used to generate cDNA in the initial PCR using the degenerate primers, which resulted in

only two successful amplifications for the liver and one for the intestine (Figure 5-4). The

RT reaction was reperformed at 500C, but this did not seem to increase the number of

amplicons. Thus, it was unlikely that the problem was in the reverse transcription step.

 When the degenerate primers were rerun for cDNA originating from the intestine of

another channel catfish (AT17), and cDNA from largemouth bass (M~icropterus

salmoides) liver, a larger number of amplicons (of expected sizes) were generated (Figure

5-25). In addition, the bands representing these various amplicons were more intense.

The UGT cloned from catfish intestine derived from AT17 and not AT45. These findings

indicate that the quality of the original RNA prepared from catfish AT45 intestine (and

possibly liver) was not satisfactory. One reason for the difference between the RNA of

both intestinal samples could have been due to the fact that while the intestinal RNA that

was used originated from mucosa that had been scraped off the smooth muscle wall of

the intestine, the AT17 intestinal RNA was derived from a tissue sample that was

processed without separation of the mucosa from the underlying muscle. It is possible

that the process of scraping the mucosa, even though this was done on ice and only lasted

for a few minutes, caused the degradation of a significant proportion of the UGT mRNA

population, resulting in the generation of a limited cDNA library. This may occur due to

stimulation of the secretion of proteases, nucleases, and other hydrolytic enzymes. On

one previous occasion, the process of scraping the catfish intestinal mucosa resulted in

poor quality cDNA for a CYP450 cloning study. Subsequently, the CYP450 was

successfully cloned from a sample that was derived from RNA originating from a

transverse section of the intestine (Dr.David Barber, personal communication).