Evolution of patterns of gene expression in Hawaiian picture-wingedDrosophila

Summary The tissue and stage specificity of expression of five enzymes was examined by electrophoretic analysis of relative enzyme levels in extracts of 13 larval and adult tissues in 27 species of Hawaiian picture-wingedDrosophila. The developmentally regulated patterns of enzyme expression thus characterized were compared to a modal standard phenotype. About 30% of the pattern features analyzed differed significantly from the standard in one or more species. Many of these regulatory differences are essentially qualitative, with tissue specific differences in enzyme activity in excess of 100 fold for some species pairs. The adaptive significance of these pattern differences is unknown, but the results provide strong direct evidence for rapid evolution of new patterns of gene regulation in this group of organisms.     

Expression of the paternal genes for lactate dehydrogenase, glutamate dehydrogenase and acetylcholinesterase in the development of hybrid fish between species from the families of Cobitidae and Cyprinidae]

The time of expression of the paternal genes of glutamate dehydrogenase (GDH), lactate dehydrogenase (LDH) and acetylcholine esterase (AChE) was investigated in the development of fish hybrids. The species which differed by the thermostability of homologous enzymes were selected as parental pairs. The appearance of differences in the thermostability of homologous enzymes between the hybrids and the maternal species suggested the beginning of paternal enzyme synthesis in the hybrid embryos. Differences in the AChE thermostability appeared simultaneously with the enzyme activity at the stage of first muscle contractions (35 hrs of development), differences in the mitochondrial GDH thermostability appeared at the stage of hatching (50-60 hrs) and those in the LDH thermostability 12-17 days after hatching. The total activity of AChE and GDH sharply increased during the period of the paternal enzyme appearance whereas the activity of LDH suffered practically no changes. Differences in the AChE thermostability between the hybrids and the maternal species are the same for both the total AChE (in supernatant, 15,000 gX10 min.) and the solubilised AChE (in supernatant, 130,000 gX60 min.). AChE of the parental species and the hybrids have the same electrophoretic mobility. The differences in the thermostability of enzymes are preserved following the electrophoresis in polyacrilamide gel.     

6-Phosphogluconate dehydrogenase (PGD) genetics in the mouse: Linkage with metabolically related enzyme loci

An electrophoretic polymorphism of 6-phosphogluconate dehydrogenase (PGD) has been observed in the subspecies Mus musculus musculus from northern Denmark. M. m. musculus is interfertile with inbred strains of mice, and F1 hybrids with C57BL/6J show a three-banded phenotype. This pattern is consistent with a dimeric enzyme structure with codominant expression of alleles. In backcrosses and the F2 generation, PGD segregated as a singly autosomal gene, designated Pgd, closely linked to Gpd-1 on chromosome 4(1.7 +/- 1.1%). Both gene products are dimers, both require NADP, and these enzymes catalyze sequential steps in metabolism.     

Modulation and mapping of a human plasminogen activator by cell fusion

Neoplastic cells, transformed cells and some normal mammalian cells secrete large amounts of plasminogen activator (PA), an arginine-specific protease which converts plasminogen to plasmin. To study the regulation of PA, we have obtained two classes of mouse-human somatic cell hybrids. PG19, a mouse PA⁺ cell line, was fused with C32 (human PA⁺) or human diploid fibroblasts (PA^?). All hybrids secreted PA. Human- and mouse-specific forms of PA were distinguished in these hybrids by electrophoretic methods. While all hybrids produced the murine PA, many produced the human PA and some did not. All hybrids which produced human PA had chromosome 6 in common. The absence of each of the other human chromosomes did not affect PA expression, while the absence of chromosome 6 correlated with the lack of human PA. We conclude that chromosome 6 carries the structural gene for human PA. These experiments also show that the fusion of mouse PA⁺ cells with human PA-cells results in the activation of the human PA gene.     

Activation of production of mouse liver enzymes in rat hepatoma-mouse lymphoid cell hybrids

The production of four liver-specific enzymes (tyrosine aminotransferase or TAT, alanine aminotransferase, aldolase B, and alcohol dehydrogenase) has been analyzed in rat hepatoma-mouse lymphoid cell hybrids containing a 1s or 2s complement of rat chromosomes. The hybrid clones which retain a nearly 2s complement of rat chromosomes show high activity of all four enzymes; those which contain a 1s rat complement show partial or complete extinction of these enzymes. The tyrosine aminotransferase produced by most of the hybrid clones is identifiable as being of both rat and mouse origin, based upon criteria of temperature sensitivity and electrophoretic mobility; antiserum to the rat liver enzyme was used to distinguish the pseudo-TAT produced by the lymphoid parent from liver-TAT produced by hepatoma and hybrid cells. By the criteron of electrophoretic mobility, the liver form (B) of aldolase, produced by only some of the hybrid clones, appears to be composed of both rat and mouse subunits. We conclude that when extinction of tissue-specific proteins does not occur or is only partial in hybrid cells (due to gene dosage effects), the genomes of both parents may be active in directing synthesis of these proteins.     

Allelic expression changes in Medaka (Oryzias latipes) hybrids between inbred strains derived from genetically distant populations

Variations in allele expressions between genetically distant populations are one of the most important factors which affects their morphological and physiological variations. These variations are caused by natural mutations accumulated in their habitats. It has been reported that allelic expression differences in the hybrids of genetically distant populations are different from parental strains. In that case, there is a possibility that allelic expression changes lead to novel phenotypes in hybrids. Based on genomic information of the genetically distant populations, quantification and comparison of allelic expression changes make importance of regulatory sequences (cis-acting factors) or upstream regulatory factors (trans-acting modulators) for these changes clearer. In this study, we focused on two Medaka inbred strains, Hd-rR and HNI, derived from genetically distant populations and their hybrids. They are highly polymorphic and we can utilize whole-genome information. To analyze allelic expression changes, we established a method to quantify and compare allele-specific expressions of 11 genes between the parental strains and their reciprocal hybrids. In intestines of reciprocal hybrids, allelic expression was either similar or different in comparison with the parental strains. Total expressions in Hd-rR and HNI were tissue-dependent in the case of HPRT1, with high up-regulation of Hd-rR allele expression in liver. The proportion of genes with differential allelic expression in Medaka hybrids seems to be the same as that in other animals, despite the high SNP rate in the genomes of the two inbred strains. It is suggested that each tissue of the strain difference in trans-acting modulators is more important than polymorphisms in cis-regulatory sequences in producing the allelic expression changes in reciprocal hybrids.     

Dissociation of expression patterns of homeodomain transcription factors in the evolution of developmental mode in the sea urchins Heliocidaris tuberculata and H. erythrogramma

The direct-developing sea urchin species Heliocidaris erythrogramma has a radically modified ontogeny. Along with gains of novel features, its entire ectoderm has been reorganized, resulting in the apparent absence of a differentiated oral ectoderm, a major module present in the pluteus of indirect-developing species, such as H. tuberculata. The restoration of an obvious oral ectoderm in H. erythrogrammaxH. tuberculata hybrids, indicates the action of dominant regulatory factors from the H. tuberculata genome. We sought candidate regulatory genes based on the prediction that they should include genes that govern development of the oral ectoderm in the pluteus, but play different roles in H. erythrogramma. Such genes may have a large effect in the evolution of development. Goosecoid (Gsc), Msx, and the sea urchin Abd-B-like gene (Hox11/13b) are present and expressed in both species and the hybrid embryos. Both Gsc and Msx are oral ectoderm specific in H. tuberculata, and show novel and distinct expression patterns in H. erythrogramma. Gsc assumes a novel ectodermal pattern and Msx shifts to a novel and largely mesodermal pattern. Both Gsc and Msx show a restoration of oral ectoderm expression in hybrids. Hox11/13b is not expressed in oral ectoderm in H. tuberculata, but is conserved in posterior spatial expression among H. tuberculata, H. erythrogramma and hybrids, serving as a control. Competitive RT-PCR shows that Gsc, Msx, and Hox11/13b are under different quantitative and temporal controls in the Heliocidaris species and the hybrids. The implications for the involvement of these genes in the rapid evolution of a direct developing larva are discussed.     

Evidence for natural reassortants of human rotaviruses belonging to different genogroups.

Of 335 rotavirus isolates associated with diarrheal disease in Bangladesh that were culture adapted and subsequently characterized for electropherotype, subgroup, and serotype, 9 had properties that suggested they may be natural reassortants between human rotaviruses belonging to different "genogroups." Two of these were examined in greater detail by RNA-RNA hybridization with prototype strains representative of each of the three proposed human rotavirus genogroups. One subgroup II isolate, 248, with a "long" electrophoretic pattern was neutralized by hyperimmune antisera to both serotype 2 and 4 strains. Consistent with these results, seven RNA segments of this isolate formed hybrids with human strains belonging to the Wa genogroup and four segments hybridized with strains belonging to the DS-1 genogroup. The second isolate examined, 456, belonged to subgroup II and had a long electrophoretic pattern but was found to be a serotype 2 strain. This isolate also appeared to be an intergenogroup reassortant because three of its segments formed hybrids with strains belonging to the Wa genogroup and eight hybridized with viruses of the DS-1 genogroup. On the basis of the relative migration rates of these RNA-RNA hybrids during gel electrophoresis, a suggested origin for each gene segment was proposed which was consistent with the results expected from electrophoretic, subgroup, and serotypic analyses.     

曲霉和青霉属内杂种和亲株的蛋白质电泳图谱比较

曲霉属内黑曲霉(Aspergitlus niger)与米曲霉(A.oryzae)具有特征明显不同的可溶性蛋白质电泳图谱,其种间杂种具有双亲的部分或全部电泳带并与黑曲霉相近。来自杂种Ⅰ的多数分离子电泳带与黑曲霉相近,只有一个分离子产生米曲霉的电泳带并具有米曲霉的遗传特性。青霉属内产黄青霉(Penicillium chrysogenum)与展青霉(P.patulum)种间及种内不同菌株间的电泳图谱基本相同,种内或种间杂种具有双亲的电泳带。结果讨论了蛋白质图谱分析的意义。     

Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification.

We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.     

DNA restriction enzyme fragment polymorphism as a tool for rapid differentiation ofAspergillus flavus fromAspergillus oryzae

Aspergillus flavus andAspergillus oryzae are difficult to differentiate using standard morphologically based characteristics. This survey, using several restriction enzymes, showed thatSmaI digestions of total DNA could be used to differentiate these two closely related species ofAspergillus. A different electrophoretic pattern was associated with each of the two species, while within a species the pattern remained constant. CsCl banding of DNA from one isolate of each species indicated the DNA that produced the species-specific pattern was associated with the nuclear DNA fraction.     

Allelic expression at the sorbitol dehydrogenase and glucosephosphate isomerase loci in an interspecific hybrid ricefish

1. The expression of alleles encoding the enzymes sorbitol dehydrogenase (SDH; EC 1.1.1.14) and glucosephosphate isomerase (GPI; EC 5.3.1.9) was investigated in Oryzias melastigma, O. javanicus and in O. melastigma female x O. javanicus male hybrids by acrylamide gel electrophoresis. 2. It was not possible to investigate the expression of SDH or GPI in reciprocal hybrids as these fry failed to develop past the stage of embryonic body formation. 3. The delay in appearance of isozymes of paternal SDH subunit composition, and paternal and maternal GPI-B subunit composition is in keeping with observed effects of gene regulatory divergence where alleles of maternal origin interact more effectively with maternal cytoplasmic regulatory factors than do alleles of paternal origin.     

ELECTROPHORETIC STUDY OF 5-HYDROXYTRYPTOPHAN DECARBOXYLASE FROM BRAIN AND LIVER IN SEVERAL SPECIES

—An electrophoretic investigation in acrylamide gels of 5-hydroxytryptophan decarboxylase, obtained mostly from mouse, rat, and beef brain and also from beef and human liver, showed electrophoretic differences between species. With the exception of the rat, only one molecular species was found (the same in beef brain and liver). In the rat, polymers form spontaneously and are, at least in part, disaggregated by urea and by triton. Mouse-rat or beef-rat molecular hybrids form in the admixtures. No electrophoretic differences were found in five mice strains that were investigated. Techniques of electrophoretic analysis and of assay of 5-hydroxytryptophan decarboxylase are described, which can be easily applied to other enzymes, provided a substrate is available in radioactive form.     

Clonal variations in keratin : Intermediate filament expression by human somatic cell hybrids

The intermediate filament composition of differentiated vertebrate cells provides a stable phenotype which appears to be specifically regulated in each cell type. In order to analyse the regulation of intermediate filament expression we have constructed human somatic cell hybrids from the fusion of the HeLa-derived cell line HEB7A and a normal human diploid fibroblast, GM2291. These parental cells differ with respect to the presence or absence of keratin intermediate filaments. Isolation of independently arising clones produced two classes of hybrids. One class expresses keratin in a stable manner and the other class lacks keratin altogether. Indirect immunofluorescence of hybrid cells using antikeratin antiserum demonstrates that there are variations in the intensity and organization of cytoskeletal keratin staining. SDS-PAGE comparisons of cell extracts from these hybrids indicates that there are quantitative differences in the relative amounts of individual keratin polypeptides as well. These clonal variations have allowed us to begin assessing the consequences of genetic interactions between cell types that are normally capable of closely regulating different subsets of intermediate filament genes.     

Glucose 6-phosphate dehydrogenase from brook,lake, and splake trout: An isozymic and immunological study

There are five G6P-specific G6PD isozymes in both brook and lake trout. The most anodal isozyme in each species has the same electrophoretic mobility; however, the five lake trout isozymes are more widely spaced on polyacrylamide gels than are those from brook trout. In hybrids, i.e., splake trout, nine forms of G6PD can be resolved. These results can be explained by a model in which we assume that each isozyme is a tetramer and that two different subunit types are produced. In splake trout, three electrophoretically distinct subunits yield 15 tetramers. That only nine are detected is a consequence of coincident electrophoretic mobility of some of the possible subunit combinations. Our results indicate that the G6PD isozymes in these trout are the products of two codominant autosomal gene loci. The hypothesis that G6PD and H6PD arose from a common ancestral type G6PD is supported by microcomplement fixation data which show an immunochemical relatedness between these enzymes. The relationship of G6PD and H6PD in trout is discussed from an evolutionary standpoint.This research was supported in part by National Science Foundation Grant GB-7271 and by United States Public Health Predoctoral Fellowship 4 FO1 GM 43657-03.     

Allele-specific expression patterns reveal biases and embryo-specific parent-of-origin effects in hybrid maize

We employed allele-specific expression (ASE) analyses to document biased allelic expression in maize (Zea mays). A set of 316 quantitative ASE assays were used to profile the relative allelic expression in seedling tissue derived from five maize hybrids. The different hybrids included in this study exhibit a range of heterosis levels; however, we did not observe differences in the frequencies of allelic bias. Allelic biases in gene expression were consistently observed for approximately 50% of the genes assayed in hybrid seedlings. The relative proportion of genes that exhibit cis- or trans-acting regulatory variation was very similar among the different genotypes. The cis-acting regulatory variation was more prevalent and resulted in greater expression differences than trans-acting regulatory variation for these genes. The ASE assays were further used to compare the relative expression of the B73 and Mo17 alleles in three tissue types (seedling, immature ear, and embryo) derived from reciprocal hybrids. These comparisons provided evidence for tissue-specific cis-acting variation and for a slight maternal expression bias in approximately 20% of genes in embryo tissue. Collectively, these data provide evidence for prevalent cis-acting regulatory variation that contributes to biased allelic expression between genotypes and between tissues.