An attempt at genetic transformation in chickens through cock sperm irradiation

Summary Two experiments were conducted to verify the real possibility for use of the genetic transformation technique as described by Pandey and Patchel in chickens in commercial poultry breeding. Multiple recessive and multiple dominant marker stocks were employed, as well as a tester and a donor line. Recipient tester females were first inseminated with dominant donor semen which was irradiated with doses of ⁶⁰Co gamma irradiation ranging from 100 to 800 Gy (control group) and 24 h later were reinseminated with unirradiated, normal semen of the recipient strain (experimental group). One genetic transformed chicken was found in the first experiment; no genetic transformed event occurred in the second experiment but one embryo was present in the 600 Gy irradiated group with parthenogenetic development capable of giving a live chick. One hundred Gy was observed not to be enough to destroy completely sperm fertilizing ability. An increased frequency of parthenogenetic development was found in all groups after insemination with irradiated semen. There were 11 individuals with developmental abnormalities from the total of 1264 analysed embryos which died after the 18th day of incubation. We concluded that egg transformation is a rare event in domestic fowl and further research for use of this technique in commercial poultry breeding is needed.     

Callus induction and plant regeneration in Vetiveria zizanioides

Callus induction was obtained from basal parts of Vetiveria zizanioides Stapf. leaves cultured on Murashige and Skoog (MS) medium supplemented with 9.0 M 2,4-dichlorophenoxyacetic acid (2,4-d), 5.7 M indoleacetic acid (IAA) and 4.6 M kinetin. Calli were maintained on MS medium with the addition of 0.9 M 2,4-d and 2.3 M kinetin. Shoot formation was obtained from fast growing 14-day-old callus on the same basal medium supplemented with 0.9 M 2,4-d and 9.3 M kinetin. Embryo-like structures were observed. When transferred to basal medium, shoots readily developed roots. Fully developed regenerated plants were then successfully established in soil.     

Mutants of Arabidopsis thaliana with altered phototropism

Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and second positive phototropism. However, while the amplitude for first positive phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in first positive phototropism are shifted to higher fluence by a factor of 20–30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 mol·m^-2 to 2700 mol·m^-2, but shows normal gravitropism. Strain JK345 shows no first positive phototropism, and reduced gravitropism and second positive phototropism. Strain JK229 shows no measurable first positive phototropism, but normal gravitropism and second positive phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. first positive and second positive phototropism contain at least one common element; and 3. first positive phototropism can be substantially altered without any apparent alteration of second positive phototropism.Abbreviation WT wild-type     

Shuttle transfer (or retrotransfer) of chromosomal markers mediated by plasmid pULB113

Summary The IncP1 plasmid pULB113 (RP4::miniMu) not only mediates the transfer of chromosomal markers in the classical direction (i.e. from the donor to the recipient cell) but also in the opposite direction (i.e. from the recipient bacterium to the donor). This phenomenon of retrotransfer was observed in homologous matings with Pseudomonas fluorescens, Alcaligenes eutrophus and Salmonella typhimurium. Retrotransconjugants could be discriminated from direct transconjugants by appropriate chromosomal and plasmid markers used to distinguish the mating partners not bearing pULB113. Retrotransfer of chromosomal markers occurred at frequencies equal to, or sometimes greater than, those observed for the direct mobilization, thus allowing the recovery of recipient recessive markers in the donor with linkage values similar to those found in the normal direction. Retrotransfer was also observed in heterospecific matings involving A. eutrophus and pULB113 bearing P. fluorescens: R-primes carrying different selected and unselected markers were recovered in both bacteria. Retrotransfer of shuttle transfer seems to be a specific trait of IncP1 plasmids.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Charles Darwin's theory of evolution: A review of our present understanding

The paper characterizes Darwin's theory, providing a synthesis of recent historical investigations in this area. Darwin's reading of Malthus led him to appreciate the importance of population pressures, and subsequently of natural selection, with the help of the wedge metaphor. But, in itself, natural selection did not furnish an adequate account of the origin of species, for which a principle of divergence was needed. Initially, Darwin attributed this to geographical isolation, but later, following his work on barnacles which underscored the significance of variation, and arising from his work on botanical arithmetic, he supposed that diversity allowed more places to be occupied in a given region. So isolation was not regarded as essential. Large regions with intense competition, and with ample variation spread by blending, would facilitate speciation. The notion of place was different from niche, and it is questioned whether Darwin's views on ecology were as modern as is commonly supposed. Two notions of struggle are found in Darwin's theory; and three notions of variation. Criticisms of his theory led him to emphasize the importance of variation over a range of forms. Hence the theory was populational rather than typological. The theory required a Lamarckian notion of inheritable changes initiated by the environment as a source of variation. Also, Darwin deployed a use/habit theory; and the notion of sexual selection. Selection normally acted at the level of the individual, though kin selection was possible. Group selection was hinted at for man. Darwin's thinking (and also the exposition of his theory) was generally guided by the domestic-organism analogy, which satisfied his methodological requirement of a vera causa principle.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

A substitution of cytosine for thymine in codon 110 of the human β-globin gene is a novel cause of β-thalassemia phenotypes

Summary We have described a novel human globin gene mutation that produced in a Japanese family the -thalassemia phenotype through a post-translational mechanism. Substitution of proline for leucine at position 110 in the G-helix of the -globin chain greatly reduced the molecular stability of the -globin subunit, leading to total destruction of the variant globin chains by proteolysis and hence to the -thalassemia phenotype. The mutation could be identified after MspI digestion. This detection of the mutation on the gene level is valuable for diagnostic purposes.     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

Factors determining the abundance and distribution of rodents in a shrub-steppe ecosystem: the role of shrubs

Summary This study addressed the relative importances of shrub resources on a rodent community in a sagebrush dominated shrub-steppe ecosystem in southwestern Wyoming. Direct effects of shrubs (i.e., providing rodents with food and cover) were assessed by removing shrubs from a 1.25 ha study plot and monitoring both rodent populations and their food resources. Shrub architecture and shrub-related food resources were found to be unimportant to deermice (Peromyscus maniculatus), Great Basin pocket mice (Perognathus parvus) northern grasshopper mice (Onychomys leucogaster) and Uinta ground squirrels (Spermophilus armatus), as shrub removal caused no significant changes in population sizes, sex ratios or age structure. Least chipmunks (Eutamias minimus) responded to shrub removal by leaving the plot and moving into adjacent shrubland. The montane vole (Microtus montanus) population showed a slight increase following shrub removal. Shrub removal did not alter the abundance of major rodent food resources on the plot (percent cover of herbaceous vegetation, soil seed reserves and ground-dwelling arthropods). Micrometeorological data suggested that shrubs did not significantly ameliorate a nocturnal rodent's micro-climate, but may have affected diurnal rodents' thermal loading rates by removing shade. While shrub architecture and food resources do not directly affect most of the rodents in this shrub-steppe ecosystem, shrubs may be important to rodents in a long-term time frame. Shrubs provide safe sites for germination and growth of herbaceous vegetation, thereby enhancing the diversity of the potential rodent food resources.     

Effect of liquid culture on the growth and development of miniature rose (Rosa chinensis Jacq. ‘Minima’)

The growth of miniature rose (Rosa chinensis Jacq. Minima) shoots cultured on liquid medium was greater relative to those cultured on two-phase (solid + liquid) medium or solid medium alone. Shoot multiplication ratio (number of multiple shoots per explant per subculture) on liquid medium was higher with 17.8–26.6 M 6-benzyladenine at compared to that at 0–8.9 M. Shoots grown on 30 ml or more of liquid medium had a higher multiplication ratio than those grown on 10 or 20 ml. The growth and multiplication ratio increased when the culture period was extended from 3 to 6 weeks, although plantlets began to exhibit some chlorosis by the 6^th week. These conditions were maintained over four subcultures for cultivars Baby Katie, Lavender Jewel, Red Sunblaze and Royal Sunblaze, with no significant change in multiplication ratio over time.Abbreviations BA 6-benzyladenine - NAA 1-naphthaleneacetic acid     

Cell wall localization of dihydroflavonol-glucoside β-glucosidase in flowers of Petunia hybrida

Limbs of flower buds from Petunia hybrida were investigated for -glucosidase activity with dihydroflavonol-glucosides and 4-methyl-umbelliferyl--D-glucoside as substrates. Dihydroflavonol-glucoside -glucosidase is localized in the cell wall. This activity has an acid pH optimum and is also active toward 4-methyl-umbelliferyl--glucoside. Besides this activity a neutral -glucosidase is present. This activity is soluble and is not active toward dihydroflavonol-glucosides. Using starch gel electrophoresis it was shown that no difference in -glucosidase activity is present between mutants able to convert dihydroflavonols into anthocyanins and mutants accumulating dihydroflavonol-glucosides. It is concluded that -glucosidase activity is not involved in anthocyanin synthesis.Abbreviations 4MU--glc 4-methylumbelliferyl--D-glucopyranoside - dHQ-7-g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside - dHM-4-g dihydromyricetin-4-glucoside Deceased     

Visual edge fixation and negative phototaxis in the mealworm beetle Tenebrio molitor

The visual fixation response of the mealworm beetle Tenebrio molitor, elicited by black stripes upon a bright background is studied in an arena and by means of the Y-maze technique. In the arena the distribution n() of the beetle's angular position is measured at different distances from the centre, which is also the starting point. If the black stripe is narrow, the maximum of n() coincides with the centre of the stripe (centre-fixation Figure 1a). If one half of the panorama is black, the distribution n() has two maxima, which are near the borders between the black and white regions (edge-fixation Figure 1b). In the Y-maze experiments the beetle is tethered, but its head is free to move. The black stripes elicit turning tendencies F(), the strength of which depends upon the angular distance between the centre of the stripe and the animal's body axis. If the black stripe is narrow, the stable zero crossing of F() lies at =0, in agreement with the centre fixation in the arena (Fig. 3). If the stripe is 180° wide, two stable zero crossings are obtained near the border lines between the black and white regions, provided that the panorama is rotated around the animal with an angular velocity w larger than about 0.08°/s (Fig. 4). Below this value of w only one stable zero crossing at =0 exists (Fig. 6). Thus the tethered beetle's response underlies a transition between centre resp. edge fixation at a critical angular velocity of the drum. Some implications of this surprising phenomenon with respect to the mechanism of fixation and negative phototaxis are discussed but at present it is considered primarily a challenge for further investigation.Supported in part by Deutsche Forschungsgemeinschaft     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Genetic polymorphism at the κ chain locus in mice: Comparisons of restriction enzyme hybridization fragments of variable and constant region genes

Variable (V) and constant (C) region genes of the mouse kappa light chain have been compared in inbred strains and in geographically isolated or genetically separated populations of mice by Southern blot analysis of endonuclease-restricted germline DNA. In most cases, the C gene is found on a single restriction fragment while the V genes of the V19 and V21 groups are each found on several (6–18) fragments. The restriction fragment (RF) patterns of V19 and V21 groups are both polymorphic when compared among inbred mouse strains. Southern blot patterns of V21 and V19 of inbred strains are also found among some geographically isolated populations of mice, suggesting that inbred strains acquired kappa loci from different subspecies. Some populations of geographical isolates show V21, V19, and C contexts similar to inbred mice while more distantly related species within the genus Mus and laboratory rats show no apparent similarity in context to inbred strains. Variable region genes determining the RF patterns of V19 and V21 appear to be linked to each other and to the C and Lyt-3 loci.     

Heterogeneity in the molecular basis of three types of hereditary persistence of fetal hemoglobin and the relative synthesis of the Gγ and Aγ types of γ chain

Restriction endonuclease analyses of DNA from one Black ^GA-HPFH homozygote and four Black and one Indian ^GA-HPFH heterozygotes have identified three different HPFH types which are the result of large deletions including the and genes. Two of the types are comparable to those characterized previously, but the third, which is present in the Indian heterozygote, shows a distinct difference in the size of the deletion. The 5 end point of the deletion in this type III ^GA-HPFH extends 0.5–1.0 kb beyond the 5 end point of one of the Black types of HPFH (type I). Each of the three types is associated with a distinct ratio between the ^G and the ^A chains, an observation supported by family data. The highest ratio is found in the heterozygote with the Indian type III ^GA-HPFH, with 69.3% ^G chains, while the averages for the other types were 50.7% ^G (type I) and 32.3% ^G (type II).This research was supported in part by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution 0764 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912. P. K. Sukumaran was on leave from the Bai Jerbai Wadia Hospital for Children, Parel, Bombay, India.     

GENETICS OF DIELDRIN AND γ-BHC RESISTANCE IN THE HOUSEFLY

A factor for dieldrin resistance in a -BHC selected strain of Uruguayan origin was located in linkage group 4 (Hiroyoshi's numbering system) and showed approximately 40% crossover to the marker ct in F₁ females. Resistance to -BHC is partly due to the same factor. Factors on other linkage groups play an important part in resistance to -BHC, but when studied separately only the fifth linkage group showed a small effect. It is concluded that several factors on different chromosomes must contribute to the total resistance to -BHC.

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Genetik der dieldrin- und -cyclohexan bei der stubenfliege

Zusammenfassung Ein Faktor für Dieldrin-Resistenz in einem -Cyclohexan-selektierten Stamm uruguayischer Herkunft ist in der Koppelungsgruppe 4 (Hiroyoshi's Zählungssystem) gelegen und zeigt bei F₁-Weibchen annähernd 40% Crossing-over gegenüber dem Markierungsgen ct. Die Resistenz gegenüber -Cyclohexan beruht teilweise auf dem gleichen Faktor. Faktoren in anderen Koppelungsgruppen spielen eine wichtige Rolle bei der -Cyclohexan-Resistenz, aber bei getrennter Untersuchung zeigte nur die 5. Koppelungsgruppe eine schwache Wirkung. Es wird daraus geschlossen, daß an der Gesamtresistenz unterschiedliche Faktoren auf verschiedenen Chromosomen beteiligt sein müssen.

     

Enzymatic β-galactosidation of β-xylopyranosides

Summary The disaccharides formed by enzymatic transfer of the -D-galactopyranosyl residue fromo-nitrophenyl -d-galactopyranoside to -d-xylopyranosides have been identified. The influence of different factors on the yields of the disaccharides obtained was evaluated. Significant changes in selectivity were observed when -galactosidase fromE. coli was used instead of -galactosidase fromA. oryzae.     

Extracellular matrix proteins,integrin receptors (VLA-β1, VLA-α2 and VLA-α5) and growth fraction in atypical macroregenerative nodules of the liver: an immunocytochemical case study

A case of cirrhotic liver harbouring three atypical macroregenerative nodules and an hepatocellular carcinoma was immunocytochemically investigated for the expression of VLA- 1, VLA-2 and VLA-5 integrins and for different extracellular matrix (ECM) components (collagen I, collagen IV, laminin, fibronectin and tenascin). In addition, the proliferative activity within the nodules was evaluated, using the MIB 1 monoclonal antibody (MAb). The cirrhotic liver disclosed a continuous staining pattern of the ECM proteins investigated, as well as a sinusoidal immunostaining of VLA-1, VLA-2 and VLA-5. The macroregenerative nodules showed a discontinued immunoreactivity for ECM proteins while maintaining a VLA-1 sinusoidal immunostaining, coupled with intercellular immunostaining. VLA-2 and VLA-5 expression was lacking. The growth fraction was low in both the above pathological conditions. The hepatocellular carcinoma was devoid of any ECM immunostaining. VLA-1 immunoreactivity exhibited a honeycomb pattern of staining, whereas VLA subunits were absent. MIB1 expression was high, being present in 30% of neoplastic nuclei. A possible relationship between atypical macroregenerative nodules and hepatocellular carcinoma is discussed.This paper was partly presented at the 35th Symposium of the Society for Histochemistry, Gargellen, Austria, Sept. 29–Oct. 3, 1993     

Inactivation of voltage-dependent calcium current in an insulinoma cell line

We have studied the mechanism of Ca current inactivation in the -cell line HIT-T15 by conventional and perforated patch recording techniques, using two pulse voltage protocols and a combination of current and tail current measurements. In 5 mM Ca, from a holding potential of - 80 mV, the maximum current showed a complex time course of inactivation: a relatively fast, double exponential inactivation (_h1 12 ms and _h2 60 ms) and a very slowly inactivating component ( > 1 s). The faster component (_h1) was due to the voltage-dependent inactivation of a low-threshold-activated (LVA), T-type current, which deactivates more slowly ( 3–5 ms) than the other components ( 0.2–0.3 ms). The intermediate component (_h2) was due to the Ca-dependent inactivation of a portion of the high-threshold-activated (HVA) current. A saturating dose of the dihydropyridine (DHP) nifedipine (10 M) did not affect the LVA current, but inhibited by 68 ± 5% the transient, Ca-sensitive portion of the HVA current and by 33 ± 12% the long lasting component. We suggest that three components of the calcium current can be resolved in HIT cells and the main target of DHPs is a HVA current, which inactivates faster than the DHP-resistant HVA component and does so primarily through calcium influx. Correspondence to: C. Marchetti