Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin.

Human mammary cells present on the cell surface a polymorphic epithelial mucin (PEM) which is developmentally regulated and aberrantly expressed in tumors. PEM carries tumor-associated epitopes recognized by the monoclonal antibodies HMFG-1, HMFG-2, and SM-3. Previously isolated partial cDNA clones revealed that the core protein contained a large domain consisting of variable numbers of 20-amino acid repeat units. We now report the full sequence for PEM, as deduced from cDNA sequences. The encoded protein consists of three distinct regions: the amino terminus consisting of a putative signal peptide and degenerate repeats; the major portion of the protein which is the tandem repeat region; the carboxyl terminus consisting of degenerate tandem repeats and a unique sequence containing a transmembrane sequence and a cytoplasmic tail. Potential O-glycosylation sites (serines or threonines) make up more than one-fourth of the amino acids. Length variations in the tandem repeat result in PEM being an expressed variable number tandem repeat locus. Tandem repeats appear to be a general characteristic of mucin core proteins.     

Structure and chromosomal location of the human granulin gene.

Granulins are a family of cysteine rich polypeptides some of which have growth modulatory activity. We showed previously that the granulins are encoded within the same precursor consisting of seven granulin domains arranged in tandem. Here we report the chromosomal location and structural organization of the protein coding region of the granulin gene. The granulin gene was assigned to chromosome 17 using DNA from human-hamster somatic cell hybrids. The protein-coding region of the granulin gene was shown to comprise 12 exons covering about 3700 bp. Each tandem granulin repeat is encoded by two non-equivalent exons, a configuration unique to the granulins that would permit the formation of hybrid granulin-like proteins by alternate splicing.     

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.     

Titins in C.elegans with unusual features: coiled-coil domains,novel regulation of kinase activity and two new possible elastic regions

We report that there are previously unrecognized proteins in Caenorhabditis elegans that are similar to the giant muscle proteins called titins, and these are encoded by a single approximately 90kb gene. The gene structure was predicted by GeneMark.hmm and then experimentally verified. The Ce titin gene encodes polypeptides of 2.2MDa, 1.2MDa and 301kDa. The 2.2MDa isoform resembles twitchin and UNC-89 in that it contains multiple Ig (56) and FnIII (11) domains, and a single protein kinase domain. In addition, however, the 2.2MDa isoform contains four classes of short, 14-51 residue, repeat motifs arranged mostly in many tandem copies. One of these tandem repeat regions is similar to the PEVK regions of vertebrate and fly titins. As the PEVK region is one of the main elastic elements of the titins and is also composed of short tandem repeats, this suggests that the repeat motifs in the Ce titins may have a similar elastic function. An interesting aspect of the two largest Ce titin isoforms, is that in contrast to other members of the twitchin/titin family, there are multiple regions which are likely to form coiled-coil structure. In transgenic animals, the first approximately 100 residues of the largest isoforms targets to dense bodies, the worm analogs of Z-discs. Anti-Ce titin antibodies show localization to muscle I-bands beginning at the L2-L3 larval stages and this pattern continues into adult muscle. Ce titins may not have a role in early myofibril assembly: (1) Ce titins are too short to span half a sarcomere, and the onset of their expression is well after the initial assembly of thick filaments. (2) Ce titins are not localized to I-bands in embryonic or L1 larval muscle. The Ce titin protein kinase domain is most similar to the kinase domains of the twitchins and projectin. The Ce titin kinase has protein kinase activity in vitro, and this activity is regulated by a novel mechanism.     

Changes in repeat number, sequence, and reading frame in S-antigen genes of Plasmodium falciparum.

The S antigens from different isolates of Plasmodium falciparum exhibit extensive size, charge, and serological diversity. We show here that the S-antigen genes behave as multiple alleles of a single locus. The size heterogeneity results from different numbers, lengths, and/or sequences of tandem repeat units encoded within the S-antigen genes. Two genes studied here encode antigenically different S antigens but nevertheless have closely related tandem repeat sequences. We show that antigenic differences can arise because repeats are translated in different reading frames.     

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.     

Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: differential expression of tau protein mRNAs in human brain.

We have isolated cDNA clones encoding a 383-amino acid isoform of the human microtubule-associated protein tau. It differs from previously determined tau sequences by the presence of an additional repeat of 31 amino acids, giving four, rather than three, tandem repeats in its carboxy-terminal half. The extra repeat is encoded by a separate exon. Probes derived from cDNA clones encoding the three (type I) and four repeat (type II) tau protein isoforms detected mRNAs for both forms in all adult human brain areas examined. However, in foetal brain only type I mRNA was found. Type I and type II mRNAs were present in pyramidal cells in cerebral cortex. In the hippocampal formation, type I mRNA was found in pyramidal and granule cells; type II mRNA was detected in most, though not all, pyramidal cells but not in granule cells. These observations indicate that tau protein mRNAs are expressed in a stage- and cell-specific manner. Tau protein is found in the protease-resistant core of the paired helical filament, the major constituent of the neurofibrillary tangle in Alzheimer's disease. Taken in conjunction with previous findings, the present results indicate that both the three and four repeat-containing tau protein isoforms are present in the core of the paired helical filament.     

Multiple copies of a retroposon interrupt spliced leader RNA genes in the African trypanosome, Trypanosoma gambiense.

The 140-nucleotide spliced leader (SL) RNA, involved in mRNA maturation in the African trypanosomes and in other kinetoplastida, is encoded by a tandem array of spliced leader genes. We show that the 1.4-kb SL gene repeat unit in Trypanosoma gambiense is organized in tandem arrays confined to two large (minimum size 350-450 kb) restriction fragments. SL genes in both arrays are interrupted by a total of eight conserved insertion elements. Cleavage of genomic DNA at restriction sites present within the insertion element but not in the SL gene repeat, releases variable numbers of SL genes from the tandem array. Since the insertion element contains a terminal poly(A) track of 36 bases and because a 49-bp duplication of target DNA has occurred at the integration site, we conclude that it is a retroposon. This retropson is uniquely associated with the SL gene clusters. These retroposons presumably originated from a single insertion event after which their copy number increased, possibly through unequal sister chromatid exchange.     

Molecular evolution of ubiquitin genes

Ubiquitin is a singular protein with multiple functions. It is probably the most slowly evolving protein known, is encoded by genes with a unique structure, and provides an intriguing case study for various aspects of molecular evolution. In particular, the multiple ubiquitin-coding repeats which have been characterized in man, yeast and a slime mould graphically illustrate the dynamics of concerted evolution, but cast doubts on the effectiveness of this process for unlinked arrays in this repeat family.     

Structural characterization of the fusion of two pentapeptide repeat proteins, Np275 and Np276, from Nostoc punctiforme: resurrection of an ancestral protein

The Nostoc punctiforme genes Np275 and Np276 are two adjacently encoded proteins of 98 and 75 amino acids in length and exhibit sequences composed of tandem pentapeptide repeats. The structures of Np275 and a fusion of Np275 and Np276 were determined to 2.1 and 1.5 A, respectively. The two Nostoc proteins fold as highly symmetric right-handed quadrilateral beta-helices similar to the mycobacterial protein MfpA implicated in fluoroquinolone resistance and DNA gyrase inhibition. The sequence composition of the intervening coding region and the ability to express a fused protein by removing the stop codon for Np275 suggests Np275 and Np276 were recently part of a larger ancestral pentapeptide repeat protein.     

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.     

Three divergent mitochondrial genomes from California populations of the copepod Tigriopus californicus

Previous work on the harpacticoid copepod Tigriopus californicus has focused on the extensive population differentiation in three mtDNA protein coding genes (COXI, COXII, Cytb). In order to get a more complete understanding of mtDNA evolution in this species, we sequenced three complete mitochondrial genomes (one from each of three California populations) and compared them to two published mtDNA genomes from an Asian congener, Tigriopus japonicus. Several features of the mtDNA genome appear to be conserved within the genus: 1) the unique order of the protein coding genes, rRNA genes and most of the tRNA genes, 2) the genome is compact, varying between 14.3 and 14.6 kb, and 3) all genes are encoded on the same strand of the mtDNA. Within T. californicus, extremely high levels of nucleotide divergence (>20%) are observed across much of the mitochondrial genome. Inferred amino acid sequences of the proteins encoded in the mtDNAs also show high levels of divergence; at the extreme, the three ND3 variants in T. californicus showed >25% amino acid substitutions, compared with <3% amino acid divergence at the previously studied COXI locus. Unusual secondary structures make functional assignments of some tRNAs difficult. The only apparent tRNA(trp) in these genomes completely overlaps the 5' end of the 16S rRNA in all three T. californicus mtDNAs. Although not previously noted, this feature is also conserved in T. japonicus mtDNAs; whether this sequence is processed into a functional tRNA has not been determined. The putative control region contains a duplicated segment of different length (from 88 to 155 bp) in each of the T. californicus sequences. In each case, the duplicated segments are not tandem repeats; despite their different lengths, the distance between the start of the first and the start of the second repeat is conserved (520 bp). The functional significance, if any, of this repeat structure remains unknown.     

Identification of 3- and 4-repeat tau isoforms within the PHF in Alzheimer''s disease.

The microtubule associated protein tau is incorporated into the pronase resistant core of the paired helical filament (PHF) in such a way that the repeat region is protected from proteases, but can be released as a major 12 kDa species from the PHF core by formic acid treatment and by boiling in SDS. This fragment retains the ability to aggregate in the presence of SDS. Detailed sequence analysis of the 12 kDa species shows that it consists of a mixture of peptides derived from the repeat region of 3- and 4-repeat tau isoforms comigrating as a single electrophoretic band. However, the 4-repeat isoforms released from the core lack either the first or the last repeat. The pronase-protected region of tau within the PHF core is therefore restricted to three repeats, regardless of isoform. The alignment of cleavage sites at homologous positions within tandem repeats after protease treatment indicates that the tau-core association is precisely constrained by the tandem repeat structure of the tau molecule.     

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates.

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five polymorphisms differed by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations.     

Dual intracellular localization and targeting of aminoimidazole ribonucleotide synthetase in cowpea

De novo purine biosynthesis is localized to both mitochondria and plastids isolated from Bradyrhizobium sp.-infected cells of cowpea (Vigna unguiculata L. Walp) nodules, but several of the pathway enzymes, including aminoimidazole ribonucleotide synthetase (AIRS [EC 6.3.3.1], encoded by Vupur5), are encoded by single genes. Immunolocalization confirmed the presence of AIRS protein in both organelles. Enzymatically active AIRS was purified separately from nodule mitochondria and plastids. N-terminal sequencing showed that these two isoforms matched the Vupur5 cDNA sequence but were processed at different sites following import; the mitochondrial isoform was five amino acids longer than the plastid isoform. Electrospray tandem mass spectrometry of a trypsin digest of mitochondrial AIRS identified two internal peptides identical with the amino acid sequence deduced from Vupur5 cDNA. Western blots of proteins from mitochondria and plastids isolated from root tips showed a single AIRS protein present at low levels in both organelles. (35)S-AIRS protein translated from a Vupur5 cDNA was imported into isolated pea (Pisum sativum) leaf chloroplasts in vitro by an ATP-dependent process but not into import-competent mitochondria from several plant and non-plant sources. Components of the mature protein are likely to be important for import because the N-terminal targeting sequence was unable to target green fluorescent protein to either chloroplasts or mitochondria in Arabidopsis leaves. The data confirm localization of the protein translated from the AIRS gene in cowpea to both plastids and mitochondria and that it is cotargeted to both organelles, but the mechanism underlying import into mitochondria has features that are yet to be identified.     

Inverted repeat domains in membrane proteins

With the upsurge in known membrane protein structures, common structural themes have started to emerge. One of these is the inverted repeat, a tandem of alpha-helical domains that have similar tertiary folds but opposite membrane orientations. In all previously known examples, both repeat units were encoded in a single continuous polypeptide. Recent structures of a bacterial multidrug transporter, EmrE, revealed an inverted repeat membrane protein wherein the two repeat units are assembled from two polypeptides with the same primary sequence. Here, we speculate on some of the implications of the EmrE structure with regards to our understanding of membrane protein evolution and topogenesis.