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PubMed1985

Structure and expression of the cell division genes ftsQ, ftsA and ftsZ.

The essential cell division genes ftsQ, ftsA and ftsZ map in a cluster of cell envelope genes located at two minutes on the Escherichia coli genetic map and appear to constitute an atypical operon. These closely clustered genes are all transcribed in the same direction and yet each of these genes was independently cloned on a multicopy plasmid and found to be expressed. Tn5 insertion mutagenesis of this region confirmed that these genes were independently expressed, indicating that each of these genes has its own promoter. However, maximum expression of the distal ftsZ gene required the promoter for the proximal ftsQ gene. The proximal ftsZ promoter was located within the ftsA structural gene and the proximal ftsA promoter was located within the ftsQ structural gene. No transcription terminators were evident from the DNA sequence analysis of this region. The sequence analysis also revealed that the termination codon for ftsQ overlapped the initiation codon of the ftsA gene and that the ftsA and ftsZ genes were separated by 60 base-pairs. The ftsQ gene product has a calculated Mr of 31,432 and the ftsA gene product has a calculated Mr of 45,327. The structure and expression of these genes are discussed.

Autoradiography↗

PubMed1987

Organization and sequence of the hsd genes of Escherichia coli K-12.

The nucleotide sequence of the hsdR and M genes, together with that for hsdS comprises an 8400 base segment spanning the entire hsd region of Escherichia coli K-12. The three hsd genes are transcribed in the same direction, but from two promoters. hsdR and hsdM are separated by 492 base-pairs, whereas the termination codon of hsdM overlaps the initiation codon of hsdS. pres precedes hsdR, and our data indicate a transcription termination signal in the interval between hsdR and pmod, as expected if transcription of hsdM and S is dependent on pmod. Transcription from pres is not influenced by the products of the hsdM and S genes, and the mechanism whereby restriction is prevented when the hsd region is transferred to a modification-deficient cell remains to be elucidated. A segment of the predicted amino acid sequence of the M polypeptide shares homology with a variety of adenine methylases and may identify part of the active site for methylation of specific adenine residues. The R polypeptide shows homology with a variety of ATPases, and pronounced regions of alpha-helical structure are predicted, one of which is amphipathic.

Amino Acid Sequence↗

PubMed1991

Modification of GP63 genes from diverse species of Leishmania for expression of recombinant protein at high levels in Escherichia coli.

Toward the future development of a defined subunit vaccine against leishmaniasis is, high levels of recombinant GP63 for diverse species of Leishmania were produced in Escherichia coli. Several features of Leishmania GP63 genes were simultaneously modified with the polymerase chain reaction (PCR) using either cloned genes or total genomic DNA from Leishmania as template DNA for the PCR amplification reactions. The PCR products included only the coding region for the predicted mature form of GP63 that occurs on the surface of Leishmania, flanked by the appropriate translation signals and cloning sites for the production of recombinant GP63 as nonfusion protein in E. coli. When the codon usage in the GP63 gene was modified to reduce the guanine and cytosine content for the codons adjacent to the ATG initiation codon, rGP63 represented about 50% of total protein in E. coli. Mouse monoclonal antibodies raised against purified Leishmania major rGP63 had equivalent immunoblotting characteristics for native GP63 and recombinant GP63 with respect to linear determinants on GP63 expressed in diverse species of Leishmania. Human T cell lines and clones were derived from a patient infected with Leishmania braziliensis panamensis using rGP63 purified from an L. major GP63 expression clone as antigen.

Animals↗

PubMed1992

A complementary DNA for human choline acetyltransferase induces two forms of enzyme with different molecular weights in cultured cells.

Complementary DNA (cDNA) clones containing the entire coding region of human choline acetyltransferase (ChAT) were isolated from cDNA libraries prepared from the autopsied spinal cord. In the human cDNA, the ATG codon assigned to the putative initiation codon for pig, rat and mouse ChAT cDNAs was replaced by ACG. The human cDNA contained an in-frame ATG codon 324 nucleotides upstream of the ACG codon. Therefore, human ChAT cDNA should code for a 748 amino acid polypeptide of 82.6 kDa. This deduced molecular weight was larger than that of ChAT protein purified from the human brain and placenta (64-70 kDa). The human ChAT cDNA containing the entire coding region was ligated to an expression vector and introduced into African green monkey kidney (COS) cells and Chinese hamster ovary (CHO) cells. The cells expressed high ChAT activity and produced two protein bands immunostained with an antibody to monkey ChAT. The molecular weight of the proteins was estimated to be approximately 70 and 80 kDa by polyacrylamide-SDS gel electrophoresis. When partial cDNAs that lacked the first ATG but contained the replaced ACG codon were introduced into COS cells, the cells expressed moderate ChAT activity and an immunoreactive protein band of 70 kDa. These results indicate that translation of human ChAT mRNA starts at two sites and produces two enzyme proteins with different molecular weights. It might be that the larger form of ChAT molecule is an enzyme precursor for processing or that the N-terminal extrapeptide is needed for subcellular localization of the enzyme.

Amino Acid Sequence↗

PubMed1999

Microsomal GST-I: genomic organization, expression, and alternative splicing of the human gene.

In this paper we report the genomic organization of the human microsomal GST-I gene. This gene spans 18 kb, and contains seven exons. Sequences that encode the 155 amino acid open reading frame are present in Exons II, III, IV, the 5'-untranslated region is present in Exons Ia, Ib, Ic, Id, and II, and the 3'-untranslated region is present in Exon IV. Exons Ia, Ib, Ic, Id, and III are alternatively spliced to generate at least six different mGST-I transcripts. The results of EST and PCR analysis show that most mGST-I transcripts terminate within Exon Ib, and primer extension analysis shows these transcripts initiate at three major sites located at 79, 81, and 88 nucleotides upstream of the ATG initiation codon. Sequences surrounding the putative initiation sites are G-C rich, and several Sp1 consensus binding sites were identified. Northern analysis shows that the human GST-I gene is preferentially expressed as a 1.0 kb transcript in liver, and in several other tissues. Finally, a comparison of the mGST-I and PIG12 sequences with those of FLAP, LTC4 synthase, mGST-II, and mGST-III suggests that these proteins are the related products of a dispersed microsomal GST gene superfamily.

Alternative Splicing↗

PubMed2000

Characterization of the mouse and human PRSS17 genes, their relationship to other serine proteases, and the expression of PRSS17 in developing mouse incisors.

The human PRSS17 (serine protease 17) gene, which is located on chromosome 19q in a cluster of genes encoding serine proteases, has been variously designated enamel matrix serine proteinase 1 (EMSP1), prostase, KLK4, and KLK-L1. We have cloned and characterized the mouse and human PRSS17 genes. Both have six exons and five introns. The mouse PRSS17 gene sequence is 10134bp; the human sequence is 7115bp. Computer analysis of the mouse PRSS17 gene sequence upstream of the translation initiation codon identified two potential transcription initiation sites, at nucleotides 2878 and 2336. The first nucleotide of the reported mouse PRSS17 cDNA sequence corresponds to position 2352 on the gene, only 16 bases downstream from one of the putative transcription initiation sites. Repetitive DNA sequences from the MSR1 family are found in both the mouse and human PRSS17 genes. Additionally, the human PRSS17 gene contains Tigger2, MER8, and Alu repetitive sequences. Phylogenetic analyses of human and rodent proteases suggest that the PRSS17 protein is not a member of the kallikrein family of serine proteases but that the PRSS17 gene may have originated prior to the divergence of the kallikrein and trypsin families of proteases. To better characterize the timing of PRSS17 expression in developing teeth, we performed in-situ hybridization on postnatal day 3 developing mouse mandibular incisors. PRSS17 mRNA was not detected in secretory stage ameloblasts but could be detected in odontoblasts, while transition-stage and maturation-stage ameloblasts were strongly positive. This pattern supports a role for the PRSS17 protein in the degradation of enamel proteins.

Amino Acid Sequence↗

PubMed1988

Translational activation of the lck proto-oncogene.

The lck proto-oncogene, a member of the src gene family, encodes a lymphocyte-specific protein tyrosine kinase (p56lck) that is implicated in the pathogenesis of lymphoid neoplasia. We report here that 5' lck sequence elements, containing AUG codons, significantly reduce the in vivo efficiency of p56lck translation from the normal messenger RNA. This result provides a quantitative explanation for the overexpression of p56lck in two retrovirally-induced murine lymphomas that express abnormal lck transcripts from which the physiological 5' translational control region has been deleted and non AUG-containing viral sequences substituted. In contrast to other mammalian genes, most proto-oncogenes contain AUG codons 5' to the authentic initiation codon, suggesting that cells can regulate translational start sites in these mRNAs. Abrogation of translational control may be a general mechanism of proto-oncogene activation in malignancy.

Animals↗

PubMed1984

Structure linkage, and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor.

We have cloned and sequenced a fragment of the chicken genome approximately 9 kilobases in length that comprises the genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor. The two genes are homologous and have identical structures: both consist of 12 exons, some of which precisely correspond to predicted structural domains of the receptor subunits. The delta and gamma subunit genes are encoded by the same DNA strand and are very closely linked, there being only 740 base pairs between the last codon of delta and the initiator codon of gamma. Blot analysis demonstrates that the genes we describe are unique in the genome. Comparison of the predicted protein sequence for the corresponding subunits of chicken and of the elasmobranch Torpedo reveals a high degree of conservation in some but not all of the protein domains.

Amino Acid Sequence↗

PubMed2004

Tissue- and stage-specific RNA editing of rps 14 transcripts in moss (Physcomitrella patens) chloroplasts.

The C to U editing event that converts an ACG codon to an AUG translation initiation codon in the chloroplast rps 14 transcript is unique to the moss Physcomitrella patens and has not been found in other species. The efficiency of RNA editing was 80% in the young protonemata and decreased to approximately 20% in old protonemata and fully developed leafy shoots. This indicates that RNA editing of this site is regulated in a tissue- and stage-specific manner. In this study, a novel C to U RNA editing site has been identified at the -1 position relative to the AUG. Because the editing site is localized in the mRNA 5' untranslated region, it may affect the efficiency of rps 14 mRNA translation.

Base Sequence↗

PubMed2004

A unique role of an amino terminal 16-residue region of long-type GATA-6.

Human GATA-6 mRNA utilizes two Met-codons in frame as translational initiation codons in cultured mammalian cells. An internal ribosome entry site (IRES) is not present in front of the coding region for short-type GATA-6. The 5'-upstream sequence with a short upstream open reading frame (uORF) did not affect the production of either long- or short-type GATA-6. Introduction of a canonical Kozak sequence around the upstream Met-codon resulted in predominant synthesis of long-type GATA-6, suggesting that the translation of short-type GATA-6 could be due to leaky scanning of the Met-codon by ribosomes. We found that at least the sequence comprising the 90th to 139th nucleotide bases from the first letter of the upstream Met-codon plays a positive role in the expression of long-type GATA-6. This was confirmed by insertion of the corresponding sequence in frame at the site of deletion (the 38th to 304th nucleotide residues). However, insertion of the sequence comprising the 92nd to 141st bases did not suppress the negative effect of the deletion. These results suggest that the translation of this region (Glu-31-Cys-46) could be critical for the apparent production of long-type GATA-6. We also demonstrated that long-type GATA-6 is potentially more active than the short-type.

Amino Acid Motifs↗

PubMed1988

The existence of two genes between infB and rpsO in the Escherichia coli genome: DNA sequencing and S1 nuclease mapping.

A number of genes encoding proteins involved in transcription and translation are clustered between 68 and 69 minutes on the Escherichia coli genome map and are transcribed clockwise as two operons: the metY operon, containing metY, P15A, nusA, infB; and about a kilobase further downstream, the rpsO and pnp operon. The DNA sequence between infB and rpsO was determined and two open reading frames were detected which code for proteins of 15,200 (P15B) and 35,091 (P35) daltons. Maxicell analysis showed a relatively strong expression of P15B whereas P35 was synthesized more weakly. An overlap of the termination codon of P15B and the initiator codon for P35 suggests that translation of P15B and P35 may be coupled. S1 nuclease mapping of in vivo transcripts between infB and rpsO provided no evidence for major promoters but detected a moderately efficient rho-independent terminator between infB and P15B. The results indicate that P15B and P35 are expressed as part of the metY operon, but that some transcriptional read through into the rpsO operon also occurs, thereby, functionally linking the expression of these two complex systems.

Amino Acid Sequence↗

PubMed1991

RNA editing makes mistakes in plant mitochondria: editing loses sense in transcripts of a rps19 pseudogene and in creating stop codons in coxI and rps3 mRNAs of Oenothera.

An intact gene for the ribosomal protein S19 (rps19) is absent from Oenothera mitochondria. The conserved rps19 reading frame found in the mitochondrial genome is interrupted by a termination codon. This rps19 pseudogene is cotranscribed with the downstream rps3 gene and is edited on both sides of the translational stop. Editing, however, changes the amino acid sequence at positions that were well conserved before editing. Other strange editings create translational stops in open reading frames coding for functional proteins. In coxI and rps3 mRNAs CGA codons are edited to UGA stop codons only five and three codons, respectively, downstream to the initiation codon. These aberrant editings in essential open reading frames and in the rps19 pseudogene appear to have been shifted to these positions from other editing sites. These observations suggest a requirement for a continuous evolutionary constraint on the editing specificities in plant mitochondria.

Amino Acid Sequence↗

PubMed2006

Plastid marker-gene excision by transiently expressed CRE recombinase.

We report plastid marker-gene excision with a transiently expressed CRE, site-specific recombinase. This is a novel protocol that enables rapid removal of marker genes from the approximately 10,000 plastid genome copies without transformation of the plant nucleus. Plastid marker excision was tested in tobacco plants transformed with a prototype polycistronic plastid vector, pPRV110L, designed to express multiple genes organized in an operon. The pMHB10 and pMHB11 constructs described here are dicistronic and encode genes for herbicide (bar) and spectinomycin (aadA) resistance. In vector pMHB11, expression of herbicide resistance is dependent on conversion of an ACG codon to an AUG translation initiation codon by mRNA editing, a safety feature that prevents translation of the mRNA in prokaryotes and in the plant nucleus. In the vectors, the marker gene (aadA) is flanked by 34-bp loxP sites for excision by CRE. Marker excision by a transiently expressed CRE involves introduction of CRE in transplastomic leaves by agro-infiltration, followed by plant regeneration. In tobacco transformed with vectors pMHB10 and pMHB11, Southern analysis and PCR identified approximately 10% of the regenerated plants as marker-free.

Biotechnology↗

PubMed2005

Further extension of mammalian GATA-6.

Mammalian GATA-6, which has conserved tandem zinc fingers (CVNC-X(17)-CNAC)-X(29)-(CXNC-X(17)-CNAC), is essential for the development and specific gene regulation of the heart, gastrointestinal tract and other tissues. GATA-6 recognizes the (A/T/C)GAT(A/T)(A) sequence, and interacts with other transcriptional regulators through its zinc-finger region. The mRNA of GATA-6 uses two Met codons in frame as translational initiation codons, and produces L- and S-type GATA-6 through leaky ribosome scanning. GATA-6 is subjected to cAMP-dependent proteolysis by a proteasome in a heterologous expression system. These protein-based characteristics of GATA-6 will be helpful for the identification of target genes, together with determination of the in vivo binding sites for GATA-6 and understanding of the complex network of gene regulation mediated by GATA-6.

Amino Acid Sequence↗

PubMed1991

Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product.

Using in vitro-constructed deletions and subcloned DNA fragments, we have identified a new gene, amiC, which regulates expression of the inducible Pseudomonas aeruginosa aliphatic amidase activity. The DNA sequence of the gene has been determined, and an open reading frame encoding a polypeptide of 385 amino acids (molecular mass, 42,834 Da) has been identified. A search of sequence libraries has failed to find homologies with other published sequences. The amiC translation termination codon (A)TGA overlaps the initiation codon for the downstream amiR transcription antitermination factor gene, implying that the amiCR operon is coordinately regulated. Disruption of the amiC open reading frame by insertion and deletion leads to constitutive amidase synthesis, suggesting that AmiC is a negative regulator. This is confirmed by the finding that a broad-host-range expression vector carrying amiC (pSW41) represses amidase expression in a series of previously characterized P. aeruginosa amidase-constitutive mutants. The AmiC polypeptide has been purified from PAC452(pSW41), and N-terminal amino acid sequencing has confirmed the gene identification.

Amidohydrolases↗

PubMed2000

Essential and dispensable virus-encoded replication elements revealed by efforts To develop hypoviruses as gene expression vectors.

We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, could serve as gene expression vectors. The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. Analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication. However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5'-proximal open reading frame (ORF), ORF A, was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B. Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization. These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences.

Amino Acid Sequence↗

PubMedDate not supplied

Comparison of functional peptide encoded in the Escherichia coli 23S rRNA with other peptides involved in cis-regulation of translation.

A new approach for studying functional rRNA fragments has been developed based on using a plasmid library expressing random fragments of rRNA. A 34 nucleotide long fragment of Escherichia coli 23S rRNA has been identified that renders cells resistant to erythromycin, when expressed in vivo. The rRNA fragment contains a five codon long open reading frame, initiating at GUG and terminating at UAA, with a Shine-Dalgarno sequence located at an appropriate distance from the initiator codon. Translation of this mini-gene is required for the observed erythromycin resistance. Experiments with in vitro translated, or synthetic, peptide indicate the ribosome as a likely target for the action of the identified rRNA-encoded peptide, which apparently remains associated with the ribosome after completion of its translation. The known properties of the rRNA-encoded peptide are compared with information about other functionally active short peptides that can be involved in regulation of translation.

Amino Acid Sequence↗

PubMed2001

Reverse transcription slippage over the mRNA secondary structure of the LIP1 gene.

The secondary structures in mRNA often cause early termination during the synthesis of cDNA. In an attempt to determine the 5'-untranslated region (UTR) of the gene LIP1 using the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we found that reverse transcriptases skipped over the LIP1 RNA secondary structures and continued the DNA synthesis through RNA adapter sequences without early termination. A fragment of only three nucleotides upstream of the LIP1 translation initiation codon was obtained from the initial RACE-PCR, which was much shorter than the 57-nucleotide fragment obtained from the cDNA library screening. Analysis of the 5' end sequence indicates the presence of high G+C content and stem-loop secondary structures. Therefore, optimizations of the reaction with high temperature (70 degrees C) and a thermostable reverse transcriptase were performed to synthesize the first-strand cDNA, which was determined to have 73 nucleotides in the 5'-UTR. These results suggest that, under cDNA synthesis conditions at 42 degrees C and 60 degrees C, the reverse transcriptases skipped over the stem-loop structures of LIP1 mRNA and continued the cDNA synthesis until they reached the RNA adapter sequences. Thisfinding draws attention to adopting optimized conditions for cDNA synthesis on G+C-rich RNA templates.

5' Untranslated Regions↗