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C Rada

Publications and source records attributed to C Rada.

16 recordsLinked to original sources
PubMed2001

Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination.

Isotype switching involves a region-specific, nonhomologous recombinational deletion that has been suggested to occur by nonhomologous joining of broken DNA ends. Here, we find increased donor/acceptor homology at switch junctions from PMS2-deficient mice and propose that class switching can occur by microhomology-mediated end-joining. Interestingly, although isotype switching and somatic hypermutation show many parallels, we confirm that PMS2 deficiency has no major effect on the pattern of nucleotide substitutions generated during somatic hypermutation. This finding is in contrast to MSH2 deficiency. With MSH2, the altered pattern of switch recombination and hypermutation suggests parallels in the mechanics of the two processes, whereas the fact that PMS2 deficiency affects only switch recombination may reflect differences in the pathways of break resolution.

Adenosine Triphosphatases↗
PubMed2001

The intrinsic hypermutability of antibody heavy and light chain genes decays exponentially.

Somatic hypermutation, essential for the affinity maturation of antibodies, is restricted to a small segment of DNA. The upstream boundary is sharp and is probably related to transcription initiation. However, for reasons unknown, the hypermutation domain does not encompass the whole transcription unit, notably the C-region exon. Since analysis of the downstream decay of hypermutation is obscured by sequence-dependent hot and cold spots, we describe a strategy to minimize these fluctuations by computing mutations of different sequences located at similar distances from the promoter. We pool large databases of mutated heavy and light chains and analyse the decay of mutation frequencies. We define an intrinsic decay of probability of mutation that is remarkably similar for heavy and light chains, faster than anticipated and consistent with an exponential fit. Indeed, quite apart from hot spots, the intrinsic probability of mutation at CDR1 can be almost twice that of CDR3. The analysis has mechanistic implications for current and future models of hypermutation.

Animals↗
PubMed2001

In vivo and in vitro studies of immunoglobulin gene somatic hypermutation.

Following antigen encounter, two distinct processes modify immunoglobulin genes. The variable region is diversified by somatic hypermutation while the constant region may be changed by class-switch recombination. Although both genetic events can occur concurrently within germinal centre B cells, there are examples of each occurring independently of the other. Here we compare the contributions of class-switch recombination and somatic hypermutation to the diversification of the serum immunoglobulin repertoire and review evidence that suggests that, despite clear differences, the two processes may share some aspects of their mechanism in common.

Animals↗
PubMed2000

Memory in the B-cell compartment: antibody affinity maturation.

In the humoral arm of the immune system, the memory response is not only more quickly elicited and of greater magnitude than the primary response, but it is also different in quality. In the recall response to antigen, the antibodies produced are of higher affinity and of different isotype (typically immunoglobulin G rather than immunoglobulin M). This maturation rests on the antigen dependence of B-cell maturation and is effected by programmed genetic modifications of the immunoglobulin gene loci. Here we consider how the B-cell response to antigen depends on the affinity of the antigen receptor interaction. We also compare and draw parallels between the two processes, which underpin the generation of secondary-response antibodies: V gene somatic hypermutation and immunoglobulin heavy-chain class switching.

Animals↗
PubMed1998

Automated detection of point mutations using fluorescent sequence trace subtraction.

The final step in the detection of mutations is to determine the sequence of the suspected mutant and to compare it with that of the wild-type, and for this fluorescence-based sequencing instruments are widely used. We describe some simple algorithms forcomparing sequence traces which, as part of our sequence assembly and analysis package, are proving useful for the discovery of mutations and which may also help to identify misplaced readings in sequence assembly projects. The mutations can be detected automatically by a new program called TRACE_DIFF and new types of trace display in our program GAP4 greatly simplify visual checking of the assigned changes. To assess the accuracy of the automatic mutation detection algorithm we analysed 214 sequence readings from hypermutating DNA comprising a total of 108 497 bases. After the readings were assembled there were 1232 base differences, including 392 Ns and 166 alignment characters. Visual inspection of the traces established that of the 1232 differences, 353 were real mutations while the rest were due to base calling errors. The TRACE_DIFF algorithm automatically identified all but 36, with 28 false positives. Further information about the software can be obtained from http://www.mrc-lmb.cam.ac.uk/pubseq/

Algorithms↗
PubMed1998

Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting.

Likely creation of mismatches during somatic hypermutation has stimulated interest in the effect of mismatch repair deficiency on the process. Analysis of unselected mutations in the 3' flank of VH rearrangements in germinal center B cells revealed that MSH2 deficiency caused a 5-fold reduced mutation accumulation. This might reflect ectopic effects of the Msh2 disruption; indeed, the mice exhibit other perturbations within the B cell compartment. However, that MSH2 (or factors dependent upon it) plays a role in the mechanism of mutation fixation is indicated by a strikingly increased focusing of the mutations on intrinsic hot spots. We propose two phases to hypermutation targeting. The first is hot spot focused and MSH2 independent; the second, MSH2-dependent phase yields a more even spread of mutation fixation.

Animals↗
PubMed1998

Monitoring and interpreting the intrinsic features of somatic hypermutation.

We have used both normal and transgenic mice to analyse the recruitment and targeting of somatic hypermutation to the immunoglobulin loci. We compare methods for analysing hypermutation and discuss how large databases of mutations can be assembled by PCR amplification of the rearranged V-gene flanks from the germinal centre B cells of normal mice as well as by transgene-specific amplification from transgenic B cells. Such studies confirm that hypermutation is preferentially targeted to the immunoglobulin V gene with the bcl6 gene, for example, escaping this intense mutational targeting in germinal centre B cells. We review our data concerning the nature of the hypermutation domain and the targeting of hotspots within that domain. We consider how enhancer-mediated recruitment of hypermutation to the immunoglobulin loci operates in a clonally maintained fashion and illustrate how both the degree of expression and demethylation of the transgene broadly correlate with its mutability.

Animals↗
PubMed1997

The 5' hypermutation boundary of kappa chains is independent of local and neighbouring sequences and related to the distance from the initiation of transcription.

The hypermutation of antibody genes targets 1-2 kb of DNA which includes the rearranged V(D)J gene segments. The precise nature, location and limits of the targeted region are of considerable interest in terms of the mechanism of hypermutation. We have analyzed the frequency and distribution of mutations in the 5' region of immunoglobulins using several modified kappa transgenes. We found that the position of the boundary, relative to the transcription initiation site, is not affected by the sequence of the V segment or by substituting the kappa chain promoter for a beta-globin promoter. Furthermore, the deletion of the leader intron (containing the hypermutation boundary) does not affect hypermutation per se, but shifts the boundary from the leader intron to the V region such that the distance between the boundary and the site of initiation of transcription remains constant. These results show that the position of the hypermutation boundary (about 185 bases downstream of the site of initiation of transcription) is not defined by the nucleotide sequence but rather by the distance to a fixed upstream position. Although mutations are also observed in the region upstream of the boundary, the frequency at which they occur is one order of magnitude lower relative to the frequency observed in the V segment. Nonetheless this upstream mutation rate remains more than two orders of magnitude higher than that of somatic genes. We discuss possible mechanisms explaining the nature and position of the boundary in the context of an error-prone DNA repair model.

Animals↗
PubMed1996

The targeting of somatic hypermutation.

Somatic hypermutation does not occur randomly within immunoglobulin V genes but, rather, is preferentially targeted to certain nucleotide positions (hot spots) and away from others (cold spots). Cold spots often coincide with residues essential for V gene folding. Hotspots, which appear to be strategically located to favour affinity maturation, are most frequently located in the CDRs (particularly CDR1) though conserved hotspots are also found at the base of FR3. Hotspots are in part created by local DNA sequence and the strong biases of codon usage in V genes indicate that the genes have evolved such that somatic hypermutation is targeted to those parts of the V where it is likely to prove most useful. These features of mutational hotspots and biased codon usage are also evident in V genes of lower animals suggesting that diversification by strategic targeting of non-templated mutation may have evolved early in antigen receptor evolution.

Animals↗
PubMed1994

The 5' boundary of somatic hypermutation in a V kappa gene is in the leader intron.

The maturation of the immune response involves the hypermutation of antibody genes and the selection of B cells expressing receptors with improved antigen binding properties. Somatic hypermutation of antibody genes is targeted to a small region approximately 1 kb surrounding the rearranged V gene. The precise definition of the 5' limit is not yet clear since the data base of somatic mutations upstream of the V region is very restricted. The available data suggest that it lies close to the promoter region and this has been used to implicate transcription in the mechanism leading to hypermutation. Here we present an extensive analysis of mutations in the 5' region of a single kappa light chain gene. A large data base from highly mutated sequences was obtained from anti-oxazolone hybridomas expressing the V kappa Ox1-J kappa 5 light chain and from polymerase chain reaction-derived clones from splenic and Peyer's patches of transgenic mice expressing the same V kappa Ox1-J kappa 5 gene combination. Although mutations were found in the 5'-flanking segment, the rate of mutation in the V-J segment was about 20-fold higher. A sharp decline between those two mutation rates is evident but the boundary was found in the leader intron of the V kappa Ox1 gene, about 150 bases downstream of the initiation of transcription site.

Amino Acid Sequence↗
PubMed1993

Affinity maturation leads to differential expression of multiple copies of a kappa light-chain transgene.

Transgenic animals containing rearranged heavy or light chains are used to study the process of hypermutation, which characterizes the maturation of the antibody response. LK6 mice contain five copies of a transgene coding for a light chain produced in response to the hapten 2-phenyloxazolone. We have selected hybridomas from secondary responses that express the transgene as the only light chain. Some of these hybridomas contain transgene copies carrying mutations known to improve antibody affinity. We have analysed the expression of the five transgene copies in those hybridomas. We report here that the somatic hypermutation process can affect the successful expression of antibody light-chain transgenes. When mutations that improve the antibody affinity appear in one transgene copy, antigenic selection favours cells that downregulate the other copies at multiple levels of gene expression, including examples where nonsense mutations correlate with a drop in messenger RNA level.

Amino Acid Sequence↗
PubMed1993

Passenger transgenes reveal intrinsic specificity of the antibody hypermutation mechanism: clustering, polarity, and specific hot spots.

We have analyzed somatic hypermutation in mice carrying an immunoglobulin kappa transgene in order to discriminate mutations that reflect the intrinsic specificity of the hypermutation mechanism from those highlighted by antigenic selection. We have immunized animals with three different immunogens. With one immunogen, the antigen-specific B cells express a transgenic kappa chain, which does not form part of the antibody; the transgene is a passenger free to accumulate unselected mutations. With the other two immunogens, the transgenic kappa chain constitutes the light chain of the expressed antibody. A comparison of the transgene mutations obtained under these different circumstances allows us to identify common features that we attribute to the intrinsic specificity of the hypermutation process. In particular, it yields only base substitutions and leads to hot spots occurring in individual positions (e.g., the second base of the Ser-31 codon). The mutations preferentially accumulate around the first complementarity-determining region. The process exhibits specific base substitution preferences with transitions being favored over transversions. We propose that these substitution preferences can be used to discriminate intrinsic from antigen-selected hot spots. We also note that hypermutation distinguishes between the coding and noncoding strands since pyrimidines (particularly thymidines) mutate less frequently than purines.

Amino Acid Sequence↗
PubMed1991

Mutation and selection during the secondary response to 2-phenyloxazolone.

The most characteristic feature of the mouse antibody response to the hapten 2-phenyloxazolone is the recurrent expression of the light-chain variable region Igk-VO chi 1 gene in its germ-line or mutated configuration. The analysis of somatic mutants of the Igk-VO chi 1 gene reported here indicates that, as found during the primary response, hypermutation is also activated during the secondary response. Somatic mutations in the Igk-VO chi 1 gene increased in sequences obtained at day 14 and day 21 in the primary response and again in the secondary response at days 3, 5, and 7. The ratio of replacement to silent mutations also increased, particularly between days 5 and 7, suggesting that a stage of negative selection operates on new somatic mutants generated in the secondary response. Most Igk-VO chi 1 mutants isolated in the secondary response had the features of selected memory clones (i.e., they carried mutations known to increase binding affinity for the hapten). However, some clones had chain-termination codons, and others had mutations predicting a nonfunctional light chain. At least three and possibly five of these clones also expressed the mutation characteristic of the memory response to 2-phenyloxazolone (His-34----Asn-34/Gln-34). We conclude that after a second antigenic challenge, new somatic variants, including some leading to the loss of antigen binding, are generated by hypermutation of cells derived from the memory pool.

Amino Acid Sequence↗
PubMed1990

Concerted evolution of class I genes in the major histocompatibility complex of murine rodents.

Full-length cDNA sequences of two class I major histocompatibility complex molecules from the DA strain of Rattus norvegicus are reported. One codes for the classical class I restriction element RT1.Aa, which maps to the locus in the rat major histocompatibility complex homologous to H-2K in the mouse. The other probably codes for a soluble nonclassical class I molecule present in DA rat serum; a short deletion in the fifth exon implies that the translated product will terminate in the membrane-spanning region. These sequences have been compared with mouse classical class I sequences as well as with three published rat class I cDNA partial sequences. The results show, first, that "locus-specific" substitutions from the H-2K, H-2D, and H-2L data set are scrambled in the RT1.Aa molecule; a majority of these substitutions have H-2D/L-specific features. Second, the data show that the four rat sequences are strikingly similar to one another regardless of locus or haplotype of origin; they share a number of apparently species-specific features that distinguish them all from mouse classical class I sequences, which likewise share distinctive features of their own. The results suggest that segmental sequence exchange plays a major role in determining the evolution of sequence in class I major histocompatibility complex molecules.

Amino Acid Sequence↗
PubMedDate not supplied

The response of the portal vein to an oral glucose load.

Changes of total blood flow, velocities, and cross-sectional area in the portal vein before and after oral glucose administration were studied using image-directed Doppler ultrasonography. Portal vein cross-sectional area, flow velocity, and total blood flow increased significantly compared with baseline studies. Relative increase in these values compared with baseline studies (95% confidence intervals) are presented, and future applications of this method are discussed.

Administration, Oral↗