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PubMed1986

Expression of the human salivary alpha-amylase gene in yeast and characterization of the secreted protein.

Recombinant plasmids were constructed in which the human salivary alpha-amylase gene, with or without the N-terminal signal sequence for secretion, was placed under control of the APase (PHO5) promoter of Saccharomyces cerevisiae. In yeast cells transformed with the alpha-amylase gene having the human signal sequence for secretion, the gene was expressed and the enzyme was secreted into the medium in three different glycosylated forms. The amylase gene without the signal sequence was also expressed in yeast, but the products were neither secreted nor glycosylated. Determination of the N-terminal amino acid (aa) sequence revealed that the 15-aa signal sequence had been cleaved from the secreted enzyme, and that the N-terminal residue, glutamine, had been modified into pyroglutamate, as is commonly observed with the mammalian salivary alpha-amylase. Thus, the human salivary alpha-amylase signal sequence for secretion was correctly recognized and processed by the yeast secretory pathway. The C-terminal residue was identified as leucine, which is predicted from the nucleotide sequence data to be located at position 511 in front of the termination codon. Therefore, there is no post-translational processing in formation of the C terminus.

Acid Phosphatase↗

PubMed1987

IS50L as a non-self transposable vector used to integrate the Bacillus thuringiensis delta-endotoxin gene into the chromosome of root-colonizing pseudomonads.

Insertion sequence IS50L of transposon Tn5 was used as a non-self transposable vector to integrate the delta-endotoxin gene (tox) from Bacillus thuringiensis subsp. kurstaki HD-1 into the chromosome of two corn-root colonizing strains of Pseudomonas fluorescens (112-12 and Ps3732-3-7). A DNA fragment containing the KmR gene from Tn5 and tox was inserted into an IS50L element (IS50L-tox) contained on a suicide plasmid. Transposition of IS50L-tox into the chromosome of P. fluorescens 112-12 and Ps3732-3-7 occurred by selecting for KmR transconjugants and supplying transposase in cis from a linked IS50R element. A frameshift mutation in the transposase gene of the IS50L-tox element was also constructed to decrease the likelihood that suppression or a spontaneous reversion at the UAA (ochre) termination codon of IS50L would create an active transposase. The inability of IS50L-tox to transpose further minimizes the potential for horizontal gene transfer of the tox gene to other bacterial species. Expression of the Tox protein in strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta).

Bacillus thuringiensis↗

PubMed1988

Synthesis of a gene coding for an insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors.

We have explored the possibility of improving baculovirus pesticides by incorporating an insect-specific neurotoxin gene into a baculovirus genome. A 112-bp gene (BeIt) encoding insectotoxin-1 of the scorpion Buthus eupeus was synthesized and cloned in Escherichia coli. For expression, BeIt was transferred to the DNA genome of Autographa californica nuclear polyhedrosis virus (AcMNPV). Three different recombinant AcMNPVs, carrying BeIt under the control of the strong AcMNPV polyhedrin promoter, were constructed and expression of BeIt was monitored upon infection of Spodoptera frugiperda (Sf) cells. Toxin expression was low using a recombinant virus in which BeIt was inserted 6 nucleotides (nt) downstream from the intact polyhedrin mRNA leader. More expression was observed when a signal-peptide was attached in-frame to the N terminus of BeIt. The highest level of expression was observed with a fusion gene comprised of the 58 N-terminal codons of polyhedrin fused to BeIt; however, the level of expression was ten- to twenty-fold below that for polyhedrin. Polyhedrin promoter-directed transcripts of all three recombinants accumulated to levels similar to those of wild-type polyhedrin transcripts, indicating that the limitation to expression of unfused BeIt was not at the level of transcription but rather at the posttranscriptional level including translation or protein stability. Paralytic activity of the toxin products was not detected.

Amino Acid Sequence↗

PubMed1995

Detection of a functional promoter/enhancer in an intron-less human gene encoding a glutamine synthetase-like enzyme.

A human genomic clone, psi GS, containing an intron-less glutamine synthetase (GS)-encoding pseudogene, was isolated by screening a human library. A sequence of 3004 bp, containing the GS coding region and both the 5' and 3' flanking sequences, was identified that exhibits all the characteristics of a processed pseudogene. The coding region shows 93% identity with the human GS cDNA (hGS) sequence and contains two frame-shifts and two termination codons. The coding sequence is flanked by a 9-bp AT repeat and a putative polyadenylation site, AATAAA, at the 3' end. Primer extension analysis and S1 nuclease mapping showed a transcription start point (tsp) 62 bp upstream from the start codon indicating a shorter untranslated region than hGS. Transfection of HeLa cells with cat constructs containing portions of the 5' flanking sequence showed the presence of a functional promoter/enhancer within 200 bp of the tsp, independent of its orientation.

Amino Acid Sequence↗

PubMed1995

A high-level prokaryotic expression system: synthesis of human interleukin 1 alpha and its receptor antagonist.

Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene. Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression.

Amino Acid Sequence↗

PubMed1988

Molecular structure and sequence homology of a gene related to alpha 1-antitrypsin in the human genome.

A 7.7-kb EcoRI genomic DNA fragment highly homologous to the human alpha 1-antitrypsin (AAT) gene has been cloned. This antitrypsin-related sequence is physically linked to the authentic AAT gene and both are present in a single cosmid clone. Nucleotide sequencing of the AAT-related genomic fragment demonstrated extensive homology with the authentic AAT gene in the introns as well as in the exons. The conservation of all RNA splice sites and lack of internal termination codons in the exonic regions suggest that it may not be a classical pseudogene. If expressed, it could result in a protein of 420 amino acid residues exhibiting a 70% overall homology with human alpha 1-antitrypsin. The signal peptide sequence is well conserved in the related gene, but the active site for protease inhibition of Met-Ser in alpha 1-antitrypsin has been changed to Trp-Ser. These data suggest that the putative protein encoded by the AAT-related gene is a secretory serine protease inhibitor with an altered substrate specificity. Interestingly, even the intronic regions in the related gene exhibit a 65% overall nucleotide sequence homology with those of the authentic AAT gene. These results suggest that the AAT-related gene is derived from a recent duplication of the authentic AAT gene and represents a new member of the serine protease inhibitor superfamily.

Amino Acid Sequence↗

PubMed1990

Human U1-70K ribonucleoprotein antigen gene: organization, nucleotide sequence, and mapping to locus 19q13.3.

We have isolated and sequenced the gene encoding the human U1-70K snRNP protein. U1-70K is an RNA-binding protein that is a specific component of the U1 small nuclear ribonucleoprotein complex (snRNP) and constitutes the major anti-(U1) RNP autoimmune antigen. We have mapped the U1-70K gene to the distal portion of chromosome 19, at band q13.3. The gene is greater than 44 kb in size and consists of 11 exons. The general structure of the gene has been completely conserved during vertebrate evolution and accounts for the production of several different U1-70K mRNA species by alternative pre-mRNA splicing. Comparison of the predicted amino acid sequences of animal U1-70K proteins reveals a high degree of conservation, particularly in the region of the RNP consensus domain. Even more striking is the complete conservation of the nucleotide sequence of an alternative included/excluded exon containing an in-frame translational termination codon. This conservation also includes significant portions of the downstream intervening sequence. This extraordinary conservation at the nucleotide sequence level suggests that alternative splicing of this exon serves an important function, perhaps in regulating the production of functional U1-70K protein.

Amino Acid Sequence↗

PubMed1990

Isolation and chromosomal localization of the human glutathione peroxidase gene.

We have isolated cDNA clones for the gene, termed GPX1, encoding the major human selenoprotein, glutathione peroxidase. Sequence analysis confirmed previous findings that the unusual amino acid seleno-cysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPX1 cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPX1 gene to a single site on chromosome 3, at region 3q11-13.1. Thus, three genomic sites bear sequence homology to the GPX1 cDNA, and the one most homologous maps to 3q11-13.1.

Base Sequence↗

PubMed1992

Strategy for detecting cellular transcripts promoted by human endogenous long terminal repeats: identification of a novel gene (CDC4L) with homology to yeast CDC4.

Several families of repetitive sequences related to integrated retroviruses have been identified in the human genome. The largest of these families, the RTVL-H family, has close to 1000 members in addition to a similar number of solitary long terminal repeats (LTRs) dispersed on all chromosomes. Previous work has shown that the expression of genomic RTVL-H elements is driven by their LTRs and that some LTRs can promote expression of a reporter gene. These observations suggest that some endogenous RTVL-HLTRs may naturally regulate the transcription of adjacent cellular genes or that rearrangements involving these elements may cause aberrant gene expression. To investigate this possibility, we have used a differential screening strategy to identify chimeric cDNA clones derived from LTR-promoted transcripts. Here we report the identification and analysis of four such clones isolated from an NTera2D1 (teratocarcinoma) cDNA library. Two of the clones, AF-1 and AF-2, contain termination codons in all reading frames. Another clone, AF-4, contains LTR sequences linked in the genome to a CpG island. The fourth clone, AF-3, contains an 862-bp open reading frame representing part of a novel gene (CDC4L) with homology to the yeast cell division cycle gene CDC4. These findings indicate that RTVL-H elements may be involved in the regulation of diverse cellular transcripts in human cells.

Amino Acid Sequence↗

PubMed1992

XY sex reversal associated with a nonsense mutation in SRY.

Sex determination in humans is mediated through the expression of a testis-determining gene on the Y chromosome. In humans, a candidate gene for the testis-determining factor (TDF) that encodes a protein with a putative DNA-binding motif and has been isolated is termed SRY. Here we describe an XY sex-reversed female with pure gonadal dysgenesis who harbors a de novo nonsense mutation in the SRY open reading frame (SRY-orf). This single-basepair substitution results directly in the formation of a termination codon in the putative SRY DNA-binding motif, presumably leading to a nonfunctional gene product. This brings the number of reported XY sex-reversed females with de novo mutations in the known SRY-orf to three, each occurring in the putative DNA-binding domain. This provides further evidence to support SRY being TDF in humans and also indicates the functional importance of the putative DNA-binding domain of the SRY protein.

Base Sequence↗

PubMed1995

A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III.

4-Hydroxyphenylpyruvic acid dioxygenase (HPD; EC 1.13.11.27) is an important enzyme in tyrosine catabolism in most organisms. Decreased activity of 4-hydroxyphenylpyruvic acid dioxygenase in the liver of mouse strain III is associated with tyrosinemia. We report a nucleotide substitution that generates a termination codon in exon 7 of the 4-hydroxyphenylpyruvic acid dioxygenase gene in III mice. This mutation is associated with partial exon skipping, and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Mouse strain III is a model for human tyrosinemia type 3 (McKusick 276710), and this strain together with recently established models for tyrosinemia type 1 will facilitate studies of hereditary tyrosinemias.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed1993

Mutations of androgen receptor gene in androgen insensitivity syndromes.

The androgen receptor belongs to the family of steroid-thyroid hormone-retinoid nuclear receptors. It contains 3 major domains: a hormone-binding region, a DNA-binding region and an amino-terminal region. Cloning of the cDNA encoding the androgen receptor and elucidation of the androgen receptor gene structure enabled the characterization of the molecular defects associated with androgen insensitivity. Mutations of the androgen receptor in 46,XY individuals cause a spectrum of androgen insensitivity syndromes, ranging from female phenotype (testicular feminization) to minor degrees of undervirilization or infertility. Reports on androgen receptor gene structure in patients with complete or partial forms of androgen insensitivity demonstrate that gene deletions are very rare. Several categories of mutations have been reported and are reviewed in this paper. Nucleotide substitutions in the androgen-binding domain or the N-terminal region that cause insertion of premature termination codons result in failure to form a functional protein. Missense mutations within the androgen-binding domain are responsible for a decrease or absence of receptor-binding activity. Mutations within the DNA-binding domain are associated with a positive receptor-binding form of androgen insensitivity. Analysis of described mutations indicates that they are spread throughout the gene, either associated with partial or complete androgen insensitivity. Furthermore, the same point mutation was reported to be associated with variable phenotypic expression of androgen insensitivity syndrome. It is thus difficult to define a genotype/phenotype relationship. However, mutations causing androgen insensitivity will certainly yield important new insights into the molecular basis of androgen action.

Androgen-Insensitivity Syndrome↗

PubMed1993

Genetic analysis of human placental aromatase deficiency.

Placental aromatase deficiency, which was characterized by maternal and fetal virilization and by a low level of estrogen excretion into urine during pregnancy, was studied by biochemical and molecular genetical techniques. Among enzymes participating in the electron transport system of the patient's placental microsomes, only aromatase activity was observed to be reduced (< 3% of normal levels). Northern and Western blotting analyses showed that the transcription of the aromatase gene and the translation of its mRNA seemed to proceed normally in the patient's tissue. However, the aromatase cDNA isolated from the patient was found to contain an extra DNA fragment of 87 base pairs (bp) which encoded 29 amino acids in frame but no termination codon. The insertion was located at the splicing point between exon 6 and intron 6 of the normal aromatase gene. The extra DNA fragment represented the first part of intron 6 except that its initial GT was altered to GC. These findings indicated that, in the patient's aromatase gene, the splicing between exon 6 and intron 6 did not occur at the normal position. This reflected the presence of one point mutation in its consensus sequence which caused the next cryptic consensus sequence 87 bp downstream, to be used according to the canonical GT/AG rule. The protein molecule thus translated contained an extra 29 amino acids. Furthermore, the patient's aromatase cDNA was observed to produce a protein molecule with a trace of activity in the transient expression system of COS-7 cells and in the high level expression system of baculovirus-insect cells. Direct DNA sequencing of aromatase genes from the patient and parents confirmed that this deficiency is a hereditary disease with an autosomal recessive inheritance pattern. The patient and parents are homozygote and heterozygotes, respectively, for this mutation.

Amino Acid Sequence↗

PubMed1995

The human 11 beta-hydroxysteroid dehydrogenase type II enzyme: comparisons with other species and localization to the distal nephron.

Effective glucocorticoid inactivation is currently thought to be an indispensable feature of mineralocorticoid target cells. The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) inactivates glucocorticoids and prevents them from binding to the non-selective mineralocorticoid receptor. In the kidney it is the NAD dependent high affinity isoform (11 beta-HSD2) which is thought to endow specificity on the receptor. The recent cloning of the human, sheep and rabbit 11 beta-HSD2 enzymes permits a comparison of the enzyme from the three species. Human and rabbit enzymes are 87% identical and of similar length, while the human and sheep enzymes have only 75% identity. The last 12 residues in all three species were found to be highly divergent, but most of the ovine dishomology can be accounted for by the deletion of a single nucleotide toward the C-terminus of the protein resulting in a shift in reading frame generating a protein 27 residues longer than the human isoform. Numerous other deletions were also observed in this region of the sheep cDNA sequence. Furthermore, the rabbit cDNA also displayed a large degree of dishomology with the human sequence a short distance downstream from the termination codon. Conserved overlapping cytoplasmic translocation signals were observed in all three species, suggesting a topology whereby the enzyme is anchored into the endoplasmic reticulum by multiple hydrophobic regions in the N-terminus and the bulk of the 11 beta-HSD2 peptide is sited in the cytoplasm. A polyclonal antibody generated against the C-terminus of human 11 beta-HSD2 was used to localize the enzyme within the kidney. A high level of immunoreactive was observed in distal tubules and collecting ducts, localizing the enzyme to the same part of the nephron as the mineralocorticoid receptor. Moderate levels of staining were also seen in vascular smooth muscle cells. These results support the notion that 11 beta-HSD2 is an autocrine protector of the mineralocorticoid receptor and that it plays an important role in cardiovascular homeostatic mechanisms.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1993

The cDNA and deduced protein sequence of house fly NADPH-cytochrome P450 reductase.

Antisera to purified house fly NADPH-cytochrome P450 reductase were used to select cDNA clones from an expression library of abdomens of phenobarbital-treated house flies. A partial cDNA of 1841 bp containing a TAG termination codon, a consensus polyadenylation site and 269 bp of 3' untranslated sequence was obtained. Sequencing of a genomic clone coupled with mRNA sequencing yielded the complete coding sequence including the starting ATG. The resulting open reading frame of 2013 nucleotides codes for a protein of 671 residues. The native reductase apoprotein has a molecular weight of 76,366 and the deduced molecular weight of the holoenzyme (i.e. with 1 mol of FAD and FMN) is 77,608. The sequence of the house fly P450 reductase protein is highly similar to that of rabbit liver, the overall amino acid positional identity is 54.5% and the overall identity among eukaryotic P450 reductases is about 25%. The P450 reductase gene of 19-23 kb was located on chromosome III, as shown by comparison of RFLP-patterns of the P450 reductase gene in two house fly strains and their hybrids.

Amino Acid Sequence↗

PubMed2006

mRNA made during heat shock enters the first round of translation.

To determine whether mRNA synthesized during a heat shock is translated at least once in spite of the strong inhibition of translation by heat shock, we used nonsense-mediated decay (NMD) as an assay since NMD requires a round of translation. As NMD substrate we used the human psigammaE-crystallin gene, which contains a premature termination codon, and as control, its close relative, the human gammaD-crystallin gene, both placed under control of the Hsp70 promoter. We show that no spliced psigammaE-crystallin mRNA can be detected in heat shocked cells, suggesting that NMD resumes as soon as splicing is restored. We further show that newly synthesized mRNAs co-sediment with the 40S ribosomal subunits, indicating that the transcripts are recruited to the translation machinery but are stalled at the translation initiation stage. Using fluorescence loss in photobleaching (FLIP) we show that cytoplasmic EGFP-CBP20 is immobile in heat shocked cells. CBP20 is part of the cap binding complex which is thought to direct the first round of translation. Together our data suggest that all mRNAs made during heat shock enter the pioneer round of translation.

Blotting, Western↗

PubMed2004

A small heat shock protein from Artemia franciscana is phosphorylated at serine 50.

Encysted embryos of Artemia franciscana are exceptionally resistant to stress and an important part of this tolerance involves p26, a small heat shock protein which functions as a molecular chaperone. Cloning and sequencing of randomly selected p26 cDNAs produced by RT-PCR with poly(A)(+) mRNA from encysted embryos as template yielded 10 clones encoding identical polypeptides. The noncoding nucleotide sequences extending from the termination codon to the poly(A) tail of each clone were also identical. These data indicated a single p26 gene is expressed during embryo development. However, two-dimensional gel electrophoresis showed that purified p26 consisted of four isoforms, providing evidence for posttranslational modification of the protein, a possibility supported by mass spectrometry and immunoprobing of Western blots. The major isoform observed in two-dimensional gels, termed a, is the primary gene product, whereas isoform c is phosphorylated at serine 50, a residue located in a protein kinase C reactive site. Isoforms b and d were generated posttranslationally, but by unknown processes. The results represent the first description of posttranslationally modified small heat shock proteins in crustaceans and they expand the phylogenetic range of organisms that possess phosphorylated isoforms of these proteins. At least two small heat shock proteins from other organisms contain serine residues equivalent in position to serine 50 of p26, but neither is phosphorylated.

Amino Acid Sequence↗

PubMed2005

The role of SMG-1 in nonsense-mediated mRNA decay.

SMG-1, a member of the PIKK (phosphoinositide 3-kinase related kinases) family, plays a critical role in the mRNA quality control system termed nonsense-mediated mRNA decay (NMD). NMD protects the cells from the accumulation of aberrant mRNAs with premature termination codons (PTCs) that encode nonfunctional or potentially harmful truncated proteins. SMG-1 directly phosphorylates Upf1, another key component of NMD, and this phosphorylation occurs upon recognition of PTC on post-spliced mRNA during the initial round of translation. At present, a variety of tools are available that can specifically suppress NMD, and it is possible to examine the contribution of NMD in a variety of physiological and pathological conditions.

Animals↗