Régulation des excédents dans le développement du bourgeon de membre de l'embryon d'oiseau. Analyse expérimentale de combinaisons xénoplastiques caille/poulet

Summary In order to support the demonstration of the regulative capacity of the chick limb bud, already stressed by one of us (Kieny, 1964, 1967), heterospecific combinations were made between chick and quail tissues, the cells of the latter bearing a distinctive nuclear marker. A Japanese quail whole limb bud (stage-18 to 21 of H. H., wing or leg) was grafted distally onto the prospective zeugopod of a chick (stage-22) wing bud sectioned at the prospective wrist level. Thus, from a heterospecific surplus recombinant containing five prospective limb segments (stylopod and zeugopod from the chick host; stylopod, zeugopod and autopod from the quail graft), it was possible to obtain a normally shaped appendage that comprised either upper arm, lower arm and hand in the case of a wing bud graft, or heteromorphic upper arm, lower leg and foot in the case of a hind-limb bud graft. In these cases, regulation for excess appeared to take place mainly within the host tissues. The three proximal segments of the recombinant, namely the chick stylopod and zeugopod of the host's stump and the quail stylopod of the graft, became reorganized and gave rise to a single stylopodial segment, which usually contained a double stylopodial bone element, one of chick, the other of quail origin.The absence of development of the squeezed prospective zeugopod can be interpreted as follows: owing to an interaction with the stylopodial graft tissues, the zeugopodial cells of the juxtaposed stump boundary have shifted proximally their originally more distal positional values, so that they changed their prospective pattern of differentiation to that of stylopod. These reset zeugopodial cells combine with the stylopodial cells of host and graft and form a huge composite stylopod, in which, due to an asynchronous determination in the two species, chick and quail tissues do not cooperate fully for the development of a single bone.

Ce travail a été effectué avec l'aide de la D.G.R.S.T. (Action complémentaire coordonnée: Biologie de la reproduction et du développement, convention n^o 73-7-1661)     

Expression of the short stature homeobox gene Shox is restricted by proximal and distal signals in chick limb buds and affects the length of skeletal elements

SHOX is a homeobox-containing gene, highly conserved among species as diverse as fish, chicken and humans. SHOX gene mutations have been shown to cause idiopathic short stature and skeletal malformations frequently observed in human patients with Turner, Leri-Weill and Langer syndromes. We cloned the chicken orthologue of SHOX, studied its expression pattern and compared this with expression of the highly related Shox2. Shox is expressed in central regions of early chick limb buds and proximal two thirds of later limbs, whereas Shox2 is expressed more posteriorly in the proximal third of the limb bud. Shox expression is inhibited distally by signals from the apical ectodermal ridge, both Fgfs and Bmps, and proximally by retinoic acid signaling. We tested Shox functions by overexpression in embryos and micromass cultures. Shox-infected chick limbs had normal proximo-distal patterning but the length of skeletal elements was consistently increased. Primary chick limb bud cell cultures infected with Shox showed an initial increase in cartilage nodules but these did not enlarge. These results fit well with the proposed role of Shox in cartilage and bone differentiation and suggest chick embryos as a useful model to study further the role of Shox in limb development.     

Analysis of Cyp26b1/Rarg compound-null mice reveals two genetically separable effects of retinoic acid on limb outgrowth

The role of retinoic acid (RA) in limb development is unclear, although it has been suggested to be a proximalizing factor which plays a morphogenetic role in pattern formation. Exogenous RA produces a teratogenic effect on limb morphology; similarly, changes in the endogenous distribution of RA following genetic ablation of the RA-metabolizing enzyme, CYP26B1, result in phocomelia accompanied by changes in expression of proximo-distal (P-D) patterning genes, increased cell death, and delayed chondrocyte maturation. Here we show that disruption of RA receptor (RAR) gamma in a Cyp26b1^−/− background is able to partially rescue limb skeletal morphology without restoring normal expression of proximo-distal patterning genes. We further show that embryos deficient in CYP26B1 exhibit early localized domains of mesenchymal cell death, which are reduced in compound-null animals. This model reveals two genetically separable effects of RA in the limb: an apoptotic effect mediated by RARγ in the presence of ectopic RA, and a P-D patterning defect which is uncovered following the loss of both CYP26B1 and RARγ. These data provide genetic evidence to clarify the roles of both RA and CYP26B1 in limb outgrowth and proximo-distal patterning.     

大豆结瘤突变体成分的初步分析及其对硝酸还原酶活性和结瘤的影响

超结瘤大豆（Ｇｌｙｃｉｎｅ ｍ ａｘ （Ｌ．） Ｍｅｒｒ．） ｎｔｓ ３８２ 和不结瘤大豆Ｎｏｄ ４９ 的叶和根组织水提取物经Ｓｅｐｈａｄｅｘ Ｇ２５ 过滤、洗脱,再根据洗脱物对硝酸还原酶（ＮＲ）活性的影响可划分为４ 个组分（ｆｒａｃｔｉｏｎ）样品,即ｎｔｓ ３８２（Ｎｏｄ ４９） Ｆ１、ｎｔｓ ３８２（Ｎｏｄ ４９） Ｆ２、ｎｔｓ ３８２（Ｎｏｄ ４９） Ｆ３ 和ｎｔｓ ３８２（Ｎｏｄ ４９） Ｆ４。其中, ｎｔｓ３８２ Ｆ２ 和Ｆ４ 抑制ＮＲ 活性作用在接种ＵＳＤＡ１１０ 后明显下降, 但接种的ｎｔｓ ３８２ Ｆ２ 却能提高大豆Ｂｒａｇｇ 的结瘤数达一倍, 而接种的ｎｔｓ ３８２ Ｆ３ 和Ｆ４ 的作用不明显。ＮＲ 活性抑制因子不是刺激结瘤的因子, 刺激结瘤的因子主要分布在接种的ｎｔｓ３８２ Ｆ２ 部分中。与这一现象相反, Ｎｏｄ ４９ Ｆ２ 和Ｆ４ 抑制ＮＲ活性的作用在接种后更强, 且也抑制大豆ｎｔｓ ３８２ 的结瘤, 其中Ｎｏｄ ４９ Ｆ４ 抑制结瘤的作用基本不能逆转。抑制结瘤因子主要分布在接过种的Ｎｏｄ ４９ Ｆ４ 部分中     

Extra tarsal joints and abnormal cuticular polarities in various mutants ofDrosophila melanogaster

Summary The legs of flies from 16 different mutant strains ofDrosophila melanogaster were examined for abnormal cuticular polarities and extra joints. The strains were chosen for study because they manifest abnormal cuticular polarities in some parts of the body (10 strains) or because they have missing or defective tarsal joints (6 strains). All but three of the stocks were found to exhibit misorientations of either the bristles, hairs, or “bract-socket vectors” on the legs. The latter term denotes an imaginary vector pointing from a hairlike structure called a “bract” to the bristle socket with which it is associated. On the legs of wild-type flies nearly all such vectors point distally, as do the bristles and hairs. In the mutant flies, the most common vector misorientation is a 180° reversal. When the bract-socket vectors of adjacent bristle sites in the same bristle row point toward one another, the distance between the sites is frequently abnormally large, whereas when the vectors point in opposite directions, the interval is frequently abnormally small. This correlation is interpreted to mean that bristle cells actively repel one another via cytoplasmic extensions that are longer in the direction of the bract-socket vector than in the opposite direction. Repulsive forces of this kind may be responsible for “fine-tuning” the regularity of bristle spacing in wild-type flies. Extra tarsal joints were found in eight of the 16 strains. A ninth strain completely lacking tarsal joints appears in some cases to have an extra tibia-basitarsus joint in its tibia. Whereas the tarsi of wild-type flies contain four joints, the tarsi ofspiny legs mutant flies contain as many as eight joints. In this extreme extra-joint phenotype, four of the joints correspond to the normal wild-type joints, and there is an extra joint in every tarsal segment except the distal-most (fifth) segment. Nearly all such ectopic extra joints have inverted polarity. In other strains the extra tarsal joints are located mainly at the wild-type joint sites, and joints of this sort have wild-type polarity. The alternation of normal and inverted (extra) joints inspiny legs resembles the alternation of normal and inverted (extra) body segment boundaries in the embryonic-lethal mutantpatch, suggesting that tarsal and body segmentation may share a common patterning mechanism.     

用PCR直接筛选和鉴定虎纹捕鸟蛛毒素Ⅰ的重组阳性克隆

以合成的两段插入序列为上、下游引物用PCR法直接筛选插入有虎纹捕鸟蛛毒素Ⅰ（HWTX－Ⅰ）cDNA的重组阳性克隆。并用PCR法快速鉴定重组体中插入片段的正、反连接方向,扩增用引物是以位于克隆位点上游的一段载体序列上游引物,以插入序列为下游引物。对100个单克隆进行了上述两次PCR筛选鉴定,选取2个有靶片段插入并且为正向连接的重组子进行测序,其结果证实了插入片段及其方向的正确性。     

Antenna and all gnathal appendages are similarly transformed by homothorax knock-down in the cricket Gryllus bimaculatus

Our understanding of the developmental mechanisms underlying the vast diversity of arthropod appendages largely rests on the peculiar case of the dipteran Drosophila melanogaster. In this insect, homothorax (hth) and extradenticle (exd) together play a pivotal role in appendage patterning and identity. We investigated the role of the hth homologue in the cricket Gryllus bimaculatus by parental RNA interference. This species has a more generalized morphology than Oncopeltus fasciatus, the one other insect besides Drosophila where homothorax function has been investigated. The Gryllus head appendages represent the morphologically primitive state including insect-typical mandibles, maxillae and labium, structures highly modified or missing in Oncopeltus and Drosophila. We depleted Gb’hth function through parental RNAi to investigate its requirement for proper regulation of other appendage genes (Gb’wingless, Gb’dachshund, Gb’aristaless and Gb’Distalless) and analyzed the terminal phenotype of Gryllus nymphs. Gb’hth RNAi nymphs display homeotic and segmentation defects similar to hth mutants or loss-of-function clones in Drosophila. Intriguingly, however, we find that in Gb’hth RNAi nymphs not only the antennae but also all gnathal appendages are homeotically transformed, such that all head appendages differentiate distally as legs and proximally as antennae. Hence, Gb’hth is not specifically required for antennal fate, but fulfills a similar role in the specification of all head appendages. This suggests that the role of hth in the insect antenna is not fundamentally different from its function as cofactor of segment-specific homeotic genes in more posterior segments.     

Susceptibility of Oncomelania hupensis formosana recombinants and hybrids with Oncomelania hupensis nosophora to infection with Schistosoma japonicum

Chiu J.-K., Ong S.-J., Yu J.-C., Kao C.-Y. and Iuima T. 1981. Susceptibility of Oncomelania hupensis formosana recombinants and hybrids with Oncomelania hupensis nosophora to infection with Schistosoma japonicum. International Journal for Parasitology11: 391–397. Three generations of Oncomelania hupensis formosana recombinants were produced by mating the Kaohsiung race with the Ilan and Changhua races of the snails. F₂ and F₃ recombinants were produced by back-crossing F₁ and F₂ with Kaohsiung O. h. formosana. Subsequently, susceptibility of recombinants to infection with the original strain of Schistosoma japonicum, Ilan or Changhua strain, was investigated. Results indicated that susceptibility of recombinants declined steadily generation by generation. Marked decline of infectivity was observed for Kaohsiung-Ilan recombinants as compared with Kaohsiung-Changhua recombinants. For example, the overall infection rate of Kaohsiung-Ilan F₁ recombinants was 7.3 % with a 51.4 % of control snails. The same figures for F₂ were 4.2 and 52.6%, and 1 and 40.3 % for F₃. On the other hand, the overall infection rate of Kaohsiung-Changhua F₁ recombinants was 21.9% with a 46.9% of control snails; and 11.9 and 50.3 % for F₂; and 7.6 and 33.2% for F₃. The F₃ hybrids of Oncomelania hupensis nosophora from Japan and O. h. formosana from Kaohsiung and Ilan were also produced, and susceptibility with the Japanese strain of S. japonicum was studied. A highly significant decline of susceptibility was observed among hybrids (4.4%) in contrast with control snails (85.6 %).Feasibility of applying O. h. formosana in biological control of S. japonicum was discussed. One must determine in the laboratory, prior to application, which race of O. h. formosana should be used depending on the Oncomelania snails of an endemic area. For S. japonicum prevalent in Yamanashi, Japan, the Ilan race of O. h. formosana was found to be better choice than the Kaohsiung race of the snails.     

一种碱裂解菌液直接电泳快速筛选重组子的方法

目的：从碱裂解法提取质粒DNA的原理．经过实验摸索获得一种快速、经济、结果可靠的菌液直接碱裂解电泳筛选重组子的方法。方法：不需提取质粒DNA．只需将细菌培养液碱裂解后直接进行普通琼脂糖凝胶电泳分析,就可以快速筛选出转化重组子。结果：结果和提取质粒酶切鉴定鉴定结果一致。结论：经实验证明菌液直接碱裂解电泳筛选重组子是一种快速、经济、可靠的方法。     

The role of hind limb flexor muscles during swimming in the toad, Bufo marinus

Most work examining muscle function during anuran locomotion has focused largely on the roles of major hind limb extensors during jumping and swimming. Nevertheless, the recovery phase of anuran locomotion likely plays a critical role in locomotor performance, especially in the aquatic environment, where flexing limbs can increase drag on the swimming animal. In this study, I use kinematic and electromyographic analyses to explore the roles of four anatomical flexor muscles in the hind limb of Bufo marinus during swimming: m. iliacus externus, a hip flexor; mm. iliofibularis and semitendinosus, knee flexors; and m. tibialis anticus longus, an ankle flexor. Two general questions are addressed: (1) What role, if any, do these flexors play during limb extension? and (2) How do limb flexors control limb flexion? Musculus iliacus externus exhibits a large burst of EMG activity early in limb extension and shows low levels of activity during recovery. Both m. iliofibularis and m. semitendinosus are biphasically active, with relatively short but intense bursts during limb extension followed by longer and typically weaker secondary bursts during recovery. Musculus tibialis anticus longus becomes active mid way through recovery and remains active through the start of extension in the next stroke. In conclusion, flexors at all three joints exhibit some activity during limb extension, indicating that they play a role in mediating limb movements during propulsion. Further, recovery is controlled by a complex pattern of flexor activation timing, but muscle intensities are generally lower, suggesting relatively low force requirements during this phase of swimming.     

Assessing the complex architecture of polygenic traits in diverged yeast populations

Phenotypic variation arising from populations adapting to different niches has a complex underlying genetic architecture. A major challenge in modern biology is to identify the causative variants driving phenotypic variation. Recently, the baker's yeast, Saccharomyces cerevisiae has emerged as a powerful model for dissecting complex traits. However, past studies using a laboratory strain were unable to reveal the complete architecture of polygenic traits. Here, we present a linkage study using 576 recombinant strains obtained from crosses of isolates representative of the major lineages. The meiotic recombinational landscape appears largely conserved between populations; however, strain-specific hotspots were also detected. Quantitative measurements of growth in 23 distinct ecologically relevant environments show that our recombinant population recapitulates most of the standing phenotypic variation described in the species. Linkage analysis detected an average of 6.3 distinct QTLs for each condition tested in all crosses, explaining on average 39% of the phenotypic variation. The QTLs detected are not constrained to a small number of loci, and the majority are specific to a single cross-combination and to a specific environment. Moreover, crosses between strains of similar phenotypes generate greater variation in the offspring, suggesting the presence of many antagonistic alleles and epistatic interactions. We found that subtelomeric regions play a key role in defining individual quantitative variation, emphasizing the importance of the adaptive nature of these regions in natural populations. This set of recombinant strains is a powerful tool for investigating the complex architecture of polygenic traits.     

Reinterpreting polarity and cancer: The changing landscape from tumor suppression to tumor promotion

Cell polarity is a fundamental property used to generate asymmetry and structure in all cells. Cancer is associated with loss of cell and tissue structure. While observations made in model system such as Drosophila, identify polarity regulators as tumor suppressors that cause inappropriate cell division, studies in mammalian epithelia do not always support such a causative contribution. Our analysis of published cancer dataset shows that many polarity genes, including PARD6B, SCRIB, PRKCI, DLG1, DLG2, DLG5 and LLGL2, are frequently amplified in multiple cancers raising the possibility that mammalian epithelia may have evolved to use polarity proteins in multiple ways where they may have tumor promoting functions. In this review, we reinterpret the published results and propose a modified perspective for the role of polarity regulators in cancer biology. In addition to the traditional form of cell polarity, which is involved establishment of maintenance of normal cell structure and asymmetry, we propose that some mammalian polarity proteins also regulate subcellular polarity (intracellular asymmetry), which can improve cellular fitness to carry out functions such as proliferation, apoptosis, stress adaptation, stemness and organelle biology. Here, we define subcellular polarity and discuss evidence that supports a role for subcellular polarity in biology.     

Characterisation of wheat-rye recombinants with RFLP and PCR probes

Summary The introgression of genetic material from alien species into wheat has become an important tool in modern wheat breeding. Ideally, only the trait of interest and no flanking material should be transferred. Random recombination between the genetic material is therefore of paramount importance. In a model system, we examined 17 recombinants putatively between chromosome 1D of wheat and 1R of rye with 60 random RFLP and three PCR markers. The recombinants had been generated by removing the normal effect of the Ph1 gene in the wheat background. Amongst the nine short-arm recombinants, three breakpoints were identified but no differentiation could be made between the five proximal recombinants. For the eight long-arm recombinants analysed only two breakpoints were identified with 36 markers. However, only a single RFLP marker was able to differentiate between the recombinants. Indeed the long-arm results are consistent with the possibility that only the rye telomeric region had been transferred. These results indicate either a strong clustering of the RFLP markers near the centromere or else imply that recombination induced between wheat and rye in the absence of the normal effect of the Ph1 gene occurs at only restricted sites. The results allow new primary recombinants to be selected for intercrossing to generate secondary recombinants which are expected to have a smaller interstitial rye segment than that present in DR-A1.     

Ectopic BASL Reveals Tissue Cell Polarity throughout Leaf Development in Arabidopsis thaliana

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The apical ectodermal ridge (AER) can be re-induced by wounding, wnt-2b, and fgf-10 in the chicken limb bud

Little effort has been made to apply the insights gained from studies of amphibian limb regeneration to higher vertebrates. During amphibian limb regeneration, a functional epithelium called the apical ectodermal cap (AEC) triggers a regenerative response. As long as the AEC is induced, limb regeneration will take place. Interestingly, similar responses have been observed in chicken embryos. The AEC is an equivalent structure to the apical ectodermal ridge (AER) in higher vertebrates. When a limb bud is amputated it does not regenerate; however, if the AER is grafted onto the amputation surface, damage to the amputated limb bud can be repaired. Thus, the AER/AEC is able to induce regenerative responses in both amphibians and higher vertebrates. It is difficult, however, to induce limb regeneration in higher vertebrates. One reason for this is that re-induction of the AER after amputation in higher vertebrates is challenging. Here, we evaluated whether AER re-induction was possible in higher vertebrates. First, we assessed the sequence of events following limb amputation in chick embryos and compared the features of limb development and regeneration in amphibians and chicks. Based on our findings, we attempted to re-induce the AER. When wnt-2b/fgf-10-expressing cells were inserted concurrently with wounding, successful re-induction of the AER occurred. These results open up new possibilities for limb regeneration in higher vertebrates since AER re-induction, which is considered a key factor in limb regeneration, is now possible.     

Production of polyhedral inclusion bodies from Helicoverpa armigera larvae infected with wild-type and recombinant HaSNPV

We report on the yield of Polyhedral Inclusion Bodies (PIBs) from first to fifth instar Helicoverpa armigera larvae inoculated with wild-type Helicoverpa armigera nucleopolyhedrovirus (HaSNPV-WT) or with one of two HaSNPV recombinants, one in which the ecdysteroid UDP-glucosyltransferase (egt) gene was deleted (HaSNPV-EGTD) and a second in which the egt gene was replaced by a scorpion toxin (AaIT) gene (HaSNPV-AaIT). A significant linear relationship between the logarithm of cadaver weight and the logarithm of the number of PIBs per cadaver was observed for all three virus types. The increase of the number of PIBs with larval weight was significantly greater for HaSNPV-WT than for the recombinant viruses. For each of the three HaSNPVs, PIB yield per cadaver was significantly affected by larval instar at death and by time to death, with later instars and longer surviving larvae producing a greater number of PIBs. As both recombinants caused host larvae to die at earlier instars than HaSNPV-WT, their virus yields were significantly reduced. Virus yield per larva, inoculated with HaSNPV-AaIT in the first, second, third, fourth or fifth larval stage was 23, 32, 41, 44 and 47% of the yield of HaSNPV-WT, respectively. For HaSNPV-EGTD, virus yield per larva inoculated in first through fifth instar, respectively, was 41, 55, 63, 54 and 82% of the yield of HaSNPV-WT. These results provide a basis for optimizing the production regime of recombinant HaSNPVs in larvae and for modeling the behavior of these viruses in agro-ecosystems     

The nucleotide sequence of the araC regulatory gene in Salmonella typhimurium LT2

The nucleotide sequence of the araC regulatory gene of Salmonella typhimurium LT2 has been determined. This sequence and the predicted araC translational product are compared to their counterparts in Escherichia coli. The two genes code for similar products although the S. typhimurium protein is eleven amino acids shorter than the E. coli protein. The predicted amino acid sequences are 92% conserved and the DNA sequences are 82% conserved for the common regions of the two genes.     

Changes in the electrical polarity of tobacco cells following the application of weak external currents

Weak externally applied electric currents changed the natural electrical pattern surrounding cells from tobacco (Nicotiana tabacum L.) suspension cultures. The artificial currents were applied transversely to short filaments of cells placed between a microelectrode lose to the filament surface and a large platinum electrode some distance away. The natural current patterns before and after electrical treatment were measured with a vibrating probe. Significant effects were confined to the cell adjacent to the microelectrode. Currents with densities of 100 A · cm^–2 at the cell surface applied for 10 min or 3 A · cm^–2 for several hours caused a localized increase in the natural current entering the part of the cell which had been nearest the positive electrode. There was no corresponding local increase in current leaving from the opposite side of the cell. Instead, the extra current appeared to leave over a relatively large area. The overall effect was a tendency for the cell to repolarize transversely with a greater proportion of its transcellular currents flowing in the direction of the current applied. The effect was measurable for several hours after the external current was discontinued and may be evidence for a natural mechanism by which neighbouring cells entrain one another's polarities during differentiation. The effect of external currents on cells growing in a 2,4-dichlorophenoxyacetic acid (2,4-D) medium (which suppresses differentiation) was qualitatively the same as on cells in an indole-3-acetic acid medium (which promotes differentiation). If anything, the response was greater in 2,4-D, implying that the disruptive effect of 2,4-D on cell and tissue polarization is not a consequence of it preventing cells sensing the transcellular currents of their neighbours.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid The authors are indebted to the Agricultural and Food Research Council of the U.K. for financial support and to the Royal Society for the provision of the vibrating probe.     

Incorporation of cholesterol into serum high density lipoprotein apoprotein and recombinants

The uptake of cholesterol (CHL) by serum high density lipoprotein (HDL) delipidated apoproteins and phospholipid-apoprotein recombinants has been studied with two methods; by incubation with Celite-dispersed cholesterol or with cholesterol crystals. The apoproteins bind very small amounts of cholesterol with a maximum of about 6 micrograms/mg apoprotein. Recombinants with dimyristoyl phosphatidylcholine (DMPC) or egg phosphatidylcholine (EPC) as phospholipid component gave similar values for cholesterol uptake. The initial rate for uptake from Celite-cholesterol by recombinants was high (0.1 mol cholesterol/mol phospholipid/h) and somewhat higher than that for phospholipid vesicles. The maximal uptake was by gel filtration shown to depend on the size of the complexes with values about 0.95 mol cholesterol per phospholipid for vesicular complexes, 0.75 for discoidal complexes and between 0.5 and 0.2 for small 'protein-rich' complexes. During the incubation of recombinants with cholesterol there was considerable decomposition of discoidal complexes and formation of larger ones. The results show that phospholipid-apoprotein complexes are efficient acceptors for cholesterol but also that about 25% of the phospholipid in the discoidal complexes is excluded from interaction with cholesterol by interaction with apoprotein.