Molecular genetics of human erythrocyte MiIII and MiVI glycophorins. Use of a pseudoexon in construction of two delta-alpha-delta hybrid genes resulting in antigenic diversification

Human glycophorins alpha and delta (or A and B) specify the MNSs blood group antigens; they exhibit considerable structural variation among populations. We show that two variant phenotypes of Miltenberger class III and VI are encoded by similar hybrid glycophorin genes in a delta-alpha-delta arrangement. Restriction mapping identified altered fragments unique to the MiIII and MiVI genes. Genomic sequences spanning exons 2 to 4 of the two genes were obtained by allele-specific polymerase chain reaction. Restriction analysis and direct sequencing of the amplified DNA revealed that MiIII and MiVI genes are identical to the delta gene except that, in both, an internal segment of the delta gene has been replaced by its homologous counterpart of the alpha gene, resulting in a delta-alpha-delta hybrid structure. In the process of hybrid formation a portion of alpha exon 3 and intron 3, that carries a functional 5' splicing signal, has been fused to an exon-like sequence in the delta gene that retains a 3' but lacks a 5' splicing signal. These rearrangements created a composite exon resulting in the expression of the ordinarily unexpressed delta gene sequence and conferred the hybrid proteins with new antigenic specificities. The expression of this sequence in MiIII glycophorin is directly demonstrated by protein sequencing. MiIII and MiVI genes differ in the location of upstream (delta-alpha) and downstream (alpha-delta) breakpoints and in the length of sequence replacement. The delta-alpha breakpoints of the two genes occur at different locations within a 35-base pair sequence of exon 3 that is clustered with multiple inverted repeats, whereas the alpha-delta breakpoints reside downstream in two dissimilar blocks of sequences of intron 3. The minimal length of the delta gene sequence that has been replaced by the alpha gene is 55 base pairs in the MiIII gene and 131 base pairs in the MiVI gene. Such segmental DNA transfers may have proceeded unidirectionally through the mechanisms of gene conversion.     

Molecular analysis of a hybrid gene encoding human glycophorin variant Miltenberger V-like molecule

The genomic structure of a human glycophorin variant, Miltenberger class V-like molecule (MiV*), was examined. Southern blot analysis of total genomic DNA revealed that the 5' half of the MiV* gene derived from glycophorin A (GPA) gene whereas the 3' half derived from glycophorin B (GPB) gene. This structure is reciprocal to another glycophorin variant, Sta, which has a GPB-GPA hybrid structure. The genomic sequences around the crossing-over point were amplified by polymerase chain reaction, and the sequences were determined. Comparison of the nucleotide sequences of the GPA, GPB, and MiV* genes indicates that the crossing-over point is located in the region around the 3' end of intron 3 of the GPA gene. This place is different from the crossing-over point for Sta, which was found to be highly homologous to that for haptoglobin-related genes. However, the nucleotide sequences within the presumptive crossing-over point for the MiV* gene were found to be homologous in a reverse orientation to the crossing-over point proposed for haptoglobin-related genes. These results suggest strongly that homologous recombination through unequal crossing over can be facilitated by specific genomic elements such as those in common for formation of MiV*, Sta, and haptoglobin-related genes. The present study also localized the gene of the third glycophorin, GPE, at chromosome 4, q31.1 band, the same locus as for the GPA and GPB genes. The results indicate that GPE was not involved in generating MiV* or Sta hybrid gene despite the fact that it is localized adjacent to the GPA and GPB genes.     

Identification of the crossing-over point of a hybrid gene encoding human glycophorin variant Sta. Similarity to the crossing-over point in haptoglobin-related genes

One of the human glycophorin variants, Stones (Sta), has been shown to be the product of a hybrid gene of which the 5'-half derived from the glycophorin B (GPB) gene whereas the 3'-half derived from the glycophorin A (GPA) gene. The present study reveals the crossing-over point of this hybrid gene from the analysis of polymerase chain reaction products. The genomic sequences encompassing the region corresponding to exon 3 to exon 4 of GPA were amplified by polymerase chain reaction with oligonucleotide primers synthesized according to GPA and GPB genomic sequences (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623). After subcloning the products, the nucleotide sequences derived from GPA, GPB, and putative Sta genes were determined. Comparison of the nucleotide sequences of GPA, GPB, and Sta genes indicate that the crossing-over took place 200 base pairs upstream from the first nucleotide of exon 4. Intriguingly, the nucleotide sequence surrounding the putative crossing-over point is homologous to the crossing-over point proposed for haptoglobin genes (Maeda, N., McEvoy, S.M., Harris, H.F., Huisman, T.H.J., and Smithies, O. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7395-7399). These results suggest strongly that homologous recombination through unequal crossing-over can be facilitated by specific genomic elements, such as those in common in these two crossing-over events. The present study also revealed that this Sta individual has a variant GPA gene; substitution of adenine for guanine at the nucleotide for codon 39 results in substitution of lysine for arginine at amino acid 39, and loss of an SstI restriction site.     

Gene conversion confined to a direct repeat of the acceptor splice site generates allelic diversity at human glycophorin (GYP) locus.

The glycophorin locus (GYP) on the long arm of chromosome 4 encodes antigens of the MNSs blood group system and displays considerable allelic variation among human populations. The genomic structure and organization of a variant glycophorin allele specifying a novel Miltenberger (Mi)-related phenotype, MiX, were examined. This variant probably arose from a gene conversion event involving a direct repeat of the acceptor splice site. Southern blot analysis indicated that MiX gene derived its 5' and 3' portions from glycophorin B or delta gene but its internal part from glycophorin A or alpha gene. Genomic sequences encompassing the rearranged regions of the MiX gene were amplified by single copy polymerase chain reaction. Direct DNA sequencing showed that during the formation of MiX gene, a short stretch of alpha exon III with a donor splice site has replaced a silent sequence in the delta gene containing a cryptic acceptor splice site. The upstream delta-alpha breakpoint is flanked by the direct repeats of the acceptor splice site, whereas the down-stream alpha-delta breakpoint is located in the adjacent intron. This segmental transfer produced a new composite exon whose expression not only transactivated a portion of silent sequence but also created intraexon and interexon hybrid junctions that characterize the antigenic specificities of MiX glycophorin. The identification of MiX as yet another delta-alpha-delta hybrid different from MiIII and MiVI in gene conversion sites suggests that shuffling of expressed and unexpressed sequences through particular genomic DNA motifs has been an important mechanism for shaping the antigenic diversity of MNSs blood group system during evolution.     

The use of monoclonal antibodies to quantify the levels of sialoglycoproteins alpha and delta and variant sialoglycoproteins in human erythrocyte membranes.

By using radioiodinated monoclonal antibodies we have estimated that there are about 600 000 copies of sialoglycoprotein alpha (synonym glycophorin A) and 80 000 copies of sialoglycoprotein delta (synonym glycophorin B) per normal human erythrocyte. Erythrocytes expressing the product of only one alpha gene contain about 300 000 copies of alpha/cell. Two erythrocyte types containing alpha-delta hybrid molecules were studied. Those with heterozygous expression of the (alpha-delta)Mi.V gene contain about 100 000 alpha-delta copies per cell, whereas those with heterozygous expression of the En(UK) gene contain about 80 000 alpha-delta copies/cell. Erythrocyte types containing delta-alpha hybrid molecules were also studied. About 200 000 copies of (delta-alpha)Dantu were measured in cells with heterozygous expression of the (delta-alpha)Dantu gene (donor M.P.), whereas about 315 000 copies of the putative (delta-alpha)Dantu hybrid were found on the erythrocytes of donor J.O. [which also have heterozygous expression of the putative (delta-alpha)Dantu gene]. The erythrocytes of donor M.P. have normal levels of alpha, whereas those of donor J.O. have only about half-normal levels. It is proposed that the hybrid sialoglycoprotein of donor J.O. is of alpha-delta-alpha composition [(alpha-delta-alpha)Dantu] rather than delta-alpha and results from a double cross-over analogous to that which gives rise to haemoglobin Parchman.     

Unequal crossing-over within the B duplication of Drosophila melanogaster: a molecular analysis.

The B mutation is associated with a tandem duplication of 16A1-16A7. It is unstable, mutating to wild type and to a more extreme form at a frequency of one in 1000 to 3000. The reversion to wild type is associated with the loss of one copy of the duplication, whereas the mutation to extreme B is associated with a triplication of the region. The instability of B has been attributed to unequal crossing-over between the two copies of the duplication. Recent molecular data show that there is a transposable element, B104, between the two copies of the duplication and support the hypothesis that this element generated the duplication via a recombination event. These data suggest that unequal crossing-over within the duplication may not be the cause of the instability of B. Instead, the instability may be caused by a recombination event involving the B104 element. This issue was addressed using probes for the DNA on either side of the B104 element at the B breakpoint. All of the data indicate that the B104 element is not involved in the instability of B and support the original unequal crossing-over model.     

Illegitimate recombination is a major evolutionary mechanism for initiating size variation in plant resistance genes

Current models for the evolution of plant disease resistance (R) genes are based on mechanisms such as unequal crossing-over, gene conversion and point mutations as sources for genetic variability and the generation of new specificities. Size variation in leucine-rich repeat (LRR) domains was previously mainly attributed to unequal crossing-over or template slippage between LRR units. Our analysis of 112 R genes and R gene analogs (RGAs) from 16 different gene lineages from monocots and dicots showed that individual LRR units are mostly too divergent to allow unequal crossing-over. We found that illegitimate recombination (IR) is the major mechanism that generates quasi-random duplications within the LRR domain. These initial duplications are required as seeds for subsequent unequal crossing-over events which cause the observed rapid increase or decrease in LRR repeat numbers. Ten of the 16 gene lineages studied contained such duplications, and in four of them the duplications served as a template for subsequent repeat amplification. Our analysis of Pm3-like genes from rice and three wheat species showed that such events can be traced back more than 50 million years. Thus, IR represents a major new evolutionary mechanism that is essential for the generation of molecular diversity in evolution of RGAs.     

Intergenic exchange maintains identity between two human lambda light chain immunoglobulin gene intron sequences.

Evidence for gene conversion or unequal double crossover in the human lambda light chain immunoglobulin locus is presented. The high level of J2C2-J3C3 intron cross-hybridization, the identity of the J lambda and J lambda 3 coding and intron sequences, the presence of multiple base differences between the C lambda 2 and C lambda 3 coding regions, and the presence of both the unconverted and converted alleles in the normal gene pool, suggest that a recombinational event has resulted in the conversion of the J lambda 2 coding and intron sequences to those of J lambda 3 and its flanking sequences. Intergenic exchanges, such as the one described here, may provide a mechanism to maintain sequence homogeneity and functionality among the duplicated members of the human lambda gene family.     

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.     

Hybrid glycophorins from human erythrocyte membranes. Isolation and complete structural analysis of the novel sialoglycoprotein from St(a+) red cells

Human red cells from donor Pj carry the Sta blood group antigen and an unusual sialoglycoprotein of 24 kDa molecular mass tentatively identified as a hybrid molecule of the anti-Lepore type [Blanchard et al. (1982) Biochem. J. 203, 419-426]. This component is resistant towards proteinase treatment and was purified from trypsin-treated and chymotrypsin-treated Pj erythrocytes. The molecule is composed of 99 amino acid residues whose alignment was established following manual and automatic sequencing of cyanogen bromide, trypsin, chymotrypsin and V8 proteinase peptides. The polypeptide chain comprises residues 1-26/28 of glycophorin B and residues 59/61-131 of glycophorin A. The sugar composition resembles that of glycophorin B, indicating the absence of an N-glycosidic chain. Identical sequences were obtained from analyses of the 24-kDa component purified from unrelated St(a+) donors. These results support the hypothesis that glycoprotein Pj represents a B-A hybrid molecule which is encoded by a new gene product resulting from an unequal crossing-over between the genes coding for the polypeptide chains of the glycophorins A and B. The novel molecule carries both N and Sta blood group antigens. The N activity is clearly understandable from the sequence of the five N-terminal residues (Leu and Glu at positions 1 and 5 respectively). Inhibition studies with the untreated and chemically modified hybrid glycoprotein indicate that the Sta determinant is located within residues approximately 25-30 of the molecule, which corresponds to the newly formed sequence found neither in glycophorin A nor in glycophorin B.     

Within- and between-individual length heterogeneity of the rDNA-IGS in Miscanthus sinensis var. glaber (Poaceae): phylogenetic analyses.

The variability in the intergenic spacer (IGS) region between 17S and 25S rRNA genes of ribosomal DNA (rDNA) gene family was surveyed in Miscanthus sinensis var. glaber. Length heterogeneity, with sizes from 1782 to 2212 base pairs, of the IGS resulted from the variation of copy numbers of the A and B subrepeats. These repeated elements were located upstream of the presumptive polymeraseII promoter, which was the region corresponding to the nontranscribed spacer (NTS). Length heterogeneity was detected both within and between individuals of Miscanthus sinensis var. glaber. Neighbor-joining analyses of repetitive A elements indicated that both unequal crossing-over and preferential conversion may have affected the hot-spot regions of the IGS in concert. Within-individual polymorphism and the reconstructed phylogeny suggested that interspecific hybridization has also contributed to length heterogeneity.     

Homology Requirements for Unequal Crossing over in Humans

To gain insight into mechanisms of unequal homologous recombination in vivo, genes generated by homologous unequal crossovers in the human beta-globin gene cluster were examined by nucleotide sequencing and hybridization experiments. The naturally occurring genes studied included one delta-beta Lepore-Baltimore fusion gene, one delta-beta Lepore-Hollandia fusion gene, 12 delta-beta Lepore-Boston genes, one A gamma-beta fusion Kenya gene, one A gamma-G gamma fusion (the central gene of a triplication) and one G gamma-A gamma fusion. A comparison of the nucleotide sequences of three Lepore-Boston genes indicates that they were derived from at least two independent homologous but unequal crossover events, although the crossovers occurred within the same 58-bp region. Nine additional Lepore-Boston genes from individuals of various ethnic origins were shown, by hybridization to specific oligonucleotide probes, to have been generated by a crossover in the same region as the sequenced genes. Evidence for gene conversion accompanying a homologous unequal crossover event was found in only one case (although some of the single nucleotide differences observed in other genes in this study may be related to the crossover events in ways that we do not presently understand). Thus, as judged by this limited sample, concurrent gene conversions are not commonly associated with homologous but unequal exchange in humans in vivo. Classification of the recombinant chromosomes by their polymorphic restriction sites in the beta-globin gene cluster indicated that the Lepore-Boston genes are found in at least six different haplotype backgrounds. Therefore the total number of independent examples in this study is at least 6, and at most 12. We have shown that in at least six cases of genes that have arisen by homologous but unequal crossing over in vivo, each event occurred in a relatively extensive region of uninterrupted identity between the parental genes. This preference cannot be explained by a mechanism whereby crossovers occur at random within misaligned related but not identical genes. In general, crossovers occur in regions that are among the largest available stretches of identity for a particular pair of mismatched genes. Our data are in agreement with those of other types of studies of homologous recombination, and support the idea that sequence identity, rather than general homology, is a critical factor in homologous recombination.     

A deletion involving Alu sequences in the beta-hexosaminidase alpha-chain gene of French Canadians with Tay-Sachs disease

French Canadians living in eastern Quebec are carriers of a severe type of Tay-Sachs disease, known as the classic form, 10 times more often than the general population. The alpha-chain of beta-hexosaminidase A, a lysosomal enzyme composed of two chains (alpha, beta), bears the mutation in this inherited disorder. We previously reported that the 5' end of the alpha-chain gene was deleted in two such patients (Myerowitz, R., and Hogikyan, N.D. (1986) Science, 232, 1646-1648). The present study reports the size, precise location, and environment of the deletion. A clone encompassing the deletion was isolated from a genomic library constructed in lambda EMBL3 with DNA from a patient's fibroblasts. Comparison of the restriction maps of the clone with that of the normal gene (Proia, R.L., and Soravia, E. (1987) J. Biol. Chem. 262, 5677-5681) showed that the deletion was 7.6 kilobases long and included part of intron 1, all of exon 1 and extended 2000 base pairs upstream past the putative promotor region of the alpha-chain gene. These data are consistent with the inability to detect mRNA and immunoprecipitable alpha-chain protein in this mutant. Sequence analysis of the deletion junction in the mutant and corresponding regions of the normal gene demonstrated the presence of similarly oriented Alu sequences at the 5' and 3' deletion boundaries. The data are in accord with the possibility that the deletion may have arisen during homologous recombination from unequal crossing over between Alu sequences.     

Genetic instabilities in (CTG.CAG) repeats occur by recombination.

The expansion of triplet repeat sequences (TRS) associated with hereditary neurological diseases is believed from prior studies to be due to DNA replication. This report demonstrates that the expansion of (CTG.CAG)(n) in vivo also occurs by homologous recombination as shown by biochemical and genetic studies. A two-plasmid recombination system was established in Escherichia coli with derivatives of pUC19 (harboring the ampicillin resistance gene) and pACYC184 (harboring the tetracycline resistance gene). The derivatives contained various triplet repeat inserts ((CTG.CAG), (CGG.CCG), (GAA.TTC), (GTC.GAC), and (GTG.CAC)) of different lengths, orientations, and extents of interruptions and a control non-repetitive sequence. The availability of the two drug resistance genes and of several unique restriction sites on the plasmids enabled rigorous genetic and biochemical analyses. The requirements for recombination at the TRS include repeat lengths >30, the presence of CTG.CAG on both plasmids, and recA and recBC. Sequence analyses on a number of DNA products isolated from individual colonies directly demonstrated the crossing-over and expansion of the homologous CTG.CAG regions. Furthermore, inversion products of the type [(CTG)(13)(CAG)(67)].[(CTG)(67)(CAG)(13)] were isolated as the apparent result of "illegitimate" recombination events on intrahelical pseudoknots. This work establishes the relationships between CTG.CAG sequences, multiple fold expansions, genetic recombination, formation of new recombinant DNA products, and the presence of both drug resistance genes. Thus, if these reactions occur in humans, unequal crossing-over or gene conversion may also contribute to the expansions responsible for anticipation associated with several hereditary neurological syndromes.     

Nucleotide sequences of switch regions of immunoglobulin C epsilon and C gamma genes and their comparison

Immunoglobulin class switch involves a unique recombination event that takes place at the switch (S) region which is located 5' to each constant region (C) gene of the heavy (H) chain. For example, differentiation of the B lymphocyte from a mu-chain producer to an epsilon-chain producer is mediated by the switch recombination between the S mu and S epsilon regions. In order to elucidate the molecular mechanism for the switch recombination, we have determined nucleotide sequences surrounding the class switch recombination sites of the C epsilon and C gamma 3 genes and those in the 5' flanking regions of the C gamma 2a and C delta genes. The results indicate that the 5' flanking regions of all the CH genes except for the C delta gene contain the S regions which comprise tandem repetition of short unit sequences in agreement with the previous analyses of the S gamma 1, S gamma 2b, S mu, and S alpha regions. Comparison of the nucleotide sequences of all the S regions revealed that length as well as nucleotide sequences of the S regions vary among different classes of the CH gene, but they share short common sequences, (G)AGCT and TGGG(G). The nucleotide sequence of the S mu region is homologous to those of the other S regions in the decreasing order of the S epsilon, S alpha, S gamma 3, and (S gamma 1, S gamma 2b, s gamma 2a) regions. We have compared the nucleotide sequences immediately adjacent to the recombination sites of seven rearranged genes and have always fund tetranucleotides TGAG and/or TGGG, except for one case. Such tetranucleotides may constitute a part of the recognition sequence of a putative recombinase. These results provide further support for our previous proposal that the switch recombination may be facilitated by short common sequences dispersed in all the S regions.     

The mitochondrial genome of wild-type yeast cells. VII. Recombination in crosses.

Two Saccharomyces cerevisiae wild-type strains were crossed, and 26 diploid clones were obtained from (1) mass mating; (2) individual buds in zygote lineages; (3) individual zygotes. The mitochondrial DNAs from these diploids were investigated in their recombination and segregation by analyzing their restriction fragment patterns.Recombinant mitochondrial genomes were present in 75% of the diploid clones. Such recombinant genomes had unit sizes different from, yet within ± 5% of, the parental ones and showed EcoRI and HindII + III fragment patterns of parental types, two strong indications that both the gene complement and the gene order were very largely preserved in the progeny.Fragment patterns produced by HpaII and HaeIII were characterized by (1) fragments originating from the DNAs of both parents; and (2) new fragments, namely fragments absent in either parent. The new fragments appear to arise from unequal crossing-over events occurring in the spacers of allelic parental genetic units and usually have preferential localizations in the genome.These results provide the first evidence for physical recombinations of mitochondrial DNA in crosses of wild-type yeast cells, indicate that recombination is very frequent in crosses, and shed some light on mitochondrial segregation. They also have interesting implications for recombination phenomena in interspersed systems of unique and repetitive nucleotide sequences.