Origin of rat beta-globin haplotypes containing three and five genes

We have reported in rat three adult beta-gene haplotypes containing either five or three genes. Detailed sequence analysis reveals that the leftmost gene is the major gene and that at the opposite end downstream lies the minor gene. All of the genes lying between them are minor-major hybrids indicating their origin by unequal crossing-over. In two haplotypes beta-globin genes were found with an L1(1) element inserted directly into IVS2. The described results allow the formulation of a pathway of mutational events leading from the ancient two-beta-gene rodent ancestor through a three-gene haplotype to five-gene haplotypes, one of which is postulated to have arisen in common laboratory strains since their capture in the wild.     

Further evidence for recombination between mouse hemoglobin beta b1 and b2 genes based on the nucleotide sequences of intron, UTR, and intergenic spacer regions

Nucleotide sequences of the intron regions and UTRs (Untranslated regions) of the hemoglobin beta adult genes, b1 and b2, and of the intergenic spacer region were determined for mouse strains representing the d, p, and w1 hemoglobin haplotypes defined by protein electrophoretic analyses. The hypothesis of recombination of the b1 and b2 genes between the d and w1 haplotypes previously reported in the cDNA nucleotide sequences was confirmed by neighbor-joining analyses of the intron regions and UTRs within the b1 and b2 genes, suggesting that all of the structures of hemoglobin beta adult genes support the hypothesis that the p haplotype was established by hybridization between d and w1 haplotype mice. The resultant recombinant of the p haplotype was found to have a d-like b1 gene and a w1-like b2 gene. In addition to the possible recombination, a break point was suggested around 2-3 kb downstream of the b1 gene within the intergenic spacer region, despite the absence of clear properties that could stimulate the recombination machinery. Some large insertions or deletions (indels) specific to the p or d haplotypes were located within the intergenic spacer region, in which the 1010-bp indel specific to the p haplotype was shared by all examined strains representing the p haplotype.     

The E beta hot spot of recombination in wild-derived natural recombinant MHC haplotypes. Cross-over site mapping and the identification of a 1.0-kb E beta deletion in the p and w14 haplotypes

Detailed molecular analysis of three wild-derived MHC haplotypes provided evidence for an important role of the E beta recombinational hot spot in the recent evolution of the mouse I region. Examination of RFLP and restriction maps of cloned DNA permitted the mapping of the natural cross-over events in the haplotypes carried by strains B10.GAA37 (w21) and B10.KPB128 (w19) to a fragment of DNA not exceeding 4.1 kb, which lies almost entirely within the intron separating the beta 1 and beta 2 exons of the E beta gene. In the w14 haplotype (strain B10.STC77), which appears to be a natural recombinant between a p-like parental haplotype and another wild-derived haplotype, the site of crossing over can be mapped to a segment between the beta 2 exon of the E beta gene (left border) and the E beta 2 gene (right border). This segment containing the cross-over site in the w14 haplotype includes the E beta hot spot. In addition, the w14 haplotype as well as the standard p haplotype contain a deletion of approximately 1.0 kb in the second intron of the E beta gene, which may represent the product of an unequal cross-over event in a E beta recombinational hot spot.     

Evidence supporting a single origin of the beta(C)-globin gene in blacks.

In order to characterize the origin(s) of the beta C-globin gene in blacks, 25 chromosomes bearing this gene were characterized at eight polymorphic restriction sites within the beta-globin gene cluster. Twenty-two of the 25 chromosomes were identical at all sites and possessed a haplotype seen only infrequently among beta A-bearing chromosomes in black Americans. Two different haplotypes were observed among the three exceptional chromosomes. These haplotypes were identical to the most common beta C allele in the 3' end of the beta-globin gene cluster, but differed in the 5' region. Partial haplotype analysis on an additional 14 beta C alleles demonstrated complete association with the typical beta C-associated polymorphisms in the 3' region of the cluster. These data can be most easily explained by a single origin of the mutation followed by spread of the mutation to other haplotypes through meiotic recombination 5' to the beta-globin gene.     

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.      

Two mouse early embryonic beta-globin gene sequences. Evolution of the nonadult beta-globins

We have determined the complete nucleotide sequence of two early embryonic beta-globin genes of the BALB/c mouse: beta h0 and beta h1 X beta h1 codes for the embryonic z protein, while the beta h0 gene may be a minor early embryonic beta-globin gene. The general sequence organization of both genes is entirely analogous to other functional globin genes. There is, however, a 220-base pair insertion of unique sequence within the first intron of beta h0 X beta h0 and beta h1 are 96% homologous for 260 base pairs 5' to the AUG initiation codon, and 93% homologous throughout their coding regions. Analysis of the 5'-flanking sequence demonstrates that these genes are more nonadult-like than adult-like. The sequences show evidence for gene conversions among the mouse nonadult beta-globin genes that were limited to individual exons, presumably by the presence of non-homologous introns. We propose that this arrangement has the beneficial evolutionary effect of allowing gene conversion to act independently on regions of the protein with different structural or functional responsibilities. beta h0 and beta h1 are evolutionary homologs to the human fetal and rabbit beta 3 genes, while their manner of expression is similar to rabbit beta 3 and dissimilar to human fetal expression. The evolutionary history of the human beta-globin genes, therefore, includes the recruitment of an embryonic gene to fetal developmental control.     

Beta-thalassemia genes in French-Canadians: haplotype and mutation analysis of Portneuf chromosomes.

beta-Thalassemia minor occurs at approximately 1% frequency in French-Canadians--in families residing in Portneuf County (population approximately 40,000) of Quebec province. We found eight different RFLP haplotypes at the beta-globin gene cluster in 37 normal persons and in 12 beta-thalassemia heterozygotes from six families. beta-Thalassemia genes in these families associated with two haplotypes only: Mediterranean I and Mediterranean II. There were two different beta-thalassemia mutations segregating in the Portneuf population: an RNA processing mutation (beta(+)IVS-1,nt110) on haplotype I (five families) and a point mutation leading to chain termination (beta(0) nonsense codon 39) on haplotype II (one family). The distribution of 5' haplotypes on normal beta A Portneuf chromosomes compared with other European populations was most similar to that in British subjects (data for French subjects have not yet been reported). Genealogical reconstructions traced the ancestry of carrier couples to settlers emigrating from several different regions of France to New France in the 17th century. These findings indicate genetic diversity of a greater degree among French-Canadians than recognized heretofore.     

Isolation and nucleotide sequence analysis of the beta-type globin pseudogene from human, gorilla and chimpanzee

The beta-globin gene cluster of human, gorilla and chimpanzee contain the same number and organization of beta-type globin genes: 5'-epsilon (embryonic)-G gamma and A gamma (fetal)-psi beta (inactive)-delta and beta (adult)-3'. We have isolated the psi beta-globin gene regions from the three species and determined their nucleotide sequences. These three pseudogenes each share the same substitutions in the initiator codon (ATG----GTA), a substitution in codon 15 which generates a termination signal TGG----TGA, nucleotide deletion in codon 20 and the resulting frame shift which yields many termination signals in exons 2 and 3. The basic structure of these psi beta-globin genes, however, remains consistent with that found for functional beta-globin genes: their coding regions are split by two introns, IVS 1 (which splits codon 30, 121 base-pairs in length) and IVS 2 (which splits codon 104, 840 to 844 base-pairs in length). These introns retain the normal splice junctions found in other eukaryotic split genes. The three hominoid psi beta-globin genes show a high degree of sequence correspondence, with the number of differences found among them being only about one-third of that predicted for DNA sites evolving at the neutral rate (i.e. for sites evolving in the absence of purifying selection). Thus, there appears to be a deceleration in the rate of evolution of the psi beta-globin locus in higher primates.     

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.     

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.     

Hemoglobin E in Europeans: further evidence for multiple origins of the beta E-globin gene

We have determined haplotypes for the known restriction site polymorphisms in the beta-globin gene cluster in two families of European ancestry containing individuals who are heterozygous for hemoglobin E. In both families, the beta E mutation is associated with a haplotype not previously found among the haplotypes of beta E chromosomes in Southeast Asia. Moreover, in one family, the mutation is present in a beta-gene framework not found in beta E chromosomes of Southeast Asia. These data provide further evidence of multiple independent origins of the beta E mutation in human populations.     

Beta-globin gene haplotypes and alpha-thalassemia analysis in Babinga pygmies from Congo-Brazzaville

We analyzed beta-globin gene cluster haplotypes and deletional alpha+-thalassemia (-alpha3.7kb) in 54 Babinga pygmy subjects from Congo-Brazzaville. The beta(S)-globin gene frequency was 0.065 and that of the deletional alpha-globin gene (-alpha3.7kb) was 0.29. Eighty-five percent of the beta(S) chromosomes and 13% of the beta(A) chromosomes were associated with the Bantu haplotype, 10% of beta(A) chromosomes with the Senegal haplotype, and the remaining beta chromosomes with atypical haplotypes. None of the chromosomes were of the Benin haplotype. These results are clearly of anthropological and evolutionary interest. They also support earlier observations that alpha+-thalassemia is prevalent at a high frequency in African populations.     

Molecular characterization of seven beta-thalassemia mutations in Asian Indians

To characterize systematically the mutations which produce beta-thalassemia in Asian Indians, we first determined the DNA polymorphism haplotype in the beta-globin gene cluster of 44 beta-thalassemia chromosomes in the ethnic group. Nine different haplotypes were observed. Upon molecular cloning and partial DNA sequencing of one beta-gene from each of eight haplotypes and two from the ninth, seven different mutations were found. None of these have been identified in Mediterranean patients, even among the five haplotypes which appeared identical in the two groups. Asian Indian mutations included one nonsense and three frameshift mutations, one deletion affecting an acceptor splice site, and two mutations affecting a donor splice site. The correlation of a specific mutation with a specific haplotype was high but not invariant. Two mutations were associated with more than one haplotype but, in each instance, the mutation spread to a new haplotype could be explained most simply by recombination 5' to the beta-globin gene. In addition, four mutations, one reported here and three others previously reported, have been observed on two chromosome backgrounds that are identical except for the status of a polymorphic HinfI site 5' to the beta gene. This HinfI site does not show significant linkage disequilibrium with markers both 5' and 3' to it, suggesting that it lies within a region of relative sequence randomization.     

Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6.

The CYP2D gene cluster on human chromosome 22 containing the functional cytochrome P450 gene CYP2D6 and two or three highly homologous pseudogenes is involved in a clinically important variation in the inactivation of drugs and environmental chemicals. Several mutant haplotypes of CYP2D6 have been identified by restriction analysis and by PCR-based allele-specific amplification. To understand the evolutionary sequence of mutational events as well as recently discovered interracial differences, we analyzed the arrangement of the CYP2D haplotype containing a common mutant allele of CYP2D6 associated with a XbaI 44-kb fragment. This haplotype contains four CYP2D genes instead of three. Comparison of the sequences of these genes with those of previously characterized haplotypes suggests that an early point mutation was followed by a crossover and a gene conversion event, the latter found preferentially in Caucasians. These data are consistent with the rapid evolution of this locus during "plant-animal warfare" with practical consequences for present-day defense of the organism against environmental adversity.     

Nonallelic members of the cytochrome c multigene family of the rat may arise through different messenger RNAs

We determined the nucleotide sequences of three nonallelic cytochrome c genes (from recombinant clones Ch4A-RC5, 6 and 8) isolated from the rat cytochrome c gene family. In contrast with a fourth gene (from Ch4A-RC4), which has an intron and correctly encodes rat cytochrome c, these three appear to be pseudogenes and resemble mRNA molecules in two respects: they are all missing the intron of clone 4, and sequence homology with clone 4 in their 3' noncoding regions abruptly ends at two different A-rich tracts reminiscent of poly(A) tails. We also detect three cytochrome c mRNAs of sizes 1400, 1100 and 700 nucleotides in several tissues of the adult rat. The size differences among the mRNAs can be accounted for by length heterogeneity in their 3' noncoding regions. Two of the 3' ends map to the two points where the mRNA-like genes diverge from clone 4 at poly(A) tracts. Furthermore, short direct repeats flank the genes of clones 5, 6 and 8 at the positions where their sequences diverge. The observations suggest that these members of the cytochrome c multigene family may arise through insertion into the genome of DNA copies of cytochrome c mRNAs.     

Molecular basis for the defective expression of the mouse Ew17 beta gene

Four of the eleven independent H-2 haplotypes of inbred mouse strains and approximately 15% of wild mouse chromosomes 17 fail to express the E alpha E beta class II histocompatibility (Ia) Ag. These E- haplotypes are defective in the expression of the E alpha and/or the E beta chain. None of the E beta defects has previously been described at the molecular level. In this study, we report the molecular basis for the defective expression of the E beta gene from the w17 haplotype of the H-2 congenic strain B10.CAS2, derived from wild Mus musculus castaneus. Comparison of the Ew17 beta genomic sequence to those of the functional Eb beta and Ed beta genes reveals a single base insertion in the RNA donor splice site of the first intron. By DNA shuffling, we have corrected the single base mutation, and we show by FACS analysis and 2-D PAGE of immunoprecipitates that the corrected Ew17 beta is expressed in L cells when co-transfected with an Ed alpha gene. Conversely, an Eb beta gene construct containing the mutant RNA splice site from Ew17 beta is not expressed. We conclude that the single base insertion in the first RNA splice donor site is the sole molecular defect in the Ew17 beta gene.