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991.
Protocol for DNA extraction from potato tubers 总被引:3,自引:0,他引:3
E.?G.?WulffEmail author S.?Torres E.?Gonzales?Vigil 《Plant Molecular Biology Reporter》2002,20(2):187-187
A method to extract high-quality DNA from potato tubers was developed and tested on 3 wild potato species (Solanum raphanifolium, S. megistracrolobum, S. bukasovii) and on the tetraploid B3 bred population, (population number 393228, derived fromS. tuberosum subsp.tuberosum). The average yield of extracted DNA varied from 10–30 μg of DNA per gram of processed tissue. The DNA was pure and suitable
for ligation-mediated polymerase chain reaction (LM-PCR) amplification, producing clear, distinctive, and reproducible banding
patterns in polyacrylamide gels. 相似文献
992.
Anton?S.?CallawayEmail author Rita?Abranches Jeffery?Scroggs George?C.?Allen William?F.?Thompson 《Plant Molecular Biology Reporter》2002,20(3):265-277
Transgene copy number affects the level and stability of gene expression. Therefore, it is important to determine the copy
number of each transgenic line. Polymerase chain reaction (PCR) is widely employed to quantify amounts of target sequences.
Although PCR is not inherently quantitative, various means of overcoming this limitation have been devised. Recent real-time
PCR methods are rapid; however, they typically lack a suitable internal standard, limit the size of the target sequence, and
require expensive specialized equipment. Competitive PCR techniques avoid these problems, but traditional competitive methods
are time consuming. Here we apply mathematical modeling to create a rapid, simple, and inexpensive copy number determination
method that retains the robustness of competitive PCR. 相似文献
993.
994.
995.
Thompson CI Munford JW Buell EH Karry RJ Lee CT Morgan BL Radnovich AJ 《Canadian journal of physiology and pharmacology》2002,80(3):180-192
Two studies compared the glucose responses of 9-day-old rats given subcutaneous insulin, either continuously or via daily injection, for 10 days. In Experiment 1, implanted pellets released a total of 0, 1.9, or 5.7 U insulin/kg the first 24 h. Injected doses were larger, 0 or 8 U/kg. Injections caused no deaths, but insulin-releasing pellets caused high mortality within 24 h. Pups surviving the pellets were normoglycemic by treatment day 8. In Experiment 2, pups received 0.184 U of insulin daily, approximately 8 U/kg at 9 days, via either injection or osmotic minipump. All pups survived. Injected pups were hypoglycemic 2 h postinjection through treatment day 10, whereas pups with insulin minipumps were normoglycemic by day 5. Insulin injections, but not minipumps, lowered plasma triglycerides on day 10. To examine age differences in response to insulin, additional pups and adults received daily injections of 0 or 8 U/kg for 10 days. All survived. Insulin lowered plasma glucose more in pups than in adults and reduced triglycerides in pups but not in adults. The rapid development of normoglycemia in pups with insulin minipumps, compared with pups injected daily with the same dose, suggests that continuous early insulin may produce insulin resistance. 相似文献
996.
997.
Tör M Manning K King GJ Thompson AJ Jones GH Seymour GB Armstrong SJ 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2002,104(2-3):165-170
Cnr (Colourless non-ripening) is a dominant pleiotropic ripening mutation of tomato (Lycopersicon esculentum) which has previously been mapped to the proximal region of tomato chromosome 2. We describe the fine mapping of the Cnr locus using both linkage analysis and fluorescence in situ hybridisation (FISH). Restriction fragment length polymorphism
(RFLP)-, amplified restriction fragment polymorphism (AFLP)-, and cleaved amplified polymorphic sequence (CAPS)-based markers,
linked to the Cnr locus were mapped onto the long arm of chromosome 2. Detailed linkage analysis indicated that the Cnr locus was likely to lie further away from the top of the long arm than previously thought. This was confirmed by FISH, which
was applied to tomato pachytene chromosomes in order to gain an insight into the organisation of hetero- and euchromatin and
its relationship to the physical and genetic distances in the Cnr region. Three molecular markers linked to Cnr were unambiguously located by FISH to the long arm of chromosome 2 using individual BAC probes containing these single-copy
sequences. The physical order of the markers coincided with that established by genetic analysis. The two AFLP markers most-closely
linked to the Cnr locus were located in the euchromatic region 2.7-cM apart. The physical distance between these markers was measured on the
pachytene spreads and estimated to be approximately 900 kb, suggesting a bp:cM relationship in this region of chromosome 2
of about 330 kb/cM. This is less than half the average value of 750 kb/cM for the tomato genome. The relationship between
genetic and physical distances on chromosome 2 is discussed.
Received: 11 January 2001 / Accepted: 30 April 2001 相似文献
998.
In the present work we have modelled and optimized the reaction mechanism of the triose phosphate isomerase (TIM) enzyme (E.C.
5.3.1.1). For this purpose we have used an approach that combines the S-system representation within the power law formalism
and linear programming techniques. By this means we have explored those rate constants whose alterations are likely to improve
the catalytic efficiency of the enzyme and investigated the available room for optimization in different metabolic conditions.
The role and plausibility of the different types of mutations on the evolution of this enzyme have also been considered.
Steady state sensitivity analysis was carried out and a new set of aggregated logarithmic gains was defined in order to quantify
the responses of the system to changes in groups of rate constants that could be explained in terms of mutations affecting
the catalytic properties of the enzyme. Evaluation of these logarithmic gains at different levels of saturation and disequilibrium
ratios enabled us to reach conclusions about the meaning and role of the diffusion limitation terms.
The catalytic efficiency of the monoenzymatic system was optimized through changes in the kinetic rate constants within different
sets of restrictions ranging from thermodynamic or kinetic to evolutionary ones. Results showed that, at very different conditions,
there is still room for improvement in the TIM enzyme. Thus, in a wide range of metabolically significant values of the disequilibrium
ratio there is a minimal variation in the optimal profile that yields 2.1 times the velocity of the basal states. Though most
of this increase is accounted for by the increase of the second order constants (that could have already reached a theoretical
maximum) significant increases (10%) in catalytic efficiencies are obtained by changes of the internal steps only. Besides
these new findings our optimization approach has been able to reproduce results obtained with other approaches. 相似文献
999.
The activities of four immobilized lipases for glycerolysis of a commercially available fish oil (TG500) rich in eicosapentaenoic residues (>58%, w/w) have been characterized in solvent-free systems. The effects of the mole ratio of TG500 to glycerol and temperature have been investigated. The highest conversion was obtained at 60°C with a Candida antarctica fraction B lipase (Chirazyme L-2) and a mole ratio of TG500 (based on fatty acid equivalents) to glycerol of 1.5 to 1. 相似文献
1000.
Two monoclinic (P2(1)) crystal forms of human serum amyloid P component (SAP) in complex with the 4,6-pyruvate acetal of beta-D-galactose (MObetaDG) were prepared. Structure analysis by molecular replacement and refinement at 2.2A resolution revealed that crystal form 1 (a=95.76A, b=70.53A, c=103.41A, beta=96.80 degrees) contained a pentamer in the asymmetric unit with a structure very similar to that of the published search model. The mode of ligand co-ordination was also similar except that four of the five subunits showed bound ligand with an additional H-bond between O1 of the galactose and the side-chain of Lys79. One sub-unit showed no bound ligand and a vacant calcium site close to a crystal contact. The 2.6A resolution structure of crystal form 2 (a=118.60A, b=109.10A, c=120.80A and beta=95.16 degrees ) showed ten sub-units in the asymmetric unit, all with two bound calcium ions and ligand. The most extensive protein-protein interactions between pentamers describe an AB face-to-face interaction involving 15 ion pairs that sandwiches five molecules of bound MObetaDG at the interface. 相似文献