Ecdysteroid-inducible polypeptides in a Drosophila cell line

In the Drosophila melanogaster cell line Kc-H, ecdysteroid hormone treatment causes increased relative synthesis of three ecdysteroid-inducible polypeptides (EIPs), named according to their molecular weights (in kilodaltons) EIP 40, EIP 29 and EIP 28. Increased synthesis of the EIPs is detectable within 45 min (EIP 28) or 75 min (EIPs 40 and 29), is maximal at 4-8 hr and continues for almost 2 days. During this period no other major changes in protein synthesis are discernible using one-dimensional gels. At maximum, EIP 28 synthesis is elevated at least 10 fold above its basal level, and EIPs 40 and 29 somewhat less. EIP induction is ecdysteroid-specific and is detectable in the presence of 10(-8) M 20-hydroxyecdysone. It does not occur in hormone-resistant cells. Apparently identical polypeptides are inducible in another ecdysteroid-responsive cell line, Schneider's line 3. Because EIP synthesis is an early and substantial response to ecdysteroids, this is a promising system for the study of steroid hormone action.     

Gene Expression in Developing Zea mays Embryos: Regulation by Abscisic Acid of a Highly Phosphorylated 23- to 25-kD Group of Proteins

We have earlier identified a set of proteins of 23 to 25 kilodaltons (kD), covering an isoelectric point (pI) range of 6.2 to 8.2, which accumulate gradually during normal embryogenesis of Zea mays and disappear in early germination. These polypeptides can be induced prematurely in immature embryos by abscisic acid (ABA) treatment. We report here that the more acidic protein forms are due to post-translational phosphorylation of at least two polypeptides of 23 kD, pI 8.2 and 25 kD, pI 8.0. A polyclonal antiserum was obtained which recognizes all forms of both the 23-kD and 25-kD polypeptides. Recovery of cDNA clones corresponding to these proteins was accomplished by hybridization with cDNA made from size-selected mRNA enriched for these sequences. Hybrid selection experiments demonstrate that clone MA12 specifically hybridizes with mRNAs encoding the 23-kD and 25-kD protein set which are recognized by the antiserum. By Northern hybridization analysis, the RNA encoded by clone MA12 is shown to accumulate in mature embryos and to be induced in young embryos upon ABA incubation.     

Effects of juvenile hormone on the ecdysone response of Drosophila Kc cells

Drosophila Kc cells are ecdysone-responsive: hormone treatment leads rapidly to increased synthesis of several ecdysone-inducible polypeptides (EIPs) and to commitment to eventual proliferative arrest. Later, the treated cells undergo morphological transformation, cease to proliferate, and develop new enzymatic activities, notably, acetylcholinesterase (AChE) activity. These responses have proven useful as models for studying ecdysone action. Here we report the sensitivity of Kc cells to another important insect developmental regulator--juvenile hormone (JH). We find that JH inhibits some, but not all, aspects of the ecdysone response. When Kc cells are treated with ecdysone in the presence of either natural JHs or synthetic analogues, the morphological and proliferative responses are inhibited and AChE induction is blocked. Most striking is that JHs protect the cells from the rapid proliferative commitment induced by ecdysone alone. The JH effects exhibit reasonable dose-response curves with half-maximal responses occurring at very low JH concentrations. Nonetheless, even at high JH concentrations the inhibitory effects are incomplete. It is interesting that EIP induction appears to be refractory to JH. It seems clear that JH is not simply a generalized inhibitor of ecdysone-induced responses.     

Identification of barley and wheat cDNA clones related to the high-Mr polypeptides of wheat gluten

We have identified 3 cDNA clones related to the high-M_r group of storage proteins in barley endosperm, the D-hordeins. A cDNA library has been constructed from wheat endosperm poly(A⁺)-RNA and screened using one of the D-hordein cDNA clones. Two wheat clones which cross-hybridised to the barley clone have been identified, by hybrid-release translation and nucleotide sequence analysis, as partial copies of mRNAs encoding the high-M_r gluten polypeptides of wheat.     

The early genetic response to light in the green unicellular alga Chlamydomonas eugametos grown under light/dark cycles involves genes that represent direct responses to light and photosynthesis

In the green unicellular alga Chlamydomonas eugametos, cellular division is readily synchronized by light/dark cycles. Under these conditions, light initiates photosynthetic growth in daughter cells and begins the G1 phase. Genes whose expression is regulated upon illumination are likely to be important mechanisms controlling cell proliferation. To identify some of those genes, two cDNA libraries were prepared with poly(A)⁺ extracted from cells either stimulated with light for 1 h or held in darkness (quiescent cells) during the same period. To restrict our analysis to those genes that are part of the primary response, cells were incubated in presence of cycloheximide. Differential screening of approximately 40 000 clones in each library revealed 44 clones which hybridize preferentially with a [³²P] cDNA probe derived from RNA of light-stimulated cells and 15 clones which react selectively with a [³²P] cDNA probe synthesized from poly(A)⁺ RNA of quiescent cells. Cross-hybridization of these clones identified 4 independent sequences in the light-induced (LI) collection and 2 in the uninduced (LR) library. Four of these cDNAs correspond to mRNAs that are positively or negatively regulated upon activation of photosynthesis. One clone represents a mRNA that accumulates transitorily at both transitions. Finally, LI818 cDNA identifies a new chlorophyll a/b-binding (cab) gene family whose mRNA accumulation is controlled by light and a circadian oscillator. The endogenous timing system controls LI818 mRNA accumulation so that it precedes the onset of illumination by a few hours. On the other hand, light affects LI818 mRNA levels independently of active photosynthesis.     

CulturedAedes albopictus mosquito cells synthesize hormone-inducible proteins

Summary To provide a framework for biochemical investigation of ecdysteroid action inAedes albopictus mosquito cells, we examined the effect of 20-hydroxyecdysone on cell growth and morphology, synthesis of inducible proteins (EIPs), and expression of a transfected gene regulated by a synthetic ecdysteroid response element. When cells were cultured in the continuous presence of 10⁻⁶ M 20- hydroxyecdysone, the rate of growth decreased and subtle changes in cell morphology were observed. In bothAedes aegypti andA. albopictus cells, synthesis of a small number of radiolabeled proteins, which appeared as minor bands on sodium dodecyl sulfate-polyacrylamide gels, was induced by treatment with 20-hydroxyecdysone. On two-dimensional polyacrylamide gels, 11 EIPs, ranging in size from approximately 22 to 52 kDa, were identified inA. albopictus C7-10 cells. Ten inducible proteins were localized in the cytoplasmic fraction; EIP28 and EIP31 were detected in both cytoplasmic and nuclear extracts, and EIP29 was detected only in the nucleus, at a very low level. None of these proteins corresponded to small heat shock proteins, whose genes are 20-hydroxyecdysone-inducible in someDrosophila cell lines. The juvenile hormone analog, methoprene, induced expression of a 25 kDa protein in C7-10 cells. Although 20-hydroxyecdysone sustained the synthesis of this methoprene-inducible protein, synthesis did not occur in the presence of 20-hydroxyecdysone alone. In transfectedA. albopictus cells, expression of a recombinant DNA construct containing two tandem synthetic ecdysteroid regulatory elements based on aD. melanogaster small heat shock protein gene was modestly induced by 20-hydroxyecdysone.     

cDNA cloning of mRNAs induced in resistant barley during infection by Erysiphe graminis f.sp. Hordei

Summary Near-isogenic cultivars of Hordeum vulgare which differ for the Mlp gene for resistance to Erysiphe graminis f.sp. hordei were inoculated with race 3 of this pathogen and in vitro translation products of mRNA populations compared by 2-dimensional gel electrophoresis and fluorography. This revealed the presence of new mRNA species in infected leaves compared to non-inoculated controls. These new mRNA species were more abundant in resistant leaves than susceptible leaves. A cDNA library was prepared from poly(A)⁺RNA isolated from infected leaves carrying the Mlp gene for resistance (cvMlp). The library was screened by differential hybridization using [³²P]-labelled cDNA prepared from poly(A)⁺RNA of both control and infected leaves. Six cDNA clones showing greater hybridization to cDNA prepared from infected leaves were selected. These six cDNA clones hybridized to DNA isolated from barley leaves but not to DNA from conidia of the fungus. In Northern blot analysis of RNA from infected leaves the six cDNA clones each hybridized to mRNA species of different size. Translation products for three of the cDNA clones corresponded to infection-related translation products identified on 2-dimensional fluorograms. The cDNA clones were used to study the kinetics of host mRNA induction during infection of the near-isogenic cultivars of barley. The host mRNA species corresponding to the cDNA clones were induced prior to 24 h after inoculation during the primary penetration processes. In addition the mRNAs corresponding to four of the cDNA clones increased to greater amounts in cvMlp than in the near-isogenic susceptible cultivar (cvmlp) over a 2-d period following inoculation. These results suggest that the Mlp gene has a regulatory role in host gene expression resulting in enhanced expression of several host mRNA species following infection by the powdery mildew fungus.     

Isolation and characterization of cDNA clones for cytochromes P-450 immunochemically related to rat hepatic P-450 form PB-1

Rat hepatic cytochrome P-450 PB-1 is a prominent constitutive P-450 form whose levels increase approximately 2-3 fold upon phenobarbital administration. Antibodies raised against this protein recognized two major proteins in immunoblots of rat liver microsomal proteins and precipitated comparable amounts of two electrophoretically separable hepatic mRNA translation products. The levels of the two mRNAs encoding these polypeptides were increased substantially upon phenobarbital administration. The anti-PB-1 antibodies were used to screen a cDNA library, and two distinct cDNA clones, pTF-1 and pTF-2, were isolated. These clones contain inserts of 1227 and 410 base pairs, respectively, and show 80% nucleic acid sequence homology in their region of overlap. The DNA sequences of these clones show 54% sequence homology to the corresponding portions of the mRNA encoding P-450 PB-4, a major phenobarbital-inducible form of rat liver P-450, and can be optimally aligned with the PB-4 sequence without introducing insertions or deletions. The level of hepatic mRNA which hybridizes to clone pTF-2 increases approximately 2-4-fold after phenobarbital treatment, whereas mRNA which hybridizes to pTF-1 does not change in concentration after this treatment. mRNA, which hybridizes to pTF-1, is, however, 4-fold more abundant in livers of female rats than in livers of male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some somatic sequences are absent or exceedingly rare in Xenopus oocyte RNA

A variety of Xenopus laevis cDNA clones derived from somatic cell RNAs were hybridized to oocyte pA⁺ RNA separated on Northern gels. We were unable to detect oocyte pA⁺ sequences complementary to three undefined tadpole cDNA clones. With one of these clones, a complex pattern of bands appears during embryogenesis. With the other two clones, a single band appears. Two additional tadpole clones hybridize to both oocyte and tadpole RNA, but yield a more complex RNA pattern from embryos than from oocytes. One of these additional tadpole clones has complementarity to actin DNA, suggesting that the additional RNA band which appears during embryogenesis is α-actin mRNA (E. A. Sturgess, J. E. M. Ballantine, H. R. Woodland, P. R. Mohun, C. D. Lane, and G. J. Dimitriadis, 1980, J. Embryol. Exp. Morphol.58, 303–320). We have also failed to detect hybridization to oocyte pA⁺ RNA with one vitellogenin and three adult globin cDNA clones. Reconstruction experiments with purified globin mRNA from anemic adult blood cells set the lower level of sensitivity for globin mRNA at one part in 10⁶. The data suggest that some Xenopus mRNA sequences are absent or very rare in the oocyte pA⁺ RNA population.     

The structure of anchorin CII, a collagen binding protein isolated from chondrocyte membrane

cDNA clones for anchorin CII (Mr = 34,000), a collagen-binding protein, were isolated from a lambda gt 11 cDNA library prepared from chick cartilage mRNA. Several overlapping clones were characterized which gave rise to an open reading frame coding for 329 residues and a 3'-untranslated segment of 500 base pairs. The clones were identified as coding for anchorin by hybrid select translation analysis and by comparing the deduced amino acid sequence with the sequence of 10 tryptic peptides of the protein. A hydrophobic domain of 25 residues interrupted with 3 polar residues was identified with the carboxyl-terminal portion. There was no evidence for an aminoterminal signal peptide. Northern analysis revealed that the 5' probe hybridizes to a single 1.7-kilobases (kb) mRNA species, whereas the 3' probe hybridizes to two mRNA species of 1.7 kb and 5 kb, which are present in many cells including chondrocytes, crop cells, and fibroblasts. The level of anchorin mRNA in chick embryo fibroblasts was increased by infection with Rous sarcoma virus.     

Cell type-specific expression of bovine keratin genes as demonstrated by the use of complementary DNA clones

Cytokeratins are a family of polypeptides that form the intermediate-sized filament characteristic of epithelial cells. The cytoskeletons of different types of epithelial cells have been reported to possess specific combinations of the members of this protein family. Therefore, we have sought to examine the correspondence between such differential protein expression and the expression of cytokeratin genes at the nucleic acid level. A library of recombinant plasmids carrying cDNA sequences synthesized from bovine epidermal mRNAs was constructed. Clones of about 10(3) base-pairs coding for all the major epidermal keratins of molecular weights of 50,000, 54,000, 59,000, 60,000 and 68,000 were identified by means of hybridization-selection, followed by one and two-dimensional gel electrophoresis of products of translation in vitro. Under stringent conditions, each of these clones hybridizes specifically with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for the other keratins, including those belonging to the same subfamily. Using these clones in RNA blot hybridization analysis, we have studied the expression of keratin genes in diverse bovine epithelial tissues (muzzle epidermis, cornea, esophagus, bladder urothelium, liver) and cultured cell lines from kidney (MDBK) and mammary gland (BMGE + H, BMGE -H). In each case we have found a correlation between the respective keratin polypeptides and the corresponding mRNAs. Whereas mRNA coding for keratins Ia and VIb have been found only in epidermis, genes coding for other epidermal keratins are expressed also in certain non-epidermal epithelia and in cells of the BMGE + H line. In contrast, epidermal keratin mRNA sequences have not been detected in liver or bladder tissue, nor in cultured kidney cells (MDBK) or mammary gland cells of the BMGE - H line, which all express a set of cytokeratin polypeptides entirely different from those of epidermis. In all cases, only one mRNA size species has been found, suggesting that in different cell types the same mRNA species is synthesized from the same keratin gene. We conclude that the mechanisms controlling the cell type-specific synthesis of the diverse keratin genes act at a pre-translational level.     

生态产业园的复合生态效率及评价指标体系

生态产业园的建设在国内取得了丰富的成果,已成为我国循环经济建设和可持续发展的重要载体之一。与国外不同,我国的生态产业园最终将演化为生态城市,其功能也从工业生产功能发展为融生产、生活和生态为一体的复合功能。生态效率是评价一个研究对象投入产出效益的量化工具,将生态效率的理念融入生态产业园,从园区作为区域可持续发展的载体、作为生态城市演化的一个特定阶段的视角,提出了复合生态效率的概念,构建了园区复合生态效率评价指标体系,并以郑州经济技术开发区为例,说明了复合生态效率指标在产业园中的应用。     

SV40 large tumor antigen can regulate some cellular transcripts in a positive fashion

Eleven cDNA clones identified from a cDNA library prepared from the mRNA fraction of SV40 transformed cells detected, by hybridization, higher levels of cellular mRNA in SV40-transformed cells than in nontransformed cells. Three of these cDNA clones detected levels of cellular mRNA that were more than 100-fold greater in SV40tsA transformed cell lines grown at the permissive temperature than in those grown at the nonpermissive temperature. Northern blot hybridizations confirmed these results and in some cases detected RNA species of multiple sizes that were regulated in a temperature-dependent fashion in SV40tsA transformed cell lines. Infection of 3T3 cells with SV40 stimulated the levels of RNAs complementary to these cDNA clones. The results demonstrate that the SV40 large T antigen can regulate the steady state levels of some cellular RNA species.