Evidence for an elevated frequency of in vivo somatic cell mutations in ataxia telangiectasia.

Somatic cell mutation frequency in vivo was measured in individuals with high cancer risk who were from ataxia telangiectasia (A-T) families. The assay for somatic mutation measures the frequency of variant erythrocytes which are progeny of erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Samples from 14 of 15 A-T homozygotes showed high frequencies of GPA gene expression-loss variant cells with normal expression of only one of the two alleles at the GPA locus (i.e., GPA hemizygous variant cells). The mean elevation of the frequency of hemizygous variant cells over those in normal controls and unaffected family members was 7-14-fold. A-T homozygotes also showed an increase in the frequency of cells in which one allele at the GPA locus had lost expression and in which the remaining allele was expressed at a homozygous level (i.e., GPA homozygous variant cells). Family members who are obligate A-T heterozygotes did not appear to have a significantly elevated frequency of GPA hemizygous or homozygous variant cells. These indications of elevated in vivo frequencies of variant erythrocytes in A-T homozygotes support a causal link between susceptibility to somatic mutation and susceptibility to cancer.     

Frequency of variant erythrocytes at the glycophorin-A locus in two Bloom's syndrome patients

Blood type MN is determined by a glycoprotein termed glycophorin A (GPA) which exists on the surface of erythrocytes, and the difference between the M and N types is derived from the presence of 2 different amino acids in the amino-terminal portion (Furthmayer, 1978). Using a pair of fluorescence-labeled monoclonal antibodies specific to each GPA, somatic mutations in erythrocytes of MN heterozygotes at the GPA-M and -N alleles can be quantitatively determined using a flow sorter (Langlois et al., 1986). Our results for 2 Bloom's syndrome (BS) patients showed that variants either lost expression of one allele (simple gene inactivation or loss) or expressed only one allele at twice the normal level (most probably somatic recombination) occurring at a frequency of about 1-3 per 10(3) erythrocytes. The flow cytometric patterns of erythrocytes from the BS patients showed a typical smear of variants bearing intermediate levels of expression of one GPA allele, indicating that the real variant frequency is even greater than that measured. On the other hand, the parents heterozygous for the BS gene showed variant frequencies (1-8 x 10(-5)) within the normal range. These data strongly support the hypothesis that the cancer proneness of BS patients is due to their increased frequency of spontaneous mutations and somatic recombination.     

A variant of glycophorin A resulting from the deletion of exon 4

We have isolated a variant form of glycophorin A which has a 39 bp deletion corresponding to nucleotides 233 to 270 of the coding sequence, which is exon 4 of the glycophorin A gene. The remainder of the sequence is identical to that of the M phenotype of glycophorin A.     

The chimpanzee M blood-group antigen is a variant of the human M-N glycoproteins

Chimpanzee erythrocytes express strong M but weak, occasional N blood-group activity, as detected by anti-M and anti-N reagents. We have found that the M activity is carried by a major membrane glycoprotein that is similar but not identical to the human MM glycoprotein (glycophorin A). We have isolated and characterized this glycoprotein from erythrocyte membranes of four individual chimpanzees. The purified glycoproteins strongly inhibited agglutination of M cells by rabbit anti-human M sera and only weakly inhibited the agglutination of N cells by rabbit anti-human N sera. They also displayed medium-to-strong inhibitory activity against chimpanzee iso- and crossimmune antisera tested with chimpanzee erythrocytes of various V-A-B-D and W^c specificities, which are known as chimpanzee extensions of the human type M-N system and the Miltenberger counterpart, respectively. Each glycoprotein was cleaved with CNBr into three fragments, whose size, solubility, and composition were analogous to those obtained by similar treatment of the human M-N antigens. The amino-terminal fragment was found to be a glycooctapeptide whose amino acid composition and partial sequence indicated that it is an intermediate form of the human M and N glycooctapeptides. Its carbohydrate content comprised two threonine-linked saccharide units that, although similar in composition to the human threonine-linked units, were fewer in number than the three units found in the corresponding human glycooctapeptides. Structural similarities to the human antigens strongly suggest that the amino terminus bears the major antigenic determinants of the molecule, and the occurrence in this region of numerous, albeit rare, variants among humans and in chimpanzees indicates that the corresponding coding sequence of the structural gene is particularly susceptible to mutational events. We conclude that the chimpanzee M gene product is a variant of the human type and that the chimpanzee gene is an allele of the human polymorphic M-N locus.This research was supported by National Institutes of Health Grants GM 16389 and HL 19011 and March of Dimes Grant 1-661.     

The Mz variety of the St(a+) phenotype--a variant of glycophorin A exhibiting a deletion

The NeuNAc level of erythrocyte membranes from two related donors exhibiting the Mz variety of St(a+) phenotype within the MNSs blood group system was found to be decreased by about 16%. The quantity of glycophorin A was decreased by about 38%, whereas that of glycophorin B was not significantly different from normal. Mz erythrocyte membranes were also found to contain an abnormal component (molar ratio to glycophorin A about 0.89:1.0) with an apparent molecular mass of about 24,000 Da. Immunoblotting experiments and amino-acid sequence analysis revealed that the novel component (and glycophorin A in one of the donors) carries blood group M activity. Blood group N activity was demonstrable for glycophorin A and glycophorin B from both donors. Amino-acid sequence analysis of chymotryptic, tryptic and cyanogen bromide peptides demonstrated that the novel molecule exhibits the typical structure of a Sta-active molecule. However, since it exhibits blood group M activity, it appears to represent a variant of glycophorin A lacking the residues 27-58 (encoded by exon three of the glycophorin A gene) rather than a glycophorin B-glycophorin A-hybrid molecule of the anti-Lepore type. Since one of the Mz heterozygotes was found to exhibit both M- and N activity on glycophorin A, the Mz gene complex appears to encode a blood group N-active glycophorin A apart from the novel component and a blood group s-active glycophorin B, although the level of glycophorin A in the erythrocyte membranes is decreased by about half.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Two monoclonal antibodies highly specific for the blood group N determinant

Two monoclonal IgM antibodies, 179K and 35/5F, obtained following immunization of mice with A₂,MN or O,MN human erythrocytes, agglutinate NN and MN red cells strongly, and MM erythrocytes weakly. As shown by hemagglutination inhibition and solid phase ELISA, both antibodies are highly specific for the blood group N determinant. They react with N glycoprotein, its amino-terminal glycopeptides and with Ss glycoprotein (glycophorin B), which carries the blood group N determinant. They fail to react with M glycoprotein, M glycoprotein-derived glycopeptides, or with internal glycopeptides derived from N glycoprotein. Reaction of the antibodies with N glycoprotein is abolished by desialylation, periodate oxidation/borohydride reduction, orN-acetylation of the glycoprotein. Thus, the antibodies are specific for an epitope which includes sialylated oligosaccharide chain(s) and is located in the region of the amino-terminal leucine residue of N glycoprotein. MMU^– erythrocytes, lacking both blood group N and Ss glycophorin are non-reactive. Agglutination of MMU⁺ erythrocytes by the anti-N antibodies occursvia interaction with glycophorin B and correlates with the Ss phenotype of red cells MM,S erythrocytes are usually more strongly, agglutinated than MM,ss cells. The agglutination of MM erythrocytes decreases markedly as the pH is increased from 6 to 8, while agglutination of NN red cells is much less affected by shifts in pH over this range. As a result, both monoclonal antibodies are highly anti-N specific typing reagents when the agglutination assay is carried out at pH 8.     

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.     

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.     

Membrane glycophorins of Dantu blood group erythrocytes

Glycophorins of erythrocytes of two unrelated individuals who exhibit the Dantu blood group phenotype were studied. Immunoblots indicated that erythrocytes of each individual contained a complement of a normal alpha-glycophorin (glycophorin A) and a variant N-glycophorin. delta-Glycophorin (glycophorin B) was present in one donor's cells but not the other's; the s and N phenotypes of the latter's erythrocytes may derive from the variant glycophorin. The variant glycophorin is of a smaller size, does not bind to Lens culinaris lectin agarose, and lacks residues approximately 40-60 of alpha-glycophorin and its single asparagine-linked carbohydrate; it contains approximately 2 less O-glycosidically bound units whose structures are identical to those found in alpha-glycophorins. All these properties are characteristic of delta-glycophorin. The variant is related to alpha-glycophorin in the carboxyl-terminal region as shown by reaction with a specific antiserum. Sequence analyses of a mixture of chymotryptic peptides of a CNBr fragment of the variant glycophorin identified the sequence Val-His-Arg-Phe-Thr-Val-Pro-Glu-Ile-Thr-Leu-Ile-Ile that contains the junction point of delta- and alpha-glycophorins spanning residues 33-38/39 of delta-glycophorin and residues 71/72-77 of alpha-glycophorin. Sequence analysis of a mixture of CNBr fragments allowed us to conclude that the variant originates from delta-s- rather than delta-S-glycophorin. The quantity of the variant Dantu glycophorin when compared to alpha-glycophorin differed in the two individuals, the ratio being 2/1 in one individual's cells and 0.5/1 in the other's. This may reflect that the two donors belong to different varieties of Dantu phenotypes. Together, the evidence indicates that both donors' erythrocytes contain a (delta-alpha) variant glycophorin, whose amino terminus originates from delta-s-glycophorin and the carboxyl end from alpha-glycophorin with a junction point around residues 39 of delta- and 71 of alpha-glycophorins. The results suggest that the unique junction region may be characteristic of the Dantu phenotype.     

Flow cytometric characterization of normal and variant cells with monoclonal antibodies specific for glycophorin A

Quantitative immunofluorescence measurements were performed on erythrocytes labeled with monoclonal antibodies to glycophorin A (GPA) to assess the level of binding of these antibodies to normal and variant cell types. The seven antibodies used in this study include two that bind preferentially to the M form of GPA, three that bind preferentially to the N form, and two that bind equally well to both. Flow cytometric analysis of mixtures of cells differing in M,N type showed binding specificities of greater than 100-fold for most of the antibodies, and showed that three antibodies bind cell-bound GPA with an affinity of approximately 10(9) M(-1). These data also showed that the level of expression of GPA varies by less than 10% from cell to cell and from individual to individual. Flow measurements were also done on human erythrocytes with the following variant forms of glycophorin: Mc, Mg, Mk, En(F), En(UK), Mi-I, Mi-II, Mi-III, S-s-U-, Tn+, and St(a+). Other cell types analyzed included erythrocytes from chimpanzee, rhesus, African green, and capuchin monkeys, and cells from the human erythroleukemia cell line, K562. Flow analysis with our seven antibodies showed these cell types have distinctive labeling patterns consistent with the known or inferred altered glycophorins presented on these cells. In most cases, variant alleles were expressed at normal levels. Our results support other observations that the variants En(UK) and St(a+) contain hybrid GPA-GPB proteins, and suggest that their level of expression is largely determined by the 3' end of the hybrid genes.     

Alteration of the genes for glycophorin A and B in glycophorin-A-deficient individuals

Glycophorins A and B are homologous glycoproteins of the red cell membrane which carry the blood-group MN and Ss antigens, respectively, and are encoded by two distinct genes closely linked on chromosome 4, which are probably derived from each other by duplication during evolution. The lack of glycophorin A is associated with the rare phenotype En(a-), indicating individuals who are defective for MN antigens, as well as for the Ena antigens, also located on this glycoprotein. The En(a-) condition is heterogenous and includes two categories of variants exemplified by the Finnish and the English types referred to as En(Fin) and En(UK), respectively. By Southern blot and preliminary genomic clone analyzes we have compared the status of the genes for glycophorins A and B, as well as that of the gene encoding glycophorin C, another unrelated red cell membrane glycoprotein, in the En(a-) variants and in the En(a+) control donors. Our data indicate that the En(Fin) variant is homozygous for a complete deletion of the glycophorin A gene without any detectable abnormality of the genes encoding glycophorins B or C. In the genome of the En(UK) variant, with the presumed genotype Mk/En(UK), and where the Mk condition abolishes the expression of MN and Ss antigens, we have identified several abnormalities of the glycophorin A and B genes, but the glycophorin C gene was unaffected. Our results strongly support the view that in Mk chromosome the glycophorin A and B genes are largely deleted, whereas the En(UK) chromosome probably contains a gene fusion product encoding a hybrid glycoprotein AM-B, composed of the N-terminal portion of a blood group M-type glycophorin A and of the C-terminal portion of glycophorin B. The determination of the 5' and 3' limits of the hybrid gene and elucidation of the mechanism involved will require sequencing of the rearranged DNA of the variant and a full knowledge of the organization of the glycophorin A and B genes.     

The Dantu erythrocyte phenotype of the NE variety. II. Serology, immunochemistry, genetics, and frequency

Red cells (RBC) possessing the low-frequency MNSs antigen Dantu from 36 Black individuals (21 propositi) were found to exhibit the NE variety of this phenotype, as judged from the electrophoretic glycophorin (GP) pattern, described in an accompanying article, and/or from the polybrene test which detects the decreased NeuAc level of these RBC. All known DantuNE RBC (53) exhibit the phenotype M+N+. This finding as well as family studies and immunochemical investigations demonstrate that the DantuNE allele encodes a blood group M-specific GP A. Thus, the strongly decreased GP A level of RBC from DantuNE heterozygotes represents the product of the Dantu allele and its normal counterpart. It is suggested that the formation of a complex with the anion channel protein (band 3) represents the prerequisite for optimum incorporation of GP A into normal RBC membranes. The hybrid GP in DantuNE RBC, produced in large quantity, might suppress the incorporation of GP A in a cis and trans manner via the formation of a complex with band 3. The hybrid GP in DantuNE RBC lacks U activity, but expresses N activity and a qualitatively altered s antigen, thus proving its GP B-GP A hybrid nature in conjunction with data described in the accompanying article. Screening of ficin-treated RBC with Vicia lectin revealed that the Dantu phenotype exhibits a frequency of about 0.005 in American Blacks and less than 0.001 in Germans.     

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.     

Somatic events unmask recessive cancer genes to initiate malignancy

A heritable mutation predisposes an individual to certain childhood malignancies, such as retinoblastoma and Wilms' tumor. The chromosomal locations of the genes responsible for the predisposition are known by linkage with chromosomal deletions and enzyme markers. A study of these tumors in comparison to the normal constitutional cells of the patients, using enzyme and DNA markers near the predisposing genes, has shown that these genes are recessive to normal wild-type alleles at the cellular level. Expression of the recessive phenotype (malignancy) involves the same genetic events that were observed in Chinese hamster cell hybrids carrying recessive drug resistance genes. In both the experimental and clinical situations, the wild-type allele is most commonly eliminated by chromosome loss with duplication of the mutant chromosome. Simple chromosome loss and mitotic recombination have been documented in both systems. In the remaining 30% of cases, inactivation or microdeletion of the wild-type allele are assumed to be responsible for expression of the recessive phenotype. Osteosarcoma is a common second tumor in patients who have had retinoblastoma. Studies with markers in osteosarcoma show that these tumors also result from unmasking of the recessive phenotype by loss of the normal allele at the retinoblastoma locus, whether or not the patient had retinoblastoma. Subsequent chromosomal rearrangements and amplification of oncogenes that occur in these homozygous tumors provide progressive growth advantage. In other malignancies, in which studies have so far focused on oncogene amplification and chromosomal rearrangements, unmasking of recessive mutations may also be the critical initiating events.     

Polymorphism in the DNA repair enzyme XRCC1: utility of current database and implications for human health risk assessment

Genetic polymorphisms are increasingly recognized as sources of variability not only in toxicokinetic but also in toxicodynamic response to environmental agents. XRCC1 is involved in base excision repair (BER) of DNA; it has variant genotypes that are associated with modified repair function. This analysis focuses on four polymorphisms: three in the coding region that affect protein structure and one in an upstream regulatory sequence that affects gene expression. The Arg399Gln variant is the most widely studied with evidence supporting a quantitative effect of genotype on phenotype. The homozygous variant (Gln/Gln) can have 3-4-fold diminished capacity to remove DNA adducts and oxidized DNA damage. This variant is relatively common in Caucasians and Asians where approximately 10% are homozygous variant. In contrast, the Arg194Trp variant appears to protect against genotoxic effects although the degree to which DNA repair is enhanced by this polymorphism is uncertain. The homozygous variant is rare in Caucasians and African Americans but it is present at 7% in Asians. A third coding region polymorphism at codon 280 appears to decrease repair function but additional quantitative information is needed and the homozygous variant is rare across populations studied. A polymorphism in an upstream promoter binding sequence (-77T>C) appears to lower XRCC1 levels by decreasing gene expression. Based upon genotype effect on phenotype and allele frequency, the current analysis finds that the codon 399 and upstream (-77) polymorphisms have the greatest potential to affect the toxicodynamic response to DNA damaging agents. However, the implications for risk assessment are limited by the likelihood that polymorphisms in multiple BER genes interact to modulate DNA repair.     

Membrane glycophorins in Sta blood group erythrocytes

Structural and immunochemical studies of glycophorins isolated from erythrocytes of an individual homozygous for the M Sta blood group phenotype are described. Reactivities with specific monoclonal antibodies indicated that two major M and N glycophorins were present. The M and N Sta glycophorins were resolved by Lens culinaris lectin affinity chromatography. The N species was not held on the lectin but the M species, like control alpha glycophorins, was retained and could be eluted with alpha-methylmannoside. The two proteins were present in almost equimolar amounts. Studies of the CNBr fragments provided evidence that the structure of M Sta glycophorin is the same as that of the usual M alpha glycophorin but that the N Sta glycophorin is a variant. The amino-terminal octapeptides of the M and N species were similar in amino acid and carbohydrate composition to those isolated, respectively, from M and N alpha glycophorins. The studies focused on CNBr glycopeptide B that, in control alpha glycophorins, extends from amino acid residues 9 to 81. The fragment from the M species exhibited properties identical to those of the corresponding fragment of control alpha glycophorins in terms of size, chromatographic behavior, amino acid and carbohydrate contents and compositions, the presence of O-glycosidically linked saccharides and a single Asn-linked carbohydrate unit. The structures of the O-linked units were inferred experimentally to be NeuAc(alpha 2,3)Gal-(beta 1,3)GalNAc and NeuAc(alpha 2,3)Gal(beta 1,3) [NeuAc(alpha 2,6)]GalNAc, present in a ratio similar to that found in controls; and the Asn-linked unit also appeared to be as in the control. The tryptic glycopeptide pattern of the M Sta glycophorin CNBr fragment B was identical to the pattern of the corresponding control fragment, and the composition of the tryptic peptides suggested sequence identity with the control fragment. In contrast, the N Sta glycophorin yielded two CNBr glycopeptides B; both contained fewer amino acid residues and virtually lacked Man and GlcNAc, indicating the absence of the Asn-linked carbohydrate. The much decreased levels of these carbohydrates in the intact N protein, corroborated the latter finding. The O-glycosidic saccharides appeared similar to those found in control alpha glycophorins. However, the tryptic glycopeptide pattern of the variant differed from control M or N alpha glycophorins, suggesting a deletion of a large segment of the molecule near residues 40-61 and/or a substitution of methionine for a residue upstream from residue 40.(ABSTRACT TRUNCATED AT 400 WORDS)     

Origin of thymidine kinase deficient (TK-) haploid frog cells via an intermediate thymidine transport deficient (TT-) phenotype

Wild-type cultured cells of the frog cell line ICR 2A give rise to 5-bromodeoxyridine (BUdR)-resistant colonies only when the selecting concentration of the drug is 5 × 10^?5 M or lower. The progeny of these colonies multiply in 10^?4 M BUdR; resistance is correlated with the absence of a thymidine (TdR)-specific transport reaction with a K^m in the range of 2–7 × 10^?4 M. All of the TdR transport-deficient (TT-) isolates examined (25) had TdR kinase activity (4% to 100% of wild-type). Variants deficient in TdR kinase activity (5% of wild-type) were obtained by exposing TT-cultures to 10^?3 M BUdR. The TK - variants multply continuously in 10^?3 M BUdR and retain the phenotype after prolonged culture in the absence of the drug. The frequency with which they occur is increased 20 to 50 fold by prior treatment of the culture with ICR 191, an acridine mustard mutagen. In haploid cells, it would be expected that TK- variants would arise in equal numbers from wild-type and TT- cultures if loss TdR kinase occurred independently of loss of the transport reaction. However, wild-type cells give no colonies resistant to 10^?3 M BUdR under conditions the give 1 to 50 colonies per million TT- cells. The TT- phenotype seems to be a required intermediate state in the origin of the TK- phenotype. Therefore, the TK- clones described above are unlikely to be products of mutation at a single genetic locus.     

Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).