Construction and characterisation of a yeast artificial chromosome library containing three haploid maize genome equivalents

We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded leaf protoplasts of the maize inbred line UE95. This library contains 79 000 clones with an average insert size of 145 kb and should therefore represent approximately three haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. Forty-one pools of DNA from 1920 individual clones have been prepared for rapid screening of the library by the polymerase chain reaction (PCR). Using this approach, together with conventional colony hybridisation, we have been able to identify between one and eight positive clones for every probe used.     

Directional Ca2+ effect on stimulation of mucin secretion from chicken trachea in vitro

Chicken tracheal mucosa in vitro transported and incorporated radioactive precursors into mucins, which were secreted at a steady rate into the tracheal lumen. Secretion of mucins labelled with ³⁵S and ³H after pulse-labelling of the mucosal layer with Na₂³⁵SO₄ and d-[1-³H]glucosamine as precursors was an energy-dependent process, as it was strongly inhibited by the action of respiratory-chain inhibitors, an uncoupler of oxidative phosphorylation, a metabolic blocker and a temperature shift from 41°C to 5°C. On the other hand, both cholinergic and parasympathomimetic agents considerably increased the secretion of dual-radiolabelled mucins when applied on the submucosal side of the trachea. The effect of Ca²⁺ was directional, since only high submucosal (3.6 or 18mm) or low luminal (zero or 0.18mm) Ca²⁺ massively enhanced the secretion of radiolabelled mucin compared with the mucin output measured under physiological Ca²⁺ conditions (1.8mm). Whereas application of ionophore A23187 on either side of the trachea significantly increased mucin output, its presence in the appropriate tracheal compartment and under appropriate Ca²⁺ conditions further accentuated the output of radiolabelled mucins. Addition of acetylcholine under appropriate conditions also had an additive effect on the Ca²⁺-stimulated secretion of mucins. Ca²⁺ stimulation of mucin secretion appears to be dependent on the metabolic integrity of the mucosal cells. Mucins secreted in response to high submucosal and low luminal [Ca²⁺] appear to consist of a number of different types of glycoproteins, as judged from their ion-exchange-chromatographic behaviour.     

Characterization of stable conformational variants of erythrocyte carbonic anhydrases.

Limiting viscosity numbers of bovine and ovine erythrocytes carbonic anhydrase variants were calculated by the objective method of comparing viscosimetric data obtained from low-activity-human erythrocyte carbonic anhydrase and its natural variant. Shifts of mobilities and isoelectric points are shown for all species variants, but variations of limiting viscosity numbers were only detected for human and bovine variants. Results of the study are consistent with the observation that variants arise by deamidation of erythrocyte carbonic anhydrases, and that deamidation is responsible for changes in structure and hydration (i. e. "conformational" modifications). Thus, all the variants so far investigated are stable conformational variants or erythrocyte carbonic anhydrases.     

Capacités reproductrices et descendance de poulets ayant subi un transfert de cellules germinales primordiales durant la vie embryonnaire

Résumé Des cellules germinales primordiales de Dindon, transférées par injection intravasculaire à des embryons de Poulet préalablement stérilisés, peuvent subir une maturation complète dans les gonades de l'hôte et fournir des gamètes plus ou moins aptes à la fécondation.Les spermatozoïdes résultants ont fécondé des oeufs de poule avec une fréquence plus grande que les spermatozoïdes normaux de Dindon, mais sans permettre un développement embryonnaire plus important ou plus normal. Cependant, on n'est pas parvenu à leur faire féconder des oeufs de dinde.Les ufs résultants ont montré parfois un vitellus anormal et n'ont été pondus que durant 7 mois. Mis en présence de spermatozoïdes de coq, ils ont été quelquefois fécondés (ou peutêtre simplement activés), mais ils n'ont jamais fourni d'embryons. Fécondés par des spermatozoïdes de Dindon, ils ont fourni des embryons, parfois anormaux, qui ont atteint dans le meilleur cas le 15è jour d'incubation (stade 38 HH). Certainespraepennae de l'embryon qui a atteint ce stade montraient une pigmentation rouge brun qui ne peut pas être déterminée par le génotype du zygote (celui d'un Dindon de race blanche) et qui rappelle le phénotype de la mère nourricière (une poule rouge brun).A la suite de transferts intraspécifiques de cellules germinales primordiales, on a pu obtenir la maturation d'ovocytes de Rhode Island Red à l'intérieur d'un ovaire de Wyandotte Blanche (chez deux poules) et vice versa (chez une poule). La ponte a également été possible, mais à une fréquence souvent inférieure à la normale.Lorsqu'une poule Wyandotte Blanche, portant des ovocytes de Rhode Island Red a été accouplée à un coq Rhode Island Red normal, le duvet de leurs descendants s'est montré plus clair que celui des poussins Rhode Island Red dans un cas, mais il a été remplacé ensuite par un plumage rouge brun conforme au génotype.Lorsqu'une poule Rhode Island Red, portant des ovocytes de Wyandotte Blanche a été accouplée à un coq Wyandotte Blanche normal, le duvet de leurs descendants n'a jamais été conforme au génotype. Il présentait toujours du pigment noir plus ou moins étendu et, dans un cas, du pigment rouge brun, qui sont tous deux présents chez la mère nourricière. On ne connaît ni l'origine ni le mécanisme d'un tel transfert de pigments, qui ne représente peut-être qu'un effet transitoire affectant le duvet de la première génération. La crête, quant à elle, s'est toujours montrée conforme au génotype d'origine.

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Reproductive capacity and offspring of chickens submitted to a transfer of primordial germ cells during embryonic life

Summary Turkey primordial germ cells transfered by intravascular injection to previously sterilized chick embryos can undergo complete maturation inside the host's gonads and can give rise to gametes which are more or less suitable for fertilization.The resulting spermatozoa fertilized hen eggs at a higher frequency than normal turkey spermatozoa, but without allowing a longer or a more normal development. However, it was impossible to fertilize turkey eggs with them.The resulting eggs sometimes had an abnormal-looking yolk and were laid during the first 7 months only. Brought in contact with chicken spermatozoa, they were fertilized (or perhaps merely activated), but they never gave rise to embryos. Fertilized by turkey spermatozoa, they developed into embryos, sometimes abnormal, which in the best case reached the 15th day of incubation (stage 38 HH). Somepraepennae of the latter embryo showed a red-brown pigment which cannot be determined by the genotype of the zygote (a white turkey's) and which resembled the phenotype of the foster mother (a red-brown hen).After intraspecific transfer of primordial germ cells, maturation of Rhode Island Red oöcytes inside a Wyandotte White ovary (in two hens) and vice versa (in one hen) was achieved. Laying was also possible but often at a lower frequency than normal.When a Wyandotte White hen bearing Rhode Island Red oöcytes was mated with a normal Rhode Island cock, the down of their offspring looked brighter than Rhode Island Red chicken's in one case, but it was subsequently replaced by red-brown feathers according to the genotype.When a Rhode Island Red hen bearing Wyandotte White oöcytes was mated with a normal Wyandotte White cock, the down of their offspring was never in agreement with the genotype. It always showed a black pigment over more or less large areas and, in one case, a red-brown pigment, both of which were present in the foster mother. The origin and the mechanism of such a transfer of pigments are not understood. It might represent merely a temporary effect acting upon the down of the first generation. As far as the comb is concerned, it was always in agreement with the original genotype.

     

A Novel Piggybac Transposon Inducible Expression System Identifies a Role for Akt Signalling in Primordial Germ Cell Migration

In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.     

Review of Canadian species of the genera Gnathusa Fenyes,Mniusa Mulsant & Rey and Ocyusa Kraatz (Coleoptera,Staphylinidae, Aleocharinae)

Four species of Gnathusa Fenyes (G. alfacaribou Klimaszewski & Langor, G. caribou Lohse, G. eva Fenyes, and G. tenuicornis Fenyes) occur in the Nearctic and in Canada. Three species of Ocyusa Kraatz (O. asperula Casey, O. californica Bernhauer, O. canadensis Lohse), and three species of Mniusa Mulsant and Ray (M. minutissima (Klimaszewski & Langor), M. yukonensis (Klimaszewski & Godin), and M. odelli Klimaszewski & Webster, sp. n.), are known from the Nearctic and all but O. californica occur in Canada. The recently described Gnathusa minutissima Klimaszewski and Langor and Ocyusa yukonensis Klimaszewski and Godin, are transferred here to the genus Mniusa Mulsant & Rey. New provincial and state records are reported for: G. eva (Alberta), G. tenuicornis (Alberta, Oregon, and New Brunswick), O. canadensis (New Brunswick and Newfoundland), M. minutissima (New Brunswick), and M. yukonensis (Nova Scotia, New Brunswick, Quebec, and British Columbia). The female of M. yukonensis was discovered and is illustrated for the first time. The genus Mniusa is reported for the first time from Canada and represents the first confirmed generic record for North America. Keys for identification of all Canadian species, images of body and genital structures, maps showing distribution mainly in Canada, and new bionomics data are provided.