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PubMed1989

Mature apolipoprotein AI and its precursor proApoAI: influence of the sequence at the 5' end of the gene on the efficiency of expression in Escherichia coli.

Apolipoprotein AI (ApoAI) plays a central role in the regulation of lipid metabolism. Initial attempts to express human apoAI cDNA in Escherichia coli did not yield detectable levels of the mature protein. By analyzing the efficiency of expression of apoAI-lacZ gene fusions, we have been able to show that the sequence at the 5' end of the ApoAI-coding region is a critical parameter. Indeed, silent changes in the codons for the first 8 residues of ApoAI, which did not alter the amino acid sequence, affected expression dramatically. Analysis of the corresponding mRNA steady-state levels suggested a role for differential mRNA stability in the control of apoAI expression in this system. Among all the possible alternative sequences, we have identified an optimal sequence which, when reinserted in the original expression plasmid, yields high level production of mature ApoAI. This procedure has been extended to the production of the natural variant ApoAI-Milano and the precursor proApoAI. Availability of these recombinant molecules would allow the investigation of their structural and biological features. In addition, the methodology used to optimize ApoAI expression is of general interest in assuring high expression of heterologous proteins in E. coli.

Amino Acid Sequence↗

PubMed1990

A generalized information function applied to the genetic code.

The problem of the partitioning of the degeneracy of the codons in the genetic code is considered in the framework of a generalized information function IG = c sigma kpk(ln pk + G(Ek] where k represents the number of codons in a specific degeneracy class and G(Ek) is an arbitrary real valued function. For G(Ek) = 0 the Shannon information function is recovered. For a particular choice of G(Ek) that takes the dominance of even degeneracies into account, it is found by direct numerical calculations that the correct degeneracy partitioning appears as optimal values of the Ig function. This results is also supported by optimization calculations in which the generalized information function is regarded as a continuous function in the degeneracy variables.

Amino Acids↗

PubMed1992

Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.

AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.

Alleles↗

PubMed1990

Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis.

The nucleotide sequence of the Synechococcus sp. PCC7942 glgB gene has been determined. The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206. Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp. glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa). In contrast, the N-terminal portions shared little homology. The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase. This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp. is different from that described for E. coli. A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp. glgB gene. The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1989

A general approach to isolating Plasmodium falciparum genes using non-redundant oligonucleotides inferred from protein sequences of other organisms.

We have constructed a number of oligonucleotide probes and tested their utility in identifying various genes in Plasmodium falciparum. The probe sequences were based on known conserved regions of proteins from other organisms, coupled with an analysis of the codon usage of the parasite. By using long single oligonucleotides, we have successfully isolated the DHFR-TS gene, two actin genes and two tubulin genes from the K1 (Thailand) isolate of P. falciparum. We compare these single probes to multiply-redundant short oligonucleotide probes and to heterologous probes. We also present a detailed quantitative analysis of optimal probe design, and of how this approach can best be implemented as a general method of isolating plasmodial genes.

Animals↗

PubMed1991

Characterization of an Escherichia coli gene encoding betaine aldehyde dehydrogenase (BADH): structural similarity to mammalian ALDHs and a plant BADH.

An open reading frame of 1476 nucleotides, cloned from a region of the Escherichia coli genome encoding betaine biosynthesis functions, was shown to encode a betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8). Either of two adjacent codons (5'-GTGATG) could function as a start codon, producing a presumptive polypeptide of 491 or 490 amino acids. The deduced primary structure of the E. coli BADH showed 39-43% positional identity, over its entire length, to aldehyde dehydrogenases (ALDH: EC 1.2.1.3) of mammalian origin. This similarity increased to 75-77% when conservative aa substitutions were also taken into consideration. Spinach BADH was also similar to the bacterial BADH, showing 38% identity and 80% overall similarity. Other homologs included a fungal and a putative bacterial ALDH. Although E. coli BADH was specific for the substrate, betaine aldehyde, it showed the highest levels of similarity to the prototype human ALDH-2. Only one gap in each sequence had to be introduced for optimal alignment. The conservation between E. coli BADH and the ALDHs was also evident in the predicted secondary structures and hydrophilicity profiles of the polypeptides, suggesting a similarity in the overall folding patterns of ALDH and BADH. These observations suggest a common ancestry for BADH and ALDH, preceding prokaryote-eukaryote divergence.

Aldehyde Dehydrogenase↗

PubMed1992

Cloning and sequence determination of a cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase.

A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.

Amino Acid Sequence↗

PubMed1979

Triphasic concentration effects of gentamicin on activity and misreading in protein synthesis.

Gentamicin is shown to exert a triphasic concentration effect on peptide synthesis in vitro with natural messengers. Low concentrations (up to 2 micron) caused slowing and a decrease in total synthesis, but little misreading (assayed with extracts lacking Glu-tRNA); the inhibition was greater with an initiating system (with phage RNA as messenger) than with pure chain elongation on purified endogenous polysomes of Escherichia coli. Moderate concentrations (up to 100 micron) slowed synthesis less, markedly increased its duration in the noninitiating system, and strongly stimulated misreading; at optimal concentrations total synthesis was even greater than normal. Moreover, with phage RNA these concentrations increased the synthesis of large polypeptides. We conclude that binding of gentamicin to its first site causes inhibition but little misreading; binding to additional site(s) partly reverses the inhibition by first-site binding and markedly stimulates misreading, and the misreading appears to favor "readthrough" of termination codons. In the third phase (greater than 100 micron) synthesis is slowed again but the pattern of misreading does not appear to be altered; this effect need not involve a specific further action on the ribosome.

Bacterial Proteins↗

PubMed1992

The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens.

The human prostate tumor cell line LNCaP contains an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induce reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.

Binding Sites↗

PubMed1989

Cloning and expression of the branching enzyme gene (glgB) from the cyanobacterium Synechococcus sp. PCC7942 in Escherichia coli.

Using the glgB gene from Escherichia coli as a hybridization probe, the gene encoding the branching enzyme of the cyanobacterium Synechococcus sp. PCC7942 has been identified on a 3.9-kb PstI fragment which was cloned into plasmid pUC9. Two types of plasmids have been isolated. Plasmid pKVN1 was expressing the Synechococcus sp. gene as was shown by complementation of the glgB mutation of E. coli KV832. Plasmid pKVN2, which carried the same insert in the opposite orientation was unable to complement E. coli KV832, indicating that the promoter of the cloned gene was either absent or was not recognized in E. coli. Determination of branching activity in extracts of Synechococcus sp. and E. coli KV832[pKVN1] showed that the enzyme was optimally active at approximately 35 degrees C. No significant activity was present at temperatures higher than 55 degrees C, reflecting the mesophilic nature of the cloned enzyme. In a cell-free coupled transcription-translation system the cloned gene specified two proteins of 84 kDa and 72 kDa, respectively, which are probably translated independently from the same gene by initiation at two different start codons.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1987

A standardized vector system for manipulation and enhanced expression of genes in Escherichia coli.

Different families of cloning and expression vectors were engineered on a standard plasmid. They contain several regulatory signals for transcription and/or translation initiation and termination. The plasmids in each series differ only in the number, type, and order of unique restriction cleavage sites clustered in front of a transcription terminator. The pLK30 plasmids are general cloning vectors and the corresponding pLK50 plasmids carry the lambda pL promoter. The pLK60 vectors carry the lambda pR promoter and translation initiation signals of the cro gene containing the Shine-Dalgarno sequence and initiation codon. The pLK70 series is similar to pLK60 except that additional 5'-translated cro sequences are included. The pLK80 plasmids have a lacZ gene fragment suitable for the construction of hybrid genes. The presence of translational stop signals in the pLK90 series facilitates the manipulation of genes truncated at the 3' end. This standardized pLK vector system offers great versatility in gene manipulation and in optimization of gene expression under the control of strong regulatable promoters. Measurement of expression levels under repressed conditions permits the identification of optimal promoter-gene configurations in constructions directing high-level expression.

Base Sequence↗

PubMed1987

Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite.

The DNA sequence containing the start of the Escherichia coli nirB gene is reported. The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point. Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon. By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208. Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth. Optimal activity was found only after anaerobic growth in the presence of nitrite. The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.

Bacterial Proteins↗

PubMed1988

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

Base Sequence↗

PubMed2026

The proteomic origin of the genetic code.

INTRODUCTION: The origin and evolution of the genetic code is a central problem in molecular biology. Classical models have emphasized stereochemistry, frozen accidents, or adaptive optimization, often treating proteins as passive products of preexisting codes. More recent views instead portray the code as a dynamic, coevolving system shaped by reciprocal interactions among amino acids, RNA, and early catalysts. AREAS COVERED: Here, I review efforts of phylogeny reconstruction of the history of tRNA, protein structural domains, and dipeptide sequences in proteomes. These complementary approaches allow exploration of the entry of amino acids and codons into the code, and the transition from an operational RNA code in the tRNA acceptor arm to the canonical code in the anticodon loop. Evidence for ancestral synthetase enzymes with dual functions in aminoacylation and peptide-bond formation, as well as early bidirectional (sense-antisense) coding reflected in dipeptide-antidipeptide emergence is also discussed. EXPERT OPINION: The genetic code is best viewed as a proteome-driven, evolvable system in which early peptides actively shaped coding rules by stabilizing structure, expanding chemical diversity, and enhancing catalysis. This perspective connects origin-of-life studies with modern efforts of code expansion, translational engineering, and peptide-based therapeutics, highlighting the impact of the code's proteomic origin.

Genetic Code↗

PubMed1989

Effect of deletions in the 5'-noncoding region on the translational efficiency of phosphoglycerate kinase mRNA in yeast.

Deletions of various sizes were introduced into the region of the yeast PGK gene encoding the 5'-nontranslated portion of the phosphoglycerate kinase (PGK) mRNA. The effect of these deletions on the translational efficiency of the mutant transcripts was analysed by assaying the levels of mutant PGK mRNA and PGK protein in cells transformed with the mutant genes. Quantification of transcript levels by either Northern analysis or a reverse transcription assay demonstrated that there were no significant differences in the levels of mutant PGK mRNA between the various mutants. Thus, the leader sequence does not appear to play a role in determining the relatively long half-life of yeast PGK mRNA. Analysis of PGK protein levels in the various mutants revealed no effect when the length of the leader was reduced from 45 to 27 nucleotides (nt). Protein levels dropped by about a factor 2, however, upon a further decrease to 21 nt. Additional shortening did not cause a further dramatic reduction in translational yield. Even an mRNA containing a leader of only 7 nt was still translated at about 50% of the optimal rate. Therefore, while optimal translation of a yeast mRNA requires a leader length of at least some 30 nt, shorter leaders still allow considerable translation to take place.

Base Sequence↗

PubMed1988

Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine-plus-cytosine contents.

An obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, produces multiple extracellular amylases. In addition to one of the amylase genes, amyA, which we previously cloned and characterized, we have cloned two additional genes, amyB and amyC, coding for amylases of this thermophile, into Escherichia coli and determined their nucleotide sequences. The two amylase genes were expressed under the control of E. coli promoters. Almost all activity was detected in the intracellular fraction in the E. coli cells. The molecular mass and NH2-terminal amino acid sequence of the AmyB enzyme, which was purified from an E. coli transformant containing the amyB gene, confirmed that the reading frame of amyB consisted of 562 amino acids (Mr 67,000). The molecular mass of the AmyC enzyme, estimated by activity staining of a crude extract of E. coli containing amyC, confirmed that AmyC consisted of 498 amino acids (Mr 59,000). The optimal temperatures for AmyB and AmyC activities on soluble starch were 80 degrees C and 70 degrees C, respectively. Both AmyB and AmyC showed a pH optimum of 5.5. AmyB and AmyC showed a different pattern of starch hydrolysis when examined by thin-layer chromatography. Some homology in the amino acid sequences with the functional regions of Taka-amylase A was found in both AmyB and AmyC. The codon usage in the amyA, amyB and amyC genes was highly biased, which reflects the fact that the guanine-plus-cytosine (G + C) content of DNA of D. thermophilum is 29 mol%. The distribution of G and C at each position of the codons was non-random; the G + C content of the first position of codons is significantly high, whereas that of the third position is somewhat low. In addition, codons consisting only of A and T were preferentially used in this thermophile.

Amino Acid Sequence↗

PubMed1992

Lipofectin enhances cellular uptake of antisense DNA while inhibiting tumor cell growth.

A natural DNA oligomer (15-mer) was synthesized with a sequence complementary to the translation initiation codon region of the human TGF-alpha mRNA and mixed with Lipofectin to form unilamellar complexes. It was found that tumor cell growth was inhibited when HCT116 cells were treated with Lipofectin-DNA oligomer complexes or with Lipofectin alone. Uptake of 32P-labeled 15-mers into colon tumor cells was compared in the presence and absence of Lipofectin. The amount of labeled oligomer found in cells that received optimal ratios of Lipofectin to DNA was 4- to 10-fold higher than the amount found in cells that received 32P-labeled DNA alone. Although Lipofectin-antisense DNA oligomer treatment of HCT116 cells caused a dose-dependent inhibition of cell growth, there was a subsequent rise in target mRNA product. Because the mechanism of growth inhibition could not involve an inhibition of TGF-alpha expression, it was concluded that Lipofectin probably exerts a nonspecific, detergent-like effect upon the cell membrane, producing an enhancement of TGF-alpha processing and release.

Base Sequence↗

PubMedDate not supplied

Compositional patterns in vertebrate genomes: conservation and change in evolution.

The evolution of vertebrate genomes can be investigated by analyzing their regional compositional patterns, namely the compositional distributions of large DNA fragments (in the 30-100-kb size range), of coding sequences, and of their different codon positions. This approach has shown the existence of two evolutionary modes. In the conservative mode, compositional patterns are maintained over long times (many million years), in spite of the accumulation of enormous numbers of base substitutions. In the transitional, or shifting, mode, compositional patterns change into new ones over much shorter times. The conservation of compositional patterns, which has been investigated in mammalian genomes, appears to be due in part to some measure of compositional conservation in the base substitution process, and in part to negative selection acting at regional (isochore) levels in the genome and eliminating deviations from a narrow range of values, presumably corresponding to optimal functional properties. On the other hand, shifts of compositional patterns, such as those that occurred between cold-blooded and warm-blooded vertebrates, appear to be due essentially to both negative and positive selection again operating at the isochore level, largely under the influence of changes in environmental conditions, and possibly taking advantage of mutational biases in the replication/repair enzymes and/or in the enzyme make-up of nucleotide precursor pools. Other events (like translocations and changes in chromosomal structure) also play a role in the transitional mode of genome evolution. The present findings (1) indicate that isochores, which correspond to the DNA segments of individual or contiguous chromatin domains, represent selection units in the vertebrate genome; and (2) shed new light on the selectionist-neutralist controversy.

Animals↗