Structure and expression of the human polymorphic epithelial mucin gene: an expressed VNTR unit

The human polymorphic epithelial mucin (PEM) is expressed apically by glandular epithelium and by the carcinomas that develop from these tissues. Previously isolated cDNA clones revealed that the core protein contained a large domain consisting of variable numbers of 60 bp tandem repeats (TR), making it an expressed minisatellite. We now report the full genomic sequence of the PEM gene, including 803 bp of 5' flanking sequence. The gene is composed of 7 exons and varies in size from approximately 4 to approximately 7 kb, depending on the number of tandem repeats in exon 2. Expression of PEM was obtained from a genomic clone in an Epstein-Barr virus based vector, after transfection into a human epithelial cell line, indicating the presence of effective regulatory sequences in this clone.     

Cloning and sequencing of a human pancreatic tumor mucin cDNA

A monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin was used to select human pancreatic mucin cDNA clones from a lambda gt11 cDNA expression library developed from a human pancreatic tumor cell line. The full-length 4.4-kilobase mucin cDNA sequence included a 72-base pair 5'-untranslated region and a 307-base pair 3'-untranslated region. The predicted amino acid sequence for this cDNA revealed a protein of 122,071 daltons containing 1,255 amino acid residues of which greater than 60% were serine, threonine, proline, alanine, and glycine. Approximately two-thirds of the protein sequence consisted of identical 20-amino acid tandem repeats which were flanked by degenerate tandem repeats and nontandem repeat sequences on both the amino-terminal and carboxyl-terminal ends. The amino acid sequence also contained five putative N-linked glycosylation sites, a putative signal sequence and transmembrane domain, and numerous serine and threonine residues (potential O-linked glycosylation sites) outside and within the tandem repeat position. The cDNA and deduced amino acid sequence of the pancreatic mucin sequence was over 99% homologous with a mucin cDNA sequence derived from breast tumor mucin, even though the native forms of these molecules are quite distinct in size and degree of glycosylation.     

Reactivity of anti-human milk fat globule antibodies with synthetic peptides

The nucleotide sequence of partial cDNA clones coding for the core protein of a human polymorphic epithelial mucin has recently been obtained, this mucin consists of a highly conserved 60 bp tandem repeat and the amino acids commonly found are PDTRPAPGSTAPPAHGVTSA. We synthesized three peptides, 1) P1.24 containing the 20 amino acids and four amino acids (PDTR) of the adjoining repeat; 2) P1.15 consisting of the first fifteen (PDTRPAPGSTAPPAH) and P1.09 the second nine amino acids (GVTSAPDTR) of peptide P1.24. The reactivities of the synthetic peptides with mAb known to react with breast cancer (BC1, BC2, BC3, HMFG-1, 3E1.2, and RCC-1) were studied. The synthetic peptide, P1.24, corresponding to the antigenic sequence predicted from the tandem repeat reacted with antibodies BC1, BC2, and BC3 (known to react with human milk mucin and mucin expressed in breast cancer) and the antibody HMFG-1 which was used to select the cDNA clones. In addition, the epitopes recognized by BC1, BC2, and BC3 appear to be in the same region of the molecule represented by their reactions with the nine amino acids in peptide P1.09 (GVTSAPDTR). By contrast, other antibodies such as 3E1.2 which reacts only weakly with components of human milk, and RCC-1 that detects a low Mr component (95 kDa) in breast cancer, had no specific reaction with the synthetic peptides, indicating that their epitopes are distinct from those of BC1, BC2, BC3, and HMFG-1. Inasmuch as the antibodies HMFG-1, BC1, BC2, and BC3 react with the fully processed milk mucin, it is likely that some of the peptide is exposed, even in the fully glycosylated molecule. Identification of the different epitopes could lead to the development of "second generation" mAb with enhanced specificity for breast carcinoma using the appropriate synthetic peptides as immunogens.     

Molecular cloning and analysis of the mouse homologue of the tumor-associated mucin, MUC1, reveals conservation of potential O-glycosylation sites, transmembrane, and cytoplasmic domains and a loss of minisatellite-like polymorphism.

We present here the full-length cDNA sequence and genomic structure of the mouse homologue of the tumor-associated mucin, MUC1. This mucin (previously called polymorphic epithelial mucin) is present at the apical surface of most glandular epithelial cells. The mouse gene, Muc-1, encodes an integral membrane protein with 40% of its coding capacity made up of serine, threonine, and proline, a composition typical of a highly O-glycosylated protein. The mucin core protein consists of an amino-terminal signal sequence, a tandem repeat domain encoding 16 repeats of 20-21 amino acids, and unique sequence containing transmembrane and cytoplasmic domains. Homology with the human protein is only 34% in the tandem repeat domain, mainly showing conservation of serines and threonines, presumed sites of O-linked carbohydrate attachment. Homology rises to 87% in the transmembrane and cytoplasmic domains, suggesting that these regions may be functionally important. The pattern of expression of the mouse mucin is very similar to that of its human counterpart and accordingly the two promoter regions share high homology, 74%, although previously identified potential hormone-responsive elements are not conserved. Interestingly, the mouse homologue, unlike its human counterpart does not exhibit a variable number tandem repeat polymorphism. We present evidence that suggests that the mouse gene was at one time polymorphic but has mutated away from this state.     

A serine, threonine and proline-rich region near the carboxyl-terminus of a rat intestinal mucin peptide.

Further sequencing of a cDNA encoding the C-terminal region of a rat intestinal mucin peptide reveals a region corresponding to 258 amino acids enriched in serine, threonine and proline, but no typical mucin-like tandem repeat structures. Between this region and a previously described stretch of 4.5 degenerate S,T,P-rich tandem repeats, there is a 42 amino acid cysteine-rich segment. The discontinuity of cysteine-rich and S,T,P-rich areas near the C-terminus has not been observed in other mammalian mucin structures reported to date.     

Intraspecific variability of the tandem repeats in Nicotiana putrescine N-methyltransferases

The putrescine N-methyltransferase (PMT) cDNA clone previously isolated from tobacco encodes a spermidine synthase-like protein with an 11 amino acid element repeated four times in tandem at the amino terminus. Genomic Southern blot analyses indicated that this N-terminal repeat array is found in tobacco PMTs but absent in Hyoscyamus and Atropa PMTs. A truncated tobacco PMT in which this repeat array was entirely removed still retained full enzymatic activity when expressed in Escherichia coli. Three PMT genes (NsPMT1, NsPMT2, NsPMT3) isolated from Nicotiana sylvestris encode two, five, and nine tandem repeats, respectively, in the first exon, but otherwise encode highly conserved proteins. Analysis of PCR fragments amplified from the genomes of N. tabacum and its two probable progenitors shows that one of the nine repeat elements in NsPMT3 was precisely deleted in the corresponding N. tabacum gene. These results indicate that direct tandem repeats of a 33 bp sequence that encodes 11 amino acids of no obvious function were added to the ancestral Nicotiana PMT gene, and that the tandem repetition was genetically very unstable, contracting or expanding during evolution of the Nicotiana species.     

Evaluation of MUC6 mucin tandem repeats

The MUC6 mucin was originally isolated from stomach mucus and is one of the major secreted mucins of the digestive tract. A full-length cDNA has not been isolated for this large molecule (greater than 15 kb) and it remains poorly studied. To circumvent the lack of reagents for investigating MUC6, we isolated a cDNA clone from a human fetal pancreatic duct cDNA library that encodes 282 amino acids of the MUC6 tandem repeat. A blast search with the sequence of this cDNA clone showed 90% homology with the original MUC6 (L07517) derived from a human stomach cDNA library and 95% homology both with AK096772, a MUC6-related protein isolated from a human prostate cDNA library and the human genome project clone AC083984. The MUC6 partial cDNA clone isolated from fetal pancreas was inserted into an epitope-tagged MUC1 mucin molecule in place of the native tandem repeat. This chimeric mucin was expressed in human pancreatic (Panc1) and colon (Caco2) carcinoma cell lines and purified for analysis of O-glycosylation by fast atom bombardment mass spectrometry (FAB-MS). The FAB-MS spectra showed O-glycans that had been detected previously on chimeric mucins carrying different tandem repeats, though the spectra for MUC1F/6TR mucins expressed in the Panc1 and Caco2 cells were very different. There was a paucity of O-glycosylation in Panc1 cells in comparison to Caco2 cells where many more structures were evident, and the most abundant glycans in Panc1 cells were sialylated.     

A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats

The nucleotide sequences of partial cDNA clones coding for the core protein of a human polymorphic epithelial mucin were determined, and a large domain was found to consist of a 60-base pair tandem repeat sequence. The cDNA clones were originally selected (Gendler, S. J., Burchell, J. M., Duhig, T., Lamport, D., White, R., Parker, M., and Taylor-Papadimitriou, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6060-6064) using three monoclonal antibodies which show differential reactivity with the mucin produced by normal and malignant breast. Two of the epitopes are exposed in the normally processed and cancer-associated mucin, while one epitope is unmasked only in the cancer-associated mucin (Burchell, J. M., Durbin, H., and Taylor-Papadimitriou, J. (1983) J. Immunol. 131, 508-513; Burchell, J., Gendler, S., Taylor-Papadimitriou, J., Girling, A., Lewis, A., Millis, R., and Lamport, D. (1987) Cancer Res. 47, 5476-5482). We show here that all three antibodies react with a synthetic peptide with an amino acid sequence corresponding to that predicted by the tandem repeat. Identification of the epitopes preferentially expressed on the cancer-associated mucin should allow a directed approach to the development of tumor-specific antibodies using synthetic peptides as immunogens.     

The central domain of bovine submaxillary mucin consists of over 50 tandem repeats of 329 amino acids. Chromosomal localization of the BSM1 gene and relations to ovine and porcine counterparts.

We previously elucidated five distinct protein domains (I-V) for bovine submaxillary mucin, which is encoded by two genes, BSM1 and BSM2. Using Southern blot analysis, genomic cloning and sequencing of the BSM1 gene, we now show that the central domain (V) consists of approximately 55 tandem repeats of 329 amino acids and that domains III-V are encoded by a 58.4-kb exon, the largest exon known for all genes to date. The BSM1 gene was mapped by fluorescence in situ hybridization to the proximal half of chromosome 5 at bands q2. 2-q2.3. The amino-acid sequence of six tandem repeats (two full and four partial) were found to have only 92-94% identities. We propose that the variability in the amino-acid sequences of the mucin tandem repeat is important for generating the combinatorial library of saccharides that are necessary for the protective function of mucins. The deduced peptide sequences of the central domain match those determined from the purified bovine submaxillary mucin and also show 68-94% identity to published peptide sequences of ovine submaxillary mucin. This indicates that the core protein of ovine submaxillary mucin is closely related to that of bovine submaxillary mucin and contains similar tandem repeats in the central domain. In contrast, the central domain of porcine submaxillary mucin is reported to consist of 81-amino-acid tandem repeats. However, both bovine submaxillary mucin and porcine submaxillary mucin contain similar N-terminal and C-terminal domains and the corresponding genes are in the conserved linkage regions of the respective genomes.     

Second-order repeats in Xenopus laevis finger proteins

The primary structure of 342 finger repeats encoded in 42 different cDNA clones isolated from Xenopus laevis oocyte and gastrula cDNA libraries has been determined. Comparative sequence analysis of the predicted protein sequences results in a consensus repeat sequence that has an extended conserved segment of 16 amino acid residues, including the evolutionary conserved H/C link element, connected to a highly variable segment that is located in the finger loop region. Groups of tandem finger repeats are found to be organized in distinct higher-order structural units, with a pair of mutually distinct fingers being the most frequently observed second-order repeat unit. Structural features observed are discussed in respect to existing models for Zn finger structure and function.     

Isolation and characterization of two wound-regulated tomato extensin genes

Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library byin situ plaque hybridization with extensin DNA probes. Tom J-10 encoded an extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro₄-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro₄-Tyr₃-Lys-Ser-Pro₄-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro₄ repeat units, a central domain with repeats of the consensus sequence Ser-Pro_2–5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro₄-Ser-Pro-Ser-Pro₄-Thr-Tyr_1–3. RNA blot hybridization analysis with the Tom L-4 extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.     

Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.

A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.     

Identification and characterization of tandem repeats in exon III of dopamine receptor D4 (DRD4) genes from different mammalian species

In this study we have identified and characterized dopamine receptor D4 (DRD4) exon III tandem repeats in 33 public available nucleotide sequences from different mammalian species. We found that the tandem repeat in canids could be described in a novel and simple way, namely, as a structure composed of 15- and 12- bp modules. Tandem repeats composed of 18-bp modules were found in sequences from the horse, zebra, onager, and donkey, Asiatic bear, polar bear, common raccoon, dolphin, harbor porpoise, and domestic cat. Several of these sequences have been analyzed previously without a tandem repeat being found. In the domestic cow and gray seal we identified tandem repeats composed of 36-bp modules, each consisting of two closely related 18-bp basic units. A tandem repeat consisting of 9-bp modules was identified in sequences from mink and ferret. In the European otter we detected an 18-bp tandem repeat, while a tandem repeat consisting of 27-bp modules was identified in a sequence from European badger. Both these tandem repeats were composed of 9-bp basic units, which were closely related with the 9-bp repeat modules identified in the mink and ferret. Tandem repeats could not be identified in sequences from rodents. All tandem repeats possessed a high GC content with a strong bias for C. On phylogenetic analysis of the tandem repeats evolutionary related species were clustered into the same groups. The degree of conservation of the tandem repeats varied significantly between species. The deduced amino acid sequences of most of the tandem repeats exhibited a high propensity for disorder. This was also the case with an amino acid sequence of the human DRD4 exon III tandem repeat, which was included in the study for comparative purposes. We identified proline-containing motifs for SH3 and WW domain binding proteins, potential phosphorylation sites, PDZ domain binding motifs, and FHA domain binding motifs in the amino acid sequences of the tandem repeats. The numbers of potential functional sites varied pronouncedly between species. Our observations provide a platform for future studies of the architecture and evolution of the DRD4 exon III tandem repeat, and they suggest that differences in the structure of this tandem repeat contribute to specialization and generation of diversity in receptor function.     

Molecular cloning of XTP, a tau-like microtubule-associated protein from Xenopus laevis tadpoles

The microtubules of the mammalian nervous system are stabilised by several microtubule-associated proteins (MAPs), including the tau and MAP-2 protein families. The most prominent feature of mammalian tau and MAP-2 proteins is a common and highly homologous microtubule-binding region consisting of three or four imperfect tandem repeats. In this paper we report the cloning and characterisation of a Xenopus laevis tau-like protein (XTP) from tadpole tails. This protein encompasses two isoforms of 673 or 644 amino acids with four tandem repeats that are highly homologous to mammalian tau repeats. Both isoforms share a common amino terminal half, whereas the carboxyl terminus downstream of the repeat region is unique for each isoform. Northern blot analysis revealed that both isoforms are preferentially expressed in the tail of X. laevis tadpoles, whereas a shorter version of XTP is expressed in the head. Recombinant proteins of both XTP isoforms were able to bind microtubules. The longest isoform, however, was more effective at promoting tubulin polymerisation, indicating that sequences downstream of the repeat region affect the microtubule assembling capacity. These results demonstrate that tau-like proteins are found in non-mammalian vertebrate species, where they may support the stability of microtubules.     

Allelic polymorphism in the hamster oviductin gene is due to a variable number of mucin-like tandem repeats

Oviductins are high-molecular-weight glycoproteins specifically secreted by the oviduct. These proteins bind to the zona pellucida of the ovulated oocyte and remain associated with the embryo during its transit in the oviduct. They may be involved in fertilization and early embryonic development. In order to explore their putative biological function, the cDNA sequence corresponding to oviductin in the golden hamster was determined. We found that the deduced amino acid sequence of this heavily O-glycosylated protein presents characteristics typical of mucins, including serine- or threonine-rich tandem repeats. Analysis of several cDNA clones and of genomic DNA revealed the presence of a single copy gene with two frequent alleles differing in the number of repeats. Comparison with oviductin sequences from other mammals indicates a high degree of conservation amongst species, except for the repeat region which shows divergence, notably in the number of repeats. Based on its biochemical and genetic properties, hamster oviductin can now be classified as a secretory mucin. This concept provides a new insight in the elucidation of its biological role: oviductin could possibly provide the oviduct and the oocyte with a protective coating ensuring normal tubal function and embryonic development. © 1995 wiley-Liss, Inc.     

Subunit structure of porcine submaxillary mucin

The structure of a high molecular weight fraction of porcine submaxillary mucin was studied by using degradative techniques. Reduction of disulfide linkages released mucin subunits together with an associated protein(s) of approximately 140 kDa. The molecular weights of the subunits ranged from approximately 0.5 x 10(6) to 2.5 x 10(6). Trypsinization of subunits generated glycosylated domains and small, poorly glycosylated or nonglycosylated tryptic peptides. The glycosylated domains, which have an average molecular weight of approximately 270K, possess an unusual amino acid composition containing only nine different amino acids. The minor amino acids which are absent from the glycosylated domains but which are consistently present in both the mucin and the mucin subunits were recovered in the tryptic peptides. Pronase digestion of the glycosylated domains generated smaller fragments of approximately 17 kDa. Comparing these results to the partial cDNA sequence for porcine submaxillary mucin reported by Timpte et al. [(1988) J. Biol. Chem. 263, 1081-1088] suggests that the glycosylated domains consist of variable numbers of the 81 amino acid tandem repeat observed in the cDNA sequence. Further, the fact that porcine submaxillary mucin contains subunits, link proteins, and glycosylated domains suggests that its structure is similar to that described for cervical and intestinal mucins. Intact mucin, mucin "subunits", and the glycosylated domains are all polydisperse with respect to molecular weight, indicating that mucin polydispersity is due to variability in the number of units linked together as well as to variability in the size of the units.     

Mutation patterns of amino acid tandem repeats in the human proteome

Background

Amino acid tandem repeats are found in nearly one-fifth of human proteins. Abnormal expansion of these regions is associated with several human disorders. To gain further insight into the mutational mechanisms that operate in this type of sequence, we have analyzed a large number of mutation variants derived from human expressed sequence tags (ESTs).

Results

We identified 137 polymorphic variants in 115 different amino acid tandem repeats. Of these, 77 contained amino acid substitutions and 60 contained gaps (expansions or contractions of the repeat unit). The analysis showed that at least about 21% of the repeats might be polymorphic in humans. We compared the mutations found in different types of amino acid repeats and in adjacent regions. Overall, repeats showed a five-fold increase in the number of gap mutations compared to adjacent regions, reflecting the action of slippage within the repetitive structures. Gap and substitution mutations were very differently distributed between different amino acid repeat types. Among repeats containing gap variants we identified several disease and candidate disease genes.

Conclusion

This is the first report at a genome-wide scale of the types of mutations occurring in the amino acid repeat component of the human proteome. We show that the mutational dynamics of different amino acid repeat types are very diverse. We provide a list of loci with highly variable repeat structures, some of which may be potentially involved in disease.     

Molecular cloning and characterization of cDNAs coding for apo-polysialoglycoprotein of rainbow trout eggs. Multiple mRNA species transcribed from multiple genes contain diverged numbers of exact 39-base (13-amino acid) repeats

Polysialoglycoprotein (PSGP) of unfertilized eggs of rainbow trout (Salmo gairdneri) consists of tandem repeats (about 25) of a glycotridecapeptide, Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly (* denotes the attachment site of a polysialoglycan chain) (Kitajima, K., Inoue, Y., and Inoue, S. (1986) J. Biol. Chem. 261, 5262-5269). By using oligodeoxynucleotide probes based on the above sequence, we isolated a genomic clone for apoPSGP which contains 39-base pair repeats (5'-GACGACGCCACCTCTGAAGCT-GCGACCGGCCCGTCTGGC-3') encoding the tridecapeptide. Using a fragment of this genomic DNA as a probe, we next screened a cDNA library constructed with mRNA from immature ovaries of rainbow trout. Nucleotide sequencing analyses of cDNA clones thus obtained revealed that apoPSGP is encoded by multiple mRNA species consisting of diverged numbers (6-32) of the 39-base repeat encoding the tridecapeptide unit and homologous 5'- and 3'-bordering regions. The encoded protein consists of three distinct regions: the N-region consisting of a putative signal peptide and a pro-peptide, the R-region containing diverged numbers of the tandem repeat of 13-amino acid residues, and the C-region with six amino acid residues. Southern blot analysis showed that multiple mRNAs are transcribed from multiple genes for apoPSGP containing diverged numbers of the 39-base pair repeat. Thus, the genes for apoPSGP constitute a multigene family. Expression of the mRNAs is stage and organ specific, i.e. they are expressed only in immature ovaries and not in mature ovaries or in any other organ.