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To increase sensitivity and to improve normalization of RNA levels in Northern blot analysis, a comparatively inexpensive optical scanner was utilized for digitizing photonegatives of ethidium bromide stained gels and autoradiograms. The optical scanner captures the image with a maximum resolution of 300 dots per inch by assigning one of 256 gray levels (8-bit) to each dot in the image. With the use of the public domain NIH Image program (requires a Macintosh II and an 8-bit video card), gel or autoradiogram bands in the digitized image are selected and their average gray scale density measured. We found that the digitized image of a photonegative of a TAE (Tris-acetate/EDTA) agarose gel, loaded incrementally with 50-1500 ng total RNA, produced a linear response over a 4-fold range down to 100 ng (R2 greater than 0.950). In utilizing "quantification" gels like this, RNA samples that are too dilute or too small for traditional spectrophotometric techniques can be normalized and loaded uniformly onto subsequent Northern gels. Results from autoradiogram scans demonstrate highly linear gray scale responses over a 4-fold range of total RNA (R2 greater than 0.950) that are reproducible with different blots and probe types (e.g., riboprobe, cDNA and oligonucleotide). In addition, we describe a normalization technique using a 30-mer oligonucleotide probe for rat 28S ribosomal RNA as a measure of total RNA loaded per gel lane. Altogether, this scanning, ribosomal RNA normalization system allows the measurement of relative changes between 20% and 400% using standard autoradiographic methods. 相似文献
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Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews
(genus Sorex) for the region between the tRNA(Pro) and the conserved
sequence block-F revealed variable numbers of 79-bp tandem repeats. These
repeats were found in all 19 individuals sequenced, representing three
subspecies and one closely related species of the masked shrew group (Sorex
cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an
outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an
adjacent 76-bp imperfect copy of the tandem repeats. One individual was
heteroplasmic for length variants consisting of five and seven copies of
the 79-bp tandem repeat. The sequence of the repeats is conducive to the
formation of secondary structure. A termination-associated sequence is
present in each of the repeats and in a unique sequence region 5' to the
tandem array as well. Mean genetic distance between the masked shrew taxa
and the pygmy shrew was calculated separately for the unique sequence
region, one of the tandem repeats, the imperfect repeat, and these three
regions combined. The unique sequence region evolved more rapidly than the
tandem repeats or the imperfect repeat. The small genetic distance between
pairs of tandem repeats within an individual is consistent with a model of
concerted evolution. Repeats are apparently duplicated and lost at a high
rate, which tends to homogenize the tandem array. The rate of D- loop
sequence divergence between the masked and pygmy shrews is estimated to be
15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid
sequence evolution in shrews may be due either to their high metabolic rate
and short generation time or to the presence of variable numbers of tandem
repeats.
相似文献
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大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育 总被引:1,自引:0,他引:1
本实验用免疫组织化学ABC法研究了大鼠胼胝体内神经肽Y免疫反应阳性(NPY-IR)纤维的生后发育。结果发现,许多NPY-IR纤维在大鼠出生时便存在于胼胝体内。NPY-IR胼胝体纤维的密度在生后1周内继续逐渐增高,在第2周内达到最高峰。之后,NPY-IR胼胝体纤维的密度逐渐下降,至第3周末时接近成年时的水平,即仅有少量NPY-IR纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多NPY-IR胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。 相似文献
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N D Griggs C H Pontzer M A Jarpe H M Johnson 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(8):2516-2521
Multiple binding sites on the staphylococcal enterotoxin A (SEA) molecule which interact with class II MHC Ag have been suggested by previous studies comparing SEA binding with that of another superantigen, toxic shock syndrome toxin-1. Using the synthetic peptide approach we have identified multiple regions of the SEA molecule which are responsible for binding to HLA Ag on Raji cells. Overlapping peptides were synthesized corresponding to the complete amino acid sequence of SEA: SEA(1-45), SEA(39-66), SEA(62-86), SEA(83-104), SEA(102-124), SEA(121-149), SEA(146-173), SEA(166-193), SEA(187-217), and SEA(211-233). Like the native SEA molecule, all of the peptides exhibited relatively high beta-sheet and low alpha-helical structure as determined by circular dichroism spectroscopy. A direct competition assay was employed with peptide blockage of 125I-SEA binding to MHC Ag. SEA(1-45), SEA(39-66), SEA(62-86), and SEA(121-149) but none of the other peptides blocked binding to Raji cells. The relative potency of the peptides in blocking SEA binding was determined with SEA(39-66) much greater than SEA(1-45) = SEA(62-86) = SEA(121-149). Peptide competition was seen at concentrations as low as 55 microM. Further, antibodies were produced to all of the peptides and tested for their ability to bind to SEA and inhibit SEA binding to HLA. Consistent with the direct inhibition of binding, antisera to SEA(1-45), SEA(39-66), and SEA(62-86) reduced the ability of SEA to bind Raji cells, whereas, antisera to the remaining peptides failed to block binding. The data suggest that the binding of the superantigen SEA to MHC molecules involves several N-terminal regions on SEA as well as an additional internal domain. This allows for the presence of multiple binding sites in an extended N-terminal region of the SEA molecule or a discontinuous binding epitope. 相似文献
8.
Nigel J. Kilby David L. Griggs Stephanie F. Berry 《Plant Cell, Tissue and Organ Culture》1992,28(3):275-280
Intra-population quinoline alkaloid profiles surveying quinine, quinidine, cinchonine and cinchonidine were determined for each of five populations of Cinchona ledgeriana grown as shoot-culture for 125 days. No significant difference in respect of mean alkaloid content between populations was detected. In contrast, there was considerable between-seedling variation in alkaloid content within each population. When nutrients were delivered to shoot-cultures in droplet form by means of an aerosol spray (as compared to the supply of nutrients direct from agar-or liquid-based reservoirs) alkaloid profile was greatly perturbed; most notable in this respect was a four-fold increase in the production of cinchonidine concomitant with a four-fold decrease in the production of cinchonine. These data are discussed with reference to the optimisation of quinoline alkaloid production by juvenile shoot-cultures of Cinchona ledgeriana. 相似文献
9.
J Webb K A Kirk W Niedermeier J H Griggs M E Turner T N James 《Bioinorganic chemistry》1976,5(3):261-266
The pattern recognition procedure of discriminant analysis has been used to characterize the trace metal profiles created by the concentrations of 8 trace metals in 15 anatomic sites of beef heart tissue. Metals analyzed were copper, tin, lead, molybdenum, strontium, cesium, barium, and aluminum. Anatomic sites sampled included main pulmonary artery, aorta, mitral and tricuspid valves, left and right coronary arteries, os cordis, right atrium, left atrial appendage, crista supraventricularis, left bundle branch, free wall of the right and left ventricles, interventricular septum, and papillary muscle of the left ventricle. The striking features of the data were: (1) All specimens of the mitral valve, tricuspid valve, and os cordis were ambiguously described by their trace metal profiles; (2) the four blood vessels constituted two groups of two tissues each (aorta, main pulmonary artery; left and right coronary arteries); (3) tissues derived from ordinary and specialized myocardium were quite different from blood vessels, heart valves and os cordis. Using these profiles, 85% of the specimens analyzed were correctly classified by discriminant analysis with respect to their anatomic origin. 相似文献
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