Developmentally regulated alternative splicing of Drosophila integrin PS2 alpha transcripts

We report the characterization of a chromosomal integrin gene that encodes the Drosophila PS2 alpha subunit. The gene is composed of 12 exons spanning 31 kb. By employing a novel method for directed cDNA cloning, we have analyzed over 300 independent cDNA clones for the existence of alternate RNA products. Two forms of PS2 alpha mRNA are frequently observed: a canonical (C) form and a form lacking the 75 nucleotide exon 8 (m8). The relative ratio of these two forms varies widely during development. Although region A, derived from exon 8 and the adjacent 25 amino acids, shows weak conservation among the sequences of alpha subunits that bind to different ligands, it is highly conserved in the homologous PS2 alpha gene of the distantly related Mediterranean fruitfly. We suggest that the variable region A may be important in determining the specificity and affinity of integrin receptors for their ligands.     

Developmentally regulated expression of alpha 6 integrin in avian embryos.

The distribution pattern of the avian alpha 6 integrin subunit was examined during early stages of development. The results show that this subunit is prevalent in cells of the developing nervous system and muscle. alpha 6 is first observed on neuroepithelial cells of the cranial neural plate and trunk neural tube. With time, immunoreactivity becomes prominent near the lumen and ventrolateral portions of the neural tube, co-distributing with neurons and axons, particularly notable on commissural neurons. The alpha 6 expression pattern is dynamic in the neural tube, with immunoreactivity peaking by embryonic day 6 (stage 30) and decreasing thereafter. The ventral roots and retina exhibit high levels of immunoreactivity throughout development. In the peripheral nervous system, alpha 6 immunoreactivity first appears on a subpopulation of sympathoadrenal cells around the dorsal aorta and later in the dorsal root ganglia shortly after gangliogenesis. Immunoreactivity appears on prospective myotomal cells as the somites delaminate into the dermomyotome and sclerotome, remaining prominent on myoblasts and differentiated muscle at all stages. The mesonephros also has intense immunoreactivity. In the periphery, alpha 6 immunoreactive regions often in proximity to laminin, which is thought to be the ligand of alpha 6 beta 1 integrin.     

Heterogeneity of alpha 2u-globulin gene products in different translational systems, plasma and urine

alpha 2u-Globulin, an androgen dependent rat urinary protein, displays considerable microheterogeneity. To explore whether this microheterogeneity of alpha 2u-globulin in male rat urine is related to the heterogeneity at the level of the genes encoding this protein, or whether it is due to post-translational processing we studied the alpha 2u-globulin mRNA translation products in rabbit reticulocyte and Xenopus oocytes. Comparison of the alpha 2u-globulin species produced in these two heterologous systems with those observed in plasma and urine indicates that the heterogeneity of this protein in urine is mainly due to heterogeneity at the level of the corresponding mRNAs.     

Developmentally regulated expression of the calcitonin gene related peptide (CGRP) in rat lung endocrine cells

Calcitonin (CT) and the calcitonin gene-related peptide (CGRP) are generated by alternative RNA processing from a single CT/CGRP gene. Recently, we reported the existence of CGRP-immunoreactivity and CGRP mRNA in endocrine cells or Kulchitsky (K) cells of human and rat lung [Wada et al. 1987b]. In this report, an examination was made of developmental changes in the expression of the CGRP gene in rat lungs by immunohistochemistry, radioimmunoassay (RIA) and Northern hybridization. CGRP-positive K-cells in lung tissue appeared on the 18th day of gestation. Their number was greatest on the 20th day of gestation and then decreased postnatally. The level of CGRP in rat lung was found to be highest in a 1-day-old neonate by RIA. In the Northern hybridization of rat lung using the CGRP 3' non-coding region (exon 6) of the first human CT/CGRP gene as the probe, 1.0 kilobase (kb) CGRP mRNA was found to be abundant on the 20th day of gestation and in a 1 day-old neonate. It thus appears that CGRP in rat lung is essential for pulmonary adaptation at birth and/or from the last intrauterine stage to the early neonatal period.     

B-Cell responses to male-specific antigen(s) in mice

It has been found that B-cell responses to male-specific antigen(s) can be clearly demonstrated by reversed plaque assays. Female mice injected with syngeneic male spleen cells showed significant increases (greater than 100 × in some strains) in the number of immunoglobulin-secreting cells in lymph nodes draining the injection site. There was a variation in B-cell responsiveness between strains and this correlated only partially with previously reported T-cell responsiveness to the H-Y antigen. C57BL (H-2 ^b ) mice were among the most responsive, while CBA (H-2 ^k ), (CBA × C57BL)F₁, and BALB/c (H-2 ^d ) were all much less responsive. These results apparently open up a new approach to the investigation of B-cell responses to male-specific antigen(s).     

Transcobalamin II expression is regulated by transcription factor(s) binding to a hexameric sequence (TGGTCC) in the promoter region of the gene

An isoenzyme of glutathione S-transferase (adGST) was purified from liver intestine of the seashell (Asaphis dichotoma) by GST-Sepharose 4B affinity chromatography followed by reverse-phase HPLC. The enzyme has a pI value of 4.6 and is composed of two subunits each with a molecular weight of 23kDa. It exhibits different catalytic activities toward the substrates 1-chloro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, ethacrynic acid, and p-nitrophenyl acetate and, fascinatingly, shows high activity toward CDNB. The amino acid composition of adGST was determined and found to be very similar to the Sloane squid GSTs. N-terminal analysis of the first 15 residues of adGST revealed that it has 73% sequence identity with the pig roundworm GSTs. The adGST shows characteristics similar to those of class sigma GSTs, as was indicated by its substrate specificity, N-terminal amino acid sequence, and amino acid composition.     

Developmentally regulated expression of a sunflower 11S seed protein gene in transgenic tobacco

Summary The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae. The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase. The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form. By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining. Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested. Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide. Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.Abbreviations PRA N-(5'-phosphoribosyl)anthranilate - InGP indole-3-glycerol phosphate - SDS sodium dodecyl sulfate     

Silent alpha(2C)-adrenergic receptors enable cold-induced vasoconstriction in cutaneous arteries

Cold constricts cutaneous blood vessels by increasing the reactivity of smooth muscle alpha(2)-adrenergic receptors (alpha(2)-ARs). Experiments were performed to determine the role of alpha(2)-AR subtypes (alpha(2A)-, alpha(2B)-, alpha(2C)-ARs) in this response. Stimulation of alpha(1)-ARs by phenylephrine or alpha(2)-ARs by UK-14,304 caused constriction of isolated mouse tail arteries mounted in a pressurized myograph system. Compared with proximal arteries, distal arteries were more responsive to alpha(2)-AR activation but less responsive to activation of alpha(1)-ARs. Cold augmented constriction to alpha(2)-AR activation in distal arteries but did not affect the response to alpha(1)-AR stimulation or the level of myogenic tone. Western blot analysis demonstrated expression of alpha(2A)- and alpha(2C)-ARs in tail arteries: expression of alpha(2C)-ARs decreased in distal compared with proximal arteries, whereas expression of the glycosylated form of the alpha(2A)-AR increased in distal arteries. At 37 degrees C, alpha(2)-AR-induced vasoconstriction in distal arteries was inhibited by selective blockade of alpha(2A)-ARs (BRL-44408) but not by selective inhibition of alpha(2B)-ARs (ARC-239) or alpha(2C)-ARs (MK-912). In contrast, during cold exposure (28 degrees C), the augmented response to UK-14,304 was inhibited by the alpha(2C)-AR antagonist MK-912, which selectively abolished cold-induced amplification of the response. These experiments indicate that cold-induced amplification of alpha(2)-ARs is mediated by alpha(2C)-ARs that are normally silent in these cutaneous arteries. Blockade of alpha(2C)-ARs may prove an effective treatment for Raynaud's Phenomenon.     

Developmentally regulated expression and functional role of alpha 7 integrin in the chick embryo

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.