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.     

Multiple origins of the human glycophorin Sta gene. Identification of hot spots for independent unequal homologous recombinations.

Human glycophorin Sta (HGpSta), one of the structural variants of erythrocyte membrane sialoglycoproteins, is encoded by a delta-alpha hybrid gene that arose from a single unequal crossover between the parent HGpB(delta) and HGpA(alpha) genes. We report here the identification of two new HGpSta genes (type A and type B) in four unrelated Sta heterozygotes from two ethnic groups. These Sta genes represent distinct genetic isoforms that differ from the previously reported Sta gene (type C) in the location of crossing-over sites. Comparison of nucleotide sequences among HGpB(delta), HGpA(alpha), and HGpSta type A genes revealed that the delta-alpha unequal crossover for the Sta type A gene occurred 110-246 base pairs downstream from pseudoexon III. In the crossing-over site of this Sta gene, an AT-rich sequence lying 3' to a nonameric palindrome was found to be highly similar to the lambda phage attachment site, att B, in inverted orientation. In the Sta type B gene, the delta-alpha crossing-over point was localized to an AG-rich sequence that is 302-490 base pairs downstream from pseudoexon III. Multiple lambda chi-like elements were identified at the crossover boundaries and within the breakpoint of this Sta gene. These results suggest strongly that recurrent and independent unequal recombination events have occurred in the formation of multiple Sta genes and that particular genomic sequences are important in defining the recombination sites for these homology-driven processes.     

A family study of multiple mutations of alpha and delta glycophorins (glycophorins A and B)

Summary Glycophorins alpha and delta are the carriers of the antigens of the MNSs blood system; this report documents the presence of three glycophorin mutations in two individuals of a 16 member family. Erythrocytes were examined by serology, sodium dodecyl sulfate electrophoresis, and immunoblotting. The inheritance pattern and immunoblot profile revealed: (1) A variant Dantu glycophorin showed properties consistent with a delta-alpha glycophorin hybrid structure, previously noted in other individuals. The gene responsible for the Dantu glycophorin in this family is linked to a gene coding for an M-specific alpha glycophorin. (2) Another variant glycophorin, Mi-III glycophorin, was first revealed by immunoblotting and subsequently confirmed by erythrocyte antigen typing. This autosomal dominant trait is associated with N blood group activity and the inheritance pattern indicates that it could be a variant of delta glycophorin. (3) In the individuals with both Dantu and Mi-III glycophorins a delta glycophorin deficiency was observed suggesting that a deletion or alteration of delta gene may exist cis to the Dantu gene. Our findings that document clustering of multiple mutations in MNSs gene loci in the propositus family are very unusual as such variants are relatively rare.     

An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype

Recently, we have found an allelic deletion of the secretor alpha(1,2)fucosyltransferase (FUT2) gene in individuals with the classical Bombay phenotype of the ABO system. The FUT2 gene consists of two exons separated by an intron that spans approximately 7 kb. The first exon is noncoding, whereas exon 2 contains the complete coding sequence. Since the 5' breakpoint of the deletion has previously been mapped to the single intron of FUT2, we have cloned the junction region of the deletion in a Bombay individual by cassette-mediated polymerase chain reaction. In addition, the region from the 3' untranslated region of FUT2 to the 3' breakpoint sequence has been amplified from a control individual. DNA sequence analysis of this region indicates that the 5' breakpoint is within a free left Alu monomer (FLAM-C) sequence that lies 1.3 kb downstream of exon 1, and that the 3' breakpoint is within a complete Alu element (AluSx) that is positioned 1.5 kb downstream of exon 2. The size of the deletion is estimated to be about 10 kb. There is a 25-bp sequence identity between the reference DNA sequences surrounding the 5' and 3' breakpoints. This demonstrates that an Alu-mediated large gene deletion generated by unequal crossover is responsible for secretor alpha(1,2)fucosyltransferase deficiency in Indian Bombay individuals.     

A novel gene member of the human glycophorin A and B gene family. Molecular cloning and expression

A new gene closely related to the glycophorin A (GPA) and glycophorin B (GPB) genes has been identified in the normal human genome as well as in that of persons with known alterations of GPA and/or GPB expression. This gene, called glycophorin E (GPE), is transcribed into a 0.6-kb message which encodes a 78-amino-acid protein with a putative leader peptide of 19 residues. The first 26 amino acids of the mature protein are identical to those of M-type glycophorin A (GPA), but the C-terminal domain (residues 27-59) differs significantly from those of glycophorins A and B (GPA and GPB). The GPE gene consists of four exons distributed over 30 kb of DNA, and its nucleotide sequence is homologous to those of the GPA and GPB genes in the 5' region, up to exon 3. Because of branch and splice site mutations, the GPE gene contains a large intron sequence partially used as exons in GPA and GPB genes. Compared to its counterpart in the GPB gene, exon 3 of the GPE gene contains several point mutations, an insertion of 24 bp, and a stop codon which shortens the reading frame. Downstream from exon 3, the GPE and the GPB sequences are virtually identical and include the same Alu repeats. Thus, it is likely that the GPE and GPB genes have evolved by a similar mechanism. From the analysis of the GPA, GPB and GPE genes in glycophorin variants [En(a-), S-s-U- and Mk], it is proposed that the three genes are organized in tandem on chromosome 4. Deletion events within this region may remove one or two structural gene(s) and may generate new hybrid structures in which the promoter region of one gene is positioned upstream from the body of another gene of the same family. This model of gene organization provides a basis with which to explain the diversity of the glycophorin gene family.     

Impairment of yeast pre-mRNA splicing by potential secondary structure-forming sequences near the conserved branchpoint sequence.

The absolutely conserved TACTAAC box within introns of RNA polymerase II-transcribed genes of the yeast Saccharomyces cerevisiae serves an indispensable role in lariat formation. We show in this report that rather short palindromic sequences inserted into the yeast actin gene intron immediately 3' to the TACTAAC box block the second but not the first splicing step. In contrast, a palindromic sequence inserted some 23 bp 3' of the TACTAAC box did not affect correct and efficient splicing. The data suggest that hairpin structures that might form adjacent to the branchsite sequence interfere with some necessary alteration of the spliceosome required for 3' intron cleavage and exon ligation.     

鉴定9个新的RHD基因mRNA可变剪接体

为了研究各种RHD基因mRNA可变剪接体的基因结构, 应用逆转录聚合酶链反应(RT-PCR)检测正常人脐血样本RHD mRNA, 对RHD cDNA进行TA克隆和序列分析, 对各可变剪接体的剪接位点进行DNA序列分析, 并将RHD mRNA进行表达序列标签(ESTs)分析。结果在28个阳性克隆中, 除全长RHD cDNA外, 共检测到12种(包括9种新的)RHD可变剪接体, 发现外显子遗漏、5′和3′剪接位点变异3种剪接形式, 涉及外显子2~9, 其中6种新的剪接体同时存在RHD和RHCE基因同源杂交现象。ESTs分析还检索到内含子保留形式的剪接体。研究表明, RHD基因mRNA存在复杂的可变剪接机制, 除已报道的剪接体外, 检测到9种新的RHD可变剪接体, 并发现了可变剪接和同源杂交并存现象。     

The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.     

Amino acid sequence of an alpha-delta-glycophorin hybrid. A structure reciprocal to Sta delta-alpha-glycophorin hybrid

In this report we examine the primary sequence of a variant glycophorin obtained from erythrocytes of an individual who exhibits an unusual MNSs blood group phenotype. We show that this protein is a hybrid molecule constructed from sequences of alpha- and delta-glycophorins (glycophorins A and B) in a alpha-delta arrangement. Serological typing revealed that the donor's phenotype was M+N+S+s+U+; yet his erythrocytes reacted with some but not all examples of anti-S antisera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a variant glycophorin band, and immunoblotting and reaction with N-glycanase suggested that its amino terminus resembled that of M-alpha-glycophorin but that its carboxyl terminus did not. A preparation highly enriched in the variant was obtained and used to generate peptide fragments for sequencing. The sequence revealed that the variant was a hybrid molecule whose amino terminus corresponded to M-alpha-glycophorin and whose carboxyl terminus corresponded to S-delta-glycophorin. CNBr cleavage of the variant glycophorin yielded four peptides. The sequence of the amino-terminal CNBr peptide (residues 1-8) was identical to the amino-terminal octapeptide of M-alpha-glycophorin. The proceeding peptide (residues 9-61) contained a segment identical to residues 9-58 of alpha glycophorin, but its carboxyl-terminal sequence had the Gly-Glu-Met sequence from S-delta-glycophorin (residues 27-29). The other two peptides, insoluble in aqueous solutions, contained highly hydrophobic sequences, identical to residues 30-52 and 53-68 of delta-glycophorin. Sequences of overlapping peptides generated by trypsin and V8 protease confirmed the hybrid nature of the variant glycophorin: residues 1-58 were identical to residues 1-58 of M-alpha-glycophorin, and residues 59-100 were entirely identical to residues 27-68 of S-delta-glycophorin. The variant glycophorin is expected to have 4 additional residues at its carboxyl terminus that correspond to the carboxyl-terminal residues 69-72 of delta-glycophorin. The amino acid sequence arrangement of the variant alpha-delta-glycophorin is an exact reciprocal of that found in another hybrid glycophorin, Sta, that is a delta-alpha hybrid. We propose that the two hybrid glycophorins represent the two possible products resulting from a reciprocal recombination event.     

A single nucleotide difference at the 3'' end of an intron causes differential splicing of two histocompatibility genes.

The murine histocompatibility class I genes, H-2 Kb and Kk, display considerable homology at their 3' ends. In fact, from exon 5 to the termination codon, only two nucleotides differ between the two genes, one at the 5' end and the other at the 3' end of intron 7. Despite this similarity, the gene products have distinctly different mol. wts as determined by SDS-PAGE. By constructing two hybrid genes, pC2 and pC4, we demonstrated that it is the cytoplasmic parts of the antigens (encoded by exons 6-8) which are responsible for the major difference in mol. wt. We have used site-directed mutagenesis to change the two nucleotides in intron 7 of the H-2 Kk gene to those present in the H-2 Kb gene. S1 nuclease mapping has been used to identify the actual splice site of the authentic Kb and Kk genes, the hybrid genes and the mutagenized genes. We have shown that it is the 3' nucleotide difference, nine nucleotides upstream of the 3' splice site, which causes the different excision of intron 7 of the Kb gene. The 5' nucleotide difference does not alter the splicing. The choice of branch points and 3' splice signals for intron 7 of five H-2 class I genes, is discussed.     

Complete nucleotide sequence of the chicken alpha A-crystallin gene and its 5' flanking region

The chicken alpha A-crystallin gene and 2.6 kb of its 5' flanking sequence have been isolated and characterized by electron microscopy and sequencing. The structural gene is 4.5 kb long and contains two introns, each approx. 1 kb in length. The first intron divides codons 63 and 64, and the second intron divides codons 104 and 105, as in rodents. There is little indication that the insert exon of rodents (an alternatively spliced sequence) is present in complete form in the chicken alpha A-crystallin gene; small stretches of similarity to this sequence were found throughout the gene. The 5' flanking sequence of the chicken alpha A-crystallin gene shows considerable sequence similarity with other mammalian alpha B-crystallin genes. In addition, one consensus sequence (GCAGCATGCCCTCCTAG) present in the 5' flanking region of the chicken alpha A-crystallin gene was found in the 5' flanking region of most reported crystallin genes.     

Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon.

Two alternative exons, BEK and K-SAM, code for part of the ligand binding site of fibroblast growth factor receptor 2. Splicing of these exons is mutually exclusive, and the choice between them is made in a tissue-specific manner. We identify here pre-mRNA sequences involved in controlling splicing of the K-SAM exon. The short K-SAM exon sequence 5'-TAGGGCAGGC-3' inhibits splicing of the exon. This inhibition can be overcome by mutating either the exon's 5' or 3' splice site to make it correspond more closely to the relevant consensus sequence. Two separate sequence elements in the intron immediately downstream of the K-SAM exon, one of which is a sequence rich in pyrimidines, are both needed for efficient K-SAM exon splicing. This is no longer the case if either the exon's 5' or 3' splice site is reinforced. Furthermore, if the exon inhibitory sequence is removed, the intron sequences are not required for splicing of the K-SAM exon in a cell line which normally splices this exon. At least three elements are thus involved in controlling splicing of the K-SAM exon: suboptimal 5' and 3' splice sites, an exon inhibitory sequence, and intron activating sequences.     

LDL受体基因内含子15结构分析及其在家族性高胆固醇血症基因诊断中的应用

为了解人类ＬＤＬ受体基因内含子１５的遗传背景,利用长链ＰＣＲ和锚定ＰＣＲ分离了ＬＤＬ受体基因外显子１５－内含子１５－外显子１６和内含子１５的３‘末端片段。利用Ｄｙｎａｌｂｅａｄｓ固相单链分离ＰＣＲ产物直接测序法测定了内含子１５ ３’末端１２２２个碱基序列。序列显示：３‘末端含有由１６个碱基组成的典型３’末端剪接位点;３‘端上游第３１个碱基处含有经典分支位点,除了经典分支位点外,在３’末端上游第２０     

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.     

Gaucher disease: A G+1----A+1 IVS2 splice donor site mutation causing exon 2 skipping in the acid beta-glucosidase mRNA.

Gaucher disease is the most frequent lysosomal storage disease and the most prevalent Jewish genetic disease. About 30 identified missense mutations are causal to the defective activity of acid beta-glucosidase in this disease. cDNAs were characterized from a moderately affected 9-year-old Ashkenazi Jewish Gaucher disease type 1 patient whose 80-year-old, enzyme-deficient, 1226G (Asn370----Ser [N370S]) homozygous grandfather was nearly asymptomatic. Sequence analyses revealed four populations of cDNAs with either the 1226G mutation, an exact exon 2 (delta EX2) deletion, a deletion of exon 2 and the first 115 bp of exon 3 (delta EX2-3), or a completely normal sequence. About 50% of the cDNAs were the delta EX2, the delta EX2-3, and the normal cDNAs, in a ratio of 6:3:1. Specific amplification and characterization of exon 2 and 5' and 3' intronic flanking sequences from the structural gene demonstrated clones with either the normal sequence or with a G+1----A+1 transition at the exon 2/intron 2 boundary. This mutation destroyed the splice donor consensus site (U1 binding site) for mRNA processing. This transition also was present at the corresponding exon/intron boundary of the highly homologous pseudogene. This new mutation, termed "IVS2 G+1----A+1," is the first splicing mutation described in Gaucher disease and accounted for about 3.4% of the Gaucher disease alleles in the Ashkenazi Jewish population. The occurrence of this "pseudogene"-type mutation in the structural gene indicates the role of acid beta-glucosidase pseudogene and structural gene rearrangements in the pathogenesis of this disease.