Human MUC4 mucin cDNA and its variants in pancreatic carcinoma

The human MUC4 gene is not expressed in normal pancreas; however, its dysregulation results in high levels of expression in pancreatic tumors. To investigate the tumor-associated expression, MUC4 cDNA was cloned from a human pancreatic tumor cell line cDNA expression library using a polyclonal antibody raised against human deglycosylated mucin and RT-PCR. Pancreatic MUC4 cDNA shows differences in 12 amino acid residues in the non-tandem repeat coding region with no structural rearrangement as compared with tracheal MUC4. The full-length MUC4 cDNA includes a leader sequence, a serine and threonine rich non-tandem repeat region, a central large tandem repeat domain containing 48 bp repetitive units, regions rich in potential N-glycosylation sites, two cysteine-rich domains, EGF-like domains, and a transmembrane domain. We also report the presence of a new EGF-like domain in MUC4 cDNA, located in the cysteine-rich region upstream from the first EGF-like domain. Four distinct splice events were identified in the region downstream of the central tandem repeat domain that generate three new MUC4 cDNA sequences (sv4, sv9, and sv10). The deduced amino acid sequences of two of these variants lack the transmembrane domain. Furthermore, two unique forms of MUC4 (MUC4/Y and MUC4/X) generated as a result of alternative splicing lack the salient feature of mucins, the tandem repeat domain. A high degree of polymorphism in the central tandem repeat region of MUC4 was observed in various pancreatic adenocarcinoma cell lines, with allele sizes ranging from 23.5 to 10.0 kb. MUC4 mRNA expression was higher in differentiated cell lines, with no detectable expression in poorly differentiated pancreatic tumor cell lines.     

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.     

Alternative splicing generates a family of putative secreted and membrane-associated MUC4 mucins.

The MUC4 mucin gene encodes a putative membrane-anchored mucin with predicted size of 930 kDa, for its 26.5-kb allele. It is composed of two regions, the 850-kDa mucin-type subunit MUC4alpha and the 80-kDa membrane-associated subunit MUC4beta. In this study, we cloned and characterized unique MUC4 cDNA sequences that differ from the originally published sequence. Eight alternative splice events located downstream of the central large tandem repeat array generated eight new, distinct cDNAs. The deduced sequences of these MUC4 cDNAs (sv1-MUC4 to sv8-MUC4, the full length cDNA being called sv0-MUC4) provided seven distinct variants, five secreted forms and two membrane-associated forms. Furthermore, two other alternative splicing events located on both sides of the tandem repeat array created two variants, MUC4/Y and MUC4/X, both lacking the central tandem repeat. Therefore, MUC4 can be expressed in three distinct forms, one membrane-bound, one secreted, and one lacking the hallmark feature of mucin, the tandem repeat array. Although no specific function has yet been discovered for the family of proteins putatively produced from the unique MUC4 gene, we suspect that the MUC4 proteins may be implicated in the integrity and renewal of the epithelium.     

Depression in gene expression for poly(ADP-ribose) synthetase during the interferon-gamma-induced activation process of murine macrophage tumor cells

A 2.7-kb cDNA clone coding for bovine poly(ADP-ribose) synthetase was isolated from a lambda gt11 expression library by direct immunological screening with an antiserum to the enzyme. The cDNA hybridizes to an approximately 3.8-kb bovine thymus polyadenylated RNA, which translates an immunoprecipitable 120-kDa protein with the antibody to the enzyme. The partial DNA sequence of the cDNA was determined and portions of the predicted amino acid sequence matched the sequence of 26 amino acids at the N terminal of the 41-kDa alpha-chymotryptic fragment and two cyanogen-bromide-cleaved peptides of the enzyme. A subcloned fragment from the coding region of the cDNA was used as a probe to estimate the level of mRNA for the enzyme during the interferon-gamma-induced activation process of the murine macrophage tumor P388D1 cell line. The amount of mRNA for the enzyme decreased nearly completely within 24 h after incubation in a medium containing interferon-gamma, while mRNA of the Ia antigen, one of the major histocompatibility gene products, was increased in the macrophage tumor cells by interferon-gamma as confirmed by the I-A beta cDNA as a probe. These results suggest that the gene expression for poly(ADP-ribose) synthetase is depressed during the interferon-gamma-induced activation process of macrophage tumor cells.     

MUC genes: mucin or not mucin? That is the question

Mucins are macromolecules lying the cells in contact with external environment and protect the epithelium against constant attacks such as digestive fluids, microorganisms, pollutants, and toxins. Mucins are the main components of mucus and are synthesized and secreted by specialized cells of the epithelium (goblet cells, cells of mucous glands) or non mucin-secreting cells. Human mucin genes show common features: large size of their mRNAs, large nucleotide tandem repeat domains, complex expression both at tissular and cellular level. Since 1987, 21 MUC symbols have been used to designate genes encoding O-glycoproteins containing tandem repeat domains rich in serine, threonine and proline. Some of these genes encode true mucins while others encode non mucin adhesion O-glycoproteins. In this paper, we propose a classification based on sequence similarities and expression areas. Two main families can be distinguished: secreted mucins or gel-forming mucins (MUC2, MUC5AC, MUC5B, MUC6), and membrane-bound mucins (MUC1, MUC3, MUC4, MUC12, MUC17). Muc-deficient mice will provide important models in the study of functional relationships between these two mucin families.     

Cloning and nucleotide sequence of the murine interleukin-3 gene

Southern hybridization analysis using a probe derived from a murine interleukin-3 (IL-3) cDNA clone revealed the presence of a single IL-3 gene in the haploid murine genome. An 8600-base-pair (8.6-kb) murine genomic EcoRI fragment containing the IL-3 gene was isolated by screening a library of size-fractionated genomic EcoRI fragments cloned in lambda gtWES X lambda B. The nucleotide sequence of a 3.5-kb region of the cloned DNA encompassing the IL-3 gene was determined. The gene contains four introns of 96, 993, 135 and 122 base pairs (bp), located within the coding region. The large intron contains 12 copies of a 14-15-bp tandem repeating sequence which resembles a human cellular homologue of a BKV enhancer sequence. The nucleotide sequence of the exons agrees exactly with that of an IL-3 cDNA cloned from WEHI-3, a tumorigenic cell line which over-produces IL-3, establishing that the unprocessed primary structure of IL-3 is identical in WEHI-3 and in BALB/c mice. Southern hybridization has revealed genomic alteration in the vicinity of the IL-3 gene in WEHI-3 cells.     

Localization of the active gene of aldolase on chromosome 16, and two aldolase A pseudogenes on chromosomes 3 and 10

Summary Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and >30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, >30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the >30-kb fragment and is probably localized on chromosome 3 with the >30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and >30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes, the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.     

MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.     

Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4.

We report here the full coding sequence of a novel mouse putative membrane-associated mucin containing three extracellular EGF-like motifs and a mucin-like domain consisting of at least 20 tandem repeats of 124-126 amino acids. Screening a cosmid and a BAC libraries allowed to isolate several genomic clones. Genomic and cDNA sequence comparisons showed that the gene consists of 25 exons and 24 introns covering a genomic region of approximately 52 kb. The first intron is approximately 16 kb in length and is followed by an unusually large exon (approximately 9.5 kb) encoding Ser/Thr-rich tandemly repeated sequences. Radiation hybrid mapping localized this new gene to a mouse region of chromosome 16, which is the orthologous region of human chromosome 3q29 encompassing the large membrane-anchored mucin MUC4. Contigs analysis of the Human Genome Project did not reveal any other mucin on chromosome 3q29 and, interestingly, our analysis allowed the determination of the genomic organization of the human MUC4 and showed that its exon/intron structure is identical to that of the mouse gene we cloned. Furthermore, the human MUC4 shares considerable homologies with the mouse gene. Based on these data, we concluded that we isolated the mouse ortholog of MUC4 we propose as Muc4. Expression studies showed that Muc4 is ubiquitous like SMC and MUC4, with highest levels of expression in trachea and intestinal tract.     

Variable number tandem repeat polymorphism of the mucin genes located in the complex on 11p15.5

A family of four genes that encode major secreted mucins (MUC6, MUC2, MUC5AC and MUC5B) map to within 400kb on chromosome 11p15.5. These genes contain long stretches of tandem repeats of sequence that encode serine- and threonine-rich domains but that otherwise show no similarity from gene to gene, and regions of unique sequence domains that do show evidence of sequence homology. We have previously reported the existence of polymorphism in three of these genes but the extent and nature of this allelic variation is now described here in detail. Variable number tandem repeat polymorphisms of MUC6, MUC2 and MUC5AC are predicted to encode mucin polypeptides that differ in length. In the case of MUC2 and MUC6 these length differences are substantial (up to twofold). MUC5B in contrast does not show common allele length variation. Three MUC2 mutations are reported, none of which are associated with the meiotic recombinations previously observed in this region of chromosome 11. Received: 24 July 1997 / Accepted: 13 November 1997     

Evolution of the large secreted gel-forming mucins

Mucins, the major component of mucus, contain tandemly repeated sequences that differ from one mucin to another. Considerable advances have been made in recent years in our knowledge of mucin genes. The availability of the complete genomic and cDNA sequences of MUC5B, one of the four human mucin genes clustered on chromosome 11, provides an exemplary model for studying the molecular evolution of large mucins. The emerging picture is one of expansion of mucin genes by gene duplications, followed by internal repeat expansion that strictly preserves frameshift. Computational and phylogenetic analyses have permitted the proposal of an evolutionary history of the four human mucin genes located on chromosome 11 from an ancestor gene common to the human von Willebrand factor gene and the suggestion of a model for the evolution of the repeat coding portion of the MUC5B gene from a hypothetical ancestral minigene. The characterization of MUC5B, a member of the large secreted gel-forming mucin family, offers a new model for the comparative study of the structure-function relationship within this important family.     

肿瘤MUC1/Y的克隆及其胞外段在大肠杆菌中的可溶性表达、纯化和鉴定

为获得MUC1 Y全长cDNA及其胞外段蛋白 ,以用于进一步的生物学功能及肿瘤生物学治疗的研究 .利用RT PCR从HeLa细胞中扩增MUC1 Y全长cDNA ;PCR扩增其胞外段 ,克隆到原核表达载体pGEX 2T ,转化DH5a菌 ,诱导表达 ;亲和层析纯化 ;凝血酶酶切、GST活性及N端蛋白测序鉴定 ;免疫家兔制备多克隆抗体 .所得MUC1 YcDNA的开放读框为 759bp ,登录于GenBank(AF12 552 5) .其信号肽编码序列缺失 9bp ,第 3 3 1位发生G A转换 ,造成缬氨酸突变为蛋氨酸 .表达获得约 4 0kD融合蛋白GST Yex ,占菌体总蛋白 2 5%～ 3 0 % ,其中 70 %～ 80 %为可溶性 ,经亲和层析一步纯化 ,纯度 >90 % ,GST比活性为 0 2 1U μg .凝血酶酶切后的N端蛋白序列测定表明与已知序列完全一致 ,抗血清ELISA效价为 1∶2 50 0 0 0 .结果表明 ,克隆到发生碱基缺失和突变的MUC1 Y全长cDNA ,获得MUC1 Y胞外段蛋白及其多抗 ,可进一步用于相关研究 .     

Cloning of the am (glutamate dehydrogenase) gene of Neurospora crassa through the use of a synthetic DNA probe

In a previous study the alteration in the amino acid sequence of Neurospora crassa NADP-specific glutamate dehydrogenase (GDH) resulting from two mutually compensating frameshift mutations was used to deduce the first 17 nucleotides of the coding sequence of the am gene. In the work reported here, a synthetic 17-mer corresponding to the deduced sequence was shown to hybridize strongly to a 9-kb HindIII fragment from N. crassa wild-type DNA but not to any corresponding fragment from the DNA of a mutant strain known to be deleted for most or all of the gene. Wild-type HindIII fragments were fractionated for size and a fraction centering around 9 kb was cloned in vector λL47. Two clones carrying the strongly hybridizing fragment were identified. The hybridization to the 17-mer was localized within a 2.7-kb BamHI fragment and, within this, to a 700-bp BamHI-BglII subfragment. 5' end-labelled polyadenylated RNA isolated from wild-type mycelium hybridized to the 2.7-kb BamHI fragment and not appreciably to flanking fragments. The partial sequence analysis of the BamHI-BglII fragment has confirmed that the 17-mer probe matches the coding sequence at the 5' end of the gene and has also revealed an intervening sequence 67 bp in length, interrupting codon 15. Both the 9-kb HindIII fragment and the 2.7-kb BamHI fragment have been shown to be capable of transforming the deletion mutant to prototrophy and ability to produce GDH. Analysis of one transformant showed that the am gene was integrated, together with a part of the long arm of the lambda vector, at an unusual locus. This transformant, in which the am gene does not show its normal linkage to the linkage group 5 marker inl, was found to produce GDH to about 20% of the normal level.     

Molecular cloning and chromosomal localization of a novel human tracheo-bronchial mucin cDNA containing tandemly repeated sequences of 48 base pairs.

A lambda gt11 cDNA library constructed from human tracheo-bronchial mucosa was screened with a polyclonal antiserum raised to chemically deglycosylated pronase glycopeptides from human bronchial mucins. Out of 20 positives clones, one partial cDNA clone was isolated and allowed to map a novel human tracheo-bronchial mucin gene. It contains 48 nucleotide tandem repeats quite perfectly identical which encodes a protein containing about 50% of hydroxy amino-acids. This clone hybridized to polydisperse messages produced by human tracheo-bronchial and human colonic mucosae. The gene (proposed name MUC 4) from which cDNA is derived maps to chromosome 3.     

Cysteine-rich regions of pig gastric mucin contain von willebrand factor and cystine knot domains at the carboxyl terminal(1).

In order to sequence the cysteine-rich regions of pig gastric mucin (PGM), we used our previously identified pig gastric mucin clone PGM-2A to screen a pig stomach cDNA library and perform rapid amplification of cDNA ends to obtain two cysteine-rich clones, PGM-2X and PGM-Z13. PGM-2X has 1071 base pairs (bp) encoding 357 amino acids containing five serine-threonine-rich 16 amino acid tandem repeats, downstream from a cysteine-rich region similar to human and mouse MUC5AC. PGM-Z13 encodes the complete 3'-terminus of PGM and is composed of 3336 bp with a 2964 bp open reading frame encoding 988 amino acids with four serine-threonine-rich tandem repeats upstream from a cysteine-rich region similar to the carboxyl terminal regions of human and rat MUC5AC and human MUC5B. This region is homologous to von Willebrand factor C and D domains involved in acid induced polymerization, and to the carboxyl terminal cystine-knot domain of various mucins, TGF-beta, vWF and norrin, which is involved in dimerization. These newly sequenced cysteine-rich regions of pig gastric mucin may be critical for its gelation and for its observed increased viscosity induced by low pH.     

Two coding regions closely linked to the rat apolipoprotein E gene: nucleotide sequences of rat apolipoprotein C-I and ECL cDNA.

Restriction fragments isolated from a 17-kb rat genomic DNA clone containing the gene for apolipoprotein (apo) E were radiolabeled and used to screen a rat liver cDNA library. A cDNA clone hybridizing to a 6-kb genomic DNA fragment was isolated and the nucleotide sequence of the cDNA insert determined. The sequence was homologous to the sequence for human apo C-I and was used to derive the corresponding amino acid sequence. Unlike human apo C-I, mature rat apo C-I contains histidine, lacks valine, and has alanine at the C terminus and aspartate as the N terminus. Screening the rat liver cDNA library with a radiolabeled 1.9-kb restriction fragment from the genomic DNA clone containing the rat apo E gene identified another cDNA clone (ECL cDNA). Nucleotide sequencing yielded a derived 75-amino-acid sequence for the ECL protein with a hydrophobicity profile similar to that of rat apo C-I. Northern analysis demonstrated a 0.50-kb band for ECL mRNA. The tissue-specific expression of the gene is similar to that of rat apo C-I. This study indicates that the rat apo C-I and ECL genes are closely linked, about 4.5 and 12 kb downstream of the apo E gene, respectively.