VTechWorks Repository :: Browsing by Author "Johnson, J.L."

Browsing by Author "Johnson, J.L."

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Autocrine mechanisms of action of insulin-like growth factor-I (IGF-I) and hormonal regulation of expression of IGF-finding proteins in mammary epithelial cells
Romagnolo, Donato (Virginia Tech, 1993)
Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) genes in mammary epithelial cells. To test the hypothesis that IGF-I affects growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, several cell lines were developed expressing an ovine exon-2 containing IGF-I cDNA under the control of the mouse mammary tumor virus-long terminal repeat (pMMTV-IGF-I), early simian virus (pSV40-IGF-I), and herpes simplex thymidine kinase (pTK-IGF-I) promoters. Stably transfected clones were generated by cotransfection of clonal MAC-T cells with the IGF-I expression vectors and a plasmid conferring resistance to hygromycin-B (HYG-B), using a calcium phosphate precipitation procedure. Induction of the MMTV-LTR with the glucocorticoid dexamethasone (DEX) was required for enhanced expression of IGF-I in MD-IGF-I (MD=Mammary Derived) cells, whereas SV40-IGF-I cells constitutively expressed the highest levels of IGF-I, followed by TK-IGF-I cells. Activity of the MMTV promoter in MD-IGF-I cells was coordinately regulated by lactogenic hormones and extracellular matrix. Acute secretion of DEX-induced recombinant IGF-I by MD-IGF-I cells stimulated cell proliferation through an autocrine/paracrine pathway and triggered the expression of IGFBP-3. Neither acute nor constitutive expression of IGF-I affected expression of type 1 IGF receptor mRNAs, but down-regulated cell surface receptor levels, in the order SV40-> TK- > MD-IGF-I. Secretion of IGF-I-induced IGFBP-3 potentiated the mitogenic actions of IGF-I as evidenced by enhancement of [³H]thymidine uptake into DNA of parental MAC-T cells. This study provides evidence that local production of IGF-I can stimulate cell proliferation of bovine mammary epithelial cells through an autocrine/paracrine mode of action. We suggest that secretion of IGF-I-induced IGFBP-3 by bovine mammary epithelial cells enhances cell responsiveness to IGF-I, but does not prevent down-regulation of the IGF-I receptor in cells constitutively expressing IGF-I.
A biochemical and physiological characterization of coenzyme F420-reducing hydrogenase from Methanobacterium formicicum
Baron, Stephen Francis (Virginia Polytechnic Institute and State University, 1988)
The coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme formed aggregates (1,000 kd) of a coenzyme F₄₂₀-active monomer (109 kd) composed of 1 each of a, β, and γ subunits (43.6, 36.7, xy and 28.8 kd, respectively). It contained 1 mol of FAD, 1 mol of nickel, 12-14 mols of iron, and 11 mols of acid-labile sulfide per mol of the 109 kd species, but no selenium. The amino acid sequence I---P--R-EGH-----EV was conserved in the N-terminus of a subunit of the enzyme and the largest subunits of nickel-containing hydrogenases from Methanobacterium thermoautotrophicum, Desulfovibrio baculatus, and Desulfovibrio gigag. FAD dissociated from the coenzyme F42O-reducing hydrogenase during reactivation with H2 and coenzyme F₄₂₀, unless KCl was present, yielding coenzyme F₄₂₀-inactive apoenzyme. The hydrogenase catalyzed H₂ production at a rate 3-fold less than that for H2 uptake. Specific antiserum inhibited the coenzyme F₄₂₀ dependent activity but not the methyl viologen-dependent activity of the purified enzyme. Cell extract of M. formicicum contained a coenzyme F₄₂₀-mediated formate hydrogenlyase system. Formate hydrogenlyase activity was reconstituted with coenzyme F₄₂₀-reducing hydrogenase, coenzyme F₄₂₀-reducing formate dehydrogenase, and coenzyme F₄₂₀, all purified from M. formicicum. The reconstituted system required FAD for maximal activity (kinetic Kd= 4 μM). without FAD, the formate dehydrogenase and hydrogenase rapidly lost coenzyme F₄₂₀-dependent activity relative to methyl viologen-dependent activity. Immunoadsorption of the formate dehydrogenase or hydrogenase from cell extract greatly reduced formate hydrogenlyase activity; addition of the purified enzymes restored activity. Formate hydrogenlyase activity of cell extract and the reconstituted system was reversible. The coenzyme F₄₂₀-reducing hydrogenase and formate dehydrogenase of M. formicicum were shown to be located at the cytoplasmic membrane using immunogold labeling of thin sectioned, Lowicryl-embedded cells. Neither enzyme was released from whole cells by osmotic shock treatment.
Characterization of the carbohydrate receptors of the Clostridium difficile enterotoxin
Tucker, Kenneth D. (Virginia Tech, 1990)
Clostridium difficile causes pseudomembranous colitis in humans and a similar ileocecitis in hamsters. This organism can colonize the intestines after antibiotic therapy disrupts the normal intestinal microflora. Once established in the intestines, the organism causes disease by producing two toxins, designated toxin A and toxin B. Only toxin A is active on intestinal epithelium, thus toxin A is the cause of the initial tissue damage in the intestines. In order for a toxin to affect a cell, it must first bind to the cell. Toxin A has been shown to bind to Galα1- 3Galβ 1-4GIcNAc on the intestinal epithelium of hamsters. I provide evidence that toxin A can use this trisaccharide as a functional receptor on cell lines, and that the expression of the carbohydrate receptor increases the sensitivity of the cells to toxin A. Furthermore, the intestinal epithelium of infant hamsters bound less toxin A at 37C than did the adult tissue, and infants are less sensitive to the disease caused by C. difficile than are adults. This provides further evidence that the activity of toxin A is increased by the binding of the toxin to Galα1-3Galβ1- 4GlcNAc. Even though Galα1-3Galβ 1-4GlcNAc was a receptor for toxin A on animal cells, it probably is not a receptor for toxin A in humans, because people do not normally express this carbohydrate. Instead, I found that toxin A bound to the carbohydrate antigens designated I, X, and Y, which are present on the intestinal epithelium of humans. These carbohydrates could be receptors for toxin A. The possible significance of these receptors is discussed.
Cis and trans signals for the replication of bovine parvovirus
Metcalf, John Brockway (Virginia Tech, 1990)
The cis and trans signals important in BPV replication were identified using a transient replication assay, the mobility shift assay, and a comparison between the BPV and LPV genomes. Replication of deleted BPV genomic clones, which contain the natural left (3’ OH end of the viral minus strand) and right (5’ PO, end of the viral minus strand) BPV termini, defined the minimum size of the BPV origin of replication (ori) to be the terminal 171 nucleotides of each terminus. Clones containing duplicate termini or altered left ends were also shown to replicate. The BPV ori was determined to have two domains identified by a computer analysis of homologus regions between these termini. Three proteins were identified that bind to the left terminal 171 nucleotides in the hairpin conformation. Inhibition of the formation of the DNA-protein complexes with competitor DNA localized two potential binding sites that correspond to the domains mentioned above. Two of the DNA-protein complexes were formed by BPV-coded proteins as determined by inhibition of the complex by anti-BPV antibodies. The third complex resulted from binding of a host cell S-phase protein that is a likely candidate for the S-phase factor required for autonomous parvovirus replication. The BPV ori thus appears to function by binding both cellular and viral proteins for the initiation of DNA synthesis from the hairpinned termini. The comparison of the BPV and LPV genome sequence suggest that the genomic organization of LPV may be more like BPV than that of the rodent parvovirus minute virus of mice; and therefore, LPV may contain similar cis signals.
Genetic analysis and phenotypic characterization of Lon mutants of Escherichia coli K-12
Torres-Cabassa, Angel S. (Virginia Polytechnic Institute and State University, 1982)
A systematic study of a collection of Lon⁻ mutants has been made in order to determine whether their pleiotropic phenotype is due to mutations affecting one or more genes. A fine structure map of the lon locus was constructed by Pl mediated generalized transduction. The lon⁻ mutations were found to map in two "clusters" within the region. Phenotypic characterization of a set of isogenic Lon⁻ strains derived from these experiments indicated that all Lon-associated phenotypes (e.g. sensitivity to UV irradiation, decreased ability to inherit plasmid and prophage, abnormal polypeptide degradation and regulation of capsular polysaccharide biosynthesis) are differentially expressed in Lon⁻ strains. A direct correlation exists between the intracistronic ordering of the lon⁻ alleles and the degree of expression the Lon⁻ phenotypes in each strain. All isogenic Lon⁻ strains exhibit conditional lethality upon a nutritional shift-up. However, some filamenting Lon⁻ mutants are not able to overcome this defect when exposed to growth conditions known to promote cell division in Lon⁻ strains. Evidence was obtained that suggest a role for nucleotide pools in the control of cell division and capsular polysaccharide production. Reversion studies indicated that all lon⁻ mutations studied are point mutations. The failure to generate deletions of the lon region in χ573, an F' strain carrying the lac to minE region on the plasmid, and the inability to cure F' strains carrying a lon⁻ mutation on the plasmid suggest that the lon gene product may be indispensable for the cell's survival. From transductional crosses, two intermediate phenotypic classes: UV-resistant, mucoid (UV^RMuc), (Class A) and UV-sensitive, nonmucoid (UV^SRou) (Class B), were obtained that did not segregate colonies of the opposite morphology. Genetic analysis of these strains by back-transduction into a proC⁻ lon⁺ background, indicated that complete genetic separation of all Lon-associated phenotypes tested was not achieved, although differences in the expression of some of these persisted. Data obtained from complementation analysis ruled out the presence of two genes at the lon locus. The patterns of complementation observed were compatible with the existence of one lon gene, having at least two distinct domains, and whose product is a multifunctional polypeptide.
Improvement of expression of recombinant human protein C in the milk of transgenic animals using a novel transgene construct
Russell, Christopher G. (Virginia Tech, 1993)
Past studies of mammary tissue specific expression of transgenes using the murine whey acidic protein (WAP) promoter have shown widely variable, position-dependent and copy number-dependent expression. This study evaluates a series of three WAP transgenes containing the cDNA of human protein C (hPC) for the expression of human protein C in the milk of mice. In two of the transgenes studied, the cDNA of (hPC) was inserted at the translational start site of a 7.8 kbp mouse WAP genomic DNA Eco RI fragment containing 2.6 kbp of 5’ flanking, 3.9 kbp WAP coding (exons and introns), and 1.3 kbp 3’ untranslated region (UTR) and flanking sequences (designated WAPPC1 and WAPPC2). A third transgene consisted of only the 2.6 kbp of WAP 5’ UTR and flanking DNA, 1.4 kbp hPC cDNA, and 1.3 kbp of 3’ WAP UTR and flanking DNA with no linker sequences (designated WAPPC3). The WAPPC1 and WAPPC2 transgenes expressed up to about 10 μg/ml recombinant hPC in mouse milk while WAPPC3 expressed 30-300 (n=10, n=5, n=11, number of founder lines evaluated for each transgene, respectively). In contrast to past studies with WAP-cDNA fusion transgenes where the maximal expression was about 5% of endogenous WAP expression, the WAPPC3 transgene gave maximal expression which was about 30% of endogenous WAP expression. Thus, results from the combination in WAPPC3 of intact 5’ and 3’ WAP UTR with the cDNA of hPC suggests that introns are not necessary to enable high level expression in the mammary gland when using WAP regulatory elements. Relative specific transcript and protein levels in the transgenic animals studied suggest that the rates of translation initiation may be different for the mRNAs of each of the transgenes studied.
Insertion sequence IS1141: discovery, characterization, and association with Mycobacterium intracellulare colonial variation
Via, Laura Ellen Akers (Virginia Tech, 1993)
Mycobacterium avium and Mycobacterium intracellulare, (M. avium complex, MAC) are human pathogens causing disease in individuals with acquired immunodeficiency syndrome (AIDS) or with thoracic abnormalities. MAC bacteria are difficult to kill because of the resistance of the pathogens to chemotherapeutic agents. One factor affecting treatment of MAC disease is the presence of interconvertible colonial variants. Transparent (T) variants have greater resistance to antibiotics and higher pathogenicity; opaque (O) variants are more susceptible to antibiotics and less pathogenic. The overall goal of this study was to investigate the mechanism for colonial variation. Based on an observation that T variants of M. intracellulare strain Va14 contained a plasmid which was 6 kb smaller than the 68 kb plasmid in O variants, it had been suggested that a transposable element might be responsible for colonial variation. The first objective was to clone the unique DNA fragment present in the 68 kb plasmid but absent from the 62 kb plasmid. The second and third objectives were to determine if the unique fragment contained a transposable element and to analyze the role of that element in the mechanism of colonial variation in M. intracellulare strain Va14. The fourth objective was to determine the distribution of IS1141 in MAC isolates. Fragments containing copies of the putative element were sequenced and a region 1596 basepairs in length with 23 basepair imperfect inverted repeats was designated as insertion sequence IS1141. IS1141 is the first insertion sequence identified in M. intracellulare. Data base searches using open reading frames (ORF) of IS1141, identified ORFb as significantly similar to the transposases of the IS3 family. The presence or absence of IS1141 in strain Va14 plasmids appeared unrelated to colonial variation, but IS1141 was present in another plasmid and the chromosome of the Va14 variants. Hybridization studies with IS1141 identified three chromosomal copies in O variants and two chromosomal copies in T variants. Va14 T variants each had a common IS1141 restriction fragment length polymorphism (RFLP) pattern which was different than the single RFLP pattern found in opaque variants. Based on these differences, it appears that IS1141 may integrate into the gene(s) responsible for the T phenotype preventing their expression. A survey of 64 James River basin non-AIDS, clinical and James River environmental MAC isolates identified 4 of 24 (17%) M. intracellulare isolates as containing IS1141. IS1141 has not been detected in any clinical or environmental M. avium or Mycobacterium species X isolates and may be limited to M. intracellulare.
Isolation and characterization of plasmids from human and environmental isolates of mycobacteria
Meissner, Paul Scott (Virginia Polytechnic Institute and State University, 1984)
Human clinical (n=131) and environmental (n=226) isolates of the Mycobacterium avium-intracellulare and M. scrofulaceum (MAIS) complex were screened for plasmids in an effort to increase knowledge about the genetics and epidemiology of these pathogenic bacteria. Approximately 50% of the clinical MAIS isolates from New York, Maryland, Virginia, South Carolina, and Georgia contained one or more plasmids. On the basis of plasmid content, aerosol MAIS isolates more closely resembled human MAIS isolates than did MAIS isolates from the other environmental sources examined (dust, soil, sediment, and water). Plasmid profiles were remarkably heterogenous, and isolates with identical profiles were rarely encountered. However, a 115 megadalton (Md) plasmid was detected in 15 mercury resistant human and environmental isolates. In one of these isolates (M. scrofulaceum W262) the presence of the 115 Md plasmid was shown to correlate with the presence of an NAD(P)H dependent mercuric reductase. Plasmids with molecular weights of 8.8, 11.2, 14.2, 16.9, 17.9, and 18.3 Md were also common among both human and environmental isolates. On the basis of molecular weight, 36 distinct plasmids were detected; their sizes ranged from 7 to 230 Md. It was concluded that human and environmental MAIS isolates share a number of plasmids with identical molecular weights and that plasmids can serve as useful entities in genetic and epidemiologic studies of this group of extremely slow-growing, poorly understood human and animal pathogens.
Isolation and characterization of the messenger RNA and the gene coding for a proline-rich zein from corn endosperm
Wang, Shu-Zhen (Virginia Polytechnic Institute and State University, 1985)
Gamma-zein, a proline-rich protein from corn endosperm, was investigated at the molecular level. Immunological and electrophoretic data indicated that gamma-zein was deposited into protein bodies in corn endosperm. Both isolated polysomes and poly(A)⁺ mRNA were found to direct in vitro synthesis of gamma-zein in a wheat germ system. In vitro synthesized gamma-zein was immunoprecipitated from the total in vitro translation products. A cDNA expression library was constructed by reverse transcription of total poly(A)⁺ mRNA using pUC8 plasmid as vector and E. coli strain DH1 as host. The library was screened for the expression of gamma-zein and alpha-zein by specific antibodies. The library was also screened with ³²P-labeled gamma-zein and alpha-zein cDNA probes. The results indicated that gamma-zein and its fragments were readily expressed in E. coli while alpha-zein was not. Seven independently selected clones, six of which were selected by antibody and one by a cDNA probe, were sequenced. A comparison of sequence information from seven clones revealed that their overlapping regions were identical. This suggests that gamma-zein is encoded by a single U gene. This finding is in conflict with what was expected on the basis of extensive charge heterogeneity of gamma-zein in isoelectric focusing. Individual bands cut from an IEF gel were rerun and shown to give several bands suggesting that the charge heterogeneity of gamma-zein may be an artifact. Sequence information of gamma-zein indicated that the gene encodes a mature protein whose primary structure includes 204 amino acids and has a molecular weight of 21,824 daltons. There are eight essentially identical tandem repeats of the hexapeptide Pro-Pro-Pro-Val-His-Leu and two of the octapeptide Gln-Pro-His-Pro-Cys-Pro-Cys-Gln in the N-terminal one-half of the polypeptide. The codon specifying the third proline in the hexapeptide repeating unit is identical, CCG, in all eight repeats. It is likely that these highly conserved tandem repeats are of critical importance to the function of gamma-zein which is presently unknown. Alternatively, it is conceivable that selective pressures responsible for conserving these tandem repeats may be operating at the nucleic acid level.
Minor form of human 5.8s ribosomal RNA
Smith, David (Virginia Polytechnic Institute and State University, 1986)
Part One: The Minor Form of Human 5.8S rRNA An elongated form of 5.8S rRNA has been found in a wide range of eukaryotes from yeast to rodents. This minor form of 5.8S rRNA is about six nucleotides longer than the major form and composes from 10% to 30% of the total 5.8S rRNA found in yeast and rodents respectively. The minor form of 5.8S rRNA (pCCGAUA-) found in mice and rats may be generated by the formation of a secondary cleavage site caused by heterogeneity in the rRNA genes. The insertion of an adenylic acid residue in the precursor rRNA generates this additional cleavage site, i.e. -ACGA- or -ACCGA for the major and minor forms respectively. There is also heterogeneity with respect to the degree of methylation in rodent 5.8s rRNA. The conformation of the two chain length isomers is influenced by 2'-O-methylation of the uridylic acid residue at position 14, i.e. the most compact conformation is not ribose methylated in that position. The molecules which are methylated in the 14th position cannot adopt the most compact conformation. In the present study I have discovered a minor form of 5.8S rRNA in human placenta and I have determined its sequence; it differs from the major form of human 5.8S rRNA in having an additional sequence (CUCGUA) on the 5'-terminus. The sequence of the major rodent 5.8S rRNA is completely conserved in the major human 5.8S rRNA but the elongation on the 5'-ends of the minor 5.8S rRNAs from the two species are only 50% conserved. Human minor 5.8S rRNA was completely methylated at the uridylic acid residue at 14 making it the first 5.8S rRNA found to be completely methylated. Part Two: Purification and Characterization of a Ribose Transmethylase from Ehrlich Ascites Cells A filter binding assay was developed for measuring ribose transmethylase activity in cell extracts and was used to quantify ribose and base transmethylase in Ehrlich ascites cells and normal mouse liver. Ribose and base transmethylase activities were elevated two-fold in Ehrlich ascites cells compared to normal mouse liver when methyl-deficient mouse tRNA was used as substrate but base transmethylase activity was elevated tenfold in Ehrlich ascites cells when E. coli tRNA was used as substrate. E. coli tRNA did not serve as a methyl acceptor for ribose transmethylases. The ribose transmethylase was purified 910-fold from Ehrlich ascites cell extracts and complete elimination of base transmethylase was achieved in one experiment. This purified ribose transmethylase was found to have an apparent Km_tRNA of 20uM tRNA and an apparent Km_SAM of 12.8uM SAM. The apparent molecular weight of the ribose transmethylase, as determined by gel filtration chromatography, was 240, 000 daltons. SDS-PAGE of the purified ribose transmethylase showed a predominant protein band of approximately 60,000 daltons.
Molecular cloning and analysis of the genome of bovine parvovirus
Shull, Bruce Colin (Virginia Polytechnic Institute and State University, 1987)
The genome of bovine parvovirus (BPV) has been cloned by blunt end ligation of double-stranded virion DNA into the plasmid pUC8. The resulting genomic clones were infectious after transfection into bovine fetal lung (BFL) cells. Sequencing of the plasmids demonstrated that deletions were common at both ends of the cloned BPV genome. Deletions of up to 34 bases at the 3’ end lowered but did not abolish infectivity, while a deletion of 52 bases eliminated infectivity, End label analysis demonstrated the repair of deletions of up to 34 bases at the 3’ end or 35 bases at the 5’ end to the wild type length. Mutually inverted sequence orientations of the palindromic termini, known as the flip and flop forms, can occur during replication of parvovirus DNA. Cloning of BPV terminal sequences permitted the identification of the 3’ flop sequence inversion as a natural component of BPV DNA. This is the first report of sequence inversions within the 3’ end of an autonomous parvovirus. Clones with the 3’ flop or flip conformations were equally infectious. Wild type virion DNA was shown to have predominantly the 3’ flip conformation but a significant amount of 3’ flop was also detected. At the 5’ end, both the flip and flop sequence conformations were identified in nearly equal amounts. The progeny virion DNA from transfection of genomic clones had the same ratio of flip to flop as did wild type at both the 3’ and 5’ ends, regardless of the starting terminal conformations of the genomic clone. These data suggest that, while sequence inversion occurs at both termini during BPV DNA replication, some mechanism exists for the preferential replication of the 3’ flip conformation. Replicative form DNA from BPV infected cells had the same ratio of flip and flop at each end and the same termini as virion DNA. A set of deletion and frameshift mutants affecting each of the coding regions of BPV was constructed using one of the genomic clones. None of these mutants was infectious when transfected into BFL cells, which demonstrates that all three of the major open reading frames are essential for the production of infectious virus.
Molecular cloning, characterization, and expression of 3-hydroxy-3-methylglutaryl coenzyme a reductase gene from tomato
Park, Hee-Sung (Virginia Tech, 1990)
In plants, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme regulating biosynthesis of phytosterols, plant growth regulators, carotenoids, antimicrobial defense compounds, and numerous other isoprenoids. To initiate molecular studies of HMGR in relation to defense responses in plants, we utilized yeast HMGR cDNA sequences to isolate tomato genomic sequences encoding HMGR. The nucleic acid sequence and gene structure was determined. The tomato HMGR gene (HMG2) contains four exons separated by three introns and encodes a polypeptide of 602 amino acid residues (about 64,714 Da). Two membrane-spanning regions are contained in the NH₂-terminus of the HMGR polypeptide. The COOH-terminus shares significant homology with HMGR sequences from different species. Genomic Southern hybridization analyses reveals that tomato contains 3 to 4 HMGR genes. The HMGz2 gene cross-hybridizes to mRNA of about 2.7 kb which is highly induced in tomato cells treated with fungal elicitors and in stems, leaves, or roots stressed by wounding suggesting that the HMGz2 is a defense-related gene in tomato. Hybridization with a gene specific probe indicates that the HMG2 gene is induced specifically during defense responses and is distinct from the gene(s) expressed during fruit development and ripening.
Molecular mechanisms of pathogenesis incited by Erwinia carotovora subsp. carotovora
Roberts, Daniel Paul (Virginia Polytechnic Institute and State University, 1985)
Erwinia carotovora subsp. Carotovora (Ecc) incites soft-rot on many plants. It is believed that soft-rot is due to the concerted activity of extracellular enzymes. Recombinant DNA techniques were used to study the molecular basis of pathogenesis incited by Ecc. Specifically, a clone library of Ecc strain EC14 DNA in plasmid pBR322 was constructed and transformed into Escherichia coli strain HB101. Some of the E. coli strains that contain these hybrid plasmids produce pectinases or cellulase(s). Plasmid pDR1 contains a 3.4 kilobase (kb) EC14 DNA fragment and mediates the production of endo-pectate lyases with isoelectric points (pI) of 9.5 and 7.5 in strain HB101. The pI 9.5 enzyme is believed to be the major extracellular pectolytic enzyme in soft-rot while the pI 7.5 enzyme has no documented counterpart in EC14. Subclone and transposon tn5 analyses of pDR1 indicate that 1.5 kb is necessary for the production of the pI 9.5 and pI 7.5 enzymes and that these enzymes are produced independently of other EC14 pectate lyase enzymes. Plasmid pDR30 contains a 2.1 kb EC14 DNA insert that mediates the production of an endo-polygalacturonase and an exo-pectate lyase in HB101. The exo-pectate lyase encoded by pDR30 produces an inducer of endo-pectate lyase synthesis as a reaction product. The endo-polygalacturonase encoded by pDR30 is thought to play a role in plant cell wall pectic polymer degradation. Restriction endonuclease and Southern hybrididizatian analyses indicate that the EC14 genes on plasmids pDR1 and pDR30 are not part of the same operon. Escherichia coli strain HB101 containing plasmid pDR1 or plasmid pDR30 is unable to macerate potato tuber slices. However, HB101 containing plasmids pDR1 and pDR30 can cause limited maceration of potato tuber slices. There appears to be a genetic interaction between plasmids pDR1 and pDR30 in maceration of potato tuber tissue. However, the EC14 gene(s) contained on plasmid pDR1 are transcribed independently of the EC14 genes contained on plasmid pDR30. It is possible that transcription of certain pectolytic enzymes independent of other pectolytic enzymes provides a flexible system for plant cell wall pectic polymer degradation.
Nutritional requirements of Treponema denticola and Treponema vincentii
Van Horn, Kenneth George (Virginia Polytechnic Institute and State University, 1982)
Treponema denticola and Treponema vincentii were grown in a medium supplemented with 0.4% (wt/vol) alpha globulin in place of whole serum. Other serum fractions did not support growth. The growth factors in alpha globulin were destroyed by trypsin and by lipase. Lipid extraction of alpha globulin showed that both a protein and a lipid fraction were required for growth. Sodium salts of either oleic acid (cis-18:1) or elaidic acid (trans-18:1), added to 0.4% delipified alpha globulin supplemented media at a final concentration of 0.04 mg/ml, replaced the alpha globulin lipids required for optimal growth of these two oral treponemes. Tween 80 (polysorbitan monooleate) also supported growth in a medium containing protein. Short chain fatty acids plus 25 µg/ml thiamine pyrophosphate, added to either a basal medium or a medium containing 0.4% albumin, supported limited growth. The principal cellular fatty acids of T. denticola grown in an oleate medium were myristic, pentadecanoic, and palmitic acids. Treponema denticola appears capable of limited synthesis of cellular fatty acids from oleate. Fifty percent of the total protein content of commercial alpha globulin was found to be albumin. The protein required for T. denticola growth was separated from the other alpha globulin proteins by Affi-Gel Blue (Bio-Rad Laboratories) affinity chromatography which selectively adsorbed albumin. Serum albumin, added to a medium containing oleate, substituted for the alpha globulin protein required by these two treponemes. Trypsin destroyed the growth promoting activity of albumin. A weight ratio of albumin to sodium oleate of 50:1 (0.4% delipified albumin - 0.08 mg/ml oleate) supported optimal growth of T. denticola and T. vincentii. Starch, added to media containing oleate, could not replace albumin for optimal growth. Serum albumin solutions tightly bound added thiamine pyrophosphate (TPP). Optimal growth was achieved only when the TPP concentrations in albumin-oleate media were sufficient to provide excess TPP, unbound to albumin. Whole cells of T. denticola were shown to have proteolytic activity toward casein and alpha globulin proteins. Alpha globulin proteins were also found avidly attached to T. denticola cells that had been suspended in alpha globulin.
Purification and characterization of Clostridium perfringens iota toxin
Stiles, Bradley G. (Virginia Polytechnic Institute and State University, 1987)
Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting "iota-like" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (i_b), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i_a) antigen, had little to no biological activity. When fractions containing i_a and i_b were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with i_a or i_b, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of i_a and i_b, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of i_a and i_b were mixed. Monospecific antisera against purified i_a and i_b neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i_a and i_b proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i_a protein is heat stable (85° C/15 min) while i_b lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i_a, but not i_b, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor.
Studies on the carbon monoxide dehydrogenase enzyme complex present in acetate-grown Methanosarcina thermophila strain TM-1
Terlesky, Katherine C. (Virginia Polytechnic Institute and State University, 1989)
The carbon monoxide dehydrogenase complex was purified from acetate-grown Methanosarcina thermophila. This complex made up greater than 10% of the cellular protein and the native enzyme formed aggregates with a Mr of approximately 1,000,000. The enzyme contained five subunits of different molecular weight suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The electron paramagnetic resonance spectrum of CO-reduced enzyme at 113K contained g values of 2.073, 2.049, and 2.028. Isotopic substitution with ⁶¹Ni, ⁵⁷Fe, or ¹³Co resulted in broadening of the spectrum consistent with a Ni-Fe-C spin-coupled complex. Acetyl-CoA caused a perturbation of the signal that was not caused by acetyl-phosphate or mercaptoethanol indicating acetyl-CoA is a physiological substrate. Cell extracts from acetate-grown M. thermophila contained CO-oxidizing:H₂-evolving activity 16-fold greater than extracts of methanol-grown cells. CO-oxidizing:H₂-evolving activity was reconstituted upon combination of: (i) CO dehydrogenase complex, (ii) a ferredoxin, and (iii) purified membranes with associated hydrogenase and b-type cytochrome. The ferredoxin was a direct electron acceptor for the CO dehydrogenase complex. The molecular weight of the isolated protein was 16,400, and the apparent minimum molecular weight was 4,900. The ferredoxin contained 2.8 ± 0.56 Fe atoms and 1.98 ± 0.12 acid-labile sulfide. UV-visible absorption maxima were 395 and 295 nm with a A₃₉₅/A₂₉₅ ratio range of 0.80 to 0.88. The N-terminal amino acid sequence revealed a 4-cysteine cluster, similar to other Fe:S centers that coordinate a Fe:S center. A CH₃-B₁₂:HS-CoM methyltransferase activity was characterized in extracts of acetate- and methanol-grown cells. The activity from extracts of acetate-grown M. thermophila was stable at 70°C for 30 minutes. The activity in cell extracts of acetate- and methanol-grown cells was fractionated with ammonium sulfate treatment and FPLC phenyl superose chromatography. Two peaks of methyltransferase activity were observed in each cell extract sample following phenyl superose fractionation.
Studies on the structure and function of various nif and nif- associated gene products encoded within the Azotobacter vinelandii nif gene cluster
Brigle, Kevin Eugene (Virginia Polytechnic Institute and State University, 1989)
The present study investigates the structural and functional roles of the metalloclusters present within the MoFe protein of nitrogenase from Azotobacter vinelandii. A gene replacement strategy was developed for oligonucleotide-directed mutagenesis of these proteins and the resulting biological and biochemical effects of these changes were examined. Identification of structurally important regions in the MoFe protein subunits and assignment of specific amino acid residues as potential metal cluster ligands were based upon several criteria: i. metallocluster extrusion requirements; spectroscopic properties of the MoFe protein; interspecies and intersubunit comparisons; iv. comparison of the MoFe protein subunit sequences to iron-molybdenum cofactor biosynthetic gene products. This mutagenesis strategy has permitted the construction of thirty-three mutant strains having specific amino acid substitutions within the MoFe protein subunits. Based on the diazotrophic growth characteristics and substrate reduction capabilities of these mutant strains, a model is presented in which potential metallocluster binding sites within the MoFe protein subunits are defined. In addition to analysis of the MoFe protein subunits, this site-directed mutagenesis and gene replacement strategy can be used to place specific mutations into any gene product encoded within the A. vinelandii nif gene cluster. Finally, nucleotide sequence analysis of the regions flanking the nifEN genes revealed the presence of three nif genes (nifT, nifY, and nifX) and four open reading frames (ORF1, ORF2, ORF3, and ORF4). Two of these genes, nifX and ORF3, were shown to be under nif control and synthesis of their products elevated in response to a demand for fixed nitrogen. Mutant strains with deletions in ORF3 appeared to accumulate an excess amount of MoFe protein when compared to wild type. The ORF3 gene product has been overproduced in E. coli. This provides an important step toward characterizing the protein and elucidating the molecular basis for its control of nifDK gene expression.
A taxonomic study of the genus Campylobacter
Roop, Roy Martin (Virginia Polytechnic Institute and State University, 1985)
One hundred and eighteen (118) Campylobacter strains were studied by DNA homology experiments and characterized phenotypically. These strains formed eleven (11) distinct DNA homology groups (species) corresponding to C. fetus, C. "hyointestinalis", C. jejuni, C. coli, C. laridis, C. nitrofigilis, C. sputorum, C. mucosalis, C. concisus, and two unnamed groups currently referred to as the aerotolerant campylobacters and the "catalase-negative or weak" (CNW) strains. For practical reasons, we propose retaining the subspecies fetus and venerealis designations for C. fetus. In addition, we propose that the subspecies sputorum and bubulus designations for C. sputorum be dropped and replaced with biovars sputorum, bubulus and fecalis, the latter biovar including the catalase-positive strains formerly known as C. “fecalis". Biotyping schemes are also presented for C. jejuni and C. coli. Growth at 25 and 42°C, sensitivity to nalidixic acid and cephalothin, growth in semisolid medium containing 1% glycine, 1% oxgall or 3.5% NaCl, growth in a semisolid minimal medium (MM), anaerobic growth in 0.1% trimethylamine-N-oxide (TMAO), H₂S production in Sulfide-Indole-Motility (SIM) medium, or on triple sugar iron (TSI) agar slants, hippurate hydrolysis, aerobic growth on agar plates, a requirement for H₂ or formate for microaerophilic growth or H₂ or formate and fumarate for anaerobic growth, alkaline phosphatase activity, and deoxyribonuclease (DNase) activity proved to be the most useful phenotypic characteristics for identifying these strains at the species, subspecies and biovar levels.
Use of cell wall-hydrolytic enzymes in studies of the reticuloendothelial-stimulatory properties of Propionibacterium acnes
Stimpson, Stephen Anthony (Virginia Polytechnic Institute and State University, 1982)
Vaccines prepared from whole cells of heat-killed Propionibacterium acnes were treated with a variety of enzymes. Only two enzymes, lysozyme and a bacteriolytic enzyme from the common European limpet, Patella vulgata, were able to abrogate the splenomegaly-inducing activity of vaccines. Inactivation of vaccine occurred without lysis of bacteria, only at high concentrations of lysozyme, and was reversed by subsequent treatment with trypsin, suggesting that lysozyme inactivation was due to a non-enzymatic adsorption of lysozyme to the bacterial surface. The bacteriolytic enzyme from limpets was purified over 150-fold by preparative isoelectric focusing and named Patella vulgata lytic (PVL) enzyme. PVL enzyme activity in crude extracts could lyse many bacteria not lysed by lysozyme. The purified PVL enzyme had an isoelectric point of 8.3 and was a glycosidase which hydrolyzed the glycan backbone of peptidoglycan. Treatment of vaccine with PVL enzyme abolished the splenomegaly-inducing activity of vaccine. An assay was developed to measure the ability of vaccine to inhibit the development of a transplantable tumor in BALB/c mice. Treatment of vaccine with PVL enzyme also abolished the antitumor activity of vaccine. Since PVL enzyme hydrolyzed peptidoglycan, it was concluded that intact peptidoglycan was essential to the splenomegaly-inducing and antitumor activities of P. acnes vaccine. Formamide-extracted vaccines were as active as untreated vaccines in antitumor assays, and were also sensitive to lysis by lysozyme. Treatment of formamide-extracted vaccines with lysozyme abolished antitumor activity, indicating that peptidoglycan was responsible for the antitumor activity of formamide-extracted vaccines. Trichloroacetic acid-extracted cell wall polysaccharide (TCA-PS) was compared with PVL enzyme-released cell wall polysaccharide (ERPS). Although antigenically similar, the ERPS had a higher molecular weight than TCA-PS, indicating that the TCA-PS had been hydrolyzed somewhat during acid-extraction and that ERPS is representative of the native cell wall polysaccharide. TCA-PS contained glucose, galactose, mannose, glucosamine, galactosamine, and small amounts of glycine and serine. ERPS contained the TCA-PS components and in addition, a small amount of lysine, and the peptidoglycan components muramic acid, alanine, glutamic acid, and diaminopimelic acid, and was therefore a complex of polysaccharide and peptidoglycan.

Browsing by Author "Johnson, J.L."

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