Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA

We have previously isolated a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase (alpha(1,3)-GT) cDNA (Larsen, R. D., Rajan, V. P., Ruff, M. M., Kukowska-Latallo, J., Cummings, R. D., and Lowe, J. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8227-8231). This enzyme constructs the terminal alpha(1,3)-galactosyl linkage within the epitope Gal alpha 1----3Gal. This epitope is expressed by New World monkeys and many nonprimate mammals but generally not by Old World primates, anthropoid apes, or man. To investigate the molecular basis for the apparent species-specific absence of this enzyme and its oligosaccharide product, we have sequenced a human genomic DNA fragment homologous to the murine alpha(1,3)-GT cDNA. This fragment contains a 703-nucleotide region that shares 82% identity with a region of the murine cDNA encoding part of the enzyme's catalytic domain. The human sequence, however, has suffered deletion of single nucleotides at two separate positions, relative to the murine sequence. These frameshift mutations disrupt the translational reading frame that would otherwise maintain a 76% amino acid sequence identity between the human sequence and the murine alpha(1,3)-GT. Moreover, nonsense mutations exist within this disrupted reading frame that would truncate the human polypeptide, relative to the murine enzyme. We therefore propose that this human sequence represents a pseudogene and cannot determine expression of Gal alpha 1----3Gal epitopes on human cells.     

The molecular basis for galalpha(1,3)gal expression in animals with a deletion of the alpha1,3galactosyltransferase gene

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes alpha1,3galactosyltransferase (alpha1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galalpha(1,3)Gal carbohydrate epitope was not observed as Galalpha(1,3)Gal staining was still present in tissues from GGTA1(-/-) animals. This shows that, contrary to previous dogma, alpha1,3GT is not the only enzyme able to synthesize Galalpha(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1(-/-) mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Galalpha(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galalpha(1,3)Gal epitope. Galalpha(1,3)Gal synthesized by iGb3S, in contrast to alpha1,3GT, was resistant to down-regulation by competition with alpha1,2fucosyltransferase. Moreover, Galalpha(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 (-/-) mice. Galalpha(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.     

Isolation of a porcine UDP-GalNAc transferase cDNA mapping to the region of the blood group EAA locus on pig chromosome 1

In our studies of the genes constituting the porcine A0 blood group system, we have characterized a cDNA, encoding an alpha(1,3)N-acetylgalactosaminyltransferase, that putatively represents the blood group A transferase gene. The cDNA has a 1095-bp open reading frame and shares 76.9% nucleotide and 66.7% amino acid identity with the human ABO gene. Using a somatic cell hybrid panel, the cDNA was assigned to the q arm of pig chromosome 1, in the region of the erythrocyte antigen A locus (EAA), which represents the porcine blood group A transferase gene. The RNA corresponding to our cDNA was expressed in the small intestinal mucosae of pigs possessing EAA activity, whereas expression was absent in animals lacking this blood group antigen. The UDP-N-acetylgalactosamine (UDP-GalNAc) transferase activity of the gene product, expressed in Chinese hamster ovary (CHO) cells, was specific for the acceptor fucosyl-alpha(1,2)galactopyranoside; the enzyme did not use phenyl-beta-D-galactopyranoside (phenyl-beta-D-Gal) as an acceptor. Because the alpha(1,3)GalNAc transferase gene product requires an alpha(1,2)fucosylated acceptor for UDP-GalNAc transferase activity, the alpha(1,2)fucosyltransferase gene product is necessary for the functioning of the alpha(1,3)GalNAc transferase gene product. This mechanism underlies the epistatic effect of the porcine S locus on expression of the blood group A antigen. ABBREVIATIONS: CDS: coding sequence; CHO: Chinese Hamster Ovary; EAA: erythrocyte antigen A; FCS: foetal calf serum; Fucalpha(1,2)Gal: fucosyl-alpha(1,2)galactopyranoside; Gal: galactopyranoside; GGTA1: Galalpha(1,3)Gal transferase; PCR: polymerase chain reaction; phenyl-beta-D-Gal: phenyl-beta-D-galactopyranoside; R: Galbeta1-4Glcbeta1-1Cer; UDP-GalNAc: uridine diphosphate N-acetylgalactosamine     

Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases

The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.     

Expression of alpha-gal epitopes on HeLa cells transduced with adenovirus containing alpha1,3galactosyltransferase cDNA

Alpha1,3galactosyltransferase (alpha1,3GT) synthesizes alpha-gal epitopes (Gal(alpha)1-3Galbeta1-4GlcNAc-R) on glycoconjugates in nonprimate mammals but not in humans. Transduction of alpha1,3GT gene into human HeLa cells by an adenovirus vector allowed for accurate kinetics studies on the appearance of alpha1,3GT and of its product, the alpha-gal epitope, in the transduced cells. Mouse alpha1,3GT cDNA was inserted into a replication-defective adenovirus vector. This viral vector, designated Ad(alpha)GT, could be propagated in human 293 cells that have the viral E1 complementing gene. Transduction of HeLa cells resulted in immediate penetration of approximately 20 Ad(alpha)GT copies into each cell and the appearance of alpha1,3GT mRNA after 4h. Catalytic activity of alpha1,3GT was first detected in the cells after 6 h. The initial appearance of alpha-gal epitopes (approximately 6 x 10(4)/cell) on cell surface glycoconjugates was detected 10 h posttransduction, whereas 24 h posttransduction each cell expressed 2 x 10(6) epitopes. The activity of alpha1,3GT in cells transduced with approximately two copies of Ad(alpha)GT was eightfold lower than that in cells transduced with approximately 20 Ad(alpha)GT copies; however, the number of alpha-gal epitopes/cell remained closely similar. This implies that increased alpha1,3GT activity above a certain saturation level does not result in a corresponding increase in the carbohydrate product, possibly because of competing glycosyltransferases.     

Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney

Galalpha1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-beta-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the beta-galactosidic linkage in the Galalpha1-3Galbeta1-4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-beta-galactosidase from Flavobacterium keratolyticus and beta-1,3-glucanases. The enzyme expressed in Escherichia coli removed the alpha-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the alpha-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-beta-galactosidase C is a valuable aid in xenotransplantation.     

Molecular cloning of a fourth member of a human alpha (1,3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x, and difucosyl sialyl Lewis x epitopes.

We and others have previously described the isolation of three human alpha (1,3)fucosyltransferase genes which form the basis of a nascent glycosyltransferase gene family. We now report the molecular cloning and expression of a fourth homologous human alpha (1,3)fucosyltransferase gene. When transfected into mammalian cells, this fucosyltransferase gene is capable of directing expression of the Lewis x (Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc), sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4 [Fuc alpha 1-->3]GlcNAc), and difucosyl sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3 Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc) epitopes. The enzyme shares 85% amino acid sequence identity with Fuc-TIII and 89% identity with Fuc-TV but differs substantially in its acceptor substrate requirements. Polymerase chain reaction analyses demonstrate that the gene is syntenic to Fuc-TIII and Fuc-TV on chromosome 19. Southern blot analyses of human genomic DNA demonstrate that these four alpha (1,3)fucosyltransferase genes account for all DNA sequences that cross-hybridize at low stringency with the Fuc-TIII catalytic domain. Using similar methods, a catalytic domain probe from Fuc-TIV identifies a new class of DNA fragments which do not cross-hybridize with the chromosome 19 fucosyltransferase probes. These results extend the molecular definition of a family of human alpha (1,3)fucosyltransferase genes and provide tools for examining fucosyltransferase gene expression.     

Localization of LewisX, sialyl-LewisX and alpha-galactosyl epitopes on glycosphingolipids in lens tissues

Mammalian lens contains several neutral and acidic glycosphingolipids, the core structures of which are ganglio-, neolacto-, globo-, and isoglobo-series sugar chains. Old World monkey lens shows glycosphingolipid compositions similar to those of human cataractous lens, in particular the presence of Lewisxand sialyl-Lewisxepitopes and the absence of alpha-galactosyl epitope. Dog and pig lenses contain globotriaosylceramide and the sialyl-Lewisxcontaining neolactotetraosylceramide, respectively, which were found in primate lens, together with the alpha-galactosyl epitope containing neolactotetraosylceramide. Thin-layer chromatography immunostaining revealed the enrichment of some neolacto-series glycosphingolipids in the cortical and nuclear fibers, but not in lens epithelia, of dog, pig, and Japanese monkey lenses. Immunohistochemical studies confirmed the expression of Lewisx, sialyl-Lewisx, and alpha-galactosyl epitopes in the inner cortical and nuclear fibers, in association with the differentiation and maturation of lens epithelial cells to lens fibers. Glycobiological approaches thus suggested that some neolacto-series glycosphingolipids are involved in lens fiber development, in which the physiological roles of the alpha-galactosyl epitope are evolutionarily replaced by the Lewisxand sialyl-Lewisxepitopes in Old World monkeys and humans.      

Primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase determined by mass spectrometry sequence analysis and molecular cloning. Evidence for a protein motif in the sialyltransferase gene family.

The Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase forms the NeuAc alpha 2,3Gal beta 1,3(4)GlcNAc sequences found in terminal carbohydrate groups of glycoproteins and glycolipids. High energy collision-induced dissociation analysis of tryptic peptides from only 300 pmol of the purified Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase provided 25% of the total amino acid sequence and led to the successful cloning of this enzyme. The peptide sequence information was used to design short degenerate primers for use in the polymerase chain reaction. A long specific cDNA fragment was amplified which was used to isolate a clone from a rat liver cDNA library. The cloned cDNA encodes a 374-amino acid protein containing an amino-terminal signal-anchor sequence characteristic of all cloned glycosyltransferases and produced sialyltransferase activity when transiently expressed in COS-1 cells. When compared with two other cloned sialyltransferases, the primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase revealed a homologous region in all three enzymes consisting of a stretch of 55 amino acids located in their catalytic domains. This feature together with lack of homology in the remaining 85% of the sequence of the three sialyltransferases defines a pattern of sequence homology not found in cloned cDNAs of other glycosyltransferase families.     

Regulation of the expression of Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids in kidney

Previous studies (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1369-1373; Galili, U., Shohet, S. B., Korbrin, E., Stults, C. L. M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762) have established that there is a unique evolutionary distribution of glycoconjugates carrying the Gal alpha 1-3Gal beta 1-4GlcNAc epitope. These glycoconjugates are expressed by cells from New World monkeys and non-primate mammals, but not by cells from humans, Old World monkeys, or apes. The lack of expression of this epitope in the latter species appears to result from the suppression of gene expression for the enzyme UDP-galactose:nLc4Cer alpha 1-3-galactosyltransferase (alpha 1-3GalT) (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Although many non-primate species are known to express this carbohydrate epitope, the nature (i.e. glycoprotein or glycosphingolipid) of the glycoconjugate carrying this epitope is only known for a few tissues in a few animal species. Furthermore, it is not known whether all animal species express this epitope in the same tissues. We have investigated these questions by analyzing the glycosphingolipids in kidney from several non-primate animal species. Immunostained thin layer chromatograms of glycosphingolipids from sheep, pig, rabbit, cow, and rat kidney with the Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipid-specific monoclonal antibody, Gal-13, demonstrated that kidney from all of these species except rat contained Gal alpha 1-3Gal beta 1-4GlcNAc neutral glycosphingolipids. A lack of expression of Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids in rat may be due to the lack of expression of the enzyme (alpha 1-3GalT) which catalyzes the formation of the Gal alpha 1-3Gal nonreducing terminal sequence of these compounds or to the lack of expression of glycosyltransferases which are necessary for the synthesis of the neolacto core structure of these compounds. These possibilities were evaluated in two ways. First, the three enzymes (UDP-N-acetylglucosamine:LacCer beta 1-3-N-acetyl-glucosaminyltransferase, UDP-galactose:Lc3Cer beta 1-4-galactosyltransferase, and alpha 1-3GalT) involved in the synthesis of the Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids were assayed using an enzyme-linked immunosorbent assay-based assay system and carbohydrate sequence-specific monoclonal antibodies. Second, TLC immunostaining was done to determine if the glycosphingolipid precursors (i.e. Lc3Cer and nLc4Cer) are expressed in rat kidney. Interestingly, rat kidney had a relatively high level of alpha 1-3GalT activity compared with the other animals tested.(ABSTRACT TRUNCATED AT 400 WORDS)     

反义RNA对猪α-1,3-半乳糖苷转移酶活性的影响

 α 1,3 半乳糖表位是猪 人异种移植超急性排斥反应的主要抗原 ,由α 1,3 半乳糖苷转移酶催化合成 .用RT PCR方法扩增中国实验用小型猪α 1,3 半乳糖苷转移酶cDNA的前 582bp ,测定碱基序列并构建其反义表达载体pLXRN ,将其转染入猪主动脉内皮细胞 .NorthernBlotting表明α 1,3 半乳糖苷转移酶mRNA减少 .检测α 1,3 半乳糖苷转移酶活性表明 ,反义RNA可使其活性下降32 2 % .研究结果表明可能通过反义RNA来抑制猪 人异种移植超急性排斥反应     

The human protein S locus: identification of the PS alpha gene as a site of liver protein S messenger RNA synthesis

The protein S locus, situated on chromosome 3, consists of two protein S genes. Here, we report the cloning and complete nucleotide sequence of the 3'-untranslated region of the two genes designated PS alpha and PS beta. Both regions span approximately 1,200 nucleotides. They show a high degree (-97%) of homology, with deviations caused by small deletions, insertions and point mutations. Comparison of PS alpha and PS beta with the reported protein S liver cDNAs, shows that the latter all originate from the PS alpha gene. The PS alpha gene therefore is marked as the major site of synthesis of liver protein S mRNA. Sequence comparison with the bovine protein S cDNA reveals that the PS beta gene has accumulated a few more mutations than the PS alpha gene since duplication of the ancestral protein S gene that seems to have occurred recently during primate evolution.     

The alpha-gal epitope and the anti-Gal antibody in xenotransplantation and in cancer immunotherapy

The alpha-gal epitope (Galalpha1-3Galbeta1-(3)4GlcNAc-R) is abundantly synthesized on glycolipids and glycoproteins of non-primate mammals and New World monkeys by the glycosylation enzyme alpha1,3galactosyltransferase (alpha1,3GT). In humans, apes and Old World monkeys, this epitope is absent because the alpha1,3GT gene was inactivated in ancestral Old World primates. Instead, humans, apes and Old World monkeys produce the anti-Gal antibody, which specifically interacts with alpha-gal epitopes and which constitutes approximately 1% of circulating immunoglobulins. Anti-Gal has functioned as an immunological barrier, preventing the transplantation of pig organs into humans, because anti-Gal binds to the alpha-gal epitopes expressed on pig cells. The recent generation of alpha1,3GT knockout pigs that lack alpha-gal epitopes has resulted in the elimination of this immunological barrier. Anti-Gal can be exploited for clinical use in cancer immunotherapy by targeting autologous tumour vaccines to APC, thereby increasing their immunogenicity. Autologous intact tumour cells from haematological malignancies, or autologous tumour cell membranes from solid tumours are processed to express alpha-gal epitopes by incubation with neuraminidase, recombinant alpha1,3GT and with uridine diphosphate galactose. Subsequent immunization with such autologous tumour vaccines results in in vivo opsonization by anti-Gal IgG binding to these alpha-gal epitopes. The interaction of the Fc portion of the vaccine-bound anti-Gal with Fcgamma receptors of APC induces effective uptake of the vaccinating tumour cell membranes by the APC, followed by effective transport of the vaccinating tumour membranes to the regional lymph nodes, and processing and presentation of the tumour-associated antigen (TAA) peptides. Activation of tumour-specific T cells within the lymph nodes by autologous TAA peptides may elicit an immune response that in some patients will be potent enough to eradicate the residual tumour cells that remain after completion of standard therapy. A similar expression of alpha-gal epitopes can be achieved by transduction of tumour cells with an adenovirus vector (or other vectors) containing the alpha1,3GT gene, thus enabling anti-Gal-mediated targeting of the vaccinating transduced cells to APC. Intratumoral delivery of the alpha1,3GT gene by various vectors results in the expression of alpha-gal epitopes. Such expression of the xenograft carbohydrate phenotype is likely to induce anti-Gal-mediated destruction of the tumour lesion, similar to rejection of xenografts by this antibody. Opsonization of the destroyed tumour cell membranes by anti-Gal IgG further targets them to APC, thus converting the tumour lesion, treated by the alpha1,3GT gene, into an in situ autologous tumour vaccine.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Immunogenicity of influenza virus vaccine is increased by anti-gal-mediated targeting to antigen-presenting cells

This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the alpha-Gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing alpha-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fc gamma receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in alpha 1,3galactosyltransferase (alpha 1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of alpha-Gal epitopes on carbohydrate chains of PR8 virus (PR8(alpha gal)) was catalyzed by recombinant alpha1,3GT, the glycosylation enzyme that synthesizes alpha-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8(alpha gal) displayed much higher numbers of PR8-specific CD8(+) and CD4(+) T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking alpha-Gal epitopes. Mice immunized with PR8(alpha gal) also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines.     

Production of alpha 1,3-galactosyltransferase-knockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase

The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galalpha 1,3-Gal epitope, which is synthesized by alpha 1,3-galactosyltransferase (alpha1,3-GT). The Galalpha 1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galalpha 1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galalpha 1,3-Gal antigen is the most attractive option. In this study, the 5' end of the alpha 1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the alpha 1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an alpha 1,3-GT knockout allele and express a randomly inserted human alpha 1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous alpha 1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the alpha 1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection.     

Primate evolution of the alpha-globin gene cluster and its Alu-like repeats

The arrangement of alpha-globin genes in Old World and New World monkeys and a prosimian, galago, has been determined by restriction mapping. Recombinant DNAs containing galago and Old World monkey alpha-globin genes have been isolated and subjected to a partial sequence determination for comparison to alpha-globin genes in human, chimpanzee and non-primate mammals. The results of this extensive structural analysis are relevant to several topics concerning the evolution of primate alpha-globin genes and Alu family repeats. All orders of higher primates (i.e. Old and New World monkeys, chimpanzee and human) have the same arrangement of alpha-globin genes. In contrast, the arrangement and correction of galago alpha-globin genes differ from those of higher primates, but are similar to those of non-primate mammals. The 5' and 3'-flanking regions of the human alpha 1 gene are orthologous to the corresponding region in galago, identifying the human alpha 2 gene as the more recently duplicated gene. The human psi alpha 1 gene is found to be inactivated after divergence of the human and galago lineages but prior to the divergence of human and monkey. Orthologous Alu family members in human and monkey DNAs indicate that the dispersion of some Alu repeats occurred prior to the divergence of these lineages. However, the Alu-like repeats of prosimian and higher primates result from entirely independent events giving rise to different repeat elements inserted at distinct genomic positions.