Medicine and Health Sciences Commons^™

Marek's Disease Virus (Mdv) Encodes An Interleukin-8 Homolog (Vil-8): Characterization Of The Vil-8 Protein And A Vil-8 Deletion Mutant Mdv, Mark S. Parcells, Su-Fang Lin, Robert L. Dienglewicz, Vladimir Majerciak, Dan R. Robinson, Hua-Chien Chen, Zining Wu, George R. Dubyak, Peter Brunovskis, Henry D. Hunt

Dartmouth Scholarship

Chemokines induce chemotaxis, cell migration, and inflammatory responses. We report the identification of an interleukin-8 (IL-8) homolog, termed vIL-8, encoded within the genome of Marek's disease virus (MDV). The 134-amino-acid vIL-8 shares closest homology to mammalian and avian IL-8, molecules representing the prototype CXC chemokine. The gene for vIL-8 consists of three exons which map to the BamHI-L fragment within the repeats flanking the unique long region of the MDV genome. A 0.7-kb transcript encoding vIL-8 was detected in an n-butyrate-treated, MDV-transformed T-lymphoblastoid cell line, MSB-1. This induction is essentially abolished by cycloheximide and herpesvirus DNA polymerase inhibitor phosphonoacetate, indicating …

Sars, A Sara Homolog Repressible By Agr, Is An Activator Of Protein A Synthesis In Staphylococcus Aureus, Ambrose L. Cheung, Katherine Schmidt, Brian Bateman, Adhar C. Manna

Dartmouth Scholarship

The expression of protein A (spa) is repressed by global regulatory loci sarA and agr. Although SarA may directly bind to the spa promoter to downregulate spa expression, the mechanism by which agr represses spa expression is not clearly understood. In searching for SarA homologs in the partially released genome, we found a SarA homolog, encoding a 250-amino-acid protein designated SarS, upstream of the spa gene. The expression of sarS was almost undetectable in parental strain RN6390 but was highly expressed in agr and sarA mutants, strains normally expressing high level of protein A. Interestingly, protein A …

Circadian Clock-Specific Roles For The Light Response Protein White Collar-2, Michael A. Collett, Jay C. Dunlap, Jennifer J. Loros

Dartmouth Scholarship

To understand the role of white collar-2 in theNeurospora circadian clock, we examined alleles ofwc-2 thought to encode partially functional proteins. We found that wc-2 allele ER24 contained a conservative mutation in the zinc finger. This mutation results in reduced levels of circadian rhythm-critical clock gene products, frq mRNA and FRQ protein, and in a lengthened period of the circadian clock. In addition, this mutation altered a second canonical property of the clock, temperature compensation: as temperature increased, period length decreased substantially. This temperature compensation defect correlated with a temperature-dependent increase in overall FRQ protein levels, with the …

Characterization Of Sarr, A Modulator Of Sar Expression In Staphylococcus Aureus, Adhar Manna, Ambrose L. Cheung

Dartmouth Scholarship

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sar and agr). The sar locus is composed of three overlapping transcripts (sar P1, P3, and P2 transcripts from P1, P3, and P2 promoters, respectively), all encoding the 372-bp sarA gene. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of sar promoters. We previously partially purified a ∼12 kDa protein with a DNA-specific column

A Study On The Effects Of The N-Terminal Amino Acid Sequence On The Activation Of Human T-Cell Leukemia Virus Type 1 Protease, Hidayah Muhammad Kendall

Chemistry & Biochemistry Theses & Dissertations

Human T-cell leukemia virus type 1 (HTL V-1) is dependent upon the enzymatic activity of its protease for maturation. Maturation of the protease is facilitated by cleavage of specific amino acid residues, followed by dimerization. The effects of the amino acid sequence located N-terminally to the cleavage site on the ability of the protease to become active were the focus of the current study. These amino acid sequences were contributed by the plasmid vector into which the protease gene was inserted.

Surface probability analyses (SPAs) of the vectors, as well as for native sequences which produce the mature protease and …

F(C)Gammari-Targeted Fusion Proteins Result In Efficient Presentation By Human Monocytes Of Antigenic And Antagonist T Cell Epitopes, Chunlei Liu, Joel Goldstein, Robert F. Graziano, Jia He, Jeremiah K. O'Shea, Yashwant Deo, Paul M. Guyre

Dartmouth Scholarship

A major challenge for using native or modified T cell epitopes to induce or suppress immunity relates to poor localization of peptides to antigen presenting cells (APCs) in vivo. In this study, we demonstrate enhanced presentation of antigenic and antagonistic peptides by targeting them to the type I Fc receptor for IgG (F(c)gammaRI, CD64) on human monocytes. A Th epitope of tetanus toxoid, TT830, and the antagonistic peptide for TT830, TT833S, were genetically grafted into the constant region of the heavy chain of the humanized anti-CD64 mAb 22 and expressed as monovalent fusion proteins, Fab22-TT830 and Fab22-TT833S. These CD64-targeted peptides …

The Product Of The Saccharomyces Cerevisiae Rss1 Gene, Identified As A High-Copy Suppressor Of The Rat7-1 Temperature-Sensitive Allele Of The Rat7/Nup159 Nucleoporin, Is Required For Efficient Mrna Export, Veronica Del Priore, Christine A. Snay, Andre Bahr, Charles N. Cole

Dartmouth Scholarship

RAT7/NUP159 was identified previously in a screen for genes whose products are important for nucleocytoplasmic export of poly(A)+ RNA and encodes an essential nucleoporin. We report here the identification of RSS1 (Rat Seven Suppressor) as a high-copy extragenic suppressor of the rat7-1 temperature-sensitive allele. Rss1p encodes a novel essential protein of 538 amino acids, which contains an extended predicted coiled-coil domain and is located both at nuclear pore complexes (NPCs) and in the cytoplasm. RSS1 is the first reported high-copy extragenic suppressor of a mutant nucleoporin. Overexpression of Rss1p partially suppresses the defects in nucleocytoplasmic export of poly(A)+ RNA, rRNA …

Cloning Of The Mammalian Type Ii Iodothyronine Deiodinase. A Selenoprotein Differentially Expressed And Regulated In Human And Rat Brain And Other Tissues., Walburga Croteau, Jennifer C. Davey, Valerie Anne Galton, Donald L. St Germain

Dartmouth Scholarship

The deiodination of thyroid hormones in extrathyroidal tissues plays an important role in modulating thyroid hormone action. The type II deiodinase (DII) converts thyroxine to the active hormone 3,5,3'-triiodothyronine, and in the rat is expressed in the brain, pituitary gland, and brown adipose tissue (BAT). Complementary DNAs (cDNAs) for the types I and III deiodinases (DI and DIII, respectively) have been isolated and shown to code for selenoproteins. However, information concerning the structure of the mammalian DII remains limited, and the pattern of its expression in human tissues is undefined. We report herein the identification and characterization of rat and …

Differential Expression Of The Toxr Regulon In Classical And E1 Tor Biotypes Of Vibrio Cholerae Is Due To Biotype-Specific Control Over Toxt Expression., Victor J. Dirita, Melody Neely, Ronald K. Taylor, Paul M. Bruss

Dartmouth Scholarship

The two major disease-causing biotypes of Vibrio cholerae, classical and El Tor, exhibit differences in their epidemic nature. Their behavior in the laboratory also differs in that El Tor strains produce two major virulence factors, cholera toxin (CT) and the toxin coregulated pilus (TCP), only under very restricted growth conditions, whereas classical strains do so in standard laboratory medium. Expression of toxin and TCP is controlled by two activator proteins, ToxR and ToxT, that operate in cascade fashion with ToxR controlling the synthesis of ToxT. Both biotypes express equivalent levels of ToxR, but only classical strains appear to express ToxT …

Protein-Peptide Interactions Analyzed With The Yeast Two-Hybrid System, Meijia Yang, Zining Wu, Stanley Fields

Dartmouth Scholarship

The yeast two-hybrid system was used to screen a library of random peptides fused to a transcriptional activation domain in order to identify peptides capable of binding to the retinoblastoma protein (Rb). Seven peptides were identified, allof which contain the Leu-X-Cys-X-Glu motif found in Rb-binding proteins, although their activity in the yeast assay variedover a 40-fold range. Mutagenesis of the DNA encoding two of these peptides followed by screening in the two-hybrid systemallowed the delineation of residues apart from the invariant Leu, Cys and Glu that affect binding to Rb. Binding affinities of a peptide and one of its variants …

Molecular Cloning And Expression Of A Unique Receptor-Like Protein-Tyrosine-Phosphatase In The Leucocyte-Common-Antigen-Related Phosphatase Family, Wei-Ren Zhang, Naotake Hashimoto, Faiyaz Ahmad, Wendi Ding, Barry J. Goldstein

Department of Medicine Faculty Papers

Protein-tyrosine-phosphatases (PTPases) have been implicated in the regulation of certain tyrosine kinase growth factor receptors in that they dephosphorylate the activated (autophosphorylated) form of the receptors. In order to identify PTPases that potentially act on receptor targets in liver, we used the human leucocyte common antigen-related PTPase (LAR) cDNA [Streuli, Krueger, Hall, Schlossman and Saito (1988) J. Exp. Med. 168, 1523-1530] and isolated two closely related transmembrane PTPase homologues from a rat hepatic cDNA library. Both PTPases had large extracellular domains that contained three immunoglobulin-like repeats and eight type-III fibronectin repeats. Both enzymes had tandem homologous PTPase domains following a …

A Thyroid Hormone-Regulated Gene In Xenopus Laevis Encodes A Type Iii Iodothyronine 5-Deiodinase., Donald L. St Germain, Robert Schwartzman, Walburga Croteau, Akira Kanamori, Zhou Wang, Donald D. Brown, Valerie Galton

Dartmouth Scholarship

The type III iodothyronine 5-deiodinase metabolizes thyroxine and 3,5,3'-triiodothyronine to inactive metabolites by catalyzing the removal of iodine from the inner ring. The enzyme is expressed in a tissue-specific pattern during particular stages of development in amphibia, birds, and mammals. Recently, a PCR-based subtractive hybridization technique has been used to isolate cDNAs prepared from Xenopus laevis tadpole tail mRNA that represent genes upregulated by thyroid hormone during metamorphosis. Sequence analysis of one of these cDNAs (XL-15) revealed regions of homology to the mRNA encoding the rat type I (outer ring) 5'-deiodinase, including a conserved UGA codon that encodes selenocysteine in …

Subunit Dynamics In Escherichia Coli Preprotein Translocase., John C. Joly, Marilyn R. Leonard, William T. Wickner

Dartmouth Scholarship

SecY, SecE, and band 1 copurify as the SecY/E integral membrane domain of Escherichia coli preprotein translocase. To measure the in vivo association of these polypeptides and assay possible exchange, plasmid-borne secY and secE genes were placed under control of the ara regulon and fused to DNA encoding the influenza hemagglutinin epitope. Cells were incubated with [35S]methionine, grown for a "chase" period, and then induced with arabinose to express epitope-tagged, nonradioactive SecY and SecE. Both the wild-type and epitope-tagged polypeptides assembled into functional, heterotrimeric SecY/E complex. However, immunoprecipitation with antibody to the epitope tag did not cross-precipitate radiolabeled SecY or …

Insulin Receptor And Epidermal Growth Factor Receptor Dephosphorylation By Three Major Rat Liver Protein-Tyrosine Phosphatases Expressed In A Recombinant Bacterial System, Naotake Hashimoto, Wei-Ren Zhang, Barry J. Goldstein

Department of Medicine Faculty Papers

Protein-tyrosine phosphatases (PTPases) play an essential role in the regulation of signal transduction mediated by reversible protein-tyrosine phosphorylation. In order to characterize individual rat hepatic PTPases that might have specificity for autophosphorylated receptor tyrosine kinases, we isolated cDNA segments encoding three PTPases (PTPase 1B, LAR and LRP) that are expressed in insulin-sensitive liver and skeletal muscle tissue, and evaluated their catalytic activity in vitro. The intrinsic PTPase activities of the full-length PTPase 1B protein and the cytoplasmic domains of LAR and LRP were studied by expression of recombinant cDNA constructs in the inducible bacterial vector pKK233-2 using extracts of …

Characterization Of The Formate (For) Locus, Which Encodes The Cytosolic Serine Hydroxymethyltransferase Of Neurospora Crassa., C. Robertson Mcclung, Cynthia R. Davis, Karen M. Page, Sylvia A. Denome

Dartmouth Scholarship

Serine hydroxymethyltransferase (SHMT) occupies a central position in one-carbon (C1) metabolism, catalyzing the reaction of serine and tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate. Methylenetetrahydrofolate serves as a donor of C1 units for the synthesis of numerous compounds, including purines, thymidylate, lipids, and methionine. We provide evidence that the formate (for) locus of Neurospora crassa encodes cytosolic SHMT. The for+ gene was localized to a 2.8-kb BglII fragment by complementation (restoration to formate-independent growth) of a strain carrying a recessive for allele, which confers a growth requirement for formate. The for+ gene encodes a polypeptide of 479 amino acids which shows …

Laminin Potentiates Differentiation Of Pcc4uva Embryonal Carcinoma Into Neurons, T. M. Sweeney, Roy C. Ogle, C. D. Little

Medical Diagnostics & Translational Sciences Faculty Publications

The embryonal carcinoma PCC4uva differentiates into neurons in response to treatment with retinoic acid and dbcAMP. We used this in vitro model system to study the effects of laminin on early neural differentiation. Laminin substrata markedly potentiate neural differentiation of retinoic acid and dbcAMP-treated cultures. Only laminin induced more rapid neural cell body clustering, neurite growth and neurite fasciculation as compared to type IV collagen, type I collagen, and fibronectin substrata. Exogenous laminin substrata promoted greater cell attachment, cellular spreading and growth to confluence than type IV collagen, type I collagen, fibronectin and glass substrata. Laminin-induced effects were inhibited by …

Fibril In Senile Systemic Amyloidosis Is Derived From Normal Transthyretin., Per Westermark, Knut Sletten, Bjorn Johansson, Gibbons G. Cornwell

Dartmouth Scholarship

The amyloid fibril in senile systemic amyloidosis (SSA), like that of familial amyloidotic polyneuropathy, is derived from transthyretin (TTR). SSA, however, is a common disease, affecting to some degree 25% of the population greater than 80 years old. In familial amyloidotic polyneuropathy, the amyloidogenesis has been considered to depend on point mutations leading to TTR variants. We show that the TTR molecule in SSA, on the other hand, has a normal primary structure. Factors other than the primary structure of TTR must therefore be important in the pathogenesis of TTR-derived amyloid.

Osteogenesis Imperfecta. The Position Of Substitution For Glycine By Cysteine In The Triple Helical Domain Of The Pro Alpha 1(I) Chains Of Type I Collagen Determines The Clinical Phenotype., Barbra J. Starman, David Eyre, Harry Charbonneau, Maria Harrylock, Mary Ann Weis, Lester Weiss, John M. Graham Jr., Peter H. Byers

Dartmouth Scholarship

Skin fibroblasts grown from three individuals with osteogenesis imperfecta (OI) each synthesized a population of normal type I collagen molecules and additional molecules that had one or two alpha 1(I) chains that contained a cysteine residue within the triple-helical domain, a region from which cysteine normally is excluded. The patients had very different phenotypes. One patient with OI type I had a population of alpha 1(I) chains in which glycine at position 94 of the triple helix was substituted by cysteine; a patient with OI type III had a population of alpha 1(I) chains in which glycine at position 526 …

Molecular Cloning And Sequence Analysis Of The Plasmodium Falciparum Dihydrofolate Reductase-Thymidylate Synthase Gene., David J. Bzik, Wu-Bo Li, Toshihiro Horii, Joseph Inselburg

Dartmouth Scholarship

Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein. The TS domain was more conserved than the DHFR domain and both P. falciparum domains …