Wild-type Stocks Research Articles

Sex-Linkage of the Black Caudal-Peduncle and Red Tail Genes in the Tuxedo Strain of the Guppy, Poecilia reticulata

Abstract Two color patterns of the Tuxedo guppy strain commercially cultured in Singapore were subjected to genetic analyses. Gene control of the black caudal-peduncle and red tail color patterns was elucidated by reciprocal crosses between the Tuxedo (TUX) strain and wild-type (WT) stock. F1 progenies were produced by single-pair crossing between TUX and WT, while the F2 generation was obtained from full-sib mating between F1 males and females. F1 and F2 data were segregated according to phenotypes and sex, and tested by chi-square analyses. Both color patterns show single gene inheritance, and are dominantly expressed in both sexes, sex-linked and determined by different loci on the X- and Y-chromosomes. Alleles for the black caudal-peduncle (Bcp) and red tail (Rdt) loci, are dominant over that of the wild-type, Bcp+ and Rdt+, which do not display these color patterns. The typical genotypes for TUX guppies are proposed to be XBcp,RdtYBcp,Rdt for males and XBcp,RdtXBcp,Rdt for females. Heterozygous TUX m...

Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2.

We demonstrate the rapid and reliable quantification of physical AAV-2 (adeno-associated virus type 2) particles via a novel ELISA based on a monoclonal antibody which selectively recognizes assembled AAV-2 capsids. Titration of a variety of recombinant AAV-2 (rAAV) preparations revealed that at least 80+percent of all particles were empty, compared with a maximum of 50percent in wild-type AAV-2 stocks, indicating that the recombinant genomes were less efficiently encapsidated. This finding was confirmed upon titration of CsCl gradient fractions from recombinant and wild-type AAV-2 stocks. ELISA-based measurement of capsid numbers revealed a large number of physical particles with low densities corresponding to empty capsids in the recombinant, but not in the wild-type AAV-2 preparations. Moreover, additional expression of VP proteins during rAAV production was found to result in an excessive capsid formation, whilst yielding only minor increases in DNA-containing or transducing rAAV particles. We conclude that encapsidation of viral genomes rather than capsid assembly can be limiting for rAAV production, provided that a critical level of VP expression is maintained. The feasibility of quantifying AAV-2 capsid numbers via the ELISA allows determination of physical to DNA-containing or infectious particle ratios. These are important parameters which should help to optimize and standardize the production and application of recombinant AAV-2.

Genetic Basis of the Variegated Tail Pattern in the Guppy, Poecilia reticulata

Abstract Variegated patterns on the caudal fin are a common and popular trait in guppy strains commercially cultured in Singapore. Gene control of this highly variable mosaic pattern of black spots and patches of different shapes and sizes on a brightly colored tail fin was elucidated by reciprocal crosses between the Green Variegated (GV) strain and wild-type (WT) stock. F1 progenies were produced by single-pair crossing between GV and WT, while the F2 generation was obtained from full-sib mating between F1 males and F1 females. Data for the F1 and F2 generations were segregated according to phenotypes and sex, and tested by chi-square analyses. Inheritance of variegated tail patterns appears to be determined by a single locus on the X- and Y-chromosomes. Genotypes of males and females of the GV strain are proposed to be X VarYVar and XVarXVar, respectively. The allele for variegated tail patterning, Var, is dominant over that of the wild-type, Var+, which does not exhibit these patterns. Recombination f...

Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression.

Abscisic acid (ABA) is a plant hormone synthesized during seed development that is involved in the induction of seed dormancy. Delayed germination due to seed dormancy allows long-term seed survival in soil but is generally undesirable in crop species. Freshly harvested seeds of wild-type Nicotiana plumbaginifolia plants exhibit a clear primary dormancy that results in delayed germination, the degree of primary dormancy being influenced by environmental culture conditions of the mother plant. In contrast, seeds, obtained either from ABA-deficient mutant aba2-s1 plants directly or aba2-s1 plants grafted onto wild-type plant stocks, exhibited rapid germination under all conditions irrespective of the mother plant culture conditions. The ABA biosynthesis gene ABA2 of N. plumbaginifolia, encoding zeaxanthin epoxidase, was placed under the control of the constitutive 35S promoter. Transgenic plants overexpressing ABA2 mRNA exhibited delayed germination and increased ABA levels in mature seeds. Expression of an antisense ABA2 mRNA, however, resulted in rapid seed germination and in a reduction of ABA abundance in transgenic seeds. It appears possible, therefore, that seed dormancy can be controlled in this Nicotiana model species by the manipulation of ABA levels.

How is developmental stability sustained in the face of genetic variation?

The number and arrangement of scutellar bristles on the thorax of Drosophila melanogaster is largely invariant in wild-type stocks. This character therefore appears to be buffered against changes in phenotype, and has previously been described as a canalized character. Mutations that do alter this phenotype increase the variability in bristle number and can reveal otherwise cryptic genetic differences at other loci. This phenomenon is examined and possible mechanisms contributing to stability of this developmental event are discussed, but the notion that the character is canalized is found not to be heuristic.

Utilization of a Photosystem by Drosophila melanogaster Larvae (Diptera: Drosophilidae)

Foraging-stage third-instar larvae from most wild-type (normal) Drosophila melanogaster stocks are generally repelled by light. To identify factors that affect the larval photoresponse, we elucidated the effects of age, temperature, and time on the photoresponse of larvae from a wild-type Canton-S stock. In addition, we analyzed the larvae from the LI2 isofemale line, which are unresponsive to light in a photoassay. To determine whether LI2 larvae behave abnormally on other behavioral paradigms, in comparison to Canton-S controls, we tested larvae in taste and olfactory assays and observed them to determine whether they dispersed in a food source. Like Canton-S larvae, LI2 larvae and other isofemale lines whose progenitors were collected from the same natural population are responsive to taste and olfactory stimuli. Moreover, LI2 larvae disperse in the food source, as do Canton-S larvae tested in the dark. Larvae expressing parasblmutations, which respond normally to light but not to chemical stimuli, do not disperse normally in the food source, suggesting that dispersal may be mediated by perception of chemical cues.

Role of the fowlpox virus thymidine kinase gene for the growth of FPV recombinants in cell culture.

Fowlpox virus (FPV) insertion plasmids were constructed that, upon integration into the viral genome via in-vivo recombination, inactivate the viral thymidine kinase (tk) gene. Using this approach, no wild-type virus-free stocks of recombinant virus could be obtained. In contrast, either integration of foreign genes into the intergenic region of the intact FPV tk gene and the open reading frame located downstream, or the functional substitution of the inactivated FPV tk gene by an intact vaccinia virus tk gene resulted in the predicted stable recombinants that were free of wild-type virus. Our results suggest that in already highly attenuated poxvirus strains an intact tk gene is essential for efficient growth of the virus in cell culture.

The Polio Eradication Effort: Should Vaccine Eradication Be Next?

The World Health Organization (WHO) has implemented a plan to eradicate poliovirus that is widely viewed as having made enormous progress. If all goes as planned, polio will be eradicated on this planet by the year 2003. However, there is a debate, as highlighted in a pair of Policy Forums in this issue, over when vaccination against polio can be stopped. Dove and Racaniello believe that the reliance of the WHO on the live Sabin oral poliovirus vaccine (OPV) means that there will be a continuing threat of release of potentially pathogenic virus into the environment. They are also concerned that the planned destruction of all wild-type polio stocks will not be possible. They therefore recommend a switch to the inactivated polio vaccine (IPV). In response, Hull and Aylward explain why a switch from OPV is not necessary and describe the studies being sponsored by the WHO to determine how and when immunization can safely be ended.

Ending polio immunization.

The World Health (WHO) has implemented a plan to eradicate poliovirus that is widely viewed as having made enormous progress. If all goes as planned, polio will be eradicated on this planet by the year 2003. However, there is a debate, as highlighted in a pair of Policy Forums in this issue, over when vaccination against polio can be stopped. Dove and Racaniello believe that the reliance of the WHO on the live Sabin oral polio virus vaccine (OPV) means that there will be a continuing threat of release of potentially pathogenic virus into the environment. They are also concerned that the planned destruction of all wild-type polio stocks will not be possible. They recommend a switch to the inactivated polio vaccine. In response, Hull and Aylward set out the reasons for thinking that a switch from the OPV is not necessary and describe the studies being sponsored by the WHO to determine how and when immunization can safely be ended.

Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions.

Using grafting procedures, we investigated the transmission of co-suppression of nitrate reductase and nitrite reductase host genes and transgenes and of post-transcriptional silencing of a uidA transgene encoding glucuronidase in tobacco. We demonstrate that silencing is transmitted with 100% efficiency from silenced stocks to non-silenced scions expressing the corresponding transgene. Transmission is unidirectional from stock to scion, transgene specific, locus independent and requires the presence of a transcriptionally active transgene in the target scion. The transmission of co-suppression occurs when silenced stocks and non-silenced target scions are physically separated by up to 30 cm of stem of a non-target wild-type plant. Taken together, these results suggest that a non-metabolic, transgene-specific, diffusable messenger mediates the propagation of de novo post-transcriptional silencing through the plant.

Y-chromosome effects onDrosophilageotaxis interact with genetic or cytoplasmic background

Previously, all of the major fruit fly,Drosophila melanogaster, chromosomes (I, II and III) have been shown to be associated with geotaxis, but the Y chromosome has not. Using two methods (back-crossing and chromosome substitution), Y chromosomes from lines that have evolved stable, extreme expressions of geotaxis were placed into different genetic and cytoplasmic backgrounds to test the resulting males for geotaxis. The results of the back-crossing do not support the interpretation of Y-chromosome effects on geotaxis. These tests do not have sufficient statistical power, however, to detect small genetic effects. In the chromosome substitution experiment, the geotaxis-line Y chromosomes were placed into high- and low-selected lines, Canton-S and Champaign wild-type backgrounds. The results of the chromosome substitution experiment provide evidence for a Y-chromosome effect on geotaxis in selected geotaxis lines, but not in wild-type stock, backgrounds. These results suggest that the Y chromosome has a small effect on geotaxis, whose detection depends on genetic and/or cytoplasmic background. The implications of these results are discussed for behaviour–genetic analysis ofD.melanogasterand for issues of statistical power in detecting small genetic effects.

Lack of response to sex-peptide results in increased cost of mating in dunce Drosophila melanogaster females

Drosophila melanogaster females homozygous for the dunce mutation have been reported to mate more often than females of a wild type stock (Canton-S). We have investigated the basis of this increased receptivity, by examining the response of dunce females, females of a revertant stock with the same genetic background and Canton-S females to injection with a component of the male accessory fluid (the ‘sex-peptide’, SP). dunce virgin females injected with SP showed no decline in receptivity towards courting males, whereas receptivity in control females was inhibited. dunce females also showed no inhibition of remating associated with the transfer of any non-sperm components at mating (the ‘copulation effect’) but did show a loss of receptivity due to the receipt of sperm (the ‘sperm effect’). The high remating rate of dunce females can therefore be attributed at least in part to their aberrant response to the receptivity-inhibiting SP. dunce females were no more susceptible than control females to the effects of a single mating; there was no significant difference in lifespan between dunce females mated once soon after eclosion and virgin dunce females. Twice-mated dunce females, however, had significantly shorter lifespans than virgin dunce females; this effect was not apparent in control dunce + revertant females. The high cost of mating incurred by dunce females is therefore due to a combination of their elevated remating rate and an increase in sensitivity to the effects of a fixed number of matings.

Flowering in Pisum: Identification of a new ppd allele and its physiological action as revealed by grafting

The late flowering, quantitative long day habit of wild type pea (Pisum sativum L.) is conferred by the joint presence of dominant genes Sn, Dne and Ppd. Grafting studies have shown that flowering in wild type plants is delayed under short days by formation of a graft‐transmissible inhibitor and that the early flowering, day neutral mutants sn and dne are deficient in this inhibitor. However, the physiological action of the Ppd gene has not been examined by grafting and the possibility exists that the ppd mutation causes early flowering and a day neutral habit by blocking response to, rather than synthesis of, the inhibitor. We here identify a second, more severe (probably null) mutant allele (ppd‐2) at the Ppd locus and show that flowering was delayed by 4 nodes in a ppd‐2 shoot grafted to a wild type stock, and promoted by 13 nodes in a wild type shoot grafted to a ppd‐2 stock. Thus a ppd‐2 shoot can respond to inhibitor donated by a wild type stock but a ppd‐2 stock is unable to provide sufficient inhibitor to prevent early flower initiation in a wild type shoot. We conclude genes Sn, Dne and Ppd each control steps in the synthesis of the flower inhibitor. Grafts among the sn, dne and ppd mutants gave an indication that the three genes may act in the sequence Sn, Ppd, Dne, but possible cases of physiological complementation need to be tested using null mutants in the same genetic background.

Proposed Schizophrenia-Related Gene Polymorphism: Expression of the Ser9Gly Mutant Human Dopamine D3Receptor with the Semliki Forest Virus System

Homozygotes for aBalI polymorphism resulting in the Ser9Gly mutation in the human dopamine D3receptor gene are suggested to display a twofold higher risk of schizophrenia. The cDNAs for this mutant and the wildtype receptor were introduced into the pSFV1C vector and recombinant Semliki Forest virus particles generated. CHO cells infected with SFV-D3virus resulted in high level expression of recombinant receptor. Double infections with wildtype and Ser9Gly dopamine D3SFV stocks generated an artificial heterozygote in CHO cells. Receptor binding analysis of several structurally different dopamine D3ligands showed similar pharmacological properties for all compounds tested except for dopamine and the D3-selective ligand GR99841. A significantly higher dopamine binding afffinity for the Ser9Gly homozygote was detected. The heterozygote binding did not differ from the wildtype. However, both the homo- and heterozygote for Ser9Gly showed significantly higher binding activity for GR99841 compared to the D3wildtype.

Highly Branched Phenotype of the Petunia dad1-1 Mutant Is Reversed by Grafting.

The recessive dad1-1 allele conditions a highly branched growth habit resulting from a proliferation of first- and second-order branches. Unlike the wild-type parent, which has lateral branching delayed until the third or fourth leaf node distal to the cotyledons, dad1-1 initiates lateral branching from each cotyledon axil. In addition to initiating lateral branching sooner than the wild type, dad1-1 sustains branching through more nodes on the main shoot axis than the wild type. In keeping with a propensity for branching at basal nodes, dad1-1 produces second-order branches at the proximal-most nodes on first-order branches and small shoots from accessory buds at basal nodes on the main shoot axis. Additional traits associated with the mutation are late flowering, adventitious root formation, shortened internodes, and mild leaf chlorosis. Graft studies show that a dad1-1 scion, when grafted onto wild-type stock, is converted to a phenotype resembling the wild type. Furthermore, a small wild-type interstock fragment inserted between a mutant root stock and a mutant scion is sufficient to convert the dad1-1 scion from mutant to a near wild-type appearance. The recessive dad1-1 phenotype combines traits associated with cytokinin overexpression, auxin overexpression, and gibberellin limitation, which suggests a complex interaction of hormones in establishing the mutant phenotype.

Branching in Pea (Action of Genes Rms3 and Rms4).

The nonallelic ramosus mutations rms3-2 and rms4 of pea (Pisum sativum L.) cause extensive release of vegetative axillary buds and lateral growth in comparison with wild-type (cv Torsdag) plants, in which axillary buds are not normally released under the conditions utilized. Grafting studies showed that the expression of the rms4 mutation in the shoot is independent of the genotype of the root-stock. In contrast, the length of the branches at certain nodes of rms3-2 plants was reduced by grafting to wild-type stocks, indicating that the wild-type Rms3 gene may control the level of a mobile substance produced in the root. This substance also appears to be produced in the shoot because Rms3 shoots did not branch when grafted to mutant rms3-2 rootstocks. However, the end product of the Rms3 gene appears to differ from that of the Rms2 gene (C.A. Beveridge, J.J. Ross, and I.C. Murfet [1994] Plant Physiol 104: 953-959) because reciprocal grafts between rms3-2 and rms2 seedlings produced mature shoots with apical dominance similar to that of rms3-2 and rms2 shoots grafted to wild-type stocks. Indole-3-acetic acid levels were not reduced in apical or nodal portions of rms4 plants and were actually elevated (up to 2-fold) in rms3-2 plants. It is suggested that further studies with these branching mutants may enable significant progress in understanding the normal control of apical dominance and the related communication between the root and shoot.

Translocation and exudation of tumor metabolites in crown galled plants.

Crown gall tumors are induced on susceptible plants by pathogenic strains of Agrobacterium. These neoplastic plants cells produce metabolites, called opines, which provide a source of nutrients to colonizing agrobacteria. Opine production previously has been shown to influence microbial communities in the immediate vicinity of the tumor. We have obtained evidence for opine translocation to and exudation from distal uninfected regions of the plant host. Grafted plants made from an opine-producing transgenic scion with a wild-type stock, or with a wild-type scion and an opine-producing stock accumulate opines in the wild-type portions of the plant. Moreover, opines were detected in root, stem, and leaf tissues of nontransgenic plants on which stem crown galls had been induced by pathogenic strains of Agrobacterium. These plants exuded opines from their roots as a component of their root exudates. Translocation of opines from the tumor to other parts of the plant, and their exudation from roots, indicates that these biologically active compounds are available to opine-catabolizing microbes that have not induced the tumors but are present in the rhizosphere or on portions of the plant distant from the site of the gall.

Nutritional reserves of autogenous and anautogenous selected strains of Aedes albopictus (Diptera: Culicidae).

Autogeny in Aedes albopictus (Skuse) females was increased from 5% in wild-type parental stock to 84% by selection through 20 generations and was decreased to 0% by selection through nine generations. Concomitantly, the mean number of eggs per female that matured autogenously increased from 6.4 to 18.3 in sugar-fed mosquitoes that developed eggs. Although the duration of the larval period of the two strains was not different, autogenous females emerged with greater amounts of metabolizable protein and lipid and total available energy reserves than did anautogenous females, but with lesser amounts of glycogen. Dry weights did not differ significantly. The difference in the energy reserves between strains was slightly more than the total energy invested in 20 autogenous eggs. Autogenous females survived 1.3 d longer than did anautogenous females when starved from emergence.

Branching Mutant rms-2 in Pisum sativum (Grafting Studies and Endogenous Indole-3-Acetic Acid Levels).

Isogenic lines of pea (Pisum sativum L.) were used to determine the physiological site of action of the Rms-2 gene, which maintains apical dominance, and its effect on endogenous free indole-3-acetic acid (IAA) levels. In mutant rms-2 scions, which normally produce lateral branches below node 3 and above node 7, apical dominance was almost fully restored by grafting to Rms-2 (wild-type) stocks. In the reciprocal grafts, rms-2 stocks did not promote branching in wild-type shoots. Together, these results suggest that the Rms-2 gene inhibits branching in the shoot of pea by controlling the synthesis of a translocatable (hormone-like) substance that is produced in the roots and/or cotyledons and in the shoot. At all stages, including the stage at which aerial lateral buds commence outgrowth, the level of IAA in rms-2 shoots was elevated (up to 5-fold) in comparison with that in wild-type shoots. The internode length of rms-2 plants was 40% less than in wild-type plants, and the mutant plants allocated significantly more dry weight to the shoot than to the root in comparison with wild-type plants. Grafting to wild-type stocks did not normalize IAA levels or internode length in rms-2 scions, even though it inhibited branching, suggesting that the involvement of Rms-2 in the control of IAA level and internode length may be confined to processes in the shoot.

Sex determination in the fly Megaselia scalaris, a model system for primary steps of sex chromosome evolution.

The fly Megaselia scalaris Loew possesses three homomorphic chromosome pairs; 2 is the sex chromosome pair in two wild-type laboratory stocks of different geographic origin (designated "original" sex chromosome pair in this paper). The primary male-determining function moves at a very low rate to other chromosomes, thereby creating new Y chromosomes. Random amplified polymorphic DNA markers obtained by polymerase chain reaction with single decamer primers and a few available phenotypic markers were used in testcrosses to localize the sex-determining loci and to define the new sex chromosomes. Four cases are presented in which the primary male-determining function had been transferred from the original Y chromosome to a new locus either on one of the autosomes or on the original X chromosome, presumably by transposition. In these cases, the sex-determining function had moved to a different locus without an obvious cotransfer of other Y chromosome markers. Thus, with Megaselia we are afforded an experimental system to study the otherwise hypothetical primary stages of sex chromosome evolution. An initial molecular differentiation is apparent even in the new sex chromosomes. Molecular differences between the original X and Y chromosomes illustrate a slightly more advanced stage of sex chromosome evolution.