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At least 19 recordsLinked to original sources
PubMed2025

Spectrum of genetic alterations in patients with peroxisome biogenesis defects in the Iranian population: a case series study.

Peroxisomal disorders are a group of hereditary metabolic disorders that happen when peroxisomes are defective. Around 80% of individuals affected by peroxisomal disorders are classified within the spectrum of Zellweger syndromes with autosomal recessive inheritance pattern that results from mutations in one of the 13 PEX genes. Clinical exome sequencing plays a vital role in the diagnosis where the symptoms are atypical. In the current study, we used this technique to find the underlying genetic cause in 14 Iranian patients with peroxisomal disorders. PEX1 variants were detected in five patients. PEX2, PEX5, PEX6 and PEX7 variants were detected in three, one, one, and two cases, respectively. Finally, ACOX1 variants were identified in two cases. All cases except two cases were homozygote for the suspected variants in Zellweger syndrome-related genes. Two cases were compound heterozygote for variants in the PEX1 gene. In total, two novel variants were identified, including c.313 C > T (p.Gln105*) and c.961 A > T (p.Ile321Phe) in the PEX1 and ACOX1 genes, respectively. The present research expands the range of genetic variations observed in Iranian individuals diagnosed with various forms of Zellweger spectrum disorders.

Humans↗
PubMed1988

Two distinct genes for ADP/ATP translocase are expressed at the mRNA level in adult human liver.

Several clones hybridizing with a bovine ADP/ATP translocase cDNA were isolated from an adult human liver cDNA library in the vector pEX1. DNA sequence analysis revealed that these clones encode two distinct forms of translocase. In particular, two clones specifying the COOH-end-proximal five-sixths of the protein exhibit a 9% amino acid sequence divergence and totally dissimilar 3' untranslated regions. One of these cDNAs is nearly identical in sequence to an ADP/ATP translocase clone (hp2F1) recently isolated from a human fibroblast cDNA library [Battini, R., Ferrari, S., Kaczmarek, L., Calabretta, B., Chen, S. & Baserga, R. (1987) J. Biol. Chem. 262, 4355-4359], with three amino acid changes and a few differences in the 3' untranslated region. Another clone isolated from the pEX1 library contains a reading frame encoding the remaining, NH2-end-proximal, 37 amino acids of the translocase. This sequence differs significantly (14% amino acid sequence divergence) from the corresponding segment of hp2F1, and the 5' untranslated regions of the two clones are totally dissimilar. RNA transfer hybridization experiments utilizing the clones isolated from the pEX1 library revealed the presence in HeLa cells of three distinct mRNA species. The pattern of hybridization and the sizes of these mRNAs suggest a greater complexity of organization and expression of the ADP/ATP translocase genes in human cells than indicated by the analysis of the cDNA clones.

Adult↗
PubMed1986

Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1.

Antibodies induced against mammalian single-stranded DNA binding protein (ssDBP) UP I were shown to be cross-reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti-ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re-evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Anti-RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro-beta-galactosidase-70K fusion protein reacted with various anti-RNP patient sera, a rabbit anti-70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3'-untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full-length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed-charge amino acid clusters have been identified at the carboxyl-terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.

Amino Acid Sequence↗
PubMed1987

Human ribophorins I and II: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins.

Ribophorins I and II represent proteins that are postulated to be involved in ribosome binding. They are abundant, highly-conserved glycoproteins located exclusively in the membranes of the rough endoplasmic reticulum. As the first step in the further characterization of the structure and function of these proteins, we have isolated and sequenced full-length human cDNA clones encoding ribophorins I and II using probes derived from a human liver expression library cloned into pEX1. The authenticity of the clones was verified by overlaps in the protein sequence of N-terminal and several internal fragments of canine pancreatic ribophorins I and II. The cDNA clones hybridize to mRNA species of 2.5 kb in length, and encode polypeptides of 68.5 and 69.3 kd, respectively. Primary sequence analysis, coupled with biochemical studies on the topology, indicates that both ribophorins are largely luminally disposed, spanning the membrane once and having 150 and 70 amino acid long cytoplasmically disposed C termini, respectively. Both are synthesized as precursors having cleavable signal sequences of 23 (ribophorin I) and 22 (ribophorin II) amino acids. The topology suggested by the primary structure has been confirmed biochemically using proteolytic enzymes and anti-ribophorin antibodies. Proteolysis of intact microsomes with a variety of enzymes resulted in a reduction in the apparent mol. wt of ribophorin I that would correspond to a loss of its 150-amino acid cytoplasmic tail. In the case of ribophorin II, it is completely resistant to such proteolysis which is consistent with its luminal disposition and fairly hydrophobic C terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Isolation of cDNA clones corresponding to the Mr = 150,000 subunit of chick type VI collagen.

Type VI collagen is a disulfide-bonded protein with an unusual structure in that the molecule contains three short triple-helical domains and very extended non-collagenous regions. The molecule is a heterotrimer composed in the chick of two polypeptides of similar apparent size in SDS-PAGE (Mr = 140- and 150,000) but different structure, and a third component that is much larger (Mr = 260,000) than the other two chains. We report here on the isolation of several overlapping cDNA clones from a chicken aorta mRNA expression library in the plasmid vector pEX1. Antibodies affinity purified onto the fusion proteins recognized the chick type VI collagen Mr = 150,000 subunit. Northern blots using the cDNA inserts from the above clones revealed a single RNA species of about 4,600 nucleotides sufficient to code for a protein with the size of the Mr = 150,000 subunit.

Animals↗
PubMed1992

Construction of dystrophin fusion proteins to raise targeted antibodies to different epitopes.

For the study of the structure and function relationship of dystrophin, defective in DMD, and for diagnostic purposes it is important to dispose of antibodies against different parts of the protein. We have made five different constructs for the expression of fusion proteins containing parts of the four domains of dystrophin. Two different recombinant expression vectors, pATH2 and pEX1, were used. Rabbits were immunized with the fusion products and several polyclonal antibodies were raised. At a later stage, monoclonal antibodies were also raised to some of the fusion proteins. One polyclonal antibody, named P20 AB, is directed against the region covering amino acid sequence 1749-2248 or the nucleotide sequence 5456-6953 of the mRNA, which corresponds to the major deletion-prone region of the DMD gene. We show the particular value, sensitivity and specificity of the P20 AB in dystrophin analysis.

Antibody Formation↗
PubMed1989

The MIC2 gene product: epitope mapping and structural prediction analysis define an integral membrane protein.

The MIC2 locus is located in the pseudoautosomal (pairing) region of human X and Y chromosomes (Goodfellow et al., Science 234, 740-743, 1986). Despite extensive molecular analysis of MIC2 (see Darling et al., Cold Spring Harb. Symp. quant. Biol. 51, 205-211, 1986), study of the gene product has been limited (Banting et al., EMBO J. 41, 1967-1972, 1985). Here we report the combined use of monoclonal antibodies, plasmid expression vectors and structural prediction analysis to define the MIC2 gene product as an integral membrane protein. Random overlapping fragments of a cDNA, corresponding to the MIC2 locus, were cloned into the plasmid expression vector pEX1 (Stanley and Luzio, EMBO J. 3, 1429-1434, 1984) to produce "epitope libraries". Six different monoclonal antibodies, known to recognize the extracellular region of the MIC2 gene product, were used to screen these libraries. Clones recognized by these antibodies were sequenced and their sequences aligned with one another and with the complete MIC2 cDNA sequence. All antibodies tested recognized adjacent and/or overlapping epitopes in the same region of the molecule. These results complement data from a hydropathy plot of a conceptual translation of the MIC2 sequence, which demonstrated the presence of a single long hydrophobic region in the mature protein. Since the antibodies recognize the extracellular portion of the molecule, we were able to determine the orientation in the plasma membrane. This method of analysis is generally applicable where antibodies and cloned cDNAs are available.

Amino Acid Sequence↗
PubMed1988

Cloning and expression of Taenia ovis antigens in Escherichia coli.

Double stranded DNA complementary to poly(A)+ mRNA from the tapeworm Taenia ovis was cut with Sau 3A to an average length of about 300 bp and inserted into the Bam HI site of the expression plasmids pEX1, pEX2 and pEX3. These plasmids express a hybrid protein derived from a fusion of the cro gene with the lac Z gene (truncated at its 5' end by 53 bp) of phage lambda. Cloning sites lie downstream from the gene fusion. Escherichia coli infected with another plasmid (pCI857) bearing the temperature sensitive repressor of phage lambda was transformed with the pEX plasmids into which T. ovis DNA had been inserted; recombinants were selected by growth at 30 degrees C in the presence of ampicillin at 100 micrograms ml-1. Replicas were made and hybrid protein expression induced in recombinants by transferring them to 42 degrees C. Several recombinants expressing antigenic determinants of T. ovis were detected with T. ovis infected sheep serum that had been absorbed to remove antibodies to E. coli. Of five selected for further study, three expressed hybrid proteins of between 165 and 170 kDa of which the T. ovis component contributed between 48 and 55 kDa; in the other two, the tapeworm contribution was between 0.5 and 1.5 kDa. These antigenic determinants may be of some interest with respect to vaccine development since they are expressed during the normal course of T. ovis infection in sheep, and they are also present in the oncosphere - the infective larva of the parasite which stimulates immunity in sheep. The native antigens in adult worms and oncospheres that correspond to the antigenic determinants produced by the recombinant clones comprise a number of species ranging from 92.5 to 180 kDa. Tests with affinity purified antibodies indicate that the expressed products of the clones represent different epitopes on the same subset of polypeptides in both adult worms and oncospheres.

Animals↗
PubMed1992

P-glycoprotein genes in the winter flounder, Pleuronectes americanus: isolation of two types of genomic clones carrying 3' terminal exons.

In mammals, P-glycoprotein (P-gp) is encoded by two or more highly conserved genes that differ in their abilities to transport drugs. One isoform class (class I) is consistently associated with the multidrug resistance phenotype, while the other (class III) is not. This study was designed to enumerate the P-gp genes in fish and determine how they are related to the two functional classes already defined in mammals. Southern blot analysis using a conserved single exon from the 3' terminal region of hamster P-gp cDNA (pEX1-172) as a probe indicated that there were two P-gp genes in right-eye flounders. Subsequently, two sets of clones were isolated from a winter flounder genomic library that correspond to the 3' ends of the two flounder P-gp genes. Sequence analysis was done on two key areas: the 3' ATP binding site and the 3' terminal exon, both of which were found to be homologous with their mammalian counterparts. Despite high levels of sequence identity in the predicted coding regions of the gene fragments it has not been possible to use these sequences to relate the homologs to particular mammalian classes of P-gp genes, perhaps because of gene conversion between mammalian P-gp genes. These cloned sequences are the first set of P-gp genes reported in lower vertebrates and will be useful for delineating the expression of P-gp genes in fish and understanding the role of P-gp in fish physiology.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

Isolation of a cDNA clone for chick intestinal apolipoprotein AI (Apo-AI) and its use for detecting apo-AI mRNA expression in several chick tissues.

Three cDNA clones for chick apolipoprotein AI (Apo-AI), the major protein component of plasma high-density lipoproteins, have been isolated. The identity of the clones has been established first by screening a cDNA library in the pEX1 expression vector with anti-Apo-AI antibodies, second by Western blot analysis of the proteins expressed by positive clones. The use of the clone containing the largest, presumably full-size, cDNA insert (apo5C12) in molecular hybridization experiments confirms that apo-AI mRNA is expressed mainly in chick small intestine and liver. Furthermore, we provide evidence that brain, heart and skeletal muscle also synthesize significant amounts of apo-AI mRNA. The Southern-blot hybridization pattern of the restriction-enzyme-digested chick DNA with the apo5C12 DNA is consistent with there being a single copy of the apo-AI gene.

Animals↗
PubMed1987

Repeating structure of chick tropoelastin revealed by complementary DNA cloning.

A cDNA library was constructed from chick aorta poly(adenylic acid)-containing RNA in the expression vector pEX1. Several clones were identified by screening the library with a polyclonal antiserum raised against chick tropoelastin and confirmed by DNA sequencing. Analysis of the deduced amino acid sequence, corresponding to the mature tropoelastin and most of the signal peptide, revealed that the molecule is composed of at least 8, and possibly 13, repeating units. The common features of each unit include an N-terminal region composed largely of alanines and lysines and ending with an aromatic amino acid, followed by a GAG span and then a C-terminal region consisting mostly of valines, prolines, and glycines often present in several copies of the sequence (VPGV). This structure is discussed in terms of the functional properties of the molecule.

Amino Acid Sequence↗
PubMed1985

The primary structure of human hemopexin deduced from cDNA sequence: evidence for internal, repeating homology.

We have cloned and analyzed a cDNA containing the coding sequence for human hemopexin. We have first identified, by immunological screening of 30.000 colonies of a liver cDNA library in the expression vector pEX1, a clone carrying an insert 1170 base pairs long that shows 100% homology with a known human hemopexin peptide. The complete sequence coding for hemopexin was isolated from a liver cDNA library in the vector pAT218. The DNA insert of 1523 base pairs shows an open reading frame coding for 439 amino acids, a 3' noncoding region of 159 nucleotides long, followed by a poly(A) tail. The insert spans the entire coding region and from which the primary structure of the protein was deduced. By computer assisted analysis of the amino acid sequence, it was possible to recognize a core unit, of about 45 amino acids, which is repeated 8 or possibly even 10 fold along the polypeptide chain. This feature suggests that the gene might have evolved through a series of duplications. This characteristic, together with prediction of secondary structure, suggest a rough model for the tridimensional folding that allows some speculations on the function of hemopexin. Blot hybridization of total RNA from human liver with nick translated hemopexin cDNA detected a message of about 1600 nucleotides. Southern blot experiments to identify the hemopexin gene (s) suggest that it is not a large multi-gene family, but that there is only one or at most a few genes in the human genome.

Amino Acid Sequence↗
PubMed1986

Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy.

We describe a method for the construction of large DNA fragment libraries in plasmid vectors, in which complementary, single-stranded extensions are ligated onto both vector and insert DNA using un-phosphorylated adaptor oligonucleotides. Special consideration has been taken of the requirements of expression screening as follows: cDNA synthesis using random oligonucleotide primers is described which maximises the probability of obtaining open reading frame fragments from large mRNA molecules, the adaptors use codons found in high abundance E. coli proteins to minimise problems of premature termination when using strong promoters, and the sequence encoded by the adaptors, when cloned into the bacterial expression vector pEX1, promotes a surface location for the foreign antigenic determinant where it is accessible to antibodies used for screening.

Bacterial Proteins↗
PubMed1987

Expression of bovine rotavirus neutralization antigen in Escherichia coli.

A 646 bp fragment derived from a full length cDNA clone of genomic segment 9 of bovine rotavirus (NCDV strain) was inserted into Escherichia coli expression plasmid pEX1. The fragment encodes amino acids 50 to 265 of the major vital neutralization antigen VP7, a 326 amino acid long outer shell glycoprotein. Several transformed bacterial clones were isolated in which the recombinant plasmid directed the synthesis of a cro-beta-galactosidase-VP7 fusion protein that was recognized by rabbit polyclonal antibodies against NCDV rotavirus. Sera from rabbits immunized with the fusion protein specifically reacted with VP7 among NCDV virion polypeptides. The chimeric polypeptide was also specifically recognized by two monoclonal antibodies against UK strain rotavirus VP7 that exhibited virus-neutralizing activity. However, immune sera to the chimeric polypeptide showed no neutralizing activity against bovine rotavirus. These results are discussed in view of a recent report that a fusion VP7-beta-galactosidase polypeptide comprising 35 more amino acids at the carboxy terminus was able to induce neutralizing antibodies in mice to simian rotavirus SA11.

Animals↗
PubMed1989

Characterization of native and recombinant 75-kilodalton immunogens from Chlamydia trachomatis serovar L2.

A 75-kilodalton (kDa) immunogen from Chlamydia trachomatis serovar L2 was characterized. The 75-kDa protein was localized in the cytoplasm of chlamydiae and was shown to be a protein synthesized early in the developmental cycle of chlamydiae. A gene library was made by the recombinant DNA technique, using the expression vectors pEX1, pEX2, and pEX3. From this library one clone was found which reacted with a monoclonal antibody against the 75-kDa immunogen of C. trachomatis. The 75-kDa protein produced by the recombinant Escherichia coli was expressed independently of the promoter for the hybrid protein cro-betagalactosidase. Thus it is not produced as a fusion protein. Epitope mapping of the 75-kDa protein from C. trachomatis L2 and from the recombinant E. coli performed by Staphylococcus aureus V8 protease digestion showed that the two proteins are identical. Furthermore, patient sera reacted with both proteins.

Animals↗
PubMed1987

Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans.

A genomic clone bank of Paracoccus denitrificans DNA has been constructed in the expression vector set pEX1, pEX2, and pEX3. Screening of this clone bank with antibodies raised against P. denitrificans methanol dehydrogenase resulted in the isolation of a clone, pNH3, that synthesized methanol dehydrogenase cross-reactive proteins. The nucleotide sequence of the P. denitrificans DNA fragment inserted in this clone has been determined and shown to contain the full methanol dehydrogenase structural gene. DNA cross-hybridization was found with DNA fragments which have been reported to contain the methanol dehydrogenase structural genes from Methylobacterium sp. strain AM1 and Methylobacterium organophilum.

Alcohol Oxidoreductases↗
PubMed1987

Cloning of Mycoplasma pneumoniae DNA and expression of P1-epitopes in Escherichia coli.

A genomic library of Mycoplasma pneumoniae was constructed by cloning partial Sau3A-digested genomic DNA into the expression plasmids pEX1 to pEX3. The recombinant clones were screened for production of M. pneumoniae P1-antigen by an in situ colony enzyme-linked immunosorbent assay (ELISA) blot method with a monospecific rabbit antiserum raised against the surface protein P1. The length of the translated P1-sequence and the size of the inserted DNA were determined. By comparison it was shown that six clones contained DNA fragments coding for an internal part of the P1-protein and eight clones code for the C-terminal part of terminated P1-protein. In reactions against sera from patients suffering from M. pneumoniae infection and sera from healthy persons, one of the internal clones and five of the C-terminal clones reacted with one or two of the patient sera, but only one clone reacted with all patient sera.

Antibodies, Bacterial↗