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PubMed1992

Intra- and interchain disulfide bridges of the human J chain in secretory immunoglobulin A.

All intra J chain disulfide bridges of human sIgA, the disulfide bonds between the J chain and the two IgA monomers, and one inter IgA monomer disulfide bridge were determined. sIgA was isolated from colostrum of healthy women and digested with IgA1-specific protease followed by cyanogen bromide cleavage. This procedure generated fragments of 140 kDa, 60 kDa, and 28 kDa. The 28-kDa polypeptide comprised the complete J chain covalently bound to two alpha 1 chain octapeptides derived from the C-termini of two alpha 1 chains. The 28-kDa fragment was digested with trypsin. The resulting peptides were purified by RP-HPLC, and subsequently characterized by amino-acid analysis, mass spectrometry, and gas phase sequencing. These data unequivocally show that the J chain cysteines C1-C6, C4-C5, and C7-C8 form intra chain disulfide bridges. The second (C2) and the third (C3) J chain cysteines are disulfide linked to two alpha chain cysteines (C17) joining the two IgA monomers of sIgA tail to tail. The remaining two alpha chains of the two monomers are directly bound to each other via their ultimate cysteines (C17-C17). A new model for the J chain in sIgA is presented.

Amino Acid Sequence↗
PubMed1999

Phylogenetic analysis of Cryptosporidium isolates from captive reptiles using 18S rDNA sequence data and random amplified polymorphic DNA analysis.

Sequence alignment of a polymerase chain reaction-amplified 713-base pair region of the Cryptosporidium 18S rDNA gene was carried out on 15 captive reptile isolates from different geographic locations and compared to both Cryptosporidium parvum and Cryptosporidium muris isolates. Random amplified polymorphic DNA (RAPD) analysis was also performed on a smaller number of these samples. The data generated by both techniques were significantly correlated (P < 0.002), providing additional evidence to support the clonal population structure hypothesis for Cryptosporidium. Phylogenetic analysis of both 18S sequence information and RAPD analysis grouped the majority of reptile isolates together into 1 main group attributed to Cryptosporidium serpentis, which was genetically distinct but closely related to C. muris. A second genotype exhibited by 1 reptile isolate (S6) appeared to be intermediate between C. serpentis and C. muris but grouped most closely with C. muris, as it exhibited 99.15% similarity with C. muris and only 97.13% similarity with C. serpentis. The third genotype identified in 2 reptile isolates was a previously characterized 'mouse' genotype that grouped closely with bovine and human C. parvum isolates.

Animals↗
PubMed1988

Hybrid genes between HLA-A2 and HLA-A3 constructed by in vivo recombination allow mapping of HLA-A2 and HLA-A3 polymorphic antigenic determinants.

HLA-A2 and -A3 genes have been modified in their third exon (second domain) by using in vivo recombination. In this method Escherichia coli are transfected with a plasmid which contains two highly homologous sequences (e.g., the third exons of HLA-A2 and -A3) and has been linearized by cleavage between these two sequences. Circularization takes place in the bacteria by homologous recombination leading to hybrid A2-A3 sequences. The analysis by DNA sequencing of a number of such recombinants shows that they indeed occur by homologous recombination (no insertions or deletions) and that the probability of crossing over decreases as the distance from the free end of DNA in the homologous region increases. No double recombinants were observed. These hybrid exons were reinserted into either HLA-A2 or HLA-A3 genes, thus generating a panel of functional hybrid genes containing one or several HLA-A2 specific substitutions in an HLA-A3 background or vice versa. These genes were expressed by transfection into murine P815-high transfection efficiency recipient cells. Serologic analysis leads to the conclusion that expression of polymorphic antigenic determinants specific for HLA-A2 (detected with M58, A2A28M1, and CR11.351 mAb) is linked to the presence of threonine residue (amino acid (AA) 142) and/or histidine residue (AA 145) and valine residue (AA 152). The expression of specific HLA-A3 polymorphic determinants (recognized by GAP-A3 mAb) is correlated with the existence of a asparagine residue (AA 127) and a aspartic residue (AA 161). But aspartic residue 161 contributes with glutamic acid residue 152 in the formation of the A3 epitope recognized by the anti-A3 mAb X1.23.2.

Antibodies, Monoclonal↗
PubMed1989

Human interstitial retinoid-binding protein. Gene structure and primary structure.

Interstitial retinoid-binding protein (IRBP) is synthesized and secreted by rod photoreceptor cells into the interphotoreceptor matrix and is known to bind retinoids and fatty acids. We have used cDNA clones encoding human IRBP to isolate a 15-kilobase genomic fragment that encompasses the complete human IRBP gene. The IRBP gene spans more than 11 kilobases and is interrupted by three introns, all of which are positioned near the 3'-end of the coding sequence. The 3741-base pair coding region of IRBP appears to have been generated by quadruplication of an approximately 900 base pair long ancestral gene. The deduced amino acid sequence predicts a mature protein of 1,230 residues (calculated molecular weight 133,000). The protein sequence can be aligned into four homologous segments, each consisting of about 300 residues. Sequence similarity between segments is as high as 60% when conservative substitutions are taken into account. Two putative N-linked glycosylation sites are located in highly conserved domains in the center of the first and second segment of IRBP. A domain consisting of 41 residues at the COOH-terminal end of the third segment has 15 matching residues (38%) with an intradiscal loop of rhodopsin, a retinal-binding protein in rod photoreceptors.

Amino Acid Sequence↗
PubMed1988

Topology of the Mr 27,000 liver gap junction protein. Cytoplasmic localization of amino- and carboxyl termini and a hydrophilic domain which is protease-hypersensitive.

Hydropathy analysis of the Mr 27,000 rat liver gap junction protein sequence deduced from a cDNA clone has suggested the presence of four transmembrane segments (Paul, D. L. (1986) J. Cell Biol. 103, 123-134). In the present report, several features of the molecular topology of the protein were investigated by microsequence analysis of peptides generated by treatment of isolated gap junctions with a variety of proteases. Under the experimental conditions used, the proteases had access only to the portion of the Mr 27,000 protein that was originally (in vivo) the cytoplasmic surface of the gap junction. Microsequencing of the peptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the amino terminus of the protein is disposed at or near the cytoplasmic surface of the gap junction, and that this surface also contains a protease-hypersensitive hydrophilic sequence between residues 109 and 123, presumably connecting the second and third transmembrane segments. Immunocytological localization of binding of monoclonal antipeptide antibodies demonstrates that the carboxyl terminus of the protein is also localized to the cytoplasmic surface of the gap junction. No protease sensitivity was found in the hydrophilic sequences thought to connect either the first and second transmembrane segments or the third and fourth segments, supporting the model's prediction that these sequences face the narrow intercellular gap which cannot be penetrated by proteases.

Amino Acid Sequence↗
PubMed1995

Linkage and candidate gene analysis of X-linked familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disorder characterized by avascularity of the peripheral retina, retinal exudates, tractional detachment, and retinal folds. The disorder is most commonly transmitted as an autosomal dominant trait, but X-linked transmission also occurs. To initiate the process of identifying the gene responsible for the X-linked disorder, linkage analysis has been performed with three previously unreported three- or four-generation families. Two-point analysis showed linkage to MAOA (Zmax = 2.1, theta max = 0) and DXS228 (Zmax = 0.5, theta max = 0.11), and this was further confirmed by multipoint analysis with these same markers (Zmax = 2.81 at MAOA), which both lie near the gene causing Norrie disease. Molecular genetic analysis further reveals a missense mutation (R121W) in the third exon of the Norrie's disease gene that perfectly cosegregates with the disease through three generations in one family. This mutation was not detected in the unaffected family members and six normal unrelated controls, suggesting that it is likely to be the pathogenic mutation. Additionally, a polymorphic missense mutation (H127R) was detected in a severely affected patient.

Base Sequence↗
PubMed1973

"Private" genetic variants and the frequency of mutation among South American Indians.

Electrophoretic studies were performed on 15 proteins of blood serum and of erythrocytes, from blood specimens from 72 villages of six relatively unacculturated and genetically pure Indian tribes of South America, for a total of 56,237 determinations. At least 10 different "private" variants were encountered, in 131 people. Two previously recognized genetic polymorphisms of these 15 proteins were also encountered. On the assumption that these variants are neutral from the standpoint of natural selection, and that only one-third of amino-acid substitutions in proteins result in electrophoretically detectable variants, the mutation rate is estimated from a formulation of Kimura and Ohta to be about 8 x 10(-5) per locus per generation. The calculation involves several approximations which can be improved by further investigations; if confirmed, then for this class of mutations Indian mutation rates are roughly an order of magnitude higher than commonly envisioned.

Amino Acid Sequence↗
PubMed2002

Structural analysis of the chimeric CYP21P/CYP21 gene in steroid 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. More than 90% of CAH cases are caused by mutations of the CYP21 gene. Approximately 75% of the defective CYP21 genes are generated through intergenic recombination, termed "apparent gene conversion," from the neighboring CYP21Ppseudogene. A chimeric CYP21P/CYP21gene with its 5' end corresponding to CYP21P and 3' end corresponding to CYP21 has been identified. This type of gene is nonfunctional because it produces a truncated protein. We found two distinct chimeric genes in CAH patients. Both genes had a sequence with -300 nucleotides of the 5' head as the CYP21P gene. The coding region consisted of a fusion molecule with the CYP21P gene in two different regions. One of the junctions was located in the chi-like sequence of GCTGGGC in the third intron and the other was in the minisatellite consensus TGGCAGGAGG of exon 5 of the CYP21P gene. In addition, analysis of restriction fragment length polymorphism for these two 3.3-kb chimeric molecules showed that these sequences arose as a consequence of unequal crossover between the CYP21Pand CYP21 genes. It is plausible that both consensus sequences are responsible for the gene conversion of these two chimeric genes.

Adrenal Hyperplasia, Congenital↗
PubMed1995

Abnormal processing of the human cholecystokinin receptor gene in association with gallstones and obesity.

BACKGROUND & AIMS: Cholesterol gallstone disease and obesity are often associated and share the potential, yet unreported, common etiology of cholecystokinin (CCK) dysfunction. While cloning the human CCK-A receptor complementary DNA (cDNA), we found predominance of a 262-base pair coding region deletion in a cDNA library prepared from a patient with this phenotype. The aim of this study was to determine the abundance, functional significance, and mechanism for generating this gene product. METHODS: Relative abundance of CCK receptor gene products was determined using polymerase chain reaction and hybridization analysis. Constructs were expressed in COS cells and studied for radioligand binding and intracellular calcium responses. A human genomic clone for this receptor was sequenced, and the critical regions were compared with those of the patient. RESULTS: Ninety-three percent of the patient's CCK receptor transcripts contained the 262-base pair deletion, whereas only 1.5% +/- 0.9% of control patients had the deletion. This encoded a receptor that did not bind or signal. The deletion corresponded with the third exon; however, this sequence and flanking introns were normal in the patient. CONCLUSIONS: Abnormality of processing an apparently normal CCK receptor gene yields the predominant product with an absent third exon and encoding a nonfunctional receptor, probably reflecting a defective trans-acting splicing factor. An atypical lariat region in the third intron may explain the presence of small amounts of this product in control patients.

Adult↗
PubMed2006

The Plasmodium falciparum RhopH2 promoter and first 24 amino acids are sufficient to target proteins to the rhoptries.

The rhoptry secretory organelles of the malaria parasite, Plasmodium falciparum, contain a RhopH complex, which is composed of the proteins RhopH1, RhopH2, and RhopH3. RhopH1 is encoded by the rhoph1/clag multi-gene family, whereas RhopH2 and RhopH3 are encoded by single-copy genes. The precise function of the RhopH complex has not been identified, but it has been shown that the component proteins are involved in erythrocyte binding and perhaps participate in the formation of the parasitophorous vacuolar membrane. In this study, we have isolated pfrhoph2 promoter plus the signal peptide encoding sequence and generated transgene expression constructs to evaluate a trafficking and the RhopH complex formation in transgenic P. falciparum parasite lines. Interestingly, we found that the N-terminal 24 amino acids of RhopH2, including signal peptide sequence, were sufficient to target GFP to the rhoptries under the rhoph2 promoter. Because it was previously shown that the timing of the expression alone could not target proteins to the apical organelles, this targeting is likely mediated via a unique mechanism that is dependent on N-terminal 24 amino acids of RhopH2 early in the secretory pathway. The N-terminal one third of Clag3.1, which contains a distinct conserved domain with Toxoplasma gondii RON2, can not associate the RhopH complex as a GFP chimera, but a c-Myc-Clag3.1 chimera lacking the C-terminus successfully associates the RhopH complex indicating that cooperation of middle region is likely required but the C-terminus is not necessary.

Amino Acid Sequence↗
PubMed1994

Noninvasive angiography in preoperative evaluation of complicated pediatric renal masses using phase contrast magnetic resonance angiography.

OBJECTIVES: To report a new technique called "phase contrast magnetic resonance angiography" (PC MRA) utilizing phase contrast magnetic resonance imaging, for assessing the renal vasculature in relation to complicated pediatric renal masses. METHODS: Three-dimensional phase contrast gradient refocused pulse sequences were obtained on four pediatric patients with complicated renal masses. Findings were compared to ultrasound and computed tomographic images and correlated with actual surgical anatomy. RESULTS: In all four cases PC MRA provided standard angiographic quality detail of the renal vascular anatomy. The PC MRA findings had a 100% correlation with surgical exploration and MRA proved to be approximately one third the cost of the standard angiographic technique. CONCLUSIONS: Our experience with the use of PC MRA in children suggests that it can be helpful in evaluating the vasculature of complicated pediatric renal masses. PC MRA utilizes current generation magnetic resonance units, is noninvasive, and does not require anesthesia. It may be especially useful in patients with hypersensitivity to contrast agents, in those with renal failure in whom contrast agents may be contraindicated, and is less expensive than standard angiography.

Child, Preschool↗
PubMed1988

Human aldolase A gene. Structural organization and tissue-specific expression by multiple promoters and alternate mRNA processing.

The complete nucleotide sequence of the human aldolase A isoenzyme gene is reported. The cloned gene sequence, spanning 7530 bp, includes twelve exons and occurs as a single copy per haploid human genome. The structural organization of the gene is quite complex: eight exons containing the coding sequence are common to all mRNAs extracted from human and other mammalian sources; four additional exons are present in the 5' untranslated region, of these one is contained in the ubiquitous type of mRNA, the second is in the muscle-specific type of mRNA and the third and fourth are in a minor species of mRNA found in human liver tissue. Furthermore, the determined sequence includes 1000 nucleotides upstream from the first exon (exon I) in the 5' flanking region, and 400 nucleotides, which include the polyadenylation signal, downstream from the termination codon. S1-nuclease-protection analysis of the 5' end of mRNA extracted from human cultured fibroblasts, muscle and hepatoma cell lines indicates the existence of four different transcription-initiation sites. The latter are also supported by the presence of conventional sequences for eukaryotic promoters. Therefore, the four promoters on the same gene generate different tissue-specific transcripts, which share the translated sequence, but each has a unique 5' untranslated region as a result of differential mRNA processing. The nucleotide homology at the coding region and the intron-exon organization of the three human and mammalian aldolase A, B and C genes confirm that they arose from a common ancestral gene, and that aldolase B diverged first.

Base Sequence↗
PubMed2000

Generation of 10,154 expressed sequence tags from a leafy gametophyte of a marine red alga, Porphyra yezoensis.

A total of 10,154 5'-end expressed sequence tags (EST) were established from the normalized and size-selected cDNA libraries of a marine red alga, Porphyra yezoensis. Among the ESTs, 2140 were unique species, and the remaining 8014 were grouped into 1127 species. Database search of the 3267 non-redundant ESTs by BLAST algorithm showed that the sequences of 1080 species (33.1%) have similarity to those of registered genes from various organisms including higher plants, mammals, yeasts, and cyanobacteria, while 2187 (66.9%) are novel. Codon usage analysis in the coding regions of 101 non-redundant EST groups showing significant similarity to known genes indicated the higher GC contents at the third position of codons (79.4%) than the first (62.2%) and the second position (45.0%), suggesting that the genome has been exposed to high GC pressure during evolution. The sequence data of individual ESTs are available at the web site http://www.kazusa.or.jp/en/plant/porphyra/EST/.

Algorithms↗
PubMed1994

[Genetic diagnosis of phenylketonuria. IV. Mutations of phenylalanine hydroxylase gene in Caucasian and Gypsy populations in Czech and Slovakia Republics].

Direct sequencing was conducted on the regions of the exon 7 and 12 in the phenylalanine hydroxylase (PAH) gene amplified by the polymerase chain reaction, using solid-phase technology involving the biotin streptavidin system. A novel mutation and seven previously known mutations were identified in the PAH genes among 15 Caucasians and 10 Gypsies in the Czech and Slovakia republics, affected with classical phenylketonuria (PKU). Two of these substitutions (R243X and G272X) resulted in the generation of a premature stop codon, and a single base transition of G to A at codon 261 resulted in the substitution of Arg for Gln (R261Q). These three mutations together accounted for 16.7% of PKU alleles among 15 Caucasians. The R252W mutation was detected in these two groups: two Caucasians were compound heterozygous for the P281L or R408W mutations (6.7% of all mutant alleles). However, all 10 Gypsy PKU patients were homozygous for the R252W transition. The R408W mutation accounted for 43% of PKU alleles in 15 Caucasian PKU patients. A novel heterozygous C-to-G transversion at the third base of codon 263 in the exon 7, resulting in the substitution of Phe for Leu (F263L), was detected in a Caucasian PKU patient (3.3% of all mutant alleles). In this study, we revealed a novel PKU mutation of the F263L in Caucasian populations and a high-risk PKU mutation of the R252W in Gypsy populations.

Base Sequence↗
PubMed1990

Generation of the neutrophil-activating peptide NAP-2 from platelet basic protein or connective tissue-activating peptide III through monocyte proteases.

We studied the origin of the neutrophil-activating peptide NAP-2, a presumed 70 amino acid cleavage product of platelet basic protein (PBP) and connective tissue-activating peptide III (CTAP-III). Purified human blood monocytes or lymphocytes were cultured with or without stimuli (LPS or PHA) in the presence or absence of platelet-release supernatant, and the formation of NAP-2 and other neutrophil-activating peptides was monitored. NAP-2 was generated whenever monocytes and platelet release supernatant were present. When a monocyte stimulus was added, NAF/NAP-1 was also formed, and in the presence of LPS a third, less potent neutrophil-stimulating fraction, consisting of NAP-2 variants with 73, 74, and 75 residues, also appeared. Monocytes alone did not yield NAP-2 and no neutrophil-activating peptide was generated by lymphocytes. The monocyte-conditioned medium was found to cleave purified CTAP-III into NAP-2 through proteinases that were highly sensitive to PMSF, moderately sensitive to leupeptin and insensitive to EDTA.

Amino Acid Sequence↗
PubMed2000

L1 (LINE-1) retrotransposon evolution and amplification in recent human history.

L1 (LINE-1) elements constitute a large family of mammalian retrotransposons that have been replicating and evolving in mammals for more than 100 Myr and now compose 20% or more of the DNA of some mammals. Here, we investigated the evolutionary dynamics of the active human Ta L1 family and found that it arose approximately 4 MYA and subsequently differentiated into two major subfamilies, Ta-0 and Ta-1, each of which contain additional subsets. Ta-1, which has not heretofore been described, is younger than Ta-0 and now accounts for at least 50% of the Ta family. Although Ta-0 contains some active elements, the Ta-1 subfamily has replaced it as the replicatively dominant subfamily in humans; 69% of the loci that contain Ta-1 inserts are polymorphic for the presence or absence of the insert in human populations, as compared with 29% of the loci that contain Ta-0 inserts. This value is 90% for loci that contain Ta-1d inserts, which are the youngest subset of Ta-1 and now account for about two thirds of the Ta-1 subfamily. The successive emergence and amplification of distinct Ta L1 subfamilies shows that L1 evolution has been as active in recent human history as it has been found to be for rodent L1 families. In addition, Ta-1 elements have been accumulating in humans at about the same rate per generation as recently evolved active rodent L1 subfamilies.

3' Untranslated Regions↗
PubMed2007

TAMGeS: a Three-array Method for Genotyping of SNPs by a dual-colour approach.

BACKGROUND: Many of the most effective high-throughput protocols for SNP genotyping employ microarrays. Genotypes are assessed by comparing the signal intensities that derive from the hybridization of different allele-specific probes labelled either by using four fluorescent dyes, one for each base, or by using only two dyes and investigating the polymorphic alleles two by two on separate arrays. The employment of only two dyes makes it possible to use a dual-laser scanner, which has the advantage of being present in every microarray laboratory. However, this protocol may present some drawbacks. To infer all the six possible genotypes it is necessary to compare signals from two arrays, but this comparison not always is successful. A number of systematic errors in the experimental protocol, in fact, may differently affect signal intensities on separate arrays. Here we present TAMGeS (Three-Array Method for Genotyping of SNPs), an exhaustive method for SNP genotyping through SBE (Single Base Extension) and dual-colour microarrays, which makes the comparison of signals on distinct arrays reliable by using a third array and a data handling method for signal normalization based on bilinear regression theory. RESULTS: We tested the effectiveness of the proposed method by evaluating the results obtained from the direct comparison of the two arrays or by applying TAMGeS, both on experimental and synthetic data. With synthetic data, TAMGeS reduced the frequency of errors by an order of magnitude, when the incidence of systematic errors was not negligible. With the experimental data, produced by genotyping 25 SNPs in 437 subjects, TAMGeS reduced the percentage of missing genotypes from 54% (Two-Array Method) to 14.5%. Allelic and genotypic call rates were 99.3% and 99.5%, respectively. The normalization procedure takes into account also systematic errors, which can be generated by a time-delayed assay, thus making the protocol more flexible. CONCLUSION: TAMGeS represents an innovative method, which proved to be very effective in producing reliable SNP genotyping data by dual-colour microarrays. The requirement of a third array is well balanced by the strong enhancement in data quality and by the greater flexibility of the experimental protocol.

Base Sequence↗
PubMed1995

Functionally different isoforms of the human calcitonin receptor result from alternative splicing of the gene transcript.

Two subtypes of the human calcitonin receptor (hCTR) have been described which differ from one another by the presence or absence of a 16-amino acid insert in the first intracellular loop. Both isoforms were stably expressed in baby hamster kidney cells to compare their ligand binding and second messenger coupling. The binding affinity and the on/off rate of binding for salmon CT were identical for the two receptor isoforms. However, the presence of the insert significantly reduced the ability of the receptor to couple to both adenylate cyclase and phospholipase C. Stimulation of a transient calcium response was only observed with the insert-negative receptor. Similarly, the ED50 for the cAMP response is 100-fold higher for the insert-positive form compared with the insert-negative form of the receptor. However, the maximal cAMP response was equivalent for both receptor isoforms. The rate of internalization of the insert-positive form of the receptor is significantly impaired relative to the insert-negative receptor, which suggests that this process may be dependent on the stimulation of a second messenger pathway. Cloning and characterization of the relevant portion of the hCTR gene revealed that these isoforms are generated by alternative splicing. We also discovered a third isoform of the hCTR, which can be generated by alternative splicing at the same position. The presence of a stop codon in this newly described alternative exon would lead to premature termination of the receptor at the C-terminal end of the first transmembrane domain.

Alternative Splicing↗