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The affinity of hapten binding of monoclonal antibodies (MAbs) specific for 4-hydroxy-3-nitrophenylacetyl (NP) has been investigated at the molecular level by both site-specific mutagenesis and recombinant antibody construction, followed by expression in myeloma cells. We have shown that a single point mutation (trp----leu at codon 33) in the variable region of the heavy chain (VH) is sufficient to endow a primary-response, germline-encoded antibody with an affinity for antigen typical of a secondary-response antibody carrying the same mutation. We have also demonstrated that mutations additional to the trp----leu exchange in the heavy chain and further mutations in the light chain are irrelevant to the high-affinity phenotype of secondary-response antibodies. Since some of these are "parallel" mutations common to clonally unrelated antibodies, this suggests that the mutation rate is not constant across the entire immunoglobulin variable region. Although antibodies with a trp----leu exchange at position 33 are positively selected because of improved hapten binding affinity, we have found that, under rare circumstances, other patterns of mutations may be selected through particular D-JH combinations; we have demonstrated one case where this has generated an antibody with very efficient hapten binding ability.
[This corrects the article on p. 2459 in vol. 5.].
Although the Xenopus immunoglobulin heavy chain locus is structurally and functionally similar to mammalian IgH loci, Xenopus antibodies are limited in heterogeneity, and they mature only slightly in affinity during immune responses. During the antibody response of isogenic frogs to DNP-KLH, mu and upsilon cDNA sequences using elements of the VH1 family were cloned, sequenced and compared with germline counterparts. There were zero to four mutations per sequence, mostly single base substitutions, in the framework and CDRs 1 and 2 of VH. No mutations were found in JH. Since the point mutation rate was only 4- to 7-fold lower than that calculated for mice, affinity maturation does not seem to be limited by mutant availability. Because of a relatively low ratio of replacement to silent mutations in the CDRs and a very high ratio of GC to AT base pairs altered by mutation, it is suggested that the problem results from the absence of an effective mechanism for selecting mutants, which in turn might be related to the absence of germinal centers in Xenopus.
Paroxysmal nocturnal haemoglobinuria (PNH), an acquired clonal blood disorder, is caused by the absence of glycosyl phosphatidylinositol (GPI)-anchored surface proteins due to a defect in a specific step of GPI-anchor synthesis. The cDNA of the X-linked gene, PIG-A, which encodes a protein required for this step has recently been isolated. We have carried out a molecular and functional analysis of the PIG-A gene in four cell lines deficient in GPI-linked proteins, obtained by Epstein-Barr virus (EBV) transformation of affected B-lymphocytes from PNH patients. In all four cell lines transfection with PIG-A cDNA restored normal expression of GPI-linked proteins. In three of the four cell lines the primary lesion is a frameshift mutation. In two of these there is a reduction in the amount of full-length mRNA. The fourth cell line contains a missense mutation in PIG-A. In each case the mutation was present in the affected granulocytes from peripheral blood of the patients, but not in normal sister cell lines from the same patient. These data prove that PNH is caused in most patients by a single mutation in the PIG-A gene. The nature of the mutation can vary and most likely occurs on the active X-chromosome in an early haematopoietic stem cell.
Recently, microsatellite instability (MI) has been demonstrated in some types of human cancers. In this study, we attempted to determine the frequency of MI in endometrial cancers and evaluate whether replication error (RER)-positive phenotype is correlated with known genetic mutations or the aberrations of other pathways in endometrial cancers. Seventy-two primary endometrial cancers were examined for microsatellite instability. Eleven tumors (15%) had RERs at two or more microsatellite loci, suggesting that generalized MI may be a molecular manifestation of endometrial cancers. We next examined whether the MI was associated with changes in the K-ras protooncogene, p53 tumor suppressor gene, and 18q LOH, which were frequently detected in endometrial cancers. The MI did not confer the potential to produce point mutations in the K-ras gene or 18q LOH, whereas the data were insufficient to identify the correlation between MI and p53 mutations in the cancers. These results suggest the presence of multiple mutation subsets that act in a complementary fashion in endometrial cancer development.
Among highly proliferating tissues the intestinal tissue is of particular interest. Techniques are available that permit an insight into how intestinal crypts as the basic macroscopic tissue unit are regenerated from a small population of self-maintaining stem cells. However, neither the precise number of these stem cells nor their properties are known. We have recently suggested a model of stem cell organization which explains the life cycle of murine intestinal crypts, their birth (by crypt fission) and extinction rates, as well as their size distribution on a quantitative basis (Loeffler & Grossman, 1991). The model assumptions involve two stochastic branching processes, one for the growth of several independent indistinguishable stem cells and a second for a threshold dependent crypt fission process. New data have now become available challenging the above concept. They relate to the conversion of crypts to monoclonal phenotypic expression after mutagenic events, presumably taking place in single stem cells. A detailed analysis of these data is shown here utilizing a more elaborate version of the above model. The new data are consistent with this model within the range of parameters predicted previously. We conclude that the cellular regeneration of intestinal crypts can be explained on the basis of several indistinguishable stem cells which can replace each other.
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