Antibody Diversity: Is It All Coded for by the Germ Line Genes?

We present a statistical method for detection of palindromes in mRNA or DNA, starting from the protein sequence. Analysis of immunoglobulin genes by this method demonstrates that palindromic se uences are not randomly distributed. They are located at each side of the hypervariable regions in the variable (V) genes, whereas no such regular design is observed in the constant (C) genes. In addition, palindromic sequences overlap the V-C junction in all immunoglobulin classes and significant palindromes are present near residue 216 of the heavy chain, which is the end of deletions in many heavy chain diseases. The relevance of these palindromes to gene translocation and generation of diversity in antibodies is discussed.A palindromic DNA sequence possesses a 2-fold rotational axis of symmetry perpendicular to the-helix and, as a consequence, the base sequences of the two strands are identical when read in the same polarity:Such DNA sequences are universally distributed and have been shown to exist in bacteriophages (1, 2), in Escherichia coli plasmids (3), and in eukaryotic DNA (4). They can be rather long (300-1200 nucleotide pairs), and some are able to form hairpin-like structures in single-stranded DNA (4). They can also be found in heterogeneous nuclear RNA (5), which is characterized by its resistance to RNase. It has been suggested (6) that their structure (cruciform-like DNA) might be a recognition site for proteins acting at the DNA level; on this basis, molecular models for genetic recombination have also been proposed (7). These speculations are supported by several findings: the Lac operator is double stranded and about 27 base pairs long; and the sequence of its RNA transcription copy has been determined (8) and contains a partial 2-fold rotational symmetry. In phage X, the gene cl is surrounded by three left operators and three right operators. Each operator has an axis of partial 2-fold symmetry (9). Inverted repeats have been found in transposable DNA segments of bacteria (10,11 pointed out that palindromic sequences may occur near the V-C junction and that these sequences could be recognition sites for enzymes involved in the translocation process (14).In this paper, we present a method for detection of palindromes in mRNA, starting from the protein sequences. The method is applied to the case of immunoglobulins, and we show that the palindromic sequences are not randomly distributed but are located preferentially at the boundary of hypervariable regions and near the V-C junction. METHODS The searching procedure is an extension of Fitch's polypeptide comparison method (15): the protein sequence is translated into its degenerate mRNA sequence, which is then analyzed for the presence of palindromes. This analysis is performed by matching, in a sliding process, each subsequence of 11 nucleotides (polarity 5'-3') with all the possible consecutive subsequences of 11 nucleotides (polarity 3'-5'). For each match, a complementation index and its probability for occurring by chance are computed and ...

Section: S=0mentioning

confidence: 95%

“…The palindromic structures that are also preferred sites for recombination (31) could promote exchange of hypervariable segments between the new and old gene sets. This can conserve variability during V gene expansion (32) and may also explain the sharing of similar hypervariable regions by antibodies of different framework sequences (30).…”

Section: S=0mentioning

confidence: 99%

On the location of palindromes in immunoglobulin genes.

Wuilmart¹,

Urbain²

1977

“…2). The light (5-7) and the heavy chains (8,9) (13) have now been found in a mouse f3-globin (14,15), rabbit globin (16), avian ovalbumin (17,18), viral proteins (19)(20)(21)(22)(23)(24)(25), some ribosomal DNA genes in Drosophila (26)(27)(28)(29), and tRNA genes in yeast (30).…”

mentioning

confidence: 99%

Variable region genes for the immunoglobulin framework are assembled from small segments of DNA—A hypothesis

Kabat

Bilofsky³

1978

Sequences of each of the four framework segments FRI, FR2, FR3, and FR4 of the variable regions (V-regions) of light and heavy chains of immunoglobulins were grouped into sets with identical sequences. Sets contained from 1 to 18 members. When each V-region was traced from one FR to the next, it was seen that members of the same set in FRI could be associated with different sets in FR2, FR3, and FR4. This suggests that the framework for the light and heavy chain V-regions is assembled during embryonic development from sets of minigenes for each FR segment. FR4 The variable region (V-region) of immunoglobulin light and heavy chains may be divided into four framework (FR) and three complementarity-determining [hypervariable (1)] regions or segments (CDR). In the V-region of light chains (VL), FR1, FR2, FR3, and FR4 comprise residues 1-23,35-49,57-88, and 98-107, respectively, and CDR1, CDR2, and CDR3 comprise residues 24-34, 50-56, and 89-97, respectively (1, 2). CDR1 and CDR3 may vary in length due to insertions (1-3, cf. 4). In the V-region of heavy chains (VH), the FR are 1-30, 36-49, 66-94, and 103-113 and CDR1, CDR2, and CDR3 are residues 31-35B, 50-65, and 95-102; CDR1, CDR2, CDR3, and FR3 may also have insertions (cf. 2). The light (5-7) and the heavy chains (8, 9) have been divided into subgroups based essentially on sequences in FR1. Each framework sequence for a subgroup is considered to represent a single structural gene. Recently, Potter (10) (11), for which the nucleic acid of the mouse VA gene was sequenced (12) from 12-day-old mouse embryo DNA, can be interpreted as having been assembled from FRI and FR3 from MOPC 315 (a VATI) and FR2 (except for Glu vs. Gln as discussed below) from MOPC 104E (a VAT); CDR1 had the same sequence in both VAT and VAIT whereas CDR2 and CDR3 had sequences of both VAT and VxIT. In line with recent findings in viral and mammalian genes, the present analysis predicts that intervening sequences will be found between the FR fragments and that the CDR will be recombined into the FR segments during early embryonic development with elimination of the intervening sequences to assemble a V-region gene. Intervening sequences (13) have now been found in a mouse f3-globin (14,15), rabbit globin (16), avian ovalbumin (17, 18), viral proteins (19-25), some ribosomal DNA genes in , and tRNA genes in yeast (30). RESULTSThe data base (2) of V-region sequences supplemented by additional published data (31-46) contained complete and partial sequences of 509 light and 225 heavy chains. Sequences of human VJT, mouse VK, rabbit VK, and human and mouse VHIIT, the only groups with sufficient data, were sorted so that identical sequences of FRI, FR2, FR3, and FR4 were located; Glx and Asx were accepted as equivalent to Gln or Glu and Asn or Asp in assembling sets. These were grouped further and exAbbreviations: V-region, variable region; VL, VH, variable regions of light and heavy chains, respectively; V,>, variable regions of K light chains; VXA, VAII, variable regions of two subgro...

“…Another inbred mouse strain was shown to produce many different antibody molecules against DNP (dinitrophenyl), of which more than 500 crossreact strongly with TNP (trinitrophenyl) (2). The occurrence of species-specific residues in the V-regions of antibody polypeptide chains and the mendelian inheritance of rabbit a allotypes point against a germ line theory (3). Nevertheless, controversial ideas of evolutionary versus somatic generation of antibody diversity have continued to coexist (3,4).…”

mentioning

confidence: 99%

“…The occurrence of species-specific residues in the V-regions of antibody polypeptide chains and the mendelian inheritance of rabbit a allotypes point against a germ line theory (3). Nevertheless, controversial ideas of evolutionary versus somatic generation of antibody diversity have continued to coexist (3,4). The direct approach to solving this controversy is to count the number of antibody V-genes present in the DNA of one cell.…”

mentioning

confidence: 99%

Evidence for Somatic Generation of Antibody Diversity

Tonegawa¹,

Steinberg²,

Dube

et al. 1974

114

RNA preparations containing 70-80% mouse K-chain mRNA have been prepared. The remainder consists of many RNA species, each of which represents a small fraction of the total RNA. The K-chain mRNA preparation hybridizes with mouse liver DNA with biphasic kinetics, indicating that it consists of two fractions -"unique" and "reiterated." Competition hybridization experiments show that the homology among the unique fractions from different mRNAs is the same as the homology among the amino acid sequences of the corresponding K-chains. Hence, in addition to the C-region (constantregion) sequences, (most of) the V-region (variableregion) sequences are also derived from unique germ line genes. The reiterated fractions from different K-chain mRNAs show essentially complete homology with each other. This fraction seems to consist mostly of sequences which do not code for amino-acid sequences of the secreted polypeptide chain, i.e., the "external" section of the mRNA molecule. It is concluded that the number of germ line genes is too small to account for the observed diversity of antibody molecules.One of the most fascinating problems of immunology is posed by the enormous diversity of the antibody molecules that one animal is able to synthesize. One inbred strain of mice has been shown to be capable of producing eight thousand different antibody molecules against the hapten NIP (4-hydroxy-5-iodo-3-nitrophenacetyl) (1). Another inbred mouse strain was shown to produce many different antibody molecules against DNP (dinitrophenyl), of which more than 500 crossreact strongly with TNP (trinitrophenyl) (2). The occurrence of species-specific residues in the V-regions of antibody polypeptide chains and the mendelian inheritance of rabbit a allotypes point against a germ line theory (3). Nevertheless, controversial ideas of evolutionary versus somatic generation of antibody diversity have continued to coexist (3, 4). The direct approach to solving this controversy is to count the number of antibody V-genes present in the DNA of one cell. To this end, we have analysed the kinetics of hybridization of a mouse K-chain mRNA to mouse liver DNA. For the analysis to be meaningful, three points are essential:1. The physical purity of the K-chain mRNA preparation must be known; that is, the amount and nature of contaminants must be determined.Abbreviations: Cot, moles of deoxyribonucleotide,/liter of incubation X see; SSC, standard saline-citrate solution (0.15 M sodium chloride-0.015 M sodium citrate, pH 7); 2 X SSC means that the concentration of the solution used is two times that of the standard saline-citrate solution; V-region, variable region; Cregion, constant region. 40272. Since K-chain mRNA contains both a unique and a reiterated fraction (5, 6), it is necessary to assign the V-region sequences to one of these fractions.3. The proportion of all possible V-genes which would have cross-hybridized with V-region sequences of the particular K-chain mRNA used, must be determined.We have recently reported a preliminary account ...