Isolation of the regulatory regions and genomic organization of the porcine alpha1,3-galactosyltransferase gene.
ABSTRACT: BACKGROUND:Alpha1,3-galactosyltransferase (alpha1,3GT) is an enzyme that produces carbohydrate chains termed alphaGal epitopes found in most mammals, although some species of higher primates, including human, are notable exceptions. The evolutionary origin of the lost alpha1,3GT enzyme activity is not yet known, although it has been suggested that the promoter activity of this gene in the ancestors of higher primates was inactivated. METHODS:We used 5'-or 3'-RACE, GenomeWalking, reverse transcriptase polymerase chain reaction (RT-PCR) and dual Luciferase reporter assay for identification of the full-length cDNA, which includes the transcription initiation site and the promoter region of porcine alpha1,3GT gene. RESULTS:The region around exon 1 is guanine and cytosine (GC)-rich (about 70%), comprising a CpG island spanning more than 1.5 kbp. The 5'-flanking region of exon 1 contains multiple transcription factor consensus motifs, including GC-box, SP1, AP2, and GATA-box sites, in the absence of TATA or CAAT-box sequences. The entire gene consists of three 5' noncoding and six coding region exons spanning more than 52 kbp. Detailed analysis of alpha1,3GT transcripts revealed two major alternative splicing patterns in the 5'-untranslated region (5'-UTR) and evidence for minor splicing activity that occurs in a tissue-specific manner. Interspecies comparison of 5'-UTR shows minimal homology between porcine and murine sequences except for exon 2, which suggests that the regulatory regions differ among species. CONCLUSIONS:These observations have important implications for experiments involving genetic manipulation of the alpha1,3GT gene in transgenic animals in terms of promoter utilization, and particularly in genetically engineering cells for the animal cloning technology by nuclear transfer.
Project description:Galactose-alpha1,3-galactose (alphaGal) epitopes, the synthesis of which requires the enzyme product of alpha1,3-galactosyltransferase (alpha1,3GT), are sugar chains on the cell surface of most mammalian species. Notable exceptions are higher primates including Old World monkeys, apes, and humans. The alphaGal-negative species as well as mice with deletion of the alpha1,3GT gene produce abundant anti-alphaGal antibodies. The evolutionary loss of alphaGal epitopes has been attributed to point mutations in the coding region of the gene. Because no transcripts could be found in the higher primate species with Northern blot analysis, a potential alternative explanation has been loss of upstream regulation of the gene. Here, we have demonstrated that the rhesus promoter is functional. More importantly, a variety of full-length transcripts were detected with sensitive PCR-based methods in the tissues of rhesus monkeys, orangutans, and humans. Five crucial mutations were delineated in the coding region of the human and rhesus and three in the orangutan, any one of which could be responsible for inactivation of the alpha1,3GT gene. Two of the mutations were shared by all three higher primates. These findings, which elucidate the molecular basis for the evolutionary loss of alphaGal expression, may have implications in medical research.
Project description:The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose alpha (1,3) galactose (alphaGal) present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s) with bioartficial liver devices (BALs), composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine alphaGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH) immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta) were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies.Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH) cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has not been previously identified, and encodes xenoantibodies at later time points post-transplant. Sequencing of IgG clones revealed increased usage of the monkey germline progenitor most similar to human IGHV3-11 and the onset of mutations.The small number of IGVH genes encoding xenoantibodies to porcine hepatocytes in non-human primates and humans is highly conserved. Rhesus monkeys are an appropriate preclinical model for testing novel reagents such as those developed using structure-based drug design to target and deplete antibodies to porcine xenografts.
Project description:Lecithin:retinol acyltransferase (LRAT) catalyzes the synthesis of retinyl esters in many tissues and is crucial for the transport and intracellular storage of vitamin A. LRAT expression is highly regulated in the liver. In this study, we have cloned and sequenced the full-length LRAT mRNA from human liver and identified its 5'- and 3'-ends. Full-length LRAT mRNA comprises 5023 nt with a predicted ORF of 230 amino acids, a short 5'UTR, and a relatively long 3'UTR of 4 kb containing several polyadenylation signals and AU-rich regions. Based on alignment of this mRNA with human genomic DNA in the GenBank database, the human LRAT gene spans about 9.1 kbp and consists of two exons and a relatively long 4-kbp intron. Further analysis of normal liver revealed a minor alternative splicing variant which lacks a 103 nt polynucleotide contained in the 5'UTR of the full-length LRAT transcript. This variant predicts that the LRAT gene is organized into three exons and two introns, as reported for LRAT cloned from retinal pigment epithelium (RPE) cells. These two LRAT mRNA variants are also present in testis, which is known to express LRAT and contain retinyl esters. Major and minor transcription start sites for human liver LRAT mRNA were identified and the sequence of the upstream proximal promoter region was retrieved from the GenBank database and physically analyzed for the presence of putative cis-acting elements essential for basal transcription. This region contains a TATA box, CCAAT box and Sp1 site, which are apparently conserved in mouse and rat LRAT genes. Our results provide evidence that multiple LRAT mRNA transcripts, which are expressed in a tissue-specific manner, may result from several mechanisms including differential splicing of the 5'UTR region and the use of multiple polyadenylation signals in the 3'UTR.
Project description:The human DEAD-box Y (DBY) RNA helicase (aka DDX3Y) gene is thought to be the major azoospermia factor a (AZFa) gene in proximal Yq11. Men with its deletion display no somatic pathologies, but suffer from complete absence of germ cells. Accordingly, DDX3Y protein is expressed only in the germline in spermatogonia, although the transcripts were found in many tissues. Here, we show the complex transcriptional control of a testis-specific DDX3Y transcript class with initiation at different sites upstream of the gene's open reading frame (5'Untranslated Region; UTR) and with polyadenylation in their proximal 3'UTR. The most distal transcriptional start site (TSS; ?1 kb upstream) was mapped in MSY2, a Y-specific minisatellite. As this testis-specific 5'UTR was subsequently processed by three alternative splicing events, it has been tentatively designated 'exon-T'(estis). The MSY2 sequence unit was also found upstream of the mouse Ddx3y gene. However, only after its tandem amplification on the Y chromosome of Platyrrhini (new world monkeys) and Catarrhini (old world monkeys) did MSY2 become part of a novel distal promoter for DDX3Y expression in testis tissue and provides a second transcriptional start site (T-TSS-II) in Catarrhini. We therefore suggest that the development of a novel distal DDX3Y promoter in primates, which is activated only in testis tissue, is probably part of the gene's germline translation control.
Project description:We have isolated and characterized the gene encoding mouse synexin, which consists of 14 exons and spans approximately 30 kbp of genomic DNA. The protein's unique N-terminal domain is encoded by six exons, and the C-terminal tetrad repeat, the site of the membrane-fusion and ion-channel domain, is encoded by seven exons. The first exon encodes the 5'-untranslated region. Analysis of synexin-gene expression in different mouse tissues shows that mRNA with exon 6 is only present in brain, heart and skeletal muscle. mRNA lacking exon 6 is expressed in all tissues we have examined. The initiation site for transcription was determined by primer-extension analysis and S1 nuclease mapping. Sequence analysis of the 1.3 kb 5'-flanking region revealed that the promoter has a TATA box located at position -25 and a number of potential promoter and regulatory elements. A CCAAT motif was not observed but CCATT is located in an appropriate position for the CCAAT motif upstream from the transcription-initiation start site. In addition, the 5'-flanking region contains two sets of palindromic sequences. Finally, we have determined that the functional synexin gene (Anx7) is located on mouse chromosome 14 and that a pseudogene (Anx7-ps1) is located on chromosome 10.
Project description:We have examined the genomic organization and the transcription unit for the human link protein gene from genomic clones and RNA prepared from human cartilage over a wide age range. Five exons cover the gene which is greater than 60 kbp. Primer extension and S1 nuclease protection analysis revealed transcription initiation to be 315 bases upstream from the translation initiation codon in RNA derived from cartilage samples ranging from fetal to 53 years of age. The first exon size therefore is 289 bp and examination of the 5' flanking sequence indicated a lack of a TATA box in close proximity to the transcription start, although a TATAA-like motif (TCTAA) was present at -75 bp. Such a sequence at a similar distance can serve as a promoter in the chicken link protein gene. The large first exon of 289 bp is similar to that of the chicken but contrasts with that described previously for human (96 bp) and rat (62 bp). We also analysed human link protein mRNA by PCR for the presence of an alternatively spliced exon that is present in rat mRNA in low abundance, but could not detect such transcripts. Equine and porcine mRNA contained this spliced form but the results suggested that this was expressed as a rare transcript.
Project description:The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, ?-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine ?-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine ?-casein gene. Sequence inspection of the 5'-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP ?. In addition, the first intron of the porcine ?-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP ?, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5'-proximal region with or without intron 1 of the porcine ?-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5'-proximal region with intron 1 of the porcine ?-casein gene. The ?-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine ?-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine ?-casein gene activity.
Project description:Preformed and elicited Ab's against the Galalpha1,3Gal terminating carbohydrate chains (alphaGal Ab's) are the primary cause of hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation. alphaGal Ab's are produced by long-lived Ab-producing cells that are not susceptible to pharmacological immunosuppression. We reasoned that antigen-specific elimination of alphaGal Ab's might be achieved in vivo by systemic administration of nonimmunogenic polyvalent alphaGal structures with high avidity for alphaGal Ab's. We devised GAS914, a soluble trisaccharide-polylysine conjugate of approximately 500 kDa that effectively competes for alphaGal binding by alphaGal IgM (IC(50), 43 nM) and IgG (IC(50), 28 nM) in vitro. Injections of GAS914 in cynomolgus monkeys, at the dose of 1 mg/kg, resulted in the immediate decrease of more than 90% of circulating alphaGal Ab's and serum anti-pig cytotoxicity. In baboons, repeated injections of GAS914 effectively reduced both circulating alphaGal Ab's and cytotoxicity over several months. Studies with [(14)C]GAS914 in rhesus monkeys and Gal(-/-) mice indicate that GAS914 binds to circulating alphaGal Ab's and that the complex is quickly metabolized by the liver and excreted by the kidney. Remarkably, posttreatment alphaGal Ab titers never exceeded pretreatment levels and no sensitization to either alphaGal or the polylysine backbone has been observed. Furthermore there was no apparent acute or chronic toxicity associated with GAS914 treatment in primates. We conclude that GAS914 may be used therapeutically for the specific removal of alphaGal Ab's.
Project description:The gene for the hemocyanin subunit e of the tarantula Eurypelma californicum has been isolated from a genomic phage library by using a corresponding cDNA clone as a probe. The transcriptional unit spans a chromosomal region of about 55 kilobase pairs (kbp). The gene consists of nine exons that are separated by large introns. The intron/exon boundaries were determined by direct comparison of genomic and cDNA sequences. A putative promoter region ("TATA" box, reversed "CAAT" box) 100 bp 5' to the translational initiation codon strongly suggests the presence of a functional gene. The 3' flanking region carries the polyadenylylation signal (AATAAA) and several conserved structures for the 3' splicing of the pre-mRNA. A comparison of the gene architecture of the subunit e gene with the three-dimensional structure of the arthropod hemocyanin subunit shows a good correspondence with the division of the subunit into three domains (two exons coding for the first, three coding for the second, and four coding for the third domain). The relationship to molluscan hemocyanins, different tyrosinases, and the larval serum proteins is discussed.
Project description:Aldehyde dehydrogenase class 3 (ALDH3) constitutes 20-40% of the total water-soluble proteins in the mammalian cornea. Here, we show by Northern blot analysis that ALDH3 expression in the mouse is at least 500-fold higher in the cornea than in any other tissue examined, with very low levels of expression detected in the stomach, urinary bladder, ocular lens, and lung. Histochemical localization reveals that this exceptional level of expression in the mouse cornea occurs in the anterior epithelial cells and that little ALDH3 is present in the keratocytes or corneal endothelial cells. A 13-kbp mouse ALDH3 promoter fragment containing >12 kbp of the 5' flanking sequence, the 40-bp untranslated first exon, and 29 bp of intron 1 directed cat reporter gene expression to tissues that express the endogenous ALDH3 gene, except that transgene promoter activity was higher in the stomach and bladder than in the cornea. By contrast, when driven by a 4.4-kbp mouse ALDH3 promoter fragment [1,050-bp 5' flanking region, exon 1, intron 1 (3.4 kbp), and 7 bp of exon 2] expression of the cat reporter gene was confined to the corneal epithelial cells, except for very low levels in the liver, effectively reproducing the corneal expression pattern of the endogenous ALDH3 gene. These results indicate that tissue-specific expression of ALDH3 is determined by positive and negative elements in the 5' flanking region of the gene and suggests putative silencers located in intron 1. We demonstrate regulatory sequences capable of directing cornea-specific gene expression, affording the opportunity for genetic engineering in this transparent tissue.