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21.
Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction 总被引:83,自引:0,他引:83
Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences. 相似文献
22.
Summary To extend the available information on the significance of the interactions between glycolytic enzymes and the actin component of the cellular ultrastructure, investigations into the compositional characteristics of the actin binding site on one of the major glycolytic enzymes, aldolase, have been undertaken. As the electrostatic nature of the association has been previously reported indicative of a cationic region on the enzyme involved in the binding, these studies have investigated the possibility of the involvement of histidine residues in this binding region. By the use of the histidine specific reagent, diethylpyrocarbonate, we have been able to establish a difference in nature of an actin binding domain and the active site domain which does contain an essential histidine. The results have been discussed in relation to the significance of this finding with respect to the binding of aldolase to subcellular structure. 相似文献
23.
D R Collins T J Knott R J Pease L M Powell S C Wallis S Robertson C R Pullinger R W Milne Y L Marcel S E Humphries 《Nucleic acids research》1988,16(17):8361-8375
Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins. 相似文献
24.
Summary Of ten, mainly oleaginous, yeasts examined for the ability to use glucose and xylose simultaneously, only one,Candida curvata D, was found which could do so. This yeast was examined further in a single-stage chemostat wherein it produced similar biomass yields, lipid contents and fatty acids on glucose plus xylose mixed in varying proportions. This oleaginous yeast would therefore be capable of growing on hydrolysed wood and straw wastes as a potential source of single cell oil. 相似文献
25.
Stephen P. Baker Kelly M. Standifer Christopher J. Kalberg Josef Pitha Colin Sumners 《Journal of neurochemistry》1988,50(4):1044-1052
The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium-dependent, high-affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose- and time-dependent manner (IC50, 6.5 microM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN-induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19-fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmax or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible beta-antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synaptosomes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium-dependent uptake of [3H]NE (IC50, 5.6 microM) with no effect on sodium-independent uptake. After pretreatment of cultures with 30 microM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24-h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system. 相似文献
26.
Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato
F. Stuart Chapin III David T. Clarkson John R. Lenton Colin H. S. Walter 《Planta》1988,173(3):340-351
The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO
3
-
absorption increased two-to five fold within 2 d of being deprived of NO
3
-
supply. Nitrogen-starved barley roots continued to maintain a high potential for NO
3
-
absorption, whereas NO
3
-
absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO
3
-
solution, roots of both species transported more NO
3
-
and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO
3
-
stores took precedence over NO
3
-
transport to the xylem. Consequently, as N stress became more severe, transport of NO
3
-
and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L
p) and exudate volume (J
v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L
p and J
v were increased, but NO
3
-
absorption and NO
3
-
flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO
3
-
absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO
3
-
absorption and transport and L
p had been observed (wild-type tomato); (2) changes in NO
3
-
absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO
3
-
transport to those observed in the wild type.Abbreviation and symbols ABA
abscisic acid
- Jv
exudate volume
- Lp
root hydraulic conductance 相似文献
27.
Growth response of barley and tomato to nitrogen stress and its control by abscisic acid,water relations and photosynthesis 总被引:14,自引:0,他引:14
Barley (Hordeum vulgare L.) and tomato Lycopersicon esculentum Mill.) were grown hydroponically and examined 2, 5, and 10 d after being deprived of nitrogen (N) supply. Leaf elongation rate declined in both species in response to N stress before there was any reduction in rate of dryweight accumulation. Changes in water transport to the shoot could not explain reduced leaf elongation in tomato because leaf water content and water potential were unaffected by N stress at the time leaf elongation began to decline. Tomato maintained its shoot water status in N-stressed plants, despite reduced water absorption per gram root, because the decline in root hydraulic conductance with N stress was matched by a decline in stomatal conductance. In barley the decline in leaf elongation coincided with a small (8%) decline in water content per unit area of young leaves; this decline occurred because root hydraulic conductance was reduced more strongly by N stress than was stomatal conductance. Nitrogen stress caused a rapid decline in tissue NO
3
-
pools and in NO
3
-
flux to the xylem, particularly in tomato which had smaller tissue NO
3
-
reserves. Even in barley, tissue NO
3
-
reserves were too small and were mobilized too slowly (60% in 2 d) to support maximal growth for more than a few hours. Organic N mobilized from old leaves provided an additional N source to support continued growth of N-stressed plants. Abscisic acid (ABA) levels increased in leaves of both species within 2 d in response to N stress. Addition of ABA to roots caused an increase in volume of xylem exudate but had no effect upon NO
3
-
flux to the xylem. After leaf-elongation rate had been reduced by N stress, photosynthesis declined in both barley and tomato. This decline was associated with increased leaf ABA content, reduced stomatal conductance and a decrease in organic N content. We suggest that N stress reduces growth by several mechanisms operating on different time scales: (1) increased leaf ABA content causing reduced cell-wall extensibility and leaf elongation and (2) a more gradual decline in photosynthesis caused by ABA-induced stomatal closure and by a decrease in leaf organic N.Abbreviation and symbols ABA
abscisic acid
- ci
leaf internal CO2 concentration
- Lp
root hydraulic conductance 相似文献
28.
NAD+-dependent propan-1-ol and propan-2-ol dehydrogenase activities were detected in cell-free extracts of Rhodococcus rhodochrous PNKb1 grown on propane and potential intermediates of propane oxidation. However, it was unclear whether this activity was mediated by one or more enzymes. The isolation of mutants unable to utilize propan-1-ol (alcA-) or propan-2-ol (alcB-) as sole carbon and energy sources demonstrated that these substrates are metabolized by different alcohol dehydrogenases. These mutants were also unable to utilize propane as a growth substrate indicating that both alcohols are intermediates of propane metabolism. Therefore, propane is metabolized by terminal and sub-terminal oxidation pathways. Westernblot analysis demonstrated that a previously purified NAD+-dependent propan-2-ol dehydrogenase (Ashraf and Murrell 1990) was only synthesized after growth on propane and sub-terminal oxidation intermediates (but not acetone), and not propan-1-ol or terminal oxidation intermediates. Therefore, our evidence suggest that another dehydrogenase is involved in the metabolism of propan-1-ol and this agrees with the isolation of the alcA- and alcB- phenotypes. The previously characterized NAD+-dependent propan-2-ol dehydrogenase from R. rhodochrous PNKb1 is highly conserved amongst members of the propane-utilizing Rhodococcus-Nocardia complex. 相似文献
29.
Donna Robbins Colin A. Chapman Richard W. Wrangham 《Primates; journal of primatology》1991,32(3):301-305
Gibbons and spider monkeys have similar diets, body size, and locomotor patterns. They are therefore expected to be subject
to similar socioecological rules. However their grouping patterns differ. Gibbons live in small stable groups, whereas spider
monkey form unstable sub-groups that vary from small to large during different seasons. If similar principles apply to the
two species, food abundance should vary more for spider monkeys than for gibbons; food density should be similar for the two
species when spider monkey sub-groups are the same size as gibbon groups; and the highest level of food abundance should be
higher for spider monkeys than for gibbons. These predictions are upheld for a comparison of particular populations ofHylobates muelleri andAteles geoffroyi. 相似文献
30.
Colin K. W. Watts Malcolm L. Handel Roger J. B. King Robert L. Sutherland 《The Journal of steroid biochemistry and molecular biology》1992,41(3-8):529-536
The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoRI, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance. 相似文献