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PubMed2002

Characterization of pMa025, a plasmid from the cyanobacterium Microcystis aeruginosa UV025.

The characterization of pMa025, a plasmid isolated from the unicellular, toxin-producing cyanobacterium Microcystis aeruginosa UV025, is described. A recombinant plasmid, pMaL [pMa025-pBluescript II SK(-)] was constructed for mapping, sequencing, and development of shuttle vectors capable of transforming both Escherichia coli and M. aeruginosa. pMa025 is 8,018 bp in length and has a G+C content of 62.3 mol%. Nineteen presumptive ORFs, ORF A - ORF S were identified using ATG or GTG as initiation codons. Fifteen different ORFs, ORF a - ORF o were identified using TGA as a degenerate codon for tryptophan. GTG was the start codon in two-thirds of the putative ORFs when TGA was the termination codon. GTG was the start codon in one-third of the putative ORFs when TGA was used as a codon for tryptophan. The deduced amino acid sequence from ORF j (3,114 bp) was significantly similar to that of a putative plasmid replication protein, RepA, from plasmid pUH24 of Synecho coccus sp. strain PCC7942. M. aeruginosa UV027 and E. coli were transformed to carbenicillin resistance with pMaL-D7, a 6.4-kb hybrid plasmid (3.46 kb pMa025, 2.95 kb pBluescript II) generated from the nested deletion strategy. pMaL-D7 will be used as a shuttle vector.

Base Sequence↗

PubMed1987

Sequence and structural features associated with translational initiator regions in yeast--a review.

We have compared the translational initiator regions of 131 yeast genes. 95% utilize the first AUG from the 5' end of the message as the start codon for translation. Yeast leader regions in general are rich in adenine nucleotides (nt), have an average length of 52 nt, and are void of significant secondary structure. Sequences immediately adjacent to AUG start codons are preferred, however, the bias in nucleotide distribution (5'-A-YAA-UAAUGUCU-3') does not reflect a higher eukaryotic consensus (5'-CACCAUGG-3') with the exception of an adenine nucleotide preference at the -3 position. A minority of yeast mRNAs that contain AUG codons in the leader region that do not serve as the start codon for the primary gene product differ from the majority of mRNAs by one or more of these general properties. This analysis appears to indicate that basic features associated with yeast leader regions are consistent with a general mechanism of initiation of protein synthesis in eukaryotes, as proposed by the ribosomal 'scanning' model, but perhaps only basic features associated with ribosomal recognition of an AUG start codon are intact.

Base Sequence↗

PubMed2001

Leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum.

We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S(1) nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from -50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the -1 position and a BRE element with more frequent A bases at position -29 to -32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.

5' Untranslated Regions↗

PubMed2001

Primary transcripts of ndhD of Liliaceae and Aloaceae require editing of the start and 20th codons.

About 350 nucleotide sequences around the start codon of the plastid ndhD gene were determined in six species to investigate the requirement of C to U editing of the cryptic start codons. The comparison with other sequences in databanks showed that, in contrast to grasses and similarly to dicots, Liliaceae and Aloaceae require editing of the primary transcript to generate the AUG start codon. Primary transcripts of Liliaceae and Aloaceae also show a new editing site at the 59th nucleotide, which has not been described in other plants. In some cases, transcripts showed partial editing which suggests that the synthesis of the protein may be controlled at the level of transcript processing.

Amino Acid Sequence↗

PubMed2004

[The relationship between a nucleotide substitution of S100A8 gene and aggressive periodontitis].

OBJECTIVES: To screen polymorphisms in the upstream region of S100A8 gene and to detect whether the polymorphisms were associated with aggressive periodontitis. METHODS: Thirty aggressive periodontitis patients and twenty-eight healthy controls were recruited for the study with informed consent. All subjects were of Chinese descent and systemically healthy. The regions about 800 bp upstream from the ATG start codon in exon 2 of the S100A8 gene of 10 patients and 8 controls were amplified by polymerase chain reaction (PCR) and analyzed by direct sequencing. A single nucleotide polymorphism (SNP) at 94 bp upstream from the ATG start codon was selected, and then the shorter regions (about 250 bp upstream from the ATG start codon) of the rest subjects were also amplified by PCR and analyzed by direct sequencing. The frequency of the SNP and the distribution of the genotype were detected and compared between the two groups. RESULTS: A nucleotide substitution (A-->G) at 94 bp upstream from the ATG start codon was demonstrated in Chinese, which was in a cis-acting element, named gamma interferon response element (gamma-IRE) in intron 1 of S100A8 gene. All of the subjects that carried the polymorphism were heterozygous. There was no statistically significant difference in the frequency of allele 2 (corresponding to the nucleotide G) between patients and controls (11.7% vs. 17.9%, chi2 = 0.887, P > 0.05). The prevalence of the heterozygous genotype was 23.2% and 35.7% (chi2 = 1.07, P > 0.05) in patients and controls, respectively. CONCLUSIONS: This is the first report that a nucleotide substitution of S100A8 gene was demonstrated in Chinese. The frequencies of allele 2 and heterozygous genotype were lower in patients, but there is no statistically significant difference between the aggressive periodontitis patients and healthy controls in this preliminary study.

Aggressive Periodontitis↗

PubMed1998

The p95 gene of Bombyx mori nuclear polyhedrosis virus: temporal expression and functional properties.

As part of our effort to identify baculovirus proteins acting as transcriptional regulators, we have characterized a gene, p95, of Bombyx mori nuclear polyhedrosis virus (BmNPV) that encompasses an open reading frame for a putative 95-kDa polypeptide (P95). The N-terminal half of the conceptually translated P95 contains two zinc finger-type DNA-binding motifs, and its C terminus contains a proline-rich region reminiscent of transcriptional activation regions. Northern blot analysis indicates that two mRNA species, 3.5 and 1.7 kb in size, are transcribed from the p95 gene at different times postinfection. These two mRNA species are produced by differential polyadenylation site usage. While the longer transcript can encode the P95 protein, the shorter one may encode a prematurely terminated version of the P95 polypeptide produced by ribosome frameshifting occurring at heptanucleotide "slippage" sites located near the relevant polyadenylation site. Transcription of the p95 gene is initiated at a proximal site located 70 nucleotides upstream of the translation start codon of P95, a middle site located 170 nucleotides from the start codon, and a set of three closely spaced distal sites located 385, 390, and 409 nucleotides from the translation start codon. The middle and distant initiation sites are utilized before and after BmNPV DNA replication, while transcripts initiated at the proximal site occur largely during the late and very late stages of viral infection. Transient-expression assays indicate that P95 can stimulate gene expression driven by the promoter of its own gene and the promoter of the cytoplasmic actin gene of B. mori. The P95-mediated trans activation can be further augmented by BmIE1, an immediate-early gene product of BmNPV. In contrast to the case with the actin promoter, however, the promoter of the p95 gene can be trans activated by the product of its own gene only in the presence of BmIE1. Our data suggest that proteins P95 and BmIE1 of BmNPV and, by analogy, those of other baculoviruses may interact with each other and synergize to potentiate transcription.

Amino Acid Sequence↗

PubMed2002

Characterization of overlapping XAGE-1 transcripts encoding a cancer testis antigen expressed in lung, breast, and other types of cancers.

Cancer testis (CT) antigens have an expression pattern that is predominantly restricted to testis in normal tissues, yet they are expressed in many different histological types of cancers. One previously described member of the CT antigen family, XAGE-1, was shown to be expressed in Ewing's sarcomas and rhabdomyosarcomas. Here we show that XAGE-1 is also expressed in breast cancer, prostate cancer, and different types of lung cancers, including lung squamous cell carcinoma, adenocarcinoma, small cell lung carcinoma, and non-small cell lung carcinoma. In addition, XAGE-1 mRNA was present in ovarian cancer, melanoma, glioblastoma, T-cell lymphoma, chronic myelogenous leukemia, and histiocytic lymphoma cell lines. We also characterized the XAGE-1 transcript by primer extension analysis and found that transcription of the XAGE-1 gene is initiated from two distinct start sites, resulting in two overlapping transcripts, XAGE-1a and XAGE-1b. XAGE-1a contains two in-frame ATG translational start codons; whereas XAGE-1b initiates downstream of the first ATG start codon. Our results suggest that XAGE-1b is the dominant transcript, and that translation begins with the second ATG start codon, producing a 9 kDa protein. Because XAGE-1 is expressed in such a diverse range of cancers, it has potential to be used as a target for many cancer immunotherapies.

Amino Acid Sequence↗

PubMed1987

Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity.

In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoglycosides↗

PubMed1991

Transient expression of deletion mutants of the herpes simplex virus thymidine kinase-encoding gene in mouse fibroblast cells.

Previous studies have shown that at least three polypeptides of 43, 39 and 38 kDa are translated from separate AUG codons of the thymidine kinase (TK) encoding mRNA of herpes simplex virus type 1. In addition, small tk-specific transcripts initiated within the tk coding region were observed. However, functional activity of these three proteins and their role in establishing of the TK+ cell phenotype is not yet clear. In order to locate the 5' boundary of the gene encoding functionally active TK, we constructed a set of deletion mutants with truncated 5' ends and examined their ability to provide a TK+ phenotype after microinjection into nuclei of LTK- cells. The results demonstrate that nucleotide sequences upstream from the second ATG codon can be removed without affecting the TK+ phenotype. Deletion of the second start codon and its downstream region inactivates the TK function. Those deletion mutants which contain only the third ATG codon are TK-. Thus, the 38-kDa polypeptide that initiates at the third start codon is not endowed with the TK+ activity. Constructs containing deletions up to nt +210 and lacking all 5'-end canonical and aberrant transcription control regions, as well as first start codon, can provide the TK+ function.

Animals↗

PubMed1990

Analysis of GPT activity in mammalian cells with a chromosomally integrated shuttle vector containing altered gpt genes.

The molecular mechanisms of reversion in mammalian cells were studied utilizing the pZipGptNeo shuttle vector, with the bacterial gpt gene in the vector integrated into the chromosomal DNA of mouse cells. From mutant cell lines containing gpt genes with single base changes, revertants were selected for the reappearance of GPT activity. The copy number and expression of the gpt genes in such revertants were analyzed, and the GPT activity encoded by revertant genes in both mammalian cells and bacteria characterized. Revertants with wild-type amino acid sequence had, on average, the highest levels of GPT activity. Revertants with amino acid sequences different from the original mutants but not corresponding to wild-type had, on average, approximately half the level of GPT activity as wild-type revertants. Revertants that still contained the original mutation in the gpt gene had even lower levels of activity. These revertants were found to have amplified mutant gpt genes, which, when transferred into bacteria, were seen to encode for GPT polypeptides with partial enzymatic activity. A revertant in which the original mutation that destroyed the AUG translational start codon was retained but in which there was a secondary mutation upstream of the start codon also was characterized. The second mutation generated an in-frame CUG codon that apparently functioned as an alternative, upstream translational start codon.

Animals↗

PubMed1998

Multiple promoters are responsible for transcription of the glpEGR operon of Escherichia coli K-12.

The transcriptional organization of the glpEGR genes of Escherichia coli was studied. Besides a promoter located upstream of the glpE start codon, three internal glpGR promoters were identified that express glpG and/or glpR (glp repressor). One promoter was located just upstream of the glpG start codon and two others (separated by several hundred base pairs) were located within glpG upstream of the glpR start codon. The transcriptional start points of these promoters were identified by primer extension analysis. The strengths of the individual promoters were compared by analysis of their expression when fused to a pormoter-probe vector. Analysis of the transcriptional expression of the glpEGR sequence with different combinations of the glpEGR promoters revealed no internal transcriptional terminators within the entire operon. Thus, the glpEGR genes are co-transcribed and form a single complex operon. The presence of multiple promoters may provide for differential expression of glpE, glpG and glpR. Potential regulation of the operon promoters by GlpR, catabolite repression, anaerobiosis or by FIS was studied. The glpE promoter was apparently controlled by the cAMP-CRP complex, but none of the promoters was responsive to specific repression by GlpR, to anaerobiosis or to FIS. Specific repression exerted by GlpR was characterized in vivo using glpD-lacZ and glpK-lacZ fusions. The degree of repression was correlated with the level of GlpR expression, and was inefficient when the glpD-encoded glycerol-P dehydrogenase was absent, presumably due to accumulation of the inducer, glycerol-P. This is in contrast to the previous conclusion that gpsA-encoded glycerol-P synthase tightly controls the cellular level of glycerol-P by end product inhibition.

Amino Acid Sequence↗

PubMed1999

Transcript mapping and transregulatory behavior of varicella-zoster virus gene 21, a latency-associated gene.

Gene 21 is one of at least four genes transcribed during latent infection of varicella-zoster virus (VZV) in human ganglia. It may encode a nucleocapsid protein, but its function in lytic and latent infection is not clear. To characterize further the structure and the function of the gene 21 open reading frame (ORF 21), precise localization of its transcripts and their termini was determined by using Northern analysis, S1 nuclease or RNase protection, and primer extension assays. One abundant 3.5-kb transcript that spans ORF 21 was identified. A predominant transcription start site was defined at -78 nucleotide (nt) relative to the ORF 21 translation start codon ATG, and two potential TATA elements were identified at 26 and 83 nt upstream of the 5' end of gene 21 transcripts. Transcription was found to terminate 210 nt beyond the ORF 21 translation stop codon and immediately before the start codon of ORF 22. In transient expression assays, the ORF 21 showed no significant transregulatory activity on promoters of diverse kinetic classes. The ORF 21 promoter, however, was transactivated strongly by VZV infection or by ORF 62.

Base Sequence↗

PubMed2002

Cloning and characterization of a testis and brain-specific isoform of mouse 3'-phosphoinositide-dependent protein kinase-1, mPDK-1 beta.

3'-Phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylates and activates members of the protein kinase AGC family and plays a key role in receptor tyrosine kinase signaling. Here we report the cloning and characterization of a splice variant of mouse PDK-1, mPDK-1 beta. The cDNA encoding mPDK-1 beta contains two alternative start codons and translation from these start codons generates proteins that are, respectively, 27 or 51 amino acid residues shorter at the amino-terminus than the previously identified PDK-1 isolated from mouse liver (now renamed mPDK-1 alpha) [J. Biol. Chem. 274 (1999) 8117]. Analysis of mouse tissues shows that mPDK-1 beta is highly expressed in the testis and various functional regions of the brain. Expression of this isoform is increased in the brain of aged mice. Both mPDK-1 alpha and mPDK-1 beta are autophosphorylated at both serine and threonine residues in vitro and showed similar levels of tyrosine phosphorylation when co-expressed with either constitutively active Src or Fyn tyrosine kinases in cells. However, the mPDK-1 isoforms showed significant differences in their response to pervanadate- or insulin plus vanadate-stimulated tyrosine phosphorylation. Taken together, our findings suggest that the two PDK-1 isoforms may be differentially regulated in cells. The specific expression of mPDK-1 beta in mouse testis and brains of aged mice also suggests potential involvement of this kinase in regulating animal spermatogenesis and aging.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed1999

Poly-3-hydroxybutyrate degradation in Rhizobium (Sinorhizobium) meliloti: isolation and characterization of a gene encoding 3-hydroxybutyrate dehydrogenase.

We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a sigma54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying a lacZ transcriptional fusion to bdhA demonstrated that gene expression is growth phase associated.

Amino Acid Sequence↗

PubMed2005

Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene.

BACKGROUND: The rTS gene (ENOSF1), first identified in Homo sapiens as a gene complementary to the thymidylate synthase (TYMS) mRNA, is known to encode two protein isoforms, rTSalpha and rTSbeta. The rTSbeta isoform appears to be an enzyme responsible for the synthesis of signaling molecules involved in the down-regulation of thymidylate synthase, but the exact cellular functions of rTS genes are largely unknown. RESULTS: Through comparative genomic sequence analysis, we predicted the existence of a novel protein isoform, rTS, which has a 27 residue longer N-terminus by virtue of utilizing an alternative start codon located upstream of the start codon in rTSbeta. We observed that a similar extended N-terminus could be predicted in all rTS genes for which genomic sequences are available and the extended regions are conserved from bacteria to human. Therefore, we reasoned that the protein with the extended N-terminus might represent an ancestral form of the rTS protein. Sequence analysis strongly predicts a mitochondrial signal sequence in the extended N-terminal of human rTSgamma, which is absent in rTSbeta. We confirmed the existence of rTS in human mitochondria experimentally by demonstrating the presence of both rTSgamma and rTSbeta proteins in mitochondria isolated by subcellular fractionation. In addition, our comprehensive analysis of rTS orthologous sequences reveals an unusual phylogenetic distribution of this gene, which suggests the occurrence of one or more horizontal gene transfer events. CONCLUSION: The presence of two rTS isoforms in mitochondria suggests that the rTS signaling pathway may be active within mitochondria. Our report also presents an example of identifying novel protein isoforms and for improving gene annotation through comparative genomic analysis.

Amino Acid Sequence↗

PubMed1997

Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli.

The chloramphenicol acetyltransferase (cat) gene from Streptomyces acrimycini encodes a leaderless mRNA. Expression of the cat coding sequence as a leaderless mRNA from a modified lac promoter resulted in chloramphenicol resistance in Escherichia coli. Transcript mapping with nuclease S1 confirmed that the 5' end of the cat message initiated at the A of the AUG translational start codon. Site-directed mutagenesis of the lac promoter or the cat start codon abolished chloramphenicol resistance, indicating that E. coli initiated translation at the 5' terminal AUG of the cat leaderless mRNA. Addition of 5'-AUGC-3' to the 5' end of the cat mRNA resulted in translation occurring also from the reading frame defined by the added AUG triplet, suggesting that a 5'-terminal start codon is an important recognition feature for initiation and establishing reading frame during translation of leaderless mRNA. Addition of an untranslated leader and Shine-Dalgarno sequence to the cat coding sequence increased cat expression in a cat:lacZ fusion; however, the level of expression was significantly lower than when a fragment of the bacteriophage lambda cI gene, also encoding a leaderless mRNA, was fused to lacZ. These results indicate that in the absence of an untranslated leader and Shine-Dalgarno sequence, the streptomycete cat mRNA is translated by E. coli; however, the cat translation signals, or other features of the cat mRNA, provide for only a low level of expression in E. coli.

Chloramphenicol↗

PubMed1997

Requirements for ribosomal protein S1 for translation initiation of mRNAs with and without a 5' leader sequence.

It has previously been proposed that Escherichia coli ribosomal protein S1 is required for the translation of highly structured mRNAs. In this study, we have examined the influence of structural features at or near the start codon of different mRNAs. The requirement for ribosomal protein S1 for translation initiation was determined when (i) the ribosome-binding site (RBS) was either preceded by a 5' non-translated leader sequence; (ii) the RBS was located 5' proximal to a mRNA start codon; and (iii) the start codon was the 5' terminal codon as exemplified by leaderless mRNAs. In vitro translation studies revealed that the leaderless lambda cl mRNA is translated with Bacillus stearothermophilusribosomes, naturally lacking a ribosomal protein S1 homologue, whereas ompA mRNA containing a 5' leader is not. These studies have been verified by toeprinting with E. coli ribosomes depleted for S1. We have shown that S1 is required for ternary complex formation on ompA mRNA but not for leaderless mRNAs or for mRNAs in which the RBS is close to the 5' end.

5' Untranslated Regions↗

PubMed2005

An encoded N-terminal extension results in low levels of heterologous protein production in Escherichia coli.

BACKGROUND: The tdk gene (encoding deoxythymidine kinase) of the gamma-proteobacterium Xenorhabdus nematophila has two potential translation start sites. The promoter-distal start site was predicted to be functional based on amino acid sequence alignment with closely related Tdk proteins. However, to experimentally determine if either of the two possible start codons allows production of a functional Tdk, we expressed the "long-form" (using the promoter-proximal start codon) and "short-form" (using the promoter-distal start codon) X. nematophila tdk genes from the T7 promoter of the pET-28a(+) vector. We assessed Tdk production and activity using a functional assay in an Escherichia coli tdk mutant, which, since it lacks functional Tdk, is able to grow in 5-fluorodeoxyuridine (FUdR)-containing medium. RESULTS: Short-form Tdk complemented the E. coli tdk mutant strain, resulting in FUdR sensitivity of the strain. However, the E. coli tdk mutant expressing the long form of tdk remained FUdR resistant, indicating it did not have a functional deoxythymidine kinase enzyme. We report that long-form Tdk is at least 13-fold less abundant than short-form Tdk, the limited protein produced was as stable as short-form Tdk and the long-form transcript was 1.7-fold less abundant than short-form transcript. Additionally, we report that the long-form extension was sufficient to decrease heterologous production of a different X. nematophila protein, NilC. CONCLUSION: We conclude that the difference in the FUdR growth phenotype between the E. coli tdk mutant carrying the long-or short-form X. nematophila tdk is due to a difference in Tdk levels. The lower long-form protein level does not result from protein instability, but instead from reduced transcript levels possibly combined with reduced translation efficiency. Because the observed effect of the encoded N-terminal extension is not specific to Tdk production and can be overcome with induction of gene expression, these results may have particular relevance to researchers attempting to limit production of toxic proteins under non-inducing conditions.

Journal Article↗