Allelic constitution of the hemoglobin beta chain in wild populations of the house mouse,Mus musculus

We studied the allelic frequency of the hemoglobin beta chain (Hbb) of wild mice, Mus musculus, collected from 46 localities, mostly in Asia and surrounding areas. The wild populations in the northern part of China, Korea, and the central part of Japan exhibited an almost monomorphic distribution of Hbbp. In the southern part of Asia, the frequency of Hbbp decreased and Hbbd was predominant. Although Hbbs and Hbbd are generally found in Europe, the Hbbp allele was present in Southeastern Europe (Bulgaria). In the light of these results, the Hbbp allele might have originated in mice of northern Asia.     

Use of a new mouse beta-globin haplotype (Hbbs2) to study hemoglobin expression during development

Mice of the mutant haplotype (Hbbs2) produce a variant beta-s globin (beta-s2major) which can be distinguished from beta-smajor and beta-sminor by cellulose acetate electrophoresis and ion exchange chromatography. Mice homozygous for this mutation were used to study the relative quantities of the mutant beta-s2major and normal beta-sminor globins specified by the two adult beta-globin genes of the Hbbs2 haplotype during development. At 11.5 days of gestation, beta-s2major comprises under 20% and beta-sminor over 80% of the adult beta-globin. The relative level of beta-sminor decreases through fetal development; at birth beta-sminor represents 33.7% of the beta-globin. The adult value of 71.0% beta-s2major and 29.0% beta-sminor globin is expressed in mice 6 days after birth. In mildly anemia alpha-thalassemic heterozygotes (Hbab2(th)/Hbab;Hbbs2/Hbbs2) the level of beta-sminor globin increases from 29.0 to 37.9%, but beta-sminor is elevated only slightly (29.0 to 33.9%) in asymptomatic beta-thalassemic heterozygotes (Hbab/Hbab;Hbbd3(th)Hbbs2). The relative quantity of beta-sminor is increased significantly (29.0 to 41.4%) in doubly heterozygous alpha-thalassemic, beta-thalassemic mice (Hbab2(th)/Hbab;Hbbd3(th)/Hbbs2). The relative levels of expression of the beta 1s2major and beta 2sminor globin genes of Hbbs2/Hbbs2 mice correlates well with the expression of the beta 1dmajor and beta 2dminor globin genes of Hbbd/Hbbd mice during development and in response to hematological stress caused by thalassemia. Expression of the beta 1sminor globin gene should not have been affected by the ENU-induced base substitution in the beta 1smajor gene. Therefore, we propose that the beta 1sminor gene is also expressed in mice of the Hbbs haplotype. The results also indicated that the two adult beta-globin genes of the Hbbs2 and, presumably, of the Hbbs haplotypes are regulated independently as are the beta 1dmajor and beta 2dminor genes of the Hbbd and Hbbp haplotypes.     

Evolution of the mouse beta-globin genes: a recent gene conversion in the Hbbs haplotype

We have determined the complete nucleotide sequence of the two nonallelic adult beta-globin genes of the C57BL/10 mouse. These genes, designated beta s and beta t, show a sequence similarity of 99.6% over the region bordered by the translational start and stop codons. Both beta s and beta t encode functional polypeptide chains that are identical. A comparison of the C57BL/10 beta-globin haplotype, Hbbs, with that of the BALB/c mouse, Hbbd, suggests that the two haplotypes have distinct evolutionary histories. The two adult beta-globin genes of the Hbbd haplotype, beta dmaj and beta dmin, are 16% divergent at the nucleotide level and encode distinct polypeptides that are synthesized in differing amounts. Our analysis indicates that a gene correction mechanism has been operating on the Hbbs chromosome to keep beta s and beta t evolving in concert, whereas on the Hbbd chromosome, beta dmin has diverged considerably from beta dmaj. We suggest that gene conversion is responsible for the maintained similarity of the Hbbs genes. Furthermore, we attribute the divergence of the Hbbd genes in part to the absence of a region of simple-sequence DNA within the large intervening sequence of beta dmin. We propose that this region of DNA plays a role in facilitating gene conversion. The deletion of this area in beta dmin introduced a block of nonhomology between the beta dmaj-beta dmin gene pair and thus may have inhibited further gene correction within the Hbbd haplotype.      

A new haplotype of the beta-globin gene complex, Hbbw1, in Chinese wild mouse

A new pattern was observed in the electrophoretic survey of the hemoglobin beta chain (Hbb) in Chinese wild mice, Mus musculus. The electrophoretic mobility of the major component of the new Hbb was identical to that of Hbbs on cellulose acetate plate, although it was almost identical to that of Hbbd or Hbbp on acrylamide gel. This suggests that the major component of Chinese Hbb has a unique primary structure. A minor component of the new Hbb was completely different from that of the other three Hbb haplotypes well known. These results indicate that the Hbb-b1 and Hbb-b2 of the new Hbb haplotype, assigned Hbbw1, are unique genes in their molecular structure. So far, Hbbw1 has been observed in northwestern China.     

Chromosomal rearrangements associated with LINE elements in the mouse genome.

Two segments of DNA that have apparently inserted in the interval between the two adult beta-globin genes in BALB/c (Hbbd haplotype) but not in C57B1/10 (Hbbs haplotype) mouse strains have been described (1). These putative insertions, each about 1000 bp in length, mapped near a repetitive element. To determine the precise position of these alleged insertions, their target sites, and the nature of their boundaries, we cloned and sequenced the appropriate regions of both chromosomes. One of the two segments is not an insertion but rather a region between two independently integrated L1 repetitive elements (LINEs) (2), one in Hbbd and the other in the Hbbs chromosome. The other segment is an insertion of 940 bp which is located within the L1 element in the Hbbd chromosome. This insert is unusual in that it exists in only one copy in the BALB/c genome.     

Evidence for close linkage of a mouse light chain marker with the Ly-2,3 locus.

Light chains associated with normal serum immunoglobulin can be resolved into a finite number of discrete focusing bands by isoelectric focusing. Four distinct light chain patterns can be distinguished among the inbred mouse strains. In the present studies inheritance of the characteristic light chain patterns has been studied in the AKXL recombinant inbred lines (derived from C57L/J and AKR/J parental lines) and in the inbred Ly-2a,3a congenic line B6.PL-Ly-2aLy-3a/Cy as well as in individual backcross animals of an incipient Ly-2a,3a congenic strain. Virtually complete concordance was observed for the expression of light chains characteristic of phenotype B (AKR-J-like) and the expression of the Ly-2a,3a allele. This observation indicates that a locus controlling light chain structure and/or expression is closely linked (less than 2.6 map units) to the Ly-2,3 locus on mouse Chromosome 6. The locus controlling normal light chain IF-patterns has been designated Ef1.     

Strain distribution and linkage relationship of a mouse embryonic hemoglobin variant

Search for structural variants of three globin chains (x, y, z), synthesized only during mouse embryonic hematopoiesis, was carried out by electrophoretic analysis of blood from 12-day embryos, all with C57BL/6 mothers, and fathers from 115 inbred stocks selected for their diverse genetic origins. Structure of the -chains of adult hemoglobins differed among the tested strains, with 57 carrying the Hbb ^sallele, 56 the Hbb ^dallele, and two the Hbb ^pallele. The search revealed no x- or z-chain variants but confirmed and extended knowledge of a previously described y-chain variant. Blood of all embryos sired by males from the 57 Hbb ^sstrains contained only y¹-chains, while blood of all embryos sired by Hbb ^dor Hbb ^pmales contained y2-chains as well as the y¹-chains inherited from their C57 BL/6 mother. The locus controlling structure of the y-chain of mouse embryonic hemoglobins is thus extremely closely linked to the locus controlling structure of adult hemoglobin -chain, with maximum possible recombination frequency less than 0.019.This work was supported in part by Grants CA-01074 from the National Cancer Institute, USPHS, and GM 18684 from the National Institute of General Medical Sciences, in part by Grant ACS-VC58 from The American Cancer Society, in part by grants to the Jackson Laboratory from the Bushrod H. Campbell and Adah F. Hall Charity Fund and the Robert Sterling Clark Foundation, and in part by the Jackson Laboratory Endowment Fund. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Mouse haemoglobin beta chains. Comparative sequence data on adult major and minor beta chains from two species, Mus musculus and Mus cervicolor.

In inbred Mus musculus several different beta chains are known. In certain strains two beta chains are produced in unequal amounts by the two closely linked genes of the doublet breeding unit allele Hbb(d): Betadmaj and betadmin. One strain has a variant doublet allele, Hbb(p), which produces a variant minor beta chain, betapmin (the major beta chain, betapmaj, may not differ from betadmaj chain). Certain other strains have a singlet allele, Hbb(s), that produces only one beta chain, betas. Other species have different beta-chain patterns. In M. cervicolor two variant major beta chains are found, betacmaj (d-like) and betacmaj (s-like), both of which were found associated with minor beta chains. M. caroli has only one, 'Leporelike' beta chain, with structural features characteristic predominantly of betadmin chain in the N-terminal half and of betadmaj chain in the C-terminal half. The present paper presents sequence data on betas, betadmaj, betadmin, betapmin and betacmaj (d-like) chains. The data on betadmin chain cover almost the whole of that chain and show a minimum of nine differences from betadmaj chain and two from betapmin chain. It is suggested that the data on the beta chains of the various species show evidence for the past occurrence of double crossovers over regions within a gene coding for only one or a few amino acids, which events can be explained by the 'hybrid DNA' models of genetic recombination. Supplementary information on the amino acid sequence of the proteins has been deposited as Supplementary Publication SUP 50067 (36 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1976) 153, 5.     

Concerted evolution led to high expression of a prosimian primate delta globin gene locus

The delta globin gene in simian primates is either weakly expressed (in hominoids and New World monkeys) or silent (in Old World monkeys). In prosimian primates, however, an unequal homologous crossover between the psi eta and delta loci of lemurs produced a hybrid psi eta delta pseudogene locus, whereas in tarsier the delta locus encodes a beta-type chain found in 18% of adult tarsier hemoglobin molecules. In the present study, the nucleotide and amino acid sequences of the galago delta and beta globin genes and their encoded peptides were determined, and evidence is provided showing that the galago delta locus encodes a beta-type chain (beta 2) found in 40% of the galago fetal and postnatal hemoglobin molecules, whereas the beta locus encodes the remaining 60% of the beta-type chain (beta 1). Galago beta 1 and beta 2 chains differ from each other by only one amino acid residue. The homology between the galago delta and beta loci extends from 800 base pairs 5' of the proximal CCAAT element to near the end of exon 3 as a result of a recombination event in which beta sequence replaced delta sequence. After this initial recombination event, concerted evolution between the loci continued over their conserved coding, intron 1, and promoter regions but failed to occur between the two loci in their intron 2 and distal 5'-flanking sequences where the two loci have now diverged by 20%. Calculations based on this divergence value and on a rate of noncoding sequence evolution of 4.2 x 10(-9) to 5.5 x 10(-9) substitutions/site/year for the lorisiform lineage to galago yielded a date of 18-24 million years ago for the initial recombination event. The fact that the promoter sequences of the galago delta locus are the same as that of the galago beta locus may account for the high level of expression of the galago delta gene.     

Oxygen Association-Dissociation and Stability Analysis on Mouse Hemoglobins with Mutant α- and β-Globins

Oxygen association-dissociation and hemoglobin stability analysis were performed on mouse hemoglobins with amino acid substitutions in an alpha-globin (alpha 89, His to Leu) and a beta-globin (beta 59, Lys to Ile). The variant alpha-globin, designated chain 5m in the Hbag2 haplotype, had an high oxygen affinity and was stable. The variant beta-globin, (beta s2) of the Hbbs2 haplotype, also had an elevated oxygen affinity and in addition was moderately unstable in 19% isopropanol. Hemoglobins from the expected nine (Hbag2/Hbag2;Hbbs/Hbbs x Hbaa/Hbaa;Hbbs2/Hbbs2) F2 genotypes can be grouped into five classes of P50 values characterized by strict additivity and dependency on mutant globin gene dosage; physiologically, both globin variants gave indistinguishable effects on oxygen affinity. The hemoglobin of normal mice (Hbaa/Hbaa;Hbbs/Hbbs) had a P50 = 40 mm Hg and the hemoglobin of Hbag2/Hbag2;Hbbs2/Hbbs2 F2 mice had a P50 = 25 mm Hg (human P50 = 26 mm Hg). Peripheral blood from Hbag2/Hbag2;Hbbs/Hbbs, Hbaa/Hbaa;Hbbs2/Hbbs2 and Hbag2/Hbag2;Hbbs2/Hbbs2 mice exhibited normal hematological values except for a slightly higher hematocrit for Hbag2/Hbag2;Hbbs/Hbbs and Hbag2/Hbag2;Hbbs2/Hbbs2 mice, slightly elevated red cell counts for mice of the three mutant genotypes, and significantly lower values for the mean corpuscular volume and mean corpuscular hemoglobin for Hbag2/Hbag2;Hbbs2/Hbbs2 mice.     

Allelic exclusion at the TCR delta locus and commitment to gamma delta lineage: different modalities apply to distinct human gamma delta subsets

Expression of a beta-chain, as a pre-TCR, in T cell precursors prevents further rearrangements on the alternate beta allele through a strict allelic exclusion process and enables precursors to undergo differentiation. However, whether allelic exclusion applies to the TCR delta locus is unknown and the role of the gamma delta TCR in gamma delta lineage commitment is still unclear. Through the analysis of the rearrangement status of the TCR gamma, delta, and beta loci in human gamma delta T cell clones, expressing either the TCR V delta 1 or V delta 2 variable regions, we show that the rate of partial rearrangements at the delta locus is consistent with an allelic exclusion process. The overrepresentation of clones with two functional TCR gamma chains indicates that a gamma delta TCR selection process is required for the commitment of T cell precursors to the gamma delta lineage. Finally, while complete TCR beta rearrangements were observed in several V delta 2 T cell clones, these were seldom found in V delta 1 cells. This suggests a competitive alpha beta/gamma delta lineage commitment in the former subset and a precommitment to the gamma delta lineage in the latter. We propose that these distinct behaviors are related to the developmental stage at which rearrangements occur, as suggested by the patterns of accessibility to recombination sites that characterize the V delta 1 and V delta 2 subsets.     

Structure of the murine immune response I-A beta locus: sequence of the I-A beta gene and an adjacent beta-chain second domain exon

The murine major histocompatibility complex I region encodes two class II antigens, I-A and I-E. From a mouse spleen DNA cosmid library of the b haplotype, we isolated a clone containing the entire I-A beta gene and a separate exon encoding a beta-chain second domain (A beta 2). The A beta gene, encompassing more than 6 kb, is encoded by six exons corresponding to the different domains of the A beta polypeptide. The translated A beta amino acid sequence displays 73% homology to human DC beta chains; homologies to other subsets of human beta chains are lower, establishing that I-A corresponds structurally to DC. The A beta 2 exon is about 20 kb centromeric to the A beta gene. Its translated amino acid sequence includes all the conserved amino acids of other class II beta-chain second domains. It shows about 60% homology to each of three subsets of human beta chains available for comparison, and to the A beta chain. No A beta 2 first domain exon has been detected with A beta or DC beta probes.     

Hereditary heat-labile hexosaminidase B: a variant whose homozygotes synthesize a functional HEX A.

Homozygosity for a mutant allele at the beta-chain locus of hexosaminidase (HEX), resulting in a variant of heat-labile HEX B, is reported for the first time in two healthy children. HEX activity in their sera, leukocytes, and cultured skin fibroblasts is severely deficient when measured on the synthetic substrate 4-MU-GLcNAc. However, their cultured skin fibroblasts synthesize and process both alpha and beta chains of HEX, and their lymphoid cells hydrolyze normally the natural ganglioside GM2. This mutation is, therefore, different from at least one of the beta-chain mutations found in previously published families with heat-labile HEX B.     

Functional consequences of a T cell receptor D beta 2 and J beta 2 gene segment deletion

The TCR beta-chain locus of NZW mice carries an 8.8-kb deletion which encompasses the C beta 1, D beta 2, and all six J beta 2 gene segments. On a theoretical basis, the absence of D beta 2 and J beta 2 gene segments in this strain should result in a 70% reduction of the diversity of the TCR repertoire. To experimentally assess the effects of this deletion, we bred the NZW TCR beta-chain allele onto a BALB/c background and tested the ability of this new congenic strain to respond to a panel of 22 random Ag. T cells from BALB/c.beta NZW mice responded to all 22 Ag tested but the magnitude of the response to a large proportion of these Ag (11 of 22) was markedly reduced when compared with T cells from BALB/c mice. Responses to the remaining Ag were either comparable (9 of 22) or occasionally even enhanced (2 of 22) compared with BALB/c mice. In addition, we found that the frequency of V beta 6- and V beta 8.1-bearing T cells was increased by approximately 20% in BALB/c.beta NZW mice. These results suggest that D beta 2 and J beta 2 gene segments are required to maintain a diverse T cell repertoire and that their deletion from the genome may confer a significant selective disadvantage in the wild.     

Complex signatures of selection and gene conversion in the duplicated globin genes of house mice

Results of electrophoretic surveys have suggested that hemoglobin polymorphism may be maintained by balancing selection in natural populations of house mice, Mus musculus. Here we report a survey of nucleotide variation in the adult globin genes of house mice from South America. We surveyed nucleotide polymorphism in two closely linked alpha-globin paralogs and two closely linked beta-globin paralogs to test whether patterns of variation are consistent with a model of long-term balancing selection. Surprisingly high levels of nucleotide polymorphism at the two beta-globin paralogs were attributable to the segregation of two highly divergent haplotypes, Hbbs (which carries two identical beta-globin paralogs) and Hbbd (which carries two functionally divergent beta-globin paralogs). Interparalog gene conversion on the Hbbs haplotype has produced a highly unusual situation in which the two paralogs are more similar to one another than either one is to its allelic counterpart on the Hbbd haplotype. Levels of nucleotide polymorphism and linkage disequilibrium at the two beta-globin paralogs suggest a complex history of diversity-enhancing selection that may be responsible for long-term maintenance of alternative protein alleles. The alternative two-locus beta-globin haplotypes are associated with pronounced differences in intraerythrocyte glutathione and nitric oxide metabolism, suggesting a possible mechanism for selection on hemoglobin function.     

Inhibition of T-cell receptor beta-chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56lck.

The variable region genes of the T cell receptor (TCR) alpha and beta chains are assembled by somatic recombination of separate germline elements. During thymocyte development, gene rearrangements display both an ordered progression, with beta chain formation preceding alpha chain, and allelic exclusion, with each cell containing a single functional beta chain rearrangement. Although considerable evidence supports the view that the individual loci are regulated independently, signaling molecules that may participate in controlling TCR gene recombination remain unidentified. Here we report that the lymphocyte-specific protein tyrosine kinase p56lck, when overexpressed in developing thymocytes, provokes a reduction in V beta--D beta rearrangement while permitting normal juxtaposition of other TCR gene segments. Our data support a model in which p56lck activity impinges upon a signaling process that ordinarily permits allelic exclusion at the beta-chain locus.     

Haemoglobins of the shark, Heterodontus portusjacksoni. III. Amino acid sequence of the beta-chain

The amino acid sequence of the beta-chain of the principal haemoglobin from the shark H. portusjacksoni has been determined. The chain has 141 residues, the same as that of mammalian alpha-chains and less than the 146 residues of mammalian beta-chains or the 148 residues of the alpha-chain from the tetrameric shark haemoglobin. The sequence was deduced from the sequences of peptides obtained by digestion of the globin or its cyanogen bromide fragments with trypsin, chymotrypsin, pepsin and papain. The difference in length of the beta-chain is most readily accounted for by the absence of the D helix. This small helical section is normally present in myoglobins and beta-globins but absent in alpha-chains. The deduction that it is absent from shark beta-chain is based on consideration of homology. The beta-chain shows the insertion of histidine beta2 and the deletions corresponding to residues A17 and AB1 relative to alpha-and myoglobin chains. The reactive thiol group in shark haemoglobin was shown by radioactive labelling to be residue 51 in the beta-chain, immediately preceding the E helix. The amino acid sequence of shark beta-chain shows 92 differences from human beta-chain, significantly more differences than shown by chicken or frog beta-chains, in line with its earlier time of divergence. If the tertiary structure of the shark beta-chain is the same as that of the horse then there are two changes in the alpha1beta2 contact site in oxyhaemoglobin and an additional one in deoxyhaemoglobin. When both alpha- and beta-chain contacts are considered there is a total of nine changes in residues involved in the alpha1beta2 contacts. There is no Bohr effect in shark haemoglobin, and of the residues normally involved in this effect the C-terminal histidine residue of the beta-chain is present, but the aspartyl (FG1) residue to which it is salt-linked is not, being replaced by a glutamyl residue.     

Inheritance of idiotype expression unlinked to the H chain allotype locus. Novel features of genetic control in the Ia.7 system

We have observed a pattern of inherited idiotype expression in three mouse strains that is unexpected from the genetics of the strains: a dominant idiotype that was expressed at high levels in two parental strains was expressed only at low levels in a heavy chain allotype congenic strain derived from them. In the C3H.SW strain, the antibody response to the class II MHC Ag I-E is of limited diversity, with dominant expression of an idiotype and the V kappa 21 L chain. The C57BL/10 strain expresses the same idiotype at high levels, whereas the CWB/12 strain, which was derived by replacing the Ig H chain Igh-Cj allele of C3H.SW with the Igh-Cb allele derived from C57B1/10, has been found to express little of this dominant idiotype. CWB/12 responds, with titers equal to those of the parental strains, to the I-E epitope responsible for dominant idiotype expression, and it expresses normal V kappa 21 levels; thus deficiencies in epitope-specific responsiveness or in V kappa 21 expression cannot explain the low Id expression in CWB/12. Furthermore, Southern blot analysis of three VH families gave no evidence of recombination within the the VH locus of CWB/12, which was Igh-Vb throughout. Black-cross analysis demonstrated that expression of the dominant idiotype segregated independently of Ig allotype, and was therefore due to genes unlinked to the H chain gene locus. To our knowledge, this pattern of Id expression is unprecedented, and indicates the need for caution in the interpretation of studies using allotype congenic strains. It also demonstrates a role for genes outside the Igh locus in the control of Id expression.