Scott Monsma - Academia.edu (original) (raw)

Papers by Scott Monsma

The Journal of experimental zoology, 1988

We describe a technique for inducing localized expression of genes fused to heat-shock gene promo... more We describe a technique for inducing localized expression of genes fused to heat-shock gene promoters. We demonstrate that a localized heat-shock response can be induced in Drosophila melanogaster at any developmental stage after formation of the cellular blastoderm by contacting a region of the animal with a heated needle. The size of the induced region can be altered by varying parameters such as the temperature and size of the needle tip. The test system utilized here is a D. melanogaster strain transformed with a fusion of the Drosophila hsp26 gene and the E. coli lacZ gene; the activity of this hybrid gene is monitored in whole animals by staining for beta-galactosidase activity. Induced beta-galactosidase activity is confined to the cells in the region of heating; the beta-galactosidase activity can still be detected 48 hr after the heat shock. Given the heat inducibility of Drosophila heat-shock promoters in heterologous systems, we suggest that this technique will be useful ...

Mechanisms of Development, 1994

Acp26Aa and Acp26Ab are Drosophila male accessory gland transcripts that are tightly linked and t... more Acp26Aa and Acp26Ab are Drosophila male accessory gland transcripts that are tightly linked and transcribed from the same DNA strand. Despite their being separated by 20 base pairs, the transcripts show identical responses to several developmental signals. These observations make it important to determine whether the 26A region contains two separable genes with the same developmental expression or a single developmentally regulated transcription unit whose product is processed to yield Acp26Aa and Acp26Ab. We show that Acp26Aa and Acp26Ab are separate mRNAs using a reverse transcription-polymerase chain reaction assay and reporter gene fusions. We also show that the regulatory elements for Acp26Ab lie within a fragment containing the intergenic region and transcribed sequences of Acp26Aa and Acp26Ab.

Thermus aquaticus Y51MC23 was isolated from a boiling spring in the Lower Geyser Basin of Yellows... more Thermus aquaticus Y51MC23 was isolated from a boiling spring in the Lower Geyser
Basin of Yellowstone National Park. Remarkably, this T. aquaticus strain is able to grow
anaerobically and produces multiple morphological forms. Y51MC23 is a Gram-negative,
rod-shaped organism that grows well between 50°C and 80°C with maximum growth rate at
65°C to 70°C. Growth studies suggest that Y51MC23 primarily scavenges protein from the
environment, supported by the high number of secreted and intracellular proteases and
peptidases as well as transporter systems for amino acids and peptides. The genome was
assembled de novo using a 350 bp fragment library (paired end sequencing) and an 8 kb
long span mate pair library. A closed and finished genome was obtained consisting of a
single chromosome of 2.15 Mb and four plasmids of 11, 14, 70, and 79 kb. Unlike other
Thermus species, functions usually found on megaplasmids were identified on the chromosome.
The Y51MC23 genome contains two full and two partial prophage as well as numerous
CRISPR loci. The high identity and synteny between Y51MC23 prophage 2 and that of
Thermus sp. 2.9 is interesting, given the 8,800 km separation of the two hot springs from
which they were isolated. The anaerobic lifestyle of Y51MC23 is complex, with multiple morphologies
present in cultures. The use of fluorescence microscopy reveals new details
about these unusual morphological features, including the presence of multiple types of
large and small spheres, often forming a confluent layer of spheres. Many of the spheres
appear to be formed not from cell envelope or outer membrane components as previously
believed, but from a remodeled peptidoglycan cell wall. These complex morphological
forms may serve multiple functions in the survival of the organism, including food and
nucleic acid storage as well as colony attachment and organization.

Journal of virology, 1996

To demonstrate the essential nature of the baculovirus GP64 envelope fusion protein (GP64 EFP) an... more To demonstrate the essential nature of the baculovirus GP64 envelope fusion protein (GP64 EFP) and to further examine the role of this protein in infection, we inactivated the gp64 efp gene of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and examined the biological properties of this virus in vivo. To provide GP64 EFP during construction of the recombinant GP64 EFP-null AcMNPV baculovirus, we first generated a stably transfected insect cell line (Sf9 OP64-6 ) that constitutively expressed the GP64 EFP of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The AcMNPV gp64 efp gene was inactivated by inserting the bacterial lacZ gene in frame after codon 131 of the gp64 efp gene. The inactivated gp64 gene was cloned into the AcMNPV viral genome by replacement of the wild-type gp64 efp locus. When propagated in the stably transfected insect cells (Sf9 OP64-6 cells), budded virions produced by the recombinant AcMNPV GP64

Developmental biology, 1990

The male accessory gland ofDrosophila is an adult secretory tissue which contributes many product... more The male accessory gland ofDrosophila is an adult secretory tissue which contributes many products to the male ejaculatory fluid. The secretions of the accessory gland affect the behavior and physiology of the female fly after mating, reducing her receptivity to courtship and stimulating egg production and oviposition. We have examined the developmental and mating-stimulated expression of two accessory gland proteins in the male and their transfer to and fates in the mated female. One of these proteins, msP 355a, has features of a prohormone and contains a region with amino acid sequence similarity to the egg-laying hormone of Aplysia; the other, msP 355b, is a small acidic protein. Both proteins are first detected in the accessory gland only after eclosion, although their transcripts are already present in late pupae. Both proteins are initially detected in the two morphologically distinct secretory cell types of the accessory gland, the main cells, and the secondary cells. In the glands of aged virgin males, they are only detected in the large vesicles of the secondary cells and in the lumen of the gland. Copulation results in an increase in the mRNAs for both proteins, as well as renewed translation of the proteins at least in the main cells. Both proteins are transferred to the female genital tract during copulation, and rapidly enter the female hemolymph. msP 355a is subject to rapid and specific cleavage within the female genital tract, but not in the hemolymph; msP 355b is not cleaved in either the female genital tract or the hemolymph.

Journal of virology, 1995

Virology, 1999

GP64 is the major envelope glycoprotein from budded virions of the baculoviruses Autographa calif... more GP64 is the major envelope glycoprotein from budded virions of the baculoviruses Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). To examine the potential role of GP64 as a viral attachment protein in host cell receptor binding, we generated, overexpressed, and characterized a soluble form of the OpMNPV GP64 protein, GP64solOp. Assays for trimerization, sensitivity to proteinase K, and reduction by dithiothreitol suggested that GP64solOp was indistinguishable from the ectodomain of the wild-type OpMNPV GP64 protein. Virion binding to host cells was analyzed by incubating virions with cells at 4°C in the presence or absence of competitors, using a single-cell infectivity assay to measure virion binding. Purified soluble GP64 (GP64solOp) competed with a recombinant AcMNPV marker virus for binding to host cells, similar to control competition with psoralen-inactivated wild-type AcMNPV and OpMNPV virions. A nonspecific competitor protein did not similarly inhibit virion binding. Thus specific competition by GP64solOp for virion binding suggests that the GP64 protein is a host cell receptor-binding protein. We also examined the kinetics of virion internalization into endosomes and virion release from endosomes by acid-triggered membrane fusion. Using a protease sensitivity assay to measure internalization of bound virions, we found that virions entered Spodoptera frugiperda Sf9 cells between 10 and 20 min after binding, with a half-time of approximately 12.5 min. We used the lysosomotropic reagent ammonium chloride to examine the kinetics of membrane fusion and nucleocapsid release from endosomes after membrane fusion. Ammonium chloride inhibition assays indicated that AcMNPV nucleocapsids were released from endosomes between 15 and 30 min after binding, with a half-time of approximately 25 min.

Virology, 1995

The baculovirus GP64 envelope fusion protein (GP64 EFP) is a class I integral membrane protein th... more The baculovirus GP64 envelope fusion protein (GP64 EFP) is a class I integral membrane protein that enters the secretory pathway and is oligomerized and extensively processed during transport to the plasma membrane. The kinetics of GP64 EFP biosynthesis, oligomerization, and processing in Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV)-infected Lymantria dispar cells were examined by pulse label, pulse-chase, and immunoprecipitation experiments. Relative rates of GP64 EFP synthesis in OpMNPV-infected L. dispar cells were examined at various times throughout the infection cycle. Using pulse labeling and immunoprecipitation, GP64 EFP synthesis was detected within 2 hr p.i., and the maximal rate of synthesis was observed in the period of 24-26 hr pi., a time coincident with the onset of high level production of budded virus in OpMNPV-infected L. dispar cells. To determine the oligomeric structure of GP64 EFP, a soluble form of OpMNPV GP64 EFP was produced and examined by a combination of gel filtration chromatography, nonreducing SDS-PAGE, and mass spectrometry. Oligomeric GP64 EFP was identified as a trimeric molecule, that migrates as two discrete bands on nonreducing SDS-PAGE. Pulse-chase studies, performed at both early (12 hr pi.) and late (36 hr p.i.) stages of the infection cycle, showed that GP64 EFP oligomerization is complete within 15 min after synthesis. Efficiency of oligomerization however was relatively low, with less than 33% of the synthesized GP64 EFP converted to trimers. The majority of monomeric GP64 EFP remaining in the cell appeared to be degraded within 30 to 45 min after synthesis. Analysis of the kinetics of carbohydrate processing at early (12 hr p.i.) and late (36 hr p.i.) times postinfection showed that for beth early and late phases of infection, carbohydrate was rapidly added, and processing began between 10 and 20 min after GP64 EFP synthesis. Although carbohydrate processing was completed within approximately 90 min after synthesis during the early phase, the same process required approximately 150 min during the late phase. Thus, carbohydrate processing appeared to become less efficient as infection progressed. These studies thus show that GP64 EFP undergoes a rapid but inefficient oligomerization step that results in a homotrimeric structure for GP64 EFP. While carbohydrate addition is rapid, carbohydrate processing requires prolonged periods of time (with half-times of 45 to 75 min) and appears to become less efficient during the late phase of the infection.

Journal of Virology, 2001

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential vi... more The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc 64؊ , could be pseudotyped by introducing a heterologous viral envelope protein

Proceedings of the …, 1983

Oligodendrocytes isolated from ovine white matter according to a published procedure [Szuchet, S.... more Oligodendrocytes isolated from ovine white matter according to a published procedure [Szuchet, S., Stefansson, K., Wollmann, R. L., Dawson, G. & Arnason, B. G. W. (1980) Brain Res. 200, 151-164] were cultured for up to 35 days and their capacity to incorporate precursors into lipids was investigated. At various times, cultures were double labeled with [3H]glycerol/

Journal of Comparative Neurology, 1996

We have examined the development of the adult retina and the outer optic lobes in the moth Manduc... more We have examined the development of the adult retina and the outer optic lobes in the moth Manduca sexta. The adult retina is generated from a group of epithelial cells lying within the larval head capsule between the larval ocelli and antenna. Proliferation of these cells begins during the feeding larval stage but accelerates at the end of the final larval instar. Proliferation occurs in two zones of mitotic activity; these zones flank a furrow in the presumptive retinal epithelium. The furrow and flanking mitotic zones migrate from posterior to anterior across the presumptive retinal epithelium. Posterior to the furrow, presumptive retinal cells from clusters and extend axons into the larval optic nerve. We have also examined the temporal patterns of neuronal proliferation and cell death during genesis of the adult outer optic ganglia, the medulla and the lamina. The medulla and the lamina are generated by distinct populations of neuroblasts in the outer optic analage; the neuroblasts divide asymmetrically to generate ganglion mother cells. Ganglion mother cells later divide symmetrically to generate immature neurons. Generation of the medulla cortex starts with the onset of the final larval instar, and cell death within the medulla cortex begins after the end of the final larval instar. Generation of the lamina cortex is initiated with the arrival of retinal afferents at the optic lobes, and cell death in the lamina cortex begins 1 day later. Generation of the outer optic ganglia terminates with the abrupt cessation of mitotic activity followed by degeneration of the outer optic anlage.