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PubMed1987

Analysis of cDNA clones that code for the transmembrane forms of the mouse neural cell adhesion molecule (NCAM) and are generated by alternative RNA splicing.

The neural cell adhesion molecule (NCAM) exists in at least three different isoforms. In the mouse, NCAM proteins with apparent Mr's of 180,000, 140,000 and 120,000 have been distinguished. These are encoded by 4 to 5 different transcripts. Here we report the full amino acid sequence of an isoform which most likely represents NCAM-140. The N-terminal extracellular portion of the 829-residue polypeptide appears to be identical to all three NCAM proteins. The Mr of 91,276 is considerably smaller than the estimate based on SDS-gel electrophoresis. The 147 C-terminal residues are distinct from NCAM-120 and contain the putative transmembrane and cytoplasmic domains. The transcript encoding NCAM-140 contains almost 3.2 kb non-coding sequence with a canonical polyadenylation signal. While the 5' sequences of NCAM-140 hybridize with all NCAM mRNAs, the 3' probes recognize only the two larger transcripts of 7.4 and 6.7 kb. From S1 nuclease protection analyses and hybridization studies of several NCAM cDNA clones with genomic NCAM sequences one can conclude that the different NCAM transcripts are generated by alternative splicing. In addition to the two alternative splice sites in the sequence encoding the extracellular domains, a third one can be predicted approximately 320 nt downstream of the start of the NCAM-140-specific sequence portion. This finding is in agreement with the existence of an extra exon in the chicken NCAM-180. Comparison between mouse and chicken NCAM amino acid sequences revealed the highest homology in the second and fifth Ig-like domains and in the cytoplasmic parts suggesting that these regions serve highly conserved functions.

Amino Acid Sequence↗

PubMed1989

Evolutionary pathways of the calcitonin (CALC) genes.

Recombinant DNA techniques have made it possible to establish the structure of various genes encoding polypeptide hormones. Comparison of nucleotide sequences of the calcitonin (CALC) genes in man has revealed surprising similarities and variations. These findings and the homologies among the sequences in different species offered an opportunity for speculation about relationships between these genes and about their evolutionary origin. The first gene (CALC-I) directing the synthesis of calcitonin (CT) or CT gene-related peptide (CGRP) comprises six exons and gives rise to two mRNAs by an alternative RNA-processing mechanism. The homology between CGRP and CT reflects their common origin. The human genome contains a second gene (CALC-II) that is structurally related to the CALC-I gene. The CALC-II RNA transcripts do not appear to be differentially processed, as only preproCGRP-II mRNA and not preproCT-II is detected. The first and second CT/CGRP genes probably have evolved from a common ancestor gene early in evolution. Meanwhile, a third genomic locus containing nucleotide sequences highly homologous to exons 2 and 3 of both CALC genes was detected and probably generated by duplication of a part of CALC-II. This locus is not likely to encode a CT- or CGRP-related polypeptide hormone. The CALC genes and this last (pseudo) gene are located on the short arm of chromosome 11. Recently, islet- or insulinoma-amyloid polypeptide (IAPP) was isolated as a major constituent of amyloid present in human insulinoma and in pancreatic islet amyloid in noninsulin-dependent diabetes mellitus. IAPP shows 46% amino acid sequence homology with human CGRP-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyloid↗

PubMed1988

Proteolytic processing of pro-alpha and pro-beta precursors from human beta-hexosaminidase. Generation of the mature alpha and beta a beta b subunits.

There are two major isozymes of human lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52), hexosaminidase A, alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). The alpha subunit contains a single polypeptide chain, while the beta subunit is composed of two nonidentical chains (beta a and beta b) derived from a common pro-beta precursor. The mature subunits, like those of most lysosomal enzymes, are produced through the proteolytic processing of propolypeptides once they enter the lysosome. In order to define the structure of the alpha and beta subunits generated in the lysosome, the alpha, beta a, and beta b polypeptides of hexosaminidase A and B were separated by a combination of molecular sieve and ion exchange high performance liquid chromatography, and amino-terminal sequences were determined. These were localized to the deduced amino acid sequences of previously isolated cDNAs coding for the prepro-alpha and beta polypeptides. From this analysis, the sites of hydrolysis generating the mature alpha, beta a, and beta b chains from hexosaminidase A and B could be determined. First, the signal peptide, required for processing of the pre-propolypeptides through the rough endoplasmic reticulum was predicted from the first in-frame Met residue on the cDNA. Second, amino acid sequencing defined the amino termini of the mature polypeptide chains and identified the pro-sequences removed from both the pro-alpha and pro-beta polypeptides. Third, an internal cleavage resulted in the removal of a tetrapeptide, Arg-Gln-Asn-Lys, and tripeptide, Arg-Gln-Asn, from the pro-beta chain of hexosaminidase A and B, respectively , to generate the beta b and beta a chains. This result localized the beta b and beta a chains to the amino-terminal and carboxyl-terminal halves of the pro-beta sequence, respectively. Finally, we previously reported minimal or no carboxyl-terminal processing of the pro-beta chain in the lysosome. On the other hand, we suggest that there is trimming at the carboxyl terminus of the pro-alpha chain based on comparison of molecular weights of deglycosylated alpha with the isolated beta b and beta a chains comprising the mature beta subunit with those predicted from the cDNA. Thus, in the lysosome the pro forms of hexosaminidase A and B undergo extensive proteolytic processing which, while specific in nature, has the appearance of removing easily accessible, nonessential domains, rather than contributing to biosynthetic maturation of function.

Amino Acid Sequence↗

PubMed1989

Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome.

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.

Amino Acid Sequence↗

PubMed1992

Junctions between genes in the haptoglobin gene cluster of primates.

To investigate the nature of the recombination that generated the haptoglobin three-gene cluster in Old World primates, we sequenced the region between the second gene (HPR) and the third gene (HPP) in chimpanzees (15 kb), as well as the region 3' to the cluster in humans (14 kb). Comparison to the previously sequenced human haptoglobin (HP) and HPR genes showed that the junction point between HP and HPR in humans (junction 1) was not identical to the junction point between the HPR and HPP genes of the chimpanzee (junction 2). An Alu sequence was found at each junction, but both Alu sequences lacked short direct repeats of the flanking genomic DNA. The lack of direct repeats implies that both junction Alu sequences are the products of recombination between different Alu elements. In addition, other insertion and deletion events are clustered in the regions near the junction Alu sequences. The observation that Alu sequences define the junctions between genes in the haptoglobin gene cluster emphasizes the importance of Alu sequences in the evolution of multigene families.

Animals↗

PubMed1987

Linkage analysis using multiple Xq DNA polymorphisms in normal families, families with the fragile X syndrome, and other families with X linked conditions.

Multipoint linkage analysis was undertaken with eight Xq cloned DNA sequences which identify one or more restriction fragment length polymorphisms in 26 families. These families comprise seven phase known normal families with three or more males in the third generation, seven families segregating for haemophilia B, one large family with dyskeratosis congenita, and 11 families with the fragile X syndrome. Phase known meioses informative for three or more loci supported the order centromere--DXYS1--DXS107--DXS102, DXS51--F9--FRAXA--DXS15, DXS52, F8--Xqter in each group of families studied. One of the normal families was segregating for protan colour blindness and showed a phase known recombination which would support the order centromere--F9--DXS52--CBP--Xqter. With the exception of DXYS1, all of these sequences have been localised to Xq27----qter by in situ hybridisation or hybridisation to Xq fragment panels, and on this basis should lie within 20 cM of one another. No recombination was observed between the sequences localised to Xq28, namely DXS52, F8, and DXS15 (between DXS15 and DXS52 Z = 12.25 at theta = 0 with confidence limits of 0 to 5 cM). However, an excess of recombination was apparent in the region of FRAXA with maximal lod scores as follows: F9 versus FRAXA (Z = 2.05, theta = 0.19), DXS52 versus FRAXA (Z = 1.85, theta = 0.26), and DXS15 versus FRAXA (Z = 1.33, theta = 0.27). No consistent differences were observed in the frequency of recombination when families with the fragile X syndrome were compared with normal families or families segregating for other X linked conditions. These results are compared with other published work and support the conclusion that although measurable linkage exists between these flanking markers and FRAXA, the intervals as measured by the frequency of meiotic recombination will seriously limit their clinical usefulness.

Chromosome Mapping↗

PubMed1986

[A study of proliferative responses to host Ia antigens in fully allogeneic bone marrow chimeras in mice--sequential analysis of the reactivity and characterization of the cells involved in the response].

Irradiation bone marrow chimeras were established by reconstitution of lethally irradiated AKR mice with C57BL/10 marrow cells which had pretreated with anti-Thy-1 serum. Mixed lymphocyte reactions (MLR) were carried out serially to analyze developing reactivities of lymphocytes from such chimeras, [B10----AKR], against donor, host or third party antigens. Spleen cells of [B10----AKR] chimeras 8 weeks after reconstitution regularly showed proliferative responses when stimulated with spleen cells of the strain of the irradiated recipient (AKR). However, the responsiveness was consistently lower than that generated against third party antigens. The stimulator cells were Ia positive in both anti-recipient and anti-third party responses, and the responding splenocytes were of donor origin and showed Thy-1+, Ly-1+2-, and L3T4+ phenotypes, although a minor population of Ly-2+ and L3T4- T cells might be involved in anti-third party response. Further, we found that substantial proliferative responses to Ia antigens of the recipient strain and also to third party antigens are generated by the thymocytes obtained from the irradiation chimeras at an early stage following bone marrow reconstitution. Majority of the responding thymocytes had surface traits of PNA-, donor type Thy-1+, and L3T4+ in both anti-recipient and anti-third party MLRs. However, as for the Ly-2 antigen, Ly-2+ thymocytes seemed to be, at least partially, involved in anti-host responses. This capacity of thymus cells to mount a response to antigens of the recipient strain declined shortly thereafter. The spleen cells at the same time developed a more durable capability to exhibit anti-host reactivities. Awareness of the sequence of development of these cellular reactivities and capacities to respond to host and third party alloantigens may be crucial to understanding the underlying molecular mechanisms involved in generating a T cell repertoire one component of which is restricted to Ia antigens of the recipient strain.

Animals↗

PubMed1987

Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression.

Ferritin stores iron within a protein shell consisting of 24 subunits of two types, heavy (H) and light (L). According to Southern blotting, the rat genome contains four copies homologous to the H-subunit cDNA (H cDNA). To determine whether only one of these is expressed, H cDNAs isolated from rat liver and heart mRNAs were compared and found to share identical nucleotide sequences. Next, genomic clones for three of the four rat H-subunit loci were isolated. Two were classical processed pseudogenes, whereas the third contained an expressed gene. RNase intron mapping of this expressed gene generated the same exon protection pattern when total RNA from rat liver or heart was used, indicating that this gene accounts for most or all of the H-subunit mRNAs (H mRNAs) in these tissues. Comparison of the expressed rat H-subunit gene (H gene) structure with published sequences for other species displays considerable conservation. The coding sequence of the rat H gene predicts 95% similarity to the human amino acid sequence, thus being more highly conserved than the L-subunit sequence of these species. Near the cap region of the 5' untranslated region, the rat H mRNA displays a 28-nucleotide sequence that is almost totally conserved in the corresponding region of the human, bullfrog, and chicken H mRNA and is also faithfully represented in the rat and human L-subunit mRNAs (L mRNAs), thus making this sequence a prime candidate for involvement in the known translational regulation of both subunits by iron. In the 5' flanking region, partially conserved sequences common to H gene and L-subunit gene (L gene) of the rat may be involved in transcriptional regulation by iron, whereas those conserved only in the H gene of man and the rat imply that other factors may independently control H-subunit regulation.

Amino Acid Sequence↗

PubMed1990

Location of a highly conserved neutralizing epitope in the F glycoprotein of human respiratory syncytial virus.

Trypsin digestion of the purified F protein from human respiratory syncytial virus (Long strain) generated a set of fragments in the amino-terminal third of the F1 subunit which contained the epitope 47F involved in neutralization. Sequencing of five escape mutant viruses selected with monoclonal antibody 47F allowed us to map precisely two amino acid residues (262 and 268) of the F1 subunit which are essential for the integrity of this important epitope. The results are discussed in terms of the mechanisms involved in virus neutralization and the design of potential synthetic vaccines.

Amino Acid Sequence↗

PubMed1988

Structure of C3f, a small peptide specifically released during inactivation of the third component of complement.

C3f, a peptide presumed to be generated by the combined actions of factors I and H on fluid-phase C3b, has been isolated and sequenced. The peptide is 17 residues long and has a molecular weight of 1,847 daltons. The amino-terminal sequence is, with the exception of a single residue, identical to that deduced for the 46-kilodalton polypeptide seen transiently in the generation of iC3b from C3b, and is in full agreement with the sequence deduced from cDNA analysis. In addition, high-pressure liquid chromatography of the digestion of C3b by factor I has shown that C3f is the sole peptide released during iC3b generation.

Amino Acid Sequence↗

PubMed1991

The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain.

To determine the mechanisms by which multiple forms of fibroblast growth factor (FGF) receptors are generated, we have mapped the arrangement of exons and introns in the human FGF receptor 1 (FGFR 1) gene (flg). We found three alternative exons encoding a portion of the third immunoglobulin (Ig)-like domain of the receptor. One of these alternatives encodes a sequence that is part of a secreted form of FGFR 1. The other two encode sequences that are likely part of transmembrane forms of FGFR 1. One of these forms has not been previously reported in published cDNAs. Also, we have determined the structural organization of a portion of the human FGFR 2 gene (bek) and found a similar arrangement of alternative exons for the third Ig-like domain. The arrangement of these genes suggests that there are conserved mechanisms governing the expression of secreted FGF receptors as well as the expression of at least two distinct membrane-spanning forms of the FGF receptors. The diverse forms appear to be generated by alternative splicing of mRNA and selective use of polyadenylation signals.

Amino Acid Sequence↗

PubMed1992

Random mutagenesis of CSF-1 receptor (FMS) reveals multiple sites for activating mutations within the extracellular domain.

Retroviral vectors containing human FMS protooncogene cDNA were reconfigured to allow single-step excision and reinsertion of restriction fragments encoding short segments of the extracellular domain of the colony-stimulating factor 1 receptor (CSF-1R). Fragments ligated into M13 bacteriophages were subjected to random chemical mutagenesis on both strands and recloned into the parental vector to create libraries of FMS genes containing mutations restricted to predefined target cassettes. Transfection of retroviral vector libraries into NIH/3T3 cells gave rise to transformed foci from which cellular DNA was amplified by the polymerase chain reaction (PCR), using primers flanking the mutagenized target sequences. Amplified fragments from individual primary transformants were recloned into intact FMS vector plasmids, and those with transforming activity were subjected to nucleotide sequence analysis. Alternatively, retroviruses rescued from transformed cells by superinfection with helper virus were used to generate secondary transformants containing unique copies of proviral DNA, whose sequences were determined after PCR amplification. Novel activating mutations were identified within sequences separating the third and fourth immunoglobulin-like loops, as well as within non-covalently stabilized loop 4 of the CSF-1R extracellular domain. Thus, FMS mutations able to convert human CSF-1R to an active oncoprotein are not restricted to those previously identified at codon 301. This approach should be generally applicable for defining activating mutations in related growth factor receptors, including those for platelet-derived growth factor and Steel factor (KIT ligand), in which ligand-independent oncoprotein variants have not been identified.

3T3 Cells↗

PubMed1989

Deletion hot spots in chimeric Escherichia coli plasmids.

Deletions form frequently in chimeric plasmids composed of M13mp2, pBR322, and pC194 (B. Michel and S. D. Ehrlich, Proc. Natl. Acad. Sci. USA 83:3386-3390, 1986). They are generated by joining of the nucleotide neighboring the nick site in the M13 replication origin to a nonadjacent nucleotide. This nucleotide is most often located within particular short plasmid regions, named deletion hot spots. Three natural hot spots were present in the chimeric plasmids. Two were active only when the DNA replication initiated at the M13 origin was allowed to progress; the third was active only in the presence of wild-type amounts of DNA ligase. Three artificial hot spots were generated by creating palindromic sequences in the plasmids.

Base Sequence↗

PubMed1978

Cyanide-insensitive NADH oxidation by subcellular fractions isolated from human polymorphonuclear blood cells.

The biochemical triad, NADH oxidation, oxygen (O2) uptake and hydrogen peroxide (H2O2) formation, by subcellular fractions of human blood polymorphonuclears (PMNs) was investigated. It was found that this biochemical triad (1) was under the control of the granule-rich fraction (GRF) only; (2) was not inhibited by cyanide; (3) occurred stoichiometrically for its three components, and (4) accounted quantitatively for the respiratory burst of the stimulated PMN. It was also shown that the above biochemical triad (1) involved an enzymatic step; (2) was enhanced by acidic pH (0.5) and Mg++; (3) was inhibited by Cu++ or low concentration of Mn++; (4) was dependent on H2O2, perhydroxyl radical (HO2) and hydroxyl radical (HO) since either catalase or superoxide dismutase or scavengers of HO2 or HO were inhibitor, and (5) involved multistep reactions. Evidence is provided that the sequence of the reactions is first a generation of H2O2, (spontaneously from NADH in our incubation medium), secondly the production of HO from H2O2, thirdly the oxidation of NADH with further production of HO2,O2 uptake and H2O2 formation, probably through a chain reaction. The identification of the enzyme(s) involved in these multistep reactions needs further studies.

Cyanides↗

PubMed2026

Genomic characterization of novel human-associated CTX-M-15-producing Serratia nevei ST625 lineage infecting a vulnerable loggerhead sea turtle.

BACKGROUND: Serratia nevei is a newly classified and opportunistic bacterial species belonging to the Serratia marcescens complex (SMC). Genomic data from this species is highly relevant for public health and epidemiological tracking. OBJECTIVE: To report the first identification and genomic characterization of extended-spectrum β-lactamase (CTX-M-15)-producing S. nevei sequence type (ST) ST625 lineage infecting a vulnerable loggerhead sea turtle. METHODS: Strain BP02 was recovered from the coelomic cavity of a loggerhead sea turtle (Caretta caretta) admitted to a rehabilitation center in southeastern Brazil. MALDI-TOF MS was initially used for species identification and was further confirmed by whole-genome sequencing on the Illumina HiSeq platform, followed by ANI, dDDH, multilocus sequence typing, resistome, plasmidome, virulome, and SNP-based phylogenomic analyses. RESULTS: Strain BP02 exhibited a multidrug-resistant profile, including resistance to third- and fourth-generation cephalosporins. Genomic analyses identified BP02 as S. nevei ST625 carrying blaCTX-M-15 within the ISEcp1-blaCTX-M-15-wbuC-ΔTn2 genetic environment, in addition to multiple AMR determinants and the IncC plasmid replicon. Phylogenomic analysis demonstrated close relatedness between BP02 and human clinical ST625 strains, previously reported in São Paulo, Brazil, including a urine-derived strain isolated in 2019, differing by only 27 SNPs. Notably, all publicly available ST625 genomes were associated with human clinical sources and displayed multidrug resistance genotypes. CONCLUSION: This study expands the current knowledge regarding the ecology and genomic features of S. nevei, demonstrating the emergence of a human multidrug-resistant clone in marine wildlife. Our findings reinforce the importance of monitoring clinically relevant SMC members across distinct ecological niches within a One Health perspective.

ESBL↗

PubMed1988

Localization of the pro-sequence within the total deduced primary structure of human beta-hexosaminidase B.

The beta subunit of beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) is synthesized in the rough endoplasmic reticulum as a prepropolypeptide. After the loss of the signal peptide and formation of an enzymatically active dimer, the pro-enzyme is either secreted from the cell or transported into the lysosome for processing to its mature form. In order to characterize the early posttranslational events we have purified nearly 1 mg of pro-hexosaminidase B from the NH4Cl containing medium of fibroblasts derived from a patient with the infantile form of Tay-Sachs disease. The partial N-terminal sequence was mapped to a position 42 residues C-terminal to the first in-frame ATG (Met residue) and 79 residues N-terminal to the known mature N-terminus. This position corresponds to that predicted for the cleavage of a 17 amino acid signal peptide generated through the use of the third rather than the first in-frame ATG as the initiation site for protein synthesis.

Amino Acid Sequence↗

PubMed1992

Recognition of a tetranucleotide loop of signal recognition particle RNA by protein SRP19.

The interaction of protein SRP19 with the RNA component of human signal recognition particle (SRP) was studied by site-directed mutagenesis of the SRP RNA. The effects of nucleotide changes in the tetranucleotide loop (tetraloop) of helix 6 showed that SRP19 recognizes a tetraloop in a sequence-specific manner. Adenosine 149 at the third position of the tetraloop was essential for binding. In contrast, changes of the base at the second position had no effect. Mutations that disrupt or compensate individual SRP RNA helices were generated to investigate the importance of base pairing and to identify other binding sites. Considerable base pairing was essential in helix 6. Another SRP19-binding site was located in the distal part of helix 8. The primary sequences of the tetraloop-binding protein SR19 and of bacterial ribosomal protein S15 are shown to be similar.

Amino Acid Sequence↗

PubMed1991

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome.

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.

Animals↗