R-3 Repository :: Browsing by Author "Beckingham, Kathleen M."

Browsing by Author "Beckingham, Kathleen M."

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A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae
(JoVE, 2018) Qiang, Karen M.; Zhou, Fanli; Beckingham, Kathleen M.
Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen distribution. It is also possible that aberrant behavior of oxygen-sensing neurons could induce hypoxia-like behavior in the presence of normal oxygen levels. In D. melanogaster, development at low oxygen levels results in inhibition of growth and sluggish behavior during the larval phases. However, these established manifestations of oxygen deficit overlap considerably with the phenotypes of many mutations that regulate growth, stress responses or locomotion. As result, there is currently no assay available to identify i) cellular hypoxia induced by a mutation or ii) hypoxia-like behavior when induced by abnormal neuronal behavior. We have recently identified two distinctive behaviors in D. melanogaster larvae that occur at normal oxygen levels in response to internal detection of hypoxia. First, at all stages, such larvae avoid burrowing into food, often straying far away from a food source. Second, tunneling into a soft substratum, which normally occurs during the wandering third instar stage is completely abolished if larvae are hypoxic. The assay described here is designed to detect and quantitate these behaviors and thus to provide a way to detect hypoxia induced by internal damage rather than low external oxygen. Assay plates with an agar substratum and a central plug of yeast paste are used to support animals through larval life. The positions and state of the larvae are tracked daily as they proceed from first to third instar. The extent of tunneling into the agar substratum during wandering phase is quantitated after pupation using NIH ImageJ. The assay will be of value in determining when hypoxia is a component of a mutant phenotype and thus provide insight into possible sites of action of the gene in question.
A lov Story: A role for jim lovell in Drosophila neural development and fertility.
(2011) Bjorum, Sonia Marie; Beckingham, Kathleen M.
A forward genetic screen looking for altered gravity response identified the gene jim lovell (lov). This gene is related to the gene fruitless, which specifies male courtship ii behavior in Drosophila. Like fruitless, lovencodes a protein with BTB-POZ domain and DNA binding domain, suggesting roles in transcription and ubiquitin-mediated degradation. In order to investigate the role(s) of lov further, several approaches have been taken. Five new mutations to the locus have been generated and their effects on various behaviors and development investigated. An antibody against the Lov protein has been generated and used to determine the wild type and mutant expression pattern of Lov during embryogenesis. The effects of ectopic expression and reduction of expression of lov in components of the nervous system was analyzed using the GAIA/ VAS system. These studies establish that lov expression is limited to the nervous system beyond very early embryogenesis. In the wild type embryonic PNS, Lov is found in subsets of neurons of all three classes - External Sensory neurons, Chordotonal organs and Multiple Dendritic neurons suggesting that Lov plays a role in specifying the final identity of certain neurons. One of the mutants has reduced Lov expression in the embryonic nervous system. Results from analysis of the new mutants and from the over/under-expression studies performed using the GAIAIUAS system have identified roles of lov in several behaviors and developmental processes. The altered expression of lov in the new mutant 111 alleles of lov results in decreased levels of male courtship directed towards a courtship partner as well as an increase in non-directed courtship. Additionally, various defects in sensory inputs were identified. The defects may contribute to in part to the reduced courtship. One of the mutants, lov66, has a male-male courtship phenotype. lov66 also has a reduced fertility due to defects in spermatogenesis, egg formation, and in ability of females to use sperm effectively to fertilize eggs. Finally, both the over and underexpression of lov resulted in a reduction in courtship.
An investigation of the role of the Drosophila gene jim lovell in endopolyploid growth
(2018-07-13) Zhou, Fanli; Beckingham, Kathleen M.; Lwigale, Peter
The Drosophila gene jim lovell (lov) encodes a putative transcription factor of the BTB/POZ (Bric-a-Brac, Tramtrack, Broad/Pox virus and Zinc finger) domain class that is expressed in many elements of the developing larval nervous system. Lov has roles in innate behaviors such as larval locomotion and adult courtship. I have identified new roles of lov in controlling endopolyploid growth. Endopolyploid growth occurs when cells repeatedly duplicate the genomic DNA without cell division. This type of growth is a normal feature of growth in some plants and animal tissues and is also used by cancer cells for survival. Most of the rapid growth of the Drosophila larva is achieved by this mechanism. I discovered that decreasing lov expression in endopolyploid larval tissues such as the tracheae and the salivary gland, reduces their cell size and endoploidy levels. dMyc is a well-known regulator of both mitotic and endopolyploid growth in Drosophila, acting mainly on nucleolar functions. I have shown that Lov is a nucleolar protein and that decreasing Lov levels can partially suppress the effects of dMyc on endopolyploid growth. I have also established that Lov interacts with another BTB/POZ domain protein called Ribbon (Rib) in controlling endopolyploid growth. Lov also regulates tracheal growth by affecting transcription of the uninflatable (uif) gene, which encodes an apical plasma membrane protein. Together, my findings indicate that Lov’s role in endopolyploid growth involves interactions with dMyc, Rib, and Uif.
Analysis of physiological roles of Drosophila calmodulin through in vivo genetic and in vitro structure/function studies
(2003) Wang, Bo; Beckingham, Kathleen M.
Calmodulin (CaM), a small protein found in all eukaryotes examined, is a major component of Ca2+ signaling pathways and functions as a Ca2+ signal sensor and transducer. A wide variety of targets are regulated by CaM, including enzymes, cytoskeleton elements and ion channels. To dissect the in vivo roles of Drosophila CaM, a series of Cam mutations were previously generated in the Beckingham lab. This thesis primarily concerns investigation of two Cam mutations; Cam7, a point mutation encoding V91G mutant CaM, and a null mutation, Camn339. Cam7 causes unprecedented defects. Cam 7 mutants are sluggish as larvae and form aberrant pupal cases with highly indented rings around the body. Mutant pupae never eclose and most die as pharate adults with head defects. Expression of wild type CaM specifically in the musculature is shown here to partially rescue the Cam7 phenotype, suggesting that muscle function is primarily affected by this mutation. Further, genetic studies performed suggest that misregulation of the ryanodine receptor (RyR), a Ca2+ channel on the sarcoplasmic reticulum, may be largely responsible for the muscle defects. Muscle contraction-associated Ca2+ release is shown here to be drastically altered in the Cam7 mutant. Biochemical studies revealed that the V91G mutation has no detectable effects on Ca2+-binding or the Ca2+-induced conformation of CaM. However, the conformation of Ca2+-free CaM is altered. Examination of the interaction between CaM and the RyR CaM binding region indicates that the V91G mutation would alter regulation of the RyR so as to cause Ca2+ leaking through the RyR channel. The ability of CaM variants with the N- or C-terminal Ca 2+-binding sites inactivated to rescue the Cam7 phenotype was also investigated. Consistent with prior CaM-RyR studies, the C-terminal binding site mutant exacerbated the phenotype. However, the N-terminal binding site mutant showed an "over-rescue" effect causing muscle relaxation. In contrast, the main behavioral defect observed in Cam null mutant larvae was found to be rescued by neural, not muscle, expression of wild type CaM and neither the N-terminal nor C-terminal Ca2+-binding site mutants had any effect on this defective behavior, suggesting a different molecular pathway is affected.
Calcium binding site mutations of Drosophila melanogaster calmodulin
(1991) Maune, John Frederick; Beckingham, Kathleen M.
This thesis describes: (1) the identification of the ATG start codon-containing exon of the Drosophila melanogaster calmodulin gene, and sequencing of this region and others of genomic and cDNA clones, (2) bacterial expression of wild-type calmodulin and generation and expression of mutants in the CA$\sp{2+}$-binding sites of the protein, and (3) physical and enzymatic studies of these proteins. The exon containing the ATG start codon was isolated using a 5$\sp\prime$ cDNA probe. Sequence studies of this genomic region and other parts of both genomic and cDNA clones of calmodulin were performed. A calmodulin cDNA expression system was generated for bacterial expression of wild-type and mutant calmodulin proteins. Mutant calmodulin proteins were generated in which only a single amino acid was altered. Each single point mutation was made in the final amino acid of each of the four Ca$\sp{2+}$-binding loops. Two mutations were made in each loop, for a total eight mutant calmodulins. Extinction coefficients were determined for wild-type protein and all eight mutant proteins. All nine proteins were studied using: (1) flow dialysis to examine equilibrium Ca$\sp{2+}$-binding, and (2) fluorescence stopped-flow with the Ca$\sp{2+}$ indicator Quin 2 to determine Ca$\sp{2+}$-dissociation rates. UV difference spectra of different Ca$\sp{2+}$ bound forms were performed for wild-type and five of the mutant proteins. Near and far UV CD was used to study wild-type and four of the mutant proteins in the absence and presence of Ca$\sp{2+}$. Far UV CD was used to study the urea denaturation profiles of wild-type and four of the mutant proteins in the absence and presence of Ca$\sp{2+}$. Calcineurin activation studies were preformed with the wild-type protein and four of the mutant proteins.
Characterization of androcam: A novel calcium-binding protein and a close relative of calmodulin from Drosophila melanogaster
(1999) Lu, Qing; Beckingham, Kathleen M.
The Androcam gene was originally isolated by Fyrberg et al. (1994), using reduced stringency cDNA library screening with a calmodulin (CaM) probe from our lab. The derived protein sequence of the new gene is 85% similar to CaM. Using Northern analysis, I established that, unlike CaM, transcripts from the new gene are not seen in all developmental stages of Drosophila melanogaster. I further determined that, the expression of this novel gene is confined to the testis. We thus named the gene Androcam. In situ hybridization with a RNA probe derived from the Androcam cDNA showed that within the testis, the transcripts are only present in germ-line cells from the polar spermatocyte stage onwards. Immunolocalization with Androcam antibodies further demonstrated that Androcam is first seen on the kl-3 Y loop in the polar spermatocytes. In the cytoplasm and nucleoplasm, the level of Androcam increases dramatically thereafter and remains at high levels in the elongating spermatids. Co-localization with a DNA dye and Androcam antibodies indicates that Androcam may be present in the head region of elongated spermatids. These data suggest that Androcam has roles during various stages of the spermatogenesis and possibly also in mature gametes. We, in collaboration with Dr. Peter Bayley's lab (NIMR, Mill Hill, U.K.) have examined the biophysical properties of Androcam. These biophysical results, accompanied with sequence comparison, suggested that Androcam is a new Ca2+-signaling protein. Our studies showed that Androcam is unlike CaM in that it has only three functional Ca 2+-binding sites. Two of these sites have very high affinity to Ca 2+ and may be in Ca2+- and target-bound form in the resting state, while the third site binds Ca2+ more weakly and is potentially the regulatory site. These Ca2+-binding properties are reminiscent of those of the cardiac/slow skeletal muscular troponin C, a relative of CaM, and a protein that is also involved in Ca 2+-signaling. The finding of Androcam in Drosophila melanogaster intrigued us to investigate whether a homolog exists in other organisms. Using the Androcam antisera as the main tools, I have attained evidence that Androcam homologs exist in other insect species and mammals.
Characterization of the gravitaxis-related protein Yuri Gagarin through genetic and biochemical approaches
(2008) Texada, Michael James; Beckingham, Kathleen M.
The Drosophila melanogaster gene yuri gagarin was first identified in a screen for aberrant behavioral responses to the mechanosensory stimulus provided by the force of gravity. This novel gene encodes Yuri isoforms of four sizes; the three larger isoforms are predicted to be composed largely of coil-forming domains, which are common protein interaction domains. The four isoforms are expressed in varying ratios throughout the animal, at all stages of development. Notably, Yuri is present in muscle tissue, localized to Actin-containing regions of the sarcomere. A male-sterile deletion allele of yuri, yuriF64, was identified and sequenced. This deletion removes roughly 500 base pairs of sequence upstream of the Yuri coding region; in these animals, the mid-sized Yuri isoforms are not expressed in any tissue, and the smallest isoform is no longer expressed in the testis. In wild-type animals, Yuri protein is present throughout the testis, and it assumes a dynamic microtubule-dependent nuclear localization pattern during the process of nuclear condensation, appearing to be a component of the "dense complex" described in ultrastructural studies of spermatogenesis. A previously undescribed filamentous Actin network co-localizes with Yuri on the nuclear surface; Yuri may act as a linker between the Actin and microtubule cytoskeletons. In yuriF64 homozygotes, no Yuri-staining or filamentous Actin structures are detected on the condensing sperm nuclei, and the basal body and centriolar adjunct are mis-localized or absent. Axonemal defects are apparent in heterozygotes and homozygotes, and the Actin-dependent sperm individualization does not take place, leaving immature sperm bound in bundles of 64. Yuri physically interacts with components of the Actin cytoskeleton (Actin, Tropomyosin 1, and Troponin T) in an in vivo pull-down assay. Tropomyosin 1 (Tm1) is the interacting protein purified in greatest abundance in the assay, suggesting that it and Yuri interact directly; this coil-forming molecule wraps actin filaments, stabilizes them against enzymatic or physical degradation, and blocks the interaction of the filament with other proteins. Tm1 is present on the condensing sperm nucleus, and Yuri co-localizes with it in muscle tissue. Thus, the defects seen in yuriF64 animals may reflect aberrant Actin dynamics arising from a lack of Yuri-Tm1 interaction.
Characterization ofbicaudal, a maternal effect mutation of Drosophila melanogaster
(1998) Markesich, Diane; Beckingham, Kathleen M.
The Drosophila melanogaster mutation bicaudal (bic) is the founding member of the group of maternal effect mutations that disrupt anterior-posterior axis formation by creating mirror image duplications of posterior segments in the anterior half of the embryo. The gene affected by the bic mutation was cloned, and a recessive embryonic lethal mutation, $vr22\sp{P3},$ was shown to be an allele of the same gene, demonstrating that the gene is essential. During embryogenesis, bic gene expression was detected in mesoderm and anterior and posterior midgut, and persistent expression was found in the somatic musculature. Late in embryogenesis, transcripts were detected in the central nervous system. The bic gene was shown to be expressed in two distinct locations in ovaries: transcripts were detected in somatic cells of the germarium, and a second activation of transcription was found exclusively in the germ-line derived nurse cells of ovarian follicles. The maternal effect of the bic mutation was shown to originate in failure to express transcripts of the gene in the germ-line nurse cells during oogenesis. The bic gene was found to encode the Drosophila homolog of beta NAC (Nascent polypeptide Associated Complex), a recently discovered ribosome associated protein of eukaryotes. Beta NAC is a subunit of a heterodimer that associates with nascent polypeptides as they emerge from the ribosome. In vitro, NAC regulates the specificity of co-translational targeting of nascent chains to the Endoplasmic Reticulum. The studies of the bic mutant effects presented here demonstrated that beta NAC has other unpredicted ribosome-associated functions, being required to ensure localized expression of proteins via translational control of mRNA. In oocytes/embryos of bic mutant females, negative regulation of translation of the mRNA of nanos, the posterior determinant, fails; NANOS protein is aberrantly expressed in the anterior compartment of the early embryo, and consequently, bicaudal embryos are created. Negative regulation of nanos was thus shown to include ribosome associated events. Furthermore, these first in vivo studies of the activity of beta NAC (BIC) identified a new ribosome associated function for the protein, and established a physiologically relevant and informative molecular genetic model system for future studies of NAC.
Chemical and spectral studies of the Lac repressor protein and its trypsin resistant core
(1982) Smith, Ann; Matthews, Kathleen S.; Palmer, Graham; Olson, John S.; Beckingham, Kathleen M.
The core protein produced by mild proteolytic digestion of the lac repressor has been purified on phosphocellulose, The repressor and core proteins were reacted with the sulfhydryl specific reagents, 2-chloromercuri-4-nitrophenol and fluorescein mercuric acetate. Modification of the cysteine residues did not alter the affinity of the proteins for inducer molecules. The operator binding activity of both proteins was unaffected by the reaction with 2-chloromercuri-4-nitrophenol; however, this binding was essentially abolished upon modification with fluorescein mercuric acetate. This loss of operator DNA binding activity in response to modification supports the thesis that determinants for specific DNA binding are located in the core region of the protein. Fluorescence spectral studies on repressor modified with 2-chloromercuri-4-nitrophenol and fluorescein mercuric acetate were performed. The quenching observed upon titration of repressor with either reagent indicates that energy transfer was occurring between the protein tryptophans and the cysteine-conjugated chromophores. Inclusion of dithiothreitol during the titration prevented the labelling of the cysteines; a corresponding decrease in energy transfer was seen. The addition of of inducer produces a blue shift in the repressor emission spectrum, but did not affect the quenching process. The quenching was sensitive to dithiothreitol for the repressor-inducer system as it had been for the repressor protein alone. The spectral behavior of the core protein was essentially identical to that displayed by the repressor.
Cloning and characterization ofbicaudal, a maternal effect mutation of Drosophila melanogaster
(1993) Gajewski, Kathleen Mary; Beckingham, Kathleen M.
The Drosophila maternal effect mutant, bicaudal (bic), affects the anterior-posterior axis of the embryo. It is incompletely penetrant, producing a variety of lethal embryonic phenotypes, the most severe of which is replacement of the anterior half with a mirror image duplication of the posterior half. Genetic mapping has placed this locus within a region defined by the overlap of two chromosomal deficiencies on the second chromosome. Indirect genetic evidence has suggested that a lethal mutation that maps to the same region, $vr22\sp{P3}$ may represent a more severe allele of the bicaudal locus. A chromosome walk was performed in this region and over 65 kb of overlapping clones isolated. The location of the P-element insertion responsible for the $vr22\sp{P3}$ mutation was localized in the walk, and cDNAs corresponding to two adjacent transcription units isolated. These clones have been analyzed and sequenced, and one shows at least a 65% identity with eukaryotic release factor. Recombination work with bicaudal mutant stocks has improved penetrance of the mutation to a level allowing genetic analysis. These stocks were used in genetic rescue experiments to determine the locations of bic and $vr22\sp{P3}.$ The results of genetic rescue and polymorphism mapping provide good evidence that $vr22\sp{P3}$ and bicaudal are in fact alleles at the same locus, and the gene codes for a protein with strong homology to release factor. The bic protein most likely plays a role in negative regulation of translation of downstream maternal genes such as nanos.
Failure to Burrow and Tunnel Reveals Roles forﾠjim lovellﾠin the Growth and Endoreplication of the Drosophila Larval Tracheae
(Public Library of Science, 2016) Zhou, Fanli; Qiang, Karen M.; Beckingham, Kathleen M.
The Drosophila protein Jim Lovell (Lov) is a putative transcription factor of the BTB/POZ (Bric- a-Brac/Tramtrack/Broad/ﾠPox virus andﾠZinc finger) domain class that is expressed in many elements of the developing larval nervous system. It has roles in innate behaviors such as larval locomotion and adult courtship. In performing tissue-specific knockdown with the Gal4-UAS system we identified a new behavioral phenotype forﾠlov: larvae failed to burrow into their food during their growth phase and then failed to tunnel into an agarose substratum during their wandering phase. We determined that these phenotypes originate in a previously unrecognized role forﾠlovﾠin the tracheae. By using tracheal-specific Gal4 lines, Lov immunolocalization and aﾠlovﾠenhancer trap line, we established thatﾠlovﾠis normally expressed in the tracheae from late in embryogenesis through larval life. Using an assay that monitors food burrowing, substrate tunneling and death we showed thatﾠlovﾠtracheal knockdown results in tracheal fluid-filling, producing hypoxia that activates the aberrant behaviors and inhibits development. We investigated the role ofﾠlovﾠin the tracheae that initiates this sequence of events. We discovered that whenﾠlovﾠlevels are reduced, the tracheal cells are smaller, more numerous and show lower levels of endopolyploidization. Together our findings indicate that Lov is necessary for tracheal endoreplicative growth and that its loss in this tissue causes loss of tracheal integrity resulting in chronic hypoxia and abnormal burrowing and tunneling behavior.
Genes expressed during oogenesis in Calliphora erythrocephala and Drosophila melanogaster
(1990) de Valoir, Tamsen Vivianne; Beckingham, Kathleen M.
We were interested in identifying genes in the dipteran flies Calliphora erythrocephala and Drosophila melanogaster with a role in oogenesis and early embryogenesis. A biochemical screen was used to complement the extensive genetic screens that have been performed to identify such genes in Drosophila. Radio-labelled cDNA probes were made using poly(A)$\sp+$ RNA preparations from staged Calliphora ovaries and embryos. These probes were used to isolate clones which were strongly expressed during oogenesis but not during embryogenesis. Four Calliphora genes which are absolutely "oogenesis-specific" in their expression pattern, as defined by our screening protocol, were identified. These are called A10B, B8I, C7F and GG7K. Three of these clones are expressed in the somatically derived follicle cells of the ovary and have been identified as being homologous to the Drosophila yolk protein 1 (A10B and B8I) and a vitelline membrane protein (GG7K). Interestingly, the yolk protein homologs are expressed in a specialized subset of follicle cells known as the border cells in Calliphora. The fourth gene, (C7F) is expressed in the nurse cells, the transcripts are translocated to the oocyte proper and maintained throughout the first four hours of embryogenesis. C7F is also expressed in late pupae and adult male flies. A number of Calliphora genes were identified which, although not oogenesis-specific, were more strongly expressed in the oocyte than the embryo. These were classified as "oogenesis-differentials". C7F and the Calliphora oogenesis-differential genes were used to screen Drosophila cDNA and genomic libraries for homologs. Some characterization of these Drosophila homologs is described here. ME31B, a maternally expressed Drosophila gene from the 31B region of the second chromosome was isolated by directly screening Drosophila libraries with Calliphora cDNA probes. ME31B is expressed throughout oogenesis and the transcript is maintained in the mature egg until four hours after fertilization. The ME31B transcript is evenly distributed throughout the oocyte and egg. A 1.5 kb cDNA for ME31B was completely sequenced. Comparison of the coding sequence with a protein data bank allowed us to show that ME31B is a member of a family of NTP-dependent helicases. The possible mutant phenotype of ME31B is discussed.
Isolation and characterization of human small nuclear ribonucleoproteins
(1984) Kinlaw, Claire S.; Berget, Susan M.; Matthews, Kathleen S.; Beckingham, Kathleen M.; Bennett, George; Kellems, Rodney E.
Human small nuclear ribonucleoproteins (snRNPs) containing U snRNAs have been fractionated into three RNA-specific populations, and snRNPs containing U1 and U2 snRNAs have been isolated~by biochemical methods. U1 and U2 snRNPs remained immunoprecipitable by Systemic Lupus Erythematosus antibodies during isolation, and purified snRNPs contained polypeptides of the same molecular weights as those defined by immunoprécipitation of crude extracts. The polypeptide components of U1 and U2 snRNPs have been compared by two-dimensional gel electrophoresis and immunobinding. U1 and U2 snRNPs contained both unique and common polypeptides. Purified U1 snRNPs contained U1 RNA and 1 snRNP polypeptides of molecular weights 67, (P67), 3, (P3), 23, (P23F), 21,5 (P22), 17,5 (P18), 2 at 12,3 (P12F and P12S), 1,2 (P1), 9,1 (P9), and 8,5 (P8). Purified U2 snRNPs contained U2 RNA and 9 snRNP polypeptides including the common polypeptides P23F, P22, P12F, P12S, P1, P9, and P8 as well as two U2 specific polypeptides of 23, (P23S) and 27, (P27) daltons. Five of the common polypeptides, including P23F, P22, P12F, P12S, and P9, were basic and may be important in snRNP assembly. One common polypeptide, P1 was acidic, and the remaining common polypeptide, P8, was neutral. Two of the U1 specific polypeptides, P3 and P18, as well as the two U2 specific polypeptides, P27 and P23S, were neutral. The third Ul specific polypeptide, P67, was slightly basic. Two of the common polypeptides, P23F and P22, were present in nonstoichiometric amounts and were recognized by a monoclonal anti-Sm antibody. Only one of them is present in rodent cells (Conner et al., 1982). Thus, in human cells, P23F and P22 may be two variants of the same polypeptide, with each snRNP complex containing either P23F or P22. P12F is also recognized by the same monoclonal antibody, suggesting it may be related to P23F and P22. Because Ul and U2 snRNPs contain common and unique polypeptides, it is suggested that they serve similar but distinct functions in. vivo. Ul has been implicated in hnRNA splicing (Lerner et al., 198; Rogers and Wall, 198). U2 may be involved in another aspect of hnRNA processing.
Isolation of oogenesis-specific genes transcribed in the germ-line of Calliphora erythrocephala and Drosophila melanogaster
(1988) Tucker, Mark Allen; Beckingham, Kathleen M.
Clones containing DNA uniquely transcribed during oogenesis were isolated from genomic DNA phage libraries for the two related Dipteran flies C. erythrocephala and D. melanogaster. Poly(A)$\sp{+}$ RNA from early or mid-stage ovarian follicles of C. erythrocephala was used to generate radiolabelled oogenesis-specific cDNA probes for screening the phage libraries. A cDNA probe made from mid-stage embryo poly(A)$\sp{+}$ RNA was used as the differential screening probe. Thus plaques hybridizing to the two oogenesis-specific probes but not the mid-stage embryo probe were selected as potentially containing oogenesis-specific genes. Two further rounds of screening were used to eliminate false positives and, after plaque purification, restriction digests of the remaining clones were screened by Southern blot hybridization to identify DNA fragments transcribed in an oogenesis-specific manner. To date, 22 oogenesis-specific clones have been isolated from C. erythrocephala and two from D. melanogaster by this method. In situ hybridization to sections of ovarian follicles has been used to determine the cell types within the follicles in which the various genes are expressed. Radiolabelled RNA probes for four of the C. erythrocephala oogenesis-specific clones and the two D. melanogaster clones have been hybridized to ovarian follicles. Two of the C. erythrocephala clones and both of the D. melanogaster clones have thus been shown to be transcribed in the germ-like cells of the follicles as opposed to the somatic follicle cells. Further studies have been concentrated on the two germ-line transcribed, oogenesis-specific clones isolated form the D. melanogaster clone library. In situ hybridization of the two D. melanogaster clones to the salivary gland polytene chromosomes has established that one clone is derived from chromosome region 31B/D (clone DA) and the second clone from region 98E/F (clone DM). Region 31B/D is rich in female sterile mutations and detailed genetic mapping of the DA clone and of these mutations was performed (using deficiency chromosomes) to determine which mutations might represent the DA gene. cDNA clones have been isolated for the transcribed region of clone DA and have been used to further define the transcription unit from this region of the D. melanogaster genome.
Regulation of cell number and cell movement in Dictyostelium discoideum
(2013-09-16) Phillips, Jonathan; Gomer, Richard H.; Braam, Janet; Beckingham, Kathleen M.; Farach-Carson, Cindy; Tabor, Jeffrey J.
Little is known about how the size of a tissue is established during development and maintained subsequently. Proliferation-inhibiting signals secreted by cells within a tissue that act specifically on cells within that tissue can provide negative feedback on cell number, thus regulating tissue size. A better understanding of tissue-specific inhibitors of proliferation could be useful for designing therapies for cancer and other diseases. However, few signals of this sort have been identified, and little is known about how these signals function. Two examples of such signals are the proteins AprA and CfaD, which are secreted by the social amoeba Dictyostelium discoideum and inhibit cell proliferation in a concentration-dependent manner. Cells lacking either AprA or CfaD proliferate rapidly, and adding recombinant AprA or CfaD to cells reduces proliferation. However, little is known about the signal transduction pathways downstream of AprA and CfaD. I identified three proteins that are required for the normal function of AprA and CfaD: the kinase QkgA, the putative transcription factor BzpN, and the putative kinase PakD. Cells lacking any one of these proteins proliferate rapidly, and adding AprA or CfaD to cells lacking these proteins does not cause reduced proliferation, indicating that these proteins are involved in AprA/CfaD signal transduction. I also found that, in addition to its proliferation-inhibiting activity, AprA also functions as an autocrine chemorepellant. Colonies of cells lacking AprA expand less rapidly than wild-type colonies, despite the fact that individual cells lacking AprA show a random motility like that of wild-type cells. Further, two independent assays demonstrate that cells show a biased movement away from a source of AprA. The chemorepellant activity of AprA requires CfaD, QkgA, and PakD, but not BzpN, indicating that AprA affects proliferation and chemorepulsion through distinct but overlapping pathways. These results suggest that AprA functions as a readout of local cell density, to which cells respond by slowing proliferation and chemotaxing to regions of lower cell density, where nutrients are more likely to be present. The study of human AprA, CfaD, QkgA, BzpN, and PakD orthologs may serve to guide therapeutic approaches that modulate chemorepulsive or antiproliferative processes.
Spectral triangulation: a 3D method for locating single-walled carbon nanotubes in vivo
(Royal Society of Chemistry, 2016) Lin, Ching-Wei; Bachilo, Sergei M.; Vu, Michael; Beckingham, Kathleen M.; Weisman, R.Bruce; Smalley-Curl Institute
Nanomaterials with luminescence in the short-wave infrared (SWIR) region are of special interest for biological research and medical diagnostics because of favorable tissue transparency and low autofluorescence backgrounds in that region. Single-walled carbon nanotubes (SWCNTs) show well-known sharp SWIR spectral signatures and therefore have potential for noninvasive detection and imaging of cancer tumours, when linked to selective targeting agents such as antibodies. However, such applications face the challenge of sensitively detecting and localizing the source of SWIR emission from inside tissues. A new method, called spectral triangulation, is presented for three dimensional (3D) localization using sparse optical measurements made at the specimen surface. Structurally unsorted SWCNT samples emitting over a range of wavelengths are excited inside tissue phantoms by an LED matrix. The resulting SWIR emission is sampled at points on the surface by a scanning fibre optic probe leading to an InGaAs spectrometer or a spectrally filtered InGaAs avalanche photodiode detector. Because of water absorption, attenuation of the SWCNT fluorescence in tissues is strongly wavelength-dependent. We therefore gauge the SWCNT–probe distance by analysing differential changes in the measured SWCNT emission spectra. SWCNT fluorescence can be clearly detected through at least 20 mm of tissue phantom, and the 3D locations of embedded SWCNT test samples are found with sub-millimeter accuracy at depths up to 10 mm. Our method can also distinguish and locate two embedded SWCNT sources at distinct positions.
Structural and Functional Studies on the Infectious Salmon Anemia Virus Nucleoprotein
(2013-10-25) Zheng, Wenjie; Tao, Yizhi Jane; Beckingham, Kathleen M.; Nikonowicz, Edward P.; Suh, Junghae; Wang, Qinghua
Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses which are able to cause infectious disease, and even worldwide pandemics. The genome of Orthomyxovirus consists of 6-8 negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes which perform multiple essential functions throughout the virus life cycle. To better understand the structural features of orthomyxovirus RNPs that allow them to be specifically packaged, we performed structural/functional studies of the nucleoprotein (NP), the major protein component of the RNPs, from the infectious salmon anemia virus (ISAV). The crystal structure of the ISAV-NP was determined to 2.7Å resolution. The ISAV-NP possesses a 112-aa N-terminal domain and a bi-lobular core structure that strongly resembles the structure of the influenza virus NP. Because the ISAV-NP forms homogenous dimers that are stable in solution, I was able to study the NP:RNA binding affinity as well as stoichiometry with fluorescence polarization, using recombinant proteins and synthetic oligos. Surprisingly, the RNA binding analysis revealed that each NP binds ~12 nts of RNA, shorter than the 24-28 nts originally estimated for the influenza A virus NP. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. These results suggest that free RNA exists in the orthomyxovirus RNPs, and selective RNP packaging is likely accomplished through direct RNA-RNA interactions.
Structure of androcam supports specialized interactions with myosin VI
(National Academy of Sciences, 2012) Joshi, Mehul K.; Moran, Sean; Beckingham, Kathleen M.; MacKenzie, Kevin R.
Androcam replaces calmodulin as a tissue-specific myosin VI light chain on the actin cones that mediate D. melanogaster spermatid individualization. We show that the androcam structure and its binding to the myosin VI structural (Insert 2) and regulatory (IQ) light chain sites are distinct from those of calmodulin and provide a basis for specialized myosin VI function. The androcam N lobe noncanonically binds a single Ca2þ and is locked in a “closed” conformation, causing androcam to contact the Insert 2 site with its C lobe only. Androcam replacing calmodulin at Insert 2 will increase myosin VI lever arm flexibility, which may favor the compact monomeric form of myosin VI that functions on the actin cones by facilitating the collapse of the C-terminal region onto the motor domain. The tethered androcam N lobe could stabilize the monomer through contacts with C-terminal portions of the motor or recruit other components to the actin cones. Androcam binds the IQ site at all calcium levels, constitutively mimicking a conformation adopted by calmodulin only at intermediate calcium levels. Thus, androcam replacing calmodulin at IQ will abolish a Ca2þ-regulated, calmodulin-mediated myosin VI structural change. We propose that the N lobe prevents androcam from interfering with other calmodulin- mediated Ca2þ signaling events. We discuss how gene duplication and mutations that selectively stabilize one of the many conformations available to calmodulin support the molecular evolution of structurally and functionally distinct calmodulin-like proteins.
Studies of the roles of the individual calcium binding sites of Drosophila calmodulin in conformational change and target interaction
(1995) Mukherjea, Poushali; Beckingham, Kathleen M.
The binding of calcium (Ca$\sp{2+}$) to the four EF-hands of the ubiquitous, eukaryotic protein, calmodulin (CaM) generates the biologically active conformation of the protein. The roles of the four individual Ca$\sp{2+}$-binding sites of Drosophila CaM had previously been examined using mutants in which the conserved glutamic acid in each site has been mutated to glutamine. In this thesis, the molecular mechanism of activation of target enzymes by CaM has been studied by the use of these mutants. Our principal finding is that most of these mutant proteins form complexes with target peptides which are conformationally distinct from the wild-type complex. However, mutation of the four sites differ significantly in the severity of their effects. This demonstrates the unique contributions of the individual Ca$\sp{2+}$-binding events to the conformational changes required for target interaction. Studies of the peptide pattern generated by different site-specific proteases have indicated that loss of Ca$\sp{2+}$-binding leads to conformational differences between the proteins. Multiple binding site mutants of CaM, including mutants in which sites 1+2, 1+3, 2+4, 3+4, or all four sites have been mutated have also been examined. Ca$\sp{2+}$-induced changes have been studied by UV difference spectroscopy and circular dichroism (CD) spectra in the near and far UV regions. Several lines of evidence indicate that the environment of the single tyrosine residue (Tyr-138 in the fourth Ca$\sp{2+}$-binding loop) is influenced by both N- and C-terminal mutations implying the existence of interdomain interaction. To examine N-terminal conformation changes on Ca$\sp{2+}$-binding to the C-terminus, the mutant F65W was created with a unique tryptophan (Trp) residue in the second Ca$\sp{2+}$-binding site. This Trp mutation has been combined with the Q mutants in the first, third and fourth Ca$\sp{2+}$-binding sites and Ca$\sp{2+}$-induced changes in the fluorescence of the Trp residue have been monitored. The activation pattern of one CaM-regulated enzyme, skeletal muscle myosin light chain kinase has indicated that for these mutant proteins the presence of the target protein could not compensate for lack of Ca$\sp{2+}$-binding and an active conformation of the protein could not be generated.
The Drosophila BTB Domain Protein Jim Lovell Has Roles in Multiple Larval and Adult Behaviors
(Public Library of Science, 2013) Bjorum, Sonia M.; Simonette, Rebecca A.; Alanis, Raul Jr.; Wang, Jennifer E.; Lewis, Benjamin M.; Trejo, Michael H.; Hanson, Keith A.; Beckingham, Kathleen M.
Innate behaviors have their origins in the specification of neural fates during development. Within Drosophila, BTB (Bric-abrac, Tramtrack, Broad) domain proteins such as Fruitless are known to play key roles in the neural differentiation underlying such responses. We previously identified a gene, which we have termed jim lovell (lov), encoding a BTB protein with a role in gravity responses. To understand more fully the behavioral roles of this gene we have investigated its function through several approaches. Transcript and protein expression patterns have been examined and behavioral phenotypes of new lov mutations have been characterized. Lov is a nuclear protein, suggesting a role as a transcriptional regulator, as for other BTB proteins. In late embryogenesis, Lov is expressed in many CNS and PNS neurons. An examination of the PNS expression indicates that lov functions in the late specification of several classes of sensory neurons. In particular, only two of the five abdominal lateral chordotonal neurons express Lov, predicting functional variation within this highly similar group. Surprisingly, Lov is also expressed very early in embryogenesis in ways that suggests roles in morphogenetic movements, amnioserosa function and head neurogenesis. The phenotypes of two new lov mutations that delete adjacent non-coding DNA regions are strikingly different suggesting removal of different regulatory elements. In lov47, Lov expression is lost in many embryonic neurons including the two lateral chordotonal neurons. lov47 mutant larvae show feeding and locomotor defects including spontaneous backward movement. Adult lov47 males perform aberrant courtship behavior distinguished by courtship displays that are not directed at the female. lov47 adults also show more defective negative gravitaxis than the previously isolated lov91Y mutant. In contrast, lov66 produces largely normal behavior but severe female sterility associated with ectopic lov expression in the ovary. We propose a negative regulatory role for the DNA deleted in lov66.

Browsing by Author "Beckingham, Kathleen M."

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