Cosmid Library Of DNA Research Articles

Identification of an additional member of the secretin superfamily of proteins in Pseudomonas aeruginosa that is able to function in type II protein secretion.

Pseudomonas aeruginosa is a prolific exporter of virulence factors and contains three of the four protein secretion systems that have been described in gram-negative bacteria. The P. aeruginosa type II general secretory pathway (GSP) is used to export the largest number of proteins from this organism, including lipase, phospholipase C, alkaline phosphatase, exotoxin A, elastase and LasA. Although these exoproteins contain no sequence similarity, they are specifically and efficiently transported by the secretion apparatus. Bacterial homologues of XcpQ (GspD), the only outer membrane component of this system, have been proposed to play the role of gatekeeper, by presumably interacting and recognizing the exported substrates to allow their passage through the outer membrane. While determining the phenotype of nonpolar deletions in each of the xcp genes, we have shown that a deletion of the P. aeruginosa strain K xcpQ does not completely abolish protein secretion. As the proposed function of XcpQ should be requisite for secretion, we searched for additional factors that could carry out this role. A cosmid DNA library from a PAK strain deleted for xcpP-Z was tested for its ability to increase protein secretion by screening for enhanced growth on lipid agar, a medium that selects for the secretion of lipase. In this manner, we have identified an XcpQ homologue, XqhA, that is solely responsible for the residual export observed in a deltaxcpQ strain, although it is not required for efficient secretion in wild-type P. aeruginosa. We have also demonstrated that this protein is capable of recognizing all of the exoproteins of P. aeruginosa, arguing against the proposed role of members of the secretin family as determinants of specificity.

UDPGT cDNA expression and UDPGT1 in human liver.

Following expression of UDPGTh1 and UDPGTh2 in Cos-1 cells, each isoform metabolized three types of dihydroxy- or trihydroxy-substituted ring structures, including the 3,4-catechol estrogen (4-hydroxyestrone), estriol and 17-epiestriol, and hyodeoxycholic acid (HDCA), but the UDPGTh2 isozyme was 100-fold more efficient than UDPGTh1. UDPGTh1 and UDPGTh2 are 86% identical overall (76 differences out of 528 amino acids), including 55 differences in the first 300 amino acids of the amino terminus, a domain which confers isoform substrate specificity. The data indicate a high level of conservation in the amino terminus is not required for the preservation of substrate specificity. Analysis of glucuronidation activity encoded by UDPGTh1/UDPGTh2 chimeric cDNAs constructed at their common restriction sites, Sac I (codon 279), Nco I (codon 385), and Hha I (codon 469), showed that nine amino acids between residues 385 and 469 are important for catalytic efficiency, suggesting that this region represents a domain which is critical for catalysis but distinct from that responsible for aglycon selection. Screening of leukocyte DNA cosmid library with human UDPGT-Br1 (1-470 bps) or UDPGT-Br2 (1-450 bps) resulted in three overlapping clones, which were isolated and mapped by endonucleases. Construction of subclones and DNA sequencing, Southern blot analysis revealed that a cluster of 4 exons (132, 88, 220, 1032 bps in one clone) encodes the entire region of 3' identity shared between human UDPGT-phenol, human UDPGT-Br1 and human UDPGT-Br2. A similar strategy but using probes which correspond to the unique regions of human UDPGT-Br1 and human UDPGT-Br2 showed that the exon 1 of UGT1A and UGT1D encodes the unique region of human UDPGT-Br1, and human UDPGT-Br2 and is located 5.6 and 49 Kb, respectively, upstream of the 4 common exons.

Toxoflavin is an Essential Factor for Virulence of Burkholderia glumae Causing Rice Seedling Rot Disease.

Mutants of Burkholderia glumae, the causal agent of rice seedling rot and grain rot diseases, were isolated by transposon (Tn) mutagenesis with Tn4431 harboring a tetracycline (Tc) resistance gene, which was carried on the suicide vector pUCD623. The resulting Tc-resistant colonies were screened for a loss of toxin production as well as for a lack of pathogenicity to rice seedlings. One of the screened mutants, the mutant No.19, was shown to have a single copy of Tn insertion on the genomic DNA by Southern hybridization test. Restriction enzyme fragments containing Tn4431-flanking sequences from the genomic DNA of mutant No.19 were cloned and used as probes for colony hybridization of the genomic DNA cosmid library from a wild strain of B. glumae. Six recombinant clones showing homology to the probes were isolated and conjugated into mutant No.19 by triparental mating. The recombinant cosmid pNP147, one of six clones, was able to complement the mutant No.19 for toxin production and pathogenicity. The toxin, purified from a culture of mutant No.19 with pNP147, was identified as toxoflavin by NMR and Mass spectra analyses. These results suggest that toxoflavin is a critical factor in disease development of B. glumae, since the nontoxigenic mutants were unable to induce seedling rot disease to rice plants.

A 37-kb restriction map of the human immunoglobulin lambda variable locus, VB cluster, harboring four functional genes and two non-coding V<FONT FACE=Symbol>l</font> sequences

The human immunoglobulin lambda variable locus (IGLV) is mapped at chromosome 22 band q11.1-q11.2. The 30 functional germline v-lambda genes sequenced untill now have been subgrouped into 10 families (V<FONT FACE="Symbol">l</font>1 to V<FONT FACE="Symbol">l</font>10). The number of V<FONT FACE="Symbol">l</font> genes has been estimated at approximately 70. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes (V) responsible for the synthesis of lambda-type Ig light chains, and the J<FONT FACE="Symbol">l</font>-C<FONT FACE="Symbol">l</font> cluster with the joining segments and the constant genes. Recently the entire variable lambda gene locus was mapped by contig methodology and its one- megabase DNA totally sequenced. All the known functional V-lambda genes and pseudogenes were located. We screened a human genomic DNA cosmid library and isolated a clone with an insert of 37 kb (cosmid 8.3) encompassing four functional genes (IGLV7S1, IGLV1S1, IGLV1S2 and IGLV5a), a pseudogene (V<FONT FACE="Symbol">l</font>A) and a vestigial sequence (vg1) to study in detail the positions of the restriction sites surrounding the V<FONT FACE="Symbol">l</font> genes. We generated a high resolution restriction map, locating 31 restriction sites in 37 kb of the VB cluster, a region rich in functional V<FONT FACE="Symbol">l</font> genes. This mapping information opens the perspective for further RFLP studies and sequencing

Cloning and expression of the porcine obese gene

The product of the obese gene, leptin, may be an important regulator of adiposity via its regulation of feed intake and energy metabolism. Probes were developed using the polymerase chain reaction to analyze gene expression and determine the structure of the porcine ob gene. Porcine ob was expressed in adipose tissue as a 3,100 bp mRNA. Finished pigs (136 kg) had higher (P<.01) levels of ob mRNA (per unit of 6‐actin mRNA) in subcutaneous adipose tissue than did growing pigs (60 kg). Obese gene expression was not detected in tissues other than adipose depots. A genomic DNA fragment containing the ob gene was isolated from a cosmid DNA library. Sequence analysis indicates that the ob gene has three exons. A short untranslated sequence was identified as exon 1 and the amino acid coding sequence was located in the second and third exons. The gene structure, intron/exon boundaries, and the amino acid sequence was highly conserved in mammalian species. The porcine leptin amino acid sequence was 95%, 92% and 89% similar to cattle, human and mouse leptin sequences, respectively.

Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172

A DNA fragment (pCHI5422) containing two genes encoding a 54-kDa and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc) 2 dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 μM (min) −1 mg −1 and 17 μM (min) −1 mg −1 on the natural swollen chitin, respectively.

Organisation of the bph gene cluster of transposon Tn4371, encoding enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds.

Tn4371 is a 55 kb transposon which encodes enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds into benzoate and 4-chlorobenzoate derivatives. We constructed a cosmid library of Tn4371 DNA. The bph genes involved in biphenyl/4-chlorobiphenyl degradation were found to be clustered in the middle of the transposon. Sequencing revealed an organisation of the bph genes similar to that previously found in Pseudomonas sp. KKS102, i.e. the bphEGF genes are located upstream of bphA1A2A3 and bphA4 is separated from bphA1A2A3 by bphBCD. Consensus sequences for sigma54-associated RNA polymerase were found upstream of bphA1 and bphEGF. Plasmid RP4::Tn4371 was transferred into a mutant of Alcaligenes eutrophus H16 lacking sigma54. In contrast to wild-type H16 exconjugants, the sigma54 mutant exconjugants could not grow on biphenyl, indicating the dependence of Tn4371 bph gene expression on sigma54. The Tn4371-encoded bph pathway was activated when biphenyl and various biphenyl-like compounds were present in the growth medium. Preliminary observations indicate the presence of a region outside the catabolic genes downstream of bphA4 which is involved in mediating at least the basal expression of BphC.

The Epimorphin Gene Is Highly Conserved among Humans, Mice, and Rats and Maps to Human Chromosome 7, Mouse Chromosome 5, and Rat Chromosome 12

A genomic DNA fragment containing the rat epimorphin gene sequence was cloned from a rat DNA cosmid library using a mouse epimorphin cDNA probe. Within the cosmid insert, nine epimorphin exons were identified and sequenced. The predicted amino acid sequence of the rat epimorphin protein exhibited 96 and 86% identity with the mouse and human epimorphin proteins, respectively. Consistent with the developmentally related expression pattern of the mouse epimorphin gene, transcripts of the rat epimorphin gene were detected in 17-day postfertilization rat embryos. The gene, designatedEpim,was assigned to rat chromosome 12 by somatic cell hybrid analysis and localized to 12q16 by fluorescencein situhybridization. The mouse and human homologs of this gene were localized on mouse chromosome 5 and human chromosome 7 by linkage analysis and chromosomalin situhybridization, respectively.

The mouse immunoglobulin kappa locus contains about 140 variable gene segments.

In continuation of our efforts to elucidate the immunoglobulin kappa locus of the mouse we analyzed 46 yeast artificial chromosomes (YACs) containing V kappa, J kappa and C kappa genes. The YACs, which were derived from DNA of C57BL/6 and C3H mice, ranged from 0.3-1.9 Mb in size. On the basis of hybridization with probes specific for the V kappa gene families a group of 13 YACs was selected for detailed analysis. The V kappa genes of the YACs were then characterized by hybridization to the family-specific probes and by the sizes of the EcoRI fragments on which they were found. This way evidence was obtained for 140 different V kappa gene signals on the YACs. Of these 63 had been characterized before on clones from a cosmid library of total mouse DNA (I. Zocher et al., Eur. J. Immunol. 1995. 25: 3326-3331) and 22 others were found now on cosmid clones derived from the YACs. Six V kappa genes of the previous study which were not found on the YACs are probably located outside of the kappa locus. The YACs were arrayed in a unique order establishing a YACs panel which most likely contains the whole kappa locus. The cosmid contigs and solitary cosmid clones which contain the 63 plus 22 V kappa gene signals mentioned above comprise about 2.0 Mb. Assuming that the remaining 55 V kappa genes are spaced at the same average distance of 24 kb, one may extrapolate to a locus size of 3.3 Mb.

Wild chromosomal variants in Aspergillus nidulans.

Pulsed-field gel electrophoresis and a chromosome-specific cosmid DNA library were used to determine the karyotypes of wild-type Aspergillus nidulans isolates from around the world. Overall, little structural variation was found, with a few major exceptions. One isolate possessed a non-essential B-chromosome of about 1.0 million base pairs (mb). Another isolate had undergone a non-reciprocal translocation of about 1.6 mb of chromosome VI onto chromosome VIII. Other than these chromosomal differences, these isolates appeared phenotypically normal. To analyze its effects on meiosis, the translocation isolate was outcrossed with another wild-type derivative that had a normal electrophoretic karyotype. This cross produced a range of phenotypes, including duplicated progeny that had a barren phenotype similar to that described for Neurospora partial disomics. The duplication was somewhat vegetatively unstable. This is the first association of sterility with chromosomal duplication in A. nidulans.

The thiostrepton-resistance-encoding gene in Streptomyces laurentii is located within a cluster of ribosomal protein operons

A common approach to identify and clone biosynthetic gene from an antibiotic-producing streptomycete is to clone the resistance gene for the antibiotic of interest and then use that gene to clone DNA that is linked to it. As a first step toward cloning the genes responsible for the biosynthesis of thiostrepton (Th) in Streptomyces laurentii (Sl), the Th resistance-encoding gene ( tsnR) was cloned as a 1.5-kb BamHI-PvuII fragment in Escherichia coli (Ec), and shown to confer Th resistance when introduced into S. lividans TK24. The tsnR-containing DNA fragment was used as a probe to isolate clones from cosmid libraries of DNA in the Ec cosmid vector SuperCos, and pOJ446 (an Ec/streptomycete) cosmid vector. Sequence and genetic analysis of the DNA flanking the tsnR indicates that the Sl tsnR is not closely linked to biosynthetic genes. Instead it is located within a cluster of ribosomal protein operons.

Isolation and characterization of five genotypic mutants of chlorate-resistant cyanobacteria unable to utilize nitrate

Spontaneous mutants of the cyanobacteriumSynechococcus PCC 7002 resistant to chlorate were isolated. Either 40mM or 400mM Na2ClO3 was used as the selective agent. Putative Chlr colonies were picked onto medium containing ammonia as the sole N source, then replicaplated to media containing either NH4+, NO2− as N sources. Of 252 putative mutants, 106 were able to use either NH4Cl or NaNO2 but not NaNO3 as their sole source of nitrogen. All of the mutant isolates had generation times similar to wild-type 7002 when grown on either ammonium (3.8–4.1 h/generation) or nitrite (4.5–4.7 h/generation). None had detectable methyl viologensupported nitrate reductase activity and are thus phenotypically NRase−. The Chlr mutants had photomediated O2 production and dark O2 uptake rates similar to the wild type and responded similarly to selected metabolic inhibitors. They expressed increased levels of phycocyanin (PC) synthesis under normal, nitrogen-replete growth conditions, but rapidly lapsed into a chlorotic state upon a shift to either medium containing nitrate or to N-free medium. Genetic analysis of the Chl4 mutants indicated that each could be rescued by direct transformation with chromosomally derived DNA from the wild-type strain. Frequencies of transformation for the mutants were characteristic for single genetic lesions in this cyanobacterium. On the basis of marker rescue by a cosmid library of wild-type DNA, the NRase− mutants could be grouped into five distinctive genotypic families.

TSPY-Related Sequences Represent a Microheterogeneous Gene Family Organized as Constitutive Elements in DYZ5 Tandem Repeat Units on the Human Y Chromosome

TSPY (testis-specific protein, Y-encoded) is encoded by members of a Y-chromosome-specific sequence family. We show here that TSPY elements are part of the DYZ5 repeat unit. We have established a cosmid library of Y-chromosomal DNA derived from the hybrid cell line 3E7 and isolated eight cosmids representing 15 TSPY elements. Interindividual variability with respect to TSPY copy number was observed. One cosmid clone was investigated in detail by subcloning and sequencing. Sequence microheterogeneity was identified among both transcribed and nontranscribed TSPY elements. Transcript variability is not restricted to single basepair exchanges. We present data for the existence of at least three differently sized TSPY transcripts, caused by different patterns of splicing.

Identification of Genomic Regions of the Herpesvirus of Turkeys (HVT) with Helper Activity for Avian Adeno-Associated Virus (AAAV)

Herpesvirus of turkeys (HVT) is a potent helper for the defective parvovirus avian adeno-associated virus (AAAV). To study the helper mechanism at the molecular level, we established a complete cosmid library of HVT DNA in a set of seven overlapping clones and transiently cotransfected secondary chicken embryo fibroblast (CEF) cells with AAAV DNA and recombinant cosmids (cBL) (individual as well as in different combinations). Using an AAAV-specific indirect immunofluorescence assay, we identified four regions on the HVT genome, represented by cBL267, cBL27, cBL33, and cBL34, which express helper functions for AAAV. As demonstrated by infection studies with extracts from cotransfected CEF cells, cBL267 promotes productive AAAV growth, while the helper effect induced by cBL27, cBL33, and cBL34 is limited to the synthesis of noninfectious AAAV antigen. In view of the data presented, possible HVT-specific helper mechanisms for AAAM are discussed.

Molecular studies on an ancient gene encoding for carbamoyl-phosphate synthetase.

1. Carbamoyl-phosphate synthetase (EC 6.3.5.5.) catalyses the synthesis of carbamoyl phosphate, the immediate precursor of arginine and pyrimidine biosynthesis, and is highly conserved throughout evolution. The large subunit of all carbamoyl-phosphate synthetases sequenced to date comprises two highly homologous halves, the product of a proposed ancestral gene duplication. The sequences of the enzymes of Escherichia coli, Drosophila melanogaster, Saccharomyces cerevisiae, rat and Syrian hamster all have duplications, suggesting that this event occurred in the progenote predating the separation of the major phylae. Until now, only limited data on carbamoyl-phosphate synthetase were available for the primitive eukaryote Dictyostelium discoideum and for the Archaea Methanosarcina barkeri MS. The DNA sequence of the D. discoideum carbamoyl-phosphate gene and additional sequence for the carbamoyl-phosphate synthetase gene of M. barkeri MS have been determined, and a duplicated structure for both is clearly demonstrated. 2. Genes with ancient duplications provide unique information on their evolution. A study of the intron/exon organization of the rat carbamoyl-phosphate synthetase I gene and the carbamoyl-phosphate synthetase hamster II gene in the CAD multi-gene complex shows that at least some of their introns are very old. Evidence is provided that some introns must have been present in the ancestral precursor before its duplication. 3. The human carbamoyl-phosphate synthetase I gene has been isolated and characterized. A human liver cDNA library was constructed and probed for carbamoyl-phosphate synthetase I. A human genomic DNA cosmid library was also probed for the carbamoyl-phosphate synthetase I gene. The cDNA sequence of the human carbamoyl-phosphate synthetase I gene has been determined, and work has been initiated to confirm that at least part of this gene is contained within two cosmids spanning 46kb. This will enable future studies to be made on mutations in this gene in the rare autosomal recessive deficiency of carbamoyl-phosphate synthetase I.

Organization and nature of fortimicin A (astromicin) biosynthetic genes studied using a cosmid library of Micromonospora olivasterospora DNA.

The cloning of five DNA segments carrying at least seven genes (fms1, fms3, fms4, fms5, fms7, fms11, and fms12) that participate in fortimicin A (astromicin) biosynthesis was described previously. These DNA fragments were used to screen a cosmid library of genomic DNA in order to examine if these biosynthetic genes are clustered in Micromonospora olivasterospora. One cosmid clone (pGLM990) was obtained, which hybridized to all the probes. Complementation analysis, using mutants blocked at various steps and chimeric plasmids subcloned from pGLM990, showed that three additional genes (fms8, fms10, and fms13) are present in pGLM990. A gene conferring self-resistance to the antibiotic, which was independently cloned in Streptomyces lividans, using the plasmid vector pIJ702 was also found to be linked to the cluster of biosynthetic genes. Thus, at least ten biosynthetic genes and a self-defense gene are clustered in a chromosomal region of about 27 kb in M. olivasterospora. Interestingly, the fms8 gene which participates in the dehydroxylation step of fortimicin A biosynthesis was found to have homology with a neomycin resistance gene nmrA from the neomycin-producing Micromonospora sp. MK50. Studies using a cell-free extract of the fms8 mutant and its parent strain showed that the enzyme encoded by fms8 phosphorylates a biosynthetic precursor, fortimicin KK1, in the presence of ATP. Thus the dehydroxylation reaction is suggested to occur via the phosphorylation of the target hydroxyl group. DNA regions homologous to fms genes were found in Micromonospora sp. SF-2098 and Dactylosporangium matsuzakiense, both producers of fortimicin group antibiotics.

Sexual development genes of Neurospora crassa.

The filamentous fungus Neurospora crassa undergoes a complex program of sexual development to form a fruiting body composed of several kinds of specialized tissue. Subtractive hybridization was used to isolate genes that are expressed preferentially during this sexual phase. Many such sexual development (sdv) genes were identified in a cosmid library of Neurospora genomic DNA. Fourteen of the sdv genes were subcloned, and their expression in mutant strains and under crossing and vegetative growth conditions was examined. All of the regulated transcripts were less abundant (and in many cases not detectable) in strains grown under vegetative (high nitrogen) conditions, suggesting that nitrogen starvation is required for their synthesis. The expression of most of the sdv genes also required a functional A mating type product, even under crossing growth conditions, suggesting that this product functions as a master control in sexual development. To determine if the products of the sdv genes play essential roles in the sexual cycle, a reverse-genetic approach (based on RIP (repeat-induced point mutation)-mediated gene disruptions) was used to create mutations in the genes. A mutant strain (asd-1) with a recessive crossing defect (apparently caused by the RIP process) was isolated; in this strain, early development is normal and may asci are formed, but ascospores are never delineated. A second recessive mutant strain (asd-2) was apparently created by ectopic integration of the transforming DNA into a gene required for the sexual process; in this strain the sexual process was blocked at an early stage, and the ascogeneous tissue underwent little development.

Mitochondrial DNA ofMarchantia polymorphaas a Single Circular Form with no Incorporation of Foreign DNA

A cosmid library and physical maps of mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, were constructed using the cosmid clones. Electrophoresis profile and the physical maps indicated that the liverwort mtDNA was approximately 183 kb long, the smallest among plant mtDNAs, and that it consisted of a single circular molecule. Southern hybridization analysis showed that genes typical to the mitochondrial genome existed in a single copy, and also that there was no incorporation of chloroplast DNA fragments into the mitochondrial genome.

Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas campestris pv. malvacearum.

In vitro-packaged cosmid libraries of DNA from the bacterium Xanthomonas campestris pv. malvacearum were restricted 200- to 1,000-fold when introduced into Mcr+ strains of Escherichia coli compared with restriction in the Mcr- strain HB101. Restriction was predominantly associated with the mcrBC+ gene in E. coli. A plasmid (pUFR052) encoding the XmaI and XmaIII DNA methylases was isolated from an X. campestris pv. malvacearum library by a screening procedure utilizing Mcr+ and Mcr- E. coli strains. Transfer of plasmids from E. coli strains to X. campestris pv. malvacearum by conjugation was enhanced by up to five orders of magnitude when the donor cells contained pUFR052 as well as the plasmid to be transferred. Subcloning of pUFR052 revealed that at least two regions of the plasmid were required for full modification activity. Use of such modifier plasmids is a simple, novel method that may allow the efficient introduction of genes into any organism in which restriction systems provide a potent barrier to such gene transfer.

Structural organization and complete sequence of the human α- N-acetylgalactosaminidase gene: Homology with the α-galactosidase A gene provides evidence for evolution from a common ancestral gene

Human α- N-acetylgalactosaminidase (α-GalNAc; EC 3.2.1.49), the lysosomal glycohydrolase that cleaves α- N-acetylgalactosaminyl moieties in glycoconjugates, is encoded by a gene localized to chromosome 22q13→qter. The deficient activity of this enzyme results in Schindler disease, an autosomal recessive disorder characterized by the increased urinary excretion of glycopeptides and oligosaccharides containing α- N-acetylgalactosaminyl moieties. Recently, the 3.6-kb full-length α-GalNAc cDNA sequence was isolated and found to have remarkable nucleotide and predicted amino acid homology (55.8 and 46.9%, respectively) with the human α-galactosidase A (α-Gal A) cDNA. To investigate the possible evolutionary relatedness of the two glycosidases, the α-GalNAc chromosomal gene was isolated and characterized. Screening of a human genomic DNA cosmid library resulted in the identification of a clone, gAGB-1, with an ∼ 35-kb insert that contained the entire α-GalNAc gene. A single ∼ 15-kb EcoRI fragment of gAGB-1, which contained the complete 3.6-kb cDNA sequence, was digested and the subcloned fragments were sequenced in both orientations. The 13,709-bp α-GalNAc gene had nine exons ranging from 95 to 2028 bp and intronic sequences of 304 to 2684 bp. All exon/intron junctions conformed to the GT AG consensus rule. Analysis of 1.4 kb of 5′ flanking sequence revealed three Sp1 and two CAAT-like promoter elements. This region was GC-rich (56%), but no HTF island was identified. The gene contained six Alu-repetitive elements, all in the reverse orientation. Comparison of the structural organization of the α-GalNAc and the α-Gal A genes revealed that all six α-GalA introns were identically positioned in the homologous α-GalNAc exonic sequence. Two additional introns, 1 and 8, were identified in the α-GalNAc gene. The predicted amino acid sequences of α-GalNAc exons 2 through 7 and those of corresponding α-Gal A exons 1 through 6 were 46.2 to 62.7% identical. In contrast, there was little, if any, similarity between the deduced amino acid sequences of α-Gal A exon 7 and α-GalNAc exons 8 and 9. The remarkable amino acid identity and the identical exonic interruption by six introns of the α-GalNAc and α-Gal A genes suggest that this region in both genes is evolutionarily related and arose through duplication and divergence from a common ancestral gene.