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Beverly Aarts
No full textBeverly Aarts passed away peacefully July 7, 2019 surrounded by her family, after 12 years of living gracefully with Multiple Myeloma
Beverly Aarts, September 9, 1936 - July 7, 2019
No full textBeverly Aarts passed away peacefully July 7, 2019 surrounded by her family, after 12 years of living gracefully with Multiple Myeloma
A pilot study of the Video Observations Aarts and Aarts (VOAA): a new software program to measure motor behaviour in children with cerebral palsy.
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52583.pdf (Publisher’s version ) (Open Access)A new computer software program to score video observations, Video Observations Aarts and Aarts (VOAA) was developed to evaluate paediatric occupational therapy interventions. The VOAA is an observation tool that assesses the frequency, duration and quality of arm/hand use in children, in particular those with cerebral palsy. Reliability studies show that the first module, designed to evaluate a forced-use programme, has an excellent content validity index (0.93) and good intra- and inter-observer reliability (Cohen's kappas ranging from 0.62 to 0.85 for the three activities tested). With the built-in statistical package, paediatric occupational therapy departments can conduct therapeutic evaluations with children with impairments in the upper extremities. Further research is recommended to apply the VOAA in clinical studies in paediatric occupational therapy
A pilot study of the Video Observations Aarts and Aarts (VOAA) a new software program to measure motor behaviour in children with cerebral palsy
No full textA new computer software program to score video observations, Video Observations Aarts and Aarts (VOAA) was developed to evaluate paediatric occupational therapy interventions. The VOAA is an observation tool that assesses the frequency, duration and quality of arm/hand use in children, in particular those with cerebral palsy. Reliability studies show that the first module, designed to evaluate a forced-use programme, has an excellent content validity index (0.93) and good intra- and inter-observer reliability (Cohen's kappas ranging from 0.62 to 0.85 for the three activities tested). With the built-in statistical package, paediatric occupational therapy departments can conduct therapeutic evaluations with children with impairments in the upper extremities. Further research is recommended to apply the VOAA in clinical studies in paediatric occupational therapy
Postmodifying Clauses in the English Noun Phrase. A Corpus-Based Study
No full textItem does not contain fulltextKU Nijmegen, 07 september 1989Promotores : Aarts, J.M.G.A., Aarts, F.G.A.M.XI, 230 p
J.G. Aarts: tussen auto's en schoensmeer
No full textIn het Brabantse leerlooiersdorp Dongen overleed eind 1929 de uitvinder en industrieel J.G. Aarts. Het plaatselijk weekblad herdacht hem als een man 'van buitengewone bekwaamheden en internationale bekendheid'. Lokaal chauvinisme of is Aarts een onterecht vergeten technicus
An En/Spm based transposable element system for gene isolation in Arabidopsis thaliana
No full textAt the start of the research described in this thesis, the main aim was to develop, study and apply an efficient En/Spm-I/dSpm based transposon tagging system in Arabidopsis thaliana to generate tagged mutants and to provide insights in the possibilities for future applications of such a transposon tagging system in studying plant gene functions. The first step was the introduction of an active En/Spm-I/dSpm system into Arabidopsis. Initially a very simple T-DNA construct was transformed, containing a nearly full En-1 element, without left and right border sequences, and with its promoter replaced by the stronger CaMV 35S promoter. As the same construct harboured a non-autonomous I/dSpm element, only one T-DNA transformation was needed. Transformation of this 'in cis two- element En/Spm-I/dSpm system' yielded one transformant with two T-DNA insertion loci, TEn2 and TEn5, each having one, respectively, five 35S -En/Spm t ransposase gene copies (Chapter 1). The transposition activity of the I/dSpm elements turned out to be surprisingly high. Instead of a germinal excision frequency, which was generally used to express the activity of heterologous transposable element systems, the term independent transposition frequency or itf was coined, as a measure accounting for the entire transposition process. Although not always easy to compare for different transposable element systems, an average itf of over 10%, as was found for this En/Spm-I/dSpm system (Chapter 1), has not been reported for any of the Ac-Ds based heterologous transposon tagging systems developed for Arabidopsis (Bancroft et al., 1992; Bhatt et al., 1996; Fedoroff and Smith, 1993; Honma et al., 1993; Long et al., 1993b; Swinburne et al., 1992). Obtaining a high transposition frequency in Ac-Ds systems is hampered by the fact that the transposase acts as an inhibitor of Ac or Ds transposition when its expression exceeds a certain level. Apparently such autoregulatory mechanism is not present in the 'in cis two-element En/Spm-I/dSpm system'.The need for a more sophisticated system diminished with the availability of this simple, but efficient En/Spm-I/dSpm transposon system and it was therefore studied in more detail to determine: 1) the ability to transpose continuously even after many plant generations; 2) the distribution of elements after transposition; 3) the ability to transpose to transcribed regions and 4) the ability to cause mutations. To start with the first issue, transposition has been studied in up to 12 generations, starting from the primary transformant. In all these generations, there was no apparent reduction in itf, demonstrating a continuous transposition of I/dSpm elements, irrespective of the generation number. The second issue, the distribution of elements after transposition, is another important aspect of a transposable element system. For maize transposons it is reported that the insertion site is preferentially physically and often genetically linked to the excision site (Dooner and Belachew, 1989; Peterson, 1970). This is not so remarkable considering that the proteins that perform the transposition steps will have a higher chance of encountering a nearby site on the genome instead of a distant DNA sequence. Like in maize (Peterson, 1970), the I/dSpm elements show a preference for insertion in genetically linked sites (Chapter 1), but the preference is not very explicit. Based on the observations of transpositions from tagged genes to sites within a few cM or only several kb away, and on the analogy to the En/Spm-I/dSpm elements in maize, the overall estimate is that about 30% of the elements transpose to sites genetically linked to the excision site. The mapped elements (Chapter 5) show a fairly even distribution over the genome, although there seems to be some clustering of elements (from different origin) to certain genomic regions, like the top of chromosome 4, the bottom of chromosome 1 and the lower half of chromosome 5.In accordance with the idea that DNA must be in an open confirmation to allow the access of transposase proteins before transposition (Zhang and Spradling, 1993), there are many indications that I/dSpm elements insert in regions of the Arabidopsis genome containing genes: a) I/dSpm flanking DNAs rarely contain repetitive sequences, but are mostly single copy sequences, as are genes (Chapters 1 and 5); b) about half of the I/dSpm elements are inserted in relatively conserved genomic regions, with no RFLPs for five restriction enzymes (Chapters 1 and 5); c) at least one third of the examined I/dSpm elements is inserted in close vicinity of transcriptionally active genomic sequences (Chapter 7). Insertion in unique, conserved and often transcribed DNA may not seem surprising for a plant species with little repetitive DNA and a small genome with a high gene density (Meyerowitz, 1989). However, a high frequency of insertion into genic regions of the genome offers the best chances for gene tagging.The most important aspect of a transposable element system is the possibility to generate tagged mutants. The En/Spm-I/dSpm system is mutagenic, with as much as 12 tagged mutants found so far. Most of these were obtained after screening for random mutant phenotypes. When screening for specific mutants, such as reduced seed dormancy, tagged alleles of the ABI3 and LEC1 genes were found (M. Koornneef et al., unpublished results). Mutants at the CER6 locus were obtained by targeted tagging, using the nearby ap1::I/dSpm allele as the I/dSpm element donor (A. Pereira, unpublished results). These selected and targeted transposon tagging experiments are illustrative examples of the feasibility to efficiently isolate tagged mutants of a special phenotypic or genotypic class.Ideally, a population saturated with different I/dSpm insertions can be made, allowing the isolation of mutants for virtually every gene. Such a 'mutation machine' can be further used for PCR based targeted gene inactivation. This novel technique, which was originally developed for Drosophila melanogaster (see O'Hare, 1990), exploits the abundance of transposons for the identification of insertions in genes with known DNA sequence, but no known mutant phenotype. In general, DNAs from multidimensional pools of individuals from a large population are used for a PCR using two primers. One primer is specific for the transposon terminus (directed outwards), the other is specific for the target gene. A fragment can only be amplified when a transposon insert is close enough to the target primer. This technique has been shown to work in Drosophila (Ballinger and Benzer, 1989; Kaiser and Goodwin, 1990), Caenorhabditis elegans (Zwaal et al., 1993), Petunia hybrida (Koes. et al., 1995) and maize (Das and Martienssen, 1995), using 'mutation machine' transposable element systems.Two I/dSpm tagged genes that have been studied in great detail are the MS2 gene (Chapters 2 and 3) and the CER1 gene (Chapter 4). Mutants for both genes display a conditional male sterile phenotype, which is the only mutant phenotype for the ms2 mutant, but for the cer1 mutant it is a pleiotropic effect of a deficiency in epicuticular wax biosynthesis. The ms2 mutants are occasionally able to self-fertilize, especially in high relative humidity and late in plant development, but seed set rarely reaches more than a few percentages of wild-type seed set. This in contrast to the cer1 mutants, which are male sterile in low relative humidity (≤50% RH) and fertile in high relative humidity (≥95% RH). Fertility cannot be completely restored by environment in the ms2 mutants due to the drastic effect of the mutation on pollen development. The MS2 gene is expressed in the tapetum around the time of microspore release from the microspore mother cells. The gene is needed for the development of a proper exine layer, protecting the microspore from harmful environmental influences. Consequently the few ms2 microspores that are produced have very feeble pollen walls, which leaves only very few pollen grains intact for fertilization.The CER1 gene acts much later in pollen development. Phenotypically cer1 and wild-type pollen cannot be distinguished, apart from a difference in gerniination ability (Chapter 4). As cer1 pollen germination is like wild type when applying CER1 pollen or by pollinating in high relative humidity, there appears to be a substance missing from the pollen coat that is required for the necessary rehydration of a pollen grain. Although essential under low relative humidity conditions, this is only a minor defect, which can be easily overcome.Besides the similarity in mutant phenotypes, the MS2 gene and the CER1 gene share the characteristic of encoding proteins with homology to enzymes in the fatty acid biosynthesis pathway. The MS2 protein has most resemblance with the wax fatty acid reductase protein from the desert shrub jojoba, which is involved in the conversion of wax fatty acids to wax alcohols (Chapter 3). The CER1 protein shares structural features of fatty acid desaturases, and it has a proposed function as a decarbonylase, converting long carbon chain aldehydes to alkanes (Chapter 4). There are more examples of a correlation between male fertility and wax biosynthesis, as also other cer mutants such as cer3, 6, 8 and 10 are known to be disturbed in male fertility. It demonstrates the general importance of fatty acid biosynthesis for male gametogenesis.The last part of this thesis has been devoted to further applications of the En/Spm-I/dSpm tagging system in Arabidopsis for the analysis of plant gene functions. The first description of transposable elements as controlling elements (McClintock, 1948), was based on the effect transposons had on the expression of maize genes. Especially (d)Spm insertions were known to cause Spm dependent or suppressible gene expression (Fedoroff, 1989). This effect is now also described for the En/Spm- I/dSpm system in Arabidopsis (Chapter 6). As in maize, an En/Spm suppressible allele contains an anti-parallel I/dSpm element insertion, which can be spliced from the mRNA. This knowledge can be further used to design an artificial gene expression system, in which an introduced gene containing an anti-parallel I/dSpm element, can be negatively controlled in the presence of an En/Spm transposase source. The reverse effect, En/Spm dependent gene expression, is now also described for Arabidopsis (Chapter 6), but the mechanism for dependence is not yet understood. The availability of the En/Spm dependent lad::I/dSpm mutant will be useful for further research.A more general way to study gene expression is the use of gene traps as detectors of gene activity. The pilot experiments described in chapter 7, have shown the possibility to adapt I/dSpm elements as gene traps. Especially their advantage in detecting genes without the need for mutation, and the possibility of studying the activity of genes which are lethal as homozygous mutants, are important additional properties of gene trap systems over "traditional" transposon tagging. In combination with its efficient transposition behaviour, an En/Spm-I/dSpm based gene trap tagging system seems an attractive alternative for the existing Ac-Ds or T-DNA based gene trap systems.Summarizing, an En/Spm-I/dSpm transposon tagging system has been well developed for Arabidopsis and many of its basic characteristics are studied and described. I/dSpm tagged mutants can be found with reasonable frequencies, either by random, selected or targeted tagging strategies. The cloning and characterization of two genes affecting male fertility has been described. Further ways to improve tagging frequencies, based on phenotypic or on genotypic selection have been discussed. In addition, the system can be exploited to study plant gene expression and gene function either by En/Spm controlled activity of I/dSpm tagged genes, or by using I/dSpm gene detector elements
Een kwestie van natuur : een studie naar de aard en het verloop van communicatie over natuur en natuurbeleid
Get PDFThis dissertation sets out to investigate recurring patterns in communication about nature and nature-policy among different stakeholders.Chapter 1 describes the context in which the research took place. The time of the study, was marked by limited acceptance of many governmental policies, including those concerning nature. This problem is associated with an increasing complexity of society, and limited involvement of citizens, administrators and politicians. As a result, we has marked increased recently attention to improving the communication between government and citizens. In this context, many speak of 'administrative renewal'.Subsequently, chapter 1 deals with several methodological concerns. The study has been carried out from an anthropological perspective. The central starting point is that society is always dynamic, with continuously changing inter-dependencies among (groups of) people. I explore three methodological problems in relation to qualitative social science research:the nature and use of concepts;the relation between researchers and their object of study;criteria for evaluating social-scientific research.These explorations lead to the formulation of methodological guidelines for qualitative research which adhered to during the research process.In chapter 2 I introduce theoretical insights which help me to connect nature, culture, communication and policy. By looking at communication as a process which takes place in a dynamic context, dimensions of the process can be identified which tend to remain obscure in more narrow approaches which look at communication as an isolated activity. I discuss the concept of culture which I describe as the dynamic mechanism through which people communicate. Starting from the assumption that communication and culture presuppose each other, I analyze communication problems in connection with existing or emerging cultural differences. In doing so, I use two theoretical perspectives: 1) Norbert Elias' established-outsider perspective which connects conflict situations with changing power-balances and processes of mutual stigmatization (we against them), and 2) autopoiesis theory, which holds that (groups of) people tend to become self-referential.Subsequently, I look at nature as a topic for communication. On the basis of an analysis of different visions on nature-conservation, I conclude that such visions can be seen as the (temporary) result of socio-cultural and political processes which determine what is to be conserved and/or produced as nature, and what not. In the context of nature-conservation, perceptions of nature are often perceptions of what is desirable societal change.In relation to the subject of policy and communication, I discuss the policy network approach as one of the operationalizations of administrative renewal. Although communication for purposes of policy making is a central theme within the policy network approach, little attention is paid to power struggles among the actors during all phases of the policy process, and consequences of such struggles for communication respectively non-communication. Associated with power struggles and shifting power balances are the (flexible) ways in which actors continuously construct arguments and identities -of themselves and others- in their interaction with others.Finally, I combine these insights in an analytical framework in which the following four themes are central:nature perceptions;inter-dependencies;the construction of identities;the construction of arguments.In each of the following three chapters, which present empirical studies on nature(policy) and communication, these themes will be elaborated upon in connection with the empirical material.Chapter 3 presents a study of communication between the government and farmers following the introduction of the Nature Policy Plan. I have explored the views and opinions of farmers about nature and nature-policy, and the way in which these views have been shaped. In this study, the theoretical notion of policy acceptance has served as an important starting point.The study indicates that most of the farmers interviewed have a very specific perception of nature. For them, nature is 'everything that grows, blossoms and lives'. Starting from this point of view, farmers assert that nature is doing well. Hence, the problem which lies at the root of the Nature Policy Plan is neither recognized nor accepted by the majority of farmers.Similarly, the policy measures which are taken in order to conserve nature are not regarded as acceptable either. The Nature Policy Plan is seen, just as one element in a series of governmental decisions which have detrimental consequences for farmers. Farmers tend to perceive the government as an enemy.Underlying the arguments that farmers use to defend their perception of nature and to denounce the government's nature policy are a few more fundamental contradictions. The central concern for farmers is the survival of their farms under reasonable conditions. However, they increasingly feel that they are confronted with developments that form a threat to their right to exist. As a result, they tend to withdraw under the motto: 'as long as we can avoid it, we don't do anything'. The attitude of farmers towards the government is characterized by negative stigmatization: the worst examples and experiences are repeated over and over again in order to support the argument that nothing good can be expected from the government. In all, the communication between farmers and the government about nature policy takes place in the context of a troubled relationship. Thus, the implementation of the Nature Policy Plan, which is explicitly based on voluntary cooperation from farmers, is seriously hampered.During the course of the research, however, the feelings of mutual dependency tend to increase, on the side of both the farmers and the government. For the government, this feeling was induced by the lack of acceptance of nature policy. The farmers, in turn, increasingly realized that nature policies are inevitable, and felt a need to alleviate the adverse conditions which confront agriculture in general. As a result, both parties made attempts to (re)engage in communication about nature policy.In chapter 4 a second empirical study is presented concerning the communication among government, farmers and nature conservationists about nature and nature policy in the Brabant Peel region. In recent decades, agricultural development in this region has been explosive, resulting in a strong and intensive agricultural sector. However, the Brabant Peel region is also known for a number of special nature and landscape values. In this context, rules and regulations concerning agricultural practice are becoming ever more strict. This development has created very severe tensions between those who represent agriculture and those who represent nature.In 1993 the Peel Environmental Cooperative (PEC) was established. In this institution farmers and horticulturists cooperate with a view to maintaining an economically viable agricultural sector in the Brabant Peel, recognizing that this will only be possible if sufficient attention is given to the preservation of nature and the environment. Right from the start, the PEC has involved itself in a network of relationships with other actors with whom it negotiates in order to further its goals. The research effort was geared mainly to investigating the way in which different relationships have influenced each other, and the consequences this has had on the functioning of the PEC with respect to its aspirations. For this purpose, I have studied the relationship of the PEC with various actors. More specifically these actors are:the government;the regional organization of nature conservationists: Working Group Save the Peel (WGSP);the agrarian community.Furthermore, the study started from a theoretical framework in which (conditions for) communication and negotiation were central concepts.The study shows that the PEC has the complex task of managing a network of relationships in which different, and often mutually exclusive, demands are made. This creates a continuous danger for the PEC to end up in a 'boundary role conflict': the different parties tend to confront the PEC with pressures and demands which are difficult to agree upon without seriously jeopardizing the opportunity to reach compromise with others. Farmers and horticulturists, for example, consider the PEC as an institution which represents their interests, and which should aim at reaching the best possible result for agriculture in its negotiations with the government. At the same time, the government and WGSP expect the PEC to contribute to a change of mentality among farmers with regard to nature and the environment. In short, the various parties have very different expectations of the PEC. In association with this, the PEC indeed takes on such differential identities in it's predominantly bilateral contacts with others. Thus, it becomes clear that the chains of interdependencies in which the PEC is involved imply unintended instabilities, which are difficult to prevent.Chapter 5 investigates the communication among interested citizens about nature and nature policy by means of a discourse-analytical study of a public debate on nature-development among a panel of 30 participants. This debate was organized by the Platform for Science an Ethics following the commotion around the implementation of the Nature Policy Plan, and the conflicts about the deliberate nature-development in particular. The debate resulted in a closing statement by the panel about nature an nature policy in the Netherlands. This closing statement has been presented to Parliament as an advice with respect to the further development and implementation of nature policy.The central starting point of discourse analysis is that language is not a neutral medium for the transfer of information, but a social activity. Wittingly or unwittingly people have a purpose with what they say, how they say it, when they say it, and to whom. Depending on their purposes, people use specific repertoires. The study focused on several repertoires, or clusters of arguments, that the participants draw upon, and on the functions these repertoires have in the debate. In addition, I have looked at whether or not, and if so how, the members refer to their background in the course of the debate.The analysis shows that the repertoires are all used for the purpose of creating maximum support for nature. In addition to several specific target-groups which explicitly serve as point of reference, the support of an undifferentiated 'general public' which participants claim for their arguments is also taken into consideration.During the discussions, the members of the panel refer to, on the one hand, scientific and policy sources, and on the other, personal experiences and feelings. In addition to these two modes of building up credibility, there is a striking third. Often, the participants refer to 'people' in general. The anonymity of those who are represented in this manner implies that such references are difficult to verify. The reference to 'people' in general can also be seen as a way of engaging and speaking on behalf of different societal groups, which serves to strengthen the public nature of the debate.Although a number of nature perceptions and desirable futures are -implicitly or explicitly- brought forward during the debate, this does not lead to making choices for or against particular types of nature in concrete situations. While this obscures the relevance of nature-development vis-à-vis other types of desirable nature, the question of type of nature which should prevail when and why remains unanswered. I relate this phenomenon to the fact that the panel was not constrained by the boundaries that in actual practice are created by politicians and stakeholders. In that sense, the debate and its closing statement are to a degree noncommittal.Finally, the study shows that the panel gradually developed into an 'insider group' as the debate proceeded. In association with this, several 'outsider groups' emerged, which were negatively referred to by the panel, and, in turn, strengthened the panel's identity as an 'insider group'. Interestingly, the outsider groups were largely created on the basis of coincidence. This supports the idea that the insider/outsider mechanism constitutes an important regulating principle that plays a role under specific circumstances.In the final chapter I make an effort to identify some of the overall insights that emerge from this research and formulate them into guidelines for effective communication about nature and nature policy. First, I describe how actors are continuously engaged, wittingly or unwittingly, in constructing arguments and identities (their own and those of other actors) in interaction with others in order to pursue certain ends. This pattern can be seen as an important characteristic of the way in which communication in negotiation processes about nature and nature policy unfold. Moreover, it appears that stakeholders, in their effort to build credibility, tend to present arguments as 'indisputable' facts. As a result, their choices -which are inherently associated with interpretations, interests, values, norms and aspirations, in short their culture- are hardly explicated during discussions with others. Thus, communication about nature tends to take place on the basis of arguments which are considered valid only by those who are already in agreement; others tend to be far less impressed. Some form of trench-warfare is the likely result. The endless discussions aboutwhat is the true perception of nature are an example.Subsequently, I re-analyze the negotiation processes which took place in the Peel and during the public debate on nature-development, and which both aimed at giving a new impulse to the further development and implementation of nature policy. In both cases the outcome was a (preliminary) compromise. In order to further analyze such compromises, I make a distinction between a reactive and a creative compromise. A reactive comprise is the result of a negotiation process that is distributive in nature. The way in which actors operate in such negotiations is characterized by a high degree of reticence with respect to one's own position, by an emphasis on positions, by overcharging in the hope of ending up in the middle, and by a lack of care for the other party. Hence, a reactive compromise is the result of reacting upon each others' proposals. A creative compromise results from an integrative process of negotiation in which actors show both a high degree of openness with respect to one's own situation and intentions, and a certain level of care for the other party in addition to self-care. This enhances the possibility that a new common problem is formulated in which the problems of the various parties are incorporated. This common problem, then, forms the basis for achieving a creative compromise.On the basis of the above, I finally elaborate on the role of communication in policy processes concerning nature. I discuss conditions which need to be fulfilled if communication and negotiation -at various points in the policy process, and in different forms- are to contribute to an acceptable nature policy. The following guidelines for supporting communication in negotiations can be formulated:reflect on the relationship between negotiators;look for 'the will' behind arguments;recognize the differences and search for the similarities;develop targets for the short term;develop a vision for the long term
Acid phosphatase-1.1, a molecular marker tightly linked to root-knot nematode resistance in tomato
No full textRoot knot nematode resistance in tomato is a genetic trait which is determined by a single dominant gene ( Mi ) on chromosome 6 of tomato. Information about the mRNA or protein product is completely lacking, which precludes the cloning of Mi by conventional strategies based on gene expression. However, an acid phosphatase-1 allozyme marker ( Aps-1 1 ) is known, which shows tight genetic linkage to the root knot nematode resistance trait. With a view to isolating Mi nucleotide sequences by a positional cloning approach, we have developed a molecular probe for the Aps-1 1 gene, using the polymerase chain reaction (PCR) and a pair of primers, corresponding to amino acid sequence information from the protein encoded by the Aps-1 1 gene.The Aps-1 1 gene product (APS-1 1) was purified from hydroponic tomato roots and tomato suspension cells using conventional low pressure column chromatographic techniques. The most striking purification was achieved by concanavalin A (Con A)-Sepharose 4B affinity column chromatography, which constituted the fourth step in our purification procedure and produced virtually pure APS-1 1. Unfortunately, however, this step caused contamination of the APS-1 1preparation with Con A released form the column. Therefore, a final Mono Q-FPLC purification step was added to remove the Con A-contamination. The resulting APS-1 1preparation was homogeneous in denaturing and non-denaturing polyacrylamide gel electrophoresis. In addition, the purified protein showed co-electrophoresis and co-elution with the APS-1 1enzymatic activity in non-denaturing PAGE and gel filtration column chromatography, respectively, thereby demonstrating its APS-1 1identity. The yield of our purification protocol was a few μg of APS-1 1protein per kg of tomato suspension cells or roots. No major loss of APS-1 1activity was observed in any of the purification steps, indicating that the low yield attained is truly reflecting the very low expression level of the Aps-1 1gene.The purified APS-1 1preparation was treated with CNBr and trypsin to produce APS-1 1peptides. Following purification by HPLC, amino acid sequence analysis of two CNBr and seven trypsin cleavage products revealed 61 residues of APS-1 1amino acid sequence.The amino acid sequence information from two APS-1 1peptides consisting of 8 and 14 amino acids respectively, allowed the synthesis of PCR primer pools to direct the amplification of a 2.4 kb Aps-1 1 fragment using a genomic DNA template. Crucial for the effectivity of these pools was the limitation of the number of different primers used to account for codon degeneracy. Restriction of the complexity of the primer pools was achieved by incorporating deoxyinosine or the most probable nucleotide(s) at ambiguous codon positions in the 5' part of the primers. On the other hand, efficient primer elongation was assured by including in the primer pool every possible combination of the three 3'-terminal codons. The 2.4 kb fragment was only amplified from a genomic template carrying the Aps-1 1 allele and was not found in using a template that carried the Aps-1 3 or Aps-1 1 + allele. Moreover, the 2.4 kb amplification product was found to reveal RFLPs between a pair of nematode resistant and sensitive nearly isogenic lines, which only differed for the Aps-1 1/Mi region. The specific amplification under the direction of the particular Aps-1 allele from which the primers had been derived and its genetic map position provide evidence showing, that the 2.4 kb PCR product represents the Aps- 1 1 target fragment. Using cDNA as a template and the same primers that directed the synthesis of the 2.4 kb genomic PCR product, a 490 bp Aps-1 1 fragment was obtained. An overlapping Aps-1 1 cDNA sequence of 550 bp was amplified using the same upstream primers but a different pool of downstream primers corresponding to a peptide that turned out to represent the C- terminus of APS-1 1. The amount of these cDNA-directed amplification products synthesized in 30 cycles of PCR was so low, that their production was only detectable by Southern blot hybridization using the 2.4 kb genomic PCR product as a probe, which provides another demonstration of the very low expression level of the Aps-1 1 gene.In addition to the 2.4 kb genomic Aps-1 1 sequence, another PCR product of about 115 bp was obtained using a different pair of primers and either a genomic or a cDNA template. This product was found to comprise several related nucleotide sequences of similar size. Because of the poor performance of the cloned products as a probe, it was not possible to establish their genetic map position by RFLP analysis.Screening of a cDNA library with the 2.4 kb putative Aps-1 1 sequence identified two clones of related nucleotide sequences, one of which apparently represented an Aps-1 1 cDNA clone, as it encoded three APS-1 1peptides (together 30 amino acids) in the orientation predicted by the PCR results.'Me nucleotide sequence of this Aps-1 1 cDNA clone revealed a stretch of 69 residues of tomato APS-1 1amino acid sequence starting from the C-terminus, and showed that peptide VII is the C-terminal tryptic peptide. Another stretch of putative APS-1 1amino acid sequence was deduced from the nucleotide sequence adjacent to the upstream primer in the 2.4 kb genomic PCR product, and comprised 56 amino acids sequence information starting with peptide IX at the N-terminal end. The amino acid sequence of tomato APS-1 1elucidated so far, did not present major sequence homology with the sequences of any other (acid) phosphatase in the GenBank or EMBL data bases. A striking sequence homology was found, however, with a vegetative storage protein from soybean, VSP-β, that accumulates to very high levels in leaves after depodding of the plants.The detection of an Aps-1 1 -related cDNA clone using the 2.4 kb genomic Aps-1 1 sequence as a probe, and furthermore, the sequence heterogeneity among the 115 bp PCR product points to the existence of a family of Aps-1 1 -related nucleotide sequences within the tomato genome. Upon comparison of four different nucleotide sequences present in the 115 bp PCR product with the Aps-1 1 cDNA sequence, it was found, that one of them corresponded with the Aps-1 1 sequence. Neither of the other three, nor the Aps-1 1 -related cDNA clone showed a higher homology at the amino acid level with soybean VSP-βthan with tomato APS-1 1, which argues against one of these sequences representing the tomato homolog of soybean VSP-β.In the isolation of an Aps-1 1 cDNA clone, we have shown that it is possible to design highly specific degenerate PCR primer pools. Therefore, whenever going from protein to gene, we recommend to try PCR first in order to obtain a highly selective probe, before turning to library screening using degenerate oligonucleotides. Furthermore, since an Aps-1 1 cDNA clone is available now, a possible starting point or a useful landmark has been provided for a chromosomal walk towards the nematode resistance gene Mi
Review Of "Syntactic Gradience" By Bas Aarts
No full textVogel R. Review Of "Syntactic Gradience" By Bas Aarts. Journal Of Linguistics. 2008;44(2):505-509
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