Molecular characterization and cell-specific expression of an ion transport peptide in the tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

The crustacean hyperglycemic hormone (CHH) peptides regulate diverse physiological processes from reproduction to metabolism and molting in arthropods. In insects, the ion transport peptides (ITP), also members of the CHH family, have only been implicated in ion transport. In this study, we sequenced a nucleotide fragment spanning the conserved A1/A2 region of the putative CHH/ITP gene. This fragment was amplified from larval cDNA of the tobacco hornworm, Manduca sexta and showed a high degree of sequence conservation with the same region from other insects and, to a lesser degree, with that of crustacean species, suggesting the presence of a Manduca-specific CHH/ITP mRNA (MasITP mRNA). CHH-like immunocytochemical analyses with two crustacean antisera (from Carcinus maenas and Cancer pagurus) identified the presence of CHH-like immunoreactivity in nervous tissue of all developmental stages, but not in the gut of M. sexta. Specifically, CHH-like peptides localized to paired type IA₂ neurosecretory cells of the pars lateralis of the brain (projecting ipsilaterallly to the corpora cardiaca-allata complex) and to neurosecretory cells and transverse nerves of the ventral nerve cord in larvae, pupae, and adults. The distribution of the putative MasITP peptide shifted during development in a manner consistent with metamorphic reorganization. A comparison of hemolymph equivalents of CHH detected by enzyme-linked immunosorbent assay with CHH-like immunoreactivity in transverse nerves provided evidence for the release of MasITP from the transverse nerves into the hemolymph at insect ecdysis. These data suggest the presence of an insect ITP in M. sexta and a role for this hormone during ecdysis. This research was funded by the National Institutes of Health (MBRS SCORE Program-NIGMS) to M.F. (grant no. 2S06 GM52588-09), by the National Center on Minority Health and Health Disparities (grant no. 5P20-MD000262), an NIH RISE graduate fellowship to A.L.D. (5 R25 GM59298), an NIH PREP fellowship to C.C.H. and M.A.U. (5 R25 GM64078), an NSF CSU LS-AMP fellowship to C.C.H. (HRD-9802113), and by NIH MBRS-MARC to M.D.P. (T34 GM08574) and NIH MA/MS-PhD Bridge Scholarship to A.L.D. and C.C.H. (5R25 GM48972).     

Mapping of nucleoside phosphorylase (Np-1) and esterase 10 (Es-10) on mouse chromosome 14

A method for detecting two alleles at Np-1 (nucleoside phosphorylase) and three alleles at Es-10 (esterase 10) from mouse blood by cellulose acetate electrophoresis is described. The allelic constitution at these loci for 44 inbred strains and stocks was determined. The location of Np-1 on chromosome 14 was established by backcross experiments in which alleles at Np-1 and Robertsonian translocations were segregating. Es-10 was shown to be linked to Np-1, and the following genetic map of Chr 14 was constructed: centromere-(8.9±4.0 cM)-[Np-1, Wc]-(10.2±1.9 cM)-Es-10-(15.5±3.7 cM)-s. The homologous human loci, NP and ES-D, are not linked.This work was supported by Contract E(11-1)-3267 with the Energy Research and Development Administration, by Contracts NO1-ES4-2156 and NO1-ES4-2159 with the National Institute of Environmental Health Sciences, and by Grants GM 19656 and GM 20919 from the National Institute of General Medical Sciences. D. A. K. was a participant in the 1975 Summer Program for College, Graduate, and Medical Students, which was supported, in part, by the Clark Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Ectopic pairing and evolution of 5S ribosomal RNA genes in the chromosomes of Drosophila funebris

5S ribosomal RNA from Drosophila melanogaster labeled with ¹²⁵I was used to locate the 5S rRNA genes in chromosomes of D. funebris by means of in situ hybridization. Silver grains were observed at three distinct sites, one of which was a recognized reverse repeat. Only one half of the reverse repeat, however, hybridizes with 5S rRNA and the significance of this phenomenon is discussed. A case of ectopic pairing between two different 5S sites in the genome is reported, and the significance of ectopic pairing is considered.The author was a Predoctoral Fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.Contribution from Oak Ridge National Laboratory, operated by the Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

Improved transferase assay for cultured fibroblasts

The sensitivity and accuracy of our previously reported transferase assay (Russell and DeMars, 1967) can be increased by lowering the pH during the chromatographic separation of the reaction product. This modification does not alter the quantitative nature of the assay.Paper # 1151 from the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin. This work was supported by the National Institutes of Health (Grants GM 08217, GM 389, and GM 06903).     

Cross-Reactive Polysaccharides from Trypanosoma cruzi and Fungi (Especially Dactylium dendroides)1

ABSTRACT. Cross-reactivity between fungal and Trypanosoma cruzi polysaccharides, owing to common residues of β-D-galactofuranose, β-D-galactopyranose, and α-D-mannopyranose, was demonstrated by using a) rabbit immune sera against T. cruzi epimastigotes and b) sera from patients with Chagas’disease. Several chagasic (Ch) sera precipitated partly purified galactomannans from Aspergillus fumigatus and from T. cruzi epimastigotes and also the galactoglucomannan from Dactylium dendroides. Reaction of one Ch serum with T. cruzi galactomannan (GM) was completely inhibited by synthetic β-D-Galf-(1 → 3)-Me α-D-Manp, and that of another Ch serum with a purified D. dendroides galactoglucomannan (GGM) was partly inhibited by (1 → 6)-linked (81%) or by (1 - 3)-linked (33%) β-D-Galf-Me α-D-Manp. The β-D-Galf-(1 → 3)-α-D-Manp epitope was present in both T. cruzi and D. dendroides polysaccharides. Rabbit anti-T. cruzi antisera precipitated A. fumigatus GM, T. cruzi antigenic extracts containing the lipopeptidophosphoglycan (LPPG), T. cruzi alkali-extracted GM, a synthetic GM, and D. dendroides GGM. Weak reactivities were obtained for a Torulopsis lactis-condensi GM containing β-D-Galp terminal residues and for baker's yeast mannan with α-D-Manp-(1 - 3)-α-D-Manp-(1- → 2)-α-D-Manp-(1 → 2) side chains. An anti-LPPG rabbit serum precipitated D. dendroides GGM—a reaction inhibited (82%) by β-D-Galf-(1 → 3)-Me α-D-Manp and, less efficiently, by a (1 → 5)-linked β-D-Galf-tetrasaccharide. Sera from mice immunized with D. dendroides whole cells reacted with CL-strain trypomastigotes as shown a) by indirect immunofluorescence, b) by a Staphylococcus adherence test, but were not lytic. Mice immunized with D. dendroides were not protected against a challenge with virulent T. cruzi trypomastigotes.     

Isozyme variability in species of the genus Drosophila. III. Qualitative comparison of the esterases of D. aldrichi and D. mulleri

Two closely related species, Drosophila aldrichi and D. mulleri, are compared on the basis of their esterase isozyme patterns after starch gel electrophoresis. Comparable esterases between the two species are identified by substrate specificity, inhibition, and enhancement of esterase activity by various agents. The extensive electrophoretic variability of most of the esterases in nature is described and data relating to the inheritance of the various enzyme forms are presented.This work was supported (in part) by a U.S. Public Health Service research grant (GM 11609 to W. S. Stone and M. R. Wheeler) and a training grant (2 T1-GM 337-07 to R. P. Wagner et al.) from the National Institutes of Health.     

A reinvestigation of the nucleolus-organizing regions in the salivary gland nuclei of Drosophila melanogaster

Comparisons were made of the morphology of the proximal region of the salivary gland X-chromosome of D. melanogaster following a number of different staining procedures. Azur B was found to be the most satisfactory staining method for identification of the nucleolus. The states of eu- and heterochromatization (sensu Prokofyeva-Belgovskaya) of the bands in the proximal region—particulary striking in the mirror-image duplication of the R(1)2, ring-X, chromosome—contribute to the variability in the banding-pattern, and consequently the refractoriness of this region to cytological investigation. No nucleolus was ever found to be associated with the group of bands presumed to be the Y-chromosome.This investigation was supported in part by a U.S. Public Health Service Research Grant, GM 15009, from the Institute of General Medical Sciences of the National Institutes of Health, and in part by a grant from the Finnish National Research Council for Sciences.     

In vitro translation of maize ADH: Evidence for the anaerobic induction of mRNA

Total cellular RNA from anaerobically stressed maize seedling roots was used to stimulate in vitro translation of authentic maize alcohol dehydrogenase (ADH) in a rabbit reticulocyte lysate system. Total products from such reactions were displayed on NEPHGE-SDS two-dimensional gels and the Adhl-specific translation products were identified by using RNA from sib seedlings segregating for Adhl charge and size variants. The application of a rapid RNA isolation procedure allowed the efficient isolation of biologically active RNAs from small amounts of seedling material. Maize ADHs translated in vitro are identical in size to in vivo ADH. Further, no ADH was detected in the products of an in vitro translation reaction stimulated by total RNA from aerobically grown seedlings. This suggests that induction of ADH protein by anaerobic stress is accomplished by production of Adh mRNA rather than activation of sequestered mRNA. The mRNAs for maize ADH1 and ADH2 are among a small class of mRNAs induced during anaerobiosis.Research was supported by NSF Grant PCM 76-11009. M.D.B. is supported by National Institute of Health Grant PHS 5 T32 GM07227-04. R.J.F. is a Predoctoral Trainee in Genetics supported by National Institute of Health Training Grants 82 and 7757 from the National Institute of General Medical Sciences.     

Satellite DNA-correlated nucleosomal proteins in Drosophila virilis

Three major satellite DNAs comprise 40–45% of the genome of Drosophila virilis. Since these satellites are not substrates for most restriction enzymes, we were able to digest D. virilis nuclei with HaeIII and micrococcal nuclease and isolate chromatin fractions containing variable levels of satellite DNA. Electrophoretic analysis of these chromatin fractions revealed that the level of the acid-soluble chromosomal protein, cp17.3, was directly related to the percentage of satellite DNA in chromatin. The correlation between cp17.3 and satellite DNA abundance suggests that cp17.3 is involved in the heterochromatic condensation of satellite DNAs. cp17.3 occurs at a frequency of one molecule per 10–20 nucleosomes. It is detected in an electrophoretically distinguishable class of mononucleosomes, provisionally identified as MN1uH2A, which contains ubiquitinated histone H2A (uH2a) but lacks histone H1. It is not detected in MN1, a second class of mononucleosomes, which lacks uH2A and H1. Since cp17.3 is correlated with satellite DNAs and present in nucleosome cores, it might be a histone variant specifically associated with satellite DNAs.This work was supported by Grant GM22138 from the National Institutes of Health. G.A.V. was a predoctoral trainee supported by Grant GM07094 from the National Institutes of Health.     

Structural homology between Drosophila melanogaster and Escherichia coli acidic ribosomal proteins

Antibodies raised against Drosophila melanogaster ribosomal proteins (r-proteins) were used to examine possible structural relationships between eukaryotic and prokaryotic r-proteins. The antisera were raised against either groups of r-proteins or individually purified r-proteins. Two antisera showed a cross-reaction with total Escherichia coli r-proteins in Ouchterlony double immunodiffusion assays: an antiserum against the D. melanogaster small subunit protein S14 (anti-S14) and an antiserum against a group of D. melanogaster r-proteins (anti-TP80). The specificity of the antisera and the identity of the homologous E. coli r-proteins were characterized by using immunooverlay and immunoblot assays. These assays indicated that anti-S14 was highly specific for protein S14 and anti-TP80 was a multispecific serum that recognized several of the D. melanogaster ribosomal proteins. The E. coli protein homologous to D. melanogaster protein S14 was identified as E. coli protein S6. By adsorption of the anti-TP80 serum, we determined that D. melanogaster protein 7/8 is homologous to the acidic E. coli protein L7/L12. D. melanogaster acidic protein 13 was also shown to be immunologically related to D. melanogaster protein 7/8.This research was supported by National Institutes of Health Grant GM23410 awarded to WYC. LMS was the recipient of a predoctoral fellowship from Molecular, Cellular, and Developmental Biology Training Grant PHS T32 CM07227. We are very grateful to Dr. Anthony Mahowald for providing us with embryos.     

Isozyme variability in species of the genus Drosophila. IV. Distribution of the esterases in the body tissues of D. aldrichi and D. mulleri adults

Tissue specificity of the esterases of two closely related species, Drosophila aldrichi and D. mulleri, is determined by starch gel electrophoresis of dissected tissues and organs and an esterase staining technique. The determination of esterase homology between the two species is supplemented by tissue specificity as an added criterion of catalytic properties. General biological functions of some of the esterases are suggested by their presence in certain tissues. The potential usefulness of knowledge of tissue specificities in evolutionary and population genetic studies is discussed.This work was supported (in part) by a U.S. Public Health Service research grant (GM 11609 to W. S. Stone and M. R. Wheeler) and a training grant (2T1-GM 337-07 to R. P. Wagner et al.) from the National Institutes of Health.     

Distribution and characterization of hexose 6-phosphate dehydrogenase in trout

Electrophoretic analysis of trout liver extracts indicates that an autosomal gene coding for hexose 6-phosphate dehydrogenase (H6P D) in lake trout, Hpd-L, is monomorphic. In brook trout, the gene is polymorphic, having at least three genetic variants termed Hpd-B ¹, Hpd-B², and Hpd-B³. F ₁ hybrid splake exhibit the three expected phenotypes resulting from Hpd-L/Hpd-B ¹, Hpd-L/Hpd-B², and Hpd-L/Hpd-B ³ genotypes. Trout H6P D is ostensibly a dimer of mol wt 230,000. The characteristics of trout H6P D, including substrate specificity, genetic polymorphism, electrophoretic characteristics, subcellular localization, and tissue distribution are in close agreement with results obtained for mammalian H6P D. We suggest that trout and mammalian H6PDs, X-linked mammalian G6P D, and autosomal avian G6P D arose from a common ancestral type G6P D.The research was supported in part by National Science Foundation grant GB-7271 and by United States Public Health Service Predoctoral Fellowship 4 FO1 GM41704-03.     

Relationships between water balance properties and habitat characteristics in the sibling Hawaiian Drosophilids,D. mimica and D. kambysellisi

Summary Four populations of Drosophila mimica and 1 population of D. kambysellisi collected at sites which differed in wetness were examined for several water balance characteristics. Net water loss per hour increased as a_v (relative humidity/100) decreased in all populations, but the rate of increase was lower in populations from dry sites. When exposed to 0.70 a_v, D. kambysellisi, which were from a rain forest, lost water faster and died sooner than did D. mimica. Two D. mimica collecting sites were divided into smaller units based on substrate type at one site and on litter wettness at the other site. The D. mimica at the first site were homogeneous with respect to the water balance properties studied here, but in the second site, there was evidence of population differentiation associated with litter wettness.Supported by research grant NSF DEB77-23370, training grants NIH 5 TO1 GM 00337-16 and NIH 5 T32 GM 07126-02 and a Grant-in-Aid from the Sigma Xi Society     

Differential fluorescent staining of Drosophila chromosomes with quinacrine mustard

The polytene and mitotic chromosomes of D. melanogaster, D. simulans, D. ananassae, and D. virilis were stained with the fluorescent dye, quinacrine mustard (QM). In all these species except D. ananassae, we have detected species-specific chromosomal loci which exhibit an extremely brilliant fluorescence. Most, but not all, of the brilliantly fluorescing areas are located in heterochromatic chromosome regions. Cytochemical and chemical methods have been employed to demonstrate that the brilliant fluorescence represents regions of acid labile non-covalent binding between DNA and QM whereas the moderate overall fluorescence is primarily due to covalent bonding (by alkylation) of the QM to DNA. The exact mode of binding of QM in the brilliant areas and the nature of the DNA in these areas are not known. The possible biological significance of the DNA in the brilliant regions is discussed.This study was supported by a Research Grant (GM 10499) from the National Institutes of Health, U.S. Public Health Service.This paper is dedicated with respect and affection to Professor Berwind P. Kaufmann on the occasion of his 74th birthday, April 23, 1971.     

A system selective for yeast mutants deficient in meiotic recombination

Summary An experimental design and rationale for detecting and recovering Saccharomyces cerevisiae mutants specifically blocked in meiotic gene conversion is presented. The system utilizes an otherwise haploid strain disomic (n+1) for chromosome III which is simultaneously heterozygous for the mating-type locus and heteroallelic at leu2. The former is an essential requirement for inducing meiotic development; i.e., DNA replication and sporulation upon transfer to acetate media, while the latter provides a convenient signal for assaying recombination at the intragenic level. Of 940 clones screened qualitatively after mutagenesis with ethyl methanesulfonate, 91 presumptive mutants were isolated. These are classed arbitrarily into four groups according to the reduction in interallelic recombination observed in quantitative tests.Supported by research grants GM: 17317 and GM: 16522 from the National Institutes of Health and a grant from the National Sciences Foundation, GB: 8534.     

Gangliosides in acetylcholine receptor-rich membranes fromTorpedo marmorata andDiscopyge tschudii

The ganglioside composition of membranes enriched in nicotinic acetylcholine receptor (AChR) from the electric raysDiscopyge tschudii andTorpedo marmorata has been determined, and compared to that of total electric organ. A ganglioside having the chromatographic mobility of GM2 constitutes the major ganglioside (60%) in totalD. tschudii electric organ, followed by a component with the mobility of GD3 (10%), and a component running just below GD1a (about 12%). Minor constituents running as GM3 (2%) and as polysialogangliosides (comprising 8–15%) were also observed. Purified native membranes ofD. tschudii andT. marmorata displayed a similar profile, except that they were richer in a GM1-like component, and the proportion of GM2-like gangliosides was lower than that in total electric organ. Using a¹²⁵I-cholera toxin overlay assay on neuraminidase-treated high-performance thin layer chromatograms, the presence of GM1, GD1a and trace amounts of GD1b and GT1 (or GQ) were detected inD. Tschudii total membranes. Immunocytochemical trechniques showed the co-localization of gangliosides GQ1c/GT1c/GP1c, recognized by the monoclonal antibody Q211, and the AChR at the ventral, innervated face of the electrocyte.     

Cat-2 gene expression

Summary Poly(A)⁴ RNA was isolated from maize scutella of different stages of post-germinative development and translated in vitro in a rabbit reticulocyte translation system. Immunoprecipitation of the translation products with CAT-2-specific antibody was used to quantitate the relative levels of translatable CAT-2 mRNA at each stage. The results show a close correlation between the developmental profile of Cat2 gene expression and the profile of CAT-2 mRNA levels. Evidence that the levels of CAT-2 mRNA are regulated by a temporal regulatory gene (Car1) is presented and the possible mechanism(s) of this regulation discussed.This work was supported by Research Grants No. GM22733 and No. GM33817 from the U.S. National Institutes of Health, Public Health Service to J.G.S. This is paper No. 9933 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695, USA     

Assignment of histidase-regulating locus to chromosome 10 of the mouse

Data from four sets of recombinant inbred strains confirm that variation at a single genetic locus is responsible for the previously observed differences in the rate of histidase synthesis in inbred mice. Linkage testing stocks were used to demonstrate linkage with steel (Sl) on chromosome 10.This research was supported in part by Grants GM 21002 and GM 18684 from the National Institutes of General Medical Sciences. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

The genetics of glutamic-pyruvic transaminase in mice: Inheritance,electrophoretic phenotypes,and postnatal changes

Glutamic-pyruvic transaminase (GPT, E.C. 2.6.1.2) from 18 inbred strains of mice was subjected to starch gel electrophoresis. Two electrophoretic phenotypes were observed: a fast-migrating pattern in 16 strains and a slower-migrating pattern in two strains. A comparison of electrophoretic patterns of F₁ and backcross progeny of two strains of mice showed that the inheritance of GPT is autosomal with two codominant alleles. The genetic locus for GPT is designated Gpt-1, and its two alleles are designated Gpt-1 ^a and Gpt-1 ^b to represent the fast-migrating (A) and slow-migrating (B) patterns. The GPT was expressed in 11 tissues with different amounts of enzyme activity. Developmental studies of GPT activity in liver showed that between 5 and 12 days after birth the mean activity was 10 units/g protein. Between 12 and 19 days, a dramatic rise in activity occurred and adult values of 300 units/g protein were reached by 26 days.This research was supported by The National Foundation (CRBS-258) and the National Institutes of Health (GM15253).Preliminary results were reported at the Annual Meeting of the American Society of Human Genetics, October 11–14, 1972, in Philadelphia.R. P. D. is an investigator of the Howard Hughes Medical Institute.     

Liver-specific lysosomal acid phosphatase deficiency (Apl) on mouse chromosome 17

Summary Apl, a gene involved in the processing of lysosomal acid phosphatase in mouse liver, has been mapped on Chromosome 17. The gene order and map distances in per cent recombination of the loci studied are T (20.6±3.4) Pgk-2 (7.4±2.2) Apl. Thus, Apl is at least 7 cM distal to H-2 on this chromosome. In addition, strain-specific allelic variants for Apl have been demonstrated on cellulose acetate gels, a quick and inexpensive method of electrophoresis.This work was supported by Contract NO1-ES42159 with the National Institute of Environmental Health Sciences, Grant 1–476 from the National Foundation, March of Dimes, and Grant GM 20919 from the National Institute of General Medical Sciences. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care