total of 71 mutations were identified by TILLING, providing multiple mutant alleles for every gene tested. Some of the mutations identified are predicted to be silent, either because they are in non-coding DNA or because they affect the third bp of a codon which does not change the amino acid encoded by that codon. However, 59% of the mutations identified are missense alleles resulting in a change in one of the amino acids in the protein product of the gene, and 3% are putative null alleles which are predicted to eliminate gene function. We compared the types of mutation identified by TILLING with those previously reported from forward EMS screens and found that 96% of TILLING mutations were G/C-to-A/T transitions, a rate significantly higher than that found in forward genetic screens where transversions and deletions were also observed. The mutation rate we achieved was 1/293 kb, which is comparable to the mutation rate observed for TILLING in other organisms. CONCLUSION: We conclude that TILLING is an effective and cost-efficient reverse genetics tool in C. elegans. It complements other reverse genetic techniques in this organism, can provide an allelic series of mutations for any locus and does not appear to have any bias in terms of gene size or location. For eight of the 10 target genes screened, TILLING has provided the first genetically heritable mutations which can be used to study their functions in vivo.

Animals↗
PubMed2004

Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.

Animals↗
PubMed1988

Testing the retrovirus hypothesis of manic depression and schizophrenia with molecular genetic techniques.

Crow's viral hypothesis of schizophrenia proposes that psychosis may be the result of mutagenesis caused by viral integration or transposition in human genomic DNA. Molecular genetic techniques can be used to systematically investigate this hypothesis. In a study of genomic lymphocyte DNA unexpected DNA polymorphisms which were probably insertions and deletions were found in specific human genomic retroviral (proviral) related sequences. However these changes were found exclusively in normal Icelandic individuals and are probably of evolutionary origin. The extent to which human retroviral insertion and deletion has taken place and the mobility of such sequences will help in understanding their evolutionary origin and might provide a source of polymorphic marker sequences that could be used in genetic linkage studies of disease.

Bipolar Disorder↗
PubMed2005

[Generation of newcastle disease virus strain ZJI isolated from an outbreak in the goose using reverse genetics technique].

The full-length cDNA clone, NDV3GM122, and the three helperplasmids pCI-NP, pCI-P and pCI-L of Newcastle disease virus strain ZJI isolated from an outbreak in the goose were cotransfected into BSR-T7/5 cell expressing T7 RNA polymerase. Meanwhile, the full-length cDNA clone NDV3GM122 and the three helperplasmids, pCIneoNP, pCIneoP and pCIneoL which were derived from NDV strain La Sota, were also cotransfected into the cell, respectively. Indiect immunofluorescence assay (IFA) was performed 48 to 96 hours post-transfection using NDV HN-specific monoclonal anbtibody (McAb) 6B1 and bright stainings were found in the transfectants, indicating that the full-length clone was functional and the HN protein was expressed. The transfected cell and the supernatant were mixed well and thereafter the mixture was inoculated into specific pathogen free (SPF) chicken eggs. The allanotoic fluid of the injected eggs gave a positive hemagglutinin( HA) titer ranging from 16 to 32 in the secondary passage and increased to 128 in the third passage, which was same to the level of parent wild-type virus. The allantoic fluid containing the recovered NDV was analyzed in hemagglutination inhibition( HI) test by using McAb 6B1 and the specific inhibition was found. The typical morphology of the produced NDV was detected in the electronic microscope. The results mentioned above demonstrated that infectious NDV of strain ZJI was successfully generated, which laid good foundation for the further related research.

Animals↗
PubMed1997

New genetic techniques for group B streptococci: high-efficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917.

Three techniques were developed to improve the genetic manipulation of group B streptococci (GBS). We first optimized a protocol for transformation of GBS by electroporation, which provided transformation efficiencies of 10(5) CFU/microgram. Variables that influenced the transformation efficiency were the glycine content of the competent cell growth media, the electric field strength during electroporation, the electroporation buffer composition, the host origin of the transforming plasmid, and the concentration of selective antibiotic at the final plating. Our transformation protocol provides an efficiency sufficient for cloning from ligation reactions directly into GBS, obviating an intermediate host such as Escherichia coli. Second, temperature-sensitive plasmids of the pWV01 lineage were shown to transform GBS, and their temperature-sensitive replication was confirmed. Lastly, the temperature-sensitive pWV01 plasmid pTV1OK, which contains Tn917, was used as a transposon delivery vector for the construction of genomic Tn917 mutant libraries. We have shown, for the first time, that Tn917 transposes to the GBS chromosome and at a frequency of 10(-3)/CFU. Furthermore, representative clones from a Tn917 library contained single transposon insertions that were randomly located throughout the chromosome. These techniques should provide useful methods for cloning, mutagenesis, and characterization of genes from GBS.

DNA Replication↗
PubMed1999

[Genetic techniques to overcome antitumor anergy].

Immunotherapy of malignant tumors is often hampered by "immune escape" phenomenons of tumor cells. Both active (e.g. induction of T-cell apoptosis, secretion of immunomodulators, -downregulators) and passive (e.g. antigen shedding, loss of MHC-class I/II molecules on the tumor cell surface) mechanisms are involved in this process. Another reason are inborn or acquired immunodeficiency syndromes. Gene technology tries to reconstitute the immunocompetent status by genetic compensation or modulation of the defect. Some of these trials have been performed already in phase I studies of different kinds of tumors and will be shortly presented here.

Clinical Trials, Phase I as Topic↗
PubMed1991

[Genetic techniques--chances--risks--limits (from the viewpoint of the legislator--ethical questions--status of medical research].

Gene technology is still a very young area of research. Advantages or disadvantages of this development cannot be tangibly predicted. Irrational desires and fears have surfaced resulting from the fact that we are now in a position of cultivate certain biological characteristics hitherto impossible to reproduce. Individual cases of genome analysis under working conditions, genome analysis as a genetic fingerprint, outdoor plant experiments and gene testing procedures in prenatal diagnoses should all be considered. Analysis uncertainty exists in all these fields, not least caused by multifunctional factors affecting genetic features. The question regarding the right to know or not to know, and the data protection problem irrevocably connected to this, is constantly being raised. It must be recorded that, in spite of the latest scientific findings, opportunities for therapy are frequently lacking. In all our efforts, the psychological conflicts of those concerned and the resulting socio-economic consequences must be taken into consideration.

Ethics, Medical↗
PubMed1997

The application of molecular genetic techniques to the study of hypertensive diseases.

The techniques of modern molecular genetics are shedding new light on hypertension and its sequelae. This article discusses techniques which have identified genes associated with hypertension and have pointed the way toward identifying the full cohort of genes operative in all forms of human hypertension. These techniques have expanded understanding of the pathophysiology of hypertension as well as its prevention.

Humans↗
PubMed2001

Plasticity and behavior: new genetic techniques to address multiple forms and functions.

As the best-studied form of vertebrate synaptic plasticity, NMDA-receptor dependent long-term potentiation (NMDAR-LTP) has long been considered a leading candidate for a cellular locus for some aspects of learning and memory. However, assigning a specific role for this form of plasticity in learning and memory has proven surprisingly difficult. Two issues have contributed to this difficulty. First, a large number of molecules have been shown to in some way mediate or modulate not only NMDAR-LTP but also many forms of plasticity. Indeed, it is increasingly clear that multiple induction and maintenance mechanisms for plasticity exist, often at the same synapse. Second, linking cellular events to behavioral function has been hindered by a lack of sufficiently precise tools. In this review, we will discuss some of the proposed mechanisms of induction and maintenance of changes in synaptic efficacy and their regulation in the context of an attempt to understand their roles in animal behavior. Further, we will discuss recently developed genetic techniques, specifically, inducible transgenic models, which now allow more precise manipulations in the study of the roles plasticity plays in learning and memory.

Animals↗