Identification of a genetic element that controls the organ-specific expression of adh1 in maize

Allozyme balances serve as markers of quantitative behavior of electrophoretically distinguishable alleles. By the use of ADH Set I allozyme balances, it is demonstrated that all Adh1-S/Adh1-F individuals from more than 20 diverse S/F families exhibit a reciprocal correlation between Adh1 quantitative behavior in two maize organs: the scutellum and primary root. Within an electrophoretic mobility class, the Adh1 allele that is relatively underexpressed in the scutellum is relatively overexpressed in the primary root, and vice versa. Segregation tests prove that this "reciprocal effect" is the property of a cis-acting site that is closely linked to or within the Adh1 structural gene, and it is not affected by diverse genetic backgrounds. Immunological and [(3)H]-leucine incorporation experiments establish that Adh1 quantitative variants differ in ADH1.ADH1 synthetic rates in the anaerobic primary root. The reciprocal-effect phenomenon suggests that the cis-acting loci controlling Adh1 quantitative expression in each respective organ are at least in close proximity, or may share common DNA sequences. We discuss the possibility that the reciprocal-effect locus is a regulatory component of the Adh1 cistron.     

Genetic heterogeneity in adenosine deaminase (ADA) deficiency: five different mutations in five new patients with partial ADA deficiency.

Complete genetic deficiency of adenosine deaminase (ADA) results in a fatal syndrome of severe combined immunodeficiency (SCID). Genetic partial deficiency of ADA, with no detectable enzyme activity in erythrocytes but with variable amounts of enzyme activity detectable in other cells, is usually associated with normal immunologic function but can give rise to a late-onset, cellular immunodeficiency syndrome. We have previously described four different mutant alleles in four such partially ADA-deficient children. We have now examined ADA in lymphoid cells from five additional newly ascertained children with partial ADA deficiency with respect to electrophoretic mobility in starch gel, isoelectric point, heat-stability, and apparent Km and Vmax. These techniques identify at least five different abnormal alleles in these five additional unrelated subjects. Three of these abnormal alleles result in expression of abnormal allelic isozymes (allozymes) different from those previously described. These are: (1) an acidic allozyme that is less acidic than the acidic allozyme we have previously reported; (2) an allozyme that is even less acidic than (1); and (3) an allozyme with apparently normal charge but which is so heat sensitive that the lability to heat can easily be detected at physiologic to febrile temperatures. Two abnormal alleles detected in these five children could correspond with previously reported mutants. These are (4) a basic allozyme that could (but probably doesn't) correspond to the basic allozyme we have previously reported and (5) a "null" allele that cannot be differentiated by these methods from any other "null" allele seen in complete ADA- -SCIDs. Three of the five new patients are genetic compounds, identified either by the presence of two electrophoretically distinguishable allozymes or by family studies that demonstrate presence of a "null" allele in addition to an electrophoretically abnormal allozyme. In three patients, one or both allozymes are phenotypically indistinguishable from an abnormal allozyme also seen in a different individual. Determination of the nucleotide sequence will be required to determine whether or not the phenotypically indistinguishable mutations are indeed genotypically identical. The newly ascertained individuals appear to share a common ethnic West Indian background, out of proportion to the frequency of this ethnic background in the newborn population from which they were ascertained, suggesting that partial ADA deficiency may confer a selective advantage to the homozygous or heterozygous phenotype.     

DNA Polymorphism at the Pgi Locus of a Wild Yam, Dioscorea Tokoro

To study the origin and maintenance mechanisms of the PGI allozyme polymorphism of a wild plant, Dioscorea tokoro, DNA sequences of the entire coding region (1701 bp) and two intronic regions (total 2049 bp) of the Pgi gene as well as a part of the Adh gene (590 bp) were analyzed. Two replacement substitutions were revealed to be responsible for the differentiation of three allozymes alleles (Pgi-a, Pgi-b and Pgi-c) that occur in natural population in intermediate frequencies. Interspecific comparison of DNA sequences identified Pgi-b as the oldest allele, from which two other alleles were derived probably within the last 150,000 years. The level of DNA polymorphism at D. tokoro Pgi locus was low. No elevated level of DNA polymorphism was detected in the close vicinity of the two replacement sites differentiating the three allozymes. Departures from the neutral mutation hypothesis were detected by Fu and Li's and MK tests. The observed patterns of DNA polymorphism are explainable by both (1) the neutral mutation hypothesis with an assumption of small effective size of D. tokoro population, and (2) the positive selection hypothesis that the allele frequencies of Pgi-a and Pgi-c have increased in a short time by their selective advantages.     

Tracing the history of an enzyme polymorphism: the case of alcohol dehydrogenase-2 (Adh-2) of the olive fruit fly Bactrocera oleae

In the olive fruit fly Bactrocera oleae, previous studies have described a one-locus three-allele electrophoretic polymorphism of the enzyme alcohol dehydrogenase and provided evidence that the polymorphism is under the influence of selection. A recent study has shown that this species carries a two-locus duplication for alcohol dehydrogenase. Here, we show that the polymorphism maps at one of the duplicated loci, Adh2, and identify the nucleotide and, therefore, the inferred amino acid differences among the three allozymes. At the amino acid level, the polymorphism is of the simplest possible form: there is no intra-allozyme variation, and interallozyme differences are restricted to one amino acid for two pairs of alleles and to two amino acids for the third pair. Consideration of the amino acid residues at the sites that segregate in B. oleae in four congeneric species and the phylogenetic trees produced from the nucleotide sequences of the Adh2 gene of these species point to the same allozyme as the ancestral form of the polymorphism. Interestingly, this allozyme comprises less than 1% of the gene pool of present-day natural populations of B. oleae, where the other two allozymes appear to form a stable polymorphism. Previous studies have shown that the frequency of the rare allozyme rises rapidly in laboratory colonies maintained on artificial diet and declines again when the artificial diet is replaced with olive fruit, the natural substrate of B. oleae. The geographical distribution of several congeneric species suggests that B. oleae originated in the Indian subcontinent, where the olive tree is practically absent. The poor performance of the ancestral allele on the olive fruit suggests the possibility that the decline of this allele and the concomitant rise of the presently common alleles might be associated with the expansion of the insect's geographical distribution to areas where the olive tree has become its main and perhaps sole host. The estimated age of the polymorphism is compatible with this hypothesis, but firmer support could be difficult to obtain.     

Further evidence of thermostability variation within electrophoretic mobility classes of enzymes

Two Drosophila melanogaster strains, each heterozygous for fast and slow alleles at the Adh locus, and each having balanced second chromosomes, were found to differ in the apparent thermostability of the slow allozyme. The two strains were crossed, and F₁heterozygotes were separated on the basis of the origin of the slow allele. After electrophoresis, the cellulose acetate strips were treated 1 1/2 min at 35 C. The putatively more sensitive allozyme showed a strikingly greater response to heat. These findings further support the conclusion that electrophoretically cryptic allelic differences exist which are expressed in thermostability differences. Further application of this approach has revealed one similar sensitive slow allozyme and three cases of a relatively resistant fast ADH allozyme in wild-caught flies.This work was supported by NIH Grant GM18967-02-03.     

Patterns of natural selection at the Alcohol dehydrogenase gene of Drosophila americana

Similar outcomes are often observed in species exposed to similar selective regimes, but it is unclear how often the same mechanism of adaptive evolution is followed. Here we present an analysis of selection affecting sequence variation in the Alcohol dehydrogenase (Adh) gene of Drosophila americana, a species endemic to a large climate range that has been colonized by D. melanogaster. Unlike D. melanogaster, there is no evidence of selection on allozymes of ADH across the sampled range. This indicates that if there has been a similar adaptive response to climate in D. americana, it is not within the coding region of Adh. Instead, analyses of a combined dataset containing 86 alleles of Adh reveal purifying selection on the Adh gene, especially within its intron sequences. Frequency spectra of derived unpreferred variants at synonymous sites indicate that these sites are affected by weak purifying selection, but the deviation from neutrality is less drastic than observed for derived variants in noncoding introns. This contrast further supports the notion that noncoding sites in Drosophila are often subject to stronger selection pressures than synonymous sites.     

Studies of Esterase-6 in DROSOPHILA MELANOGASTER. II. the Genetics and Frequency Distributions of Naturally Occurring Variants Studied by Electrophoretic and Heat Stability Criteria

Measurements of the electrophoretic mobility and thermostability of esterase-6 allozymes have been used to determine the amount of allelic variation at the esterase-6 locus in Drosophila melanogaster. We studied 398 homozygous lines obtained from four natural populations. Use of a spectrophotometric assay for esterase-6 activity has allowed precise quantitation of heat-stability variants. Using these methods, eight putative alleles were detected within the two most common electrophoretic classes. Analyses of F1 and F2 progeny show that the behavior of stability variants is consistent with the hypothesis that this variation is due to allelic variation at the Est-6 locus. Analyses of the gene-frequency distributions within and between populations show (1) that observed allele-frequency distributions do not deviate significantly from those expected for neutral variants, and (2) that there is little evidence for an increase in apparent divergence of the different populations at the genotypic or phenotypic levels when the additional variation detected is considered. These findings suggest that gene-frequency analysis alone is unlikely to resolve the question of the selective significance of allozyme variation.     

Genetic diversity among progenitors and elite lines from the Iowa Stiff Stalk Synthetic (BSSS) maize population: comparison of allozyme and RFLP data

Summary Data for restriction fragment length polymorphisms (RFLPs) of 144 clone-enzyme combinations and for 22 allozyme loci from 21 U.S. Corn Belt maize (Zea mays L.) inbreds were analyzed. The genetic materials included 14 progenitors of the Iowa Stiff Stalk Synthetic (BSSS) maize population, both parents of one missing BSSS progenitor, four elite inbreds derived from BSSS, and inbred Mo17. Objectives were to characterize the genetic variation among these 21 inbreds for both allozymes and RFLPs, to compare the results from both types of molecular markers, and to estimate the proportion of unique alleles in the BSSS progenitors. Genetic diversity among the 21 inbreds was substantially greater for RFLPs than for allozymes, but the percentages of unique RFLP variants (27%) and unique allozyme alleles (25%) in the BSSS progenitors were similar. Genetic distances between inbreds, estimated as Rogers' distance (RD), were, on average, twice as large for RFLP (0.51) as for allozyme data (0.24). RDs obtained from allozyme and RFLP data for individual line combinations were only poorly correlated (r = 0.23); possible reasons for discrepancies are discussed. Principal component analysis of RFLP data, in contrast to allozyme data, resulted in separate groupings of the ten BSSS progenitors derived from the Reid Yellow Dent population, the four BSSS elite lines, and Mo17. The remaining six BSSS progenitors were genetically rather diverse and contributed a large number of rare alleles to BSSS. The results of this study corroborate the fact that RFLPs are superior to allozymes for characterizing the genetic diversity of maize breeding materials, because of (1) the almost unlimited number of markers available and (2) the greater amount of polymorphisms found. In particular, RFLPs allow related lines and inbreds with common genetic background to be identified, but a large number of probe-enzyme combinations is needed to estimate genetic distances with the precision required.Joint contribution from Cereal and Soybean Research Unit, USDA, Agricultural Research Service, and Journal Paper No. J-14236 of the Iowa Agricultural and Home Economics Experiment Station, Projects 2818 and 2778     

Extensive amino acid polymorphism at the pgm locus is consistent with adaptive protein evolution in Drosophila melanogaster

PGM plays a central role in the glycolytic pathway at the branch point leading to glycogen metabolism and is highly polymorphic in allozyme studies of many species. We have characterized the nucleotide diversity across the Pgm gene in Drosophila melanogaster and D. simulans to investigate the role that protein polymorphism plays at this crucial metabolic branch point shared with several other enzymes. Although D. melanogaster and D. simulans share common allozyme mobility alleles, we find these allozymes are the result of many different amino acid changes at the nucleotide level. In addition, specific allozyme classes within species contain several amino acid changes, which may explain the absence of latitudinal clines for PGM allozyme alleles, the lack of association of PGM allozymes with the cosmopolitan In(3L)P inversion, and the failure to detect differences between PGM allozymes in functional studies. We find a significant excess of amino acid polymorphisms within D. melanogaster when compared to the complete absence of fixed replacements with D. simulans. There is also strong linkage disequilibrium across the 2354 bp of the Pgm locus, which may be explained by a specific amino acid haplotype that is high in frequency yet contains an excess of singleton polymorphisms. Like G6pd, Pgm shows strong evidence for a branch point enzyme that exhibits adaptive protein evolution.     

Evolutionary implications of allozyme and RAPD variation in diploid populations of dioecious buffalograss Buchloë dactyloides

Buffalograss, Buchloë dactyloides, is widely distributed throughout the Great Plains of North America, where it is an important species for rangeland forage and soil conservation. The species consists of two widespread polyploid races, with narrowly endemic diploid populations known from two regions: central Mexico and Gulf Coast Texas. We describe and compare the patterns of allozyme and RAPD variation in the two diploid races, using a set of 48 individuals from Texas and Mexico (four population samples of 12 individuals each). Twelve of 22 allozyme loci were polymorphic, exhibiting 35 alleles, while seven 10-mer RAPD primers revealed 98 polymorphic bands. Strong regional differences were detected in the extent of allozyme polymorphism: Mexican populations exhibited more internal gene diversity (H_e= 0.20, 0.19) than did the Texan populations (H_e= 0.08, 0.06), although the number of RAPD bands in Texas (n= 62) was only marginally smaller than in Mexico (n= 68). F-statistics for the allozyme data, averaged over loci, revealed strong regional differentiation (mean F_RT=+ 0.30), as well as some differentiation among populations within regions (mean F_PR=+ 0.09). In order to describe and compare the partitioning of genetic variation for multiple allozyme and RAPD loci, we performed an Analysis of Molecular Variance (AMOVA). AMOVA for both allozyme and RAPD data revealed similar qualitative patterns: large regional differences and smaller (but significant) population differences within regions. RAPDs revealed greater variation among regions (58.4% of total variance) than allozymes (45.2%), but less variation among individuals within populations (31.9% for RAPDs vs. 45.2% for allozymes); the proportion of genetic variance among populations within regions was similar (9.7% for RAPDs vs. 9.6% for allozymes). Despite this large-scale concordance of allozyme and RAPD variation patterns, multiple correlation Mantel techniques revealed that the correlations were low on an individual by individual basis. Our findings of strong regional differences among the diploid races will facilitate further study of polyploid evolution in buffalograss.     

Purification and characterization of two allozymic forms of octopine dehydrogenase from California populations ofMetridium senile

Summary In northern and southern California populations of the plumose sea anemone,Metridium senile, octopine dehydrogenase occurs in two allozymic forms and these forms are distributed in a highly population-specific manner; the frequency of the slow allele (ODH ¹⁰⁰) is 0.875 in the northern (Bodega Bay) population while the frequency of the fast allele (ODH ¹⁰³) in the southern population (Santa Barbara) is 0.125. Purification techniques resulted in an increase in purity of approximately 400 fold. The enzyme is a monomer ofM _r 35,000 to 40,000. Though there is some flexibility in the amino acid substrate which the enzyme uses (arginine and lysine react similarly), the specificity for the keto acid is limited to pyruvate.The kinetic characters of the two allozymes ofMetridium senile ODH are very different with respect to type of substrate saturation (Fig. 4 and 5) and apparent Michaelis constants (K _m) for pyruvate, lysine, arginine, and octopine (Table 5), product inhibition by octopine (Fig. 2), and optimal activity with respect to pH (Fig. 3). The properties of the slow and fast allozymes resemble the kinetic properties of cephalopod brain and muscle tissue-specific isozymes (Table 7). The kinetic data indicate that the slow allozyme would not allow a great deal of accumulation of octopine in vivo, while the fast allozyme is poised markedly towards octopine production.When the data presented in this study are compared to various physiological findings of other investigators, it becomes evident that the probable in vivo function of ODH in sea anemones is to act, in a manner analogous to vertebrate LDH, during the short-term anaerobiosis associated with muscle contraction and locomotion. The population-specific distribution and the different functional properties of the two ODH allozymes are most likely related to the different degree of tidal exposure which the two populations experience in nature. Only the slow allozyme possesses the regulatory properties which would allow a shift to the alternative anaerobic pathways utilized during these longer exposure periods.Abbreviations ODH octopine dehydrogenase - LDH lactate dehydrogenase - PHI phosphohexose isomerase     

Isozyme analysis in a genetic collection of amaranths (Amaranthus L)

In various populations of the cultivated and weedy amaranth species, the electrophoretic patterns of alcohol dehydrogenase (ADH), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and malic enzyme (Me) were studied. In total, 52 populations and two varieties (Cherginskii and Valentina) have been examined. Allozyme variation of this material was low. Irrespective of species affiliation, 26 populations and two varieties were monomorphic for five enzymes; a slight polymorphism of three, two, and one enzymes was revealed in three, nine, and fourteen populations, respectively. A single amaranth locus, Adh, with two alleles, Adh F and Adh S, controls amaranth ADH. Two alleles, common Gdh S and rare Gdh F, control GDH; no heterozygotes at this locus were found. The MDH pattern has two, the fast- and slow-migrating, zones of activity (I and II, respectively). Under the given electrophoresis conditions, the fast zone is diffuse, whereas slow zone is controlled by two nonallelic genes, monomorphic Mdh 1 and polymorphic Mdh 2 that includes three alleles: Mdh 2-F, Mdh 2-N, and Mdh 2-S. Low polymorphism of IDH and Me was also found, though their genetic control remains unknown.     

Restriction map variation at the adh locus of Drosophila melanogaster in inverted and noninverted chromosomes

Restriction map variation among 39 Standard and 40 In(2L)t chromosomes extracted from a Spanish natural population of Drosophila melanogaster was investigated for a 2.7-kb region encompassing the Adh locus with ten four-cutter restriction enzymes. A total of 20 polymorphisms were detected, representing 15 restriction site polymorphisms, 4 length polymorphisms and the allozyme polymorphism. Variation at the DNA level was compared among St-Adh(F), St-Adh(S) and t-Adh(S) chromosomes. t-Adh(S) chromosomes show a higher level of variation than St-Adh(F) chromosomes. This suggests that In(2L)t arose before the fast/slow allozyme divergence in the evolutionary history of D. melanogaster.     

Isozyme Analysis in a Genetic Collection of Amaranths (<Emphasis Type="Italic">Amaranthus</Emphasis> L.)

In various populations of the cultivated and weedy amaranth species, the electrophoretic patterns of alcohol dehydrogenase (ADH), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and malic enzyme (Me) were studied. In total, 52 populations and two varieties (Cherginskii and Valentina) have been examined. Allozyme variation of this material was low. Irrespective of species affiliation, 26 populations and two varieties were monomorphic for five enzymes; a slight polymorphism of three, two, and one enzymes was revealed in three, nine, and fourteen populations, respectively. A single amaranth locus, Adh, with two alleles, Adh F and Adh S, controls amaranth ADH. Two alleles, common Gdh S and rare Gdh F, control GDH; no heterozygotes at this locus were found. The MDH pattern has two, the fast- and slow-migrating, zones of activity (I and II, respectively). Under the given electrophoresis conditions, the fast zone is diffuse, whereas slow zone is controlled by two nonallelic genes, monomorphic Mdh 1 and polymorphic Mdh 2 that includes three alleles: Mdh 2-F, Mdh 2-N, and Mdh 2-S. Low polymorphism of IDH and Me was also found, though their genetic control remains unknown.     

Intron size polymorphism of the Adh1 gene parallels the worldwide colonization history of the Mediterranean fruit fly, Ceratitis capitata

The exon-primed intron-crossing (EPIC) PCR technique was used to analyse the size variation at the first intron of the Ceratitis capitata Adh ₁ gene. A total of 27 samples from 16 natural populations was analysed from five geographical regions in the species range: Africa, Mediterranean Basin, Latin America, Hawaii and Australia. The Adh ₁ first intron varies extensively in length with at least 18 size variants ranging from 1400 bp to 3450 bp. These variants can be grouped into four distinct size categories: short, medium, long and very long. The majority of these variants are present only in the African populations. Only a subset of the ancestral variants appear to have succeeded in migrating from Africa during the medfly colonization process. The medfly population structure inferred from the intron size polymorphism is congruent with that observed from the analysis of allozyme variation. The geographical dispersal of the medfly from its source area is associated with a gradual and great reduction in intron variability which parallels the trend of decreasing variability evaluated at 26 biochemical loci. The intron phylogenetic tree is in agreement with allozyme data in portraying the dynamic population history of the medfly. Stochastic evolutionary forces such as drift, bottleneck effects and migration seem to have played the major roles in the dispersion pattern of Adh ₁ intron variation during the colonization of the medfly.     

The effect of temperature on biochemical and molecular properties ofDrosophila alcohol dehydrogenase

The gene products of the two major alleles of alcohol dehydrogenase (ADH-F and ADH-S) have been subjected to kinetic and biochemical analyses over a range of temperatures. Although temperature was found to have a significant effect on both kinetic and biochemical properties of Drosophila ADH, no significant differential effect was observed between the major ADH allozymes. The results are discussed within the context of the selective maintenance of Adh polymorphism in natural populations.     

ASSOCIATIONS BETWEEN ALLOZYME FREQUENCIES AND SOIL CHARACTERISTICS IN GAILLARDIA PULCHELLA (COMPOSITAE)

Selection favoring different alleles in different environments frequently has been suggested as an explanation for allozyme variation within and among populations. This hypothesis predicts that allozyme frequencies will be correlated with environmental variables. Previous studies on allozyme frequency-environment covariation in plants often have relied on qualitative assessments of the environment and have emphasized highly autogamous species. We have examined allozyme frequency-soil associations in Gaillardia pulchella, an obligately outcrossed annual plant, by regressing the frequencies of 15 common allozymes representing six polymorphic enzyme loci on principal components from a set of 20 quantitative soil variables. Fifty-one populations, representing four taxonomic varieties, were included in the analysis. Among the 26 populations representing the var. pulchella, allozymes Adh-2f and Pgm-1c were significantly associated with a block of highly inter-correlated soil characteristics which serve to discriminate between soils derived from calcareous vs. non-calcareous rock types. This geographically complex pattern of allozyme frequency-soil covariation is not likely to be spurious and, thus, indicates the presence of adaptively differentiated soil races, or ecotypes. However, these results are not sufficient to conclude that the allozyme frequency divergence between ecotypes was mediated by selection, either directly or through genetic hitchhiking. The pattern of allozyme frequency-soil covariation within var. pulchella was not found among the other taxonomic varieties. Patterns of genotype-environment covariation often may be recognizable only within geographically or environmentally restricted groups of populations because of the confounding influences of other environmental variables.     

Allozyme polymorphisms within and among open-pollinated and adapted exotic populations of maize

Summary Twelve U.S. Corn Belt open-pollinated and five adapted exotic populations of maize (Zea mays L.) were assayed for allozyme (allele) variation at 13 enzyme marker loci. Extensive allozyme variability was observed in all populations studied. No locus was monomorphic over all populations. Each of the lociIdh2, Got1, Mdh2, Pgd1, andPgd2 expressed two allozymes over all populations,Adh1, Acp1, Prx1, andEst1 each had three allozymes present,Est4, Glu1, andEnp1 had five allozymes, andAcp4 had six allozymes present. Significant deviations of genotypic frequencies were detected from Hardy-Weinberg equilibrium frequencies and 94% of average Fixation Index values indicated heterozygote deficiencies, which suggested that nonrandom mating and/or natural selection favoring homozygotes were possible factors affecting the maintenance or loss of genetic variability marked by these enzyme loci. Genetic distance and cluster analyses indicated that the observed genetic variability at the 13 enzyme loci was closely related to Dent and Flint types of maize.     

Genetic analyses of two spawning stocks of the short-finned squid (Illex argentinus) using nuclear and mitochondrial data

Short-finned squid Illex argentinus (Omastrephidae) is an economically important species which supports a long-term and intensive commercial fishery in the South-West Atlantic Ocean. In this study, the genetic variability of allozymes (40 loci) and mitochondrial regions, COI (556 bp) and 16S rDNA (439 bp), were addressed in the two most important species’ spawning stocks, the Summer Spawning Stock (SSS) and the South Patagonic Stock (SPS). Five out of 20 polymorphic allozyme loci were polymorphic at 95% criterion and heterozygosity levels were low. The concatenated analysis of mitochondrial molecular markers revealed high to low values of haplotype and nucleotide diversity, respectively. Nuclear and mitochondrial data revealed no significant genetic differences between these two spawning stocks. Our results and the biological characteristics of I. argentinus deal with a no simple genetic structure of populations described as spatial and temporal chaotic patchiness. SSS and SPS spawning stocks of I. argentinus could not be elevated to the status of genetic stocks.