Cell Biology & Molecular Genetics

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Studies on the Root Growth of Willow Cuttings at Controlled Temperatures
(1931) Baker, Henry H.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md)
Different Mechanisms of ras Proto-Oncogene Overexpression Detected by a Sib-Selection Tumorigenesis Assay
(1990) Bachurski, Cindy J.; Hetrick, Frank M.; Cell Biology and Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md)
Human insulinoma and renal cell carcinoma DNAs were analysed by a sib-selection tumorigenesis assay to detect the presence of dominant acting oncogenes. Although no novel oncogenes were detected in the five tumor DNAs tested, the increased sensitivity of the tumorigenesis assay allowed detection of overexpressed ras proto-oncogenes activated during transfection. Tumorigenic overexpression of ras proteins occured by two different mechanisms: gene amplification, and increased translation efficiency of a fusion protein. Thirty to fifty fold amplification of a human N-ras gene was detected in primary and secondary mouse tumors. All tumors containing amplified copies of N-ras overexpressed p21 and the cloned gene had no coding sequence mutations. Since DNA from the original human tumor and several metastases did not show N-ras gene amplification, it was concluded that amplification had occured during the primary tumorigenesis assay. In another series of tumors co-integration of the mouse c-H -ras gene during the secondary transfection resulted in tumorigenic overexpression of ras protein without elevation of mRNA levels. Three c-H-ras cDNA clones from secondary tumor RNA contained no coding sequence mutations, but were divergent at the 3'end. Polymerase chain reaction amplification of RNA showed novel alternative splicing at intron E of mouse c-H-ras mRNA in both tumors and untransfected cells. An upstream ATG was identified that potentially initiates translation of an open reading frame (ORF) overlapping the H-ras p21 translation initiation site. A single base deletion within exon -1 of the cDNA clones placed this upstream ATG in frame with the ras coding sequence, creating a potential fusion protein. Translation of this mRNA in rabbit reticulocyte extracts and Xenopus oocytes showed exclusive production of a p23 ras fusion protein. When the upstream ATG was deleted, only p21 ras was translated in both systems. Based on these results it is proposed that in vivo recognition of the upstream ATG and translation of the ORF overlapping the p21 start site might serve to modulate the translation of p21. The single base deletion and resultant ras fusion protein may constitute a novel mechanism of ras overexpression by circumventing this translational regulation .
Production and Characterization of New Alleles of SCAR in Drosophila melanogaster
(2003-12-04) Valentine, Summer Danyse; de Cuevas, Maragaret; Mount, Stephen; Baehrecke, Eric; Cell Biology & Molecular Genetics
Germline cysts are clusters of germ cells that arise by division from a single cell and are connected to one another by stable intercellular bridges. Cysts are a highly conserved stage of pre-meiotic germ cell development, but the molecular mechanisms that control their formation are highly unknown. Developing cysts in Drosophila contain a cytoplasmic organelle called a fusome, which plays a critical role in cyst formation. The SCAR gene, which encodes an actin regulatory protein, was identified in a screen for mutations that disrupt fusomes and cyst formation. It was not clear, however, if the mutation l(2)k03107 disrupts only SCAR. To address this question, new alleles were created by excising the P-element in l(2)k03107. These alleles were then characterized molecularly and phenotypically. The results show that two alleles have deletions in the SCAR gene. Further testing must be done to determine the role of SCAR in cyst formation.
Effect of Transcriptional Parameters on the Folding of the Tetrahymena Group I Intron
(2004-01-29) Koduvayur, Sujatha P; Woodson, Sarah A; Julin, Douglas; Molecular and Cell Biology
Differential elongation rates and pausing patters of polymerases are known to affect co-transcriptional folding of RNAs. The mechanism of coupling is not well studied. This study evaluates whether these factors contribute to the 20-50 fold splicing enhancement seen in vivo for the Tetrahymena group I intron. The splicing rates of T7 and Escherichia coli (E. coli) RNA polymerase (RNAP) transcripts were compared in vitro and in E. coli. T7 RNAP is a rapid and highly processive polymerase, while E. coli RNAP elongates transcripts more slowly. In a collaborative study with Scott Jackson, the splicing of transcripts of eukaryotic polymerase I and II (pol I and pol II) were compared in Saccharomyces cerevisiae. As the stability of nascent structures can be altered by changing the sequence of transcribed RNA, I have also studied the effect of rRNA exons on the folding of this RNA by comparing the splicing rates of pre-RNAs with E. coli and Tetrahymena thermophila domain IV rRNA exons in vitro and E. coli. In yeast, S. cerevisiae and T. thermophila rRNA, and GFP mRNA exon sequences were compared. My E. coli and in vitro comparative study reveals that co-transcriptional folding is the reason for the rate enhancement seen in vivo for this RNA. For E. coli RNAP transcripts this is effected by the template-dependant, site-specific pausing of the polymerase along the template. For the pre-RNA with Tetrahymena rRNA exons this is effected by the low stability of nascent structures that enable rapid rearrangement of non-native structures into native ones. In yeast, we find that both the polymerase and the RNA processing events affect folding of this intron. Pol I transcripts splice 10-fold better than their pol II counterparts and mRNA processing events retard splicing of short pol II transcripts by 10-fold. Moreover, in yeast, only mutations that increase the fraction of misfolded intermediates are rescued but not those that destabilize the native structure. This folding facilitation is however not dependant on the presence of longer rRNA exons in yeast as it is in E. coli. This might be indicative of a different folding facilitatory mechanism in yeast from that seen in E. coli.
NOVEL EXCHANGEABLE EFFECTOR LOCI ASSOCIATED WITH THE PSEUDOMONAS SYRINGAE HRP PATHOGENICITY ISLAND: EVIDENCE FOR INTEGRON-LIKE ASSEMBLY FROM TRANSPOSED GENE CASSETTES
(2004-02-10) Charity, James Carl; Hutcheson, Steven W; Delwiche, Charles F; Stewart, Richard C; Cell Biology & Molecular Genetics
Pseudomonas syringae strains use a type III secretion system (TTSS) to translocate effector proteins that assist in the parasitism of host plant cells. Some genes encoding effector proteins are clustered in the exchangeable effector locus (EEL) associated with the hrp pathogenicity island. A PCR-based screen was developed to amplify the EEL from P. syringae strains. Of the 86 strains screened, the EEL was successfully amplified from 29 predominately North American P. syringae pv. syringae strains using hrpK and queA-derived primers. Among the amplified EEL, ten distinct types of EEL were identified that could be classified into six families distinguishable by genetic composition. These results are consistent with the hypotheses that the EEL is a source of disparate effectors involved with the pathogenicity of P. syringae strains and that the EEL evolved independently of the central conserved region of the hrp pathogenicity island, possibly by integron-like assembly of transposed gene cassettes.
Multiple Antibiotic Resistances of Enterococci from the Poultry Production Environment and Characterization of the Macrolide-Lincosamide-Streptogramin Resistance Phenotypes of Enterococcus faecium
(2004-04-26) Hayes, Joshua Richard; Joseph, Sam W.; Cell Biology & Molecular Genetics
Enterococcus spp. were collected from poultry production and processing environments across a region of the eastern seaboard of the United States. Using a microtiter plate adaptation of traditional biochemical assays, 532 isolates were identified to the species level. E. faecalis was observed to be the predominant species recovered (64%), followed by E. faecium (24%), E. hirae (6%), E. gallinarum (5%), and less than 1% of E. avium, E. casseliflavus, E. durans, and indeterminate species. All isolates were tested for susceptibility to 28 antimicrobials using broth microdilution. Antimicrobial resistance was observed among the isolates with E. faecalis more resistant to lincosamide, macrolide, and tetracycline antimicrobials and E. faecium more resistant to fluoroquinolone and penicillin antimicrobials. Resistance to multiple antimicrobials was observed among all species, with a proportionately greater diversity of resistance phenotypes among isolates of E. faecium. The prevalence of resistance to an important antimicrobial used in human medicine, the streptogramin quinupristin-dalfopristin, was demonstrated among 63% of E. faecium isolates. These observations led to the investigation of the molecular determinants among those isolates of E. faecium that contributed to resistance to the group of antibiotics known as the macrolide-lincosamide-streptogramin (MLS) superfamily. Ribotype analysis demonstrated that four ribotypes constituted 65% of the observed population, but displayed diverse antibiograms, suggestive of the acquisition of multiple resistance elements to other antimicrobials. This was consistent with the absence of geographic clustering of MLS phenotypes or ribotypes. Colony PCR screening for the streptogramin resistance determinants erm(A), erm(B), mef(A), lnu(B), msr(C), vgb(A), vat(D), and vat(E) was performed. Resistance to streptogramin antimicrobials was largely unaccounted for by PCR screening for specific resistance determinants. Erythromycin methyltransferase determinants erm(A) and erm(B) were observed among 7.5% and 5% of resistant isolates whereas the efflux gene msr(C), the streptogramin B hydrolase vgb(A), or the streptogramin A acetyltransferase genes vat(D) and vat(E) were not detected among resistant E. faecium. Mef(A) was detected in only 4% of macrolide-resistant isolates whereas the screening for lnu(B) was not successful. These results indicate that streptogramin resistance is widespread among E. faecium from the poultry production environment but the mechanisms of resistance within this population remain largely uncharacterized.
THE ORIGIN OF THE DINOFLAGELLATE PLASTID
(2004-04-29) Bachvaroff, Tsvetan Radoslavov; Delwiche, Charles F; Cell Biology & Molecular Genetics
The peridinin pigmented dinoflagellate chloroplasts are the result of a secondary endosymbiotic event between a photosynthetic eukaryote and a dinoflagellate. Dinoflagellate chloroplast and nuclear evolution were independent before this endosymbiotic event. To reconstruct the evolution of the dinoflagellate chloroplast, phylogenies were constructed with a chloroplast gene psbB. The gene phylogeny should reflect the evolution of the chloroplast and indicate the plastid donor lineage. Gene sequences derived from the dinoflagellate chloroplast were extremely divergent but suggested that the plastid donor could have been a haptophyte. In an attempt to find better genes for analysis and to further understand gene transfer about 4900 randomly selected expressed genes were sequenced from two dinoflagellates, Lingulodinium polyedra and Amphidinium carterae. From these genes, thirty typically plastid-encoded genes were found, including eight otherwise known only from plastid genomes. Based on poly-A tails, gene families, and leader sequences these genes appear to be nuclear-encoded in dinoflagellates. This result suggests that dinoflagellate chloroplasts may have the smallest protein-coding potential yet known. These genes and the partially sequenced chloroplast genome of a haptophyte were used in a phylogenetic analysis. There is strong conflict between genes encoded in the chloroplast and those in the nucleus. The chloroplast genes suggest relationship between haptophyte and dinoflagellate plastids, while the nuclear-encoded genes suggest a relationship with heterokonts. Chromophyte plastid monophyly is supported by these data but the single origin of the chromophyte plastid from red algae does not mean that the host lineages are monophyletic. These results are consistent with at least two different scenarios: either dinoflagellates and haptophytes independently acquired a plastid from the heterokonts, or dinoflagellates acquired their plastids from haptophtyes, who in turn acquired their plastids from heterokonts. The evolutionary rate of the remaining plastid-encoded genes was compared with formerly plastid-encoded genes. These relative rate tests revealed strong incongruence between minicircle genes, formerly plastid-encoded genes and genes that were likely to have been acquired from the nucleus of the plastid donor lineage.
LARSON directly represses AGAMOUS during early flower organogenesis in Arabidopsis thaliana
(2004-04-30) Bao, Xiaozhong; Liu, Zhongchi; Cell Biology & Molecular Genetics
How cells in a multicellular organism assume their developmental fates and form distinct patterns is a fundamental biological question. To address this question, I studied genetic and molecular regulation of Arabidopsis flower organ formation and identity determination. Specifically, how the expression of floral meristem and floral organ identity gene AGAMOUS (AG) was regionalized during flower organogenesis. A novel AG repressor LARSON (LSN) was previously isolated in a genetic screen. lsn loss-of-function mutations caused precocious expression of AG in the inflorescence meristem and ectopic expression of AG in sepal primordia, resulting in partial homeotic transfomation of late inflorescences into floral meristems and strong homeotic transformation of first whorl sepals into carpels. LSN encoded a homeodomain protein that directly bond to AG cis-regulatory elements in vitro. The cis-regulatory elements were conserved in 17 Brassicaceae species. LSN was expressed in a subset of cells located in the peripheral zones of inflorescence and floral meristems. LSN expression was significantly reduced in the sepal and petal primordia in wild-type flowers, indicating that repression of AG in the sepals and petals was independent of LSN transcription. LSN might establish epigenetic AG repression in the ancestral cells in the peripheral zone to specify the identities of descendant cell types in the floral organs. Therefore, floral organ identities were not only dependent upon gene expression in the organs, but were also dependent upon the histories of the cell development. Genetic and molecular analyses showed that LSN acted upstream of a putative repression complex, which, I proposed, was involved in the maintenance of AG repression in flowers. The putative repressive complex consistes of APETALA1 (AP1), LUNIG (LUG) and SEUSS (SEU) known to encode flower specific repressors of AG. Mutations in these three genes enhanced the lsn phenotypes. However, none of their proteins interacted with LSN in yeast. Instead, AP1, SEU, and LUG might form a protein complex. Genetic and molecular analyses suggested that the AG-repressive functions of the putative complex depended upon LSN activity in the peripheral zone of floral meristem. The AG-repressive function of LSN in the inflorescence meristem was independent of AP1/SEU/LUG putative complex.
Identification of autophagic cell death and implications for therapy
(2004-05-04) Alva, Ajjai Shivaram; Baehrecke, Eric H; Cell Biology & Molecular Genetics
Autophagy is an evolutionarily conserved mechanism of bulk protein and organelle degradation that requires the ATG class of genes. Although autophagy has been frequently observed in dying cells in several species, a causative role for autophagy in cell death has not been demonstrated. We show that inhibition of caspase-8 in mouse L929 fibroblast cells causes cell death with the morphology of autophagy. Autophagic cell death in L929 cells is dependent on ATG genes and involves the receptor interacting protein (RIP) and the activation of the MAP kinase kinase 7(MKK7) - Jun N-terminal kinase (JNK) - cJUN pathway. We also show that autophagy occurs in many primary human tumors including cancer of the breast, lung and pancreas. Our findings validate autophagic cell death and might explain the role of autophagy in development, viral infections, neurodegenerative diseases and cancer.
Developmental Programmed Cell Death In The Midline Glia Cells Of Drosophila Embryo
(2004-05-14) Jaligam, Vanaja; Baehrecke, Dr. Eric H; Cell Biology & Molecular Genetics
Apoptosis is conserved in worms, flies and mammals. My goal was to identify new Drosophila midline glia cell death genes. Caspase substrate Nuclear Lamin and the midline glia specific reporter slit-lacZ were in Drosophila embryonic development cell death studies. Midline glia cell death is prevented in H99 mutants that delete hid, reaper and grim, and in p35 flies that over-express the pan-caspase inhibtor. Homozygous deficiency Df(3R)E79 flies possess a defect in midline glia cell death. Df(3R)E79 midline glia appear to be phagocytosed but do not undergo Lamin degradation suggesting that a caspase regulator resides in this deleted region of the genome. Df(3R)E79 removes 112 genes, including several interesting candidates including mus309, a protein with similarity to a RING finger protease reported to regulate apoptosis in humans. Future studies will determine the nature of the cell death regulator in Df(3R)E79 and how this gene functions in apoptosis.
Human Immunodeficiency Virus Nucleocapsid Protein: Analysis of the mechanism of strand exchange and the role of the zinc fingers in nucleic acid chaperone activity.
(2004-06-15) Heath, Megan Joy; DeStefano, Jeffrey J; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The human immunodeficiency virus genome is coated by the nucleocapsid protein (NC). NC is a 55 amino acid highly basic protein. It has two zinc fingers that differ by five amino acids. NC contains nucleic acid chaperone activity that aids in the formation of highly stable nucleic acid structures by destabilizing and preventing the formation of weaker structures. This activity is important for genome dimerization and maturation, tRNA:primer binding site annealing, and many steps in reverse transcription. Annealing experiments were performed with four different RNA structures and complementary DNAs. NC enhanced annealing of all structures showing that NC enhances both unwinding of nucleic acid structure and hybridization of unstructured sequences. NC mutant proteins were used in annealing assays. 1.1 NC had two copies of the first zinc finger, 2.2 NC had two copies of the second zinc finger, and 2.1 NC had both zinc fingers with their positions switched. Experiments showed that all mutants could enhance the annealing of weakly structured nucleic acids but only 1.1 NC and 2.1 NC enhanced annealing of strongly structured nucleic acids. Results suggest that finger one is important for nucleic acid unwinding while finger two plays an accessory role in annealing. The mechanism of strand exchange, another important aspect of NC chaperone activity, was also investigated. Experiments were performed using RNA:DNA hybrids with either the DNA or RNA radioactively labeled. Hybrids were incubated with different types of RNA acceptor molecules to which the DNA could transfer. The transfer of the DNA or the displacement of the original donor RNA was monitored. Experiments showed that optimal enhancement of strand exchange by NC occurred with acceptors that had more than 22 nucleotides that could anneal to the single stranded region of the DNA. Also, experiments with acceptors that had point mutations showed that the region of the acceptor that binds to the single stranded region of the DNA should be completely complementary for optimal NC stimulation. These results indicate the annealing of the acceptor and DNA outside the donor:DNA hybrid region can be an important initiation step for NC enhanced strand exchange.
Cell Changes During Autophagic Cell Death of Larval Salivary Glands During Drosophila melanogaster Metamorphosis
(2004-06-15) Martin, Damali; Baehrecke, Eric H; Mount, Stephen; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Autophagic cell death has been implicated in human diseases such as neurodegeneration and cancer. In order to obtain a clearer picture of the mechanisms that regulate autophagic cell death, I have been studying the cell changes that occur during this process using Drosophila melanogaster larval salivary glands as a model. During Drosophila metamorphosis, larval salivary gland histolysis is triggered by a pulse of the steroid hormone 20-hydroxyecdysone (ecdysone) that occurs twelve hours after puparium formation. Ecdysone directly causes the activation of the early genes E93, BR-C and E74A, with ßFTZ-F1 serving as a competence factor for their induction. In turn, these transcription regulators activate a set of late genes rpr, hid, dronc, drice and ark that have a more direct role in the cellular changes that occur during salivary gland cell death. While dying salivary glands use typical apoptotic machinery, the morphology of their cells resembles autophagic cell death. The morphological changes seen during salivary gland autophagic cell death are a result of active caspase cleavage of structural proteins such as nuclear Lamin, alpha-Tubulin and alpha-Spectrin. However, some changes that occur during cell death are caspase-independent, indicating that other proteins are important for histolysis of these glands. Proteome analyses identified 5,313 proteins that are present before and during salivary gland cell death. These proteins are involved in numerous processes such as autophagy, the ubiquitin- mediated proteolysis, cell organization, cell growth regulation and cell cycle regulation. Further analyses of these proteins may illustrate their importance during salivary gland autophagic cell death. Forward genetic screening was also used to identify mutations in genes that affect salivary gland cell death. One such gene ctp encodes a light chain of a dynein motor, and animals with mutations in ctp have salivary glands that fail to die.
A phylogenetic and biogeographic analysis of Sanguisorbeae (Rosaceae), with emphasis on the Pleistocene radiation of the high Andean genus Polylepis.
(2004-06-16) Kerr, Malin Sofia; Delwiche, Charles F; Reveal, James L; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
A phylogenetic and biogeographic analysis of the tribe Sanguisorbeae (Rosaceae) was conducted with emphasis on the radiation of the Andean tree Polylepis. Phylogenetic analyses of coding and non-coding nuclear markers reveal a complex evolutionary history of the tribe including ancient and recent allopolyploid hybridization. Sanguisorba sensu lato is shown to be paraphyletic and split between the allopolyploid hybrid Sanguisorba and the non-hybrid Poterium and Poteridium. A monophyletic origin of the southern hemispheric subtribe Sanguisorbinae is supported, and this clade is given a phylogentic taxon name (Verruchaena). Dating analyses using the penalized likelihood method suggest that this taxon originated in the late Miocene. A biogeographic hypothesis is presented in which Verruchaena originated in the New World with subsequent transoceanic dispersals to southern Africa and Australasia. The paramo genus Polylepis most likely arose from hybridization between two Andean ancestors supporting a "vertical" rather than "horizontal" origin of this taxon.
Influence of Viral Nucleocapsid Protein and Genomic RNA Structural Intricacies on the Mechanism of HIV Recombination
(2004-06-30) Derebail, Suchitra S; DeStefano, Jeffrey J; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Internal strand transfers involve template switching during retroviral replication, within internal regions of the viral genome. Such transfers are a major source of genetic variability in retroviruses like HIV-1. An in vitro strand transfer assay that mimicked recombinational events occurring during reverse transcription in HIV-1 was used to assess the role of nucleocapsid protein (NC) and structural intricacies of genomic RNA in strand transfer. Transfers in highly structured templates from the U3 3' LTR, gag-pol frameshift region, and Rev response element (RRE) were strongly enhanced by NC. In contrast, weakly structured templates from the env and pol-vif regions transferred well without NC and showed lower enhancement. Assays conducted using NC zinc finger mutants supported a differential role for the two fingers in strand transfer with finger one (N-terminal) being more important on highly structured RNAs. The lack of strong polymerase pause sites in the weakly structured templates from the env and pol-vif regions demonstrated that non-pause driven mechanisms could also promote transfer. Mapping assays were conducted on high and low structured templates from the gag-pol and the env regions respectively (called GagPol and Env templates), to locate the point(s) of transfer in each case. The majority of transfers were located near a major pause site in the gag-pol region; in contrast, in the env region, most transfers were located towards the end of the homologous region between donor and acceptor templates. Various truncated/mutant GagPol acceptor templates were analyzed with wild type GagPol donor templates in strand transfer assays. Results indicated that destabilized acceptor templates enhance the level of transfer and cause a highly efficient 'chasing' of the pause site into transfer products. The outcome of these experiments also suggested that strand transfer in the GagPol templates is via a pause induced 'donor dissociation' method. In the Env template the mechanism of transfer was proposed to occur by a pause independent, 'acceptor invasion' method. This knowledge about the interplay of RNA structure and NC protein on recombination could help in designing antiviral vaccines and drug inhibitors.
Identification and Secretion of Effectors From the Pseudomonas syringae Type III Secretion System
(2004-08-06) Losada Bohannon, Liliana Cristina; Hutcheson, Steven W.; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The hrp pathogenicity island (PAI) of Pseudomonas syringae encodes a type III secretion system (TTSS) that translocates virulence proteins, called effectors, into plant cells. The whole array of effectors of different P. syringae stains and their activities inside the host are not known. Furthermore, the manner in which effectors are selected for secretion, and how the process is regulated are not clear in P. syringae. This study identified a novel effector from P. syringae pv. syringae Psy61 using a genomic screen. The effector was a 375 aa protein of 40.5 kDa that was designated HopPsyL. A hopPsyL::kan mutant of Psy61 exhibited strongly reduced virulence in Phaseolus vulgaris cv. Kentucky Wonder, but did not appear to act as a defense response suppressor. The ectopically expressed gene reduced the virulence of P. syringae DC3000 transformants in Arabidopsis thaliana Col-0. HopPsyL appears to be a novel TTSS-dependent effector that functions as a host-species-specific virulence factor in Psy61. In addition, this study reports that TTSS-dependent effectors are subject to the proteolytic degradation by Lon that appears to be rate-limiting to secretion. TTSS-dependent secretion of these effectors could be detected from the Lon mutants. This study found that a primary role for chaperones in P. syringae appeared to be protection of effectors from Lon-mediated degradation prior to secretion. Distinct Lon-targeting and chaperone-binding domains were identified in at least one effector. The results imply that Lon is involved at two distinct levels in the regulation of the P. syringae TTSS: regulation of assembly of the secretion apparatus and modulation of effector secretion. Interestingly, degradation of P. syringae effectors was also retarded by the presence or expression of the P. syringae TTSS. The protection from Lon-mediated degradation was not due to the assembly of the TTSS. Rather, the results suggest the existence of a stabilizing factor harbored within the hrp cluster. This study proposed that this factor functions as a general chaperone for type III secretion in P. syringae.
INVESTIGATING A PUTATIVE NON STRUCTURAL PROTEIN OF THE BIRNAVIRUS DROSOPHILA-X VIRUS
(2004-08-12) Savage, Jason Eric; Wu, Louisa; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Drosophila-X Virus (DXV) is the prototype virus of the Entomobirnavirus genera in the family Birnaviridae; its genome consists of two double-stranded RNA segments. DXV has two open reading frames on segment A, one encoding a polyprotein and the other capable of encoding a putative 27-kDa non structural protein (DXV-NS). This project investigated the existence of DXV-NS since this putative protein is unique to Birnaviridae. Research into the nature of DXV-NS was furthered by the development of a number of tools: recombinant baculovirus and a stable Drosophila cell line expressing DXV-NS; an antibody against NS; a reporter construct to test a potential -1 frameshift signal. Though in the course of this research much was learned about DXV-NS, the greater question as to whether this open reading frame is expressed by DXV or is merely an artifact remains elusive.
THE EFFECT OF THE DELETION OF OPA 5 ON THE ABILITY OF N. GONORRHOEAE STRAIN MS11 TO TRANSCYTOSE A POLARIZED T84 EPITHELIAL CELL MONOLAYER
(2004-08-18) Davis, Meredith; Stein, Daniel C; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Neisseria gonorrhoeae can express 11 different opacity proteins (Opa). Plasmids were constructed that allowed for the generation of strains containing deletions in each Opa-encoding-gene (amplified by PCR and cloned into pUC19). The Opa-encoding region was replaced with a spectinomycin resistance cassette and transformed into N. gonorrhoeae. The biological properties of one of these transformants (MS11 Opa5 delta s) was tested in a transcytosis assay. Wildtype MS11 was able to efficiently cross a polarized epithelial monolayer of T84 cells within 6 hours and occurred in both the apical to basolateral and the basolateral to apical directions. The number of cells that crossed the monolayer depended upon the dose of the inoculum. Transcytosis of MS11 Opa5 delta s occurred in the apical to basolateral direction, but was delayed. The data indicate that gonococci use multiple mechanisms of transcytosis, and the Opa 5 protein is important for rapid apical to basolateral transcytosis.
COMPLEX POLYSACCHARIDE DEGRADATION BY MICROBULBIFER DEGRADANS STRAIN 2-40: STUDIES OF THE CHITINOLYTIC SYSTEM AND CARBOHYDRASE ARCHITECTURE
(2004-10-18) Howard, Michael Barry; Hutcheson, Steven W; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Microbulbifer degradans strain 2-40 is a Gram negative marine bacterium that is able to depolymerize and metabolize a wide variety of complex polysaccharides, including chitin. Chitin is the second most abundant biopolymer in nature and is a widely available nutrient in many environments. The chitinolytic system of M. degradans consists of three chitin depolymerases (ChiA, ChiB, and ChiC), three N-acetylglucosaminidases (HexA, HexB, and HexC), a chitodextrinase (CdxA), a chitin-binding protein (CbpA), and a suite of enzymes involved in the transport and metabolism of GlcNAc and GlcNAc2. ChiB, the largest eubacterial chitinase described, includes two complete Glycoside Hydrolase family 18 catalytic domains; one is exolytic and the other is endolytic. These catalytic domains share similar reaction optima, temperature, pH, and metal ion sensitivities, and are shown to function synergistically in the depolymerization of crystalline chitin. ChiA, ChiB, and 44 other M. degradans carbohydrases contain polyserine linker regions. These linkers are composed predominantly of serine (79%), have an average length of 39 residues, and are encoded by all six serine codons without any obvious bias or pattern. Polyserine domains are found only between functional groups (e.g., catalytic, binding, or anchoring domains) and are only found in putative secreted, carbohydrate depolymerases. The sequence of the M. degradans genome was determined during the course of this work and permitted the first genomic analysis of a chitinolytic organism to be performed. Further, these experiments and subsequent analyses have lead to a greater understanding of how M. degradans is able to metabolize such a diverse collection of biopolymers.
Pseudomonas syringae pathogenesis: Regulation of type III secretion and identification of a secreted effector
(2004-11-01) Bretz, James Robert; Hutcheson, Steven W; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The Pseudomonas syringae hrp pathogenicity island encodes a type III secretion system (TTSS) which is used to translocate effector proteins into host cells to facilitate pathogenesis. Expression of the hrp TTSS is controlled by the alternative sigma factor, HrpL, whose expression in turn is positively controlled by two truncated enhancer binding proteins, HrpR and HrpS. Although a number of environmental conditions are known to modulate hrp TTSS expression, such as stringent conditions and pathogenesis, the mechanism by which the activities of these transcriptional factors are modulated had not been established. Both HrpR and HrpS were shown to be required for full expression of hrpL. hrpRS were shown to be expressed as an operon and a promoter was identified 5' to hrpR. The hrpRS promoter and coding sequence were found to be conserved among P. syringae strains. The hrpRS operon was constitutively expressed under conditions in which the hrpL promoter was inactive, indicating the involvement of a negative regulatory factor. Transposome (Tnp) mutagenesis was used to identify Lon protease as a negative regulator of hrpL expression, suggesting an effect on HrpR and/or HrpS. HrpR was observed to be unstable in wild-type P. syringae strains grown in non-inductive media. The apparent half-life of HrpR increased more than 10-fold in the P. syringae lon::Tnp mutants or upon transfer to inductive (stringent) conditions. As a result, an interaction between factors involved in the stringent response, Lon protease, and hrp regulation was also investigated. The regulatory system described above was used to develop a HrpL-dependent promoter trap to identify effectors secreted by the Hrp TTSS. One of these potential effectors, HopPtoD2, was shown to encode a protein tyrosine phosphatase that was translocated into Arabidopsis thaliana cells via the hrp-encoded TTSS. A hopPtoD2 mutant exhibited strongly reduced virulence in Arabidopsis thaliana. Expression of hopPtoD2 delayed the development of several defense-associated responses in infected plants. These results indicate that HopPtoD2 is a translocated effector with protein tyrosine phosphatase activity that modulates plant defense responses.
FUNCTIONAL ANALYSES OF ARABIDOPSIS RIBONUCLEOTIDE REDUCTASE SMALL SUBUNIT GENE FAMILY
(2004-11-23) Wang, Chunxin; Liu, Zhongchi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
A fundamental question in plant development is how cell division events are coordinately regulated in the context of growth and development. To address this question, I chose to study a pleiotropic mutant, tso2, which exhibited developmental defects including callus-like floral organs and fasciated shoot meristem. I isolated the TSO2 gene and showed it encodes the small subunit of ribonucleotide reductase (RNR). RNR catalyzes a rate-limiting step in the production of deoxyribonucleotides needed for DNA synthesis. Subsequently, I showed that tso2 mutants reduce the dNTP levels. To understand why tso2 mutants, defective in this essential process, are still viable, I identified two homologs of TSO2, R2A and R2B in Arabidopsis. Mutations in R2A and R2B were isolated using a reverse genetic approach. While r2a, r2b single mutants or r2a r2b double mutants fail to display any visible phenotype, r2a and r2b mutations can enhance tso2 genetically, resulting in seedling lethality and embryonic lethality in tso2 r2a and tso2 r2b, respectively. Overexpression of either R2A or R2B can rescue the tso2 mutants, suggesting that the three R2 genes are functionally redundant. In addition to the developmental defects, tso2 mutants were more sensitive to HU (hydroxyurea) and UV-C, indicating that TSO2 plays a major role in DNA repair. In tso2 r2a double mutant seedlings, increased DNA damage accumulates, leading to massive programmed cell death. In addition, release of transcriptional gene silencing was observed in tso2 r2a double mutants, suggesting that DNA damage can lead to epigenetic instability. To further identify regulators of RNR and novel components of plant DNA damage response pathways, 18 independent tso2 suppressors were isolated in a genetic screen. These suppressors fall into at least four different complementation groups. My genetic and molecular characterization of TSO2 is the first functional study of RNR in plants. My results indicated that plants could initiate programmed cell death in response to DNA damage. The developmental defects in tso2 mutants are caused by epigenetic instability and aberrant cell division. The isolation of potential tso2 suppressors will be crucial to the understanding of plant DNA damage response pathway, an understudied area in plant biology.

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