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PubMed1996

Noxious colorectal distention induced-c-Fos protein in limbic brain structures in the rat.

Colorectal distention is a non-invasive stimulus used to study visceral pain processing in the nervous system. In this study, immunocytochemical labeling for the immediate-early gene, c-Fos, was used to map limbic brain structures involved in processing visceral pain. Rats received noxious colorectal distention while loosely restrained or loose restraint without distention (control). The brains were immunostained and the density of Fos-labeled nuclei within areas of the brain associated with limbic function were examined. Many cortical (cingulate; retrosplenial; insular; perirhinal, entorhinal) and subcortical (periaqueductal gray; locus coeruleus; lateral parabrachial area; paraventricular, anterodorsal and centromedian thalamic nuclei; lateral septal area; dorsomedial hypothalamus; cortical amygdala; subiculum) areas were labeled in the control rats, but significantly more Fos was observed in these areas following noxious colorectal distention (CRD). Additional areas were labeled following CRD but not restraint (e.g. infralimbic and prelimbic cortices; mediodorsal thalamic nucleus; central amygdaloid nucleus). The results show that noxious visceral stimuli result in Fos expression in limbic structures that exceeds that induced by restraint stress, suggesting that different pathways and circuits are recruited by stimuli which can produce similar emotional responses.

Animals↗
PubMed2001

Prism adaptation to rightward optical deviation improves postural imbalance in left-hemiparetic patients.

Left-hemiparetic patients show predominant postural imbalance as compared to right-hemiparetic patients. The right hemisphere is crucial for generating internal maps used for perceptual and premotor processing of spatial information. Predominant postural imbalance with right-brain damage could thus result from a distortion of an internal postural map. Well-known manifestations of distorted internal maps due to right-hemisphere lesions, such as hemineglect, may show improvement following prism adaptation shifting the visual field to the right. We therefore investigated the effect of prism adaptation on postural imbalance in left-hemiparetic patients. Three groups of five patients were either adapted to prisms deviating the visual field to the right or left or exposed to neutral prisms while performing reaching movements of the right arm. Postural imbalance was reduced only following prism adaptation to the right. Thus, brief adaptation (i.e., 3 min) to rightward-shifting prisms can dramatically improve postural imbalance. This result shows that the effect of exposure to prisms that horizontally shift the visual field to the right in a reaching task generalizes to the postural system, and it suggests an interaction between horizontal and vertical reference frames. This also supports the theory that predominant postural imbalance in patients with right-brain damage may be partly related to a distortion of an internal postural map.

Adult↗
PubMed2002

Multielectrode venous catheter mapping as a high quality constraint for electrocardiographic inverse solution.

A persistent challenge in solving inverse problems in electrocardiography is the application of suitable constraints to the calculation of cardiac sources. Whether one formulates the inverse problem in terms of epicardial potentials or activation wavefronts, the problem is physically ill-posed and hence results in numerically unstable computations. Suitable physiological constraints applied with appropriate weighting can recover useful inverse solutions. However, it is often difficult to determine the best possible constraints and their optimal weighting. We have recently begun to use multielectrode catheters as a means of mapping epicardial signals in animal models. To accommodate the sparse sampling of this venous catheter based approach, we have applied statistical signal processing methods to estimate complete epicardial maps of activation time and epicardial potentials. Such measurements--and the estimated maps from them--also have the potential to provide high quality constraints for electrocardiographic inverse problems because they provide direct--albeit sparse--access to the desired solution. In this presentation we describe several approaches we have applied to extract useful constraints from sparsely sampled epicardial signals as well as a training set of epicardial maps, and use them to improve the quality of computed inverse solutions. Results suggest that combining various information sources provides valuable constraint information. Such a multimodal approach to cardiac mapping is clinically and technically viable and offers a possible means to overcome a major remaining limitation of inverse electrocardiography.

Animals↗
PubMed2006

Insertional mutagenesis in C. elegans using the Drosophila transposon Mos1: a method for the rapid identification of mutated genes.

One benefit of the nematode Caenorhabditis elegans as a model system is the ease to conduct forward genetic screens and to isolate mutants with phenotypes of interest. However, identifying the mutated genes requires positional cloning, which can be laborious and time consuming. Insertional mutagenesis with a heterologous transposon bypasses the mapping steps and expedites the process of identifying the mutated genes. The Drosophila transposon Mos1 can be mobilized in the C. elegans germline to cause mutations. Mutagenic insertions are subsequently localized within the genome using inverse polymerase chain reaction. The mutagenicity of this technique is roughly one order of magnitude lower than chemical mutagens. However, the molecular identification of the mutated genes is extremely rapid. Therefore, before using Mos1-mediated mutagenesis, one must evaluate the trade-off between time spent screening for mutants vs time spent mapping and rescuing a mutation.

Animals↗
PubMed2000

Coexistence of linear zones and pinwheels within orientation maps in cat visual cortex.

Revealing the layout of cortical maps is important both for understanding the processes involved in their development and for uncovering the mechanisms underlying neural computation. The typical organization of orientation maps in the cat visual cortex is radial; complete orientation cycles are mapped around orientation singularities. In contrast, long linear zones of orientation representation have been detected in the primary visual cortex of the tree shrew. In this study, we searched for the existence of long linear sequences and wide linear zones within orientation preference maps of the cat visual cortex. Optical imaging based on intrinsic signals was used. Long linear sequences and wide linear zones of preferred orientation were occasionally detected along the border between areas 17 and 18, as well as within area 18. Adjacent zones of distinct radial and linear organizations were observed across area 18 of a single hemisphere. However, radial and linear organizations were not necessarily segregated; long (7.5 mm) linear sequences of preferred orientation were found embedded within a typical pinwheel-like organization of orientation. We conclude that, although the radial organization is dominant, perfectly linear organization may develop and perform the processing related to orientation in the cat visual cortex.

Animals↗
PubMed1997

Atm-dependent interactions of a mammalian chk1 homolog with meiotic chromosomes.

BACKGROUND: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest. RESULTS: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1(Hu/Mo) has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22-23, a region marked by frequent deletions and loss of heterozygosity in human tumors. CONCLUSIONS: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor suppressor gene.

Amino Acid Sequence↗
PubMed2006

Functional mapping - how to map and study the genetic architecture of dynamic complex traits.

The development of any organism is a complex dynamic process that is controlled by a network of genes as well as by environmental factors. Traditional mapping approaches for analysing phenotypic data measured at a single time point are too simple to reveal the genetic control of developmental processes. A general statistical mapping framework, called functional mapping, has been proposed to characterize, in a single step, the quantitative trait loci (QTLs) or nucleotides (QTNs) that underlie a complex dynamic trait. Functional mapping estimates mathematical parameters that describe the developmental mechanisms of trait formation and expression for each QTL or QTN. The approach provides a useful quantitative and testable framework for assessing the interplay between gene actions or interactions and developmental changes.

Animals↗
PubMed2004

A nearest-neighboring-end algorithm for genetic mapping.

MOTIVATION: High-throughput methods are beginning to make possible the genotyping of thousands of loci in thousands of individuals, which could be useful for tightly associating phenotypes to candidate loci. Current mapping algorithms cannot handle so many data without building hierarchies of framework maps. RESULTS: A version of Kruskal's minimum spanning tree algorithm can solve any genetic mapping problem that can be stated as marker deletion from a set of linkage groups. These include backcross, recombinant inbred, haploid and double-cross recombinational populations, in addition to conventional deletion and radiation hybrid populations. The algorithm progressively joins linkage groups at increasing recombination fractions between terminal markers, and attempts to recognize and correct erroneous joins at peaks in recombination fraction. The algorithm is O (mn3) for m individuals and n markers, but the mean run time scales close to mn2. It is amenable to parallel processing and has recovered true map order in simulations of large backcross, recombinant inbred and deletion populations with up to 37,005 markers. Simulations were used to investigate map accuracy in response to population size, allelic dominance, segregation distortion, missing data and random typing errors. It produced accurate maps when marker distribution was sufficiently uniform, although segregation distortion could induce translocated marker orders. The algorithm was also used to map 1003 loci in the F7 ITMI population of bread wheat, Triticum aestivum L. emend Thell., where it shortened an existing standard map by 16%, but it failed to associate blocks of markers properly across gaps within linkage groups. This was because it depends upon the rankings of recombination fractions at individual markers, and is susceptible to sampling error, typing error and joint selection involving the terminal markers of nearly finished linkage groups. Therefore, the current form of the algorithm is useful mainly to improve local marker ordering in linkage groups obtained in other ways. AVAILABILITY: The source code and supplemental data are http://www.iubio.bio.indiana.edu/soft/molbio/qtl/flipper/ CONTACT: ccrane@purdue.edu.

Algorithms↗
PubMed2002

Fast MRI of RF heating via phase difference mapping.

A method is presented for the rapid acquisition of temperature maps derived from phase difference maps. The temperature-dependent chemical shift coefficients (TDCSCs) of various concentrations of aqueous cobalt and dysprosium-based compounds were measured. The largest TDCSC calculated was for 100 mM DyEDTA, which had a TDCSC of -0.09 PPM/K; 160 mM CoCl2 had a TDCSC of -0.04 PPM/K. These temperature-dependent chemical shifts (TDCSs) result in phase changes in the MR signal with changing temperature. Agarose phantoms were constructed with each paramagnetic metal. A fast gradient-echo (FGRE) MR image was acquired to serve as the baseline image. A "test" MRI procedure was then performed on the phantom. Immediately afterwards, a second FGRE MR image was acquired, serving as the probing image. Proper image processing as a phase difference map between the probing image and the baseline image resulted in an image which quantitatively described the temperature increase of the phantom in response to a particular "test" imaging experiment. Applications of this technique in assessing the safety of pulse sequences and MR coils are discussed.

Biophysical Phenomena↗
PubMed1998

The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic.

Bailie et al. [In Vitro Cell Dev. Biol. (1992) 28A, 621-624] reported that primary cultures of rat hepatocytes possess low affinity binding sites for nerve growth factor (NGF). NGF treatment of primary cultures of rat hepatocytes with a maximally effective concentration of NGF (20 ng/ml, 0.8 nM) caused acute phasic activation of Raf-1 and p42(MAPkinase), and a smaller sustained activation of B-Raf. The transient increase in Raf-1 and p42(MAPkinase) activity returned to baseline within approximately 30 min. NGF treatment of hepatocytes did not induce expression of cyclin dependent kinase (cdk) inhibitor proteins, but instead stimulated cdk2 activity and increased [3H]thymidine incorporation into DNA. In contrast to hepatocytes, NGF treatment of PC12 pheochromocytoma cells caused large sustained activations of B-Raf and p42(MAPkinase), and a lower phasic activation of Raf-1. The sustained activations of B-Raf and p42(MAPkinase) were for more than 5 h. Treatment of PC12 cells with NGF increased p21(Cip1/WAF-1) expression, reduced cdk2 activity and inhibited DNA synthesis, the opposite to the effects of NGF treatment of hepatocytes. However when p42(MAPkinase) was chronically activated in hepatocytes, via infection with an inducible oestrogen receptor-Raf-1 fusion protein, expression of p21(Cip-1/WAF1) and p16(INK4a) cdk inhibitor proteins increased, cdk2 activity decreased, and DNA synthesis decreased. Equally, treatment of hepatocytes with 50 mM ethanol elevated the basal activity of p42(MAPkinase) and temporally extended the ability of NGF treatment to activate p42(MAPkinase). Ethanol and NGF co-treatment increased expression of p21(Cip-1/WAF1) and p16(INK4a) cdk inhibitor proteins and decreased hepatocyte DNA synthesis. These data demonstrate that NGF can cause either acute/phasic or sustained activation of the MAP kinase cascade in different cell types. Acute activation of the MAP kinase cascade correlated with increased DNA synthesis. In contrast, sustained activation of the MAP kinase cascade correlated with increased expression of cdk inhibitor proteins, a reduction in cdk activity, and an inhibition of DNA synthesis. These data suggest a general mechanism exists where acute activation of the MAP kinase cascade promotes G1 progression/S phase entry and that chronic activation of the MAP kinase cascade inhibits this process.

Animals↗
PubMed2000

Dendritic cytoskeletal protein expression in mental retardation: an immunohistochemical study of the neocortex in Rett syndrome.

Many syndromes associated with mental retardation (MR) are characterized by cortical dendritic anomalies. Despite their morphological similarity, these changes appear to involve different stages of dendritic development. The neuronal cytoskeleton, which includes microfilaments, neurofilaments and microtubules, is essential for these developmental processes. Levels and phosphorylation of microtubule-associated proteins (MAPs), which stabilize microtubules, seem to determine different stages of dendritic formation with certain MAPs (e.g. MAP-2) appearing to mediate the effects of external modulators upon these processes. Early studies on neuronal cytoskeleton in MR, which have shown a selective reduction in MAP-2 expression, have focused on Rett syndrome (RS). Here, by a semiquantitative immunohistochemical analysis of the pericentral cortex, we examine the contribution of specific neuronal populations to these changes in cytoskeletal proteins. Decreased MAP-2 staining in RS was more marked in layers V-VI, while increased nonphosphorylated neurofilament immunoreactivity was found in layers II-III in RS. Age-related increases in dendritic MAP-2 immunoreactivity in layers V-VI were also absent in RS. The specificity of these cytoskeletal protein changes, their significance for RS pathogenesis and plasticity, as well as their implications for other MR-associated disorders, are also discussed.

Adolescent↗
PubMed2006

Selection of currently relevant words: an auditory verbal memory study using positron emission tomography.

Clinical and imaging studies have shown that the posterior and anterior parts of the limbic system make distinct contributions to memory processing. Whereas the medial temporal lobes and connected structures are important for learning and recognition of new information, the posterior orbitofrontal cortex appears to be crucial for the selection of currently relevant memories, that is, the ability to distinguish between memories that pertain to ongoing reality and memories that do not. Previous studies demonstrating this dissociation used repeated continuous recognition tasks with visual stimuli. In this H2O positron emission tomography study, we demonstrate that this same organizing principle also applies to auditory verbal information, that is, to the processing of auditorily presented words.

Acoustic Stimulation↗
PubMed1997

Registration of neural maps through value-dependent learning: modeling the alignment of auditory and visual maps in the barn owl's optic tectum.

In the optic tectum (OT) of the barn owl, visual and auditory maps of space are found in close alignment with each other. Experiments in which such alignment has been disrupted have shown a considerable degree of plasticity in the auditory map. The external nucleus of the inferior colliculus (ICx), an auditory center that projects massively to the tectum, is the main site of plasticity; however, it is unclear by what mechanisms the alignment between the auditory map in the ICx and the visual map in the tectum is established and maintained. In this paper, we propose that such map alignment occurs through a process of value-dependent learning. According to this paradigm, value systems, identifiable with neuromodulatory systems having diffuse projections, respond to innate or acquired salient cues and modulate changes in synaptic efficacy in many brain regions. To test the self-consistency of this proposal, we have developed a computer model of the principal neural structures involved in the process of auditory localization in the barn owl. This is complemented by simulations of aspects of the barn owl phenotype and of the experimental environment. In the model, a value system is activated whenever the owl carries out a foveation toward an auditory stimulus. A term representing the diffuse release of a neuromodulator interacts with local pre- and postsynaptic events to determine synaptic changes in the ICx. Through large-scale simulations, we have replicated a number of experimental observations on the development of spatial alignment between the auditory and visual maps during normal visual experience, after the retinal image is shifted through prismatic goggles, and after the reestablishment of normal visual input. The results suggest that value-dependent learning is sufficient to account for the registration of auditory and visual maps of space in the OT of the barn owl, and they lead to a number of experimental predictions.

Animals↗
PubMed2004

The contribution of spatial remapping impairments to unilateral visual neglect.

Left visual neglect following right hemisphere damage is a heterogeneous phenomenon, in which several underlying impairments have been identified. Despite recent advances in understanding the neural and cognitive bases of these impairments, current theories of neglect, particularly those that emphasise attentional deficits, do not explain a number of phenomena, including: 'Ipsilesional' neglect after left orienting. Positive or 'productive' manifestations. Spatial transposition errors. Mislocalisations. Revisiting behaviour during visual search. Lack of awareness for objects toward the contralesional side of space. We propose that these manifestations of neglect can be accounted for by an additional underlying disorder of spatial remapping due to parietal dysfunction. In primary visual areas, retinotopic maps are renewed and thus overwritten at each new ocular fixation. Remapping processes operating in higher-level oculocentric visual maps of the parietal cortex ensure visual integration of these successive retinal images over time and space, by creating a constantly updated representation of stimulus locations in terms of distance and direction from the fovea. They consist in the storage, refreshment and re-localization of the different components of the visual scene that are successively attended during its exploration, and provide spatial constancy of visual perception and a spatial buffer for working memory [Cereb Cortex 5 (1995) 470; Visual Cogn 7 (2000) 17]. We begin this article by reviewing theoretical and experimental arguments that have highlighted the importance of parietal remapping processes in maintaining an accurate representation of space across saccadic shifts. We then focus on findings from the double-step saccade task, [Ann Neurol 38 (1995) 739] as a basis for our model of the role of remapping impairments in many of the symptoms of neglect. From these results, remapping impairments would be demonstrated when a saccade has to be guided across the midline after having fixated an object in either the left or right visual field for patients with either left- or right-side parietal lesions. In addition, patients with right-side lesions will have remapping impairments within the left visual field following a saccade to a left-side target (see Fig. 5). In a large part of the article, we seek to build our hypothesis based on this basic model and more speculative assumptions supported with extensive evidence from the literature.

Attention↗
PubMed1989

X-ray-induced specific locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. III. Genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions by means of complementation tests.

Genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions was made by means of complementation tests on all possible pairwise combinations of 50 X-ray-induced irreparable adenine-3 mutants (designated ad-3IR). All mutants were induced in either heterokaryon 11 or heterokaryon 12 of Neurospora crassa, 2-component heterokaryons heterozygous for mutants at the 3 closely linked loci ad-3A and ad-3B and nic-2 (nicotinamide-requiring) located about 5.0 map units distal to ad-3B. The complementation tests involved mutants of the following genotypes: 15 ad-3A, 27 ad-3B, 7 ad-3A ad-3B nic-2 and 1 ad-3B nic-2. To facilitate mapping, 5 additional strains (each consisting of a gene/point mutation at the ad-3A or ad-3B locus and a separate site of closely linked recessive lethal damage in the immediately adjacent regions [designated ad-3R + RLCL]) were also included. The data from these complementation tests showed that the majority (46/50) of X-ray-induced irreparable ad-3 mutants mapped as a series of overlapping multilocus deletions that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential function) between ad-3A and ad-3B. The remaining mutants (4/50) were found to result from a series of closely linked, but separate, mutations (designated multilocus mutations) of the type ad-3IR + RLCL, different from those found in previous studies (de Serres, 1968; de Serres and Brockman, 1968). The data from the present complementation tests have expanded the process of genetic fine structure mapping of the ad-3 and immediately adjacent regions (de Serres, 1968) and defined the presence of the following 11 genetic loci: (a) 4 loci (with either known [i.e. col-1t] or unknown [i.e. unknA]) function proximal to ad-3A: unknA, unknB, col-1t, and col-2t, (b) 4 loci in the 'X' region: unknC, unknD, unknE, and unknF, (c) 2 loci distal to ad-3B: unknG, col-3t, and (d) 1 locus distal to nic-2: unknH.

Adenine↗
PubMed1975

An analysis of the spatial distribution of ciliary units in a ciliate, Euplotes minuta.

Six to eleven longitudinal ciliary rows are arrayed over the dorsal surface of the ciliate Euplotes minuta. Forty-two to 129 ciliary units are distributed among these rows. The number of rows depends on genotype, clonal age, and vegetative ancestry, while the total number of units is controlled partly by the number of rows and also by the separate action of genetic and environmental factors. 2. The pattern of distribution of units among different rows of non-dividing cells can be analysed on the basis of the percentage of the total unit complement of the cell that is found in each individual row. If the assumption is made that ciliary rows are uniformly spaced over a dorsal field whose width is independent of the number of rows, then it can be shown that units are distributed among rows according to a relatively invariant spatial pattern. The form of this pattern remains the same in the face of variation in the absolute number of rows and of units. 3. Prior to cell division new units develop anterior and posterior to old units situated within the equatorial zone of each row. About one-half of the original units are included within this zone. The cell fission line develops within this zone such that the total number of units passed to the anterior and posterior division products are about equal. 4. The pattern according to which units of different zones (proliferating and non-proliferating) are distributed among different rows has been mapped in cells that have completed the process of proliferation of units but have not yet completed cell division. The results of this mapping show that the pattern of distribution of units in the equatorial zone at the conclusion of proliferation is not the same as the overall pattern in non-dividing cells. Further analysis indicates that the geometry of proliferation can be most simply represented as a result of two superimposed processes, one of which is the recruitment of old units into the zone of proliferation, while the other is the intensity of proliferation, i.e. the number of new units formed adjacent to each old unit. Both recruitment and intensity have constant values in the central region of the dorsal field, while recruitment is higher and intensity lower near both margins. The recruitment and intensity distributions are mutually nearly reciprocal, with slight asymmetries that formally account for the more dramatic asymmetry of the pattern of non-dividing cells. 5. A dualistic hypothesis is formulated for the control of the formation of new ciliary units within ciliary rows. The position of each new unit is largely controlled locally in relation to pre-existing units, while the decision of whether or not new units will develop at all, and how many will be formed, depends on superimposed positional systems operating within the context of the entire dorsal surface.

Cell Division↗
PubMed2004

Different cortical activations for subjects using allocentric or egocentric strategies in a virtual navigation task.

Subjects were required to navigate through a virtual 3D labyrinth presented on a screen while fMRI images were obtained. Contrasting the fMRI images obtained during the navigation trials with appropriate control conditions revealed a bilateral network comprising the parietal lobe (including the intraparietal sulcus) and various lateral and medial premotor areas. The subjects using an allocentric strategy showed stronger activation in the medial temporal areas including the parahippocampal region, the hippocampus, and the thalamus. In addition, the cerebellum was also active in those subjects. We believe that this activation pattern is related to visually guided memory retrieval based on generalized spatial maps. The stronger activation in the thalamic-basal ganglia-cerebellar-loop points to a more automatic support of memory and attentional processes possibly supporting memorization of spatial maps.

Adult↗
PubMed2006

Gene identification and cloning by molecular marker mapping.

In the course of map-based cloning, mutant genes are identified through linkage to a sufficiently small region of the genetic map and subsequent DNA sequencing. This process has become fairly straightforward for Arabidopsis mutations, owing to the completed genome sequence and the discovery of many thousands of molecular markers. Initially, plants with the desired phenotype are identified in populations treated with ethylmethanesulfonate or other mutagens. Once the mutant phenotype has been discovered and confirmed, map-based identification of the mutated gene is a four-stage process: (1) mutant plants are crossed to another Arabidopsis ecotype and F2 seeds are generated; (2) approx 50 homozygous mutant F2 plants are genotyped to determine linkage to four or five molecular markers on each of the five chromosomes; (3) A larger F2 population (1000 to 2000 plants) is grown, genotyped, and phenotyped to determine fine-scale genetic linkage, ideally narrowing to a chromosomal region of about 40 kbp; and (4) sequencing of mutant and wild-type DNA is used to verify the identity of the mutated gene. Given a mutant phenotype that can be determined unambiguously in a single F2 plant, it is possible to complete an Arabidopsis map-based cloning project in about 1 yr.

Arabidopsis↗