First Order Considerations in Analyzing the Generator of Diversity

A comparison between the cloned mouse DNA segments that were found to code for the X and K light chains of immunoglobulins established that there were seven short nucleotide sequences, two of which matched 6 out of 7, two 7 out of 8, two 8 out of 9, and one 9 out of 10 bases; these sequences were located either at homologous amino acid positions or at positions displaced by four amino acids or less. They all occurred in the framework regions (FRs), five next to the complementarity-determining regions (CDRs). Three of these were unique and did not occur elsewhere in the immunoglobulin nucleotides sequenced thus far or in DNAs of phage 4X174, phage G4, or simian virus 40. Five could serve as sites of joining by recombination or insertion of CDR to FR segments, and the invariant tryptophan that is the first residue of the second FR might serve as a sixth. These sites are consistent with the minigene or insertional hypotheses for the generation of antibody diversity but could also serve as points of recognition for a mutator enzyme or could serve to limit somatic mutation to the CDRs.The vast amount of experimental data on amino acid sequences of immunoglobulin variable regions (V regions) (1) has provided a basis for trying to understand antibody diversity. From a statistical analysis of such sequences (2, 3) as they were being accumulated, the three complementarity-determining (hypervariable) regions or segments (CDRs) were recognized in the light (L) and in the heavy (H) chains and predicted to form the walls of the antibody combining site; this has been verified by x-ray crystallography in four different laboratories (refs. 4-7; see ref. 8). As a consequence of this analysis each immunoglobulin chain was divided into four framework regions (FRs) separated by the three CDRs as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. It was recognized that a simple mechanism for assembling the chains could be the insertion of nucleotides coding for the CDRs into those coding for the FR segments to assemble a complete V-region gene (2, 3). When the various FR segments for each chain were arranged into sets (9), each set being made up of segments identical in sequence, it was noted that the members of a given FR1 set could be associated with different FR2 sets, FR3 sets, and FR4 sets. This independent assortment of FR sets led to the hypothesis (9) that the V-region gene is assembled from sets of germ line minigenes for the FR segments and inferentially from sets of minigenes for the CDRs. A minigene was defined as a DNA segment coding for a portion of the V region that shows some evidence of segregation as a functional unit independent of the rest of the DNA coding for the V region (10). It was further hypothesized that the minigenes were assembled somatically during differentiation. Because assortment was demonstrated only for FR segments, it would be independent of whether one or two CDR residues assorted with a given FR. The CDRs could not be examined for independent assortment because there were too few tha...

Section: G a C T G A G G A T G A G G C A A T A T A T T T C T G T G C supporting

confidence: 87%

Some sequence similarities among cloned mouse DNA segments that code for lambda and kappa light chains of immunoglobulins.

Kabat²,

Bilofsky³

1979

“…Mouse VA sequences have been shown by Weigert and Riblet (32) to exhibit a minimal amount of variation, limited exclusively to complementarity-determining regions, from a common VA sequence. The limited substitution pattern has been interpreted (32,33) to indicate that VA diversity is generated by somatic mutation from one or at most a few germ-line genes. Mouse VH amino-terminal sequences appear to be somewhat less structurally diverse than mouse VK (34).…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of antibody diversity: Multiple genes encode structurally related mouse κ variable regions

McKean¹,

Bell²,

Potter³

1978

The complete amino acid sequences of the variable regions of three mouse Vx-21 kappa chains (A22, Till, and CB10) and one partial sequence (B32) have been determined and are compared to four previously reported variable regions. These eight K variable region sequences have, with the exception of an amide difference at residue 1, identical amino-terminal 23-residue sequences, all are of the same length, and all have extensive amino acid sequence homology throughout the variable region. When these eight variable regions are grouped by sequence homology, five different groups (Vx-21A, B, C, D, and E) are present whose members share common sets of amino acids within a group. The origins of the genetic basis of antibody diversity have been approached through amino acid sequence analyses of immunoglobulin (Ig) variable regions of human, mouse, and rabbit K (VK), X (VX), and heavy chains (VH) (1), and more recently by nucleic acid hybridization (2-4). Amino acid sequences of immunoglobulin variable (V) regions derived from a single inbred strain of mice (e.g., BALB/c) most directly reflect the Ig-producing genetic potential of the individual. In such a system the role of genetic polymorphisms is reduced to a minimum and many of the differences in Ig primary structures can be related to the genetic mechanisms of Ig diversity.With the availability of several three-dimensional models of Ig V regions that have been determined by x-ray crystallography (5, 6) it has been possible to determine the functional components of the primary sequences of heavy and light chains.The framework segments of the V region sequences determine the conformation of the variable domain and interactions with other domains. Amino acid sequence differences in the framework segments of the V region are thought to be permissive changes that apparently do not alter the ability of the polypeptide to fold into a functional domain (7). The chains fold so that the complementarity-determining regions [also referred to as hypervariable regions (8) Mouse VK sequences that differ from all other mouse VK sequences by three amino acids within the amino-terminal 23 residues have been tentatively defined as an isotype. Such sequence differences within the amino-terminal 23-residue sequences are predictive of extensive sequence differences throughout the V regions whose complete sequences have been determined (10,13,17). By this criterion, 26 VK isotypes (VK-1 to VK-26) have been distinguished among 63 available sequences (16). It is expected that the criteria used to distinguish isotypes from the primary amino acid sequence data will become more refined as additional complete VK sequences become available and enable us to determine what genetic mechanisms are involved in generating antibody diversity.This involves the sequence analysis of eight V regions that have been assigned to the VK-21 isotype (16). In 1973 we (13) compared the sequence diversity in a group of four of these proteins. With the exception of an amide difference at residue 1, the four ...

“…"Somatic mutation models" depend upon random somatic events to generate a vast repertoire of specificities from limited genetic information. Presumably, such models include combinatorial association of heavy and light chain variable regions as well as genetic recombinational and mutational events (4,(10)(11)(12)(13)15). The hallmark of this theoretical framework is a reliance upon random events; the theory dictates that divergence from germline information would depend largely on antigenic experience of the individual.…”

Section: Characteristics Of Neonatal B-cell Responses To Influenzamentioning

confidence: 99%

“…The mechanism by which so large a repertoire of clon, otypes is generated, however, remains a persistent and intriguing question. Recent analyses of the heritable DNA sequences encoding murine immunoglobulin light chains imply that, although a fairly large number of genes may be present in some cases (7), it is likely that somatic events play an integral role in the expansion and expression of the repertoire (8)(9)(10)(11)(12)(13)(14)(15).…”

mentioning

confidence: 99%

“…For example, if all individuals of a strain exhibited identical repertoires at a certain age, the likelihood that nonrandom processes account for diversification would be greatly increased. Although similarities in the repertoires of adult individuals within a murine strain have been observed (4)(5)(6)(15)(16)(17)(18), the size of the adult repertoire and the concomitant low frequency of representation of most…”

mentioning

confidence: 99%

See 1 more Smart Citation

Patterned acquisition of the antibody repertoire: diversity of the hemagglutinin-specific B-cell repertoire in neonatal BALB/c mice.

Cancro¹,

Wylie²,

Gerhard³

et al. 1979