D ISC U SSIO N

At the outset of this project it was not clear whether the lactase persistence /

non-persistence polymorphism was controlled at the level of transcription or

translation. Indeed, there was considerable evidence that, at least in some cases, an

abnormal lactase protein could be detected in lactase non-persistent individuals (Witte

etal.y 1990, Potter, J. & Swallow, D. M., personal communication). However, the significance of these observations was not clear and it was not known whether the level

of lactase mRNA in these individuals was reduced.

In this study 1 have demonstrated a low level of the lactase protein in all the 11

non-persistent individuals tested. In 9 of the 10 samples analysed a low level o f RNA

was found. An abnormal protein pattern was detected in one individual from the

patient series (no.46). This individual also had relatively low RNA. It is possible that

this pattern is due to the presence of an abnormal forni of the lactase protein in this

individual but it may be that it is somehow a by-product of delayed processing o f a

lower amount of the normal lactase protein. It is of interest to note that this individual

is a Filipino. The individual studied by W itte et at. who was shown to have an

alteration in the rate of conversion of lactase precursor to the mature foim was a Native

American (Witte etciL, 1990).

Furthermore, it of particular interest that 1 also observed one individual with a

high level of mRNA but with little detectable protein (no 29). This individual was an

Italian. This is curious in view of the fact that the group which originally published the

observation that the levels of mRNA could be high in certain lactase non-persistent

individuals studied a Neapolitan population (Sebastio et a i, 1989).

The work on rats had suggested that the use of non-dissociating conditions for

the analysis of the protein might detect the lactase protein in different conformations

were made to analyse the protein samples from the patient series using non-dissociating

conditions. This analysis was not 100% successful. In those cases that could be tested

no further evidence of variant protein patterns was obtained. Unfortunately there was

not enough material from individual 46. No evidence of differently processed lactase

protein was observed for patient 29, the individual who had a high level of lactase

mRNA but a low level of lactase protein under standard conditions.

The observation that the antibody mlac 6 would cross-react with lactase protein

in rats meant that it was possible to attempt to replicate the experiment of Nsi-Emvo et al., (1987). It is of some interest that 1 was unable to repeat the original result when analysing samples from rats. This is hard to explain except by suggesting that the

samples had been handled differently and that this was not apparent in the reported

protocol. The only other possible difference is that the rat used in this analysis was a

Sprague-Dawley rat, whereas the analysis of Nsi-Emvo and colleagues used W istar rats.

The results presented in this thesis have shown that antibodies mlac 6 and mlac

10 recognise lactase protein in dimer and precursor forms in addition to the mature

form. 1 have also shown that the some of the mlac series of antibodies cross-react with

denatured lactase in species other than man. Several of this series of antibodies

recognise native lactase. These have also been examined for cross-reaction with lactase

in other species. The preliminary results are summarised in Table 3.7.1. These

observations allow the use some of these antibodies in the analysis of the

developmental regulation of the lactase protein in different species. Specifically, the

antibody mlac 6 recognised denatured lactase protein in all species tested, whereas mlac

10 only recognised lactase in pigs and humans. A possible use for these reagents would

be to distinguish between human and mouse lactase protein in the intestine of mice

transgenic for the human lactase gene.

The cross-reaction of the antibodies with lactase of other species suggest that

species studied. It is also noteworthy that mlac 6 recognises mouse lactase even though

this antibody was raised by the hybridoma technique using mouse spleen cells. This

cross-reaction probably results from the fact that this epitope is normally cryptic in the

mlacl mlac 2 mlac 3 mlac 4 mlac 5 mlac 6 mlac 8 mlac 9 mlac 10 human + + + + + + + 9 rabbit + + + NT pig - / + + NT + + + + rat NT NT NT NT NT N T mouse NT N'l' NT NT NT NT lamb + + NT NT + NT NT NT NT Table 3.7.1.

The cross reaction o f anti human lactase antibodies with native lactase from other species as judged by enzym e im m unobinding assay and im m u nop récipitation experim en ts.

NT indicates that that these were not tested. The in fondation for rabbit was obtained

from Matiiri et al., 1992., human from Man in et a!., 1991, the results for the other species are unpublished (A.J. Collins and C.B. Harvey).