Our project on the Atlantic cod, Gadus morhua, received funding in the Fall of 1996 and I am reporting the progress made toward its goals in its third quarter.

Progress Outline:

1. Existing-Data Analysis: Existing plus new sample analysis continues.
2. Serum Sample Analysis: We are proceeding with our study of cod serum proteins.
3. New Tissue Analysis: Ovarian tissue is yielding proteins for purification.
4. Equipment Use: Data archival equipment is being used.
5. Personnel Identification and Training: A new technical assistant is being trained.
6. Publication: Hartling, Pereira and Kunkel (1997) reprints are available.

1. Existing-Data analysis: A WWW page linking to the prior flounder and current cod project descriptions has been created over the summer. Links to the NOAA/CMER page are included as acknowledgment of funding. A link to the Medline abstract of the flounder project is included on the flounder link. Entrez links do separate searches of the Medline database of Journals on Pluronectes and Gadus to keep investigators aware of recent publications:

http://www.bio.umass.edu/biology/kunkel/fish-www.html

1. Gel Permeation Analysis: We have retooled our gel permeation system to use Biorad Agarose A1.5 media to separate large yolk and serum proteins. Runs of ovarian extracts have been run with initial success. We have borrowed a fraction collector from Dr. Elita Pastra of Boston College to replace our malfunctioning fraction collector on a short term basis.
2. New Tissue Analysis: One of the 15 samples of whole cod ovaries from egg laden cod females obtained from long-line cod fishermen from the Chatham MA harbor has been studied in a concentrated manner. We have had success with applying the heat denaturation protocol (Hartling et al., 1997) and have added a fractional Ammonium Sulfate Precipitation step which is used to eliminate non-Lv material as well as produce a stabile 2/3-saturated cut which seems to contain the Lv of interest. This puts us on the verge of having a purified product with which we can immunize our rabbits.
3. Equipment Use: We have learned to use the CD writer (Pinnacle Micro 4x4) and have begun taking images of the cod ovary sections obtained from Frank Almeida.
4. Personnel Identification and training: A recent graduate of Hampshire College with experience in molecular biology is being trained in protein purification. He has already confirmed the heat stability of cod-Lv, produced a protocol for its use on ovarian extracts and is working on the Ammonium Sulfate fractionation and gel permeation protocols.
5. Publication: Hartling, Pereira and Kunkel (1997) is available in reprint form and reprint requests are coming in. The article in J Exp. Zool., is searchable from Medline. This paper on winter flounder has significance to our present project on cod because it describes the technique of purification of flounder Lv which we are now extending to cod Lv. Our initial experiments suggest that cod Lv is also heat-stabile and this procedure may be of general use for other fish Lvs including cod. The paper also describes the immunological assay developed for flounder which we also will use as a model to apply to cod.

In general we seem to be on track to having a purified Lv and an antiserum for use in the late Fall season of cod fishing.

Respectfully submitted,

Joseph G. Kunkel

jgk/hs