AUTONOMA - Nikos Anastasopoulos - Autonomy: Reflections on Scale and Complexity
… And even more complexity in the human immunoglobulin λ light-chain gene locus
1
82 out that ' . . . reductive activation may simply reflect the operation of a device which ensures that sulphydryl groups are kept in a reduced state despite the photo- synthetic generation of oxidants such as H202'. This viewpoint gains strength in view of two recent reports. Nakano and Asada s at Kyoto University have calculated that the rate of l-h(h production in illumi- nated choloroplasts is about 10 t/,M sec -1, and although most of this 14_202 is effec- tively scavenged by an active ascorbate peroxidase, some presumably manages to escape this fate and instead deactivates enzymes by oxidative formation of disul- phide bridges. Rosa and Whatley 6 at the University of Oxford have evidence for such a system from experiments with iso- lated intact chloroplasts. The CO2 fixation by these chloroplasts was lowered by 1-1202 (which was generated by broken chloro- plasts that are always found in preparations of intact chloroplasts). However, the addi- tion of small amounts of uncouplers reversed this effect of H20~, apparently because the resultant increased electron flow led to a greater rate of light-mediated enzyme activation that compensated for the increased rate of enzyme deactivation of 1-1202. TIBS - March 1 982 References 1 Nishizawa, A. N. and Buchanan, B. B. (1981) J. Biol. Chem. 256,6119--6126 2 Mohamed, A. H. and Anderson, L. E. (1981) Arch. Biochern. Biophys. 209, 606-612 3 Sugiyama, T. and Hatch,M. D. (1981) Plant Cell Physiol. 22, 115-126 4 Leegood, R. C. and Walker, D. A. (1980)Arch. Biochem. Biophys. 200, 575-582 5 Nakano, Y. and Asada, K. (1981) Plant Cell Physiol. 22, 867~80 6 Rosa,L. and Whatley,F. R. (1981) Plant Physiol. 68, 364-370 GRAHAME KELLY Departmentof Biochemistry andNutrition,University of NewEngland, Annidale,Australia . .. And even more complexity in the human immunoglobulin h light-chain gene locus In the December Journal Club I I discussed the surprising complexity that had been revealed in the gene locus coding for mouse immunoglobulin h-light chains. Now Leder's groups have reported that ~, genes form an even larger and more complex fam- ily within the human genome. lmmunoglobulin molecules consist of two light chains and two heavy chains, both of which can be divided into an N-terminal region of variable amino acid sequence, responsible for binding antigen, and a C-terminal region of reasonably constant amino acid sequence, which mediates the various effector functions of antibodies s. Variable and constant regions are encoded by distinct, distant gene segments that are brought together during lymphocyte dif- ferentiation 4. In all mammalian species there are several serologically distinct types of heavy chains and two types of light chains, K and h. Whilst ~ chains are only a minor component of serum immunoglobu- lin in the mouse, they are abundant in man. Within the human ~ chain family, there are at least four nonallelic forms (isotypes) that differ by limited amino acid substitutions within the constant region, to produce the serological markers Kern, Oz and Mcg (see Table l). Thus, the known ~. chain proteins TABLE 1. Amino acid substitutions in the constant regionof the four known human h chainisotypes Serological markers Kern Oz Mcg Critical amino acid residues 112 114 152 163 190 ÷ - Ala Set GLY Thr Arg Ala Ser Ser Thr Arg Ala Ser Ser TIu" LYS ÷ + ASN THR GLY LYS Arg are Oz Kern +, Oz Kern-, Oz÷, Kern and Mcg ÷ Oz Kern ~. Several additional h chain variants have been recognized, but it is not known whether these represent allelic variants or distinct isotypes. To isolate and examine the diversity and arrangement of human k constant region genes, Heiter et al. ~ took advantage of the cross hybridization between mouse and human immunoglobulin sequences and used a mouse C~ probe to screen a library of recombinant lambda bacteriophage con- taining overlapping fragments from human liver DNA. To verify that the clones that they isolated actually contained Cx genes, they demonstrated by Southern hybridiza- tion that the clones detected sequences that had undergone the expected rearrange- ments associated with gene activation 4 in DNA from ?~-producing lymphocytes, whereas in r-producing lymphocytes the corresponding sequences were in the same arrangement as in germline DNA. By com- parative restriction enzyme mapping they ordered most of the overlapping DNA fragments that they had isolated into a con- tiguous stretch of some 50,000 nucleotides, containing at least six h-like gene sequences, regularly spaced along the chromosome about 5000 base pairs apart. In addition to the six genes in this linkage group, there are at least three additional ~.-like genes, which Leder's group has cloned, that have not yet been linked to this major cluster. Thus, there are an extremely large number of human Cx genes. The nucleotide sequence that was deter- mined for three of the six clustered genes revealed that they corresponded to the isotypes Mcg ~ Oz- Kern +, Oz Kern and Oz ÷ Kern . The nucleotide sequences of these three genes show very strong homology, the Oz- Kern- and Oz + Kern ~) El~vier Biomedical Press 1982 0376 - 5067/82/G~00 - 0(]<}0/$02 75 genes having only eight silent base substitu- tions and one base substitution giving rise to the one amino acid difference. The Kern ÷ Oz gene has not yet been located, but is possibly one of the others within this clus- ter. Whether the other ~,-like genes (both within this cluster and distant from it) encode new isotypes or are silent 'pseudo' genes has not yet been determined. Interestingly, a region within the middle of the ~-gene cluster appears to be very labile. When cloned in Escherichia coli this region was frequently deleted, in a manner reminiscent of the behaviour of switch-recombination sites from the mouse heavy-chain locus (for example, Ref. 6). In man, this segment also appears to be un- stable. Southern hybridization analysis of DNA from 15 individuals revealed at least two examples of polymorphisms of length within this region. It is therefore tempting to speculate that the C~ locus may be able to undergo rearrangements of biological rele- vance, perhaps in a manner similar to the heavy-chain constant region locus7. References 1 Gough, N. (1981) Trends Biochem. Sci. 6, December,pp. VI-VII 2 Hieter, P. A., Hollis, G. F., Korsmeyer,G. J., Waldmann, T. A+ and Leder, P. (1981) Nature (London) 294, 536-540 3 Gaily, J. A. (1973) in The Antigens (Sela, M., ed.), Vol. 1, pp. 162-298, AcademicPress, New York 4 Gough, N. (1981) Trends Biochem. Sci. 6, 203-209 5 Fett, J. W. and Deutsch, H. F. (1975) lmmunochemistry 12,643-.652 6 Lui, C. P., Tucker, P. W., Mushinski,J. F. and Blatmer, F. R. (1980)Science 209, 1348-1353 7 Cory, S., Jackson, J. and Adams, J. M. (1980) Nature (London) 285, 450--456 NICK GOUGH European Molecular Biology Laboratory, Postfach 10.2209, 6900Heidelberg,F.R.G.
Transcript of … And even more complexity in the human immunoglobulin λ light-chain gene locus
82
out that ' . . . reductive activation may simply reflect the operation of a device which ensures that sulphydryl groups are kept in a reduced state despite the photo- synthetic generation of oxidants such as H202'. This viewpoint gains strength in view of two recent reports. Nakano and Asada s at Kyoto University have calculated that the rate of l-h(h production in illumi- nated choloroplasts is about 10 t/,M sec -1, and although most of this 14_202 is effec- tively scavenged by an active ascorbate peroxidase, some presumably manages to escape this fate and instead deactivates enzymes by oxidative formation of disul-
phide bridges. Rosa and Whatley 6 at the University of Oxford have evidence for such a system from experiments with iso- lated intact chloroplasts. The CO2 fixation by these chloroplasts was lowered by 1-1202 (which was generated by broken chloro- plasts that are always found in preparations of intact chloroplasts). However, the addi- tion of small amounts of uncouplers reversed this effect of H20~, apparently because the resultant increased electron flow led to a greater rate of light-mediated enzyme activation that compensated for the increased rate of enzyme deactivation of 1-1202.
T I B S - March 1 982
References 1 Nishizawa, A. N. and Buchanan, B. B. (1981)
J. Biol. Chem. 256, 6119--6126 2 Mohamed, A. H. and Anderson, L. E. (1981)
Arch. Biochern. Biophys. 209, 606-612 3 Sugiyama, T. and Hatch, M. D. (1981) Plant Cell
Physiol. 22, 115-126 4 Leegood, R. C. and Walker, D. A. (1980)Arch.
Biochem. Biophys. 200, 575-582 5 Nakano, Y. and Asada, K. (1981) Plant Cell
Physiol. 22, 867~80 6 Rosa, L. and Whatley, F. R. (1981) Plant Physiol.
68, 364-370 GRAHAME KELLY
Department of Biochemistry and Nutrition, University of New England, Annidale, Australia
. . . A n d even m o r e complex i ty in the h u m a n i m m u n o g l o b u l i n h l ight-chain gene locus
In the December Journal Club I I discussed the surprising complexity that had been revealed in the gene locus coding for mouse immunoglobulin h-light chains. Now Leder's group s have reported that ~, genes form an even larger and more complex fam- ily within the human genome.
lmmunoglobulin molecules consist of two light chains and two heavy chains, both of which can be divided into an N-terminal region of variable amino acid sequence, responsible for binding antigen, and a C-terminal region of reasonably constant amino acid sequence, which mediates the various effector functions of antibodies s. Variable and constant regions are encoded by distinct, distant gene segments that are brought together during lymphocyte dif- ferentiation 4. In all mammalian species there are several serologically distinct types of heavy chains and two types of light chains, K and h. Whilst ~ chains are only a minor component of serum immunoglobu- lin in the mouse, they are abundant in man. Within the human ~ chain family, there are at least four nonallelic forms (isotypes) that differ by limited amino acid substitutions within the constant region, to produce the serological markers Kern, Oz and Mcg (see Table l). Thus, the known ~. chain proteins
TABLE 1. Amino acid substitutions in the constant region of the four known human h chain isotypes
Serological markers
Kern Oz Mcg
Critical amino acid residues
112 114 152 163 190
÷ - Ala Set GLY Thr Arg Ala Ser Ser Thr Arg Ala Ser Ser TIu" LYS
÷ + ASN THR GLY LYS Arg
are Oz Kern +, Oz Kern-, Oz ÷, Kern and Mcg ÷ Oz Kern ~. Several additional h chain variants have been recognized, but it is not known whether these represent allelic variants or distinct isotypes.
To isolate and examine the diversity and arrangement of human k constant region genes, Heiter et al. ~ took advantage of the cross hybridization between mouse and human immunoglobulin sequences and used a mouse C~ probe to screen a library of recombinant lambda bacteriophage con- taining overlapping fragments from human liver DNA. To verify that the clones that they isolated actually contained Cx genes, they demonstrated by Southern hybridiza- tion that the clones detected sequences that had undergone the expected rearrange- ments associated with gene activation 4 in DNA from ?~-producing lymphocytes, whereas in r-producing lymphocytes the corresponding sequences were in the same arrangement as in germline DNA. By com- parative restriction enzyme mapping they ordered most of the overlapping DNA fragments that they had isolated into a con- tiguous stretch of some 50,000 nucleotides, containing at least six h-like gene sequences, regularly spaced along the chromosome about 5000 base pairs apart. In addition to the six genes in this linkage group, there are at least three additional ~.-like genes, which Leder's group has cloned, that have not yet been linked to this major cluster. Thus, there are an extremely large number of human Cx genes.
The nucleotide sequence that was deter- mined for three of the six clustered genes revealed that they corresponded to the isotypes Mcg ~ O z - Kern +, Oz Kern and Oz ÷ Kern . The nucleotide sequences of these three genes show very strong homology, the O z - Kern - and Oz + Kern
~ ) E l ~ v i e r B i o m e d i c a l Press 1982 0 3 7 6 - 5067 /82 /G~00 - 0(]<}0/$02 75
genes having only eight silent base substitu- tions and one base substitution giving rise to the one amino acid difference. The Kern ÷
Oz gene has not yet been located, but is possibly one of the others within this clus- ter. Whether the other ~,-like genes (both within this cluster and distant from it) encode new isotypes or are silent 'pseudo' genes has not yet been determined.
Interestingly, a region within the middle of the ~-gene cluster appears to be very labile. When cloned in Escherichia coli this region was frequently deleted, in a manner reminiscent of the behaviour of switch-recombination sites from the mouse heavy-chain locus (for example, Ref. 6). In man, this segment also appears to be un- stable. Southern hybridization analysis of DNA from 15 individuals revealed at least two examples of polymorphisms of length within this region. It is therefore tempting to speculate that the C~ locus may be able to undergo rearrangements of biological rele- vance, perhaps in a manner similar to the heavy-chain constant region locus 7.
References 1 Gough, N. (1981) Trends Biochem. Sci. 6,
December, pp. VI-VII 2 Hieter, P. A., Hollis, G. F., Korsmeyer, G. J.,
Waldmann, T. A+ and Leder, P. (1981) Nature (London) 294, 536-540
3 Gaily, J. A. (1973) in The Antigens (Sela, M., ed.), Vol. 1, pp. 162-298, Academic Press, New York
4 Gough, N. (1981) Trends Biochem. Sci. 6, 203-209
5 Fett, J. W. and Deutsch, H. F. (1975) lmmunochemistry 12,643-.652
6 Lui, C. P., Tucker, P. W., Mushinski, J. F. and Blatmer, F. R. (1980)Science 209, 1348-1353
7 Cory, S., Jackson, J. and Adams, J. M. (1980) Nature (London) 285, 450--456
NICK GOUGH
European Molecular Biology Laboratory, Postfach 10.2209, 6900 Heidelberg, F.R.G.
