The Catharanthus roseus 16-hydroxytabersonine O-methyltransferase involved in vindoline biosynthesis /

Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex dimeric anticancer alkaloids vinblastine and vincristine that are derived by coupling vindoline and catharanthine monomers. This thesis describes the novel application of carborundum abrasion (CA) technique as a tool for large scale isolation of leaf epidermis enriched proteins. This technique was used to facilitate the purification to apparent homogeneity of 16-hydroxytabersonine-16-0-methyltransferse (l60MT) that catalyses the second step in the 6 step pathway that converts tabersonine into vindoline. This versatile tool was also used to harvest leaf epidermis enriched mRNAs that facilitated the molecular cloning of the 160MT. Functional expression and biochemical characterization of recombinant 160MT enzyme showed that it had a very narrow substrate specificity and high affinity for 16-hydroxytabersonine, since other closely related monoterpene indole alkaloids (MIAs) did not act as substrates. In addition to allowing the cloning of this gene, CA technique clearly showed that 160MT is predominantly expressed in Catharanthus leaf epidermis, in contrast to several other OMTs that appear to be expressed in other Catharanthus tissues. The results provide compelling evidence that most of the pathway for vindoline biosynthesis including the 0- methylation of 16-hydroxytabersonine occurs exclusively in leaf epidermis, with subsequent steps occurring in other leaf cell types. Small molecule O-methyltransferases (OMTs) (E.C. 2.1.1.6.x) catalyze the transfer of the reactive methyl group of S-adenosyl-L-methionine (SAM) to free hydroxyl groups of acceptor molecules. Plant OMTs, unlike their monomeric mammalian homologues, exist as functional homodimers. While the biological advantages for dimer fonnation with plant OMTs remain to be established, studies with OMTs from the benzylisoquinoline producing plant, Thalictrum tuberosum, showed that co-expression of 2 recombinant OMTs produced novel substrate specificities not found when each rOMT was expressed individually (Frick, Kutchan, 1999) . These results suggest that OMTs can fonn heterodimers that confer novel substrate specificities not possible with the homodimer alone. The present study describes a 160MT model based strategy attempting to modify the substrate specificity by site-specific mutagenesis. Our failure to generate altered substrate acceptance profiles in our 160MT mutants has lead us to study the biochemical properties ofhomodimers and heterodimers. Experimental evidence is provided to show that active sites found on OMT dimers function independently and that bifunctional heterodimeric OMTs may be fonned in vivo to produce a broader and more diverse range of natural products in plants.

Second Welland Canal - Book 2, Survey Map 5 - Locks 16, 17 and 18 in Grantham

Survey map of the Second Welland Canal created by the Welland Canal Company showing the Grantham Township at Merritton. Identified structures associated with the Canal include Locks 15, 16, 17, and 18, and the towing path. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include streets and roads (ex. Hartzel Road and Macadamized Road), Lybster Cotton Mill, St. Catharines Paper Mill, J. Brown Cotton Mills, hydraulic race, a pond, several barns, and a number of structures (likely houses or cabins) belonging to: J. McNamara, M. Moran, A. Delany, T. Joyce, J. Delany, C. Blake, F. Weaver, W. Leeson, and Mrs. Aikins. Properties of note are: Concession 10 Lots 11 and 12. A number of reserved properties exist and are outlined in blue. They include three reserved properties for lock lots, and one large property reserved for a quarry.

Second Welland Canal - Book 2, Survey Map 16 - Deep Cut in Thorold

Survey map of the Second Welland Canal created by the Welland Canal Company showing the canal in the Thorold Township between Allanburg and Port Robinson. Identified structures and features associated with the Canal include the Deep Cut and the towing path. The surveyors' measurements and notes can be seen in red and black ink and pencil. Local area landmarks are also identified and include streets and roads (ex. Road to Port Allanburg), and the Spoil Bank. Properties and property owners of note are: Lots 185, 186, and 187, J. J. Church and H. Vanderburgh. Four properties adjacent to the canal are outlined in blue and labeled J through M, with L and K belonging to John Beatty, M belonging to John Coulter, and J belonging to G. Jordan (formerly belonging to John Coleman Jordan).

Second Welland Canal - Book 3, Survey Map 16 - Through Humberstone

Survey map of the Second Welland Canal created by the Welland Canal Company showing the border area of the townships of Crowland and Humberstone. Identified structures associated with the Canal include back ditches, towing path and spoil banks. Surveyor measurements and notes can be seen in red and black ink and pencil. Local area landmarks include line between the 7th Concession of Crowland and the Gore, Town Line between the Townhips of Crowland and Humberstone and the Highane and Company Store. Roads parallel to Canal include southern Road Allowance and the Road to Port Colborne. Roads perpendicular to Canal include Road Allowance between the 4th and 5th Concession. Properties and property owners are noted as Thomas Merrit, John Betty, John Brown, Charles French, James McCoppen, and S.D. Woodruff. Lots noted are: Lots Number 22, 23, 24, 5th Concession."Humberstone" - Scale: 4 Chs. per Inch "Gore"

Photograph of Irene Bell age 16 years, 1909

An unidentified girl, believed to be Irene Bell, is featured in this small black and white portrait photograph. Handwritten inscription on the reverse indicates the photo was presented in August 1909. The name of the recipient is illegible. This photo was in the possession of the Rick Bell family of St. Catharines, Ontario.According to entries in the Bell Family Bible, Irene Frances Bell, daughter of George A. Bell and Nellie F. Bell, was born on Jan. 19, 1893. This would make her 16-years-old at the time this photograph was presented in 1909.

The New-York Weekly Museum Newspaper, Vol. II, No.24- October 16, 1813

A weekly paper that was published from 1805 to 1814. War related news includes: Page 95 - Weekly Retrospect: relief effort in London, England for those affected by the war in British North America; naval battles on Lake Ontario by Commodore Chauncey in late September; movements of Major Grant, De Rottenburgh's regiment, General Wilkinson, Commodore Yoe's fleet, General Hampton; the capture of Fort Malden by William Harrison. The rest of the newspaper contains literary works (poems and translations), marriages, deaths and other anecdotes. The motto on the front page states: "Visiting Every Flower with Labour Meet, and Gathering all its Treasures, Sweet by Sweet."

The Weekly Messenger, Vol.2 No.39- July 16, 1813

A weekly newspaper that was published every Friday from 1811 to 1815.

Jacob Cochenour’s War Loss Claim, August 16, 1841.

Jacob Cochenour was an early settler of Bullock’s Corners in the Township of West Flamborough. His father, Jacob Cochenour Sr., was an American who settled in this area in 1785 on 200 acres of Crown Land along the Spencer Creek.

Portsmouth Oracle, 16 July 1814 (vol. 25, no. 43) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.

The Weekly Messenger, 5 February 1813 (vol. 2, no. 16) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.

The Weekly Messenger, 16 April 1813 (vol. 2, no. 26) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.

American Mercury, 16 August 1814 (vol. 31, 1572) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812. The 1814 Aug. issues report events of the Battle of Chippewa.

The War, 10 October 1812 (vol. 1, no. 16) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.

The War, 16 January 1813 (vol. 1, no. 30) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.