|Document Type:||Master's Thesis|
|Name:||Jennifer A. Hensley|
|Title:||Cloning and Characterization of a Gene Involved in Lipooligosaccharide Biosynthesis in Haemophilus somnus|
|Degree:||Master of Science|
|Department:||Veterinary Medical Sciences|
|Committee Chair:||Dr. Thomas J. Inzana|
|Committee Members:||Dr. Stephen M. Boyle|
|Dr. Brenda Winkel Shirley|
|Keywords:||Haemophilus somnus, lipooligosaccharide, phase variation, CAAT repeats|
|Date of defense:||May 6, 1998|
|Availability:||Release the entire work for Virginia Tech access only.
After one year release worldwide only with written permission of the student and the advisory committee chair.
Repetitive tetramers of the DNA sequence 5’-CAAT-3’ are present in several loci associated with lipooligosaccharide (LOS) phase variation in Haemophilus influenzae type b (Hib). In an attempt to identify H. somnus phase-variable LOS genes, the presence of CAAT repeats within the H. somnus 738 genome was confirmed using a (CAAT)7 probe. A 3.9 kb EcoRI fragment that reacted with the probe was cloned and sequenced. Sequence analysis confirmed the presence of 31 CAAT repeats downstream of two potential start codons, and indicated that small or large proteins would be encoded depending on the number of CAAT repeats. The larger gene products showed 46% amino acid homology to Lex2b from Hib, which influences LOS phase variation in that species. In H. somnus, this gene was named lob1 (lipooligosaccharide biosynthesis gene). Sequence analysis showed that randomly selected colonies most frequently contained 33 CAAT repeats in lob1, corresponding to a 294 amino acid product. Colonies selected for negative reactivity to mAb 5F5 were significantly more likely to have different numbers of CAAT repeats in lob1 than randomly selected colonies. The presence of lob1 in trans altered the LOS profile of a non-phase variable strain of H. somnus, and caused increased levels of reactivity to polyclonal antisera made to purified LOS from strain 738. Based on the ability of this gene to alter the LOS profile of a non-phase varying strain and the correlation of changes in CAAT repeats with mAb 5F5 reactivity, lob1 appears to be involved in LOS biosynthesis and phase variation.
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