We report the screening of thirty-one YACs with a number of markers using polymerase chain reaction (PCR) to construct a physical map of part of human chromosome 17q21.3-q22. A contig of YACs covering about 4 Mb was constructed around the TCF11 gene at 68 cM from the most telomeric marker on the p arm, localizing TCF11 telomeric to genetic marker D17S1827. Both human and mouse P1-derived artificial chromosomes (PACs) containing TCF11 were isolated and characterized. The human heterochromatin protein 1 gene, HP1Hsbeta, and its homologue in mouse, MoMOD1, were identified centromeric to TCF11.
The reviewed studies addressed the possibility of using gene transfer for correction of l-ascorbic acid biosynthesis and carbohydrate utilization in rainbow trout. Analyses of enzymatic activities in the l-AAB pathway indicated that reasons for the lack of l-AA production can be common in fish and scurvy-prone animals. Rat gulonolactone oxidase cDNA was transferred into trout. Regardless of the fact that rGLO transcription occurred in embryos, neither GLO protein, nor enzyme activity were detected. There was no production of l-AA in transgenic fish raised on vitamin C-free diets or injected with l-gulonolactone. These results indicated that the conditions required for translation or stability of rGLO were not present in trout tissues. To augment carbohydrates utilization, human glucose transporter 1 and rat hexokinase II cDNAs were tested. In the transfected embryos, HK activity, rates of hexose uptake and glucose oxidation were increased. The effect of hGLUT1 on glucose metabolism was greater than that of rHKII. Trout carrying hGLUT1 and rHKII with viral or piscine promoters were created. Though interpretation of the metabolic effects of the transgenes was complicated with mosaicism, a tendency to improved carbohydrate utilization was revealed in some of the transgenic individuals.
Animal Embryonic-Stem (ES) cells represents a unique tool in animal genetic manipulation. Though putative ES cells from several species have been reported, only those from mice proved successful. In this work, a long-term embryonic cell culture, derived from the commercial fish (Sparus aurata), is reported. These cells have been in vitro characterized for totipotency and transfected with a GFP plasmid.
An efficient selection analysis of single-stranded DNA fragments using subtractive hybridization on magnetic beads and fluorescence-based fragment analysis is described. This approach facilitates either direct cloning of captured fragments or removal of common clones from cDNA or shot-gun clone libraries.
A new method of amplifying short DNA molecules immobilized on a solid support has been developed. This method uses a solid-phase rolling circle replication reaction, termed rolling circle amplification (RCA). The probe consists of a single-stranded DNA primer anchored at the 5' terminus to a solid support and a single stranded DNA template hybridized to the immobilized primer. Here, DNA ligase was used to circularize the template, and DNA polymerase I was used to extend the immobilized primer in a rolling circle replication reaction. This method was used to identify a known polymorphism in BRCA1 exon 5. These results demonstrate that RCA offers considerable promise to facilitate effective mutation screening of DNA using a solid-phase format.
Variegation and inherited somatic mosaicism has been observed in transgenic mice carrying yeast artificial chromosomes (YACs) in which a DNAse I hypersensitive site (HS) located −12 kb upstream of the mouse tyrosinase gene had been deleted. At present, we are generating new transgenic animals with minor deletions of the HS.
High sensitivity, robustness and scalability are the three criteria which influence whether techniques for rapid mutation detection will be used in the future. PCR-SSCP, one of the most popular methods for detecting mutation, especially in the field of medical genetics, is being improved (1) to efficiently detect mutations in long stretches of PCR products; (2) to simplify data interpretation by removing PCR artifacts and (3) to minimize human involvement in the process of mutation detection by a simple post-PCR fluorescence labeling followed by separation using automated DNA sequencers.
We report a reliable method for PCR (polymerase chain reaction) amplification of genomic DNA from PET. This method uses DNA extraction with the QIAquick kit and amplification with AmpliTaq Gold. Amplification of up to 959 bp from PET was achieved with this combination which exceeds the current reported upper limit of 800 bp. In summary, the gradual activation of the AmpliTaq Gold during thermal cycling allows both for higher-fidelity and higher-throughput PCR amplification from PET. The use of the QIAquick kit for DNA purification of PET is sensitive, reproducible and suitable for management of a high number of samples.
We evaluated fish promoters as an alternative to viral promoters in the construction of DNA vaccines for aquaculture. A carp beta-actin promoter drove expression of the luciferase gene in live fish tissue to levels comparable to the CMVtk promoter.
To examine whether the utilization of protein and lipids is altered in the genetically modified, rapidly growing charr, we compared CMVOnGH1 transgenic and sibling fish. Muscle composition and rates of gas exchange were analyzed. Plasma metabolites were determined in the recently fed and post-absorptive state. No difference was found in muscle composition. At equal rates of protein accretion, the rate of NH4 excretion was 43% greater in sibling charr. The lower molar ratio of NH4 to O2 exchange implied the reduced expenditure of metabolized protein in transgenic charr. Plasma NH4 concentration in transgenic fish did not differ from that in sibling charr whereas the greater level of total CO2 indicated enhanced oxidation of non-protein nutrients. Decreased plasma triglycerides concentration and lower triglyceride to cholesterol ratio showed faster utilization of ingested lipids in transgenic charr, especially of energy-containing fraction. However, this was not accompanied with a reduced lipid content or altered fatty acid composition of muscle triglycerides or phospholipids. Comparative studies suggested that the transgenic charr had acquired features of domesticated salmonid fish. Their increased metabolic rate and enhanced utilization of dietary lipids, especially triglycerides, resembled the characteristics of domestic rainbow trout rather than wild counterparts.
In higher eucaryotes, natural bicistronic mRNA have been rarely found so far. The second cistron of constructed bicistronic mRNAs is generally considered as not translated unless special sequences named internal ribosome entry site (IRES) are added between the two cistrons. These sequences are believed to recruit ribosomes independently of a cap structure. In the present report, a new IRES found in the HTLV-1 genome is described. A systematic study revealed that this IRES, but also the poliovirus (polio) and the encephalomyocarditis virus (EMCV) IRES work optimally when they are added about 100 nucleotides after the termination codon of the first cistron. Unexpectedly, these IRES became totally inefficient when added after 300–500 nucleotide spacers. This result and others are not compatible with the admitted mechanism of IRES action. The IRES appear to be rather potent translation stimulators. Their effects are particularly emphasized in cells in which the normal mechanism of translation initiation is inhibited. For these reasons, we suggest to call IRES rescue translation stimulators (RTS).
p53 gene encodes a transcription factor with tumor suppressive properties and to date, somatic mutation of this gene is the most common genetic event in human cancer. A relational database has been developed to facilitate the retrieval and analysis of these mutations at the International Agency for Research on Cancer (IARC) and it currently contains information on over 8000 individual tumors and cell lines. Many factors may influence the detection and reporting of mutations, including selection of tumor samples, study design, choice of methods, and quality control. There is also concern that several biases may affect the way data appear in the literature. Minimizing these biases is an essential methodological issue in the development of mutation databases. In this paper, we review and discuss these main sources of bias and make recommendations to authors in order to minimize bias in mutation detection and reporting.
Nucleotide sequence analysis of arbitrarily primed PCR products from two known regions of human genome revealed that at least six contiguous bases at the 3'-end of a primer of 20 bases, were perfectly matched in the primer-template hybrids.
Improvements in modern commercial aquaculture are linked to the utilization of biotechnological methods and processes. The most visible approach has been the use of growth hormone (GH) and/or insulin-like growth factor I and II (IGF-I and II), to accelerate the growth of fish. Previously we have reported that the injection of bovine GH, (bGH) in striped bass hybrids increased the specific growth rate and food conversion efficiency without significant alteration of food consumption rate. In this paper we present the results of experiments in which growth, food consumption, conversion efficiency, ammonia excretion, and amino acid absorption were monitored for individual fish after bGH injection. The specific growth rate was stimulated by 50% without significant change in relative food consumption rate. Food conversion efficiency increased by 51%. Intestinal L-leucine absorption was increased by 25-40% at various concentrations tested. The relative N-retention was stimulated by 20% when computed raw. When a correction factor derived from the elevated amino acid absorption was introduced into the computations. the calculated relative N-retention was increased by 56%. Muscle amino acid profile was appreciably altered. We conclude GH supplementation or over-expression in aquaculture profoundly alters the physiological and nutritional conditions of fish. Nutritional profiles of fish food must be altered relative to these physiological changes in order to maximize growth.
The control of tick infestations and the transmission of tick-borne diseases remain a challenge for the cattle industry in tropical and subtropical areas of the world. Traditional control methods have been only partially successful and the parasites continue to result in significant losses for the cattle industry. Recently, vaccines containing the recombinant B. microplus gut antigen Bm86 have been developed. Our vaccine formulation (Gavac(TM), Heber Biotec S.A., Havana, Cuba) has been registered and is commercially available in Cuba, Colombia, Dominican Republic, Brazil and Mexico. In controlled pen trials, Gavac(TM) has been effective for the control of artificial infestations of B. annulatus, B. decoloratus and chemical-sensitive and resistant B, microplus strains from Australia, Africa, America and Iran. In controlled field trials in Cuba, Brazil, Argentina and Mexico, Gavac has shown a 55-100% efficacy in the control of B. microplus infestations in grazing cattle 12-36 weeks after the first vaccination. Field trials under production conditions have been conducted in Cuba, Colombia, Brazil and Mexico in pure and cross-bred cattle herds. The application of Gavac(TM) has increased the time between acaricide treatments by an average of 32 +/- 21 days (P = 0.0005) resulting in important savings for the cattle industry. In Cuba, a cost-effectiveness analysis was conducted in more than 260 000 animals. The cost-effectiveness analysis showed a 60% reduction in the number of acaricide treatments, together with the control of tick infestations and transmission of babesiosis, which resulted in savings of $23.4 animal(-1) year(-1). These results clearly demonstrate the advantage of vaccination and support the application of Gavac for the control of Boophilus spp. infestations. (C) 1999 Published by Elsevier Science B.V. All rights reserved.
A simple in situ method for scoring short tandem DNA repeat length has been developed using T4 endonuclease VII. This method measures tandem repeated simple sequences embedded in unique sequences. Single-stranded loops are formed on duplexes containing mismatched (different) numbers of tandem repeats. No single stranded loops are formed on structures containing matched (identical) numbers of tandem repeats. The matched and mismatched loop structures were distinguished and differentially labeled by enzymatic treatment with T4 endonuclease VII.
Extraction of nuclei from unmounted archived tissue has become a method widely used for molecular cytogenetic investigations. It is a suitable approach to take advantage of the large series of formalin fixed and subsequently paraffin-embedded material which has been collected in the times before interphase cytogenetic analysis has become possible. We present a new kind of assay for the extraction of nuclei from one single mounted tissue section; the extracted interphase nuclei are suitable very well for a fluorescence in situ hybridization (FISH) approach. The method described has been successfully used for the analysis of the INT2/FGF3-amplicon in 20 samples of oral squamous cell carcinoma.
Growth manipulation in fish is one of the targets of gene transfer experiments. The aim is to produce strains with improved growth performance. The transfer of growth hormone transgenes has been successful in many fish species. Now detailed knowledge of the molecular events that control growth in fish is necessary in order to efficiently manipulate this process. We have selected tilapia for our studies because these species are suitable for basic research as well as for the development of improved strains for aquaculture. Here we review the results of basic and applied research in the field of growth control and manipulation in tilapia. Our experiments produced new scientific results on growth control in tilapia. These results were used to develop a new aquacultured line with improved growth performance. Many of these results are probably applicable to other teleosts.
A homogeneous detection mechanism based on fluorescence resonance energy transfer (FRET) has been developed for two DNA diagnostic tests. In the template-directed dye-terminator incorporation (TDI) assay, a donor dye-labeled primer is extended by DNA polymerase using allele-specific, acceptor dye-labeled ddNTPs. In the dye-labeled oligonucleotide ligation (DOL) assay, a donor dye-labeled common probe is joined to an allele-specific, acceptor dye-labeled probe by DNA ligase. Once the donor and acceptor dyes become part of a new molecule, intramolecular FRET is observed over background intermolecular FRET. The rise in FRET, therefore, can be used as an index for allele-specific ddNTP incorporation or probe ligation. Real time monitoring of FRET greatly increases the sensitivity and reliability of these assays. Change in FRET can also be measured by end-point reading when appropriate controls are included in the experiment. FRET detection proves to be a robust method in homogeneous DNA diagnostic assays.
The abundance and ubiquity of ticks from ancient times long ago suggested that they have eluded host immunity. In the last 15 years, several authors have demonstrated suppression of the Th1 responses (cell-mediated immunity), and sometimes the Th2 responses (humoral immunity), subsequent to tick infestations in laboratory and natural models. Although the mechanisms to produce suppression are not well-defined yet, evidences for antigenic competition, lymphocyte cytotoxicity, presence of immuno-inhibiting substances in the saliva, and existence of modulators of cytokines in salivary extracts have been reported. Management of tick-induced immunosuppression is essential to replace tick control by acaricide application with more environmentally sound vaccination.
