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PubMed1994

Methamphetamine-induced behavioral sensitization and its implications for relapse of schizophrenia.

Vulnerability to relapse is a central issue in the biology of schizophrenia. The common neural mechanisms underlying such vulnerability can be studied using the experimental model of behavioral sensitization induced by repeated administration of low doses of methamphetamine (MAP) to rodents. This review summarizes a series of behavioral and neurochemical studies on MAP-induced behavioral sensitization from the viewpoint that the mechanisms involved in initiation (or development) of psychotic symptoms and their expression differ. The initiation of behavioral sensitization to MAP in rats requires stimulation of dopaminergic neurons, and can be blocked by SCH 23390 (a dopamine D1-receptor antagonist) and BMY 14802 (a sigma-receptor antagonist). The expression of behavioral sensitization induced by subchronic MAP pretreatment takes several forms. First, dopamine release from the cerebral dopaminergic neuron terminal containing areas in response to either to rechallenge with MAP or cocaine, or evoked by intrastriatal ouabain infusion is enhanced. Second, the behavioral responses to dopamine D2- and sigma-receptor agonists are augmented. A third form involves changes indicative of transsynaptic neural circuits, such as increased numbers of D1 receptors in the substantia nigra pars reticulata, enhanced electrophysiological responses to D1 receptor activation, the putative role of excitatory amino acid receptors and interchangeability of MAP and stress. Although MAP-induced behavioral sensitization in rodents serves as a useful animal model, the elucidation of the mechanisms involved in the vulnerability of patients with schizophrenia to relapse of psychotic episodes requires further study.

Animals↗
PubMed1989

Fine-tuning of nif and fix gene expression by upstream activator sequences in Bradyrhizobium japonicum.

The significance of Bradyrhizobium japonicum upstream activator sequences (UASs) for differential NifA-mediated fix and nif gene expression was investigated by two means: (i) hybrid fixA- and fixB-lacZ fusions were constructed by transposing a nifH-UAS cartridge in front of their promoters; and (ii) B. japonicum mutants were generated carrying specific chromosomal deletions or UAS cartridge insertions within the fixA, fixB or nifH promoter-upstream regions. Expression of fixA was not affected, and expression of fixB decreased only to 42%, when the respective fixA and fixB promoter-upstream DNAs were deleted. This shows that in B. japonicum the NifA-dependent activation of at least the fixA promoter does not require the presence of a closely adjacent UAS. Deletion of the UASs in front of the nifH gene not only reduced the expression of nifH down to 2.5% but, surprisingly, also resulted in a reduction of the fixB mRNA level to less than 20%. This suggests that the nifH-UASs may exert a long-range effect on the expression of the 3-kb-distant fixBCX operon in nif cluster I or B. japonicum. Artificial transposition of the nifH-UASs in front of the fixA and fixB promoters strongly enhanced fixA and fixB expression.

Base Sequence↗
PubMed2006

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

Animals↗
PubMed1986

A method for reconstructing patterns of somatosensory cerebral cortical activity.

An interactive graphics package was developed in order to acquire, display, and manipulate images of cerebral cortical autoradiographic data. The primary purpose for development of the system was to reconstruct accurate 2-dimensional maps of the functional activity within the somatosensory cerebral cortex. A Datacube Q-bus graphics module (QVG/QAF-123) was interfaced with the Micro PDP-11/23 to accept a standard RS170 video input signal, and autoradiographs of serial sections (each 20 microns thick) of a cerebral cortex were digitized individually to 768 X 512 X 8 bit resolution. Input look-up tables were used to standardize the autoradiographic data. Boundaries of the somatosensory cortex were entered (with a Summagraphics MM 1201 digitizer), and the image data was stored on disk file (a method of data compression was devised). A method for segmenting the image data for many (sequential) sections was developed that provided arrays from which the maps were generated. Thresholding, histogram equalization, edge detection and edge enhancement, and filters in both the spatial and frequency domains were employed to process the images of the maps. Plots of optical density values along any axis of the maps and gray level histograms of any map region could also be generated. Maps made by the described method are much higher in resolution than those produced by traditional (manual) methods, and permit analysis of the reconstructions in both the frequency and spatial domains.

Animals↗
PubMed1996

Transforming potentials of epidermal growth factor and nerve growth factor receptors inversely correlate with their phospholipase C gamma affinity and signal activation.

The exchange of nerve growth factor receptor/Trk and epidermal growth factor receptor (EGFR) phospholipase C gamma (PLC gamma) binding sites resulted in the transfer of their distinct affinities for this Src homology 2 domain-containing protein. Relative to wild-type EGFR, the PLC gamma affinity increase of the EGFR switch mutant EGFR.X enhanced its inositol trisphosphate (IP3) and calcium signals and resulted in a more sustained mitogen-activated protein (MAP) kinase activation and accelerated receptor dephosphorylation. In parallel, EGFR.X exhibited a significantly decreased mitogenic and transforming potential in NIH 3T3 cells. Conversely, the transfer of the EGFR PLC gamma binding site into the Trk cytoplasmic domain context impaired the IP3/calcium signal and attenuated the MAP kinase activation and receptor dephosphorylation, but resulted in an enhancement of the ETR.X exchange mutant mitogenic and oncogenic capacity. Our findings establish the significance of PLC gamma affinity for signal definition, the role of this receptor tyrosine kinase substrate as a negative feedback regulator and the importance of this regulatory function for mitogenesis and its disturbance in oncogenic aberrations.

3T3 Cells↗
PubMed1981

Functional organization of plasmid pKM101.

Tn5 insertion mutants and in vitro-generated deletion mutants of the mutagenesis-enhancing plasmid pKM101 have been used to identify several genetic regions on the pKM101 map. In clockwise order on the pKM101 map are: (i) the bla gene, coding for a beta-lactamase; (ii) the Slo region, responsible for retarding cell growth on minimal medium; (iii) the tra genes, enabling pKM101 to transfer conjugally; (iv) sensitivity to IKe phage (this function[s] maps within the tra region); (v) the muc gene(s), responsible for enhancing ultraviolet light and chemically induced mutagenesis in the cell; and (vi) the Rep region, essential for plasmid replication. The muc gene(s) and the Rep region are contained in a deoxyribonucleic acid region bounded by inverted repeated sequences.

Coliphages↗
PubMed2000

Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells.

We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiates the mitogenic response to insulin-like growth factor-I (IGF-I). The present study was undertaken to determine whether the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of known IGF-I receptor substrates plays an important role in the cAMP-dependent potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or dibutyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In contrast, cAMP pretreatment potentiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not affect the amount of IRS-2. We found that the IGF-I-dependent tyrosine phosphorylation of 66 kDa Shc (Src homology collagen) was markedly increased by cAMP pretreatment, and that this change was mainly due to an increase in the levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment significantly increased binding of Grb2 (growth-factor-receptor-bound protein 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-activated protein kinase) induced by IGF-I was also enhanced by cAMP. The presence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treatment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA synthesis in response to IGF-I. On the other hand, cAMP pretreatment increased binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to IRS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatment with and without TSH or dibutyryl cAMP. Our results suggest that the interaction between cAMP-dependent and IGF-I-dependent pathways leads to an augmentation of cell proliferation, which is mediated, at least in part, through the MAP kinase and PI 3-kinase signalling pathways. These effects are mediated by changes in tyrosine phosphorylation of IGF-I receptor substrates, including IRS-2 and Shc.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation.

The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 435 amino acids, of which the C-terminal 114 residues have been implicated in controlling both localization and function of this enzyme. Inspection of the sequence of the C-terminal segment reveals a number of potential sites of phosphorylation. We show that PTP1B is phosphorylated on seryl residues in vivo. Increased phosphorylation of PTP1B is seen to accompany the transition from G2 to M phase of the cell cycle. Two major tryptic phosphopeptides appear in two-dimensional maps of PTP1B from mitotic cells. One of these comigrates with the peptide generated following phosphorylation of PTP1B in vitro at Ser386 by the mitotic protein Ser/Thr kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible for the pronounced retardation in the electrophoretic mobility of PTP1B from mitotic cells has been identified by site directed mutagenesis as Ser352. The identify of the kinase responsible for this modification is presently unknown. We also show that stimulation of HeLa cells with the phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional phosphopeptide mapping reveals that the bulk of the phosphate is in a single tryptic peptide. The site, identified as Ser378, is also the site of phosphorylation by protein kinase C (PKC) in vitro. Thus the TPA-stimulated phosphorylation of PTP1B in vivo appears to result directly from phosphorylation by PKC. The effect of phosphorylation on the activity of PTP1B has been examined in immunoprecipitates from TPA-treated and nocodazole-arrested cells. TPA treatment does not appear to affect activity directly, whereas the activity of PTP1B from nocodazole-arrested cells is only 70% of that from asynchronous populations.

Alanine↗
PubMed1980

Frameshift suppression Saccharomyces cerevisiae. II. Genetic properties of group II suppressors.

Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. One of these groups (Group II: SUF1, SUF3, SUF4, SUF5 and SUF6) appears to include a set of informational suppressors in which the vehicle of suppression is glycyl-tRNA. Some of the genetic properties of Group II suppressors are described in this communication.--Corevertants of the Group II frameshift mutations his4-519 and leu2-3 have been characterized to determine the spectrum of reversion events induced by the frameshift mutagen ICR-170. Seventy-three ICR-induced corevertants were analyzed. With the exception of one cerevertant, which carried an allele of SUF1, all carried alleles of SUF3 or SUF5. SUF1, SUF3, SUF4 and SUF6 were represented among spontaneous and UV-induced corevertants. In the course of these experiments one of the suppressors was mapped. SUF5, the probable structural gene for tRNAGLY1, is located between ade2 and ade9 on chromosome XV.--SUF1, SUF4 and SUF6 have novel properties and comprise a distinct subset of suppressors. Although these suppressors show no genetic linkage to each other, they share several common features including lethality in haploid pairwise combinations, reduced tRNAGLY3 isoacceptor activity and increased efficiency of suppression in strains carrying the cytoplasmically inherited [PSI] element. In addition, strains carrying SUF1, SUF4 or SUF6 are phenotypically unstable and give rise to mitotic Suf+ segments at high frequency. These segregants invariably contain a linked, second-site mutation that maps in or adjacent to the suppressor gene itself. Strains carrying any of these suppressors also give rise to mitotic segregants that exhibit enhanced efficiency of suppression; mutations responsible for this phenotype map at two loci, upf1 and upf2. These genes show no genetic linkage to any of the group II suppressors.--Methods that permit positive selection have been devised in order to examine large numbers of variants. The importance of these interacting mutants is underscored by their potential utility in studying suppressor function at the molecular level.

Chromosome Mapping↗
PubMed1993

Comparative molecular field analysis of in vitro growth inhibition of L1210 and HCT-8 cells by some pyrazoloacridines.

In vitro screening of a number of 2-(aminoalkyl)-5-nitropyrazolo[3,4,5- kl]acridines has previously indicated (Sebolt, J.S.; et al. Cancer Res. 1987, 47, 4299-4304) that these compounds, in general, exhibit selective cytotoxicity against the human colon adenocarcinoma, HCT-8, cell line, relative to mouse leukemia L1210 cells. Comparative molecular field analysis (CoMFA) was applied to HCT-8 and L1210 growth inhibition assays (IC50s) of a series (44) of the pyrazoloacridine derivatives with the objective of predicting improved solid tumor selectivity. In the absence of crystallographic data, the 9-methoxy derivative (15), which is currently in clinical study, was selected as the template molecular model. Two different structural alignments were tested: an alignment of structures based on root mean square (RMS)-fitting of each structure to 15 was compared with an alternative strategy, steric and electrostatic alignment (SEAL). Somewhat better predictive cross-validation correlations (r2) were obtained with models based on RMS vis-à-vis SEAL alignment for both sets of assays. A large change in lattice spacing, e.g., 2 to 1 A, causes significant variations in the CoMFA results. A shift in the lattice of half of its spacing had a much smaller effect on the CoMFA data for a lattice of 1 A than one of 2 A. The relative contribution of steric and electrostatic fields to both models were about equal, underscoring the importance of both terms. Neither calculated log P nor HOMO and/or LUMO energies contribute to the model. Steric and electrostatic fields of the pyrazoloacridines are the sole relevant descriptors to the structure-activity (cross-validated and conventional) correlations obtained with the cytotoxic data for both the L1210 and HCT-8 cell lines. The cross-validated r2, derived from partial least-squares calculations, indicated considerable predictive capacity for growth inhibition of both the leukemia and solid-tumor data. Evidence for the predictive performance of the CoMFA-derived models is provided in the form of plots of actual vs predicted growth inhibition of L1210 and HCT-8 cells, respectively, by the pyrazoloacridines. The steric and electrostatic features of the QSAR are presented in the form of standard deviation coefficient contour maps of steric and electrostatic fields. The maps indicate that increases or decreases in steric bulk that would enhance growth inhibition of HCT-8 cells would likewise promote growth inhibition of L1210 cells. Contour maps generated to analyze the electrostatic field contributions of the pyrazoloacridines to growth inhibition provide an essentially similar set of results.(ABSTRACT TRUNCATED AT 400 WORDS)

Acridines↗
PubMed1998

Lipidation as a novel approach to mucosal immunization.

We describe the design and development of a novel peptide-based approach for mucosal immunization. The design contains an amplified peptide chain as multiple antigen peptide (MAP) with a cluster of lipids. Such a design would confer on lipidated MAP the ability to self-assemble in water, mimicking enveloped viral particles. The importance of lipidation for mucosal immunization was confirmed by oral immunization with lipidated MAP in phosphate-buffered saline (PBS), which induced mucosal and systemic immune responses at local and distant sites, including sera and vaginal IgG as well as secretory IgA in saliva, vaginal secretions and fecal matter. T-cell proliferative responses were found in spleen, Peyer's patches and genital lymph nodes. In addition, significant splenic cytotoxic T-cell responses were also observed. No significant immune responses were observed with non-lipidated MAPs by oral delivery in PBS. Furthermore, these responses were selectively enhanced by different regimens, systemic priming and microparticle delivery. These results demonstrate the effectiveness of lipidated MAP for mucosal immunization to evoke both systemic and mucosal immune responses without the use of carrier or extraneous adjuvant.

Administration, Oral↗
PubMed2000

Potentiation of nerve growth factor-action by picrosides I and II, natural iridoids, in PC12D cells.

Natural iridoid, picroside I (beta-D-glucopyranoside, 1a,1b,2,5a,6, 6a-hexahydro-6-hydroxy-1a-(hydroxymethyl)oxireno[4,5]cyclopenta[1, 2-c]pyran-2-yl, 6-(3-phenyl-2-propenoate)) or II (beta-D-glucopyranoside, 1a,1b,2,5a,6, 6a-hexahydro-6-[(4-hydroxy-3-methoxybenzoyl)oxy]-1a-(hydroxymethyl )ox ireno[4,5]cyclopenta[1,2-c]pyran-2-yl) alone did not exhibit neuritogenic activity, but caused a concentration-dependent (>0.1 microM) enhancement of nerve growth factor (NGF, 2 ng/ml)-induced neurite outgrowth from PC12D cells. The picroside-induced enhancing action of NGF was abolished by GF109203X (2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(indol-3-yl)maleimide) (0.1 microM), a protein kinase C inhibitor. Furthermore, PD98059 (2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one) (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, completely blocked the picroside-induced enhancement of neurite outgrowth in the presence of NGF (2 ng/ml), suggesting that picrosides activate the MAP kinase-dependent signaling pathway. Interestingly, no increase in the expression of phosphorylated MAP kinase was observed in picroside-treated (60 microM) PC12D cells in the presence of NGF (2 ng/ml). These results suggest that picroside I or II enhances NGF-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the NGF receptor-mediated intracellular MAP kinase-dependent signaling pathway.

Animals↗
PubMed2006

Tuning plasmons on nano-structured substrates for NIR-SERS.

Surface-Enhanced Raman Spectroscopy (SERS) is a very sensitive and selective technique for detecting surface species. Colloidal crystal-templated 'inverse opal' nanostructured gold films have been demonstrated to be excellent SERS substrates by various researchers around the globe. However, visible excitation laser sources commonly used in SERS experiments can cause photochemical reactions on the surface as well as fluorescence from the adsorbed molecules. A way to circumvent this possibility is the use of Near Infra-Red (NIR) laser sources. This demands appropriate design of substrates for NIR-SERS in order to obtain maximum enhancement of signals from analytes. In the current paper, we use systematic variation of sphere size and electrochemical control over film height to tune plasmons on such nanovoid substrates. We use plasmon maps as a tool for predicting NIR-SERS enhancements recorded with a 1064 nm laser source for benzenethiol as the probe molecule. Direct correlation is observed between Raman enhancements and plasmonic resonances with ingoing and outcoming radiation. Our study demonstrates the feasibility of plasmon engineering and the predictive power of their mapping on our substrates. It also demonstrates the ability to design reproducible NIR-SERS substrates and its empirical fruition.

Gold↗
PubMed2000

The proto-oncogene c-Cbl is a positive regulator of Met-induced MAP kinase activation: a role for the adaptor protein Crk.

Hepatocyte growth factor triggers a complex biological program leading to invasive cell growth by activating the c-Met receptor tyrosine kinase. Following activation, Met signaling is elicited via its interactions with SH2-containing proteins, or via the phosphorylation of the docking protein Gab1, and the subsequent interaction of Gab1 with additional SH2-containing effector molecules. We have previously shown that the interaction between phosphorylated Gab1 and the adaptor protein Crk mediates activation of the JNK pathway downstream of Met. We report here that c-Cbl, which is a Gab1-like docking protein, also becomes tyrosine-phosphorylated in response to Met activation and serves as a docking molecule for various SH2-containing molecules, including Crk. We further show that Cbl is similarly capable of enhancing Met-induced JNK activation, and several lines of experimentation suggests that it does so by interacting with Crk. We also show that both Cbl and Gab1 enhance Met-induced activation of another MAP kinase cascade, the ERK pathway, in a Crk-independent manner. Taken together, our studies demonstrate a previously unidentified functional role for Cbl in Met signaling and suggest that Met utilizes at least two docking proteins, Gab1 and Cbl, to activate downstream signaling pathways. Oncogene (2000) 19, 4058 - 4065.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Optical mapping reveals developmental dynamics of Mg2+-/APV-sensitive components of glossopharyngeal glutamatergic EPSPs in the embryonic chick NTS.

To examine whether there are any differences in functional organization between the glossopharyngeal nerve (N. IX)- and vagus nerve (N. X)-projecting areas in the nucleus of the tractus solitarius (NTS), we performed optical recording of neural responses evoked by N. IX stimulation in 5- to 9-day-old embryonic chick brain stem preparations and compared the results with those in our previous studies concerning the N. X-related NTS. First, we investigated DL-2-amino-5-phosphonovaleric acid (APV)/Mg2+ sensitivity of the glutamatergic excitatory postsynaptic potentials (EPSPs) in the N. IX-related NTS. In 7- to 9-day-old preparations, we found regional differences in the degree of both the APV-induced reduction and Mg2+-free-induced enhancement of the EPSPs. We constructed developmental maps of spatial patterns of the APV- and Mg2+-sensitive components and showed that functional expression of the N-methyl-D-aspartate (NMDA) receptor dynamically changed during development. Second, we studied initial expression of synaptic functions in the N. IX-related NTS. In 6-day-old preparations, although action potentials alone were usually detected in normal Ringer solution, small EPSPs were elicited in a Mg2+-free solution. This result suggests that the NMDA receptor-mediated synaptic function is latently generated in the N. IX-related NTS at the 6-day-old embryonic stage and that external Mg2+ regulates the onset of synaptic functions. Developmental patterns of APV/Mg2+ sensitivity and the stage of initial expression of the glossopharyngeal EPSP were similar to those of the N. X, suggesting that the developmental sequence of the synaptic function in the NTS is the same for the N. IX- and N. X-related NTS.

2-Amino-5-phosphonovalerate↗
PubMed1999

SB 203580, an inhibitor of p38 mitogen-activated protein kinase, enhances constitutive apoptosis of cytokine-deprived human eosinophils.

The role of p38 mitogen-activated protein (MAP) kinase, and extracellular-regulated protein kinase -1 and -2 in regulating constitutive apoptosis and interleukin (IL)-5-induced survival of human eosinophils have been investigated. Two populations of donors were identified whose eosinophils, in the absence of exogenous cytokines, underwent apoptosis at different rates. Eosinophils were thus arbitrarily classified as either "fast"- or "slow"-dying cells, where greater or less than 15% of the cells were apoptotic at 2 days, respectively. The selective p38 MAP kinase inhibitor, SB 203580, increased constitutive eosinophil apoptosis in both populations (EC(50) approximately 2 microM) as evinced from morphological analysis, flow cytometry, and DNA laddering. The ability of SB 203580 to kill eosinophils was not due to nonspecific toxicity or through the inhibition of prostanoid or leukotriene production. Exposure of eosinophils to IL-5, at a concentration (10 pM) that enhanced survival maximally, abolished SB 203580-induced apoptosis. In contrast PD 098059, which selectively blocks MAP kinase kinase (MEK) 1, did not affect apoptosis of fast- or slow-dying eosinophils, or the enhanced survival of cells effected by IL-5. Collectively, these results suggest that: 1) the basal activity of p38 MAP kinase may regulate the survival of cytokine-deprived eosinophils through inhibition of apoptosis, 2) the enhancement of eosinophil survival effected by IL-5 is mediated by a mechanism(s) divorced from the activation of p38 MAP kinase, and 3) neither spontaneous eosinophil apoptosis nor their enhanced survival by IL-5 involves the activation of MEK-1.

Apoptosis↗
PubMed1992

PFGE-resolved RFLP analysis and long range restriction mapping of the DNA of Arabidopsis thaliana using whole YAC clones as probes.

The cleavage patterns of 23 rare-cutting restriction endonucleases (rcREs) on high molecular weight DNA, isolated from leaves of Arabidopsis thaliana (Arabidopsis), have been analysed using pulsed field gel electrophoresis (PFGE). The DNA digested with rcREs can be used for restriction fragment length polymorphism (RFLP) analysis. We show that RFLPs are more readily identified in restriction fragments that require resolution by PFGE than in smaller restriction fragments. Taking advantage of the low dispersed repetitive DNA content of the Arabidopsis genome, whole yeast artificial chromosomes (YACs) were used as probes to PFGE resolved genomic DNA. This enabled whole YAC clones to be used as RFLP markers and long range restriction maps to be constructed. These techniques should enhance the analysis of regions of the genome of Arabidopsis (and other organisms with low levels of dispersed repetitive DNA) that are the subject of chromosome walking strategies to isolate particular loci.

Arabidopsis↗