Results of previous autopsy studies indicate that increased hepatic iron stores or hepatic iron overload is common in African Americans dying in hospitals, but there are no reports of hepatic iron content in other cohorts of African Americans.We investigated the prevalence of heavy liver iron deposition in African American adults. Using established histochemical criteria, we graded Perls' acid ferrocyanide-reactive iron in the hepatocytes and Kupffer cells of 341 consecutive African American adults who were autopsied in the coroner/medical examiner office. Heavy staining was defined as grade 3 or 4 hepatocyte iron or grade 3 Kupffer cell iron.There were 254 men and 85 women ; gender was unstated or unknown in two subjects. Approximately one-third of subjects died of natural causes. Heavy staining was observed in 10.2% of men and 4.7% of women. 23 subjects had heavy hepatocyte staining only, six had heavy Kupffer cell staining only, and one had a mixed pattern of heavy staining. 15 subjects had histories of chronic alcoholism; three had heavy staining confined to hepatocytes. We analyzed the relationships of three continuous variables (age at death in years, hepatocyte iron grade, Kupffer cell iron grade) and two categorical variables ) in all 341 subjects using a correlation matrix with Bonferroni correction. This revealed two positive correlations: hepatocyte with Kupffer cell iron grades (p < 0.01), and male sex with hepatocyte iron grade (p < 0.05). We also analyzed the relationship of steatosis, inflammation, and fibrosis/cirrhosis in 30 subjects with heavy iron staining using a correlation matrix with Bonferroni correction. There were significant positive correlations of steatosis with inflammation , and of inflammation with fibrosis/cirrhosis .The present results confirm and extend previous observations that heavy liver iron staining is relatively common in African Americans. The pertinence of these observations to genetic and acquired causes of iron overload in African Americans is discussed. Hepatic iron overload was detected by Perls' acid ferrocyanide staining and atomic absorption spectrometry at autopsy in more than one percent of African American adults who died in hospitals ,2. In onHFE-associated hemochromatosis [HFE-associated hemochromatosis.In contrast, screening programs that included cohorts of African Americans presumably representative of the general African American population identified a much lower proportion of subjects with possible iron overload -5 than iomatosis -5. In thomatosis -5. TakenThus, we graded Perls' acid ferrocyanide-reactive iron in the livers of 341 consecutive African American adults who underwent autopsy in the coroner/medical examiner office. We selected this population for study because such subjects are more representative of the general African American population than persons who died in hospital. We then compared these observations with autopsy results reported previously in African Americans who died in hospitals . The perThe performance of this study was approved by the Institutional Review Board of the University of Alabama at Birmingham and by the Coroner/Medical Examiner's Office of Jefferson County, Alabama. We evaluated formalin-fixed tissue obtained in 361 consecutive, unselected coroner/medical examiner autopsy cases of African-American adults (age >18 years) in Jefferson County, Alabama; deaths in all of the present cases occurred during the interval 1998 – 2002. Each subject was identified as African American by his/her previous medical histories or legal records, or by the coroner/medical examiner staff. Most autopsies were performed on the same day that the respective bodies were available for evaluation at the Coroner/Medical Examiner's Office; other autopsies were completed within ~ 18 hours. All tissues were placed directly in fixative at the time of collection at autopsy. Liver was not available or was not interpretable due to autolysis in 20 cases. Available records in each case were reviewed; age at death, sex, summary of known illnesses, and cause of death were tabulated for all cases for which liver tissue was available.Tissues obtained at autopsy were routinely fixed in 10% neutral buffered formalin. Triplicate sections of paraffin-embedded liver were prepared. One section was stained with hematoxylin and eosin, another with Perls' acid ferrocyanide technique to demonstrate non-heme ferric iron , and a tAll slides were simultaneously reviewed by three investigators, and the iron grades assigned in each case represent their consensus opinions. Hepatocellular iron was graded according to these criteria: grade 0 – no visible iron; grade 1 – iron visible in very few hepatocytes; grade 2 – iron visible in 5 – 10% of hepatocytes; grade 3 – iron visible in ≥ 40% of hepatocytes; and grade 4 – abundant iron visible in most hepatocytes . KupfferWe designated a gradient of stainable iron in hepatocytes from the periportal area decreasing towards the hepatic venule as present or absent; visualization of a gradient required hepatocyte iron staining of grade ≥ 2 ,10. The ®, Microsoft Corp., Redmond, WA), and a statistical program (GB-Stat® v. 10.0, 2003, Dynamic Microsystems, Inc., Silver Spring, MD). Descriptive data are displayed as enumerations, percentages, mean ± 1 S.D., medians, and ranges. Frequency values were compared using chi-square analysis or Fisher exact test, as appropriate. Mean values were compared using student t-test. Some data were analyzed using a correlation matrix with Bonferroni correction. Values of p < 0.05 were defined as significant.The present data set consisted of observations in 341 subjects and their respective livers. Analyses were performed with a computer spreadsheet , and 85 women . The mean age of men was lower than that of women (p = 0.0255). There were two subjects for whom gender was unstated or unknown. Approximately one-third of subjects died of natural causes . However, the mean hepatocyte iron grade was 0.83 ± 0.96; the mean Kupffer cell iron grade was 0.32 ± 0.32.Increased hepatocyte iron grades were observed in 64 men 25.2%) and 8 women (9.4%) (p = 0.0021). Increased Kupffer cell iron grades were observed in 20 men (7.9%) and one woman (1.2%) (p = 0.0266) (Tables 5.2% and Heavy iron staining was observed in 30 subjects (8.8%), including 10.2% of men and 4.7% of women (p = 0.1202). The mean age of men and women with heavy iron staining was similar: 45 ± 15 y and 47 ± 9 y (p = 0.7389). 24 subjects (7.1%) had grade 3 or 4 hepatocyte iron; of these, one also had grade 3 Kupffer cell iron. Seven subjects (2.1%) had grade 3 Kupffer cell iron; of these, one also had grade 3 hepatocyte iron. Altogether, 23 subjects had heavy iron staining in hepatocytes only  and two categorical variables ) using a correlation matrix with Bonferroni correction. This revealed two significant positive correlations: hepatocyte iron grade with Kupffer cell iron grade (p < 0.01), and male sex with hepatocyte iron grade (p < 0.05).These subjects were comprised of 26 men and 4 women Table . HepaticWe analyzed the relationship of hepatocyte and Kupffer cell iron grades, steatosis, inflammation, and fibrosis/cirrhosis in these 30 subjects .Three persons who had histories of chronic alcoholism also had hepatic cirrhosis. A 36 year-old man had grade 0 hepatocyte and grade 0 Kupffer cell iron. A 46 year-old man had grade 2 hepatocyte iron and grade 0 Kupffer cell iron. A 50 year-old woman had grade 4 hepatocyte iron and grade 0 Kupffer cell iron . Three had histories of chronic alcoholism (described above). One of these three subjects, a 50 year-old woman, had grade 4 hepatocyte iron and grade 0 Kupffer cell iron  [HJV on Ch1q [ALAS2 on ChX [Primary iron overload in African Americans is often associated with preferential deposition of iron in macrophages in multiple organs ,7,21. InFPN1 ,21,22. T2Y/H63D) ,21, or w on Ch1q  or the e2 on ChX . Other p2 on ChX . HoweverAcquired disorders account for increased hepatic iron deposition in some African Americans. In the present study, we did not observe any subject who had heavy liver iron staining and fibrosis or cirrhosis who did not also have hepatic steatosis or inflammation. Chronic viral hepatitis C occurs in approximately 1.8% of the overall U.S. population , and theIron overload sometimes develops spontaneously or after repeated erythrocyte transfusion in African Americans with heritable or acquired anemia, or with myelodysplasia or acute leukemia ,15,33-41Three of the five present subjects with micronodular cirrhosis had a history of chronic alcoholism. African Americans are at greater risk than whites for developing several alcohol-related conditions, including hepatic cirrhosis ,43. In tWe conclude that heavy liver iron staining is common in African American adults who were autopsied in the coroner/medical examiner office. The different histologic patterns of heavy liver iron staining we observed in the present subjects are consistent with the phenotypic and genotypic heterogeneity of primary iron overload in African Americans  and withThe author(s) declare that they have no competing interests.JCB conceived and designed the study, reviewed and graded the liver specimens, contributed to the statistical analyses of data, and contributed to writing the manuscript. RTA reviewed and graded the liver specimens, contributed to the statistical analyses of data, and contributed to writing the manuscript. AKR reviewed and graded the liver specimens and contributed to writing the manuscript. RMB provided information on the autopsy cases, provided the liver specimens, reviewed the histology of selected liver specimens, and contributed to writing the manuscript. All authors approved of the manuscript in its final form.The pre-publication history for this paper can be accessed here:


Timely and accurate information about the onset of malaria epidemics is essential for effective control activities in epidemic-prone regions. Early warning methods that provide earlier alerts  may permit control measures to interrupt transmission earlier in the epidemic, perhaps at the expense of some level of accuracy.th-degree polynomial distributed lag model. For each week, the numbers of malaria cases were predicted using coefficients obtained using all years except that for which the prediction was being made. The effectiveness of alerts generated by the prediction system was compared against that of alerts based on observed cases. The usefulness of the prediction system was evaluated in cold and hot districts.Expected case numbers were modeled using a Poisson regression with lagged weather factors in a 4The system predicts the overall pattern of cases well, yet underestimates the height of the largest peaks. Relative to alerts triggered by observed cases, the alerts triggered by the predicted number of cases performed slightly worse, within 5% of the detection system. The prediction-based alerts were able to prevent 10–25% more cases at a given sensitivity in cold districts than in hot ones.The prediction of malaria cases using lagged weather performed well in identifying periods of increased malaria cases. Weather-derived predictions identified epidemics with reasonable accuracy and better timeliness than early detection systems; therefore, the prediction of malarial epidemics using weather is a plausible alternative to early detection systems. Malaria epidemics are reported frequently and have caused high morbidity and mortality among all age groups in the African highlands -4. Earlyth percentile of cases from the same week in previous years [Early detection systems, which are used to detect epidemics once they have begun, can correctly identify periods that are defined by expert observers as "epidemic," albeit with varying specificity. A number of such systems have been proposed or implemented. For example, WHO has advocated the use of alerts when weekly cases exceed the 75us years  and otheus years -12. Howeus years  and if tus years .While early detection systems appear to provide timely information about the onset of severe epidemics, they intrinsically trigger alerts only when unusual transmission is already underway. Another approach, known as "early warning," attempts to predict epidemics before unusual transmission activity begins, usually by using weather variables that predict vector abundance and efficiency, and therefore, transmission potential ,15-18. THere, an attempt to combine these avenues of previous work is described, using modified versions of previously described models based on weather factors to provide predictions of Plasmodium falciparum cases in these 10 districts of Ethiopia, and evaluating thresholds that trigger warnings. The hypothesis tested here is that the use of predicted cases  would reduce the precision of the alert thresholds , as the price of obtaining the alerts with greater advance notice. In fact, the early warning system based on predicted cases performed slightly worse in most cases than the early detection system, but the performance was rarely much worse and occasionally slightly better. These comparisons are described and their implications for the choice of malaria prediction/detection systems in epidemic-prone areas of Africa are discussed.P. falciparum. The original data collected on the basis of Ethiopian weeks (where the number of days in each week varies between 5 and 9) were normalized to obtain mean daily cases for each Ethiopian week [Microscopically confirmed malaria cases were collected from a health facility in each of ten districts of Ethiopia over an average of 10 years; this data set has been previously described . Each ofian week .Daily meteorological data  recorded at the local weather stations nearest to the health facility were obtained from the National Meteorological Services Agency (NMSA) for the same period. These daily data were collapsed into weekly data to correspond with the weekly malaria cases. The weekly mean for minimum and maximum temperatures and the total weekly rainfall were calculated from the daily records.th-degree polynomial distributed lag (PDL) model [The expected case numbers for a given week were modeled using a Poisson regression with lagged weather factors, an autoregressive term, a time trend and indicator variables for week of the year. Biological considerations about the interrelationship between weather, mosquito and malaria parasite suggest that malaria cases should follow periods of increased temperature and increased rainfall, at defined intervals -28. ThusL) model  was fittE(stY) denotes expected value for the daily average number of malaria cases at site s on week t; , , t-iR, and st-i Yare the weekly minimum and maximum temperatures, rainfall and autoregressive term i weeks previously; s tand s Wdesignate time trend and week in a year at site s; s αrepresent the intercept, at site s.where For each week at each location in the data set, the number of cases was predicted using equation (1) and data available four weeks prior to the week for which the prediction is made. Coefficients of equation (1) were obtained using all years except that for which the prediction was being made, to avoid circularity. The prediction for week t was then made using this all-but-current-year model with weather and case data for the weeks up to week t-4. The predicted number of cases is thus estimated using the following model: represent the predicted cases for year j; , , , , ,  and  are parameter estimates  from all years except j; , , , jt,  and  are minimum and maximum temperatures, rainfall, time, week and autoregressive term respectively from year j.where In early detection algorithms, actual cases in a given time period are typically compared against some threshold level of cases to determine whether excess cases have been observed. Often, the threshold level represents an upper bound on "normal" case numbers from previous years. If this threshold level is crossed , an alert is generated . Such syIn this study of the usefulness of prediction systems for generating alerts, historically based thresholds were similarly used – weekly percentile (defined as a given percentile of the case numbers obtained in the same week) or weekly mean with standard deviation (defined as the weekly mean plus a defined number of standard deviations) algorithms as threshold levels  – but ge1. Weekly percentile, where sij T= psijQ, where psij Qrepresents the pth (p = 70, 75, 80, 85, 90, or 95) percentile of observations from week i at facility s in years other than j.Threshold is exceeded when 2. Weekly mean with standard deviation., where sij T= sij μ+ Ysijβσ, where β = 0.5, 1.0, 1.5, 2.0, 2.5 or 3.Threshold is exceeded when The effectiveness of alerts generated by our four-week-ahead prediction system was compared against that of alerts based on a detection system using actual cases. Since the prediction system generates predicted numbers of cases four weeks ahead of time, this permits implementation of control measures four weeks earlier than under a detection system. On the other hand, one would expect that the accuracy of prediction might be less than that of detection. The comparisons were designed to assess this trade-off between the ability to act earlier in possible epidemics and the possible loss of accuracy.A method previously described  was usedFor each value of each type of threshold (applied to either the predicted and observed number of cases) at each health facility, the number of PPC was transformed into a proportion (percentage), by adding the number of PPC for the alerts obtained and dividing this sum by the sum, over all weeks in the data set, of the number of potentially prevented cases. Proportion rather than actual cases were used because the numbers of malaria cases vary from district to district. To compare the performance of the predicted and observed cases on a single scale, a curve was plotted for each algorithm showing the mean percent of PPC (%PPC) over all districts versus the average number of alerts triggered per year, with each point representing a particular threshold value. Better methods of generating a warning were those that potentially prevent higher numbers of malaria cases using smaller numbers of alerts.The performance of the alerts provided by both the predicted and observed cases was compared with random and optimally timed alerts. PPC was estimated for alerts chosen on random weeks during the sampling period. To estimate the performance of optimally-timed alerts , the optimal timing of alerts were identified by retrospectively going through data if one had perfect predictive ability; the optimal week for one alert was chosen; then by going through the remaining weeks, the optimal week for a second alert was chosen, and so on. The optimal alert would serve as an upper bound curve for the best choice of alert times, given a defined alert frequency .The relative importance of weather factors in determining malaria transmission significantly depends on the climate of the area. It has recently been shown that although rainfall was significantly associated in cold and hot districts, minimum temperature contributed only in the cold districts of Ethiopia . FurtherThe prediction algorithm indicates the overall pattern of cases well, yet underestimates the height of the largest peaks. Comparisons of the predicted and observed malaria cases, for each week in six of the ten districts, are shown in Figure th percentile or mean + 1.50 standard deviations). The horizontal axis gives the number of alerts per year triggered by the particular threshold value, while the vertical axis shows the %PPC associated with that threshold value. Each point represents the mean across all 10 districts. Two different choices of the function for determining PPC (reducing cases to weekly mean: low-effectiveness, a and c, or weekly mean minus one s.d.: high-effectiveness, b and d) and the choice of window of effectiveness  were considered. The performance of the predicted number of malaria cases using the mean plus (0.5, 1, and 1.5) standard deviation algorithm (for an eight-week window of low-effectiveness) reveals that it prevented 29%/0.9 alerts, 27.3%/0.6 alerts and 24.2%/0.43 alerts per year, which compares with 31.4%/0.85 alerts, 29.8%/0.65 alerts and 27.5%/0.52 alerts per year respectively when the observed cases are used to trigger alerts  indicate that for most  districts, the model performed well qualitatively, in the sense of predicting more cases than expected from the weekly mean when such excess cases occurred, and predicting fewer when in fact fewer cases than the weekly mean occurred. This finding focuses attention on the fact that a system can give timely and accurate alerts for epidemic control, even if it is unable to provide accurate predictions of case numbers Figure . The iniStudies have shown that temperature affects transmission in cold environments more than it does in hot environments ,32. ThusNon-climatic factors such as population immunity, migration and drug resistance are believed to influence malaria transmission and have been cited as causes of malaria epidemics -36. The The model chosen for the prediction of malaria cases was based loosely on a model previously evaluated for its ability to explain seasonal variation in malaria incidence in the same data set . The forP. falciparum cases using lagged weather and case incidence data performed well in identifying periods of increased malaria cases. Furthermore, the prediction system allowed recognition of epidemic periods at an early stage, thereby facilitating interventions making epidemics preventable with adequate lead time. However, this study indicated that early warning system using weather and other predictor variables are more reliable in relatively cold than hot districts. In conclusion, it has been demonstrated that weather derived predictions identified epidemics with reasonable accuracy and better timeliness compared to early detection systems. Therefore, warning systems based on predictions derived from lagged weather variables may be a useful alternative to early detection systems for targeting resources against incipient falciparum malaria epidemics.This study showed that short-term (four-week-ahead) predictions of HDT, ML and JS conceived the study. HT and ML undertook statistical analysis. HT drafted the manuscript, which was revised by ML. JS participated in designing of the study and statistical analysis. AT initiated the study and made data available in collaboration with WHO and Ministry of Health of Ethiopia. All authors contributed to the writing of the manuscript and approved the submitted version of the manuscript.


Ambystoma mexicanum. Analysis of this data has uncovered an unusual phylogenetic distribution of the cyclin dependent kinase inhibitor 1 gene family in amphibians.An EST database has been generated for the axolotl  Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum.The ambystomatid salamander, Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians.Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online. Xenopus laevis and Silurana tropicalis.The Caudata  are a major focus of work in vertebrate evolution and speciation ,2. The sDesmognathus monticola  to nearly 70 billion base pairs for Plethodon vandykei  [Ambystoma mexicanum, a species important for studies in evolution, regeneration and development, has an estimated genome size between 21.9 billion and 48 billion base pairs [A salamander EST project is particularly important as these organisms have extremely large genomes, making a genome project unwieldy and unlikely without specialized approaches such as methylation filtration . Genome amander) . The ambse pairs ,6 and mec [A. mexicanum. We chose this species because of its role in evolutionary, developmental and regeneration studies. A. mexicanum is easily bred in the laboratory, and animals can be obtained from a large, NSF-funded colony [A. mexicanum EST information. This database is also being implemented for EST sequences from a full-length X. laevis cDNA library, and for sequences from a Canis familiaris EST project.Molecular evolution studies of salamanders have relied primarily on mitochondrial genes such as those for ribosomal RNAs and cytochrome c . The lacd colony . We haveTo generate a diverse set of sequences involved in organizing and regenerating the primary body axis, two independent cDNA libraries were used for sequencing. One was derived from dorsal regions of stage 18-22 embryos containing neural tube, somite and notochord - called the 'neural tube' library - the other from 6-day post-amputation regenerating tail blastema. From 18,432 sequencing attempts 17,522 high-quality sequences were obtained after Phred analysis . All seqThe blastema and neural tube libraries were unnormalized and unamplified. We assessed library quality and diversity on the basis of the number of redundant clones in the library. Redundancy was estimated by performing BLASTN searches  against c oxidase subunit I (469 ESTs), 12S rRNA (445 ESTs), nuclear factor 7 Zn-binding protein A33 (332 ESTs), type II keratin (274 ESTs), keratin (211 ESTs) and cytoplasmic beta-actin (206 ESTs) (Table To identify ESTs belonging to the same open reading frames (ORFs), sequences were assembled into contigs using TIGR-Assembler version 2 . The 17,A. mexicanum in the National Center for Biological Information (NCBI) EST databases prior to this work, most of which originate from a sequencing effort of the Voss laboratory , as the top hit with an E-value of 4e-07. This assignment was based on the carboxy-terminal 120 amino acids of the protein, which represents the less conserved region. When we isolated a full-length clone for Am_4671 from our library, we could confirm that it is indeed the axolotl ortholog of cyclin-dependent kinase inhibitor 1B (p27Kip1), as discussed later.To identify putative homologies to known proteins, we subjected the contigs to BLASTX searches against the non-redundant protein database (NR, NCBI) where a cutoff E-value of 1e-05 was used for parsing output files. In our annotation, we used an E-value of 1e-20 as an upper limit to assign significant homology. We note that this does not imply that such sequences are true orthologs. In addition, in cases where no significant homology was found, we used an E-value limit of 1e-05 to designate weak homology. We find this additional category of 'weak homology' useful for data mining. As most contigs do not represent full-length sequences, it is possible that only a highly divergent region of a gene sequence is available in our collection. The category of weak homology allows us to find potential homologs in such situations. For example, the BLAST search for contig Am_4671 yielded the GenBank entry NP_004055, cyclin-dependent kinase inhibitor 1B (Mus musculus), rat (Rattus norvegicus), frog (X. laevis), zebrafish (Danio rerio), fugu (Takifugu rubripes), fruitfly (Drosophila melanogaster), mosquito (Anopheles gambiae), worm (Caenorhabditis elegans), newts and the yeast species Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans were collected and the closest homolog from the above species was used to assign a putative identity.Taken together, a total of 3,718 (58%) sequences shared homology with a protein from selected model organisms in the non-redundant database and could be assigned a putative identity. The E-value distribution of the top hits in the non-redundant database is shown in Figure To estimate how many of the clones are full length we examined the BLAST alignments for the position of the alignment in respect to the database sequence. Of the 3,718 sequences with homologs, 1,107 (29.8%) could be aligned in the amino terminus . As the library was poly(dT) primed, many of these clones are likely to represent full-length inserts. Of these 199 (5.4%) could be aligned from the amino terminus to the carboxy terminus and are potential full-length sequences.A. mexicanum contigs. With the remaining set of contigs, we first performed BLASTN searches against the nucleotide non-redundant (NT) database and BLASTX searches against the EST database. Finally, we performed BLASTX searches against the fugu and human proteomes. In all cases, an E-value of 1e-05 was used to assign potentially homologous sequences. Sequences in the NT database identified an additional 134 contigs and a further 220 contigs found a hit in the EST databases. A homolog was found for 3,340 (52%) contigs in the fugu proteome and 3,698 (58%) contigs shared homology with a protein from the human proteome. In total, an additional 468 contigs identified a homolog in the selected databases beyond the original assignment from the non-redundant protein database  contigs in any of the databases searched. Because the library was poly(dT) primed, many of these sequences could represent 3' untranslated regions (3' UTRs). We determined that 953 sequences (43% of non-homologous contigs) contained no ORF and were therefore potential untranslated regions. Thirty of the sequences shared homology to an existing 4% contigFrom the homologous proteins found, contigs were assigned a biological process, molecular function and cellular component from the Gene Ontology (GO) database . The cloThe largest fraction of the contigs was assigned a cellular process in the GO category biological process (87% of annotated contigs) Figure . We spliA. mexicanum. We also compared the occurrence of those domains in other vertebrate species. For most of the common protein domains, only a fraction were found in our dataset; many of these are quite abundant compared to X. laevis or Gallus gallus. The RNA-binding domains KH and RRM especially showed high abundance in our contigs. A complete list of domains is downloadable from [To identify potential domains in the axolotl contigs, we performed RPS-BLAST searches against the conserved domain database (CDD, NCBI) ,18 usingble from .We assigned cellular functions to the identified domains and analyzed the output according to the functional distribution of contigs Figure . The mosin silico differential display has been performed for ESTs in the NCBI collection, and, as with the NCBI differential display data, we have assessed the statistical significance of the differences using Fisher's exact test. A total of 104 contigs met the cutoff value of 0.005 in Fisher's exact test and can therefore be considered differentially expressed.We were interested to see if there were strong differences in the sequence representation of the libraries that reflect the different biological processes taking place in each tissue. To this end, we compared the representation of ESTs in the two libraries. This type of Table A total of 168 contigs annotated under RNA metabolism  were more frequently sequenced or uniquely sequenced in blastema . This group included RNA metabolism, RNA processing, splicing, editing, nuclear export, binding, catabolism, cleavage, capping, rRNA modification, rRNA transcription and tRNA aminoacetylation. Forty-five contigs assigned a process in RNA metabolism were upregulated or unique in neural tube . After Fisher's exact test analysis, 24 of the clones were considered differentially regulated in the two libraries; 22 out of the 24 contigs were enriched or unique in blastema  were assigned as cell-cycle genes  and were more frequently sequenced or uniquely sequenced in the blastema library, compared with 52 in the neural tube library (2.5% and 0.8%, respectively). This category included regulation of mitosis, mitosis, cell-cycle regulation, regulation of cyclin-dependent kinase (CDK) activity, cell proliferation, DNA replication, M phase, mitotic spindle checkpoint, mitotic spindle assembly, chromosome segregation and cytokinesis. As an example, 10 different types of cyclins were found, from various stages of the cell cycle. Seven of the contigs found in cell-cycle regulation met the cutoff criteria of statistical significance in Fisher's exact test. Five out of the seven contigs were more highly represented or unique in blastema Table .Whereas proliferation-associated genes were found with a higher sequence representation in the blastema library, genes that had been electronically annotated as involved in 'cell differentiation' had a higher representation in the neural tube library. A total of 28 contigs were electronically assigned the biological process 'differentiation'. After Fisher's exact test, five contigs showed differential regulation in this group. Three out of the five contigs were found in neural tube Table . Taken tX. laevis [As mentioned earlier, the Mexican axolotl is an important model organism for a number of reasons. First, it is the premier vertebrate model for studying regeneration. Second some aspects of caudate development, for instance mesoderm involution and notochord formation, more closely resemble those found in higher vertebrates than do those in other amphibian embryological models such as . laevis . FinallyKip1, the protein phosphatase Cdc25A, as well as the anaphase-promoting complex (APC) activator proteins Cdc20 and Cdh1. Representing genes involved in developmental processes, we found transcription factors such as HoxA2, B12, C4 and C8, Pax6, as well as Cdx1 and Cdx2. Furthermore we found several genes for proteins that are part of the transforming growth factor-beta (TGF-β) signaling pathway, such as TGF-β, bone morphogenetic protein 1 (BMP-1), BMP and activin membrane-bound inhibitor, activin receptor type II, as well as the transcription factors Smad5 and Smad8. Genes for proteins such as Smad8 and BMPs might be of especial interest to the research field of embryonic development, as they have been associated with mesoderm involution [A. mexicanum [We examined our dataset for genes that are potentially useful for studying regeneration features or developmental processes. To this end, we analyzed our data for genes that are either involved in regulating the cell cycle - as would be expected for the highly proliferative tissue of a regenerating body structure - or could play an essential role during development and metamorphosis from the larval to the adult stage. A list of genes that could be assigned to either cell-cycle regulation or development is shown in Table exicanum -23. We iX. laevis and the caudate A. mexicanum grouping together compared to other vertebrates such as fish, birds and mammals  and p57 (CDKN1C). In X. laevis, however, only a single family member called p28Kix1 , which shows unusual sequence features compared to the p27 sequences from any other vertebrate species, had been described in the literature [A. mexicanum harbored the 'canonical' p27Kip1 or a p28Kix1 similar to that of Xenopus. We initially searched our A. mexicanum data for CDKN1 orthologs and, in contrast to Xenopus, we found a bona fide p27Kip1 sequence that clusters closer to vertebrate p27Kip1 sequences compared to the Xenopus p28Kix1 , p27terature ,25. We w1 Figure . Considea et al. .Kix1 orthologs were only found in amphibians . We were not able to identify a p28Kix1-like gene in any other database. These p28 orthologs group as a distinct branch in an unrooted phylogenetic tree  and CDKN1C (p57) were present in the A. mexicanum databases but were not found in either X. laevis or X. tropicalis, which have far more EST and genome sequence information  and the highly derived p28Kix1 are the only CDKN1 family members found in both X. laevis and X. tropicalis. In contrast, the ambystomatids appear to have all the members of the CDKN1-family - including p28Kix1 - assuming that the p21 gene is missing purely as a result of lack of sequence information. In addition, our data suggest that p28 is an amphibian-specific variant of the CDKN1 family. Two major questions arise from these data: first, does the amphibian-specific p28 fulfill a cellular function that is unique to this phylogenetic lineage; and second, does the genotypic difference in the gene set of the CDKN1 family in the two amphibian species account for the macroscopic differences observed in developmental mechanisms. The fact that the CDKN1 family is an essential regulator of the cell cycle opens new possibilities for experimental research along these lines.The salamander, and in particular the species tebrates . This anA. mexicanum genes are homologous to genes present in other vertebrates. Sixty-six percent of contigs gave a significant match in either the non-redundant protein or nucleotide databases, the EST databases or the human and fugu protein databases. Thirty-four percent of contigs could not be assigned a homolog in any of the searched databases, and 44% of those could not be assigned a coding sequence and are therefore considered to be part of the UTR. Nineteen percent of the contigs seem to represent novel genes that have not been found in any other organism so far.Given the estimates in the number of genes present in the human genome (20,000-50,000) , we estiThe expressed sequence tags generated in this study also provide a large source of sequence information for developmental and regeneration studies. For example, an examination of the database yielded 194 genes involved in cell proliferation, including pivotal cell-cycle genes such as those for Cdc2, 10 different cyclin family members, Cdk4 and p27. A search for developmental molecules involved in intercellular communication yielded Wnt8, Wnt5B, FGF receptor 4a (FGFR4a), Sonic hedgehog, BMP receptor (BMPR) and BMP-1, while a search for homeodomain-containing proteins yielded 11 members, including Cdx1, Cdx2, HoxA2, HoxC8 and HoxB13.The ESTs were derived from two cDNA libraries, stage 18-22 embryonic neural tube/notochord/somite tissue, and day-6 regenerating tail tissue. The embryonic library represents a developmental stage where tissue specification is occurring, whereas the blastema library represents a tissue that is undergoing dedifferentiation, rapid proliferation and cell respecification. Accordingly, we find differences in transcript representation in the two libraries. The blastema library is particularly enriched in cell-cycle genes and RNA metabolism genes, presumably reflecting the high proliferative index of the early regenerating blastema.A. mexicanum was generated to provide a comprehensive sequence dataset for the community of biologists. Forty percent of genes could still be found in singlets, which reflects a high diversity of sequences in our cDNA set. Annotation of the assembled contigs revealed a substantial difference in gene representation in the two sequence libraries, reflecting their biological source - regenerating blastema being in a highly proliferative state and embryonic neural tube being a tissue undergoing differentiation. Sequence analysis of assembled contigs revealed that 64% of genes had a putative homolog in other species; 19.4% of the contigs contained a putative coding sequence and can be considered novel genes. From this, we conclude that A. mexicanum does not contain an unusually high number of organism-specific genes. The CDK inhibitor family CDKN1 was selected for comparative phylogenetic analysis. Unlike the frogs X. laevis and X. tropicalis, ambystomatids most probably contain all members of the CDKN1 family, including the amphibian-specific protein p28Kix1/p27Xic1, which shows unusual sequence divergence compared to CDKN1 members in other vertebrate species. Such data would support the contention that A. mexicanum is closer to a basal tetrapod compared to X. laevis. The EST sequences and annotated contigs presented in this paper will be a publicly available and useful resource for research in various fields.This set of 17,352 ESTs from A. mexicanum embryos. Total RNA quality was assessed by determining the relative brightness of the 28S:18S rRNA bands (2:1). For library construction mRNA was purified and size fractionated, then poly(dT)-primed cDNA was synthesized and directionally cloned into the NotI-SalI sites of the pCMVSport6 vector. DNA was transformed by electroporation into EMDH10B-TONA bacteria (library construction performed by Invitrogen). Two separate, unnormalized libraries were produced. The blastema library contained an average insert size of 1.67 kb and 2.67 × 107 independent transformants and the neural tube library had an average insert size of 1.5 kb and 1.9 × 107 transformants. From each library 100,000 clones were arrayed into 384-well plates (Resource Zentrum/Primary Database, Berlin, Germany).Total RNA was purified using Trizol (Invitrogen) from 6-day regenerating tail blastemas and from neural tube-somite-notochord-containing tissue dissected from stage 18-22 For sequencing, single-pass reads from the 5' end of the library inserts were performed using a custom-designed SP6 primer: GCACATTAGGCCTATTTAGGTGACA. DNA from bacterial library clones was amplified using the Templiphi reaction, based on φ29 rolling-circle replication of DNA (AP Biotech). Briefly, approximately 0.5 μl of bacterial glycerol stocks were picked up using 96-pin plastic replicators (Genetix) and centrifuged into 96-well PCR plates. Five microliters of denaturing buffer was added, and samples heated to 95°C for 3 min. After cooling, 5 μl Templiphi enzyme was added and samples incubated overnight in a 30°C incubator. The Templiphi reaction provides two advantages for large-scale sequencing projects on capillary sequencers. First, the reaction proceeds to an endpoint where all nucleotide is incorporated, yielding uniform quantities of DNA from varying amounts of starting bacteria (or DNA). Second, the rolling-circle reaction results in large pieces of DNA that, in contrast to plasmid DNA, do not enter the capillary and interfere with the sequencing run.For sequencing reactions, the DNA preparation was diluted fivefold with distilled water. Sequencing reactions were performed using the DYEnamic ET Dye terminator kit diluted twofold with DYEnamic ET dilution buffer (AP Biotech). Five microliters of DNA was added to 5 μl of sequencing reaction mix with primer and cycled 30 times under the following conditions: 95°C 20 sec, 60°C 1 min. Sequencing was performed on a MegaBACE 1000 (AP Biotech). Runs were either performed at injection: 3 kV 60 sec, run: 8 kV 120 min, or injection: 3 kV 60 sec injection, run: 3 kV 360 min.The redundancy of the arrayed libraries was tested by performing BLASTN searches  against The sequences were submitted to GenBank. After quality control, individual ESTs were used to search the non-redundant protein database (release of July 2004) using the program BLASTX from the standalone NCBI-BLAST package . For ann2), the base would be false, 30 would reflect a wrongly sequenced base in 1 of 1,000 trials (103), and so forth.) Sequence and contig files can be downloaded at [Quality control of sequenced ESTs was performed using the program Phred  using a oaded at . The resoaded at . Alignmeoaded at  or Cap3 oaded at ) againstoaded at ) from thoaded at .A. mexicanum contig was an E-value of 1e-20.Based on the GO annotation of the closest annotated homolog, contigs were assigned a molecular function, biological process and cellular component from the GO database . To thisKip1 gene were sequenced in the EST collection but neither were full-length sequences. To isolate the full-length sequence, 200,000 clones of our arrayed blastema and neural tube libraries were screened by PCR. Briefly, the bacterial library clones in each 384-well plate were pooled, mini-prepped and arrayed into 96-well plates (RZPD, Berlin), resulting in 576 DNA pools. These DNA pools were screened by PCR using the custom SP6 primer (GCACATTAGGCCTATTTAGGTGACA) as a forward primer and a gene-specific p27 reverse primer (TGATTTCCAATGGCTGGTTT). Fifty nanograms of DNA from each pool was used for PCR reactions and PCR cycling was performed at the following conditions: 94°C 2 min, 30 cycles of 94°C 15 sec, 65.5°C 30 sec, 72°C 90 sec, followed by 72°C 7 min). The largest positive band (1.1 kb) was gel purified and sequenced on an ABI377 machine using the SP6 primer.Two EST sequences of the p27A. mexicanum contigs was used for tree construction. Trees were displayed using the program nj-plot [Multiple sequence alignments were done with the program ClustalX  using st nj-plot  for the  nj-plot  for the A relational database was created using the open source software MySQL as the database server to store and navigate through resulting sequence contigs and annotations. Scripts connecting the web-based front end to the database were written in the programming language Python.


Ictalurus punctatus, is invested with a high density of cutaneous taste receptors, particularly on the barbel appendages. Many of these receptors are sensitive to selected amino acids, one of these being a receptor for L-arginine (L-Arg). Previous neurophysiological and biophysical studies suggested that this taste receptor is coupled directly to a cation channel and behaves as a ligand-gated ion channel receptor (LGICR). Earlier studies demonstrated that two lectins, Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and that PHA-E inhibited the L-Arg-stimulated ion conductance of barbel membranes reconstituted into lipid bilayers.The channel catfish, Both PHA-E and RCA-I almost exclusively labeled an 82–84 kDa protein band of an SDS-PAGE of solubilized barbel taste epithelial membranes. Further, both rhodamine-conjugated RCA-I and polyclonal antibodies raised to the 82–84 kDa electroeluted peptides labeled the apical region of catfish taste buds. Because of the specificity shown by RCA-I, lectin affinity was chosen as the first of a three-step procedure designed to enrich the presumed LGICR for L-Arg. Purified and CHAPS-solubilized taste epithelial membrane proteins were subjected successively to (1), lectin (RCA-I) affinity; (2), gel filtration (Sephacryl S-300HR); and (3), ion exchange chromatography. All fractions from each chromatography step were evaluated for L-Arg-induced ion channel activity by reconstituting each fraction into a lipid bilayer. Active fractions demonstrated L-Arg-induced channel activity that was inhibited by D-arginine (D-Arg) with kinetics nearly identical to those reported earlier for L-Arg-stimulated ion channels of native barbel membranes reconstituted into lipid bilayers. After the final enrichment step, SDS-PAGE of the active ion channel protein fraction revealed a single band at 82–84 kDa which may be interpreted as a component of a multimeric receptor/channel complex.The data are consistent with the supposition that the L-Arg receptor is a LGICR. This taste receptor remains active during biochemical enrichment procedures. This is the first report of enrichment of an active LGICR from the taste system of vertebrata. The initial event in taste transduction involves recognition of taste stimuli by plasma membrane-associated receptor proteins. These proteins are concentrated at the apical end of specialized neuro-epithelial cells (taste cells) found within multicellular end-organs known as taste buds ,2. The rTo date, 7-transmembrane G protein coupled receptors (7TM-GPCR's) for three taste modalities have been identified by both molecular cloning and through searches of the human and mouse genome. Sweet taste stimuli appear to be recognized by at least one heterodimer T1R2/T1R3) of the three member family of 7TM-GPCR's, the T1R's  of the t,8-10. TwIctalurus punctatus [While these recent discoveries have markedly improved the understanding of taste transduction, it is apparent from neurophysiological, biophysical and biochemical studies that receptors and transduction processes other than the GPCR/second messenger systems are utilized by the sense of taste ,20. For unctatus ,32. LittI. punctatus. The catfish is an advantageous model system for studying taste transduction [To evaluate the role of LGICRs in taste transduction, receptors of this class need to be identified and fully described. To date, a well characterized example of a likely LGICR class of taste receptors is found on the common channel catfish, sduction  because sduction -36, and sduction ,37-40.Of these three receptor systems, the one tuned to the amino acid, L-Arg, appears to be of particular high specificity and affinity ,41. CalcThe L-Arg induced increase in intracellular calcium independent of extracellular calcium and not blocked by D-Arg may utilize a mechanism such as a GPCR-polyphosphoinositol linked pathway with IP3 releasing calcium from intracellular stores. The other L-Arg-stimulated pathway, showing intracellular changes in calcium dependent upon extracellular calcium and blocked by D-Arg, is consistent with an LGICR mechanism. The LGICR mechanism was given additional credence through studies demonstrating that membranes from barbel epithelium (that contains taste buds), when reconstituted into lipid bilayers, show L-Arg-stimulated ion channel activity inhibited by D-Arg -33.Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and both lectins labelled identical SDS-PAGE-separated bands of a barbel membrane preparation [Using membrane homogenates from catfish barbel epithelium, biochemical binding studies revealed high affinity sites for L-Arg that were inhibited by D-Arg, L-arginine methyl ester, and to a lesser extent by L-lysine and L-α-amino-β-guanidino propionic acid . Previouparation . L-Arg-sparation  and RCA-paration . When reparation .While these prior studies are consistent with the hypothesis that one of the receptors for L-Arg is an LGICR, both the neurophysiological studies and the reconstitution experiments could not definitively test this hypothesis. Without actually isolating an LGICR for L-Arg, it remains possible that a GPCR moiety for L-Arg could conceivably be tightly coupled to a separate ion channel.The purpose of the studies reported here was to investigate the possibility that an LGICR exists for L-Arg, by:1. biochemically enriching this putative LGICR to near homogeneity, and2. biophysically characterizing the L-Arg-stimulated ion channel activity of the presumed LGICR at each step of enrichment to• demonstrate that the same LGICR is being enriched with each step, andin situ.• demonstrate that the ion channel properties and kinetics of the enriched LGICR are similar to those of the presumed LGICR This enrichment and characterization are necessary steps towards eventually cloning this putative LGICR.The data are consistent with the interpretation that a receptor for L-Arg can be solubilized in an active state and can be enriched to a point where, upon denaturation and SDS-PAGE, material containing receptor-like activity elutes as a single band.I. punctatus, called "Sp" (defined in methods), revealed numerous proteins labelled by silver stain  glycoprotein motifs of the taste buds of catfish barbel . HoweverThe specificity shown by these lectins in binding inhibition, ion channel conductance inhibition, lectin blots, and lectin histochemistry, all give credence to use of lectin affinity chromatography as the first step in enrichment of L-Arg-stimulated channel activity.Since the lectins, RCA-I and PHA-E, inhibited the binding of L-Arg but not of L-alanine , the assA quantitative protein assessment of the eluted materials from the RCA-I column indicated that over 99% of the protein from fraction Sp passed through the column unbound. Only occasionally did this protein material contain minimal L-Arg-stimulated ion channel activity. In contrast, the remaining protein eluted from the affinity column by galactose and reconstituted into lipid bilayers (See below) consistently contained ion channels activated by L-Arg and inhibited by D-Arg.Silver staining of SDS-PAGE separated proteins before Fig.  and afteOn the assumption that the 82–84 kDa protein was at least a subunit of this L-ArgR, the SDS-PAGE band at 82–84 kDa was electroeluted and used to develop polyclonal antibodies from three guinea pigs (See Methods). The affinity purified and pre-treated polyclonal antibodies from two of these guinea pigs proved most specific and these were labelled, "GP1" and "GP2," respectively. In Western blots of fraction Sp Fig , both ofThe GP polyclonal antibodies were used as a confirmatory marker of the 82–84 kDa peptide during subsequent enrichment steps. Both the GP1 and GP2 antibodies labelled the 82–84 kDa protein band in Western blots of an SDS solubilized partial membrane preparation from catfish barbel and both immuno-labelled taste cells of the catfish (see ahead).The CHAPS-solubilized, dialyzed, non-denatured protein eluted from the galactose wash of the lectin column was applied to a Sephacryl S-300 HR column and eluted with Tris/CHAPS. Each protein-containing peak of the elution was assayed for L-Arg-stimulated channel activity and subjected to SDS-PAGE with silver staining and Western blotting against the GP1 antibody. Only protein from the first peak, in fractions 1 and 2,  contained L-Arg-stimulated ion channel activity (see ahead). Silver staining of material in these first two fractions run on SDS-PAGE revealed a prominent band at 82–84 kDa Fig . The broAs a final enrichment step eluted material from fractions 1 and 2 of the Sephacryl column were lyophilised and resuspended into Start Buffer (see methods) and loaded on a Hitrap Q anion exchange column. Both the pH 9 and pH 8 elution resulted in protein being released from the column, with the majority of protein eluting at pH9. However, only the pH 9 fraction contained L-Arg-stimulated ion channel activity when reconstituted into lipid bilayers.A silver stain of an SDS-PAGE of the pH 9 eluent showed a deeply staining band at 82–84 kDa along with weak staining above 200 kDa and in the 35 kDa range Fig . There wThe GP antibodies were developed against the 82–84 kDa fraction of solubilized catfish barbel membranes, since it was a band of this molecular weight that was labelled by the lectins, PHA-E and RCA-I. While it is expected that GP1 and GP2 would label an 82–84 kDa band by Western blots, the fact that the GP1 and GP2 antibodies faithfully marked each enriched fraction that exhibited L-Arg-stimulated ion channel activity (see below) suggests that they are immuno labels of the receptor. As such, their localization within the barbel may be a marker for this presumed L-ArgR.Figure LGICR enrichment was followed and verified by measuring the L-Arg-stimulated conductance of lipid bilayers (equimolar mixture of DOPS:DOPE) into which the protein fractions derived from each purification step were fused. Nearly identical L-Arg-stimulated single channel activity was observed from active fractions of all three steps: the galactose-eluted protein from the RCA lectin column, the protein of the first peak, fractions 1 & 2, of the material eluted from the Sephacryl S-300 column, and the protein of the pH 9 elution from the ion exchange column. The fact that consistent and nearly identical L-Arg-stimulated activity was observed with material from each subsequent enrichment step indicates that the enrichment procedures were sufficiently benign so as to permit the retention of LGICR-type activity and, presumably, native receptor conformation.General agonist/antagonist channel properties. Figure top panel). Addition of 10 μM L-Arg to the cis side buffer solution induced appearance of ion channels . L-Arg-stimulated channel activity in positive fractions from all columns was readily blocked by the addition of 100 μM D-Arg to the same side of the chamber wherein L-Arg had been added .  induced channel activity.)In lipid bilayers into which active material had been fused, but in the absence of added L-Arg, no spontaneous channel activity was observed Fig. top panelWhile 10 μM L-Arg was generally used in the screening and assay procedures, we estimate that the threshold for L-Arg-induced channel activity of the solubilized putative L-ArgR is about 1 μM L-Arg.In addition to inhibition by D-Arg, the lectins, RCA-I and PHA-E (not shown here, but see ), also iFigure + and Cl-. A potential of zero current  was measured after formation of a 4-fold transmembrane concentration gradient of electrolyte (100 mM NaCl at cis side and 25 mM NaCl at trans side) across the bilayer containing a few ion channels. The average reversal potential (n = 5 bilayers, ± S.D.) elicited by voltage ramps .The cation-anion selectivity of L-Arg activated channels found in material contained in fractions 1 & 2 off the Sephacryl column was determined for Nasee Fig.  was -14 The current-voltage relationships for L-Arg activated channels formed by active protein fractions from all three enrichment steps are illustrated in Figure The electrophysiological properties of ion channels formed by the protein fractions throughout the enrichment scheme described here closely resemble those measured for the native channels  see Tab. Both na1. are activated by L-Arg and inhibited by D-Arg over the same concentration ranges;2. display nearly the same unitary conductance  may be used for selected stimuli of several taste modalities. In spite of their likely role in taste, little is known about these LGICRs . The besor L-Arg ,33.The observation that L-Arg acts as a stimulus for the taste system of the channel catfish was first reported by Caprio . SubsequI. punctatus [The receptor sites for L-Arg have narrow structural requirements, with only a few structural analogs of L-Arg acting as cross-adapting stimuli . The recunctatus ,51,52 wiunctatus .in situ intracellular electrophysiological recordings, indicated that the majority of L-Arg-induced depolarizations are generated by inward currents [2+ activity observed in taste cells stimulated by L-Arg could be blocked by D-Arg [More recently, whole cell patch clamp and calcium imaging of isolated catfish taste receptor cells, along with earlier currents ,43. As pcurrents  and conscurrents  the incrStudies in our laboratory demonstrated that plasma membrane vesicles from barbel epithelium incorporated into lipid bilayers displayed L-Arg (μM)-stimulated ion channel activity that was inhibited by D-Arg ,32,35. NPreliminary reports on the localization of an L-ArgR showed that the antibodies, GP1, and the lectin, PHA-E, when incubated with intact, unfixed barbels, labelled exterior-facing epitopes on catfish barbel taste buds ,45 and SConsidering collectively the neurophysiological, biochemical, behavioural, biophysical and localization data, a putative receptor for L-Arg emerges as one of high structural specificity, with D-Arg acting as an antagonist, one of high sensitivity, and one expressed in the apical membrane of a specific sub-class of taste receptor cells. Yet, because the previous biophysical studies were carried out with intact cells, epithelial homogenates or reconstituted membrane vesicles, the data are insufficient to permit a distinction between the L-ArgR as a single LGICR macromolecular complex and the receptor as two separate entities, a recognition molecule coupled to ion channel activity. To help make this distinction, solubilization and enrichment of the receptor were required. We assumed that if receptor activity survived solubilization and increasing enrichment, and if this receptor appeared to purify as a unitary entity by SDS-PAGE, then it is likely that the major receptor for L-Arg is indeed a LGICR.This enrichment procedure was also a necessary first step in cloning the receptor, since partial amino acid sequences may be obtained from the purified product.The initial enrichment step of lectin affinity was predicated upon the observation that the lectins, RCA-I and PHA-E, inhibited the specific binding of L-Arg to its presumed receptor sites , and thaLectin affinity chromatography employing agarose bound RCA-I was therefore used to achieve an initial partial enrichment of the putative L-ArgR. RCA-I was chosen for lectin affinity because in our hands it was more stable and easier to work with than the agarose bound PHA-E. In pilot studies, lectin affinity chromatography with PHA-E led to results similar to those obtained with RCA-I.The two additional enrichment procedures using Sephacryl-S300 and Hitrap Q ion exchange were chosen to further enrich the putative L-ArgR because both could be carried out using solubilization buffers that were less likely to destroy the activity of the receptor and both are standard biochemical purification techniques. Each chromatography step that retained L-Arg-stimulated ion channel activity resulted in increasingly concentrated and increasingly purified protein of molecular weight near 82–84 kDa. This enrichment is readily seen in SDS-PAGE of Figure Throughout enrichment, the 82–84 kDa band was consistently recognized by the GP1 and GP2 antibodies in Western blots Fig.  indicateWhile the 82–84 kDa protein(s) may be a major constituent of the active ion channel complex, the actual molecular weight of the active receptor, and therefore some estimate of its quarternary composition, was difficult to determine. Attempts at running native gels led to inconsistent findings. Of the procedures used for enrichment, the Sephacryl column was the one that could, theoretically, at least, give an estimate of the size of the complex. Using this column, all of the L-Arg-stimulated ion channel activity was located within the initial eluted peak (fractions 1 & 2). Calibration of the column suggested a molecular weight of > 640 kDa for eluted material at this initial peak. However, this high apparent molecular weight may not represent the actual weight of the unitary LGICR, since, like many other LGICRs, the L-ArgR may form clusters ,56-58. TAfter these enrichment procedures, the L-Arg-stimulated ion channel activity was retained and the 82–84 kDa protein fraction was greatly purified. The correlation of these two observations suggests that protein in the 82–84 kDa range is at least part of the L-ArgR.:The biophysical characteristics of the L-Arg-stimulated channels reconstituted from each step in the enrichment scheme remained nearly unchanged from those described for the channel observed in native membrane fragments ,32:1. both channels were activated by the same range of concentration of L-Arg and blocked by the same concentration range of D-Arg Fig. ;2. neither were activated by L-alanine nor by L-proline;3. both displayed similar amplitude histograms , Phaseolus vulgaris Erythroagglutinin (PHA-E), in their biotinylated forms, the ABC kits, and rhodamine-conjugated RCA-I were purchased from Vector Lab (Burlingame, CA). The second antibody, Cy3-conjugated goat anti-guinea pig IgG, was obtained from Jackson ImmunoResearch Labs. (West Grove, PA). The 4 CN Membrane Peroxidase Substrate System was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Gels and ampholytes were purchased from BioRad (Hercules, CA). Protein was quantitated using a BioRad DC Protein Assay. Synthetic 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar Lipids, Inc. (Pelham, AL).All electrolytes, buffers and other chemicals were reagent grade from Sigma (St. Louis, MO). Water was deionized and further purified through a Milli-Q Plus PF system (Bedford, MA). 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (98%) (CHAPS), polyoxyethylenesorbitan monolaurate (TWEEN 20), phenylmethanesulfonyl fluoride (PMSF) and pepstatin A were purchased from Sigma. n-Octyl-β-D-gluco-pyranoside (n-octylglucoside) was purchased from Calbiochem (LaJolla, CA). The absolute enantiomer, L-arginine HCl, was purchased from Sigma, and the absolute enantiomer, D-arginine HCl, was a gift of the Ajinomoto Co., Tokyo, Japan. Sephacryl S-300 HR, High Range Gel Filtration Calibration Kits, and 1.0 ml Hitrap Q columns were purchased from Pharmacia Biotech (Piscataway, NJ). Agarose-bound lectins, Maxillary and mandibular barbels from approximately 50 euthanized channel catfish, ~25–40 cm long, were removed and placed in a beaker containing 100 ml of 50 mM Tris-HCl buffer (pH = 7.8), 100 mM NaCl, 1 mM EDTA ("TRIS Wash"). After one exchange of buffer, barbels were removed to a 50 ml polypropylene screw cap tube filled with TRIS Wash buffer and stored at -80°C until used.The preparation of a solubilized, plasma membrane-enriched fraction from catfish barbels was adapted from Kalinoski . A typicTo solubilize the pellets from the 21,500 × g spin each pellet was recovered by two, 1 ml rinses of 50 mM TrisHCl (pH 7.8), 50 mM NaCl ("Low Osmolar Buffer")  and transferred to a 15 ml Teflon/glass homogenizer. Twenty milligrams of CHAPS and 40 μl of protease inhibitor-mix (0.275 mM pepstatin A, 57.5 mM PMSF, in ethanol) were added to the ~4 ml suspension in the homogenizer. The suspension was homogenized by ten slow strokes of the Teflon pestle using a rotating motor drive at moderate speed. The homogenate was transferred to a 15 ml capped tube, diluted to 10 ml with Low Osmolar Buffer, and placed on a Clay Adams Nutator rocker/shaker overnight at 4°C to solubilize the proteins. After the overnight agitation, the suspension was centrifuged for 1 h at 100,000 × g. The supernatant was recovered and is referred to as "Sp." This Sp was then used in the subsequent lectin affinity chromatography step.All steps in the lectin affinity procedure were performed at 4°C. Agarose-bound RCA I affinity resin was pre-washed by adding 8 ml of gel slurry to a 125 ml conical glass tube and bringing the final volume to 14 ml with TRIS Wash. The tube was inverted several times, then centrifuged at 500 × g for 1 min. The supernatant was discarded and the wash step repeated three additional times with TRIS Wash buffer, then two more times with Low Osmolar buffer. The resulting agarose-bound RCA I gel, with a bed volume of 4 ml, was used for affinity chromatography.The 4 ml agarose-RCA gel in the 15 ml tube was combined with ~7 ml of Sp, and the mixture equilibrated on the Nutator tube rocker for 30 min. The mixture was poured into a 1 cm × 10 cm column and the effluent collected as 1 ml fractions at a flow rate of 6.0 ml/h. Effluent from the column was monitored by absorbance at 230 nm and/or 280 nm.To remove unbound protein, the column was washed with a sufficient volume (~12 ml) of Low Osmolar Buffer until no detectable protein eluted from the column. Proteins bound to the RCA resin were then eluted from the column with 10 ml of 50 mM Tris-HCl (pH = 7.8), 200 mM NaCl, 1 mM EDTA, 20 mM D-galactose. Protein from both the Low Osmolar elution step and the D-galactose elution step was reconstituted into lipid bilayers and assayed for L-Arg-stimulated ion channel activity (See ahead).The galactose-eluted protein fractions showing L-Arg-stimulated ion channel activity were pooled and dialyzed over night against 2000 ml Milli-Q water containing 0.05% CHAPS. Dialyzed samples were lyophilized and stored at -80°C.All steps in the gel filtration procedure were performed at 4°C. Lyophilized proteins from the galactose elution of the lectin column were prepared for further enrichment using size exclusion chromatography by dissolution in 300 μl of TRIS Wash with the addition of 0.2% CHAPS and 10% sucrose. This preparation was loaded onto a Sephacryl S-300 HR column (1 cm × 40 cm). Fractions of 0.4 ml were eluted from the column with a buffer composed of TRIS Wash plus 0.2% CHAPS. The column was run at 2.4 ml/hr, and the effluent absorbance monitored at 230 nm and 280 nm. Eluted fractions containing measurable protein were evaluated for L-Arg-stimulated ion channel activity by incorporation into a lipid bilayer (See ahead). In addition, SDS-PAGE (See ahead) was performed on each eluted fraction. Those fractions exhibiting L-Arg-stimulated ion channel activity were dialyzed overnight against 2000 ml of Milli-Q water containing 0.05% CHAPS, lyophilized and stored at -80°C.The column was calibrated with a Gel Filtration Calibration Kit with protein standards from 158 to 669 kDa.All steps in the ion exchange procedure were performed at 4°C. Further enrichment of the fractions exhibiting L-Arg-stimulated ion activity was achieved using an anion exchange column (Hitrap Q). The column was prepared by washing with 5.0 ml of Start Buffer (25 mM Tris-HCl, pH = 9.0), followed by 2.0 ml of Regeneration Buffer (Start Buffer plus 1.0 M NaCl) and then 5.0 ml of Start Buffer. Lyophilized proteins of the active fraction from the gel filtration column were dissolved in 300 μl of Start Buffer and applied to the column. Prior to elution, the loaded column was washed with 2.0 ml Start Buffer. Elution was at 12 ml/h in 400 μl fractions. First, 1.0 ml of First Elution Buffer (25 mM Tris-HCl, pH = 9.0, 500 mM NaCl) was applied to the column followed by (second), 2.0 ml of Start Buffer (no NaCl), followed by (third) 4.0 ml of Second Elution Buffer (25 mM Tris-HCl, pH = 8.0, 500 mM NaCl). Effluent from the column was monitored with in line UV (230 and 280 nm) and conductivity detectors. Each fraction was assayed for L-Arg-stimulated activity (See ahead.). The active fractions were pooled, dialyzed overnight against 2000 ml of Milli-Q water containing 0.05% CHAPS, lyophilized, and stored at -80°C.The procedure for development of antibodies against the catfish barbel peptides labelled by the lectins, RCA-I and PHAE has been described previously . BrieflyThe IgG fraction of GP1 and GP2 antisera was purified using an E-Z-SEP antibody purification kit (Pharmacia). To reduce non-specific binding in the immunohistochemical studies and Western blots, the GP1 and GP2 antibodies were incubated with powder derived from an acetone precipitation of a catfish brain homogenate. One ml of E-Z-SEP-purified antibody was incubated with 10 mg of powder for 40 min at 4°C. The powder was removed by centrifugation and the procedure was repeated once with fresh powder. The resulting pretreated antibodies are called simply, "GP1" and "GP2."Tris-glycine gels (4–20%, Bio-Rad) were used for SDS-PAGE. Protein of fractions before any chromatography (i.e., Sp), as well as those from each chromatography step, were denatured by mixing 10 μl from each fraction, 1:1, with sample buffer containing 125 mM Tris-HCl (pH = 8.0), 20% glycerol, 4% SDS, 4% β-mercaptoethanol and 50 μg/ml bromophenol blue, and placing the mix in a boiling water bath for 5 min. Gels were run at a constant 20–25 mA for about 1 h, using prestained broad range molecular weight markers (Bio-Rad) in 1 or more lanes. Proteins were stained with Bio-Rad Silver Stain Plus kit.For lectin blots, proteins were electrophoretically transferred to nitrocellulose sheets (Bio-Rad Mini Trans-Blot, Hercules, CA). The sheets were incubated in a blocking solution  (150 mM NaCl, 100 mM sodium phosphate, pH = 7.4)] and 0.05% Tween 20) for 2 h at room temperature, and then incubated with biotinylated RCA-I or PHA-E (10 μg/ml) overnight at 4°C. Following exposure to biotinylated lectin, nitrocellulose sheets were washed extensively with blocking solution. Lectin-bound protein bands were visualized using a Vectastain peroxidase ABC kit with a 4 CN Membrane Peroxidase Substrate system. Development was stopped after one hour using 5% glacial acetic acid.For Western blots, proteins were transferred to nitrocellulose and incubated in blocking solution (PBS, pH = 7.4, 5% non-fat dry milk, 1% goat serum and 0.05% TWEEN 20) for 2 h at ambient temperature with constant slow rocking (Nutator). The nitrocellulose was incubated overnight at 4°C (rocking) with primary antibody (GP1, 1/500) in blocking solution. The nitrocellulose was washed with blocking solution and incubated with biotinylated secondary antibody (1:250) for 1 h with slow rocking. Bands were visualized as above.Rhodamine-conjugated RCA-I (Vector Labs) was used to estimate the specificity of lectin interaction with glycoproteins of catfish barbel. To assess the localization of the antigen contained in the 82–84 kDa band from the SDS-PAGE of a membrane fraction of catfish barbel , immunohistochemistry was performed using the GP1 and GP2 antibodies pretreated as described above.I. punctatus) (the fish being 5 – 7 cm in length) and immediately placed in 4% buffered paraformaldehyde (PFA) (0.1 M sodium phosphate buffer, pH 7.2–7.4) for eight hours at 4°C. After washing out the PFA with several rinses of excess buffer, the barbels were placed successively in 10%, 20%, and 30% sucrose (in buffer) for 24 hr, all at 4°C. After the final cryoprotect sucrose step, barbels were cut into pieces of less than 1 cm and mounted with M-1 Embedding Matrix (Thermo Shandon, Pittsburgh, PA). The tissue was sectioned at 10 microns on a Microm HM500OM cryostat.Barbels were removed from euthanized albino channel catfish (For lectin histochemistry, ten micron sections of fixed barbel were incubated in the dark with rhodamine-conjugated RCA-I lectin (Vector Labs., Burlingame, CA), diluted 1/200 for 2 to 3 hr at ambient temperature. The sections were then washed quickly with Dulbecco's PBS (GIBCO/Invitrogen Corp), followed by three incubation washes of 10 min each.For immunohistochemistry, 10 micron barbel sections, pre-washed 3 times for 10 min each in Dulbecco's PBS, were first incubated at ambient temperature for 3 to 5 hr in blocking buffer consisting of 3% bovine serum albumin, 2% goat serum, 0.3% TritonX100, and 0.1% sodium azide in Dulbecco's PBS at pH 7.1. The sections were then incubated with primary antibody, GP1 or GP2, in blocking buffer for 18 hr at 4°C. The primary antibody solution was removed and the sections were then washed once quickly with Dulbecco's PBS, followed by three incubation washes with PBS of 10 min each. The sections were then incubated in the dark with second antibody, Cy3-conjugated goat anti-guinea pig IgG (Jackson ImmunoResearch Labs., West Grove, PA) at 1:1000 dilution for 60 min. at ambient temperature. The secondary antibody was removed and the sections washed once quickly with Dulbecco's PBS, followed by three incubation washes with PBS of 10 min each. Excess fluid was removed from the slides and the sections mounted under cover slips with VectaShield (Vector Labs). Sections were observed with a Nikon Microphot FXA fluorescence microscope, photographed, and images sized and enhanced using the GNU Image Manipulation Program software .Immuno-specificity was verified by running negative controls where the primary antibody was omitted from the procedure. In all cases, this control step showed no taste bud labelling Fig. . The scaReconstitution of protein fractions containing likely L-Arg-stimulated channel activity was carried out with material from the low osmolar and galactose-eluted fractions of the lectin affinity procedure, from the protein-containing fractions off the Sephacryl S-300 gel filtration column and from each fraction of the ion exchange column. Lipid vesicles were prepared by sonication of 5 mg of DOPE:DOPC (2:1) and 0.5 ml of 5 mM Tris-HCl (pH = 7.2), 300 mM NaCl and 500 mM sucrose, to which 10 μg n-octylglucoside was added. To prepare the liposome-detergent mixture for incorporation into a lipid bilayer, approximately 0.2 to 0.5 μg of presumed receptor protein was added to the liposome in a cassette dialysis unit, and the mixture dialyzed overnight against 2000 ml of the Tris/NaCl/sucrose buffer at 4°C.trans side of the bilayer.Virtually solvent-free lipid bilayer membranes were prepared as described . The mem2, 100 mM NaCl (unless otherwise stated). Fifty to 100 μl of the dialyzed liposome vesicles containing presumed receptor was added to the cis-side of the membrane. Fusion of the vesicles was initiated mechanically by gently mixing the membrane bathing solution from the cis-side using a micro-pipette. L-Arg was added to the cis side approximately 20 min after addition of vesicles. Unless otherwise stated all other additions of reagents also were made from the cis side. Channel sidedness was determined by sensitivity of the bilayer to L-Arg. The orientation of the channels was such that the L-Arg sensitive side was normally in the cis compartment. All bilayer experiments were performed at room temperature.The bilayer was bathed symmetrically with 5 mM MOPS (pH 7.2), 1 mM CaClThe current was amplified by a Dagan 3900 integrating patch-clamp amplifier (Dagan Corp., Minneapolis, MN) in the voltage clamp mode. Single channel data were digitized at 15 kHz (Digidata 1200, Axon Instruments, Foster City, CA) and analyzed using pClamp6 (Axon Instruments) and Origin 5.1  software on an IBM compatible computer.The calculated success rate of incorporation of vesicular proteoliposomes into lipid bilayers was about 25%. Success rate is defined as the ratio of the number of successful incorporation attempts to the total number of incorporation attempts (an "incorporation attempt" refers here to the formation of a new bilayer and application of putative channel protein). Over the course of these studies, the number of incorporation attempts for each fraction tested as indicated above was about ten.WG developed the enrichment procedures and drafted the manuscript. YK developed protein stabilization procedures and performed and interpreted the bilayer studies. DB performed the lectin- and immunohistochemistry and refined many of the enrichment procedures. AS developed the antibodies and performed pilot chromatography procedures. DLK initially designed the lectin procedure and carried out preliminary work on solubilization that made subsequent procedures possible. JT performed initial bilayer studies and advised YK on a continuing basis. JGB, the corresponding author and head of the laboratory, conceived the study, developed tissue preparations, designed and coordinated all phases of the study, and finalized the manuscript. All authors read and approved the final manuscript.


The clear dominance of two-gender sex in recent species is a notorious puzzle of evolutionary theory. It has at least two layers: besides the most fundamental and challenging question why sex exists at all, the other part of the problem is equally perplexing but much less studied. Why do most sexual organisms use a binary mating system? Even if sex confers an evolutionary advantage (through whatever genetic mechanism), why does it manifest that advantage in two, and exactly two, genders (or mating types)? Why not just one, and why not more than two?Assuming that sex carries an inherent fitness advantage over pure clonal multiplication, we attempt to give a feasible solution to the problem of the evolution of dimorphic sexual asymmetry as opposed to monomorphic symmetry by using a spatial (cellular automaton) model and its non-spatial (mean-field) approximation. Based on a comparison of the spatial model to the mean-field approximation we suggest that spatial population structure must have played a significant role in the evolution of mating types, due to the largely clonal (self-aggregated) spatial distribution of gamete types, which is plausible in aquatic habitats for physical reasons, and appears to facilitate the evolution of a binary mating system.Under broad ecological and genetic conditions the cellular automaton predicts selective removal from the population of supposedly primitive gametes that are able to mate with their own type, whereas the non-spatial model admits coexistence of the primitive type and the mating types. Thus we offer a basically ecological solution to a theoretical problem that earlier models based on random gamete encounters had failed to resolve. One of the most general rules in biology seems to be that sex involves the fusion of gametes  of different type. In most taxa this sexual asymmetry is reflected in the male / female distinction between mating partners and/or between mating sex cells. This paper aims to help understand why sex is asymmetric.The primary difference between male and female is anisogamy, the differential size and mobility of gametes. Anisogamy is thought to have evolved from a more primitive condition of isogamy  and fungi (e.g. yeast), the asymmetry in sexual fusion and subsequent development are regulated by a binary mating type system. Mating is only possible between cells of different mating type. Molecular analysis has revealed a remarkable and complex genetic mating type structure [Aspergillus nidulans, or may consist of individuals that are heterokaryotic for mating type (carry nuclei of both mating types) such as Podospora anserina. In the latter case sexual fusion is between different mating types at the nuclear level, but can occur within a single individual mycelium.In isogamous species without apparent male-female differentiation, like unicellular green algae  approximation. Such a comparison allows precise consideration of the kinetics of gamete encounters in the model system and emphasizes the role that spatial aspects of the kinetics might have played in mating type evolution. For detailed descriptions of both models see the The specific questions we address with both the mean-field model and the cellular automaton are the following:a) Are there reasonable parameter values that allow the coexistence of the mating types and the pan-sexual type?b) Under what (if any) circumstances is it possible that the mating types exclude the pan-sexual type?c) Does spatial structure play an important role in the outcome of the mating type competition system?r, R, R', the vegetative death rates d, D, D', the germination rate g, the sex rate σ and the finess erosion rate φ in the mean-field, and the corresponding probability parameters in the cellular automaton model. Having explored a broad range of the parameter space – with straightforward constraints on the fitness parameters (birth and death rates), i.e., with D ≤ D' ≤ d <r ≤ R' ≤ R – we found that it is the strength of the inbreeding effect (the difference of D and D' and that of R' and R) and the rate of fitness erosion φ that has the most interesting effects on coexistence. Changing the remaining parameters – the sex rate and the germination rate – within reasonable limits (σ > 0, g > 0) does not affect the results in a qualitative sense.Numerical solutions to the mean-field model and simulations with the cellular automaton reveal that the system admits a single stable equilibrium state both in the non-spatial and in the spatial setting , and ξ > 0 means a fitness difference in favour of outbred offspring.ξ – φ projection of the parameter space, for both the mean-field model . At φ = 0 (no fitness loss during vegetative multiplications), on the other hand, it is the mating types who win for any ξ > 0. At ξ = 0 = φ, the mating types and the pan-sexual type coexist, and the same applies to any parameter combination satisfying ξ ≠ 0 ≠ φ. Thus we can say that the non-spatial (mean-field) model allows coexistence for almost any parameter combination, except for the biologically less feasible margins of the parameter plane. It predicts in general that both the mating types and the pan-sexual type should have persisted, even if at variable relative frequencies. The cellular automaton model yields a different prediction, admitting the exclusion of the pan-sexual type, i.e., the victory of the two mating types on a considerable section of the parameter plane, including the ξ = 0 = φ point and its close  neighbourhood  every vegetative cell is in the post-zygote state. For the same reason the birth rates are also equal for all the vegetative cells, so the only factor that can make a difference between the cell types is the availability of empty sites: the limiting "resource" for reproduction. In the mean-field model the empty sites are equally available to any cell, so the growth rates of the pan-sexual and the mating type strains are identical in the long run, hence their coexistence. In the cellular automaton, however, each strain develops patches. The mating type strains do not have sex at all within their own patch, only at the interface with the patches of other strains. The pan-sexual strain has sex all the time everywhere in the habitat, therefore a larger part of its population is in the dormant zygote state. It is for this reason that at the interface with the mating type patches the pan-sexual strain has a smaller supply of vegetative invaders and thus a smaller chance to capture an empty site there. This results in a travelling front between a mating type patch and a pan-sexual patch and ultimately in the demise of the pan-sexual population altogether. This effect can even overcompensate a small disadvantage for the mating types arising from increasing the rate of fitness erosion φ slightly above 0, therefore the close neighbourhood of the ξ = 0 = φ point on the parameter plane belongs to the mating types as well. We think that it is exactly this mechanism that makes the mating types victorious in the spatial model at many parameter combinations that allowed for coexistence in the mean-field approximation. The elementary events at the interfaces between patches of different gamete types have a profound effect on the ultimate outcome of their competition at the larger spatial scale of the whole habitat.In the mean-field model the coexistence of mating types and the pan-sexual type at see Fig. . The detξ = 0 = φ for example (or anywhere else where the mating types take over) in the two models proves that it is not stochastic drift but a real dynamical trend that eliminates the pan-sexual strain in the cellular automaton . If the populations are viable, i.e., if they have a vegetative growth rate higher than 0, then less frequent mating (dormancy) is beneficial in terms of the average fitness of the pan-sexual population. This effect dominates at low values of φ, where the fitness advantage of sex does not vanish too fast. A comparison of Figs. φ. In all, it is quite obvious that sex rate reduction is not an efficient strategy for the pan-sexual strain to avoid exclusion by the mating types.In order to explain the net effect of sex rate reduction on the fitness, and thus on the survival chances of the pan-sexual population one has to consider two different aspects. On the one hand, sex rate reduction decreases the relative fitness of the pan-sexual strain, because it decreases the frequency of both its inbred and outbred matings, the means of keeping fitness high. This negative fitness effect is most pronounced at high rates of fitness erosion There is a logical possibility that asymmetric cell fusion has evolved for other reasons than and prior to sex and has subsequently been incorporated in the evolution of a full sexual cycle (the sequence of syngamy, karyogamy and meiosis). In that case sex would have been asymmetric from the start. This speculative idea has been analysed theoretically by   our spatial (cellular automaton) model shows the evolution towards exclusively two mating types, whereas the non-spatial model of the same system with the same parameters predicts the coexistence of the mating types and the pan-sexuals. Thus, taking for granted that sex is profitable in evolutionary terms, we offer a basically ecological answer to the question why two mating types can be better than just one. This is, however, only a solution to half of the problem of the optimal number of mating types. Could a third, a fourth, a fifth etc. mating type invade the same system? These questions arise on a very general level in relation to the origin of sexual asymmetry, and they call for a more extended theoretical approach in the future.Assuming that sexual reproduction confers some average fitness advantage compared to simple clonal multiplication, and also supposing that the more genetically different the fusing gametes are the bigger the fitness benefit of the offspring can be, we show that a population consisting of two mating types can displace a pan-sexual population which is otherwise similar to the mating types in all other respects. In the most realistic domain of its parameter space  that of its parent. Sexual fusion produces a dormant zygote which upon germination gives rise to haploid vegetative cells through meiotic division, in which the parental gamete types segregate as if determined by a mendelian pair of alleles. To these meiotic products – "post-zygote" vegetative cells -a higher fitness, i.e., a higher division rate and/or a lower death rate, is attributed than to "pre-zygote" vegetative cells not having gone through a sexual cycle in the near past. That is, we assume that sexual offspring have an immediate short-term fitness advantage over asexually derived daughter cells. The actual advantage may be dependent on whether the zygote has been produced by "outbreeding" (with at least one of the gametes involved belonging to one of the two mating types) or "inbreeding" . In general we may, but need not, assume that inbred zygotes yield vegetative cells of somewhat less (but still positive) fitness advantage than outbred zygotes. Note that here "outbreeding" and "inbreeding" mean mating between different and identical gamete types, respectively, i.e., we assume – without specifying the precise nature of this outbreeding advantage – that mating between different gamete types may result in fitter offspring on average than mating between cells of the same  gamete type. The simplest possible genetic mechanism with this effect might be the production of recombinant offspring carrying fewer (slightly) deleterious alleles than both parental genotypes. This mechanism will be operative more often in heterotypic than in homotypic matings, because among the latter a larger proportion will involve selfing  identical genotypes). The fitness advantage of sexually derived vegetative cells fades away in time during successive rounds of vegetative reproduction (fitness erosion due to the accumulation of harmful mutations), but it can be re-gained through another sexual event. This means that post-zygote cells return to the pre-zygote state when they are not involved in a new sexual cycle for a sufficiently long time.Wpre-zygote <Wpost-zygote,inbred ≤ Wpost-zygote,outbred.As for the ecology of the system, we assume that the habitat consists of a limited amount of sites that cells can occupy, and that the three cell types are competing for these sites. Death events leave empty sites behind, which can be occupied later by new offspring. The chance of a newborn cell to settle is proportional to its division rate and the number of empty sites available. In accordance with what has been said earlier about the fitness advantages of sex, three different division rates and death rates are possible: one for pre-zygote, the second for inbred post-zygote, and the third for outbred post-zygote vegetative cells. The straightforward fitness order of these three types is: The fusion of two gametes produces a zygote of double size compared to a gamete, and the zygote enters a dormant state with zero rates of division and death. Zygotes leave dormancy at a constant rate, giving rise to post-zygote vegetative cells which inherit the mating type of the gametes they are produced by, and gain fitness according to whether the mating was of the inbreeding or the outbreeding type.Fig. Based on Fig. x, y and p are the numbers of sites occupied by pre-zygote vegetative cells , Zxy, Zxp, Zyp are the sites of outbred, and Zpp are those of inbred zygotes. Similarly, X, Y and P are sites of outbred, Q are those of inbred post-zygote vegetative cells. E is the number of empty sites within the habitat. The parameters of the model are listed and described in Table where x, y and p) has three terms. The first defines the vegetative fitness of the corresponding cell type , the second is the outflow from the pre-zygote vegetative state due to sex, and the third is the inflow due to the fitness erosion of post-zygote vegetative cells. Zygotes have no vegetative fitness; the first term in their differential equations is the inflow due to sex, the second is the outflow due to germination. Post-zygote vegetative cells have a vegetative fitness different from that of pre-zygotes (first term); they form zygotes fusing  with both pre- and post-zygote cells matching in mating type (second term); their fitness advantage erodes at a constant rate resulting in an outflow into the pre-zygote state (third term), and the germination of dormant zygotes maintains an inflow from the zygote states (fourth term). The number of empty sites is increased by the deaths of vegetative cells (first three terms) and decreased by the number of sites taken by newborn vegetative offspring (fourth term). The total number of sites does not change in time, so the 12 time derivatives sum up to zero.The right-hand side of the differential equations for the sites occupied by pre-zygote vegetative cells . In all numerical calculations the initial populations were 10 pre-zygote vegetative cells of both mating types and the pan-sexual type, all other states had 0 initial abundances.With assumptions as similar to the nonspatial system as possible, we have implemented a site-based (cf. ), spatiaThe pattern is updated one randomly chosen site at a time, i.e., we use an asynchronous random updating algorithm. Any site chosen for update can be empty, occupied by a vegetative cell, or occupied by a zygote. We specify the algorithm for each of these cases in turn. A schematic diagram of a single step of updating is given in Fig. i has a chance pi to put a daughter cell into the empty site. pi depends on the vegetative reproduction parameter βI (0 ≤ βI ≤ 1)of neighbour i. βI is the spatial analogon of ri in the mean-field model, and it takes one of three possible values depending on whether i is in the pre-zygote, the inbred or the outbred post-zygote state.After updating, an empty site can be occupied by one (and only one) of the vegetatively produced offspring of the cells in the 8 neighbouring sites , or it remains empty. Each vegetative neighbour Specifically, the chance of the empty site to remain empty isi takes the site isso the probability that the offspring of neighbour βi, but only one of the candidate offspring survives. The chance of survival is proportional to the reproduction parameter of the mother cell.The rationale behind this formalism is that each neighbour attempts putting an offspring into the empty site with a probability δ of the cell occupying the site, which in turn depends on its vegetative status (pre-zygote, inbred or outbred post-zygote). With a mating partner in one of the neighbouring sites, a surviving vegetative cell may enter the sexual cycle with probability s turning itself and a randomly chosen, suitable neighbour into gametes, and mate. The result is a dormant zygote occupying the two neighbouring sites of the fused gametes. A survivor skipping sex may keep its original fitness, or – if it was a post-zygote cell – it can lose its fitness advantage with a probability f .Updating a site occupied by a vegetative cell may result in four possible outcomes: turn the site into the empty state (death), leave it as it was , change the vegetative status of the resident cell from post-zygote to pre-zygote , or produce a zygote (sex). The probability of a death event depends on the death probability γ) or germinate (with probability γ). A germinated zygote yields two vegetative cells, the mating types of which are the same as those of the gametes which produced the zygote. The vegetative status of the cells thus obtained is post-zygote, and they can be either inbred or outbred, depending on the parental gamete type combination. The daughter cells are positioned at random into the two sites the zygote had occupied.A zygote can do two things: remain dormant (with probability 1 – At time 0 we have populated 2% of the sites by pre-zygote vegetative individuals of both mating types and the pan-sexual type, assigning individuals to sites at random. All other sites were empty at time 0. The simulations were run for 10.000 generations.RH organized and coordinated the project, collected most of the literature and drafted parts of the manuscript. TC designed and built the models, drafted parts of the manuscript and the figures.