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1.
RDCs for the 14 kDa protein hen egg-white lysozyme (HEWL) have been measured in eight different alignment media. The elongated
shape and strongly positively charged surface of HEWL appear to limit the protein to four main alignment orientations. Furthermore,
low levels of alignment and the protein’s interaction with some alignment media increases the experimental error. Together
with heterogeneity across the alignment media arising from constraints on temperature, pH and ionic strength for some alignment
media, these data are suitable for structure refinement, but not the extraction of dynamic parameters. For an analysis of
protein dynamics the data must be obtained with very low errors in at least three or five independent alignment media (depending
on the method used) and so far, such data have only been reported for three small 6–8 kDa proteins with identical folds: ubiquitin,
GB1 and GB3. Our results suggest that HEWL is likely to be representative of many other medium to large sized proteins commonly
studied by solution NMR. Comparisons with over 60 high-resolution crystal structures of HEWL reveal that the highest resolution
structures are not necessarily always the best models for the protein structure in solution. 相似文献
2.
In solution NMR spectroscopy the residual dipolar coupling (RDC) is invaluable in improving both the precision and accuracy
of NMR structures during their structural refinement. The RDC also provides a potential to determine protein structure de novo. These procedures are only effective when an accurate estimate of the alignment tensor has already been made. Here we present
a top–down approach, starting from the secondary structure elements and finishing at the residue level, for RDC data analysis
in order to obtain a better estimate of the alignment tensor. Using only the RDCs from N–H bonds of residues in α-helices
and CA–CO bonds in β-strands, we are able to determine the offset and the approximate amplitude of the RDC modulation-curve
for each secondary structure element, which are subsequently used as targets for global minimization. The alignment order
parameters and the orientation of the major principal axis of individual helix or strand, with respect to the alignment frame,
can be determined in each of the eight quadrants of a sphere. The following minimization against RDC of all residues within
the helix or strand segment can be carried out with fixed alignment order parameters to improve the accuracy of the orientation.
For a helical protein Bax, the three components A
xx
, A
yy
and A
zz
, of the alignment order can be determined with this method in average to within 2.3% deviation from the values calculated
with the available atomic coordinates. Similarly for β-sheet protein Ubiquitin they agree in average to within 8.5%. The larger
discrepancy in β-strand parameters comes from both the diversity of the β-sheet structure and the lower precision of CA–CO
RDCs. This top-down approach is a robust method for alignment tensor estimation and also holds a promise for providing a protein
topological fold using limited sets of RDCs. 相似文献
3.
Conserved noncoding elements (CNCs) are an abundant feature of vertebrate genomes. Some CNCs have been shown to act as cis-regulatory modules, but the function of most CNCs remains unclear. To study the evolution of CNCs, we have developed a statistical method called the “shared rates test” to identify CNCs that show significant variation in substitution rates across branches of a phylogenetic tree. We report an application of this method to alignments of 98,910 CNCs from the human, chimpanzee, dog, mouse, and rat genomes. We find that ~68% of CNCs evolve according to a null model where, for each CNC, a single parameter models the level of constraint acting throughout the phylogeny linking these five species. The remaining ~32% of CNCs show departures from the basic model including speed-ups and slow-downs on particular branches and occasionally multiple rate changes on different branches. We find that a subset of the significant CNCs have evolved significantly faster than the local neutral rate on a particular branch, providing strong evidence for adaptive evolution in these CNCs. The distribution of these signals on the phylogeny suggests that adaptive evolution of CNCs occurs in occasional short bursts of evolution. Our analyses suggest a large set of promising targets for future functional studies of adaptation. 相似文献
4.
Eletsky A Acton TB Xiao R Everett JK Montelione GT Szyperski T 《Journal of structural and functional genomics》2012,13(1):9-14
The protein family (Pfam) PF04536 is a broadly conserved domain family of unknown function (DUF477), with more than 1,350
members in prokaryotic and eukaryotic proteins. High-quality NMR structures of the N-terminal domain comprising residues 41–180
of the 684-residue protein CG2496 from Corynebacterium glutamicum and the N-terminal domain comprising residues 35–182 of the 435-residue protein PG0361 from Porphyromonas gingivalis both exhibit an α/β fold comprised of a four-stranded β-sheet, three α-helices packed against one side of the sheet, and
a fourth α-helix attached to the other side. In spite of low sequence similarity (18%) assessed by structure-based sequence
alignment, the two structures are globally quite similar. However, moderate structural differences are observed for the relative
orientation of two of the four helices. Comparison with known protein structures reveals that the α/β architecture of CG2496(41–180)
and PG0361(35–182) has previously not been characterized. Moreover, calculation of surface charge potential and identification
of surface clefts indicate that the two domains very likely have different functions. 相似文献
5.
Finding the dynamics of an entire macromolecule is a complex problem as the model-free parameter values are intricately linked
to the Brownian rotational diffusion of the molecule, mathematically through the autocorrelation function of the motion and
statistically through model selection. The solution to this problem was formulated using set theory as an element of the universal
set —the union of all model-free spaces (d’Auvergne EJ and Gooley PR (2007) Mol BioSyst 3(7), 483–494). The current procedure
commonly used to find the universal solution is to initially estimate the diffusion tensor parameters, to optimise the model-free
parameters of numerous models, and then to choose the best model via model selection. The global model is then optimised and
the procedure repeated until convergence. In this paper a new methodology is presented which takes a different approach to
this diffusion seeded model-free paradigm. Rather than starting with the diffusion tensor this iterative protocol begins by
optimising the model-free parameters in the absence of any global model parameters, selecting between all the model-free models,
and finally optimising the diffusion tensor. The new model-free optimisation protocol will be validated using synthetic data
from Schurr JM et al. (1994) J Magn Reson B 105(3), 211–224 and the relaxation data of the bacteriorhodopsin (1–36)BR fragment
from Orekhov VY (1999) J Biomol NMR 14(4), 345–356. To demonstrate the importance of this new procedure the NMR relaxation
data of the Olfactory Marker Protein (OMP) of Gitti R et al. (2005) Biochem 44(28), 9673–9679 is reanalysed. The result is
that the dynamics for certain secondary structural elements is very different from those originally reported.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
6.
James S. Craigie Shawna L. MacKinnon John A. Walter 《Journal of applied phycology》2008,20(5):665-671
Aqueous extracts of Ascophyllum nodosum and several other brown seaweeds are manufactured commercially and widely distributed for use on agricultural crops. The
increasingly regulated international trade in such products requires that they be standardized and defined to a degree not
previously required. We examined commercially available extracts using quantitative 1H NMR and principal components analysis (PCA) techniques. Extracts manufactured over a 4-year period using the same process
exhibited characteristic profiles that, on PCA, clustered as a discrete group distinct from the other commercial products
examined. In addition to recognizing extracts made from different seaweeds, analysis of the 1H spectra in the 0.35–4.70 ppm region allowed us to distinguish amongst extracts produced from the same algal species by different
manufacturers. This result established that the process used to make an extract is an important variable in defining its composition.
A comparison of the 1H NMR integrals for the regions 1.0–3.0 ppm and 3.0–4.38 ppm revealed small but significant changes in the A. nodosum spectra that we attribute to seasonal variation in gross composition of the harvested seaweed. Such changes are reflected
in the PCA scores plots and contribute to the scatter observed within the data point cluster observed for Acadian soluble
extracts when all data are pooled. Quantitative analysis using 1H NMR (qNMR) with a certified external standard (caffeine) showed a linear relationship with extract concentration over at
least an order of magnitude (2.5–33 mg/mL; R
2 > 0.97) for both spectral regions integrated. We conclude that qNMR can be used to profile (or “fingerprint”) commercial
seaweed extracts and to quantify the amount of extract present relative to a suitably chosen standard.
Issued as NRCC no. 42,652. 相似文献
7.
Jožica Dolenc John H. Missimer Michel O. Steinmetz Wilfred F. van Gunsteren 《Journal of biomolecular NMR》2010,47(3):221-235
The C-terminal trigger sequence is essential in the coiled-coil formation of GCN4-p1; its conformational properties are thus
of importance for understanding this process at the atomic level. A solution NMR model structure of a peptide, GCN4p16–31,
encompassing the GCN4-p1 trigger sequence was proposed a few years ago. Derived using a standard single-structure refinement
protocol based on 172 nuclear Overhauser effect (NOE) distance restraints, 14 hydrogen-bond and 11 ϕ torsional-angle restraints,
the resulting set of 20 NMR model structures exhibits regular α-helical structure. However, the set slightly violates some
measured NOE bounds and does not reproduce all 15 measured 3J(HN-HCα)-coupling constants, indicating that different conformers of GCN4p16–31 might be present in solution. With the aim to resolve
structures compatible with all NOE upper distance bounds and 3J-coupling constants, we executed several structure refinement protocols employing unrestrained and restrained molecular dynamics
(MD) simulations with two force fields. We find that only configurational ensembles obtained by applying simultaneously time-averaged
NOE distance and 3J-coupling constant restraining with either force field reproduce all the experimental data. Additionally, analyses of the
simulated ensembles show that the conformational variability of GCN4p16–31 in solution admitted by the available set of 187
measured NMR data is larger than represented by the set of the NMR model structures. The conformations of GCN4p16–31 in solution
differ in the orientation not only of the side-chains but also of the backbone. The inconsistencies between the NMR model
structures and the measured NMR data are due to the neglect of averaging effects and the inclusion of hydrogen-bond and torsional-angle
restraints that have little basis in the primary, i.e. measured NMR data. 相似文献
8.
Michael S. Waterman 《Bulletin of mathematical biology》1994,56(4):743-767
Recently algorithms for parametric alignment (Watermanet al., 1992,Natl Acad. Sci. USA
89, 6090–6093; Gusfieldet al., 1992,Proceedings of the Third Annual ACM-SIAM Discrete Algorithms) find optimal scores for all penalty parameters, both for global and local sequence alignment. This paper reviews those techniques.
Then in the main part of this paper dynamic programming methods are used to compute ensemble alignment, finding all alignment
scores for all parameters. Both global and local ensemble alignments are studied, and parametric alignment is used to compute
near optimal ensemble alignments. 相似文献
9.
This paper investigates the cyclical behaviour of the wine production in Douro region during the period 1932–2008. In general,
wine production is characterised by large fluctuations which are composed of short-term and/or long-term cycles. The aim of
this paper is twofold: firstly, we decompose the wine production's variance in order to find the dominating production cycles,
i.e we try to explain whether wine production follows more long-term or short-term cycles. In the next step, we try to explain
those cycles using a dependent variable, namely the medium spring temperature (Tm_Sp) for the period 1967–2008. We estimated
a Time-Varying Autoregressive Model, which could explain 75% of the production that is characterised by 4.8- and 2.5-year
cycles. We use the Short Time Fourier Transform to decompose the link between wine production and temperature. When the temperature
was incorporated, the R
2 increased and the Akaike criterion value was lower. Hence, Tm_Sp causes a large amount of these cycles and the wine production
variation reflects this relationship. In addition to an upward trend, there is a clearly identifiable cycle around the long-term
trend in production. We also show how much of the production cycle and what cycle in particular is explained by the Tm_Sp.
There is a stable but not constant link between production and the Tm_Sp. In particular, the temperature is responsible for
5.2- and 2.4-year cycles which has been happening since the 1980s. The Tm_Sp can also be used as an indicator for the 4.8-
and 2.5-year cycles of production. The developed model suggests that stationarity is a questionable assumption, and this means
that historical distributions of wine production are going to need dynamic updating. 相似文献
10.
Svenja Stolzenburg Michael B. Lauridsen Henrik Toft Pierre A. Zalloua Dorrit Baunsgaard 《Metabolomics : Official journal of the Metabolomic Society》2011,7(2):270-277
NMR based metabolic profiling of blood samples in epidemiological studies can be used for molecular phenotyping and biomarker
discovery. Often metabolic changes in blood are more subtle and demand a high quality spectrum especially when looking at
low molecular weight compounds. In order to improve 1H NMR spectroscopic data we compared different serum sample preparation methods. Application of phosphate buffer reduces chemical
shift variation, enhances resolution of signal multiplicity, facilitates visual inspection of NMR spectra and annotation of
signals compared to traditionally used saline. For analysis of low molecular weight compounds we found that standard 1D spectra
of ultrafiltrated serum samples show enhanced spectral quality of small metabolites as compared to transverse relaxation edited
spectra (also called Carr–Purcell–Meiboom–Gill, CPMG) spectra of unfiltered serum samples due to improved signal-to-noise
ratio. Thus, NMR signals attributable to different amino acids and other small metabolites could readily be detected in spectra
of ultrafiltrated serum, but remained invisible in the corresponding CPMG spectra. An OPLS model of fasting blood glucose
showed an increase of Q2 when using spectra from ultrafiltrated serum (Q2 = 0.261) compared to using CPMG spectra (Q2 = 0.173). Similar results were observed for OPLS models of BMI (Q2 = 0.253 and Q2 = 0.216, respectively). Furthermore, a reduction in model dimensionality was observed when using ultrafiltrated serum data.
In conclusion we recommend sample preparation of serum samples in phosphate buffer instead of saline. Ultrafiltration of serum
samples prior to NMR analysis is beneficial especially for low concentrated small metabolites. 相似文献
11.
In the neural superposition eye of a dipteran fly every ommatidium has eight photoreceptors, each associated with a rhabdomere, two central and six peripheral, which altogether result in seven functional light guides. Groups of eight rhabdomeres in neighboring ommatidia have largely overlapping fields of view. Based on the hypothesis that the light signals collected by these rhabdomeres can be used individually, we investigated the feasibility of estimating 3D scene information. According to Pick (Biol Cybern 26:215–224, 1977) the visual axes of these rhabdomeres are not parallel, but converge to a point 3–6 mm in front of the cornea. Such a structure theoretically could estimate depth in a very simple way by assuming that locally the image intensity is well approximated by a linear function of the spatial coordinates. Using the measurements of Pick (Biol Cybern 26:215–224, 1977) we performed simulation experiments to find whether this is practically possible. Our results indicate that depth estimation at small distances (up to about 1.5–2 cm) is reasonably accurate. This would allow the insect to obtain at least an ordinal spatial layout of its operational space when walking. 相似文献
12.
Background
The performance of alignment programs is traditionally tested on sets of protein sequences, of which a reference alignment is known. Conclusions drawn from such protein benchmarks do not necessarily hold for the RNA alignment problem, as was demonstrated in the first RNA alignment benchmark published so far. For example, the twilight zone – the similarity range where alignment quality drops drastically – starts at 60 % for RNAs in comparison to 20 % for proteins. In this study we enhance the previous benchmark. 相似文献13.
Dithiothreitol (CH2SHCHOHCHOHCH2SH), under neutral conditions in aqueous medium, reacts readily and reversibly with vanadate to form longlived complexes.
The ligand, vanadium and proton stoichiometries were established from concentration and pH studies. The two predominant products
each contained two vanadium(V) nuclei and one dithiothreitol and carried an overall doubly negative charge. The equilibrium
shifted toward a triply negative charge with increase in pH through the pK
a range of the products. The 51V NMR spectra clearly showed two resonances for each product (–352 and –362 ppm for one and –399 and –526 ppm for the other),
thus establishing there are chemical differences in the coordination about each vanadium. A coordination scheme was proposed
for each product. The common motif proposed was the presence of a cyclic [VO]2 core as the source of a strong stabilizing interaction leading to the very favourable formation constants (overall about
107 at pH 7). The coordination shell about the individual vanadiums each contained one sulfur in the one product and one sulfur
about one vanadium and only oxygen about the other vanadium in the second product. Under neutral conditions the reduction
of V(V) to V(IV) requires in the order of 90 min. However, if hydrogen peroxide, in greater than a 2 : 1 molar ratio over
dithiothreitol, is included in the reaction medium, all the dithiothreitol is rapidly oxidized, and peroxovanadium(V) complexes
are observed. Addition of excess dithiothreitol regenerates the dithiothreitol/vanadate complexes.
Received: 2 May 1997 / Accepted: 2 July 1997 相似文献
14.
An NMR investigation of proteins with known X-ray structures is of interest in a number of endeavors. Performing these studies
through nuclear magnetic resonance (NMR) requires the costly step of resonance assignment. The prevalent assignment strategy
does not make use of existing structural information and requires uniform isotope labeling. Here we present a rapid and cost-effective
method of assigning NMR data to an existing structure—either an X-ray or computationally modeled structure. The presented
method, Exhaustively Permuted Assignment of RDCs (EPAR), utilizes unassigned residual dipolar coupling (RDC) data that can
easily be obtained by NMR spectroscopy. The algorithm uses only the backbone N–H RDCs from multiple alignment media along
with the amino acid type of the RDCs. It is inspired by previous work from Zweckstetter and provides several extensions. We
present results on 13 synthetic and experimental datasets from 8 different structures, including two homodimers. Using just
two alignment media, EPAR achieves an average assignment accuracy greater than 80%. With three media, the average accuracy
is higher than 94%. The algorithm also outputs a prediction of the assignment accuracy, which has a correlation of 0.77 to
the true accuracy. This prediction score can be used to establish the needed confidence in assignment accuracy. 相似文献
15.
S. Thangminlal Vaiphei Yuefeng Tang Gaetano T. Montelione Masayori Inouye 《Molecular biotechnology》2011,47(3):205-210
Gram-negative bacteria consist of two independent membranes, the inner cytoplasmic membrane and the outer membrane. The outer
membrane contains a number of β-barrel proteins such as OmpF, OmpC, OmpA, and OmpX. In this article, we explored to use the
condensed Single Protein Production (cSPP) system for isotope labelling of OmpA and OmpX for NMR structural study, both of
which are known to consist of eight β-strands forming a barrel in the outer membrane. Using a deletion strain lacking all
major outer membrane proteins, both OmpA and OmpX were expressed well in a 20-fold cSPP system. We demonstrated that outer
membrane fractions prepared from the cSPP system in M9 medium containing 15N–NH4Cl can be directly used for NMR structural study of the outer mebrane proteins without any further purification to get excellent
[1H–15N]-TROSY spectra. This method would be quite valuable for the study of pure proteins in their native membrane environment
without the need of purification and reconstitution. 相似文献
16.
Aisenbrey C Bertani P Henklein P Bechinger B 《European biophysics journal : EBJ》2007,36(4-5):451-460
Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to
understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy
on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide
bonds and domains relative to the bilayer normal. Using this approach the 15N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the 2H solid-state NMR spectra acquired from peptides labelled with 3,3,3-2H3-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium
NMR line shapes are highly sensitive to small variations in the alignment of the Cα–Cβ bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to
the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field
direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state
of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic
helices. Thereafter investigations of the viral channel peptides Vpu1–27 and Influenza A M222–46 are shown. Whereas the 15N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate
considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation
times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and
oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously
been performed.
Dedicated to Prof. K. Arnold on the occasion of his 65th birthday. 相似文献
17.
Background
Genomic sequence data cannot be fully appreciated in isolation. Comparative genomics – the practice of comparing genomic sequences from different species – plays an increasingly important role in understanding the genotypic differences between species that result in phenotypic differences as well as in revealing patterns of evolutionary relationships. One of the major challenges in comparative genomics is producing a high-quality alignment between two or more related genomic sequences. In recent years, a number of tools have been developed for aligning large genomic sequences. Most utilize heuristic strategies to identify a series of strong sequence similarities, which are then used as anchors to align the regions between the anchor points. The resulting alignment is globally correct, but in many cases is suboptimal locally. We describe a new program, GenAlignRefine, which improves the overall quality of global multiple alignments by using a genetic algorithm to improve local regions of alignment. Regions of low quality are identified, realigned using the program T-Coffee, and then refined using a genetic algorithm. Because a better COFFEE (Consistency based Objective Function For alignmEnt Evaluation) score generally reflects greater alignment quality, the algorithm searches for an alignment that yields a better COFFEE score. To improve the intrinsic slowness of the genetic algorithm, GenAlignRefine was implemented as a parallel, cluster-based program. 相似文献18.
Effects of elevated temperature on bacterial community structure and function in bioreactors treating a synthetic wastewater 总被引:1,自引:0,他引:1
T M LaPara A Konopka C H Nakatsu J E Alleman 《Journal of industrial microbiology & biotechnology》2000,24(2):140-145
The impact of elevated temperature on bacterial community structure and function during aerobic biological wastewater treatment
was investigated. Continuous cultures, fed a complex growth medium containing gelatin and α-lactose as the principal carbon
and energy sources, supported mixed bacterial consortia at temperatures ranging from 25–65°C. These temperature- and substrate-acclimated
organisms were then used as inocula for batch growth experiments in which the kinetics of microbial growth and substrate utilization,
efficiency of substrate removal, and mechanism of substrate removal were compared as functions of temperature. Bacterial community
analysis by denaturing gradient gel electrophoresis (DGGE) revealed that distinct bacterial consortia were supported at each
temperature. The efficiency of substrate removal declined at elevated temperatures. Maximum specific growth rates and the
growth yield increased with temperature from 25–45°C, but then decreased with further elevations in temperature. Thus, maximum
specific substrate utilization rates did not vary significantly over the 40°C temperature range (0.64 ± 0.04 mg COD mg−1 dry cell mass h−1). A comparison of the degradation of the protein and carbohydrate portions of the feed medium revealed a lag in α-lactose
uptake at 55°C, whereas both components were utilized simultaneously at 25°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 140–145.
Received 09 August 1999/ Accepted in revised form 12 November 1999 相似文献
19.
Koprowski M Przybylak R Zielski A Pospieszyńska A 《International journal of biometeorology》2012,56(1):1-10
Scots pine (Pinus sylvestris) is a very common tree in Polish forests, and therefore was widely used as timber. A relatively large amount of available
wood allowed a long-term chronology to be built up and used as a source of information about past climate. The analysis of
reconstructed indexed values of mean temperature in 51-year moving intervals allowed the recognition of the coldest periods
in the years 1207–1346, 1383–1425, 1455–1482, 1533–1574, 1627–1646, and 1694–1785. The analysis of extreme wide and narrow
rings forms a complementary method of examining climatic data within tree rings. The tree ring widths, early wood and late
wood widths of 16 samples were assessed during the period 1581–1676. The most apparent effect is noted in the dry summer of
1616. According to previous research and our findings, temperature from February to March seems to be one of the most stable
climatic factors which influenced pine growth in Poland. Correlation coefficients in the calibration and validation procedure
gave promising results for temperature reconstruction from the pine chronology. 相似文献
20.
Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids
via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid
production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one
culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were
plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength
(43 μEs−1m−2) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating.
In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration,
as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and
32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce
seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so,
indicating that the protocol is genotype-dependent. 相似文献