The polarity of axon growth in the wings of Drosophila melanogaster

We have analyzed the growth of axons in the wings of the mutants Hairy wing and hairy of Drosophila melanogaster. These mutants produce many supernumerary bristle organs and sensilla campaniformia, whose axons grow between the two wing epithelia and can be visualized in both pupal and adult stages. The sensory axons of wild-type animals follow two paths in the wing, within longitudinal veins L1 and L3, and always grow with a distal to proximal polarity. In the mutants, all axons following these two paths likewise grow with correct polarity. Axons elsewhere in the wing, however, are found to grow in many different directions, including from proximal to distal and hence directly away from the central nervous system. A variety of patterns of axon growth and fasciculation are seen in different individuals. Only if the supernumerary axons encounter the two normal paths do they reliably grow toward the base of the wing. We conclude that these two paths provide polarity information for axon growth, information which is either not used or not available elsewhere in the wing in spite of the obvious morphological polarization of every epithelial cell. The time course of neural differentiation suggests that the normal sensory cells of mutant wings, which grow axons relatively early, may be the source of polarity information for the later-differentiating supernumerary cells.     

Developmental compartments in the Drosophila melanogaster wing disc

Distribution of the enzyme aldehyde oxidase (AO) within the pouch of the mature wing disc is precise and differential. General locations of compartmental boundaries have been identified by fate mapping and studies of AO distribution. The suspected locations of the boundaries were verified by analyzing the distribution of AO-negative cells within an AO-stained background in gynandromorphs and in X-ray-induced clones of AO-negative cells. The anterior/posterior border appeared slightly anterior to the junction of the AO⁺ anterior presumptive wing surfaces and AO^? posterior wing surfaces. A narrow band of AO⁺ cells extending proximodistally on both presumptive wing surfaces belongs to the posterior compartment. Two dorsal/ventral (dor./vent.) restrictions were found. The dor./vent. restriction equivalent to the dor./vent. border found in the adult wing was located at the ventral most edge of the AO-stained presumptive wing margin. A second restriction which was less strictly obeyed was found on the dorsal edge of the wing margin. We conclude that the whole presumptive wing margin is part of the dorsal compartment. Within the anterior wing margin an intensively stained oval was also found to be clonally restrictive. Therefore, territories were found within the prospective wing margin for which no such features have been identified in the adult Drosophila melanogaster wing.     

Analysis of visual system development in Drosophila melanogaster: mutations at the Glued locus

We have analyzed several aspects of the development of flies carrying mutations at the Glued locus. Optic lobe abnormalities in individuals heterozygous for the original Glued allele were previously shown to result from an action of this mutation in the retinula cells. We have estimated when the functioning of this gene or its product is required for normal visual system development by using genetic mosaicism induced by somatic recombination and temperature shifts of a temperature-sensitive mutation at this locus. Both methods point to a period in the mid-third instar, suggesting that early events in the formation of ommatidia and/or late events in the program of retinal cells are affected. Application of a new histological stain for developing axons indicates that individuals heterozygous for Glued exhibit abnormalities in the retinula fiber projection by the late third instar. Thus, the adult phenotype is not solely the result of later cellular degeneration or rearrangement. Beneath M+ Gl+ clones which encompass the entire eye were found optic lobe abnormalities with features not seen in either other mosaics or Gl heterozygotes. The possibility that these abnormalities result from temporal asynchrony in the development of eye and and optic lobe in these individuals is discussed and the results of attempts to test this hypothesis are presented.     

Regeneration of segment boundaries in oncopeltus: cell lineage

The segment boundary of Oncopeltus is a straight interface between the cells of two segmental compartments; it coincides with an abrupt change of pigmentation and a groove in the surface of the cuticle. When a segment boundary is burnt it is regenerated by the cells migrating into the wound. We describe the cell lineage of this process: the two masses of migrating cells first contact each other on a convoluted frontier, but this soon straightens. The new boundary forms exactly at the interface between the two cell populations, without a single cell straying across the line. Even when the cautery is asymmetric and, as a consequence, the cells from the different segments meet at an abnormal position, the segment boundary is regenerated at this interface. After extirpation or transplantation ectopic boundaries can be formed within a segment. These new boundaries also act as lineage restrictions and stop mingling of cells across them. Our results support the hypothesis that the integrity and straightness of the compartment boundary depend on cell affinities; the cells in one polyclone adhering more to each other than to the cells of the neighbouring polyclone. There may be a gradient of cell affinity in each segment, with an abrupt change at the segment boundary.     

The acquisition of resistance to ecdysteroids in cultured Drosophila cells

Drosophila melanogaster K_c cells become refractory toward ecdysteroids after 4 days of exposure to the molting hormone, 20-OH-ecdysone. Associated with the appearance of hormonal insensitivity is a loss of ecdysteroid receptors. Hormone-resistant cells maintain a low level of receptor that is indistinguishable from that of responsive, hormonally naive cells. After extended periods in culture, ecdysteroid receptor content in previously exposed cells returns to that of naive control cells. The reappearance of receptor is coincident with the resumption of hormonally induced growth inhibition.     

26S and 18S rRNA synthesis in bobbed mutants of Drosophila melanogaster

For the most part, bobbed mutations of Drosophila melanogaster consist of deletions of 26S and 18S rDNA located on the X and Y chromosomes. Studies on the synthesis of rRNA of third instar larvae and one day old adult females of three severe bobbed genotypes, indicate that no decrease can be detected, compared ot wild type strains. One of the bobbed mutants studied was a rather unusual type: these flies possess a quantity of rDNA that should confer upon them a near wild type phenotype whereas they actually show an extreme bobbed phenotype. The two other bobbed mutants are of a classical type: their severe bobbed phenotype corresponds to large deletions of rDNA. Two hypotheses can be proposed to explain the extreme bobbed phenotype of the flies, in spite of the fact that rRNA synthesis occurs normally. A regulatory phenomenon may interfere at the stages studied, but in earlier stages a net decrease in rRNA synthesis may have occurred producing an irreversible effect in the tissues affected by bobbed mutations (abdominal cuticle, bristles). The second hypothesis is that the rRNA produced may not be functional, perhaps because it is specific of earlier stages.     

Drosophila melanogaster U1 and U2 small nuclear RNA genes contain common flanking sequences

The flanking sequences of three U2 genes (or pseudogenes) and one U1 gene of Drosophila melanogaster have been determined. Comparison of the sequences reveals a remarkable homology between position ?30 and ?65 upstream from the structural genes, starting with a TATA box-like sequence. The 3′ flanking regions are also conserved in all genes and contain a canonical A-A-T-A-A-A polyadenylation signal.     

The effect of fasting and refeeding on the composition and synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in rat liver

Refeeding a high-sucrose, fat-free diet to fasted rats caused drastic alterations in the fatty acid composition of hepatic diacylglycerols, triacylglycerols, and phosphatidylcholines. However, the fatty acid profile of phosphatidylethanolamines did not change significantly. These results suggest that the fatty acid composition of diacylglycerols may influence the distribution of diacylglycerols among triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. Fasting and refeeding also affected the activities in vitro of a number of enzymes responsible for the formation of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. The activity of hepatic phosphatidate phosphatase increased fourfold upon refeeding. However, fasting the rats did not affect the activity of this enzyme despite the reduced triacylglycerol synthesis in the fasted liver in vivo. Fasting and refeeding induced alterations in the activities of diacylglycerol acyltransferase, cholinephosphotransferase, and ethanolaminephosphotransferase which correlated reasonably well with the changes observed in the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in vivo, although the changes in diacylglycerol acyltransferase were too moderate. The changes in the activity of cholinephosphate cytidylyltransferase, which is suggested to catalyze the rate-limiting step in the formation of CDP-choline, ran parallel with the alterations in the synthesis of phosphatidylcholines in vivo. No such correlation was found between the activity of ethanolaminephosphate cytidylyltransferase and the rate of phosphatidylethanolamine synthesis. The present results indicate that the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines is controlled by the availability of the various substrates as well as by the activities of several enzymes involved in these processes.     

The action of ribonuclease T1 on reovirus double-stranded RNA.

A small number of nucleotides are released from highly purified reovirus double-stranded RNA by ribonuclease T₁ in the presence of 0.3 m NaCl. These nucleotides include ppGp, which is quantitatively released from the RNA, and lesser amounts of ApUpGp, Gp, and ApGp. The same products are released from each of the three size classes of double-stranded RNA segments. In experiments involving specific labeling of termini, the only demonstrable sites of hydrolysis were at the 5′ termini of the minus strands. The limited extent of ribonuclease T₁ hydrolysis and localization of its action at the 5′ termini of the minus strands are compatible with a perfect duplex structure for the double-stranded RNA segments wherein the secondary structure of the termini is less stable than that of internal regions.     

The molecular and catalytic properties of galactosyltransferase from fetal calf serum

A soluble galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyl-transferase) was purified to apparent homogeneity from fetal calf serum with an overall increase in specific activity of 19,600-fold. The enzyme exhibited the following properties: specific activity, 8.5 units/mg of protein; acceptor specificity, N-acetylglucosamine/ ovalbumin = 3.3; diffusion coefficient, 5.56; sedimentation coefficient, 3.2; and molecular weight, 47,800. Comparison of the structural and catalytic properties of the fetal calf serum enzyme with purified galactosyltransferase from bovine milk indicated that the enzymes from the two bovine sources are very similar and possibly identical.     

Specific binding of [3H]nitrendipine to membranes from coronary arteries and heart in relation to pharmacological effects. Paradoxical stimulation by diltiazem

High affinity binding sites for the calcium channel inhibitor [³H]nitrendipine have been identified in microsomes from pig coronary arteries (K_D=1.6 nM; B_max=35 fmol/mg) and in purified sarcolemma from dog heart (K_D=0.11 nM; B_max=230 fmol/mg). [³H]nitrendipine binding to coronary artery microsomes was completely inhibited by nifedipine, partially by verapamil and D600 and, surprisingly, was stimulated by d-cis-diltiazem but not by 1-cis-diltiazem, a less active isomer. Half-maximal relaxation of KCl-depolarized coronary rings occurred in a slow process at 1 nM nitrendipine or 100 nM d-cis-diltiazem. In dog trabecular strips, nitrendipine caused a negative inotropic response (ED₅₀=1μM). These results suggest that there may be multiple binding sites for different “subclasses” of calcium channel inhibitors, and that drug binding sites may be different molecular entities from the putative calcium channels.     

Putative superoxide dismutase activity of iron-EDTA: a reexamination

It has been reported that iron-EDTA complexes mimic the action of superoxide dismutase, displaying 0.01% of the activity of the enzyme (Halliwell, B., 1975, FEBS Lett., 56, 34–38). This was purportedly directly confirmed by J. G. McClune, J. A. Fee, G. A. McCluskey, and J. T. Groves, 1977, J. Amer. Chem. Soc., 99, 5220–5222. A reexamination of the behavior of this compound has demonstrated that it does not catalyze the dismutation of O₂^?, but rather inferferes with assays for superoxide dismutation activity, which are based on the reductions of nitroblue tetrazolium or of cytochrome c. The sources of this interference have been examined. Investigators engaged in searching for mimics of superoxide dismutase are urged to be wary of similar artifacts.     

Evidence for the lack of direct phosphorylation of bovine caudate tyrosine hydroxylase following activation by exposure to enzymatic phosphorylating conditions.

Highly purified bovine caudate tyrosine hydroxylase can be activated by exposure to enzymatic phosphorylating conditions. This activation is due to both a decrease in the K_m for the pterin cofactor and to some increase in V_max. The K_m for the enzyme's substrate, tyrosine, is unchanged by activation. After tyrosine hydroxylase was activated in the presence of [γ-³²P]-ATP, no incorporation of ³²P into the enzyme was observed by either immunoprecipitation studies or by sucrose gradient studies.     

Identification of the sym plasmid of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other Rhizobia and Agrobacterium tumefaciens

A plasmid of 150 Mdal from Rhizobium leguminosarum RCC1001 was found to be a Sym plasmid (pSym1) carrying genes for root nodulation and nitrogen fixation on plants of the pea vetch cross-inoculation group. The plasmid was expressed not only in different R. leguminosarum and R. trifolii hosts, but also in Agrobacterium tumefaciens and R. meliloti, although in root nodules induced by A. tumefaciens and R. meliloti hosts no nitrogen was fixed. The host range for root nodule induction appeared to be determined by pSym1 and only included plants of the pea vetch cross-inoculation group; in contrast, the host range for the induction of root hair deformations, which was found also to be determined by pSym1 was less restricted and included besides plants of the pea vetch group in addition plants of the clover group. This corroborates previous findings that host specificity for nodulation and nitrogen fixation is exerted at a stage after the induction of root hair deformations.     

An attempt to select for spontaneous locomotor activity in Drosophila melanogaster

Seven generations of selection for high and low spontaneous locomotor activity were made in the wild-laboratory strain Oregon of Drosophila melanogaster. Great care was taken to select for activity and not for reactivity. In opposition with the non totally unambiguous results obtained by another author, absolutely no response to selection could be obtained. Thus the Oregon strain of Drosophila melanogaster does not appear to possess any additive genetic variance for spontaneous locomotor activity. Yet before taking for granted that that conclusion is applicable to all strains of Drosophila melanogaster an experimental selection should be performed again using a freshly captured wild strain.     

Identification of vitelline membrane proteins in Drosophila melanogaster

In Drosophila melanogaster, proteins involved in vitelline membrane production are secreted by ovarian follicle cells during stages 9 and 10 of oogenesis. We have used SDS-PAGE and two-dimensional electrophoresis to identify six major size classes of radiolabeled components in purified vitelline membrane preparations. Analyses of in vivo labeled proteins from egg chambers of different developmental stages and stage 10 follicle cells show that components of five of these size classes are synthesized by follicle cells during the period of vitelline membrane deposition. Immunological analysis of eggshell antigens utilizing complex antisera raised to purified eggshell fragments has confirmed the identity of components of three size classes.     

The map position of Sym-plasmid regions expressed in the bacterial and endosymbiotic form of Rhizobium leguminosarum

The Sym plasmid of Rhizobium leguminosarum, which is called pRle1001a, was found to be transcribed in both cultured bacteria and in bacteroids isolated from mature pea root nodules. The transcribed regions were localized on a restriction endonuclease map of this plasmid. None of the areas expressed in the endosymbiotic form overlapped with the one that is expressed in stationary phase cultures of the bacteria. One relatively large region that is actively transcribed in nitrogen-fixing bacteroids included the DNA homologous to the structural nif genes D and H of Klebsiella pneumoniae. This transcribed segment is also highly conserved in the Sym plasmid of R. trifolii 5 and a plasmid of R. phaseoli 3622, which carries nif genes. It is assumed that this region carries the nif operon.     

A substrate for the fluorogenic assay of exo-beta-N-acetylmuramidase: synthesis and purification of 4-methylumbelliferyl N-acetylmuramide.

A fluorogenic substrate for exo-β-N-acetylmuramidase from Bacillus subtilis B was synthesized. 4-Methyl-2-oxo-1,2-benzopyran-7-yl 2-acetamido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranoside was prepared from 4-methyl-2-oxo-1,2-benzopyran-7-yl 2-acetamido-2-deoxy-β-d-glucopyranoside, condensed with dl-2-chloropropionic acid, the benzylidene residue removed by acetolysis and the 4-methyl-2-oxo-1,2-benzopyran-7-yl 2-amino-3-O-(d-1-carboxyethyl)-2-deoxy-β-d-glucopyranoside purified by chromatography on silica gel and Sephadex G-10 and by high-voltage paper electrophoresis. The identity of the product was confirmed by pmr studies, acid hydrolysis followed by chromatography of the products, and enzymic digestion.     

Carnosine binding: characterization of steric and charge requirements for ligand recognition.

The steric and charge requirements for binding of l-carnosine (β-alanyl-l-histidine) by bovine serum albumin were investigated with proton magnetic resonance (¹HMR) spectrometry. The histidinyl side chain of the dipeptide is responsible for primary recognition by the binding site. Furthermore, recognition is specific to a particular orientation of the histidinyl side chain that is determined by the other amino acid residue of the dipeptide. It was found that, although salts do not have a great effect on the binding of carnosine to bovine serum albumin, this binding cannot be measured by equilibrium dialysis in the presence of salt because of formation of a complex Donnan equilibrium. Carnosine, which has been postulated to have a role in olfaction, binds to the crude particulate fraction of nasal olfactory epithelium in the same steric orientation as it does to bovine serum albumin. Therefore, we have used the binding of carnosine to bovine serum albumin as a model system to test potential competitive inhibitors of carnosine binding that ultimately could be tested for activity in the olfactory pathway. It was found that the binding of carnosine to bovine serum albumin is a good model of nonspecific binding of carnosine to tissue preparations but not of the specific binding of carnosine to the nasal olfactory epithelium. In addition to requiring the proper conformation of the histidinyl residue, the binding to olfactory epithelium also appears to require recognition of the β-alanyl residue and of substituents on the imidazole ring. Evidence is provided that the carnosine binding by the nasal olfactory epithelium demonstrated by ¹HMR spectroscopy does not occur with the mature olfactory receptor neurons.