Identification and genetic mapping of random amplified polymorphic DNA (RAPD) markers to the sheep genome

The random amplified polymorphic DNA (RAPD) assay utilizes the polymerase chain reaction (PCR) and short primers of arbitrary nucleotide sequence to amplify DNA. In this study, the RAPD assay was used to identify and map polymorphic markers in the AgResearch International Mapping Flock (IMF) sheep pedigrees. Sires and dams of eight of the full-sib IMF pedigrees were screened with 131 different 10-mer oligonucleotide primers. An average of 85 RAPD polymorphisms was identified between each parental pair, and 53 markers were contributed to the AgResearch IMF collaboration. Forty-five of the RAPD markers were mapped in the AgResearch IMF genetic linkage map, and at least one marker was located on 17 of the 26 autosomes and both sex chromosomes. Three lines of evidence were used to check for the homology of scored polymorphisms in different pedigrees, pedigree evaluation, segregation analysis, and Southern blot analysis. These results demonstrate that the RAPD assay is a powerful approach for identifying polymorphisms that can be used as markers for constructing a sheep genetic linkage map.     

Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA

The random amplified polymorphic DNA technique (RAPD) has found wide use in molecular genetics because of its speed and ease of use. For various reasons, with this method the amplified DNA fragments are produced at different concentrations between genotypes and even between polymerase chain reaction (PCR) runs. Since the detection of the multiple amplified fragments is performed routinely by agarose gel, and seldom by acrylamide gel electrophoresis, we have found that by capillary zone electrophoresis (CZE), which is more sensitive and accurate than gel electrophoresis, it is possible to unequivocally detect amplified fragments even at low concentration, avoiding polymorphism misinterpretation. CZE is also useful to make more potentially polymorphic fragments evident per random primer used, with obvious economical benefits.     

Random amplified polymorphic DNA (RAPD) sequences as markers for canine genetic studies

Our knowledge of the concentration of growth factors in growing bone is limited. In the present study, we examined the developmental changes in the concentrations of insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-beta) in the rat femur between weanling and maturity. We show that during the rapid growth phase there is a continuous rise in bone matrix IGF-I and TGF-beta in all compartments of the femoral bone. The association between IGF-I and TGF-beta is not only temporal, but with few exceptions is also observed within the animals of each age class. These data support the hypothesis that IGF-I and TGF-beta play an important role in the growth-associated accumulation of bone mass.     

Sequence analysis of DNA randomly amplified from the Saccharomyces cerevisiae genome

OBJECTIVES: To report the features of malignancies responsible for a chest wall mass and involving the sternum, the sternocostal and/or sternoclavicular joints, the chondrocostal junction and/or the adjacent soft tissues. METHODS: The medical records of patients with a chest wall mass due to malignant disease were reviewed retrospectively. The following data were abstracted from each record: characteristics of the pain and mass, constitutional symptoms, physical findings, laboratory test results, findings from imaging studies (plain radiographs, computed tomography and magnetic resonance imaging of the chest, radionuclide bone scan), histologic features of the biopsy specimen from the chest wall mass and origin of the mass. RESULTS: Seven men and three women with a mean age of 53.1 years were included in the study. A single patient had a history of malignant disease (lymphoma); in the remaining nine patients the chest wall mass was the first manifestation of the malignancy. All ten patients had pain with a mixed time pattern. The mass was located on the sternum in half the patients and in a parasternal location in the other half. Erythrocyte sedimentation rate elevation was found in seven patients, an increased serum level of lactate dehydrogenase in one and a monoclonal immunoglobulin in three. Sternal lesions were visible on plain radiographs in four patients. Computed tomography of the chest consistently disclosed sternal or sternocostal lytic lesions with spread to the adjacent soft tissues; in five cases, enlarged lymph nodes were visible in the anterior part of the mediastinum. Magnetic resonance imaging of the chest did not add to the information provided by computed tomography. Radionuclide uptake on the bone scan was increased, decreased, or normal at the site of the lesion. The cause was Hodgkin's disease in two cases, non-Hodgkin's lymphoma in three, metastatic bone disease in two (from an adenocarcinoma of the lung and a hepatocarcinoma, respectively), multiple myeloma in one, and solitary plasmacytoma in two. CONCLUSION: A chest wall mass can be caused by a known or as yet undiagnosed malignancy. Chest wall involvement due to malignant disease in rare, however. The specific features of sternal metastases, lymphomas involving the sternum, and sternal plasmacytomas are discussed. Nonmalignant chest wall lesions that can manifest as a bulging or swelling of the chest wall are reviewed.     

Segregation analysis of random amplified polymorphic DNA (RAPD) markers in Picea abies Karst

The reliability of arbitrarily primed amplification products was tested. The segregation analysis of 266 amplification products obtained using 17 different 10-mer oligonucleotides in 34 megagametophytes from a single tree of Picea abies was carried out. Fifty-four out of the 165 variable bands fit the 1:1 segregation ratio expected for Mendelian traits. The segregation ratio of a subset of six RAPD markers in five other individuals from the same population confirmed their genetic nature. Our results strengthen the evidence previously reported that RAPDs markers can be considered Mendelian traits useful in the detection of genetic variability among both different individuals and populations.     

Randomly amplified polymorphic DNA (RAPD) for evaluating genetic relationships among varieties of guinea fowl

BACKGROUND: The aim of the present study was to investigate the existence of differences in dental status and in quantitative and qualitative salivary values between 100 patients with liver cirrhosis (LC) and a group of controls. MATERIAL AND METHODS: We analyzed the number of carious, missing and filled teeth. Also the unstimulated (UWS) and stimulated whole saliva flow rates (SWS) were determined, along with the stimulated parotid saliva flow rate (PSS) and the concentration in both UWS and SWS of sodium, potassium, total proteins and immunoglobulin A (IgA). RESULTS: A significantly higher number of carious and missing teeth was observed in the patients with cirrhosis (2.4 and 14.6, respectively) than in the control group (1.3 and 10.6, respectively), and a higher stimulated parotid flow rate with LC (0.64 and 0.44, respectively; p < 0.02) with a decrease in sodium and an increase in potassium, total proteins and IgA in patients with cirrhosis. In the LC group, caries were found to affect more teeth in those patients with alcohol-induced LC than in those with liver disease of other causes (3.9 and 1.7, respectively; p < 0.05), but in contrast, no differences were found in the saliva flow rate and the concentration in both UWS and SWS of sodium, potassium, total proteins and IgA. Finally, no relationship was observed between the dental status and functional hepatic tests. CONCLUSIONS: CH patients showed a worse dental status, a higher SPS rate and some electrolytes and proteins alterations.     

Molecular diversity and relationship within Lactococcus lactis, as revealed by randomly amplified polymorphic DNA (RAPD)

Lactococcus lactis strains are widely used in industrial dairy fermentations. Conventional phenotypic tests have been used for years to classify members of this species into two subspecies, lactis and cremoris, and play a key role in the choice of strains to be used in particular cheese fermentations. DNA hybridisation techniques have also been used for strain classification, giving rise to two genome homology groups. However, results showed discrepancies between the two methods of classification. We applied the randomly amplified polymorphic DNA fingerprinting (RAPD) technique to resolve previous contradictions in lactococcal classifications. Unlike usual RAPD methods, we use three primers to classify 113 strains and integrate the resulting information by a digitised programme used for this purpose. Our analysis revealed three major RAPD groups, designated G1, G2 and G3. G1 and G3 contain strains of the lactis subspecies, and G2 contains strains of the cremoris subspecies, as previously defined by phenotypic characteristics. Moreover, group G1 corresponds to one genome homology group, and groups G2 and G3 correspond to the second one. The taxonomic structure within L. lactis is therefore unusual: two distinct genetic groups of strains show indistinguishable phenotypes, while conversely, two phenotypically distinct groups are genetically homologous. We hypothesize that a subfamily of the subsp. lactis group gave rise to the cremoris subspecies.     

Random amplified polymorphic DNA (RAPD) typing of necrophageous insects (Diptera, Coleoptera) in criminal forensic studies: validation and use in practice

There is a heightened threat of biological weapons being used for biological warfare or bioterrorism. Many of the microorganisms and toxins that may be used as such biological weapons can easily be acquired and mass produced. Dissemination of aerosols of these biological agents can produce mass casualties. If used by a terrorist they may overwhelm our current public health system. Some biological agents, such as Bacillus anthracis (anthrax) and botulinum toxin, are considered far more likely than others to be used as biological weapons; smallpox virus was apparently produced in mass quantities by the former Soviet Union and may also be a serious threat. The release of such agents could go undetected for several hours or days and would be followed by mass illnesses and a first line of response by the public health community. Rapid epidemiological investigation to identify the nature of the disease outbreak would be critical for limiting casualties. For many, but not all, biological agents there are medical treatments that can greatly lower the mortality rate. There currently are, however, insufficient supplies of medicinals and trained personnel to cope with a massive bioterrorist or biological warfare use of biological weapons. Increasing our preparedness is critical.     

Sequencing of cDNA clones from the genetic map of tomato (Lycopersicon esculentum)

AIM: To study the impact of both audiovisual information and nurse-training on the use of budesonide Turbuhaler in preschool children who had never used a dry powder inhaler. DESIGN: A single-blind, randomized, parallel-group trial studied 72 children aged 3-5 y. All children and their parents were shown an instructional video and given written instruction. After this, peak inspiratory flow (PIF1) through Turbuhaler was measured. Children in group A (n = 36) then received individual training by a nurse while those in Group B (n = 36) did not and PIF2 was measured. Afterwards, Group B received similar individual training while Group A received no additional training, and PIF3 was measured. Group A was given a placebo Turbuhaler and encouraged to practice at home. Two weeks later, both groups returned to the clinic where PIF4 was measured. RESULTS: The number of children who were able to correctly perform PIF1, PIF2 and PIF4 in Group A was 27, 34 and 36, respectively. The corresponding numbers for Group B were 30, 29, and 29. No effect of training was seen in 3-y-old children. Individual training by a nurse was associated with a statistically significant increase in PIF2 (10 l/min; p = 0.014). Moreover, 2 weeks of home training was associated with an additional increase in PIF of 8 l/min compared with Group B (p < 0.015). After individual instruction and home training, mean PIF in children aged 4 and 5 was 56 (42-72) and 55 (41-66) l/min, respectively. CONCLUSION: After individual instruction and training at home, the vast majority of children aged 4 and 5 y can use Turbuhaler correctly. Audiovisual information and individual instruction is not sufficient in the majority of these children. Few 3-y-old children can learn the correct use of Turbuhaler.     

Digitalis-like and vasoconstrictor effects of endogenous digoxin-like factor(s) from the venom of Bufo marinus toad

Digitalis glycoside-like properties of the Bufo marinus toad crude venom and one of its constituents, bufalin, were studied in various assay systems. In concentrations 0.3-30 micrograms/ml crude venom increased the contractility of isolated electrically driven rat atria, constricted rat aortic rings, inhibited ouabain-sensitive Na+,K(+)-ATPase in rat erythrocytes and the Na+,K(+)-pump in rat aorta, and cross-reacted with antidigoxin antibody from the dissociation enhanced lanthanide fluoroimmunoassay (DELFIA). These effects were unaffected by adrenoceptor blockers and the 5-HT antagonist, deseril, but were blocked by antidigoxin antibody. Bufalin (10-30 microM) increased myocardial contractility and inhibited Na+,K(+)-ATPase in rat erythrocytes similarly to crude Bufo marinus venom. In rat aorta bufalin showed weak and delayed vasoconstrictor activity which was antagonized by 2 microM phentolamine, and had a biphasic effect on the Na+,K(+)-pump; 0.5-1.0 microM bufalin stimulated the pump, while higher concentrations inhibited its activity. Although the effects of bufalin were blocked by antidigoxin antibody, bufalin showed very low digoxin-like immunoreactivity in the DELFIA. These observations suggest that, in addition to bufalin, Bufo marinus venom contains at least one more digitalis-like steroid with significant intrinsic vasoconstrictor activity which, unlike bufalin, constricts the blood vessels acting directly via inhibition of the sodium pump in the vascular smooth muscle membrane.     

Differential display with carboxy-X-rhodamine-labeled primers and the selection of differentially amplified cDNA fragments without cloning

Pregnancy block, whereby recently mated female mice abort their pregnancies when exposed to novel (strange) males, was studied in house mice (Mus domesticus) differing in t-complex genotype; t-mutations are deleterious and +/t females avoid +/t males as mates. The results of Experiment 1, in which the genotype of the female, stud male, and strange male was systematically varied, showed that pregnancy block was most frequent when the strange male was +/+. Because this effect was not enhanced among +/t females when stud males were +/t, the results cannot clearly be explained by the hypothesis that pregnancy block is a manifestation of mate choice. Moreover, the "strange male" effect in Experiment 1 is unlikely to be a female response correlated with the risk of male infanticide, as +/+ and +/t males did not differ in their infanticidal tendencies (Experiments 2 & 3). Alternative hypotheses, including a modified version of the mate choice hypothesis, are discussed.     

Sequencing and characterization of trypsin modulating oostatic factor (TMOF) from the ovaries of the grey fleshfly, Neobellieria (Sarcophaga) bullata

Injection of crude extracts of late vitellogenic ovaries into staged females of the grey fleshfly Neobellieria (Sarcophaga) bullata inhibited oocyte development and biosynthesis of trypsin-like enzymes in the gut. Trypsin synthesis in N. bullata is cyclic and is correlated with egg development, which is discontinuous. A trypsin modulating oostatic factor (Neb-TMOF) was purified from 10,000 vitellogenic ovaries and sequenced by mass spectrometry. Neb-TMOF is a hexapeptide (NH2-NPTNLH-COOH). Injection of the hormone at physiological concentrations (10(-9) M), inhibited trypsin-like synthesis by the midgut of liver-fed female flies, and caused a reduction of the vitellogenin concentration in the hemolymph and of oocyte growth. The role of Neb-TMOF in controlling egg development and the physiological similarities with Aedes-TMOF are discussed.     

Arbitrary (RAPD) primer sequences used in insect studies

The corneal quilt is a simple, safe, inexpensive, and effective device designed to decrease the risk of intraoperative retinal phototoxicity from the operating microscope. Retinal injury is a well documented hazard, especially during prolonged procedures. The corneal quilt can be easily used as an adjunct to other methods designed to protect the retina from excessive incident light. In this study, we fashioned an approximately 6- x 5-millimeter semi-transparent corneal occluder from the nonadhesive portion of a 3M Steridrape. We determined spectrophotometrically that the corneal quilt reduced the transmission not only of visible light, but of ultraviolet and blue wavelengths as well, the wavelengths known to be the most phototoxic to the retina. However, no data were obtained actually demonstrating the degree to which light is scattered after transmission through the corneal quilt.     

浅谈改良式序列间歇反应器(MSBR)工艺

介绍了SBR工艺的基本特点，并详细介绍了MSBR工艺的工作原理、工作特点和工艺参数及在工程中的应用。     

Phylogeography of Bufo marinus from its natural and introduced ranges

The marine toad, Bufo marinus, has a broad natural distribution extending from the south-west of the USA to southern Peru and the central Amazon. It was introduced to several localities in the Caribbean and Pacific Oceans to control sugar cane pests. We sequenced 468 bp of mitochondrial DNA (mtDNA) containing the ND3 gene, and flanking tRNA genes from toads spanning the broad natural and introduced ranges. Consistent with the known history of introductions and expected effects of serial bottlenecks, mtDNA within introduced populations in Hawaii and Australia was uniform and most closely related to samples from eastern Venezuela and French Guiana. However, mtDNA nucleotide diversity in the geographic region spanning the source areas is also relative low (0.18-0.46%) and the absence of variation in the introduced populations precludes quantitative assessment of the reduction in genetic diversity. Unexpectedly, there was a large phylogeographic break (5.4% sequence divergence) within the natural range separating populations east and west of the Venezuelan Andes. We hypothesize that the two major lineages of B. marinus were isolated by the uplift of the eastern Andean cordillera which was completed approximately 2.7 Ma. Another species of the marinus group, B. paracnemis, had mtDNA paraphyletic, with marinus, being nested within the eastern lineage. Thus, at least one speciation event within the marinus group postdates the split within marinus. These findings suggest that the taxonomy of B. marinus should be re-evaluated and that the search for pathogens to control Australian populations should be conducted in populations from both lineages in the natural range.     

The genome organization of the broad bean necrosis virus (BBNV)

In this study the effect of lipoprotein lipase (LPL) on the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CE) by hepatic cells was investigated. Human HDL3 (d 1.125-1.21 g/ml) was radiolabeled with 125I in the protein moiety and with 3H in the CE moiety. LPL was prepared from bovine milk. Human hepatocytes in primary culture and human Hep3B hepatoma cells were incubated in medium containing doubly radiolabeled HDL3 with or without LPL. Without LPL, apparent HDL3 particle uptake according to the lipid tracer (3H) was in excess of that due to the protein label (125I) indicating selective CE uptake from HDL3. Addition of LPL increased selective CE uptake up to 7-fold. This stimulation of HDL3 selective CE uptake was independent of the lipolytic activity of LPL as suggested by several experimental approaches. Cell surface heparan sulfate proteoglycan deficiency decreased the LPL-mediated increase in selective CE uptake suggesting an important role for these molecules. In low density lipoprotein (LDL) receptor- or LDL receptor-related protein-(LRP)-deficient cells, LPL increased selective CE uptake as it did in normal cells yielding no evidence that these receptors play a role in the LPL effect on selective CE uptake. In summary, lipoprotein lipase increases the selective uptake of high density lipoprotein-associated cholesteryl ester by hepatic cells in culture. This effect is dependent on cell surface heparan sulfate proteoglycans but independent of lipolysis and of endocytosis mediated by low density lipoprotein receptor-related or low density lipoprotein receptors.